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Dietary proteins as precursors of dipeptidyl-peptidase IV inhibitors — allies to complement pharmacotherapy… Lacroix, Isabelle Marie Estelle 2015

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DIETARY(PROTEINS(AS(PRECURSORS(OF(DIPEPTIDYL1PEPTIDASE(IV((INHIBITORS(—(ALLIES(TO(COMPLEMENT(PHARMACOTHERAPY(IN(THE(MANAGEMENT(OF(TYPE(2(DIABETES((by##Isabelle#Marie#Estelle#Lacroix###B.Sc.,#Université#Laval,#2008###A#THESIS#SUBMITTED#IN#PARTIAL#FULFILLMENT#OF##THE#REQUIREMENTS#FOR#THE#DEGREE####DOCTOR#OF#PHILOSOPHY#in#THE#FACULTY#OF#GRADUATE#AND#POSTDOCTORAL#STUDIES#(Food#Science)####THE#UNIVERSITY#OF#BRITISH#COLUMBIA#(Vancouver)###April#2015#©#Isabelle#Marie#Estelle#Lacroix,#2015## ii#ABSTRACT'#Inhibition#of#the#enzyme#dipeptidylVpeptidase#IV#(DPPVIV)#is#a#promising#approach#for#managing#hyperglycemia#in#type#2#diabetes.#The#overall#objective#of#this#study#was#to#produce,#from#dietary#proteins,#peptides#able#to#inhibit#the#activity#of#DPPVIV#and#study#their#mechanisms#of#action.###The# potential# of# proteins# from# various# food# commodities# to# serve# as# precursors# of#DPPVIV#inhibitors#was#first#investigated#using#an# in#silico#approach.#Peptide#sequences#reported#to#have#DPPVIV#inhibitory#activity#were#found#in#all#proteins#studied,#including#those#from#milk,#a# food#product#shown#in#several#studies#to#have#beneficial#effects#on#glycemic# regulation.# Therefore,# milk# proteins# were# selected# for# hydrolysis# using#different# proteases# to# release# the# active# fragments.# Fractionation# of# the# most# active#hydrolysates,# the#peptic#digests#of#whey#protein# isolate#and#αVlactalbumin,#revealed#a#number# of# peptides# with# varying# effectiveness# and# modes# of# inhibition.# Among# the#sequences# identified,# the# βVlactoglobulinVderived# peptides# 46LKPTPEGDL54# and#46LKPTPEGDLEIL57#displayed#the#greatest#potency#(IC50#=#54#and#57#µM,#respectively)#and#inhibited#DPPVIV#in#an#unVcompetitive#manner.###Peptide#arrays#were#investigated#as#an#alternative#strategy#to#screen#food#proteins#for#the# presence# of# DPPVIV# inhibitory# peptides# within# their# sequences.# Using# SPOT#technology,# 114# decaVpeptides# spanning# the# entire# sequence# of# αVlactalbumin# were#synthesized#on#cellulose#membranes#and#their#binding#to#and#inhibition#of#DPPVIV#were#studied.# SPOTV# and# traditionallyVsynthesized# peptides# displayed# consistent# trends# in#DPPVIV# inhibitory#activity,#confirming# that#peptide#arrays#can#be#used# to#complement#or#support#the#traditional#methods#currently#used#to#identify#DPPVIV#inhibitors.##Lastly,#the#effect#of#proteinVderived#peptides#on#the#activity#of#porcine#and#human#DPPVIV,# the# two# most# commonly# used# species# to# assess# DPPVIV# activity# in# vitro,# was#compared.# The# enzymes# differed# in# their# susceptibility# to# inhibition# by# 43# of# the# 62### iii#peptides#investigated.#Generally,#porcine#DPPVIV#was#inhibited#more#strongly#than#the#human#enzyme.##Findings#from#this#research#showed#that#DPPVIV# inhibitory#peptides#can#be#generated#from# dairy# proteins.# These# natural# inhibitors,# although# less# effective# than# synthetic#drugs,# could# be# used# to# complement# pharmacotherapy# in# the#management# of# type# 2#diabetes.#Additional#research#to#evaluate#their#efficacy#in#humans#is,#however,#needed.######## ### iv#PREFACE'#The# information# presented# in# the# literature# review# sections# 2.1.1,# 2.1.2# (with# the#exception# of# section# 2.1.2.11),# 2.1.3# as# well# as# Tables# 2.1# (with# the# exception# of#information# on# sodiumVdependent# glucose# coVtransporter# 2# inhibitors)# &# 2.2# and#Figure# 2.1# has# been# published# in# the# journal#Molecular#Nutrition# and# Food#Research#(2014,#58,#61–78)#under#the#title#“Overview#of#food#products#and#dietary#constituents#with# antidiabetic# properties# and# their# putative# mechanisms# of# action:# A# natural#approach#to#complement#pharmacotherapy#in#the#management#of#diabetes”.#I#was#the#lead#author#of#this#manuscript#and#my#supervisor#E.C.Y#LiVChan#the#coVauthor.###The#material#presented#in#section#2.1.4.1#and#a#portion#of#Table#2.3#are#extracted#from#a#review# I# authored# entitled# “Investigation# of# the# putative# associations# between# dairy#consumption#and#incidence#of#type#1#and#type#2#diabetes”#published#in#Critical#Reviews#in# Food# Science# and# Nutrition# (2014,# 54,# 411–432).# E.C.Y.# LiVChan# coVauthored# this#article.###The#work#presented#in#Chapter#4#has#been#published#in#the#Journal#of#Functional#Foods#(2012,#4,# 403–422)# under# the# title# “Evaluation# of# the# potential# of# dietary# proteins# as#precursors#of#dipeptidyl#peptidase#(DPP)VIV#inhibitors#by#an# in#silico#approach”.#E.C.Y.#LiVChan#and#I#designed#the#study;# I#conducted#the# literature#review,#performed#the# in#silico# analysis# and# wrote# the# manuscript.# E.C.Y.# LiVChan# provided# feedback# on# the#manuscript#and#made#editorial#changes.##A#version#of#Chapter#5#was#published#in#the#International#Dairy#Journal#(2012,#25,#97–102)# under# the# title# “Dipeptidyl# peptidaseVIV# inhibitory# activity# of# dairy# protein#hydrolysates”.# The# introduction# of# the# published# article# was# modified# for# this#dissertation#by#the#removal#of#most#of#the#information#presented#in#the#first#and#second#paragraphs.# # E.C.Y.# LiVChan# and# I# designed# the# study;# I# performed# the# experiments,#analyzed# the# data# and# wrote# the# manuscript.# E.C.Y.# LiVChan# critically# reviewed# the#manuscript#and#made#editorial#changes.####  v A version of Chapter 6 was published in the Journal of Agricultural and Food Chemistry (2013, 61, 7500–7506) under the title “Inhibition of dipeptidyl peptidase (DPP)-IV and D-glucosidase activities by pepsin-treated whey proteins” (http://pubs.acs.org/articlesonrequest/AOR-inmJJxtEHhjrap4qGPku). The introduction of the published article was modified for this dissertation by the removal of most of the information presented in the second paragraph. E.C.Y. Li-Chan and I designed the study; I performed the experimental work, analyzed the data and wrote the manuscript. E.C.Y. Li-Chan critically reviewed the manuscript and made editorial changes.  A version of Chapter 7 and the information presented in Appendix A were published in the journal Peptides (2014, 54, 39–48), under the title “Isolation and characterization of peptides with dipeptidyl peptidase-IV inhibitory activity from pepsin treated bovine whey proteins”. The introduction of the published article was modified for this dissertation by the removal of the information presented in the second paragraph. E.C.Y. Li-Chan and I designed the study; I conducted the experiments, analyzed the data and wrote the manuscript. E.C.Y. Li-Chan critically reviewed the manuscript and made editorial changes.  The work presented in Chapter 8 and Appendix B has been published in the International Journal of Molecular Sciences (2014, 15, 20846–20858) under the title “Peptide array on cellulose-support — a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of D-lactalbumin”. E.C.Y. Li-Chan and I designed the study; I performed the experiments, analyzed the data and wrote the manuscript. E.C.Y. Li-Chan critically reviewed the manuscript and made editorial changes.  The work presented in Chapter 9 and Appendix C has been accepted for publication in the journal Peptides (2015, http://dx.doi.org/10.1016/j.peptides.2015.03.016), under the title “Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides”. E.C.Y. Li-Chan and I designed the study; I ## vi#performed#the#experiments,#analyzed#the#data#and#wrote#the#manuscript.#E.C.Y.#LiVChan#critically#reviewed#the#manuscript#and#made#editorial#changes.##Some# wordings# and# the# formatting# of# the# published# materials# described# in# this#dissertation# have# been# modified# to# allow# consistency# and# adequate# flow# of# the#information#presented#as#well#as# to#ensure#compliance#with#The#University#of#British#Columbia#thesis#and#dissertation#formatting#requirements.#### ### vii#TABLE'OF'CONTENTS'#ABSTRACT(........................................................................................................................................(ii#PREFACE(..........................................................................................................................................(iv#TABLE(OF(CONTENTS(................................................................................................................(vii#LIST(OF(TABLES(..........................................................................................................................(xiv#LIST(OF(FIGURES(.......................................................................................................................(xvii#LIST(OF(ABBREVIATIONS(..........................................................................................................(xx#ACKNOWLEDGEMENTS(...........................................................................................................(xxii#CHAPTER(1(:(INTRODUCTION(....................................................................................................(1#CHAPTER(2(:(LITERATURE(REVIEW(........................................................................................(3#2.1#DIABETES#............................................................................................................................................#3#2.1.1#Pathophysiology#of#Diabetes#..............................................................................................#3#2.1.2# Overview# of# the# Pharmacological# Therapies# for# the# Management# of#Diabetes#and#Their#Mode#of#Action#............................................................................................#9#2.1.2.1#Sulfonylureas#....................................................................................................................#9#2.1.2.2#Meglitinides#......................................................................................................................#9#2.1.2.3#Biguanides#......................................................................................................................#10#2.1.2.4#Thiazolidinediones#.....................................................................................................#11#2.1.2.5#AlphaVGlucosidase#Inhibitors#.................................................................................#11#2.1.2.6#GlucagonVLike#PeptideV1#(GLPV1)#Mimetics#....................................................#12#2.1.2.7#DipeptidylVPeptidase#IV#Inhibitors#......................................................................#12#2.1.2.8#Amylin#Analogues#........................................................................................................#13#2.1.2.9#Dopamine#D2VReceptor#Agonists#.........................................................................#13#2.1.2.10#Bile#Acid#Sequestrants#............................................................................................#14#2.1.2.11#SodiumVDependent#Glucose#CoVTransporter#2#Inhibitors#......................#14#2.1.2.12#Exogenous#Insulin#....................................................................................................#15#2.1.3#Role#of#Diet#in#the#Incidence#and#Management#of#Diabetes#..............................#15#2.1.3.1#Food#Products#and#Dietary#Constituents#that#Affect#Glucose#Uptake#..#19### viii#2.1.3.1.1#Inhibition#of#CarbohydrateVHydrolyzing#Enzymes#..............................#19#2.1.3.1.2#Inhibition#of#Intestinal#Glucose#Transport#..............................................#21#2.1.3.1.3#Inhibition#of#Hepatic#Glucose#Output#.........................................................#22#2.1.3.1.4# Delay# of# Gastric# Emptying# and/or# Intestinal# Absorption# of#Carbohydrates#........................................................................................................................#23#2.1.3.2#Food#Products#and#Dietary#Constituents#that#Affect#the#Incretin#Effect#............................................................................................................................................................#24#2.1.3.2.1#Stimulation#of#GlucagonVLike#PeptideV1#Secretion#..............................#24#2.1.3.2.2#Inhibition#of#DipeptidylVPeptidase#IV#(DPPVIV)#....................................#25#2.1.3.3# Food# Products# and# Dietary# Constituents# that# Mimic# Insulin# Action,#Affect#Insulin#Release#or#Sensitivity#...................................................................................#26#2.1.3.3.1#Inhibition#of#Protein#Tyrosine#Phosphatase#IB#.....................................#26#2.1.3.3.2#Activation#of#Peroxisome#ProliferatorVActivated#Receptor#Gamma#(PPARγ)#......................................................................................................................................#27#2.1.3.3.3#InsulinVMimetic#Action#.....................................................................................#28#2.1.3.3.4#Insulin#Secretagogues#.......................................................................................#30#2.1.3.4#Food#Products#and#Dietary#Constituents#that#Alleviate#Oxidative#Stress#............................................................................................................................................................#31#2.1.4# Putative# Association# Between# the# Consumption# of# Dairy# Foods# and# the#Incidence#of#Diabetes#....................................................................................................................#33#2.1.4.1#Dairy#Consumption#and#Type#2#Diabetes:#Possible#Mechanisms#...........#39#2.1.4.1.1#Effects#of#NonVprotein#Constituents# in#Dairy#Products#on#the#Risk#of#Type#2#Diabetes#.................................................................................................................#39#2.1.4.1.2#Effects#of#Milk#Casein#and#Whey#Proteins#on#Glucose#Regulation# 43#2.2#INCRETIN#HORMONES#...............................................................................................................#45#2.3#DIPEPTIDYLVPEPTIDASE#IV#....................................................................................................#47#2.4#BIOACTIVE#PEPTIDES#................................................................................................................#53#2.4.1#Approaches#to#Identify#Bioactive#Peptides#from#Dietary#Proteins#................#53#2.4.2#Dairy#Proteins#as#Sources#of#Bioactive#Peptides#....................................................#55#2.4.3#Dietary#Proteins#as#Sources#of#Peptides#with#DPPVIV#Inhibitory#Activity#...#57### ix#2.4.3.1#Milk#ProteinVDerived#Peptides#with#DPPVIV#Inhibitory#Activity#............#59#2.4.3.2#In#Vivo#Effect#of#Milk#Derived#Peptides#and#Dairy#Protein#Hydrolysates#on#DPPVIV#Activity#......................................................................................................................#64#CHAPTER(3(:(OBJECTIVES(AND(EXPERIMENTAL(APPROACHES(...................................(66#3.1#OBJECTIVES#....................................................................................................................................#66#3.2#OVERVIEW#OF#EXPERIMENTAL#APPROACHES#..............................................................#67#CHAPTER( 4( :( EVALUATION( OF( THE( POTENTIAL( OF( DIETARY( PROTEINS( AS(PRECURSORS( OF( DIPEPTIDYL1PEPTIDASE( IV( INHIBITORS( BY( AN( IN# SILICO(APPROACH(.....................................................................................................................................(68#4.1#SUMMARY#........................................................................................................................................#68#4.2#INTRODUCTION#............................................................................................................................#68#4.3#MATERIAL#AND#METHODS#......................................................................................................#71#4.3.1#Proteins#....................................................................................................................................#71#4.3.2#Peptides#with#DPPVIV#Inhibitory#Activity#..................................................................#71#4.3.3#In#Silico#Analysis#...................................................................................................................#71#4.4#RESULTS#AND#DISCUSSION#.....................................................................................................#72#4.4.1# Peptides# Reported# to# Present# an# Inhibitory# Activity# Against# the# Enzyme#DPPVIV#..................................................................................................................................................#72#4.4.2#Potential#of#Dietary#Proteins#to#Serve#as#DPPVIV#Inhibitor#Precursors#.......#78#4.4.2.1#DPPVIV#Inhibitory#Peptides#in#Plant#Proteins#.................................................#79#4.4.2.2#DPPVIV#Inhibitory#Peptides#in#Milk#and#Chicken#Egg#Proteins#................#87#4.4.2.3#DPPVIV#Inhibitory#Peptides#in#Bovine#Meat#and#Salmon#Proteins#.........#92#4.5#CONCLUSION#................................................................................................................................#102#CHAPTER( 5( :( DIPEPTIDYL1PEPTIDASE( IV( INHIBITORY( ACTIVITY( OF( DAIRY(PROTEIN(HYDROLYSATES(.....................................................................................................(103#5.1#SUMMARY#......................................................................................................................................#103#5.2#INTRODUCTION#..........................................................................................................................#103#5.3#MATERIALS#AND#METHODS#.................................................................................................#105#5.3.1#Materials#................................................................................................................................#105#5.3.2#In#Vitro#Simulated#Gastrointestinal#(GI)#Digestion#of#Dairy#Ingredients#...#106### x#5.3.3#Enzymatic#Hydrolysis#of#Sodium#Caseinate#and#Whey#Protein#Isolate#......#107#5.3.3.1#Enzyme#Screening#.....................................................................................................#107#5.3.3.2#Hydrolysis#Using#Selected#Enzymes#..................................................................#108#5.3.4#Ultrafiltration#of#Sodium#Caseinate#and#Whey#Protein#Isolate#Hydrolysates#...............................................................................................................................................................#108#5.3.5#Extent#of#Hydrolysis#.........................................................................................................#109#5.3.6#DPPVIV#Inhibitory#Activity#Assay#................................................................................#109#5.3.7#Statistical#Analysis#.............................................................................................................#110#5.4#RESULTS#AND#DISCUSSION#...................................................................................................#110#5.4.1# Extent# of# Hydrolysis# and# DPPVIV# Inhibitory# Activity# of# Dairy# Ingredients#During#In#Vitro#Digestion#with#Pepsin#and#Pancreatin#.................................................#110#5.4.2# Assessment# of# the# DPPVIV# Inhibitory# Activity# of# Sodium# Caseinate# and#Whey#Protein#Isolate#Hydrolysates#Obtained#Using#Various#Proteases#...............#114#5.4.3#Fractionation#of#Sodium#Caseinate#and#WPI#Hydrolysates#Showing#Potent#DPPVIV#Inhibitory#Activity#.........................................................................................................#116#5.5#CONCLUSION#................................................................................................................................#119#CHAPTER( 6( :( ( INHIBITION( OF( DIPEPTIDYL1PEPTIDASE( IV( AND(α1GLUCOSIDASE(ACTIVITY(BY(PEPSIN1TREATED(WHEY(PROTEINS(........................................................(120#6.1#SUMMARY#......................................................................................................................................#120#6.2#INTRODUCTION#..........................................................................................................................#120#6.3#MATERIALS#AND#METHODS#.................................................................................................#123#6.3.1#Materials#................................................................................................................................#123#6.3.2#Peptic#Hydrolysis#of#Whey#Proteins#..........................................................................#124#6.3.3#Extent#of#Hydrolysis#.........................................................................................................#124#6.3.4#SDSVPAGE#..............................................................................................................................#125#6.3.5#Determination#of#DPPVIV#Inhibitory#Activity#.........................................................#125#6.3.6#Extraction#of#Rat#Intestinal#αVGlucosidase#.............................................................#126#6.3.7#Determination#of#αVGlucosidase#Inhibitory#Activity#..........................................#127#6.3.8#Statistical#Analysis#.............................................................................................................#128#6.4#RESULTS#AND#DISCUSSION#...................................................................................................#128### xi#6.4.1#Extent#of#Hydrolysis#of#PepsinVTreated#Whey#Proteins#...................................#128#6.4.2#DPPVIV#Inhibitory#Activity#of#PepsinVTreated#Whey#Proteins#.......................#130#6.4.3#αVGlucosidase#Inhibitory#Activity#of#PepsinVTreated#Whey#Proteins#.........#133#6.5#CONCLUSION#................................................................................................................................#136#CHAPTER( 7( :( ISOLATION( AND( CHARACTERIZATION( OF( PEPTIDES( WITH(DIPEPTIDYL1PEPTIDASE( IV( INHIBITORY( ACTIVITY( FROM( PEPSIN1TREATED(BOVINE(WHEY(PROTEINS(......................................................................................................(137#7.1#SUMMARY#......................................................................................................................................#137#7.2#INTRODUCTION#..........................................................................................................................#137#7.3#MATERIALS#AND#METHODS#.................................................................................................#139#7.3.1#Materials#................................................................................................................................#139#7.3.2#Preparation#of#Whey#Protein#Hydrolysates#...........................................................#140#7.3.3# Isolation# of# DPPVIV# Inhibitory# Peptides# from#Whey# Protein#Hydrolysates#...............................................................................................................................................................#140#7.3.3.1#CationVExchange#Chromatography#....................................................................#140#7.3.3.2#SizeVExclusion#Chromatography#.........................................................................#141#7.3.3.3#ReversedVPhase#High#Performance#Liquid#Chromatography#(RPVHPLC)#..........................................................................................................................................................#142#7.3.4# Identification# of# Peptide# Sequences# by# Liquid# ChromatographyVElectrospray#IonisationVTandem#Mass#Spectrometry#(LC–ESIVMS/MS)#..............#142#7.3.5#Peptide#Synthesis#...............................................................................................................#143#7.3.6#DPPVIV#Inhibitory#Activity#Assay#................................................................................#143#7.3.7#Determination#of#the#Modes#of#Inhibition#...............................................................#144#7.3.8#Statistical#Analysis#.............................................................................................................#145#7.4#RESULTS#.........................................................................................................................................#145#7.4.1# Fractionation# of# PepsinVTreated# Whey# Proteins# with# DPPVIV# Inhibitory#Activity#...............................................................................................................................................#145#7.4.1.1#Fractionation#of#PepsinVTreated#Whey#Protein#Isolate#............................#145#7.4.1.2#Fractionation#of#PepsinVTreated#αVLactalbumin#.........................................#150### xii#7.4.1.3# DPPVIV# Inhibitory# Activity# of# Fractions# Isolated# from# PepsinVTreated#WPI#and#αVLactalbumin#.........................................................................................................#153#7.4.2# Identification# of# the# Peptide# Sequences# Present# in# the# Purified# Fractions#WPIV5bV3,#WPIV5bV4,#αVlaV2e#and#αVlaV2f#by#LC–ESIVMS/MS#.....................................#153#7.4.3#DPPVIV#Inhibitory#Activity#of#Synthesized#Peptides#...........................................#154#7.4.4#Modes#of#Inhibition#of#Synthesized#Peptides#.........................................................#158#7.5#DISCUSSION#..................................................................................................................................#160#7.6#CONCLUSION#................................................................................................................................#163#CHAPTER(8(:(PEPTIDE(ARRAY(ON(CELLULOSE(SUPPORT(—(A(SCREENING(TOOL(TO(IDENTIFY( PEPTIDES( WITH( DIPEPTIDYL1PEPTIDASE( IV( INHIBITORY( ACTIVITY(WITHIN(THE(SEQUENCE(OF(α1LACTALBUMIN(................................................................(164#8.1#SUMMARY#......................................................................................................................................#164#8.2#INTRODUCTION#..........................................................................................................................#165#8.3#MATERIALS#AND#METHODS#.................................................................................................#167#8.3.1#Materials#................................................................................................................................#167#8.3.2#Peptide#Array#Synthesis#..................................................................................................#167#8.3.3#Probing#of#the#MembraneVBound#Peptide#Arrays#...............................................#168#8.3.4#DPPVIV#Inhibition#Assay#on#SPOTVSynthesized#Peptides#.................................#169#8.3.5#DPPVIV#Inhibition#Assay#on#TraditionallyVSynthesized#Peptides#.................#169#8.3.6#Determination#of# the#Mode#of# Inhibition# and# the# Inhibition#Constant# (Ki)#...............................................................................................................................................................#170#8.4#RESULTS#.........................................................................................................................................#171#8.4.1#Binding#of#DPPVIV#to#DecaVPeptides#on#the#Array#...............................................#171#8.4.2#DPPVIV#Inhibitory#Activity#of#SPOTVSynthesized#DecaVPeptides#..................#173#8.4.3#Validation#of#the#DPPVIV#Inhibitory#Activity#of#the#DecaVPeptides#..............#175#8.4.4#Modes#of#Inhibition#of#DecaVPeptides#.......................................................................#177#8.5#DISCUSSION#..................................................................................................................................#179#8.6#CONCLUSION#................................................................................................................................#181#CHAPTER( 9( :( COMPARISON( OF( THE( SUSCEPTIBILITY( OF( PORCINE( AND( HUMAN(DIPEPTIDYL1PEPTIDASE(IV(TO(INHIBITION(BY(PROTEIN1DERIVED(PEPTIDES(.(182### xiii#9.1#SUMMARY#......................................................................................................................................#182#9.2#INTRODUCTION#..........................................................................................................................#183#9.3#MATERIALS#AND#METHODS#.................................................................................................#187#9.3.1#Materials#................................................................................................................................#187#9.3.2#Preparation#of#Whey#Protein#Hydrolysates#...........................................................#187#9.3.3#Porcine#(p)#DPPVIV#Inhibition#Assay#.........................................................................#188#9.3.4#Recombinant#Human#(rh)#DPPVIV#Inhibition#Assay#...........................................#188#9.3.5#Determination#of# the#Mode#of# Inhibition# and# the# Inhibition#Constant# (Ki)#...............................................................................................................................................................#189#9.3.6#Statistical#Analysis#.............................................................................................................#190#9.4#RESULTS#.........................................................................................................................................#190#9.4.1#Effect#of#Synthesized#Peptides#on#the#Activity#of#Porcine#and#Recombinant#Human#DPPVIV#................................................................................................................................#190#9.4.2#Modes#of# Inhibition#and# Inhibition#Constants#of# the#Synthesized#Peptides#Displaying# the# Strongest# Inhibitory# Activity# against# Porcine# and# Recombinant#Human#DPPVIV#................................................................................................................................#192#9.4.3#Modes#of#Inhibition#and#Inhibition#Constants#of#Whey#Protein#Hydrolysates#on#Porcine#and#Recombinant#Human#DPPVIV#...................................................................#194#9.5#DISCUSSION#..................................................................................................................................#194#9.6#CONCLUSION#................................................................................................................................#196#CHAPTER(10(:(CONCLUSION(..................................................................................................(197#10.1#MAIN#FINDINGS#........................................................................................................................#197#10.2#FUTURE#AREAS#OF#RESEARCH#.........................................................................................#199#REFERENCES(...............................................................................................................................(203#APPENDICES(...............................................................................................................................(234#APPENDIX#A#–#CHAPTER#7#SUPPLEMENTARY#TABLE#...............................................................#234#APPENDIX#B#–#CHAPTER#8#SUPPLEMENTARY#TABLES#.............................................................#238#APPENDIX#C#–#CHAPTER#9#SUPPLEMENTARY#TABLE#AND#FIGURE#....................................#241## '## xiv#LIST'OF'TABLES'#Table# 2.1# Currently# available# pharmacological# therapies# for# the# management# of#diabetes.#................................................................................................................................................................#6#Table# 2.2# Overview# of# food# products# and# dietary# constituents# reported# to# present#antidiabetes#properties#and#their#mechanism#of#action.#..............................................................#17#Table# 2.3# Observational# studies# on# the# association# between# dairy# intake# and# type# 2#diabetes.#.............................................................................................................................................................#34#Table#2.4#Some#physiological#substrates#of#dipeptidylVpeptidase#IV#.....................................#52#Table#2.5#Examples#of#biologically#active#peptides#derived#from#milk#proteins.#..............#57#Table#2.6#Protein#hydrolysates#shown#to#display#DPPVIV#inhibitory#activity.#....................#58#Table#2.7#Milk#proteinVderived#DPPVIV#inhibitory#peptides.#......................................................#60#Table#4.1#DiVpeptides#reported#in#the#literature#to#present#DPPVIV#inhibitory#activity.#73#Table#4.2#TriV#and#oligoVpeptides#reported#in#the#literature#to#present#DPPVIV#inhibitory#activity.#...............................................................................................................................................................#75#Table# 4.3# Location# of# peptide# sequences# of# plant# proteins# with# DPPVIV# inhibition#activity.#...............................................................................................................................................................#80#Table#4.4#Location#of#peptide# sequences#of# chicken#egg# and# cow’s#milk#proteins#with#DPPVIV#inhibitory#activity.#.........................................................................................................................#89#Table# 4.5# Location# of# peptide# sequences# of# bovine# meat,# Atlantic# and# chum# salmon#proteins#with#DPPVIV#inhibitory#activity.#............................................................................................#94#Table# 4.6# Occurrence# frequency# (A)# of# peptides# with# DPPVIV# inhibitory# activity# in#proteins#of#various#food#commodities.#...............................................................................................#101### xv#Table# 5.1# DPPVIV# inhibitory# activity# of# sodium# caseinate# and# whey# protein# isolate#hydrolysates#and#their#fractions#obtained#by#ultrafiltration.#...................................................#117#Table#6.1#DPPVIV#and#αVglucosidase# inhibitory# activities#of#hydrolyzed#whey#proteins#obtained#by#peptic#treatment.#................................................................................................................#132#Table# 7.1# DipeptidylVpeptidase# IV# inhibitory# activity# of# whey# protein# protein# isolate#(WPI)# and# αVlactalbumin# (αVla)# hydrolysates# obtained# by# peptic# treatment# and# the#most#active#RPVHPLC#fractions#isolated#from#them.#.....................................................................#153#Table# 7.2# DipeptidylVpeptidase# IV# inhibitory# activity# and# mode# of# inhibition# of#synthesized#peptides.#.................................................................................................................................#156#Table# 8.1# Percent# inhibition# of# DPPVIV# activity# caused# by# SPOTVsynthesized# decaVpeptides#derived# from#αVlactalbumin# (A1–K4,#Appendix#B,# Table#B.1)# and# from#other#proteins#(K5–K11,#Appendix#B,#Tables#B.1#&#B.2).#........................................................................#174#Table# 8.2# DipeptidylVpeptidase# IV# inhibitory# activity# of# SPOTV# and# traditionallyVsynthesized#decaVpeptides.#.....................................................................................................................#176#Table# 8.3# Inhibition# constant# (Ki)# and#mode# of# inhibition# of# traditionally# synthesized#decaVpeptides.#...............................................................................................................................................#177#Table# 9.1# Dietary# proteins# reported# to# be# precursors# of# peptides# able# to# inhibit# the#activity#of#porcine#or#human#DPPVIV.#..................................................................................................#184#Table#9.2# Inhibition#constant# (Ki)# and#mode#of# inhibition#of# synthesized#peptides#and#whey#protein#hydrolysates#on#porcine#(p)#and#recombinant#human#(rh)#DPPVIV.#........#193##### xvi#Table#A.1# Peptide# sequences# identified# in# the#RPVHPLC# fractions# of# the#whey# protein#isolate#(WPIV5bV3#and#WPIV5bV4)#and#αVlactalbumin#(αVlaV2e#and#αVlaV2f)#hydrolysates#with#pepsin#.....................................................................................................................................................#234#Table#B.1#Sequences#of#114#αVlactalbuminVderived#decaVpeptides#and#their#position#on#the#arrays.#.......................................................................................................................................................#238#Table# B.2# Characteristics# of# the# seven# nonVαVlactalbuminVderived# decaVpeptides#included#on#the#arrays.#..............................................................................................................................#240#Table#C.1#Sequence#and#origin#of#the#62#peptides#investigated#for#their#effect#on#porcine#and#human#DPPVIV#activity.#.....................................................................................................................#241### '## xvii#LIST'OF'FIGURES'#Figure#2.1#The#“ominous#octet”#model#displaying#the#metabolic#abnormalities#involved#in#the#pathogenesis#of#type#2#diabetes.#...................................................................................................#5#Figure#2.2#Schematic# representation#of# the#generation#of# the# incretins#GLPV1#and#GIP#from#proglucagon#and#proGIP,#respectively,#and#their#cleavage#by#the#enzyme#DPPVIV.#................................................................................................................................................................................#46#Figure#2.3#Members#of#the#DPPVIV#gene#family#and#their#distribution.#.................................#48#Figure#2.4#Ribbon#diagram#of#human#dipeptidylVpeptidase#IV.#................................................#51#Figure# 2.5# Approaches# towards# the# discovery# of# bioactive# peptides# from# dietary#proteins.#.............................................................................................................................................................#55#Figure#3.1#Overview#of#experimental#approaches.#.........................................................................#67#Figure# 5.1# Extent# of# hydrolysis# (mmol# LVleucine# equiv/g# protein)# of# dairy# protein#hydrolysates#as#a#function#of#time#course#of#digestion.#..............................................................#111#Figure#5.2#DPPVIV#inhibitory#activity#of#dairy#protein#hydrolysates#as#a#function#of#time#course#of#digestion.#.....................................................................................................................................#113#Figure# 5.3# DPPVIV# inhibitory# activity# of# sodium# caseinate# and# whey# protein# isolate#(WPI)#hydrolysates#generated#using#different#enzymes.#...........................................................#115#Figure# 6.1# Extent# of# hydrolysis# (mmol# LVleucine# equiv/g# protein)# of# whey# protein#hydrolysates#obtained#by#peptic#treatment.#....................................................................................#129#Figure# 6.2# SDSVPAGE# electrophoregrams# of# whey# protein# samples# (A)# and# their#hydrolysates#after#peptic#treatments#(B).#.........................................................................................#130#Figure#6.3#DPPVIV# inhibitory#activity#of#whey#protein#hydrolysates#obtained#by#peptic#treatment.#........................................................................................................................................................#131### xviii#Figure#6.4#αVGlucosidase# inhibitory#activity#of#whey#protein#hydrolysates#obtained#by#peptic#treatment.#..........................................................................................................................................#135#Figure#7.1#Elution#profile#(A)#and#DPPVIV#inhibitory#activity#(B)#of#whey#protein#isolate#(WPI)#hydrolysate#fractions#obtained#by#cationVexchange#FPLC.#..........................................#147#Figure# 7.2# Elution# profile# (A)# and# DPPVIV# inhibitory# activity# (B)# of# WPIV5# fractions#obtained#by#sizeVexclusion#FPLC.#..........................................................................................................#148#Figure# 7.3# Elution# profile# (A)# and# DPPVIV# inhibitory# activity# (B)# of#WPIV5b# fractions#obtained#by#reversedVphase#HPLC.#......................................................................................................#149#Figure#7.4#Elution#profile#(A)#and#DPPVIV#inhibitory#activity#(B)#of#αVlactalbumin#(αVla)#hydrolysate#fractions#obtained#by#sizeVexclusion#FPLC.#............................................................#151#Figure# 7.5# Elution# profile# (A)# and# DPPVIV# inhibitory# activity# (B)# of# αVlaV2# fractions#obtained#by#reversedVphase#HPLC.#......................................................................................................#152#Figure# 7.6# Mature# amino# acid# sequences# of# bovine# (A)# αVlactalbumin# (UniProt# KB#database# accession# number# P00711)# and# (B)# βVlactoglobulin# (UniProt# KB# database#accession#number#P02754).#....................................................................................................................#155#Figure# 7.7# LineweaverVBurk# plots# of# DPPVIV# activity# in# the# presence# of# (A)# peptide#LAHKALCSEKL#(250–1250#µM,#final#assay#concentration),#(B)#peptide#LKPTPEGDL#(63–250#µL,#final#assay#concentration)#and#(C)#peptide#LCSEKLDQ#(188–750#µM,#final#assay#concentration).#..............................................................................................................................................#159#Figure#8.1#Schematic#representation#of#peptide#array#synthesis,#binding#and#inhibition#experiments.#..................................................................................................................................................#172#Figure# 8.2# Binding# of# DPPVIV# to# decaVpeptides# derived# from# αVlactalbumin# (A1–K4,#Table#B.1,#Appendix#B)#and#from#other#protein#(K5–K11,#Appendix#B,#Tables#B.1#&#B.2).#..............................................................................................................................................................................#173### xix#Figure# 8.3# Correlation# between# binding# of# DPPVIV# to# decaVpeptides# on# the# array#(indicated# by# luminescence# intensity)# and# inhibition# of# the# enzyme# activity# by# the#peptides.#...........................................................................................................................................................#175#Figure# 8.4# Mature# amino# acid# sequence# of# αVlactalbumin# (UniProt# KB# database#accession#number#P00711).#....................................................................................................................#175#Figure# 8.5# LineweaverVBurk# plots# of# DPPVIV# activity# in# the# absence# and# presence# of#peptides.# (A)#LPEWVCTTFH,# (B)#LAHKALCSEK,# (C)#LKPTPEGDLE,# (D)#WCKDDQNPHS,#(E)#LCSEKLDQWL,#(F)#IPAVFKIDAL,#(G)#YPSKPDNPGE.#............................................................#178#Figure# 9.1# Sequence# alignment# of# human# (UniProt# KB# database# accession# number#P27487)#and#porcine#(UniProt#KB#database#accession#number#P22411)#DPPVIV.#.........#186#Figure# 9.2# Percent# inhibition# of# porcine# (p)# and# recombinant# human# (rh)# DPPVIV#activities#by#synthesized#peptides.#.......................................................................................................#191##Figure# C.1# LineweaverVBurk# plots# of# porcine# DPPVIV# (pDPPVIV)# and# recombinant#human#DPPVIV#(rhDPPVIV)#activities#in#the#presence#and#absence#of#peptides.#..............#243#### ### xx#LIST'OF'ABBREVIATIONS'#αVla# AlphaVlactalbumin#AA# Amino#acid#ACE# Angiotensin#IVconverting#enzyme#ADA# Adenosine#deaminase#AMPK# Adenosine#monophosphateVactivated#protein#kinase#ATP# Adenosine#triphosphate#βVlg# BetaVlactoglobulin#BMI# Body#mass#index#BSA# Bovine#serum#albumin#Ca# Calcium#CI# Confidence#interval#DPP# DipeptidylVpeptidase#EMFA# Erythrocyte#membrane#fatty#acid#FAP# Fibroblast#activation#protein#FDA# Food#and#Drug#Administration#FPLC# Fast#protein#liquid#chromatography#FRX# Farnesoid#X#receptor#GI# Gastrointestinal#GIP# GlucoseVdependent#insulinotropic#polypeptide#GLPV1# GlucagonVlike#peptideV1#GLUT2# Glucose#transporter#2#G6P# GlucoseV6Vphosphate#G6Pase# GlucoseV6Vphosphatase#GRPP# GlicentinVrelated#pancreatic#polypeptide#HVGlyVProVpVNA# GlyVProVparaVnitroanilide#HR# Hazard#ratio#IC50# Concentration#causing#50%#inhibition#IP# Intervening#peptide#KATP# Adenosine#triphosphateVsensitive#potassium#channel### xxi#Ki# Inhibition#constant#Km# MichaelisVMenten#constant#IRSV1# Insulin#receptor#substrate#1#Lf# Lactoferrin#LC–ESIVMS/MS# Liquid#chromatography–electrospray#ionizationVtandem#mass#spectrometry#MCFAs# MediumVchain#fatty#acids#Mg# Magnesium#Nrf2# Nuclear#factor#(erythroidVderived#2)#like#2#OR# Odds#ratio#PC# Prohormone#convertase#pI# Isoelectric#point##PPARγ# Peroxisome#proliferatorVactivated#receptor#gamma#PTP1B# Protein#tyrosine#phosphatase#1B#PYY# Peptide#YY#ROS# Reactive#oxygen#species#RPVHPLC# ReversedVphase#high#performance#liquid#chromatography#RR# Relative#risk#SACS# SVAllyl#cysteine#sulfoxide#SDSVPAGE# Sodium#dodecyl#sulfateVpolyacrylmide#gel#electrophoresis#SGLT# SodiumVdependent#glucose#coVtransporter##TCA# Trichloroacetic#acid#TFA# Trifluoroacetic#acid#T2D# Type#2#diabetes#TNBS# 2,4,6VTrinitrobenzenesulfonic#acid#TZDs# Thiazolidinediones#WP# Whey#protein#WPI# Whey#protein#isolate#### ### xxii#ACKNOWLEDGEMENTS'##I#owe#so#many#people#a#debt#of#gratitude,#and#I#hardly#know#where#to#begin.###Many#people#have#helped#me#throughout#my#doctoral#journey,#but#none#more#than#my#supervisor,#Dr.#Eunice#LiVChan.#Thank#you# for#being#so#open#and#supportive#of#all#my#ideas.# Thank# you# for# your# trust,# for# accepting,# always# patiently# and# humbly,# all# the#times#I#totally#ignored#your#advice.#Thank#you#for#your#kindness,#for#all#the#laughs#that#we#shared#and#for#all#the#other#usual#reasons,#but#especially#all#the#others.##Very#special#thanks#are#also#owed#to#Imelda#Cheung#with#whom#I#spent#so#many#hours#in#the#lab.#Thank#you#for#your#encouragements,#your#bright#ideas#and#your#assistance.#My#lab#work#skills#have#improved#tremendously#just#by#working#with#you.##I#thank#Dr.#Green#and#Dr.#Scaman,#my#supervisory#committee#members,#for#their#time#and#thoughtful#feedback#on#my#work.#I#also#would#like#to#thank#Dr.#Cliff#and#Dr.#Wasik#for#their#encouragements#and#enthusiasm#about#my#research.#Special#thanks#also#go#to#Barbara#Wakal#–#your#joyful#demeanor#never#failed#to#brighten#my#days.##I#am#thankful#for#the#help#I#received#from#Dr.#Dirk#Winkler#with#peptide#array#synthesis#as# well# as# from# Dr.# Philip# R.# Gafken# and# Ms.# Lisa# Nader# Jones# with# LC–ESIVMS/MS#analysis#and#peptide#sequencing.###I# am# extremely# grateful# for# the# financial# support# that# I# received# from# the# Natural#Sciences# and# Engineering# Research# Council# of# Canada# (NSERC),# the# Canadian# Dairy#Commission,# the# Canadian# Institute# of# Food# Science# and#Technology# (CIFST)# and# the#University#of#British#Columbia.###Lastly,# I#would# like# to# thank#my# friends# and# family# for# their# love,# care# and# rollicking#humor.# You#make# everything# seem#possible,# always.# Extra# heartfelt# thank# you# to#my### xxiii#mother# Sylvie# Lafontaine,# my# solid# ground# and# personal# superhero.# Your# boundless#love#and#faith#in#me#make#me#feel#strong#and#fearless.############# «Il#est#grand#temps#de#rallumer#les#étoiles»#Guillaume#Apollinaire##### 1#CHAPTER'1:'INTRODUCTION'#Type#2#diabetes#is#one#of#the#most#challenging#health#problems#of#the#21st#century;#the#incidence#and#prevalence#of#this#complex#endocrine#disorder#are#rising#at#an#alarming#rate# all# around# the# globe# (Sebokova# et# al.,# 2007;# International# Diabetes# Federation,#2013).##When#not#adequately#treated,#type#2#diabetes#can#lead#to#a#host#of#serious#and#debilitating# complications,# including# blindness,# amputation,# renal# failure# and#cardiovascular#diseases#(Fowler,#2008;#Inzucchi#et#al.,#2012).#Currently,#twelve#classes#of# pharmacological# therapy# are# available# for# the# management# of# diabetes# (Blonde,#2009;#Tahrani#et#al.,#2011;#Goswami#et#al.,#2014).#Among#these,#inhibitors#of#the#enzyme#dipeptidylVpeptidase# IV# (DPPVIV)#are#amid# the#newest#drugs# introduced# to# the# type#2#diabetes# pharmacopeia.# # These# orally# available# agents# exert# their# glucoseVlowering#effect#by#preventing#the#inactivation#of#the#incretins#glucoseVdependent#insulinotropic#polypeptide# (GIP)# and# glucagonVlike# peptideV1# (GLPV1),# two# gutVderived# hormones#playing#a#pivotal#role#in#glycemic#regulation#(White,#2008;#Deacon,#2011).###Dietary# proteins# are# known# to# be# instrumental# in# a# wide# range# of# nutritional# and#biological#processes#(JahanVMihan#et#al.,#2011).#Over#the#last#two#decades,#an#increasing#amount# of# research# has# shown# that# they# can# also# be# precursors# of# a# variety# of#biologically#active#peptides#that#have#the#potential#to#improve#human#health#(Shahidi#&#Zhong,# 2008).# Even# though# bioactive# peptides# have# been# found# encrypted# in# the#sequence# of# proteins# from# both# plant# and# animal# sources,# milk# proteins# are# often#considered# some# of# the#most# important# precursors# of# bioactive# peptides# (Korhonen,#2009).# In# addition# to# containing# protein# fragments# with# antiVhypertensive,# antiVbacterial,# antiVcariogenic,# antiVoxidative,# mineral# binding,# opioid# and#immunomodulating# activities# (Hartmann# &# Meisel,# 2007;# Jauregi,# 2008),# dietary#proteins,#particularly#dairy#proteins,#have#recently#been#shown#to#also#contain#within#their#sequence#peptides#able#to#inhibit#the#activity#of#the#DPPVIV#enzyme#(Dziuba#et#al.,#2009;# Tulipano# et# al.,# 2011;#Uchida# et# al.,# 2011;#Uenishi# et# al.,# 2012;#Nongonierma#&#FitzGerald,# 2013b).# This# finding# that# proteins# can# be# used# to# generate# peptides#with### 2#DPPVIV#inhibitory#activity#that#could#be#employed#as#a#complementary#approach#in#the#management# of# type# 2# diabetes# has# triggered# great# interest# in# the# bioactive# peptide#area.#Nevertheless,#investigations#on#the#production#of#DPPVIV#inhibitors#from#proteins#are#sparse#and#only#a#limited#number#of#peptides#with#DPPVIV#inhibitory#activity#have#been#isolated#and#characterized#from#food#protein#hydrolysates.#Little#is#also#known#on#the#mechanisms#by#which#proteinVderived#inhibitors#affect#DPPVIV#activity.###The#overall#objective#of#the#present#research#was,#therefore,#to#evaluate#the#potential#of#dietary#proteins,#particularly#dairy#proteins,#to#serve#as#sources#of#DPPVIV#inhibitors,#to#isolate# and# identify# the# active# peptides# and# gain# an# understanding# of# their#modes# of#action#on#the#DPPVIV#enzyme.###### '## 3#CHAPTER'2:'LITERATURE'REVIEW'#2.1'DIABETES'#Diabetes# is#one#of# the# fastest#growing#health#concerns#worldwide.# It# is#estimated#that#8.3#%#of#adults#–#382#million#people#–#are#currently#living#with#diabetes#and#that#if#the#actual# demographic# patterns# continue,# over# 592#million# people#will# be# affected#with#this#metabolic#disorder#by#2035#(International#Diabetes#Federation,#2013).###Diabetes#refers#to#a#group#of#chronic#diseases#characterized#by#hyperglycemia#resulting#from#either#the#inability#of#the#pancreatic#βVcells#to#produce#insulin#(type#1#diabetes)#or#from# defects# in# both# insulin# secretion# and# insulin# action# (type# 2# diabetes# and#gestational#diabetes).##Accounting#for#about#90%#of#all#cases#diagnosed,#type#2#diabetes#is# the#most# prevalent# form# of# diabetes# (Buchanan#&# Xiang,# 2005;# American#Diabetes#Association,#2012;#International#Diabetes#Federation,#2013).###When# improperly# treated,# hyperglycemia# can# cause# serious# and# debilitating#macrovascular# and# microvascular# complications,# including# stroke,# coronary# artery#disease#and#peripheral#arterial#disease#as#well#as#diabetic#nephropathy,#neuropathy#and#retinopathy#(Fowler,#2008).##Therefore,#effective#diabetes#management#strategies#are#of#primary#importance.###2.1.1$Pathophysiology$of$Diabetes1$#In#nonVdiabetic# individuals,# an#elevation#of#blood#glucose# levels# following# food# intake#triggers# the# synthesis# and# release# of# insulin# from# the# pancreatic#βVcells.# Once# in# the#########################################################1#A#version#of#this#section#has#been#published.#Lacroix,# I.M.E.,#LiVChan,#E.C.Y.#Overview#of# food#products#and#dietary#constituents#with#antidiabetic#properties#and#their#putative#mechanisms#of#action:#A#natural#approach#to#complement#pharmacotherapy#in#the#management#of#diabetes.#Mol.#Nutr.#Food#Res.#2014,#58,#61–78.### 4#systemic# circulation,# insulin# binds# to# receptors# in# various# organs# including# the# liver,#skeletal#muscles#and#adipose#tissues.#This#initiates#a#cascade#of#reactions#leading#to#the#suppression#of#glucose#production#from#the#liver,#the#inhibition#of# lipolysis# in#adipose#tissues,# and# the# stimulation# of# glucose# uptake# from# the# muscle# and# adipose# tissues,#which# ultimately# causes# the# return# of# blood# glucose# levels# to# within# the# normal#physiological# range# (Cheng# &# Fantus,# 2005).# Hyperglycemia# arises#when# the# body# is#unable#to#properly#metabolize#carbohydrates.#Although#this#condition#is#observed#in#all#forms#of#diabetes,# the#pathophysiological#defects#responsible# for# its#occurrence#differ.#In# type# 1# diabetes,# hyperglycemia# occurs# as# a# result# of# the# destruction# of# insulinVproducing#βVcells#of#the#pancreas#by#T#lymphocytes#(Åkerblom#et#al.,#2002),#leading#to#an# absolute# incapacity# of# the# pancreas# to# produce# the# required# insulin# to# maintain#adequate#blood# glucose# levels# (Merriman,# 2009).# Since# insulin# is# essential# for# proper#glucose# regulation,# the# treatment# of# type# 1# diabetes# always# involves# the# use# of#exogenous#insulin.###On# the#other#hand,# eight#metabolic# abnormalities# are# involved# in# the#development#of#glucose#intolerance#in#type#2#diabetes#(Figure(2.1),#with#insulin#resistance#(defined#as#a#reduced#response#of#target#organs#such#as#liver,#skeletal#muscles,#and#adipose#tissues#to#insulin# (DeFronzo# &# Tripathy,# 2009)),# and# impaired# insulin# secretion# being# the# core#pathophysiologic# defects# (DeFronzo,# 2009).# Since# multiple# hormones# and# organ#systems# are# implicated# in# the# pathogenesis# of# type# 2# diabetes,# several# classes# of#glucoseVlowering# therapies# have# been# developed# in# order# to# target# specifically# the#different#metabolic#abnormalities#causing#hyperglycemia#in#diabetic#patients.#Table(2.1#presents#an#overview#of#the#pharmacological#agents#that#are#currently#available#for#the#treatment#of#diabetes.###### 5#### #Figure(2.1(The#“ominous#octet”#model#displaying#the#metabolic#abnormalities#involved#in# the# pathogenesis# of# type# 2# diabetes.# Impaired# insulin# secretion,# and# insulin#resistance# in# the# liver# and#muscle# are# believed# to# be# the# core# defects# (adapted# from#DeFronzo,# 2009).# This# figure# was# published# in# Lacroix# &# LiVChan# (2014a).HYPERGLYCEMIA!CORE-DEFECTS!Muscle insulin resistance ⇒↓ glucose uptake!Hepatic insulin resistance ⇒↑ glucose production!Impaired pancreatic insulin secretion!Dysfunction of neurotransmitters in the central nervous system ⇒ impaired appetite regulation!Dis-regulation of pancreatic α-cells ⇒↑glucagon secretion!Impaired kidney response ⇒↑glucose re-absorption!Decreased gut incretin effect ⇒ impaired glucose response!Impaired adipocyte metabolism ⇒ ↑Lipolysis and ↓glucose uptake!!6!Table&2.1&Currently!available!pharmacological!therapies!for!the!management!of!diabetes.!!Drug class Example(s) (generic name) Example(s) (trade name) Site of actiona Cellular mechanism Physiological action(s) Route  Undesirable side effect(s) Referencesb Sulfonylureas Gliclazide Glimepiride Glipizide Glyburide Diamicon® Amaryl® Glucotrol® DiaBeta®, Glynase®  Pancreas Close KATP channels on the plasma membranes of pancreatic β-cells Stimulate insulin secretion Oral Hypoglycemia, weight gain Lebovitz (2001); Cheng & Fantus (2005); DiStefano & Watanabe (2010)  Meglitinides Nateglinide Repaglinide Starlix® Prandin® Pancreas Close KATP channels on the plasma membranes of pancreatic β-cells Stimulate insulin secretion Oral Hypoglycemia, weight gain Landgraf (2000); Hatorp (2002); McLeod (2004); Cheng & Fantus (2005) Biguanides Metformin Glucophage® Liver, (adipose tissue, muscle) Activate AMP-kinase Decrease hepatic glucose production, reduce gastrointestinal glucose absorption, improve insulin sensitivity in muscle, may help to preserve pancreatic β-cell function, may increase the endogenous concentration of   GLP-1 Oral Gastrointestinal disturbances, possible association to lactic acidosis in rare cases, vitamin B12 deficiency  Lebovitz (2001); Kirpichnikov et al. (2002); Kim et al. (2008)  Thiazolidinediones Pioglitazone Rosiglitazone Actos® Avandia® Adipose tissue, muscle, liver Activate the transcription factor PPARγ Increase insulin sensitivity in muscle and liver, enhance adipogenesis, lower circulating free fatty acids Oral Anemia, bone fracture, edema, congestive heart failure, weight gain Yki-Järvinen (2004); Cheng & Fantus (2005); DiStefano & Watanabe (2010) !7!Table&2.1&continued)Drug class Example(s) (generic name) Example(s) (trade name) Site of actiona Cellular mechanism Physiological action(s) Route  Undesirable side effect(s) Referencesb α-Glucosidase inhibitors Acarbose Miglitol Precose® Glyset® Intestinal enterocyte Inhibit the intestinal enzymatic activity of α-glucosidase Slow down the intestinal digestion and absorption of carbohydrates Oral Gastrointestinal disturbances Lebovitz (1997) Glucagon-like peptide-1 mimetics Exenatide Liraglutide Byetta® Victoza® Pancreas, (liver, brain) Activate GLP-1 receptors Increase glucose-dependent insulin secretion, suppress glucagon release, delay gastric emptying, modulate appetite, trophic effects on β-cells Injection Gastrointestinal disturbances Holst & Deacon (2004); Mentlein (2005); Addison & Aguilar (2011) Dipeptidyl-peptidase IV inhibitors Saxaglitpin Sitagliptin Vildagliptin Alogliptin Linagliptin Onglyza® Januvia® Galvus® Nesina® Tradjenta® GI tract, (pancreas) Inhibit the enzymatic activity of DPP-IV, increase the endogenous concentrations of GLP-1 & GIP  Increase glucose-dependent insulin secretion, suppress glucagon release Oral Transient and minimal side effects including dizziness, nausea and headache Lambeir et al. (2003); Tahrani et al. (2011)  Amylin analogues Pramlintide  Symlin® Brain, (pancreas) Activate amylin receptors Slow down gastric emptying, inhibit glucagon secretion, mediate satiety Injection Hypoglycemia, nausea, vomiting Schmitz et al. (2004); Adeghate & Kalász (2011)  Dopamine D2-receptor agonists Bromocriptine mesylate Cycloset ® Brain, (adipose tissue, liver) Activate dopaminergic receptors Increase dopamine levels , regulate circadian rhythm, increase insulin sensitivity Oral Nausea, fatigue, vomiting, dizziness, headache, rhinitis Keche (2010), DeFronzo (2011) ! !!8!Table&2.1&continued)Drug class Example(s) (generic name) Example(s) (trade name) Site of actiona Cellular mechanism Physiological action(s) Route  Undesirable side effect(s) Referencesb Bile acid sequestrants Colesevelam WelcholTM  GI tract, liver Bind bile acids, believed to activate the nuclear farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor Believed to increase GLP-1 and GIP levels, decreased hepatic glucose production Oral Gastrointestinal disturbances, increased triglyceride levels Ma et al. (2006); Zhang et al. (2006); Sonnett et al. (2009); Beysen et al. (2012)  Sodium-dependent glucose co-transporter 2 inhibitors Canagliflozin Dapagliflozin InvokanaTM FarxigaTM Kidney Inhibit the transporter SGLT2 in the proximal convoluted tubule Prevent glucose reabsorption and facilitate its urinary excretion Oral Uro-genital tract infections Chao & Henry (2010); Kalra (2014); Opie (2014) Insulinc Insulin lisprod Regular insuline NPH insulinf Insulin glargineg Insulin lispro protamise/ insulin lisproh Humalog® d Humulin® Re Humulin® N f Lantus® g Humalog® Mix 50/50 & 75/25h Adipose tissue, muscle, liver Activate insulin receptors Increase glucose clearance, reduce hepatic glucose production Injection Hypoglycemia, weight gain Blonde (2009)  a!Organs/tissues!in!brackets!are!secondary!targets.!b!!Representative!references,!non@exhaustive!list.!c!Including!rapid@acting!analogues,!short@acting,!intermediate@acting,!long@acting!analogues!and!combinations.!d!!Rapid@acting!analogue.!e!!Short@acting.!f!Intermediate@acting.!g!Long@acting!analogue.!h!Combination.!AMP,!adenosine!monophosphate;!GIP,!glucose@dependent!insulinotropic!polypeptide;!GLP@1,!glucagon@like!peptide@1,!KATP,!adenosine!triphosphate@sensitive!potassium!channel;!PPARγ,!peroxisome!proliferator@activated!receptor!gamma;!SGLT2,!sodium@dependent!glucose!co@transporter!2.!A!version!of!this!table!was!published!in!Lacroix!&!Li@Chan!(2014a).!!! 9!In! the! following! sections,! the! mechanisms! by! which! these! drugs! exert! their! anti9hyperglycemic!effect!are!briefly!described.!!2.1.2$Overview$of$the$Pharmacological$Therapies$for$the$Management$of$Diabetes$and$Their$Mode$of$Action2$!2.1.2.1$Sulfonylureas$!Sulfonylureas!represent!one!of!the!most!widely!used!classes!of!oral!anti9diabetes!drugs!(DiStefano!&!Watanabe,! 2010).! They! increase! endogenous! insulin! secretion! from! the!pancreas! by!binding! to! plasma!membrane! receptor! (SUR!1)! coupled! to! an! adenosine!triphosphate! (ATP)9sensitive! potassium! channel! located! on! the! plasma!membrane! of!pancreatic!β9cells.!This!interaction!results!in!closure!of!the!potassium!channels,!which!causes! inhibition! of! the! efflux! of! potassium! ions! and! depolarization! of! the! cell!membrane,! resulting! in! the! opening! of! voltage9dependent! calcium! channels.! The!calcium! influx! then! triggers! exocytosis! of! insulin! from! the! β9cells! (Cheng! &! Fantus,!2005;!DiStefano!&!Watanabe,!2010).!!!2.1.2.2$Meglitinides$$!Meglitinide! derivatives! are! a! relatively! new! family! of! insulin! secretagogues! including!nateglinide,! a! phenylalanine! derivative! and! repaglinide,! a! benzoic! acid! derivative!(Landgraf,! 2000).! Similar! to! sulfonylureas,! meglitinides! stimulate! insulin! release! by!promoting! closure! of! ATP9sensitive! potassium! channels! by! binding! to! the! SUR! 1!receptor!on!the!β9cell!membrane.!However,!some!glinides,!such!as!repaglinide,!bind!at!a!different! site! than! the! sulfonylureas! (Landgraf,! 2000;! Stumvoll! et! al.,! 2005).! The!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!2!A!version!of!this!section!has!been!published.!Lacroix,! I.M.E.,!Li9Chan,!E.C.Y.!Overview!of! food!products!and!dietary!constituents!with!antidiabetic!properties!and!their!putative!mechanisms!of!action:!A!natural!approach!to!complement!pharmacotherapy!in!the!management!of!diabetes.!Mol.%Nutr.%Food%Res.%2014,!58,!61–78.!! 10!pharmacokinetics! and! pharmacodynamics! of! nateglinide! and! repaglinide! also! differ!from!the!sulfonylureas,! the!glinides!being!absorbed!more!rapidly!and!causing!a!more!rapid!and!shorter!insulin!response!(Hatorp,!2002;!McLeod,!2004).!!!2.1.2.3$Biguanides$!Metformin! is! a! biguanide! that! has! been! used! for! more! than! 50! years! for! the!management! of! type! 2! diabetes! (Lebovitz,! 2001)! and! is! often! the! first9line! drug! of!choice! for! the! treatment! of! newly! diagnosed! type! 2! diabetes! patients! (American!Diabetes!Association,!2012).!Metformin!exerts!its!anti9hyperglycemic!effects!primarily!by!reducing!hepatic!glucose!output!and!improving!insulin!sensitivity!(Kim!et!al.,!2008).!The!exact!mechanism!by!which!metformin!decreases!hepatic!glucose!production!is!not!fully! understood,! but! it! is! believed! that! it! relies! on! the! activation! of! adenosine!monophosphate! (AMP)9activated! protein! kinase! (AMPK),! a! serine/threonine! kinase!that!is!involved!in!the!modulation!of!glucose!and!fatty!acid!metabolism.!The!activation!of! AMPK! causes! the! inhibition! of! lipogenesis! and! gluconeogenesis! while! promoting!lipolysis!as!well!as!fatty!acid!oxidation!(Kirpichnikov!et!al.,!2002;!Cheng!&!Fantus,!2005;!Kim! et! al.,! 2008).! Moreover,! the! activation! of! AMPK! by! metformin! has! also! been!reported! to! suppress! the! expression! of! the! gluconeogenic! enzymes!phosphoenolpyruvate! carboxykinase! and! glucose969phosphatase! (G6Pase),! which! has!the!effect!of!inhibiting!gluconeogenesis!(Kim!et!al.,!2008).!!!Metformin! has! also! been! suggested! to! help! preserve! pancreatic! β9cell! functions!(Marchetti! et! al.,! 2009)! and! to! increase! the! concentration! of! glucagon9like! peptide91!(GLP91),! an! incretin!hormone! that! increases! insulin! secretion! in! a! glucose9dependent!manner!(Mannucci!et!al.!2001).!! $! 11!2.1.2.4$Thiazolidinediones$$!Thiazolidinediones! (TZDs)! are! a! class! of! insulin9sensitizing! therapies! that! enhance!insulin!sensitivity!in!muscle!and!liver,!increase!adipogenesis!and!lower!circulating!free!fatty! acids.! These! drugs! act! primarily! as! ligands! for! the! peroxisome! proliferator9activated! receptor! γ! (PPARγ),! a! ligand9activated! transcription! factor! which! is!predominantly!expressed!in!adipose!tissues!(Lebovitz,!2001;!Yki9Järvinen,!2004;!Cheng!&!Fantus,!2005;!DiStefano!&!Watanabe,!2010),!and! is! involved! in!glucose!metabolism!and!fatty!acid!storage!(Cheng!&!Fantus,!2005).!The!activation!of!PPARγ!by!TZDs!causes!an! increase! in! adipocyte! differentiation! (DiStefano! &! Watanabe,! 2010).! Since! the!expression!levels!of!PPARγ!in!muscle!and!liver!are!very!low,!the!improvement!of!insulin!sensitivity!in!these!peripheral!tissues!by!TZDs!is!believed!to!result!from!their!effects!on!adipocytes! (Cheng! &! Fantus,! 2005).! In! fact,! TZDs! have! been! reported! to! lower! the!circulating!levels!of!lipids!and!the!accumulation!of!lipid!in!non9adipose!tissues,!such!as!the!skeletal!muscle!and!liver,!which,!in!turn,!help!to!prevent!fatty!acid9induced!insulin!resistance!(Ye!et!al.,!2004).!In!addition,!TZDs!have!also!been!suggested!to!decrease!the!circulating!concentrations!of!free!fatty!acids!and!of!pro9inflammatory!cytokines,!such!as!tissue! necrosis! factor9α! and! interleukin! 6,! while! increasing! the! concentration! of!adiponectin,!the!latter!being!known!to!present!insulin9sensitizing!properties!(Lebovitz,!2001;!Stumvoll!et!al.,!2005).!!!2.1.2.5$Alpha>Glucosidase$Inhibitors$!Alpha9glucosidase! inhibitors! exert! their! anti9hyperglycemic! effect! by! interfering!with!the! digestion! of! carbohydrates! in! the! gut! and! delaying! the! entry! of! glucose! into! the!systemic!circulation.!Located!in!the!brush!borders!of!the!small!intestine,!α9glucosidase!enzymes!are!responsible!for!the!hydrolysis!of!oligosaccharides!and!disaccharides! into!monosaccharides.!Compounds!such!as!acarbose!act!as!competitive!inhibitors!of!the!α9glucosidase! enzymes,! thus! delaying! and! shifting! the! hydrolysis! and! absorption! of!! 12!carbohydrates! throughout! the! small! intestine! and! colon.! This! results! in! lower!postprandial!glucose!levels!(Lebovitz,!1997).!!!!2.1.2.6$Glucagon>Like$Peptide>1$(GLP>1)$Mimetics$!The!incretin!hormone!glucagon9like!peptide91!(GLP91)! is!a!309amino!acid!polypeptide!secreted! from! intestinal! L! cells! following! food! consumption.! The! GLP91! hormone!possesses!several!glucoregulation!actions,!including!the!stimulation!of!insulin!secretion!and!the!suppression!of!glucagon!release,!both!in!a!glucose9dependent!manner!(Holst!&!Deacon,! 2004;! Mentlein,! 2005;! Addison! &! Aguilar,! 2011).! The! insulinotropic! effect!results!from!the!binding!of!GLP91!to!its!specific!receptor!on!the!β9cell!membrane,!which!causes!the!G9protein!coupled!activation!of!adenylate!cyclase!and!leads!to!formation!of!cAMP.!The!subsequent!activation!of!protein!kinase!A!and!the!cAMP9regulated!guanine!nucleotide! exchange! factor! II! (also! referred! to! as! Epac2)! generates! a! multitude! of!reactions,! including! the! enhanced! exocytosis! of! insulin9containing! granules! (Holst! &!Gromada,! 2004).! Moreover,! GLP91! is! also! known! to! retard! gastric! emptying! and! to!suppress! food! intake! and! appetite! (Holst!&!Deacon,! 2004;! Addison!&!Aguilar,! 2011),!and! it! may! also! have! trophic! effects! on! pancreatic! β9cells! (Stoffers! et! al.,! 2003;!Wajchenberg,! 2007).! However,! circulating! GLP91! has! a! very! short! half9life! (within!minutes)!due!to!renal!degradation!and!proteolytic!cleavage!by!the!enzyme!dipeptidyl9peptidase!(DPP)9IV!(EC!3.4.14.5)!(Mentlein,!2005).!Thus,!GLP91!mimetics!that!present!binding!affinity!and!glucoregulation!effects!similar! to!endogenous!GLP91,!but! that!are!resistant! to! DPP9IV! action,! represent! one! therapeutic! approach! that! can! be! taken! to!extend!the!incretin!effect!and!thus!enhance!insulin!secretion!(Mentlein,!2005).!!2.1.2.7$Dipeptidyl>Peptidase$IV$Inhibitors$!Inhibitors!of! the!dipeptidyl9peptidase!IV!(DPP9IV)!enzyme!provide!a!second!approach!to!extend!the!incretin!effect.!The!DPP9IV!enzyme!is!a!serine!protease!expressed!in!both!! 13!soluble! and!membrane! forms! in! a! number! of! cells,! particularly! on! epithelial! tissues,!such!as!intestine,!liver!and!kidney!(Lambeir!et!al.,!2003;!Lorey!et!al.,!2003;!Thoma!et!al.,!2003).! It! is! involved! in!a!number!of!biological!processes,! including! the! inactivation!of!the!incretin!hormones!GLP91!and!glucose9dependent!insulinotropic!polypeptide!(GIP),!DPP9IV!being!responsible!for!more!than!95%!of!the!degradation!of!the!former!(Thoma!et!al.,!2003).!Given!the!beneficial!effect!of!GLP91!on!glucose!homeostasis,!the!prevention!of! its! inactivation! by! use! of! agents! with! DPP9IV! inhibitory! activity,! represents! an!effective!way!to!improve!glycemic!regulation.!!!!2.1.2.8$Amylin$Analogues$$!Amylin,!also!known!as! islet!amyloid!polypeptide,! is!a!37!amino!acid!peptide!hormone!co9secreted! with! insulin! by! the! pancreatic! β9cells! (Schmitz! et! al.,! 2004;! Adeghate! &!Kalász,!2011).!Amylin!analogues,!such!as!pramlintide,!can!improve!glycemic!control!in!people!with! type! 1! diabetes! and! type! 2! diabetes! by! slowing! down! gastric! emptying,!suppressing!postprandial!glucagon!secretion,!and!mediating!satiety!and!food!intake!by!activation! of! the! area! postrema! neurons! and/or! nucleus! tractus! solitarius! (Young,!2005;!Michel!et!al.,!2007)!as!well!as!by!inhibiting!ghrelin!release!(Schmitz!et!al.,!2004;!Adeghate!&!Kalász,!2011).!!2.1.2.9$Dopamine$D2>Receptor$Agonists$!The!ergot!alkaloid!dopamine!D29receptor!agonist!bromocriptine,!which!has!been!used!for!more!than!3!decades!in!the!treatment!of!prolactinomas!and!Parkinson’s!disease,!has!recently!been!approved!by!the!United!States!Food!and!Drug!Administration!(FDA)!as!an!adjunct!to!diet!and!exercise!for!the!management!of!type!2!diabetes!(Keche,!2010).!The!circadian! neuroendocrine! rhythm! mediated! by! dopamine! and! serotonin! has! been!shown! to! play! an! important! role! in! insulin! action! and!body! fat! stores! in! vertebrates.!Bromocriptine!is!believed!to!improve!glucose!tolerance!in!insulin9resistant!individuals!! 14!by! mimicking! the! normal! peak! of! dopamine! in! the! central! nervous! system,! thereby!resetting! the! excessive! hypothalamic! drive! for! elevated! plasma! glucose,! triglycerides!and! free! fatty! acid! levels! in! fasting! and! postprandial! conditions! (DeFronzo,! 2011;!Shivaprasad!&!Kalra,!2011).!!!!2.1.2.10$Bile$Acid$Sequestrants$!Used! for! the! treatment!of!dyslipidaemia! for!about!a!decade,! the!bile!acid! sequestrant!colesevelam!hydrochloride!has!also!recently!been!approved!by!the!FDA!as!an!adjunct!therapy!for!the!treatment!of!type!2!diabetes!(Sonnett!et!al.,!2009;!Handelsman,!2011).!Although! several! clinical! studies! have! shown! the! glucose9lowering! effect! of! bile! acid!sequestrants! in! patients! with! type! 2! diabetes,! the! exact! mechanism! underlying! this!beneficial!effect!is!not!well!understood.!It!is!believed!that!bile!acids,!by!interacting!with!the! nuclear! farnesoid! X! receptor! (FXR)! and! the! G! protein9coupled! bile! acid! receptor,!play!a!role!in!glucose!and!energy!regulation!(Lefebvre!et!al.,!2009).!The!hypoglycemic!effect! of! bile! acid! sequestrants,! known! to! alter! normal! bile! acid! pool,! has! been!suggested! to! result! from! their! ability! to!modulate!FXR9dependent! signaling!pathways!involved! in! peripheral! insulin! sensitivity! (Zhang! et! al.,! 2006)! and! hepatic! glucose!production!!(Ma!et!al.,!2006).!Alternatively,!bile!acid!sequestrants!are!also!believed!to!promote! the! secretion! of! the! incretin! hormones! GIP! and! GLP91! (Suzuki! et! al.,! 2007;!Beysen!et!al.,!2012).!!!!2.1.2.11$Sodium>Dependent$Glucose$Co>Transporter$2$Inhibitors$!The!sodium9dependent!glucose!co9transporter!2!(SGLT2)!is!a!low9affinity,!high9capacity!transporter!that!catalyzes!the!active!transport!of!80990%!of!the!glucose!load!across!the!proximal! convoluted! tubule! cells! of! the! kidney! (Chao! &! Henry,! 2010;! Opie,! 2014).!Inhibitors! of! SGLT2! are! a! newly! approved! class! of! anti9diabetic! therapies! that! act! by!preventing! renal! glucose! reabsorption!and! facilitating! its! excretion! in!urine! therefore!! 15!leading! to! a! reduction! in! plasma! glucose! concentrations! (Chao!&!Henry,! 2010;!Kalra,!2014;!Opie,!2014).!!!!!!!2.1.2.12$Exogenous$Insulin$!Insulin! and! insulin! analogues! are! considered! the!most! potent! anti9diabetes! therapies!(Blonde,! 2009).! Since! patients! with! type! 1! diabetes! are! unable! to! produce! insulin,!exogenous!insulin!is!always!required!to!achieve!proper!glycemic!regulation.!In!the!case!of!type!2!diabetes! individuals,!exogenous!insulin!can!be!used!alone!or! in!combination!with!other!anti9hyperglycemic!agents,!such!as!metformin!and!thiazolidinediones.!!!2.1.3$Role$of$Diet$in$the$Incidence$and$Management$of$Diabetes3$!It! is! recognized! that! diet! plays! a! major! role! in! the! incidence! and! management! of!diabetes.! Over! the! last! few! decades,! numerous! studies! have! investigated! the!relationship! between! the! consumption! of! specific! food!products! or! nutrients! and! the!incidence!of!diabetes!as!well!as! the! impact!of!dietary!patterns!on! the!development!of!this! metabolic! disorder.! Findings! from! these! studies! have! shown! that! the! intake! of!certain!foods!such!as!fruits!and!vegetables!(Feskens!et!al.,!1995;!Ford!&!Mokdad,!2001;!Montonen!et!al.,!2005),!dairy!products!(Choi!et!al.,!2005;!Liu!et!al.,!2006;!Pittas!et!al.,!2006;!Villegas!et!al.,!2010;!Malik!et!al.,!2011;!Grantham!et!al.,!2013)!as!well!as!whole9grains!(Liu!et!al.,!2000;!Fung!et!al.,!2002;!Montonen!et!al.,!2003;!de!Munter!et!al.,!2007)!were! inversely! associated! with! the! incidence! of! diabetes,! while! the! consumption! of!other!foods!such!as!processed!meat!(van!Dam!et!al.,!2002;!Schulze!et!al.,!2003;!Fretts!et!al.,!2012),!were!positively!correlated!to!diabetes!risk.!Moreover,!compiling!data!from!in%vitro! and! animal!models,! as!well! as! clinical! studies,! have! shown! that! several! dietary!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!3!A!version!of!this!section!has!been!published.!Lacroix,! I.M.E.,!Li9Chan,!E.C.Y.!Overview!of! food!products!and!dietary!constituents!with!antidiabetic!properties!and!their!putative!mechanisms!of!action:!A!natural!approach!to!complement!pharmacotherapy!in!the!management!of!diabetes.!Mol.%Nutr.%Food%Res.%2014,!58,!61–78.!! 16!constituents!present!anti9diabetes!properties.!Many!of!these!components!are!believed!to!exert!similar!physiological!effects!to!those!of!synthetic!anti9diabetic!drugs.!!!In!the!following!sections,!the!putative!mechanisms!by!which!glycemia!can!be!regulated!are! discussed! and! an! overview! of! foods! and! dietary! constituents! displaying! anti9diabetic!properties!and/or!contributing!to!glycemic!regulation!via!those!mechanisms!is!presented! (Table! 2.2).! The! evidence! and! plausible! mechanisms! for! the! putative!association!between!dairy! food!consumption!and!the! incidence!of! type!2!diabetes!are!also!reviewed.!!!!!!17!Table&2.2&Overview!of!food!products!and!dietary!constituents!reported!to!present!antidiabetes!properties!and!their!mechanism!of!action.&Anti-diabetic property Physiological mechanism of action Example(s) of foods/food constituents References Reduction of glucose uptake Inhibition of carbohydrate-hydrolysing enzymes  Flavonoids and tannins (many fruits and vegetables), sorghum, sardine and egg white proteins Matsui et al. (1996); Kim et al. (2000); McCue et al. (2005); McDougall et al. (2005); Tadera et al. (2006); da Silva Pinto et al. (2008); Oboh et al. (2010); Gonçalves et al. (2011); Kim et al. (2011a); Yu et al. (2011); Etxeberria et al. (2012) Inhibition of intestinal glucose transport Polyphenols (tea), strawberry and apple extracts Kobayashi et al. (2000); Shimizu et al. (2000); Johnston et al. (2005); Manzano & Williamson (2010) Inhibition of hepatic glucose output Chlorogenic acid (green coffee) Henry-Vitrac et al. (2010); Charkoudian et al. (2012) Delay of intestinal glucose absorption resulting from the formation of viscous gels that slow down the diffusion of glucose in the lumen Soluble fibers (oat, barley), guar gum, high-viscosity hydroxypropylmethylcellulose Jenkins et al. (1978); Torsdottir et al. (1989); Braaten et al. (1991); Vuorinen-Markkola et al. (1992); Granfeldt et al. (1995); Hallfrisch et al. (1995); Wolever & Bolognesi (1996); Yokoyama et al. (1997); Urooj et al. (1998); Maki et al. (2007)  Enhancement of incretin effect Stimulation of GLP-1 secretion triggered by short-chain fatty acids released during bacterial fermentation of fiber or by proteins and peptides Fermentable fibers, starches and sugar alcohol (e.g. cornstarch, lactitol, Viscofiber®), corn and milk proteins, meat hydrolysate Hall et al. (2003); Gee & Johnson (2005); Keenan et al. (2006); Reimer (2006); Greenway et al. (2007); Zhou et al. (2008); Mochida et al. (2010) Inhibition of DPP-IV Peptides or hydrolysates of milk, rice bran, egg, corn and fish proteins  Van Amerongen et al. (2009); Mochida et al. (2010); Tulipano et al. (2011); Uchida et al. (2011); Hatanaka et al. (2012); Huang et al. (2012); Li-Chan et al. (2012); Tulipano et al. (2012); Uenishi et al. (2012)      !18!Table 2.2 continued Anti-diabetic property Physiological mechanism of action Example(s) of foods/food constituents References Enhancement of insulin sensitivity Inhibition of PTP1B Cinnamon, oxidized catechins (tea) Imparl-Radosevich et al. (1998); Ma et al. (2011) Activation of PPARγ Unsaturated fatty acids, isoflavones genistein and daidzein (soy), many herbs and spices (e.g. thyme, oregano, rosemary), 20(S)-protopanaxatriol, psi-baptigenin, hesperidin, apigenin, chrysin, kaempferol Desvergne & Wahli (1999); Liang et al. (2001); Belury et al. (2002); Dang et al. (2003); Han et al. (2006); Rau et al. (2006); Chacko et al. (2007); Salam et al. (2008); Christensen et al. (2009); Cho et al. (2010); Medjakovic et al. (2010); Jungbauer & Medjakovic (2012) Enhancement of insulin action Mimicking of insulin functions Cinnamon, allspice, bay leaves, nutmeg, clove, epigallocatechin gallate, epicatechin gallate, tannins, theaflavins (tea), conglutin-γ (lupin), tannic acid 1,2,3,4,6-penta-O-galloyl-d-glucopyranose   Broadhurst et al. (2000); Anderson & Polansky (2002); Anderson et al. (2004); Liu et al. (2005b); Bertoglio et al. (2011); Terruzzi et al. (2011)  Enhancement of insulin secretion Stimulation of insulin secretion Anthocyanins (e.g. cyanidin-3-glucoside), delphinidin-3-glucoside and pelargonidin-3 galactoside (fruits), S-allyl cysteine sulfoxide and diallyl trisulfide (garlic), bitter gourd Augusti & Sheela (1996); Jayaprakasam et al. (2005); Liu et al. (2005a); Yibchok-anun et al. (2006) Alleviation of oxidative stress Prevention of oxidant formation or radical chain propagation via the activation of Nrf2-mediated ARE Flavonols (fruits, vegetables, tea, wine), lipoic acid, vitamin E, β-carotene, vitamin C  Cao et al. (1998a); Cao et al. (1998b); Jacob et al. (1999); Lean et al. (1999); Johansen et al. (2005); Ziegler et al. (2004); Dembinska-Kiec et al. (2008); Akbar et al. (2011); Qin et al. (2013)  ARE,!antioxidant!response!element;!DPPCIV,!dipeptidylCpeptidase!IV;!GLPC1,!glucagonClike!peptideC1;!Nrf2,!nuclear!factor!(erythroidCderived!2)!like!2;!PTP1B,!protein!tyrosine!phosphatase!1B;!PPARγ,!peroxisome!proliferatorCactivated!receptor!gamma.!!This!table!was!published!in!Lacroix!&!LiCChan!(2014a).!!! 19!2.1.3.1%Food%Products%and%Dietary%Constituents%that%Affect%Glucose%Uptake%!2.1.3.1.1%Inhibition%of%Carbohydrate4Hydrolyzing%Enzymes%!One! of! the! effective! therapeutic! approaches! to! decrease! postprandial! hyperglycemia!consists! in! retarding! the! absorption! of! glucose! by! the! inhibition! of! carbohydrate9hydrolyzing! enzymes! such! as! α9glucosidase! and! α9amylase! in! the! digestive! tract!(Lebovitz,!1997).!In!the!past!decade,!numerous!studies!have!been!conducted!to!identify!carbohydrate9hydrolyzing! enzyme! inhibitors! from! natural! sources,! particularly! from!fruits!and!vegetables.!Raspberry!(McDougall!et!al.,!2005),!red!grape!(McCue!et!al.,!2005;!McDougall!et!al.,!2005),!green!pepper!(McCue!et!al.,!2005),!cambucci!and!cupuaçu!fruits!(De!Souza!Schmidt!Gonçalves!et!al.,!2010),!and!carrot!(McCue!et!al.,!2005),!for!example,!have!been!reported!to!inhibit!α9amylase!activity,!while!blackcurrant!(McDougall!et!al.,!2005),!blueberry!(McDougall!et!al.,!2005),!strawberry!(da!Silva!Pinto!et!al.,!2008)!and!ginger!(Oboh!et!al.,!2010)!were!able!to!inhibit!the!activity!of!α9glucosidase.!!!Polyphenolic!compounds!present!in!these!foods!have!been!suggested!to!be!responsible!for!this!inhibitory!activity.!In!fact,!several!studies!have!reported!a!positive!correlation!between!the!amount!of!phenolic!compounds!in!plant!extracts!and!their!ability!to!inhibit!carbohydrate9hydrolyzing!enzymes!(Etxeberria!et!al.,!2012).!A!high!phenolic!content!is,!however,! not! always! associated!with! a! strong! inhibitory! activity! (Oboh! et! al.,! 2010),!thus!suggesting!that!the!type!of!phytomolecules!and!the! interaction!among!them!may!be!important!factors!determining!the!α9glucosidase!and!α9amylase!inhibitory!activity!of!plant!products!(Etxeberria!et!al.,!2012).!Polyphenolic!compounds!are!believed!to!exert!their! inhibitory! activity!by!binding! to! the! carbohydrate9hydrolyzing!enzymes! (Kim!et!al.,!2011a).!Different!inhibition!modes!have!been!reported,!including!competitive!(You!et!al.,!2012)!non9competitive!and!mixed9type!(Tadera!et!al.,!2006;!Shobana!et!al.,!2009),!suggesting!that!the!enzyme!inhibition!can!occur!by!the!interaction!of!the!polyphenolic!compounds!with!different!regions!of!the!enzymes.!!!! 20!Flavonoids! are! the!most! common! group! of! polyphenolic!molecules! (Etxeberria! et! al.,!2012)!and!are!widely!distributed!in!fruits!and!vegetables!(Kim!et!al.,!2000;!Tadera!et!al.,!2006).! Kim! et! al.! (2000)! investigated! the! in# vitro# effects! of! 21! flavonoids! on! the!activities!of!α9glucosidase!and!α9amylase.!The!flavonoids!amentoflavone,! luteolin!79O9glucoside,!daidzein!and!luteolin!displayed!the!strongest!inhibitory!activity,!with!luteolin!showing! the!strongest! inhibition!with!a!potency!greater! than! that!of! the!anti9diabetic!drug! acarbose! (Kim! et! al.,! 2000).! This! finding! was,! however,! contradicted! in! a!subsequent!study!by!Matsui!et!al.!(2002),!in!which!luteolin!was!not!found!to!be!a!potent!α9glucosidase!inhibitor.!This!discrepancy!might!be!attributable!to!the!different!types!of!α9glucosidase!used!in!these!two!studies:!yeast!by!Kim!et!al.!(2000)!versus!rat!by!Matsui!et! al.! (2002).! Tedera! et! al.! (2006)! also! investigated! the! inhibitory! activity! of! several!groups! of! flavonoids! against! α9amylase! and! α9glucosidase! and! found! that! the!compound’s! structure! had! an! impact! on! its! inhibitory! activity.! Flavonoids! presenting!linkage!of!their!B!ring!at!the!third!position,!2,39double!bond!and!hydroxyl!substitution!on!their!B!ring!had!a!greater!inhibitory!activity.!!!Many! foods! of! plant! origin,! particularly! fruits,! are! also! rich! in! tannin! components!(Serrano! et# al.,! 2009).! Both! proanthocyanidins! and! hydrolysable! tannins! have! been!shown!to!inhibit!carbohydrate9hydrolyzing!enzymes,!possibly!via!their!ability!to!bind!to!the!enzymes!(McDougall!et!al.,!2005;!Gonçalves!et!al.,!2011).!!!In!addition!to!fruits!and!vegetables,!other!food!commodities!such!as!sorghum!(Kim!et!al.,!2011a),!as!well!as!peptides!derived! from!sardine!muscle! (Matsui!et!al.,!1996)!and!egg! white! protein! (Yu! et! al.,! 2011),! have! also! been! found! to! display! α9glucosidase!inhibitory! activity.! These! peptides! have! been! suggested! to! inhibit! the! enzyme! by!binding! to! its! active! site;! the! exact! binding! mechanism,! however,! has! yet! to! be!discovered.!!!! 21!2.1.3.1.2%Inhibition%of%Intestinal%Glucose%Transport%!In! addition! to! their! α9glucosidase! and/or! α9amylase! inhibitory! activity,! polyphenols!found! in! food!products,! such! as! catechins,! have! also! been! shown! to! inhibit! intestinal!glucose!transport!(Kobayashi,!2000;!Shimizu!et!al.,!2000;!Johnston!et!al.,!2005;!Kwon!et!al.,!2007;!Manzano!&!Williamson,!2010).!In!the!intestine,!glucose!is!mainly!transported!by!two!transporters:!by!the!sodium9dependent!glucose!co9transporter!1!(SGLT1)!when!the! luminal! glucose! concentration! is! low! and! by! the! facilitated! glucose! transporter! 2!(GLUT2)!when! luminal! glucose! concentration! is! high! (Manzano!&!Williamson,! 2010).!Polyphenols! in! tea,!particularly! those!having!galloyl! residues!such!as!epigallocatechin!gallate!and!epicatechin!gallate,!were!found!to!bind!to!SGLT1!and!inhibit!the!transport!of!glucose!(Kobayashi!et!al.,!2000;!Shimizu!et!al.,!2000).!Epicatechin!gallate!was!shown!to!competitively! inhibit! the! transporter,!although! it!was!not! itself! transported!by!SGLT1!(Kobayashi!et!al.,!2000;!Shimizu!et!al.,!2000).!Using!human!intestinal!Caco92!cells!under!sodium9dependent! and! sodium9free! conditions! as! a! model! system,! Johnston! et! al.!(2005)! showed! that!glucose!uptake!by!GLUT2!and!SGLT1!was! inhibited!by!aglycones!and!glycosides,!respectively,!while!both!transporters!were!inhibited!by!epigallocatechin!gallate,! epicatechin! gallate! and! epigallocatechin.! The! authors! suggested! that! the!inhibitory! activity! of! non9glycosylated! dietary! polyphenols! may! result! from! steric!hindrance! caused! by! their! incorporation! into! the! membrane! and! the! resulting!disruption! of! the! lipid! bilayer! (Johnston! et! al.,! 2005).! Moreover,! extracts! from!strawberry!and!apple!were!also!found!to!inhibit!glucose!uptake!as!well!as!transport!in!Caco92!cells,!the!inhibition!of!the!GLUT2!transporter!being!stronger!than!that!of!SGLT1!(Manzano!&!Williamson,!2010).!The!polyphenols!59caffeoylquinic!acid,!phloridzin!and!quercetin939O9rhamnoside!were!found!to!contribute!to!90%!of!the!inhibitory!activity!of!the! apple! extract,! while! pelargonidin939O9glucoside! was! responsible! for! 26%! of! the!inhibitory! activity! of! the! strawberry! extract! (Manzano! &! Williamson,! 2010).! These!findings!suggest!that!foods!rich!in!dietary!polyphenols!may!help!in!the!management!of!diabetes!by!inhibiting!intestinal!glucose!uptake.!!!! 22!2.1.3.1.3%Inhibition%of%Hepatic%Glucose%Output%%!The!liver!also!plays!a!central!role!in!glucose!homeostasis!by!regulating!the!uptake!and!storage!of!glucose!by!glycogenesis!as!well! as! the! release!of!glucose!by!glycogenolysis!and! gluconeogenesis! (Nordlie! et! al.,! 1999;! Agius,! 2007).! One! possible! approach! to!suppress! hepatic! glucose! production! and! consequently! lower! fasting! hyperglycemia!consists! of! the! inhibition! of! enzymes! involved! in! glycogenolysis! and/or!gluconeogenesis.!One!such!enzyme!is!glucose!69phosphatase!(G6Pase),!which!is!located!in! the! lumen!of! the!endoplasmic! reticulum!and!catalyzes! the! terminal! step!of!hepatic!glucose! production,! namely! the! hydrolysis! of! glucose969phosphate! (G6P)! to! glucose!(Nordlie!et!al.,!1999;!Agius,!2007).!Currently,!several!synthetic!and!natural!inhibitors!of!the!G6Pase!system!have!been!identified.!These!molecules!are!usually!classified!as!either!inhibitors!of!the!catalytic!subunit!of!G6Pase!or!inhibitors!of!the!G6P!transporter!(T1).!It!should!be!noted,!however,!that!the!inhibition!of!G6Pase!as!a!therapeutic!strategy!for!the!management!of!type!2!diabetes!has!two!main!limitations,!namely!the!increased!risk!of!severe! hypoglycemia! since! G6Pase! activity! is! needed! for! hepatic! glucose! output! by!glycogenolysis! and! gluconeogenesis! (Agius,! 2007),! and! a! risk! of! hepatic! steatosis!resulting! from! the! disturbance! of! the! expression! of! genes! implicated! in! lipogenesis!(Bandsma!et!al.,!2001).!!!!Chlorogenic!acids!are!known!to!be!natural!inhibitors!of!the!G6Pase!system!(Arion!et!al.,!1997).!These!compounds,!secondary!metabolites!of!plants,!are!particularly!abundant!in!green!coffee!beans!(Charkoudian!et!al.,!2012).! In!a!recent!study!by!Henry9Vitrac!et!al.!(2010),! Svetol®,! a!decaffeinated!green!coffee!extract!with!high!content!of! chlorogenic!acids,!was!found!to!cause!the!inhibition!of!G6P!hydrolysis!in#vitro.!The!chlorogenic!acids!caffeoylquinic! and! dicaffeoylquinic! acids! were! shown! to! be! the! active! compounds!responsible! for! the! inhibitory! activity! of! the! green! coffee! extract! on! human! hepatic!G6Pase!activity!(Henry9Vitrac!et!al.,!2010).!The!consumption!of!coffee!has!been!shown!to!be! associated!with! a! reduced! risk!of! type!2!diabetes! in! a!number!of! observational!studies!(van!Dam!&!Feskens,!2002;!Salazar9Martinez!et!al.,!2004;!Huxley!et!al.,!2009).!! 23!One!of!the!possible!mechanisms!responsible!for!the!putative!protective!effects!of!coffee!consumption!on!glycemic! regulation!could!be!related! to! the!ability!of! the!chlorogenic!acids!in!coffee!to!lower!hepatic!glucose!production!via!inhibition!of!G6P!hydrolysis.!!2.1.3.1.4%Delay%of%Gastric%Emptying%and/or%Intestinal%Absorption%of%Carbohydrates%!The! incorporation! of! soluble! fibers! from!different! sources! such! as! oat! (Jenkins! et! al.,!1978;! Braaten! et! al.,! 1991;! Granfeldt! et! al.,! 1995;! Hallfrisch! et! al.,! 1995),! barley!(Wolever! &! Bolognesi,! 1996;! Yokoyama! et! al.,! 1997;! Urooj! et! al.,! 1998),! guar! gum!(Torsdottir! et! al.,! 1989;! Vuorinen9Markkola! et! al.,! 1992),! and! high9viscosity!hydroxypropylmethylcellulose!(Maki!et!al.,!2007),!into!a!meal!has!been!shown!in!many!clinical! studies! to! reduce! postprandial! insulin! and! glucose! responses.! Fenugreek,! a!leguminous!herb!commonly!used!in!Indian!and!Middle!Easter!cuisine,!has!been!used!for!decades! in! the! treatment! of! diabetes! (Aggarwal,! &! Shishu,! 2011).! Over! 30! studies! in!animal!models!and!humans!have!reported!on!the!anti9diabetic!properties!of!this!plant’s!seeds! (Srinivasan,!2005).! !The!beneficial! effect! of! fenugreek! in! glycemia! regulation! is!believed!to!be!primarily!attributable!to!its!fiber!and!gum,!which!can!account!for!up!to!52%!of!the!seeds!content!(Srinivasan,!2005).!Soluble!dietary!fibers!are!believed!to!slow!down!the!absorption!of!digestible!carbohydrates,!and!thus!reduce!hyperinsulinemia,!by!forming! viscous! gels! in! the! gastrointestinal! tract! that! delay! glucose! diffusion! in! the!lumen.!Although!fibers!can!slow!down!gastric!emptying!(Benini!et!al.,!1995),!this!effect!has!been!reported!to!be!marginal!and!not!sufficient!to!significantly! impact!the!rate!of!glucose! absorption! (Edwards! et! al.,! 1988).! Thus,! the! main! factor! explaining! the!beneficial! effect! of! soluble! fibers! on! glycemic! regulation! is! believed! to! be! the!lengthening!of!the!digestion!and!absorption!of!carbohydrate!(Lavin!&!Read,!1995).!!!%!!!!! 24!2.1.3.2%Food%Products%and%Dietary%Constituents%that%Affect%the%Incretin%Effect%!2.1.3.2.1%Stimulation%of%Glucagon4Like%Peptide41%Secretion%!The! insulin! secretory! response! to! the! gut9derived! hormones! GIP! and! GLP91! is!responsible!for!at!least!50%!of!glucose9mediated!insulin!secretion!(Kim!&!Egan,!2008).!Dietary! resistant! starches! and!other! fermentable! fibers,!which! are!un9digested! in! the!small!intestine!but!can!be!fermented!in!the!large!intestine!(Murphy!et!al.,!2008),!have!been!reported!to!stimulate!secretion!of!GLP91!and!peptide!YY!(PYY;!a!peptide!believed!to!regulate!food!intake)!in!animals!and/or!humans!(Gee!&!Johnson,!2005;!Keenan!et!al.,!2006;!Zhou!et!al.,!2006;!Greenway!et!al.,!2007;!Zhou!et!al.,!2008;!Freeland!et!al.,!2010).!!!In! a! study! by! Keenan! et! al.! (2006),! rats! fed! fermentable! resistant! starches! (high!amylose9resistant! cornstarch)! showed! increased! plasma! levels! of! GLP91! and! PYY! as!well! as! an! increased! transcription! of! the! genes! for! PYY! and! proglucagon! (the! GLP91!precursor).!Similarly,!the!ingestion!of!the!non9digestible,!but!fermentable,!sugar!alcohol!lactitol! (49O9β9D9galactopyranosyl9D9glucitol)!was! also! associated!with! an! increase! of!postprandial! PYY! and! GLP91! levels! in! rats! in! a! study! by! Gee! &! Johnson! (2005).! In!humans,! the! consumption! of! 15! or! 20! g! of! lactitol! caused! an! increase! in! plasma!PYY!concentrations,!although!not!as!marked!as!observed!in!rats.!The!plasma!levels!of!GLP91!were,! however,! unaffected! by! lactitol! (Gee!&! Johnson,! 2005).! On! the! other! hand,! the!ingestion! for! fourteen!weeks!of!Viscofiber®,!a!high9viscosity! fermentable!dietary! fiber!from! oat! and! barley,! resulted! in! increased! fasting! levels! of! both! GLP91! and! PYY! in!healthy! overweight! and! obese! subjects! in! a! study! by! Greenway! et! al.! (2007).! The!fermentation!of!resistant!starches!or!other!fermentable!fibers!in!the!gut!is!believed!to!be! the!primary!mechanism! responsible! for! the! increased! secretion!of!GLP91! and!PYY!(Zhou! et! al.,! 2008).! As! the! resistant! starches! go! through! the! large! intestine,! they!undergo! bacterial! fermentation! yielding! short9chain! fatty! acids,! which! have! been!associated!with!increased!gene!expression!of!PYY!and!proglucagon.!!!!! 25!In!addition!to!fermentable!fibers!and!starches,!meat!hydrolysate!(Reimer,!2006)!as!well!as! dairy! (Hall! et! al.,! 2003;! Chen! &! Reimer,! 2009)! and! corn! (Mochida! et! al.,! 2010)!proteins!have!also!been!reported!to!increase!GLP91!secretion.!!!!2.1.3.2.2%Inhibition%of%Dipeptidyl4Peptidase%IV%(DPP4IV)%!Inhibition! of! the! enzyme! DPP9IV! is! now! recognized! as! an! effective! strategy! to! slow!down! the! rapid! inactivation! of! the! incretin! hormones! and! thus! enhance! insulin!secretion.! Although! a! wide! variety! of! synthetic! compounds! presenting! DPP9IV!inhibitory!activity!have!been!identified!over!the!last!decade,!it!is!only!recently!that!DPP9IV! inhibitors! have! been! identified! in! food! products.! Peptides! with! DPP9IV! inhibitory!activity!have!now!been!found!in!hydrolysates!of!the!proteins!from!milk!(Tulipano!et!al.,!2011;! Uchida! et! al.,! 2011;! Tulipano! et! al.,! 2012;! Uenishi! et! al.,! 2012),! egg! (Van!Amerongen!et!al.,!2009),!rice!bran!(Hatanaka!et!al.,!2012),!corn!(Mochida!et!al.,!2010)!and!fish!(Huang!et!al.,!2012;!Li9Chan!et!al.,!2012).!The!effect!of!the!consumption!of!these!natural! DPP9IV! inhibitors! has! not! been! studied! yet! in! humans.! However,! the! oral!administration!of!the!β9casein9derived!peptide!Leu9Pro9Gln9Asn9Ile9Pro9Pro9Leu!to!rats!in!a!glucose!tolerance!test!was!found!to!lead!to!a!significant!reduction!in!blood!glucose!concentrations!compared!with!that!in!the!placebo!group!(Uenishi!et!al.,!2012).!!!The! mechanism! by! which! DPP9IV! inhibitors! found! in! food! proteins! exert! their!inhibitory!activity!has!yet!to!be!elucidated.!Nevertheless,!several!inhibition!modes!have!been! reported! for! synthetic! non9modified! peptide9based! DPP9IV! inhibitors,! including!competitive,! non9competitive,! mixed9type! and! irreversible! (Lorey! et! al.,! 2003;! Kühn9Wache!et! al.,! 2011).! It! is! thus!plausible! that!DPP9IV! inhibitors! found! in! food!proteins!may!inhibit!the!enzyme!by!binding!either!at!the!active!site!or!outside!the!catalytic!triad!of!DPP9IV.!Alternatively,! these!naturally!occurring!DPP9IV! inhibitors! could!also!act! as!substrates!of!the!enzyme!with!low!turnover!rates,!similarly!to!the!well!known!synthetic!DPP9IV! inhibitory! peptides! diprotin! A! (Ile9Pro9Ile)! and! diprotin! B! (Val9Pro9Leu)!(Rahfeld!et!al.,!1991).!!!!! 26!In!addition!to!peptides!derived!from!dietary!proteins,!a!number!of!phenolic!compounds!present! in! fruits! and! vegetables,! including! procyanidin,! resveratrol,! apigenin! and!flavone!have!also!been!reported!to!be!able!to!inhibit!the!activity!of!DPP9IV!(González9Abuín!et!al.,!2012;!Fan!et!al.,!2013).!!!2.1.3.3% Food% Products% and% Dietary% Constituents% that% Mimic% Insulin% Action,% Affect% Insulin%Release%or%Sensitivity%!2.1.3.3.1%Inhibition%of%Protein%Tyrosine%Phosphatase%IB%!Mounting!evidence!has!associated!protein!tyrosine!phosphatase!1B!(PTP1B)!to!obesity,!insulin!resistance!and!type!2!diabetes!(Ma!et!al.,!2011).!The!enzyme!PTP1B!is!believed!to!act!as!a!negative!regulator!of!insulin!and!leptin!signal!transduction!(Goldstein,!2002;!Johnson!et!al.,!2002).!This!phosphatase!dephosphorylates! the! insulin!receptor!as!well!as!the!insulin!receptor!substrate!1!(IRS91)!in!the!insulin!signaling!pathway!(Goldstein!et!al.,!2000),!while!it!binds!and!dephosphorylates!the!tyrosine!kinase!downstream!of!the!leptin! receptor! janus! kinase! 2! (Lund! et! al.,! 2005).! The! inhibition! of! PTP1B! has! been!shown!to!increase!insulin!sensitivity,!improve!glucose!metabolism!and!protect!against!high9fat! diet9induced! obesity! (Ma! et! al.,! 2011;!Goldstein,! 2002),! thus! representing! an!interesting!approach!for!the!treatment!of!metabolic!disorders.!!A!number!of!inhibitors!of!the!PTP1B!enzyme!have!been!disclosed!in!the!literature!over!the! past! decade! (Johnson! et! al.,! 2002).! Although!most! of! these!molecules! have! been!chemically! synthesized,! a! few! natural! PTP1B! inhibitors! have! also! been! identified! in!food! products! (Imparl9Radosevich! et! al.,! 1998;! Ma! et! al.,! 2011;! Teng! et! al.,! 2011).!Cinnamon!has!been!reported!to!potentiate! insulin!activity! in#vitro! (Khan!et!al.,!1990).!Imparl9Radosevich!et! al.! (1998)! showed! that! compound(s)! in! cinnamon! inhibited! the!rat!brain!PTP1,!an!homologue!of!the!PTP1B!enzyme.!Extracts!of!black,!green!and!oolong!teas!were!also!able!to!inhibit!the!activity!of!PTP1B!and!induce!tyrosine!phosphorylation!! 27!of! cellular! proteins! in! cultured! cells! in! a! recent! study! by! Ma! et! al.! (2011).! Oxidized!catechins!in!the!tea!extracts!were!suggested!to!be!responsible!for!the!inhibitory!activity!towards!PTP1B!via! a! two9step!process! in!which! the!phenolic! compounds! first! access!the!PTP1B’s!catalytic!center!and!then!oxidize!the!cysteinyl!residue!within!the!catalytic!pocket!vital!for!enzymatic!activity!(Ma!et!al.,!2011).!!2.1.3.3.2%Activation%of%Peroxisome%Proliferator4Activated%Receptor%Gamma%(PPARγ)%!Peroxisome! proliferator9activated! receptor! gamma! is! a! ligand9activated! transcription!factor!that!is!part!of!the!superfamily!of!nuclear!hormone!receptors.!Since!PPARγ!plays!an! essential! role! in! glucose! and! lipid! homeostasis,! its! activation! represents! a! useful!approach! for! the! treatment! of! type! 2! diabetes.! In! addition! to! the! well9known! TZDs!described! in!section!2.1.2.4,!a!variety!of!other!synthetic!and!natural! compounds!have!been!shown!to!act!as!activators!of!PPARγ!(Penumetcha!&!Santanam,!2012).!!!!Various! unsaturated! fatty! acids! such! as! oleate,! linoleate,! eicosapentaenoic,!docosahexaenoic,!arachidonic!acids!(Desvergne!&!Wahli,!1999)!and!conjugated!linoleic!acid! (Belury! et! al.,! 2002)! have! been! identified! to! be! natural! ligands! for! PPARγ.!Moreover,!a!growing!body!of!evidence!seems!to!suggest!that!oxidized!unsaturated!fatty!acids! are! more! potent! ligands! than! their! unoxidized! counterparts! (Penumetcha! &!Santanam,!2012).! Similarly! to! synthetic!PPARγ! agonists,! dietary! lipids! activate!PPARγ!by!binding!to!its!ligand!binding!domain!and!causing!conformational!changes!(Itoh!et!al.,!2008;!Penumetcha!&!Santanam,!2012).!!Isoflavones! are! also! known! to! be! potent! PPARγ! agonists! (Medjakovic! et! al.,! 2010).!Genistein,! the! principal! soy! phytoestrogen,! has! been! reported! in! several! studies! to!activate!the!nuclear!receptor!(Dang!et!al.,!2003;!Chacko!et!al.,!2007;!Medjakovic!et!al.,!2010).!Moreover,! the!soy! isoflavone!daidzein!and! its!metabolite!equol!have!also!been!reported! to! be!PPARγ! agonists! by!Cho! et! al.! (2010).! The! authors! found! that! daidzein!increases! insulin9stimulated! glucose! uptake! by! enhancing! the! expression! of! insulin9! 28!responsive!glucose!transporter!4!(GLUT!4)!and!IRS91!mRNA!via!the!activation!of!PPARγ.!How! isoflavones! affect! PPARγ! is,! however,! unknown.! In! addition! to! soy! isoflavones,!several!other!plant!extracts,!such!as!thyme,!buckwheat!and!black!elder!(Christensen!et!al.,! 2009),! rosemary! and! sage! (Rau! et! al.,! 2006)! as!well! as! components! in! herbs! and!spices! including! oregano,! marjoram! and! cinnamon! (Jungbauer! &! Medjakovic,! 2012),!were!found!to!be!able!to!activate!PPARγ.!Other!PPARγ!agonists!of!culinary!plant!origin!identified!so!far!include!the!ginsenoside!20(S)9protopanaxatriol!(Han!et!al.,!2006),!the!flavonoids!psi9baptigenin,!hesperidin! (Salam!et! al.,! 2008)!as!well! as! apigenin,! chrysin!and!kaempferol!(Liang!et!al.,!2001).!!!!!!Although!it!is!clear!from!the!current!literature!that!several!foods!and!their!constituents!can! activate! PPARγ,! the! exact! mechanism(s)! by! which! these! elements! interact! with!PPARγ! and! the! physiological! effects! of! the! activation! of! PPARγ! by! these! components,!however,!remain!to!be!explored.!!!2.1.3.3.3%Insulin4Mimetic%Action%!Insulin! replacement! therapy! is! required! in!patients!with! type!1!diabetes! and! is! often!also!needed!in!type!2!diabetic!patients!when!insulin!production!declines.!The!search!for!alternative! options! to! insulin! therapy! that! would! circumvent! the! inconvenience! of!insulin! administration! and! its! side! effects! has! led! to! the! discovery! of! several! small!molecules,!such!as!demethylasterriquinone9B1!(also!known!as!L9783281)!from!a!fungal!extract!(Zhang!et!al.,!1999)!and!the!synthetic!hydroxyfuroic!acid!compound!D9410639!(Tsai! &! Chou,! 2009),! that!mimic! the! in# vivo! function! of! insulin! (García9Vicente! et! al.,!2007;! He! et! al.,! 2011).! These! compounds! trigger! insulin! signaling! by! selectively!activating! the! insulin! receptor! without! displacing! insulin! and! were! found! to! reduce!blood!glucose!levels!in!rodent!models!(Zhang!et!al.,!1999;!García9Vicente!et!al.,!2007;!He!et!al.,!2011).!!!! 29!Information! on! natural! compounds! of! food! origin! displaying! insulin9like! activity! is!sparse.! Nevertheless,! a! few! studies! have! suggested! that! some! dietary! components! in!food! may! mimic! insulin! functions.! Broadhurst! et! al.! (2000)! investigated! the! in# vitro!effect! of! various! plant! extracts! on! insulin9dependent! glucose! metabolism! using! rat!epididymal!adipocytes.!Several!botanical!products!including!cinnamon,!green!and!black!teas,!allspice,!bay!leaves,!nutmeg,!clove!and!mushroom,!were!shown!to!increase!insulin9dependent!utilization!of!glucose.!The!authors!suggested!that!for!most!of!these!products,!the!active!compounds!were!of!phenolic!nature!(Broadhurst!et!al.,!2000).!A!subsequent!study!by!Anderson!&!Polansky!(2002)!showed!that!epigallocatechin!gallate,!epicatechin!gallate,! tannins!and! theaflavins!were! the!active! components! in! tea! responsible! for! its!insulin9potentiating!activity.!Moreover,!further!investigation!on!the!anti9hyperglycemic!activity! of! cinnamon! by! Anderson! et! al.! (2004)! suggested! that! water9soluble!polyphenolic! polymers,! shown! in! the! study! to! increase! insulin9dependent! in# vitro#glucose!metabolism,!are!the!active!components!in!cinnamon!responsible!for!its!insulin9mimetic!biological!activity.!!!!Recently,!a!glycoprotein!found!in!lupin!seed,!conglutin9γ,!was!shown!to!display!insulin9mimetic!activity!(Bertoglio!et!al.,!2011;!Terruzzi!et!al.,!2011).!The!anti9diabetic!effect!of!lupin! has! been! reported! as! early! as! in! the! 1930s! in! the! traditional! pharmacopoeia!(Scarafoni!et!al.,!2007).!However,!conglutin9γ!was!only!recently!considered!as!the!active!principle!responsible!for!the!claimed!biological!activity.!In!a!glucose!overload!trial!with!murine! models! and! healthy! human! subjects! conducted! by! Bertoglio! et! al.! (2011),!conglutin9γ! was! found! to! reduce! blood! glucose! levels! but! without! affecting! insulin!concentrations.! ! The! authors! suggested! that! the! protein! acted! as! an! insulin9mimetic!compound.!Similarly,!work!by!Terruzzi!and!colleagues!(2011)!with!cell!models!showed!that! conglutin9γ! displayed! insulin9mimetic! activity! and! could! regulate!muscle! energy!metabolism,! proteosynthesis! and! muscle9specific! myosin9heavy! chain! gene!transcription!via!the!modulation!of!the!same!signaling!pathway!as!insulin.!!!!! 30!In!addition!to!conglutin9γ,!tannic!acid,!a!type!of!tannin!found!in!plant9based!foods,!such!as!green!tea,!berries,!vegetables!and!cereal!grains,!has!also!been!reported!in!a!study!by!Liu!et!al.#(2005b)!to!present!insulin9like!glucose!transport!stimulatory!activity!in!3T39L1!adipocytes.!In!a!subsequent!study,!the!authors!found!that!the!tannic!acid!component!1,2,3,4,69penta9O9galloyl9d9glucopyranose! (PGG)! was! able! to! bind! to! the! insulin!receptor!and!activate! the! insulin9mediated!glucose! transport!signaling!pathway!(Li!et!al.,! 2005).!Moreover,! PGG!was! shown! in# vivo! to! reduce! blood! glucose! concentrations!and!improve!glucose!tolerance!in!diabetic!and!obese!rodent!models!(Li!et!al.,!2005).!!2.1.3.3.4%Insulin%Secretagogues%!Insufficient! insulin! secretion! is! a! major! pathophysiology! in! the! development! of!hyperglycemia!in!diabetes!(Stumvoll!et!al.,!2005).!While!pancreatic!β9cells!are!no!longer!able! to! produce! insulin! in! type! 1! diabetic! patients,! individuals! with! type! 2! diabetes,!especially!at!the!early!stage!of!the!disease,!may!have!nearly!normal!β9cell!mass!(Liu!et!al.,!2007).!In!those!latter!diabetic!patients,!treatment!with!insulin!secretagogues!is!often!an! interesting!therapeutic!strategy.! In!addition!to! the!putative!mechanisms!presented!earlier,! dietary! polyphenols! have! also! been! suggested! to! improve! carbohydrate!regulation!by!stimulating!insulin!secretion!(Jayaprakasam!et!al.,!2005;!Hanhineva!et!al.,!2010).! In!a!study!by!Jayaprakasam!et!al.#(2005),!anthocyanins! found!in!fruits,!such!as!cyanidin939glucoside,! delphinidin939glucoside! and! pelargonidin93! galactoside,! were!shown! to! potentiate! insulin! secretion! in! rodent! pancreatic! β9cells.! Garlic! oil! and! its!sulfur9containing! compound! diallyl! trisulfide,! as! well! as! the! sulfur9containing! amino!acid! S9allyl! cysteine! sulfoxide! (SACS)! isolated! from! garlic,! were! also! suggested! to!stimulate!insulin!secretion!in!studies!by!Liu!et!al.!(2005a)!and#Augusti!&!Sheela!(1996),!respectively.! Furthermore,! when! administered! to! alloxan! diabetic! rats,! SACS!significantly! improved! diabetic! conditions! to! a! similar! extent! as! glyburide! (a!sulfonylurea!drug,!Table!2.1)!and!insulin!(Augusti!&!Sheela,!1996).!Similarly,!Yibchok9anun!and!colleagues!(2006)!showed!that!a!zinc9free!protein!extract! from!bitter!gourd!(Momordica# charantia),! one! of! the! most! studied! vegetables! for! its! anti9diabetes!! 31!properties,! was! effective! at! reducing! plasma! glucose! levels! and! increasing! plasma!insulin!secretion!in!diabetic!and!non9diabetic!rats.!!!The!mechanism!by!which!these!dietary!factors!potentiate!insulin!secretion!is!unknown.!However,!considering!their!structural!similarity!to!meglitinides,!it!can!be!hypothesized!that! some! dietary! polyphenols! may! act! analogously! to! the! benzoic! acid! and!phenylalanine!derivatives!repaglinide!and!nateglinide!(Table!2.1),!which!are!known!to!stimulate! insulin! release! by! enhancing! the! function! of! the! ATP9dependent! potassium!channels!in!pancreatic!β9cells!(Landgraf,!2000).!!2.1.3.4%Food%Products%and%Dietary%Constituents%that%Alleviate%Oxidative%Stress%!A! growing! body! of! evidence! has! suggested! that! oxidative! stress,! resulting! from! the!overproduction!and/or! insufficient! removal! of! reactive!oxygen! species! (ROS),! plays! a!pivotal!role!in!the!onset!and!progression!of!diabetes!and!its!complications!(Rösen!et!al.,!2001;! Chang! &! Chuang,! 2010).! Nutrient! excess,! dyslipidemia,! endoplasmic! reticulum!stress,! chronic! inflammation! and! over9activity! of! the! renin9angiotensin! system! are!among!the!many!factors!that!have!been!shown!to!enhance!the!production!of!oxidants!in!diabetic! patients! (Henriksen! et! al.,! 2013).! The! resulting! excess! of! ROS! has! been!suggested! to! impair! critical! signaling! pathways! involved! in! glycemia! regulation,!ultimately! leading! to! reduced! insulin! sensitivity! in! the! liver! and! muscles! as! well! as!pancreatic!β9cell!failure!(Chang!&!Chuang,!2010;!Giacco!&!Brownlee,!2010;!Henriksen,!2013).! ! Moreover,! many! studies! have! suggested! that! oxidative! stress! and! chronic!inflammation! are! interrelated! factors! involved! in! the!development!of! type!2!diabetes!and!other!chronic!diseases!(Dembinska9Kiec!et!al.,!2008).!!!!!Antioxidants! and! nutrients! with! high! antioxidant! capacity! have! been! suggested! to!reduce!oxidative!stress!through!their!ability!to!prevent!oxidant!formation!and!interfere!with!radical!chain!propagation,!possibly!through!their!ability!to!activate!the!body’s!own!antioxidant!systems!via!the!nuclear!factor!(erythroid9derived!2)!like!2!(Nrf2)!pathway!! 32!(Johansen!et!al.,!2005;!Yu!et!al.,!2012).!Several!new!lines!of!studies!have!proposed!that!phytochemical!compounds,! including!several! types!of! flavonoids! found! in!plant! foods,!could! induce! the!activation!of! the!Nrf29mediated!antioxidant!response!element!(ARE)!and!promote!the!gene!expression!of!antioxidant!species!such!as!superoxide!dismutase91,! glutathione! peroxidase! 1,! glutathione! S9transferase! and! thioredoxin,!which! in! turn!can!remove!ROS!(Chartoumpekis!&!Kensler,!2013;!Qin!et!al.,!2013).!!Moreover,!via!their!ability! to! lower! oxidative! stress,! antioxidants! have! also! been! suggested! to! reduce!chronic! inflammation! by! causing! the! inhibition! of! the! nuclear! factor9κB! pathway,! a!crucial!player!in!tissue!inflammation!(Akash!et!al.,!2013).!!Studies! in! humans! have! shown! that! the! intake! of! vegetables! and! fruits! rich! in!antioxidants! can! increase! plasma! antioxidant! capacity! (Cao! et! al.,! 1998a! &! 1998b).!Moreover,! dietary! flavonols,! which! are! found! ubiquitously! in! fruits,! vegetables! and!beverages!such!as!tea!and!wine,!have!been!reported!to!have!a!protective!effect!against!oxidative!stress! in!diabetic!patients!(Lean!et!al.,!1999).!Lipoic!acid,!a!potent! lipophilic!free!radical!scavenger!that!can!be!found!in!red!and!organ!meats,!has!also!been!shown!to!increase! insulin9mediated!glucose!uptake! (Jacob!et!al.,!1999)!and! to! improve!positive!neuropathic!symptoms!and!deficits!in!diabetic!patients!(Ziegler!et!al.,!2004).!!!Despite! some! promising! findings! on! the! effects! of! dietary! antioxidants! on! oxidative!stress,! glycemia! regulation! and! complications! associated! with! diabetes,! a! number! of!randomized! controlled! trials! have! failed! to! show! a! beneficial! effect! of! antioxidant!interventions,!including!vitamin!E,!β9carotene!and!vitamin!C!supplementations,!on!the!prevention!of!type!2!diabetes!(Chang!&!Chuang,!2010).!In!a!recent!meta9analysis,!Akbar!et!al.!(2011)!concluded!that!dietary!vitamins!C!and!E!had!no!effect!on!plasma!glucose!and! insulin! levels,! but! significantly! reduced! the! levels! of! glycated! hemoglobin! A1C,! a!recognized! surrogate! for! diabetic! complications.! These! findings! suggested! that! the!dietary! antioxidants! vitamins! C! and! E! may! not! be! effective! in! improving! glycemic!control!but!could!have!a!protective!effect!against!diabetic!complications!(Akbar!et!al.,!2011).!!! 33!2.1.4%Putative%Association%Between%the%Consumption%of%Dairy%Foods%and%the%Incidence%of%Diabetes%!Over!the!last!10!years,!the!relationship!between!the!consumption!of!dairy!foods!and!the!incidence!of!type!2!diabetes!has!been!investigated!in!a!number!of!cohort!studies!(Table!2.3).! These,! however,! have! yielded! mixed! results,! some! studies! having! reported! a!reduced!risk!of!type!2!diabetes!with!higher!consumption!of!dairy!products!(Choi!et!al.,!2005;! Liu! et! al.,! 2006;! Pittas! et! al.,! 2006;! Villegas! et! al.,! 2010;! Malik! et! al.,! 2011;!Grantham!et!al.,!2013),!whereas!others!have!found!no!association!(van!Dam!et!al.,!2006;!Elwood!et!al.,!2007;!Kirii!et!al.,!2009;!Margolis!et!al.,!2011;!Sluijs!et!al.,!2012;!Louie!et!al.,!2013;! Soedamah9Muthu! et! al.,! 2013;! Struijk! et! al.,! 2013;! von! Ruesten! et! al.,! 2013).!Investigations! on! the! consumption! of! specific! types! of! dairy! foods! have! also! given!inconsistent! results.! For! example,! while! some! epidemiological! studies! have! reported!associations!between! the! incidence!of! type!2!diabetes!and! the! intake!of! low9fat!dairy!products! (Choi! et! al.,! 2005;! Liu! et! al.,! 2006;! van!Dam! et! al.,! 2006;!Malik! et! al.,! 2011;!Margolis!et!al.,!2011),!cheese!(Liu!et!al.,!2006;!Sluijs!et!al.,!2012;!Grantham!et!al.,!2013)!and!yogurt!(Choi!et!al.,!2005;!Liu!et!al.,!2006;!Margolis!et!al.,!2011;!Sluijs!et!al.,!2012),!other! investigations! have! not! found! any! relationship! (Kirii! et! al.,! 2009;! Louie! et! al.,!2013;! Soedamah9Muthu! et! al.,! 2013;! Struijk! et! al.,! 2013;! von! Ruesten! et! al.,! 2013).!!Nevertheless,! findings! from! two! recent! meta9analyses! of! prospective! cohort! studies!suggested! that! the! consumption!of!dairy!products,! low9fat!dairy!products! and! cheese!have!a!preventive!effect!on!the!incidence!of!type!2!diabetes!(Aune!et!al.,!2013;!Gao!et!al.,!2013).!Of!the!17!studies!included!in!their!meta9analysis,!Aune!et!al.!(2013)!found!that!individuals!with! the!highest! consumption!of! dairy! foods,! compared! to! those!with! the!lowest! intake,! presented! a! lower! risk! of! diabetes! (RR! =! 0.89,! 95%! CI! 0.8290.96,! P!heterogeneity!0.05).!Similarly,!Gao!et!al.!(2013),!who!considered!15!prospective!cohort!studies!and!1!case9cohort!study!in!their!analysis,!reported!a!relative!risk!of!0.88!(95%!CI!0.8190.98,!P!heterogeneity!0.000)!for!individuals!with!high!versus!low!consumption!of! dairy! foods.! These! findings! are! in! accordance! with! results! from! previous! meta9analyses!(Elwood!et!al.,!2010;!Tong!et!al.,!2011).!!34!Table&2.3&Observational!studies!on!the!association!between!dairy!intake!and!type!2!diabetes.&&! Study Number and gender of subjects Location Design of the Cohort (follow-up, years) Dairy intake (lowest and highest amount) Results Adjustments Choi et al., 2005 41,254 men United States Health Professionals Follow-up cohort (HPFS) (12 yrs) Total dairy: <0.9 and ≥2.9 servings/d Total dairy: RR = 0.77, 95% CI 0.62-0.95, Ptrend 0.003 Age, BMI, total energy, smoking, high cholesterol, hypertension, diabetes history, alcohol, physical activity, follow-up time, certain dietary variables, fiber, glycemic load Low-fat: <0.14 and >1.58 servings/d Low-fat: RR = 0.73, 95% CI 0.59-0.89, Ptrend 0.001 High-fat: <0.38 and >1.72 servings/d High-fat: RR = 0.97, 95% CI 0.78-1.21, Ptrend 0.78 Liu et al., 2006a 37,183 women United States Women’s Health Study cohort (WHS) (10 yrs) Total dairy: <0.85 and >2.9 servings/d Total dairy: RR = 0.68, 95% CI 0.52-0.89, Ptrend 0.006 Age, BMI, total energy, smoking, high cholesterol, hypertension, diabetes history, alcohol, physical activity, hormone therapy, total fat, fiber, glycemic load, vit. D, Ca, Mg Low-fat: ≤0.27 and >2.00 servings/d Low-fat: RR = 0.69, 95% CI 0.52-0.91, Ptrend 0.007 High-fat: <0.20 and >1.329 servings/d High-fat: RR = 0.99, 95% CI 0.82-1.20, Ptrend 0.90 Pittas et al., 2006 83,779 women United States Nurses’ Health Study (NHS) (20 yrs) Total dairy: <1.0 and >3.0 servings/d Total dairy: RR = 0.89, 95% CI 0.81-0.99, Ptrend 0.008 Age, BMI, total energy, smoking, hypertension, diabetes history, physical activity, type of fat, glycemic load, retinol, caffeine, fiber, Ca, vit. D van Dam et al., 2006 41,186 black women United States Black Women’s Health Study cohort (BWHS) (8 yrs) Total dairy: 0.07 and 2.53 servings/d Total dairy: HR = 0.93, 95% CI 0.75-1.15, Ptrend 0.31 Age, BMI, total energy, smoking, physical activity, alcohol, diabetes history, education level, coffee, sugar-sweetened soft drink, meats Low-fat: 0.00 and 1.22 servings/d Low-fat: HR = 0.87, 95% CI 0.76-1.00, Ptrend 0.04 High-fat: 0.07 and 1.33 servings/d High-fat: HR = 1.03, 95% CI 0.88-1.20, Ptrend 0.94 Elwood et al., 2007 640 menb United Kingdom The Caerphilly cohort (20 yrs) Total milk: lowest versus highest quartile Total milk: RR = 0.57, 95% CI 0.20-1.63, Ptrend 0.247 Age, BMI, smoking, social class !35!Table&2.3&continued&&Study Number and gender of subjects Location Design of the Cohort (follow-up, years) Dairy intake (lowest and highest amount) Results Adjustments Kirii et al., 2009 25,877 men and 33,919 women Japan The Japan Public Health Center-based Prospective Study (JPHC) (5 yrs) Total dairy: <50 and ≥300 g/d Total dairy:  Men: OR = 1.18, 95% CI 0.90-1.56, Ptrend 0.21 Women: OR = 0.71, 95% CI 0.51-0.98, Ptrend 0.054 Age, BMI, total energy, smoking, hypertension, diabetes history, alcohol, physical activity, coffee, energy-adjusted magnesium, area Milk: <50 and ≥200 g/d Milk:  Men: OR = 1.02, 95% CI 0.85-1.24, Ptrend 0.88 Women: OR = 0.87, 95% CI 0.70-1.09, Ptrend 0.16  Cheese: 0 and ≥5 g/d Cheese:  Men: OR = 0.88, 95% CI 0.64-1.21, Ptrend 0.39 Women: OR = 1.12, 95% CI 0.80-1.57, Ptrend 0.56  Yogurt: 0 and ≥60 g/d Yogurt:  Men: OR = 1.01, 95% CI 0.75-1.136, Ptrend 0.94 Women: OR = 0.77, 95% CI 0.58-1.01, Ptrend 0.13 Malik et al., 2011 37,038 women United States Nurses’ Health Study II cohort (NHS II)    (7 yrs) Total dairy:  0.62 and 2.14 servings per 1000 kcal/d  Total dairy: RR = 0.75, 95% CI 0.55-1.02, Ptrend 0.03 Age, BMI, total energy, smoking, diabetes history, alcohol, physical activity, hormone therapy, oral contraceptive, polyunsaturated: saturated fat ration, trans fat, cereal fiber, process meat, carbonated soft drink, fruit drink, coffee, mutual adjustment: low- and high-fat dairy products Low-fat: 0.18 and 1.44 servings per 1000 kcal/d  Low-fat: RR = 0.74, 95% CI 0.55-1.01, Ptrend 0.03 High-fat: 0.19 and 1.14 servings per 1000 kcal/d  High-fat: RR = 0.72, 95% CI 0.53-0.99, Ptrend 0.03 !36!Table&2.3&continued)Study Number and gender of subjects Location Design of the Cohort (follow-up, years) Dairy intake (lowest and highest amount) Results Adjustments Margolis et al., 2011 82,076 women United States Women’s Health Initiative Observational Study (WHI-OS)             (7.9 yrs) Total dairy: 0.5 and 3.4 servings/d Total dairy: RR = 0.93, 95% CI 0.83-1.04, Ptrend 0.15 Age, race/ethnicity, BMI, total energy, smoking, systolic & diastolic blood pressure, diabetes history, education level, income, alcohol, physical activity, hormone therapy, total fat, total fiber, glycemic load, Mg, interaction of low-fat dairy and BMI, interaction of yogurt and time Low-fat: 0.05 and 2.8 servings/d Low-fat: RR = 0.65, 95% CI 0.44-0.96, Ptrend 0.003 Yogurt: < 1 serving/month and ≥ 2 servings/week Yogurt: RR = 0.46, 95% CI 0.31-0.68, Ptrend 0.004 Sluijs et al., 2012 24,475 men and women Europe European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct Study (11.7 yrs)c Total dairy: 79.7 and 628.9 g/d Total dairy: HR = 0.97, 95% CI 0.82-1.15, Ptrend 0.69 Age, center, sex, BMI, total energy, smoking, education level, alcohol, physical activity, fruit, vegetables, red meat, processed meat, sugar sweetened soft drinks, coffee, cereals, cereal products, Ca, Mg, vit. D   Milk: 0.3 and 486.1 g/d Milk: HR = 1.08, 95% CI 0.90-1.31, Ptrend 0.52  Yogurt and thick fermented milk: 0 and 190.4 g/d Yogurt and thick fermented milk: HR = 0.89, 95% CI 0.77-1.03, Ptrend 0.11  Cheese: 3.2 and 73.7 g/d Cheese: HR = 0.83, 95% CI 0.70-0.98, Ptrend 0.003   Fermented dairy: 11.6 and 220.7 g/d Fermented dairy: HR = 0.85, 95% CI 0.73-0.99, Ptrend 0.02 Louie et al., 2013 1824 men and women Australia Blue Mountains Eye Study (BMES)      (10 yrs) Total dairy: 0.5 and 3.1 servings/d Total dairy: OR = 1.50, 95% CI 0.47-4.77, Ptrend 0.568 Age, sex, BMI, total energy, smoking, physical activity, diabetes history, systolic blood pressure, HLD & total cholesterol, triglyceride, glycemic load, vegetable fiber, Ca Reduced/low-fat: 0 and 2.1 servings/d Reduced/ low-fat: OR = 1.09, 95% CI 0.57-2.09, Ptrend 0.764 Regular fat dairy: 0.1 and 1.9 servings/d Regular fat dairy: OR = 0.87, 95% CI 0.48-1.57, Ptrend 0.596 !37!Table&2.3&continued)!Study Number and gender of subjects Location Design of the Cohort (follow-up, years) Dairy intake (lowest and highest amount) Results Adjustments Grantham et al., 2013 5582 men and women Australia The Australian Diabetes Obesity and Lifestyle Study (AusDiab) (5 yrs) Total dairyd: 205 and 477 g/d Total dairy: OR = 0.71, 95% CI 0.48-1.05 Age, sex, total energy, smoking, physical activity, education level, diabetes history, triacylglycerol, HDL cholesterol, systolic blood pressure, waist & hip circumference Low-fat milkd: 0 and 375 g/d Low-fat milk: OR = 0.65, 95% CI 0.44-0.94 Full-fat milkd: 0 and 375 g/d Full-fat milk: OR = 1.18, 95% CI 0.78-1.79 Yogurtd: 3 and 114 g/d Yogurt: OR = 1.14, 95% CI 0.78-1.67 Cheesed: 4 and 29 g/d Cheese: OR = 0.78, 95% CI 0.53-1.15 Soedamah-Muthu et al., 2013 4526 men and women United Kingdom The Whitehall II Prospective Cohort Study (10 yrs) Total dairy: 246 and 575 g/d Total dairy: HR = 1.30, 95% CI 0.95-1.77, Ptrend 0.11 Age, ethnicity, BMI, employment grade, smoking, alcohol, physical activity, total energy, coronary heart disease and hypertension history, fruit, vegetables, bread, meat, fish, coffee, tea High-fat: 27 and 182 g/d High-fat: HR = 1.23, 95% CI 0.91-1.67, Ptrend 0.17 Low-fat: 28 and 458 g/d Low-fat: HR = 0.98, 95% CI 0.73-1.31, Ptrend 0.88 Total milk: 147 and 441 ml/d Total milk: HR = 0.97, 95% CI 0.71-1.32, Ptrend 0.86 Fermented dairy: 17 and 105 g/d Fermented dairy: HR = 1.17, 95% CI 0.87-1.58, Ptrend 0.31 Yogurt: 0 and 117 g/d Yogurt: HR = 1.04, 95% CI 0.77-1.42, Ptrend 0.77 Cheese: 6 and 31 g/d Cheese: HR = 1.20, 95% CI 0.88-1.64, Ptrend 0.25 !38!Table&2.3&continued)Study Number and gender of subjects Location Design of the Cohort (follow-up, years) Dairy intake (lowest and highest amount) Results Adjustments Struijk et al., 2013 5,953 men and women Denmark Inter99 Study (5 yrs) Total dairy: 47 and 578 g/d Total dairy: OR = 0.96, 95% CI 0.58-1.58, Ptrend 0.95 Age, gender, intervention group, total energy, smoking, education level, alcohol, physical activity, wholegrain cereal, meat, fish, tea, fruit, vegetables, change in diet from baseline to 5 yrs follow-up, waist circumference Low-fat: 6 and 536 g/d Low-fat: OR = 0.85, 95% CI 0.52-1.40, Ptrend 0.72 High-fat: 4 and 89 g/d High-fat: OR = 0.94, 95% CI 0.56-1.58, Ptrend 0.96 Milk & milk products: 16 and 546 g/d Milk & milk products: OR = 0.95, 95% CI 0.58-1.57, Ptrend 0.84 Cheese: 4 and 49 g/d Cheese: OR = 0.78, 95% CI 0.47-1.29, Ptrend 0.28 Fermented dairy: 13 and 260 g/d Fermented dairy: RR = 0.86, 95% CI 0.50-1.47, Ptrend 0.51 von Ruesten et al., 2013 9098 men and 14,433 women  Germany European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study (8 yrs) Low-fat dairy: per 100 g/d Low-fat dairy: HR = 1.02, 95% CI 0.96-1.09 Age, sex, BMI, energy intake, smoking, pack-years of smoking, alcohol, physical activity, education level, hypertension, high blood lipids, waist-to-hip ratio, vitamin supplementation, other food groups High-fat dairy: per 100 g/d High-fat dairy: HR = 1.00, 95% CI 0.92-1.08 Low-fat cheese: per 30 g/d Low-fat cheese: HR = 0.98, 95% CI 0.83-1.15 High-fat cheese: per 30 g/d High-fat cheese: HR = 0.96, 95% CI 0.85-1.08 a!Results!with!multivariate!model!3!adjustments.!!b!Data!from!food!frequency!questionnaire!was!available!for!2,375!men,!but!only!640!men!kept!a!sevenFday!weighed!dietary!intake!record.!c!A!total!of!340,234!EPIC!participants!were!followed!up!for!3.99!million!personFyears.!d!Dairy!intake!values!for!this!study!as!reported!in!a!metaFanalysis!of!cohort!studies!by!Aune!et!al.!(2013).!!OR,!odds!ratio;!RR,!relative!risk;!HR,!hazard!ratio;!BMI,!body!mass!index;!Ca,!calcium,!vit.!D,!vitamin!D;!Mg,!magnesium.!This!table!is!adapted!from!Lacroix!&!LiFChan!(2014b).!!! 39!2.1.4.1%Dairy%Consumption%and%Type%2%Diabetes:%Possible%Mechanisms4%!2.1.4.11%Effects%of%Non/protein%Constituents%in%Dairy%Products%on%the%Risk%of%Type%2%Diabetes%!A!number!of!milk!constituents!have!been!suggested!to!contribute!to!the!beneficial!effect!of! dairy! food! consumption!on! the! incidence!of! type!2!diabetes.! Lactose! is! the! second!major!milk! components! after!water! (Lindmark!Månsson,! 2008).!However,! despite! its!high! content! in! cow’s! milk,! the! current! knowledge! on! the! possible! roles! of! dairy!components! in! the! incidence!of! type!2!diabetes!does!not!seem!to!suggest! lactose!as!a!dominant! factor! in! the! inverse! association! between! dairy! food! consumption! and!diabetes.! In! fact,! lactose! intake! was! not! found! to! be! significantly! related! to! type! 2!diabetes!(RR!=!0.99;!95%!CI!0.80O1.22;!Ptrend!0.33)!in!a!prospective!study!by!Janket!et!al.!(2003),! in!which! the! intake!of! total!and!specific! types!of! sugar,! including! lactose,!was!investigated!in!38,480!women!of!The!Women’s!Heath!Study.!Lactose,!dairy!protein!and!fat! have! been! suggested! to! enhance! satiety! and! lower! the! risk! of! overweight! and!obesity,!known!to!be!major!risk!factors!for!type!2!diabetes,!compared!with!other!highOcarbohydrate!foods!and!beverages!(Pereira!et!al.,!2002).!However,!in!a!study!by!Pereira!et! al.! (2002),! adjusting! for! these! nutrients! had! no! significant! effects! on! the! inverse!relationship! they! observed! between! dairy! food! intake! and! the! insulin! resistance!syndrome.!!Cow’s! milk! is! known! to! be! a! major! food! source! of! calcium,! magnesium! as! well! as!vitamin! D.! Vitamin! D! deficiency! has! been! shown,! in! animal! and! human! models,! to!increase!peripheral!tissue!insulin!resistance!and!decrease!insulin!secretion!(Isaia!et!al.,!2001;! Moreira! &! Hamadeh,! 2010).! In! fact,! a! positive! correlation! between! 25Ohydroxyvitamin! D! (25(OH)D)! concentration! and! insulin! sensitivity! was! observed! in!glucoseOtolerant! individuals,! whereas! hypovitaminosis! D!was! found! to! have! negative!effect!on!pancreatic!βOcell! function! (Chiu!et! al.,! 2004).!Magnesium!deficiency!has!also!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!4!A!version!of!this!section!has!been!published.!Lacroix,!I.M.E.,!LiOChan,!E.C.Y.!Investigation!of!the!putative!association!between!dairy!consumption!and! incidence!of! type!1!and! type!2!diabetes.!Crit.'Rev.'Food'Sci.'Nutr.'2014,!54,!411–432.!! 40!been!found!to!have!adverse!effects!on!insulin!action!(LopezORidaura!et!al.,!2004;!Martini!et!al.,!2010).!Hypomagnesemia!may!alter!tyrosineOkinase!activity!at!the!insulin!receptor!level! and! may! lead! to! increased! intracellular! calcium! concentration,! and! these! two!effects! can! result! in! impaired! insulin! action! (Martini! et! al.,! 2010).!On! the!other!hand,!calcium!is!needed!for! insulinOmediated! intracellular!processes!and!changes! in!calcium!concentration! in! insulin! target! tissues!may! affect! the! insulin! signal! transduction! and!cause!peripheral!insulin!resistance!(Pittas!et!al.,!2007).!!!Although!vitamin!D,!magnesium!and!calcium!may!play!a!role!in!glucose!regulation,!the!findings!of!their!effects!on!the!risk!of!type!2!diabetes!are!conflicting!and!the!significance!of! their! possible! contribution! to! the! inverse! association! observed! between! dairy!product!intake!and!diabetes!remains!unclear.!Total!vitamin!D!and!total!calcium!intakes!were!significantly!associated!with!the!risk!of!type!2!diabetes!in!a!prospective!study!by!Pittas!et!al.!(2006).!These!associations,!however,!were!found!when!these!nutrients!were!obtained! from!supplements,! rather! than! from!dietary! sources! (Pittas!et!al.,!2006).!On!the!other!hand,!calcium!and!vitamin!D!intakes!alone!were!not!linked!to!the!risk!of!type!2!diabetes! in!a! further!study!by!Kirii!et!al.!(2009),!and!a!positive!association!between!calcium!and!type!2!diabetes!was!observed!only!in!individuals!with!a!higher!vitamin!D!intake.!Yet,!calcium!plus!vitamin!D!supplementation!was!not!shown!to!reduce!the!risk!of!diabetes!in!a!randomized!placeboOcontrolled!trial!(de!Boer!et!al.,!2008).!Calcium!and!vitamin!D!intake!were!not!found!either!to!explain!the!inverse!association!between!dairy!consumption!and!the!insulin!resistance!syndrome!observed!in!overweight!adults!of!The!Coronary! Artery! Risk! Development! in! Young! Adults! (CARDIA)! study! (Pereira! et! al.,!2002).!Vitamin!D!is!mainly!produced!endogenously!by!the!skin!during!exposure!to!the!sunlight,! and! diet! only! accounts! for! approximately! 30%! of! the! vitamin! D! obtained!(Moreira!&!Hamadeh!2010).!Thus,!dietary!intake!of!this!vitamin!alone!may!not!provide!an!accurate!indication!of!an!individual’s!overall!vitamin!D!status.!!!!!!!!!The! impact! of! dietary! fat! on! the! incidence! of! type! 2! diabetes! has! been! of! particular!interest! due! to! the! role! that! fatty! acids! play! in! glucose! regulation! by! affecting! cell!membrane! function,! insulin! signal,! genetic! expression! as! well! as! enzymatic! activity!! 41!(Risérus! et! al.,! 2009).! Several! factors! may! affect! bovine! milk! fatty! acid! composition,!including! the!breed,! the! type!of! feeding!and! the! season! (Pešek!et! al.,! 2005;!Lindmark!Månsson,! 2008),! but! in! general! bovine! milk! contains! approximately! 4.2%! of! fat,! of!which!almost!70%!is!saturated!whereas!monoOunsaturated!and!polyOunsaturated!fatty!acids! account! for! about! 25%! and! 2.3%,! respectively! (Lindmark! Månsson,! 2008).!Moreover,! approximately! 7O9%! of! milk! fat! is! composed! of! mediumOchain! fatty! acids!(MCFAs)!(Pešek!et!al.,!2005),!which!present!a!chain!length!of!8!to!12!carbons,!and!about!3O7%!of!shortOchain!fatty!acids,!which!present!a!chain!length!of!4!to!6!carbons!(Pešek!et!al.,!2005;!Lindmark!Månsson,!2008).!A!diet!characterized!by!a!high!intake!of!saturated!fat!disturbed!insulin!sensitivity!(Pfeuffer!&!Schrezenmeir,!2006;!Wein!et!al.,!2009).!On!the! other! hand,! MCFAs! have! been! reported! to! improve! insulin! sensitivity! in! some!animal! and! human! studies! (Pfeuffer!&! Schrezenmeir,! 2002;! Pfeuffer!&! Schrezenmeir,!2006;!Wein!et!al.,!2009).!Unlike!long!chain!fatty!acids,!MCFAs!do!not!require!binding!to!fatty!acidObinding!proteins! for!their! transport!within!cells!and!across!membranes!and!this!distinction!impacts!numerous!regulatory!pathways.!However,! the!effect!of!MCFAs!on!insulin!level!or!plasma!glucose!observed!in!most!human!or!animal!studies!is!not!so!clearOcut!(Pfeuffer!&!Schrezenmeir,!2006).!!Another! type! of! fatty! acid! naturally! found! in! cow’s!milk,! transOpalmitoleic! acids,!was!recently!suggested!to!have!a!preventive!effect!on!the!risk!of!diabetes!(Mozaffarian!et!al.,!2010!&!2013).!Mozaffarian!et!al.!(2010)!examined!the!association!between!blood!transOpalmitoleate! level! and! the! incidence! of! type! 2! diabetes! in! 3,736! subjects! of! the!Cardiovascular! Health! Study.! Higher! circulating! levels! of! transOpalmitoleate! were!associated!with! significant! lower! risk! of! type! 2! diabetes! (HR! 0.41,! 95%! CI! 0.27O0.64![quintiles! 4! versus! 1]! and! HR! 0.38,! 95%! CI! 0.24O0.62! [quintiles! 5! versus! 1]).! In! a!subsequent! study,! Mozaffarian! and! colleagues! (2013)! also! observed! an! inverse!association! between! circulating! transOpalmitoleate! and! type! 2! diabetes! risk! in! 2617!individuals!of!the!MultiOEthnic!Study!of!Atherosclerosis!(MESA)!(HR!0.52,!95%!CI!0.32O0.85![quintiles!5!versus!1]).!The!authors!speculated!that!transOpalmitoleate!could!partly!mimic! the! biological! function! of! adiposeOproduced! cisOpalmitoleate! which! have! been!found! in! animal! experiments! to! improve! insulin! resistance! and! related! metabolic!! 42!abnormalities!in!hepatic!and!skeletal!muscle!in!addition!to!help!in!suppressing!hepatic!fat! production.! Increased! hepatic! fat! production! has! been! suggested! to! contribute! to!nonalcoholic! steatohepatitis,! a! condition! associated! with! insulin! resistance!(Mozaffarian!et!al.,!2010).!!!In!order!to!assess!the!role!of!specific!fatty!acids!in!the!pathogenesis!of!type!2!diabetes,!Krachler! et! al.! (2008)! investigated! the! erythrocyte! membrane! fatty! acid! (EMFA)!composition,!which!provides!an!estimation!of! the! fatty!acid! intake,!of!159! individuals!with! type! 2! diabetes! and! 291! referents.! Higher! levels! of! pentadecanoic! (15:0)! and!heptadecanoic! (17:0)! in! erythrocyte! membrane,! which! were! found! to! be! positively!correlated!with! the! intake! of! highOfat! dairy! products! such! as! butter! and!3%! fat!milk,!were!associated!with!a! lower!risk!of! type!2!diabetes! (OR!=!0.65;!95%!CI!0.50O0.85;!P!0.002!and!OR!=!0.47;!95%!CI!0.35O0.63;!P!<0.001,!respectively)!(Krachler!et!al.,!2008).!Kröger!et!al.!(2011)!also!reported!a!strong!link!between!the!incidence!of!type!2!diabetes!and! the! fatty! acid! profile! of! erythrocyte!membrane! phospholipids! and! the! activity! of!desaturase! enzymes! in! 2724! subjects,! including! 673! diabetes! cases,! of! The! European!Prospective! Investigation! into! Cancer! and! Nutrition! (EPIC)OPotsdam! Study.! However,!dietary! fatty! acid!profile!was! found! to!present!only!a!modest!or! low!correlation!with!EMFA!composition!and!to!not!be!significantly!related!to!the!risk!of!diabetes!(Kröger!et!al.,!2011).!!!Although!milk!fat!may!have!some!effects!on!the! incidence!of!type!2!diabetes,! it!seems!unlikely!that!it!is!the!primary!mechanism!for!the!inverse!association!between!diabetes!and!dairy!product!consumption!observed!in!epidemiological!studies,!as!the!protective!effect!of!dairy!has!been!more!strongly!linked!to!the!intake!of!lowOfat!dairy!foods.!From!the!mixed! results! yielded! by! the! studies! that! have! assessed! the! effects! of! vitamin! D,!calcium!and!magnesium!on!the!incidence!of!type!2!diabetes,!it!also!seems!unlikely!that!these! nutrients! are! the! main! contributors! to! the! beneficial! effect! of! dairy! product!consumption!on!the!risk!of!type!2!diabetes!reported!in!observational!studies.!Moreover,!even! if! there! are! evidences! of! possible! associations! between! dairy! fat,! vitamin! D,!calcium,! magnesium! and! the! incidence! of! type! 2! diabetes,! assessing! whether! the!! 43!reported!effects!are!specific!to!the!risk!of!developing!diabetes!is!complex!because!these!components! have! also! been! suggested! to! have! antiOobesity! properties! and/or!help! in!lowering!the!risk!of!the!metabolic!syndrome,!which!are!both!in!turn!associated!with!the!risk!of!developing!type!2!diabetes!(Pfeuffer!&!Schrezenmeir,!2006).!!!!!2.1.4.1.2%Effects%of%Milk%Casein%and%Whey%Proteins%on%Glucose%Regulation%!!The! possible! effect! of! dairy! foods! on! the! incidence! of! type! 2! diabetes! has! also! been!suggested!to!be!attributable!to!the!protein!fraction!of!milk.!Dietary!proteins!are!found!to!cause! the!release!of! insulin! in!both!healthy!and!diabetic! individuals!when! ingested!with! or! without! carbohydrate.! Moreover,! dietary! proteins! are! also! known! to! induce!glucagon!secretion!(Claessens!et!al.,!2008).!!Milk!protein!seems!to!induce!an!increase!in!postprandial! insulin! response! with! a! reduction! in! postprandial! blood! glucose! levels!(Liljeberg!Elmståhl!&!Björck,!2001;!Östman!et!al.,!2001).!When!compared!with!casein!or!other! animal! or! vegetable! proteins,! whey! proteins! were! found! to! be! particularly!insulinotrophic!(Nilsson!et!al.,!2004).!This!finding!was!suggested!to!be!due!to!the!rapid!digestibility!of!whey!proteins!(Akhavan!et!al.,!2010)!and!its!high!content!of!branchedOchain! amino! acids! (particularly! leucine),! which! are! known! for! their! high! capacity! to!increase!insulin!response!(Peterson!et!al.,!2009).!Several!amino!acids!can!potentially!act!as! direct! insulin! secretagogues! (Nilsson! et! al.,! 2007),! and! increased! levels! of! amino!acids! in! postprandial! plasma! are! believed! to! be! associated! with! enhanced! insulin!response! (Calbet! &!MacLean,! 2002).! Leucine,! isoleucine,! valine,! lysine! and! threonine!were!among!the!essential!amino!acids!showing!a!pronounced!postprandial!increase!in!plasma! following! the! ingestion! of! whey! drink! and! were! also! shown! to! present! the!strongest!correlation!with!insulin!response!(Nilsson!et!al.,!2004).!!!!It!has!also!been!suggested!that!the!glycemic!regulatory!properties!of!milk!protein!may!be!related!to!its!effect!on!the!release!of!incretin!hormones!from!the!gut.!In!fact,!proteins,!such! as! whey! protein,! trigger! the! release! of! cholecystokinin,! glucagonOlike! peptideO1!(GLPO1),! glucoseOdependent! insulinotropic! polypeptide! (GIP)! and! peptide! tyrosine!! 44!tyrosine! (PYY)! from! the! intestinal! enteroendocrine!cells! (Akhavan!et!al.!2010).!These!peptides!play!an!important!role!in!stomach!emptying!and!glycemic!control.!A!proteinOstimulated! insulin!response! in!healthy! individuals!or! individuals!with! type!2!diabetes!that! did! not! mirror! the! increase! in! plasma! amino! acid! levels! was! observed! in! some!studies,!supporting!the!role!of!incretin!hormones!in!proteinOstimulated!insulin!release!(Nilsson!et!al.,!2004).!!!A!recent!study!on!the!effects!of!consumption!of!whey!protein!(WP)!and!whey!protein!hydrolysate! (WPH)! before! a! meal! on! satiety! and! on! blood! glucose! and! insulin!concentrations!in!young!adults!(Akhavan!et!al.!2010)!showed!that!only!WP!contributed!to!blood!glucose!regulation!by!insulinOdependent!and!insulinOindependent!mechanisms.!This! observation! suggests! that! noninsulintropic! mechanisms! require! stimulation!arising! from! the! digestion! of! intact! proteins.! Despite! the! fact! that! the!mechanism! of!action!by!which!premeal!WP!leads!to!postmeal!glucose!regulation!is!not!well!defined,!it!seems!probable!that!the!insulinOindependent!actions!of!premeal!consumption!of!WP!is!partly!due!to!the!effect!of!protein!on!gastric!emptying!caused!by!the!release!of!incretin!hormones.!A!mixture!of!branchedOchain!amino!acids!was!shown! to!produce! the!same!effect!as!intact!whey!protein!on!insulin,!but!not!on!gut!hormones!(Nilsson!et!al.,!2004).!This!observation,!as!well!as!the!possible!existence!of!a!threshold!for!amino!acidOinduced!GIP! release! (Nilsson! et! al.,! 2007),! may! explain! the! lack! of! effect! of! WPH! on! blood!glucose.!!Manders! et! al.! (2006)! investigated! the! effects! on! blood! glucose! concentration! of! the!consumption!of!a!beverage!containing!casein!protein!hydrolysate/leucine!mixture!after!each!main!meal.!They!observed!a!reduction!in!the!prevalence!of!hyperglycemia,!with!a!significant!reduction!of!the!average!24Oh!blood!glucose!level,!in!type!2!diabetes!patients!receiving! the! protein! hydrolysate/leucine! mixture! compared! to! those! receiving! a!placebo!mixture.!Similarly,!the!addition!to!a!glucose!drink!of!5–20!grams!of!protein!in!the!form!of!a!protein!supplement!known!as!“glycemic!index!lowering!peptide”!(GILP),!which! is! composed! of! a! blend! of! whey! peptides! and! intact! whey! protein! with! high!content!of!branchedOchain!amino!acids,!was!shown!to!cause!a!doseOdependent!decrease!! 45!in! postprandial! glycemia! in! 10! healthy! subjects! (Petersen! et! al.,! 2009).! Although! the!mechanism!by!which!GILP!causes!a!decrease!in!the!postprandial!glycemia!is!not!known,!several!hypotheses!have!been!raised,!such!as!the!effect!of!branched!chain!amino!acids!in! stimulating! insulin! response,! the! effect! of! whey! protein! on! incretin! hormone!secretion! and! the! effect! of! the! whey! peptide! fraction! as! a! source! of! inhibitors! of!dipeptidylOpeptidase!IV!(DPPOIV),!a!member!of!the!prolyl!endopeptidase!family!known!to!hydrolyse!incretin!hormones!such!as!GLPO1!(Petersen!et!al.,!2009).! Interestingly,! in!animal! studies! showing! the! effect! of! whey! protein! on! the! reduction! of! postprandial!glycemia,!higher!levels!of!intact!GIP!and!GLPO1!were!observed,!and!this!despite!the!fact!that!only!the!secretion!of!GLPO1!was!increased!(Gunnarsson!et!al.,!2006).!It!is!suggested!that! the! digestion! of! whey! protein! might! generate! protein! fragments! (diO! or! triOpeptides)!that!may!act!as!endogenous!inhibitors!of!DPPOIV,!and!thus!lead!to!decreased!degradation! of! the! incretin! hormones! and! to! higher! levels! of! intact! GIP! and! GLPO1!(Drucker,!2006;!Gunnarsson!et!al.,!2006).!!!2.1$INCRETIN$HORMONES$!The! term! incretins! refers! primarily! to! two! gutOderived!hormones:! glucoseOdependent!insulinotropic!polypeptide!(GIP),!formerly!known!as!gastric!inhibitory!polypeptide,!and!glucagonOlike!peptideO1!(GLPO1).!!!Derived! from!the! transcription!of! the!proglucagon!gene,!GLPO1! is! secreted! from!the!L!cells! in! the! distal! jejunum,! ileum! and! colon,! in! primarily! two! active! forms,! glycineOextended! GLPO17O37! and! GLPO17O36! amide! (Figure' 2.2),! the! latter! being! the! most!abundant!form!in!human!plasma!(Addison!&!Aguilar,!2011;!Tasyurek!et!al.,!2014).!GLPO1! is! released! within! minutes! of! food! consumption! and! plays! a! role! in! a! number! of!processes! involved! in! glucose! regulation.!Via! its! actions!on! the!pancreatic!βOcells,! the!GLPO1! hormone! promotes! insulin! secretion! as!well! as! insulin! gene! transcription! and!biosynthesis.!GLPO1!has!also!been!shown!to!inhibit!glucagon!release,!suppress!appetite!and!food!intake,!and!retard!gastric!emptying.!Moreover,!GLPO1!has!also!been!suggested!! 46!to!have!trophic!effects!on!pancreatic!βOcells!(Kim!&!Egan,!2008;!Phillips!&!Prins,!2011;!Tasyurek!et!al.,!2014).!!!!!!!!'Figure'2.2'Schematic!representation!of!the!generation!of!the! incretins!GLPO1!and!GIP!from!proglucagon!and!proGIP,!respectively,!and!their!cleavage!by!the!enzyme!DPPOIV.!GLPO1!is!the!product!obtained!by!the!postOtranslational!processing!of!the!proglucagon!gene!by!PC1/3! in!the! intestinal!L!cells.! It!exists! in!the!circulation! in!two!active! forms:!GLPO17O37!and!GLPO17O36ONH2.!Similarly,!GIP!is!derived!from!the!modification!of!proGIP!by!PC1/3!in!the!enteroendocrine!K!cells.!Both!GIP!and!GLPO1!are!substrates!for!the!DPPOIV!enzyme!that!hydrolyzes! them!into!shorter!and! inactive!molecules.!DPPOIV,!dipeptidylOpeptidase! IV;!GIP,! glucoseOdependent! insulinotropic!polypeptide;!GLPO1,! glucagonOlike!peptideO1;! GLPO2,! glucagonOlike! peptideO2;! GRPP,! glicentinOrelated! pancreatic!polypeptide;!IP,!intervening!peptide;!PC,!prohormone!convertase.!Adapted!from!Kim!&!Egan,!2008.!!!!!!!GRPP Glucagon IP1 GLP-1 IP2 GLP-2 PC1/3     ↓  PC1/3     ↓  C-terminal N-terminal GIP      ↑  PC1/3      ↑  PC1/3 H A E G T F T S D V S S Y L E G Q A A K E F I A W L V K G R G 7 37 H A E ! T F T S D V S S Y L E G Q A A K E F I A W L V K G R -NH2 7 36 Y A E G T F I S D Y S I A M D K I H Q Q D F V N W L L A Q K G K K N D W K H N I T Q 42 1 DPP-IV PROGLUCAGON PROGIP GLP-17-37 GLP-17-36 GIP1-42 G ! 47!GIP!is!a!42!amino!acids!long!peptide!derived!from!the!proGIP!gene!(Figure'2.2)!that!is!secreted! from!the!enteroendocrine!cells! (K!cells)!primarily! located! in! the!upper!small!intestine!(Kim!&!Egan,!2008,!Phillips!&!Prins,!2011,!Tasyurek!et!al.,!2014).!Similarly!to!GLPO1,!GIP!stimulates! insulin!secretion! in!a!glucoseOdependent!manner.! In!addition!to!its!insulinotropic!effect,!GIP!is!also!known!to!stimulate!pancreatic!glucagon!release!and!to!be!involved!in!fat!metabolism!(Tasyurek!et!al.,!2014).!!!The! incretin!hormones!are!believed! to!be!responsible! for!approximately!50%O70%!of!the!total!insulin!secreted!following!glucose!intake!(Baggio!&!Drucker,!2007).!However,!once!secreted,!they!are!rapidly!hydrolyzed!(halfOlives!of!5–7!and!1–2!mins,!for!GIP!and!GLPO1,! respectively)! by! the! enzyme! DPPOIV! into! shorter! and! inactive! molecules!(Addison!&!Aguilar,!2011).!!!!!!!!!!!!!!!!!2.3$DIPEPTIDYL4PEPTIDASE$IV$!DipeptidylOpeptidase!IV!(DPPOIV;!EC!3.4.14.5),!also!originally!known!as!the!lymphocyte!cell! surface!marker! CD26! or! as! the! adenosine! deaminase! (ADA)Obinding! protein,! is! a!110! kDa! glycoprotein! containing! 766! amino! acid! residues.! DPPOIV!mainly! exists! as! a!membraneOanchored! cellOsurface! enzyme,! but! can! also! be! found! as! a! soluble! form!(Filippatos!et!al.,!2014).!It!is!widely!expressed!in!epithelial!tissues,!capillary!endothelia!and! lymphocytes,! including! the! liver,! gastrointestinal! tract,! lymph! nodes! and! kidney!(Filippatos!et!al.,!2014)!and!is!implicated!in!a!number!of!biological!processes!as!both!a!regulatory!protease!and!a!binding!protein!(Mentlein,!1999;!Pro!&!Dang,!2004;!Zhong!et!al.,!2013).!!It!is!a!serine!protease!belonging!to!the!prolyl!oligopeptidase!family,!a!group!of! structurally! related! enzymes! that! remove! NOterminal! dipeptides! from! peptide!substrates! (Thoma! et! al.,! 2003).! As! shown! in! Figure' 2.3,! the! DPPOIV! gene! family! is!composed!of!four!enzymatic!members,!fibroblast!activation!protein!(FAP),!DPPOIV,!DPPO8! and! DPPO9,! and! two! catalytically! inactive! members,! DPPO6! and! DPPO10! (Drucker,!2007).!Sharing!52%!of!amino!acid!identity!with!the!protein!sequence!of!DPPOIV,!FAP!is!the! most! closely! related! to! the! DPPOIV! enzyme! (Mentlein,! 2005).! Although! sharing!! 48!structural! similarities,! DPPOIV! and! FAP! differ! in! their! expression! patterns,! the! latter!having! a! restricted!distribution! in! abnormal! tissues! (Mentlein,! 2005;!Yu! et! al.,! 2010).!Similarly! to!DPPOIV,! DPPO8! and!DPPO9! are! ubiquitously! distributed!whereas! the! nonOenzymatically! active! DPPO6! and! DPPO10,! which! act! as! modulators! of! voltageOgated!potassium!channels,!are!mainly!expressed!in!the!brain!(Yu!et!al.,!2010).!!'''!'Figure'2.3'Members!of!the!DPPOIV!gene!family!and!their!distribution.!DPP,!dipeptidylOpeptidase;!FAP,!fibroblast!activation!protein!(Drunker,!2007;!Yu!et!al.,!2010).!'!!!! !Enzymatic!members!Member! Distribution!DPP-9!DPP-8!FAP!DPP-IV!Non-enzymatic!members!DPP-6!DPP-10!Ubiquitously distributed!(epithelial, lymphocytes, endothelial cells)!Ubiquitously distributed!(epithelial, lymphocytes, endothelial cells)!Ubiquitously distributed!(epithelial, lymphocytes, endothelial cells)!Fibroblast, mesenchymal!Brain!Brain!! 49!The! DPPOIV! enzyme! exists! as! a! dimer,! each! of! its! monomer! subunits! consisting! of! a!short!NOterminal!cytoplasmic!section!(AA1O6),!a!transmembrane!domain!(AA7O29)!and!a!large!extracellular!domain!(AA30O766).!The!extracellular!domain,!which!is!involved!in!protein! interactions!and!enzymatic!activity,! is!composed!of!a!catalytic!region!(AA511O766)!with!an!α/β!hydrolase!fold,!and!an!eightOblade!βOpropeller!domain.!The!active!site!of!DPPOIV,!which!contains!a!catalytic!triad!composed!of!the!amino!acid!residues!Ser630,!Asp708!and!His740!(Lambeir!et!al.,!2003),! can!be!accessed!either!via!a! large!opening!between!the!propeller!and!hydrolase!domains!or!through!a!narrow!tunnel!in!the!centre!of! the! βOpropeller! domain! (Engel! et! al.,! 2003;! Rasmussen! et! al.,! 2003)! (Figure' 2.4).!Since! the! active! site! is! located! in! a! small! pocket! inside! the! large! cavity,! its! access! is!limited! to! elongated! peptides,! unfolded! or! partly! unfolded! protein! fragments! and!proteins!with!an!unfolded!NOterminal!region!(Rasmussen!et!al.,!2003).!!In'vivo,! the!enzyme!is!responsible! for!the!cleavage!of! the!amide!bond!following!the!P1!residue!of!a!number!of!molecules,!including!chemokines,!neuropeptides!and!regulatory!peptides,!such!as!the!incretin!hormones!GIP!and!GLPO1!(Table'2.4).!The!ability!of!DPPOIV! to!exert! its!enzymatic!action! is!believed! to!primarily!rely!on! the!recognition!of! the!substrate’s! positively! charged! NOterminal! by! two! glutamate! residues! (Glu205! and!Glu206)! located! within! the! enzyme! active! site! (Engel! et! al.,! 2003;! Aertgeerts! et! al.,!2004).!The!GluO205OGluO206!motif,!which! is! found! in!a!small!horizontal!helix!(AA201O207)!of! the!propeller!domain,! allows! the! stabilization!of!peptide! substrate!within! the!active!site!by!forming!salt!bridges!with!its!NOterminal.!The!horizontal!helix!containing!the!two!glutamic!acids!also!narrows!the!active!site!so!that!there!is!space!only!for!two!amino!acids!before!the!peptide!substrate!reaches!the!active!serine!residue!(Rasmussen!et! al.,! 2003).! The! substrate! specificity! of! DPPOIV! is! believed! to! primarily! rely! on! the!nature!of! the! amino!acid! sequence! surrounding! the! scissile!bond! (Engel! et! al.,! 2003).!The! best! catalytic! efficiencies! have! been! reported! for! substrates! bearing! proline! or!other!small!uncharged!residues!such!as!serine!and!alanine!at!their!P1!position!(Engel!et!al.,! 2003).! The! hydrophobic! S1! pocket,! which! is! lined! by! the! amino! acids! Val656,!Tyr631,! Tyr662,! Trp659,! Tyr666! and! Val711,! is! believed! to! dictate! this! specificity!! 50!(Aertgeerts!et!al.,!2004).!Since!the!side!chains!of!the!residues!in!P2!and!P1’!positions!do!not!make!specific!contact!with!DPPOIV,!the!enzyme!has!been!reported!to!show!little!or!no! substrate! specificity! for! the! amino! acids! in! these! two! positions! (Hunziker! et! al.,!2005).!Recent!findings!by!KühnOWache!and!colleagues!(2011)!showed,!however,!that!as!the!length!of!the!peptide!substrate!increases,!the!influence!of!the!amino!acid!residue!at!the! P1! position! on! the! substrate! turnover! rate! becomes! negligible.! The! authors!suggested! that! longer! peptides,! such! as! natural! substrates,! could! interact! with! the!enzyme!at!an!additional!binding!site!located!outside!the!catalytic!centre.!This!secondary!binding!site!interaction!was!proposed!to!involve!the!substrate’s!amino!acid!residues!in!the!P’4OP’8!position!(KühnOWache!et!al.,!2011).!!!!!!!!! '  51     Figure 2.4 Ribbon diagram of human dipeptidyl-peptidase IV. Image obtained from: http://commons.wikimedia.org/wiki/File:1PFQ.png     !52!Table&2.4&Some!physiological!substrates!of!dipeptidyl7peptidase!IV(Gorrell,!2005;!Mendieta!et!al,!2011;!Zhong!et!al,!2013).Substrate Primary expression N-terminal Function(s)/role(s) Name Abbreviation Eotaxin CCL11 Endothelium, monocytes, epithelium, T cells Gly-Pro-Gly Recruits eosinophils, involved in allergic responses Gastrin-releasing peptide GRP Brain, gut intrinsic neurons, pancreatic parasympathetic neurons Val-Pro-Leu Stimulates GLP-1 and gastrin secretion, regulates gastric acid secretion and enteric motor function Glucagon-like peptide-1 GLP-1 L cells of the colon and ileum His-Ala-Glu Stimulates pancreatic glucose-dependent insulin secretion, suppresses glucagon release, reduces appetite, slows gastric emptying Glucagon-like peptide-2 GLP-2 L cells of the colon and ileum His-Ala-Asp Intestinal growth and metabolism, reduces bone reabsorption, neuroprotection Glucose-dependent insulinotropic polypeptide GIP K cells of the duodenum Tyr-Ala-Asp Stimulates pancreatic glucose-dependent insulin secretion, promotes fatty acid metabolism Growth hormone-releasing factor GHRF Hypothalamus Tyr-Ala-Glu Stimulates the production and release of growth hormones INF-γ-induced protein-10 IP-10/ CXCL10 Endothelium, fibroblasts, monocytes, T cells, keratinocytes Val-Pro-Leu Chemoattraction of monocytes, T cells and natural killer cells, inhibits angiogenesis  Interferon-inducible T cell α chemoattractant ITAC/ CXCL11 Endothelial cells, fibroblasts, keratinocytes Phe-Pro-Met Chemoattraction of T cells, induces calcium release in activated T cells Macrophage-derived chemokine MDC/ CCL22 Epithelial cells, T cells, macrophages Gly-Pro-Tyr Chemoattraction of T cells, chemotaxis of dendritic cells, natural killer cells and monocytes Monokine induced by INF-γ Mig/ CXCL9 Keratinocytes Thr-Pro-Val Chemoattraction of activated T cells Neuropeptide Y NPY Hypothalamus Tyr-Pro-Ser Regulates energy balance, modulate memory processing Peptide histidine methionine PHM Nervous system His-Ala-Asp Stimulates insulin and glucagon release, vasodilation Peptide YY PYY Pancreas, L cells of the colon and ileum Tyr-Pro-Ile Inhibits gastric motility, increases absorption of electrolyte and water in the colon, reduces appetite Regulated on activation normal T cell expressed and secreted RANTES/ CCL5 Lymphocytes Ser-Pro-Tyr Recruits leukocytes into inflammatory sites Substance P SP Produced by nerves and inflammatory cells Arg-Pro-Lys Neurotransmitter and neuromodulator ! 53!2.4$BIOACTIVE$PEPTIDES$!Bioactive! peptides! are! defined! as! specific! protein! fragments! that! are! released! during!gastrointestinal! digestion! or! food! processing! and! that! can! have! a! positive! impact! on!human! heath! by! exerting! beneficial! effects! on! the! body’s! cardiovascular,! digestive,!immune!and!nervous!systems!(Shahidi!&!Zhong,!2008;!Kamau!et!al.,!2010).!!!Over! the! past! few! decades,! peptide! sequences! presenting! a! variety! of! biological!properties,! including! antiKhypertensive,! antiKoxidative,! antiKbacterial,! antiKcariogenic,!mineralKbinding,!opioid!and! immunomodulating!activities,!have!been! identified! in! the!sequence!of!dietary!proteins!from!plant!and!animal!sources!(Hartmann!&!Meisel,!2007;!Jauregi,!2008).!These!peptides!primarily!contain! two! to! twenty!amino!acids!and! their!activity! and!potency!are! influenced!by! their! structural! characteristics,! such!as! length,!amino!acid!composition!and!sequence!(Korhonen!&!Pihlanto,!2006).!!!Although!dairy!proteins!are!often!considered!one!of!the!most!important!precursors!of!biologically! active! peptides,! a! wide! variety! of! other! dietary! proteins! have! been!investigated!for!their!potential!to!serve!as!sources!of!bioactive!peptides,!including!eggs,!meat,! fish,! gelatin,! corn,! wheat! and! soybean! (Yamamoto! et! al.,! 2003;! Korhonen! &!Pihlanto,!2006;!Hartmann!&!Meisel,!2007;!Mine!et!al.,!2010).!!2.4.1%Approaches%to%Identify%Bioactive%Peptides%from%Dietary%Proteins%!The! traditional! approach! to! study! bioactive! peptides! from! dietary! proteins! typically!involves!a!number!of!steps!and!processes!(Figure!2.5),!starting!with!the!generation!of!bioactive!peptides!from!dietary!proteins.!Bioactive!peptides!can!be!naturally!produced!in#vivo! from!proteins!during!digestion!by! the! enzymes!present! in! the! gastrointestinal!tract.! In# vitro,! the! most! common! way! to! generate! bioactive! peptides! is! through!enzymatic! hydrolysis! of! proteins! using! digestive! enzymes! from! animal! sources! or!proteases! derived! from! plants! and! microorganisms.! Biologically! active! peptides! can!! 54!also! be! produced! by! acid! or! alkaline! hydrolysis! of! proteins! or! by! fermentation! with!proteolytic! starter! cultures! (Jauregi,! 2008;! Gokavi,! 2009;! Korhonen,! 2009).! Once!generated,!the!active!peptides!are!isolated!by!fractionation!techniques!and!their!amino!acid! sequences! determined! by! mass! spectrometry.! The! identified! peptides! are! then!chemically! synthesized! and! tested! to! validate! their! biological! activity! (Khaldi,! 2012;!Udenigwe,!2014).!Another!investigation!strategy!that!can!be!used!to!discover!bioactive!peptides! consists! of! chemically! synthesizing! peptide! fragments! found! within! the!sequence!of!dietary!proteins!based!on! their! structural! characteristics! and! similarities!with!peptides!known!to!have!biological!activities!(Udenigwe,!2014).!!!!!!To! complement! these! empirical! methods,! computerJassisted! techniques! can! also! be!used! to! help! predict! the! best! protein! precursors! of! peptides! with! biological! activity!(Udenigwe,!2014).!!!!!! 55!!Figure!2.5!Approaches!towards!the!discovery!of!bioactive!peptides!from!dietary!proteins.!$2.4.2%Dairy%Proteins%as%Sources%of%Bioactive%Peptides%!Dairy!products!contain!a!number!of!proteins!known!to!play!a!wide!range!of!nutritional,!functional!and!biological!roles.!In!the!past!two!decades,!peptides!presenting!an!array!of!biological!properties!have!been! identified! in! the! sequences!of!proteins! from!both! the!casein! and!whey! fractions! of!milk! (Table! 2.5).! Enzymatic! treatments! using! digestive!enzymes,!particularly!pepsin,!trypsin!and!chymotrypsin,!have!been!successfully!used!in!Newly discovered bioactive peptides!Screening of proteins for the presence within their sequences of known bioactive peptides!Identification of most promising proteins!In silico approach!Fractionation!Peptide identification!Peptide synthesis!Validation of activity!Synthesis of peptide fragments found in proteins selected based on their similarity to known bioactive peptides!Traditional approach!Protein hydrolysis!! 56!a! body! of! studies! to! release! peptides! displaying! antiJhypertensive,! antiJbacterial,!calcium! binding,! immunomodulating! and! opioid! activities! from! different! casein! and!whey!proteins!(e.g.!Meisel!&!FitzGerald,!2003;!Yamamoto!et!al.,!2003;!FitzGerald!et!al.,!2004;! Gobbetti! et! al.,! 2004).! ! Bioactive! peptides! have! also! been! identified! in! the!hydrolysates! of!milk! proteins! obtained! using! a! number! of! other! proteases,! including!enzymes!from!bacterial!and!fungal!sources!such!as!alcalase,!thermolysin!and!protease!K!(e.g.!Yamamoto!et!al.,!2003;!Mizuno!et!al.,!2004;!da!Costa!et!al.,!2007;!del!Mar!Contreras!et!al.,!2011).!!Currently,! most! of! the! research! in! the! area! of! biologically! active! peptides! from!milk!proteins! has! focused! on! peptides! with! inhibitory! activity! against! the! angiotensin! IJconverting!enzyme!(ACE)!(Ricci!et!al.,!2010).!Findings!from!these!studies!have!led!to!the!commercialization! of! several! dairy! products! and! dairy! ingredients! enriched! in! ACE!inhibitory!peptides!that!can!be!used!for!the!management!of!hypertension!(Ricci!et!al.,!2010).!!!More! recently,! dairy! proteins! have! also! been! found! to! contain! within! their! primary!sequences!peptides!with!DPPJIV!inhibitory!activity!suggesting!that!they!could!be!used!as!natural!sources!of!DPPJIV!inhibitors.!!!! 57!Table!2.5!Examples!of!biologically!active!peptides!derived!from!milk!proteins.!Parent protein Peptide sequence Locationa Bioactivity Reference αS1-Casein Lys-Thr-Thr-Met-Pro-Leu-Trp [193–199] ACE inhibitory Hernández-Ledesma et al. (2005) β-Casein Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu [6–14] ACE inhibitory Gobbetti et al. (2000) β-Casein Leu-Leu-Tyr [191–193] Immunostimulatory Migliore-Samour et al. (1989) κ-Casein Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe-Met [96–106] Anti-oxidative Kudoh et al. (2001) α-Lactalbumin Tyr-Gly-Leu-Phe [50–53] Opioid Pihlanto-Leppälä (2001) Lactoferrin Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe [17–41] Anti-microbial Bellamy et al. (1993) β-Lactoglobulin Leu-Asp-Ala-Gln-Ser-Ala-Pro-Leu-Arg [32–40] ACE inhibitory Pihlanto-Leppälä et al. (2000) β-Lactoglobulin Val-Ala-Gly-Thr-Trp-Thr [15–20] Anti-microbial Pellegrini et al. (2001) a!Numbering!based!on!the!mature!protein!sequence.!ACE,!angiotensin!IKconverting!enzyme.!!!!!2.4.3%Dietary%Proteins%as%Sources%of%Peptides%with%DPP?IV%Inhibitory%Activity%!It!is!only!recently!that!dietary!proteins!have!been!proposed!to!be!natural!precursors!of!peptides!with! DPPKIV! inhibitory! activity,! but! already! the! idea! that! proteins! could! be!used!to!produce!DPPKIV!inhibitors!has!triggered!a!lot!of!interest!in!the!area!of!bioactive!research.! As! shown! in!Table! 2.6,! hydrolysates! from! a! number! of! proteins,! including!milk! (Uchida! et! al.,! 2011;! Uenishi! et! al.,! 2012;! Nongonierma! &! FitzGerald,! 2013c;!Silveira! et! al.,! 2013),! fish! (Huang! et! al.,! 2012;! LiKChan! et! al.,! 2012),! pork! (Hsu! et! al.,!2013),! egg! (Van!Amerongen! et! al.,! 2009),! dryKcured! ham! (Gallego! et! al.,! 2014),! azuki!beans! (Tominaga! et! al.,! 2012),! amaranth! (VelardeKSalcedo! et! al.,! 2013),! rice! bran!(Hatanaka!et!al.,!2012)!and!corn!(Mochida!et!al.,!2010)!have!been!found!to!be!able!to!inhibit!the!activity!of!DPPKIV.!!Despite!the!knowledge!that!DPPKIV!inhibitors!can!be!generated!from!a!variety!of!dietary!proteins! by! enzymatic! treatment,! up! to! now,! only! a! few! studies! have! gone! on! to!fractionate! the!protein!hydrolysates!and!to!successfully! isolate!and! identify! the!active!! 58!peptides! responsible! for! the! observed! inhibitory! activity.! The! DPPKIV! inhibitory!peptides! GlyKProKAlaKGlu! and! GlyKProKGlyKAla! (IC50! values! of! 49.6! and! 41.9! µM,!respectively)!were!identified!in!a!FlavourzymeKtreated!salmon!skin!gelatin!sample!(LiKChan! et! al.,! 2012),!while! ProKGlyKValKGlyKGlyKProKLeuKGlyKProKIleKGlyKProKCysKTyrKGlu,!CysKAlaKTyrKGlnKTrpKGlnKArgKProKValKAspKArgKIleKArg! and! ProKAlaKCysKGlyKGlyKPheKTryKIleKSerKGlyKArgKProKGly!(IC50!values!of!116.1,!78.0!and!96.4!µM,!respectively)!were!found! in! tuna! cooking! juice! hydrolysates! obtained! by! enzymatic! treatments! with!Protease!XXIII!and!Orientase!90N!(Huang!et!al.,!2012).!Moreover,!a!diKpeptide!of!lower!potency,!IleKPro!(IC50!value!of!410!µM),!was!isolated!in!a!rice!bran!hydrolysate!prepared!using!the!enzyme!Umamizyme!G!(Hatanaka!et!al.,!2012).!!!!Table!2.6!Protein!hydrolysates!shown!to!display!DPPKIV!inhibitory!activity.!Protein source Proteolytic/microbial enzyme Reference Amaranth trypsin, pepsin, pancreatin  Velarde-Salcedo et al. (2013) Azuki bean Umamizyme G, koji mold Tominaga et al. (2012) Corn papain Mochida et al. (2010) Cow’s milk pepsin, Corolase PP, trypsin,  Uchida et al. (2011); Silveira et al. (2013); Uenishi et al. (2012); Nongonierma & FitzGerald (2013c) Dry-cured ham NAa Gallego et al. (2014) Lysozyme Newlase F, Promod 278, Alcalase, Umamizyme, pepsin Van Amerongen et al. (2009) Porcine skin gelatine Alcalase Hsu et al. (2013) Rice bran Umamizyme G, Bioprase SP Hatanaka et al. (2012) Salmon skin gelatin Flavourzyme, Alcalase, bromelain Li-Chan et al. (2012) Tuna cooking juice Protease XXIII, Orientase 90N Huang et al. (2012) a!No!enzymatic!treatment!applied.!!!!!! 59!2.4.3.1&Milk&Protein1Derived&Peptides&with&DPP1IV&Inhibitory&Activity&!Dairy!proteins!are!currently!the!food!proteins!that!have!been!the!most!studied!for!their!potential!to!serve!as!precursors!of!bioactive!peptides!with!DPPJIV!inhibitory!activity.!In!the!past! few!years,! a!number!of!dairy!protein!hydrolysates!produced!using!primarily!digestive!enzymes,!such!as!pepsin!and!trypsin,!have!been!investigated!for!their!effect!on!DPPJIV!activity.!A!summary!of!the!DPPJIV!inhibitory!peptides!that!have!been!identified!in!the!sequences!of!milk!proteins!is!presented!in!Table!2.7.!Of!these!sequences,!only!a!few! were! actually! isolated! and! identified! in! dairy! hydrolysates! and! cheese! extract!(shown!in!bold!in!Table!2.7);!most!were!chemically!synthesized!and!only!hypothesized!to! be! among! the! active! components! responsible! for! the! observed! DPPJIV! inhibitory!activity!of!dairy!protein!hydrolysates.!!As!shown!in!Table!2.7,!proteins!from!both!the!casein!and!whey!fractions!of!milk!have!been! found! to! contain! fragments! able! to! inhibit! the! activity! of!DPPJIV! in#vitro.! These!peptides! vary! from! two! to! eleven! amino! acids! in! length! and!most! contain! a! proline!and/or!hydrophobic!amino!acids!within!their!sequence.!Moreover,!they!differ!greatly!in!their!ability!to!inhibit!the!DPPJIV!enzyme,!their!IC50!values!ranging!from!3.5!to!3216!µM.!Most!of!the!diJ!and!triJpeptides!reported!in!Table!2.7!were!investigated!for!their!mode!of!action!on!DPPJIV!activity!and!reported!to!behave!as!competitive!or!nonJcompetitive!inhibitors.!However,! the!modes! of! action! of! the! longer!milk! proteinJderived! peptides!(5–11!amino!acids)!are!unknown.!!!60!Table&2.7!Milk!protein.derived!DPP.IV!inhibitory!peptides.&Protein (ID)a Sequenceb Locationc IC50 (µM) Mode of inhibition Reference  αS1-Casein GL [10-11] 2615.03 Competitive Nongonierma & FitzGerald (2013b) (P02662) LP [11-12] 712.5 Competitive Nongonierma & FitzGerald (2013d)  LPQ [11-13] 82 ND Uenishi et al. (2012)  VA [25-26]  168.24 Competitive Nongonierma & FitzGerald (2013b)  EK [35-36], [192-193] 3216.73 Competitive Nongonierma & FitzGerald (2013b)  YP [146-147], [159-160] 658.1 Competitive Nongonierma & FitzGerald (2014)  WY [164-165] 281.0 Non-competitive Nongonierma & FitzGerald (2013a)  IP [182-183] 149.6 Competitive Nongonierma & FitzGerald (2014)  LW [198-199] 993.4 Competitive Nongonierma & FitzGerald (2013a) αS2-Casein EK [23–24], [157–158] 3216.73 Competitive Nongonierma & FitzGerald (2013b) (P02663) VR [44–45], [204–205] 826.1 Non-competitive Nongonierma & FitzGerald (2013d)  VA [64–65] 168.24 Competitive Nongonierma & FitzGerald (2013b)  AL [81–82], [175–176] 882.13 Competitive Nongonierma & FitzGerald (2013b)  VPITPT [117–122] 130 ND Uenishi et al. (2012)  VPITPTL [117–123] 110 ND Uenishi et al. (2012)  FL [163–164] 399.58 Competitive Nongonierma & FitzGerald (2013b)  LP [176–177] 712.5 Competitive Nongonierma & FitzGerald (2014)  LPQ [176–178] 82 ND Uenishi et al. (2012)  WI [193–194] 138.7 Non-competitive Nongonierma & FitzGerald (2013a)  WIQP [193–196] 237.3 Non-competitive Nongonierma & FitzGerald (2013d)  IP [201–202] 149.6 Competitive Nongonierma & FitzGerald (2014) β-Casein (P02666) SL [15–16], [57–58], [69–70], [124–125],        [164–165] 2517.08 Competitive Nongonierma & FitzGerald (2013b)  EK [31–32] 3216.73 Competitive Nongonierma & FitzGerald (2013b)  YP [60–61], [114–115], [180–181] 658.1 Competitive Nongonierma & FitzGerald (2014)  YPFPGPIPN [60–68] 670 ND Uenishi et al. (2012)  FPGPIPN [62–68] 260 ND Uenishi et al. (2012)  PGPIHNSd [63–69] 1000 ND Uenishi et al. (2012)  IP [66–67], [74–75] 149.6 Competitive Nongonierma & FitzGerald (2014)  LP [70–71], [135–136], [137–138], [151–152], [171–172] 712.5 Competitive Nongonierma & FitzGerald (2014)  LPQ [70–72] 82 ND Uenishi et al. (2012)  LPQNIPP [70–76] 160 ND Uenishi et al. (2012)  LPQNIPPL [70–77] 46 ND Uenishi et al. (2012) !!61!Table&2.7&continued!Protein (ID)a Sequenceb Locationc IC50 (µM) Mode of inhibition Reference  PQNIPPL [71–77] 1500 ND Uenishi et al. (2012)  IPPLTQTPV [74–82] 1300 ND Uenishi et al. (2012)  VPPFIQPE  [84–91] 2500 ND Uenishi et al. (2012)  FL [87–88], [190–191] 399.58 Competitive Nongonierma & FitzGerald (2013b)  FLQP [87–90] 65.3 Competitive Nongonierma & FitzGerald (2013d)  HL [134–135] 143.19 Competitive Nongonierma & FitzGerald (2013b)  LPL [135–137] 241.4 Competitive Nongonierma & FitzGrald (2014)  LPLPL [135–139] 325.0 Competitive Nongonierma & FitzGrald (2014)  WM [143–144] 243.1 Non-competitive Nongonierma & FitzGerald (2013a)  VLGP [197–200] 580.4 Competitive Nongonierma & FitzGerald (2013d)  VR [201–202] 826.1 Non-competitive Nongonierma & FitzGerald (2013d) κ-Casein EK [12–13] 3216.73 Competitive Nongonierma & FitzGerald (2013b) (P02668) IP [26–27], [108–109], [119–120] 149.6 Competitive Nongonierma & FitzGerald (2014)  IPI [26–28] 3.5 Competitive Nongonierma & FitzGerald (2013a)  IPIQY [26–30] 35.2 Competitive Nongonierma & FitzGerald (2014)  YP [35–36], [58–59] 658.1 Competitive Nongonierma & FitzGerald (2014)  GL [39–40] 2615.03 Competitive Nongonierma & FitzGerald (2013b)  VA [48–49], [143–144] 168.24 Competitive Nongonierma & FitzGerald (2013b)  AL [49–50] 882.13 Competitive Nongonierma & FitzGerald (2013b)  FL [55–56] 399.58 Competitive Nongonierma & FitzGerald (2013b)  LP [56–57] 712.5 Competitive Nongonierma & FitzGerald (2014)  LPYPY [56–60] 108.3 Competitive Nongonierma & FitzGerald (2014)  YPY [58–60] 243.7 Competitive Nongonierma & FitzGerald (2014)  YPYY [58–61] 194.4 Competitive Nongonierma & FitzGerald (2014)  VR [67–68] 826.1 Non-competitive Nongonierma & FitzGerald (2013d)  WQ [76–77] 120.3 Non-competitive Nongonierma & FitzGerald (2013a)  HL [102–103] 143.19 Competitive Nongonierma & FitzGerald (2013b) α-Lactalbumin SL [22–23] 2517.08 Competitive Nongonierma & FitzGerald (2013b) (P00711) LP [23–24] 712.5 Competitive Nongonierma & FitzGerald (2014)  WV [26–27] 65.69 Non-competitive Nongonierma & FitzGerald (2013b)  GL [51–52] 2615.03 Competitive Nongonierma & FitzGerald (2013b)  WC [60–61] 420.0 Non-competitive Nongonierma & FitzGerald (2013a)  FL [80–81] 399.58 Competitive Nongonierma & FitzGerald (2013b)  WL [104–105], [118–119] 43.6 Competitive Nongonierma & FitzGerald (2013a)  AL [109–110] 882.13 Competitive Nongonierma & FitzGerald (2013b)  EK [113–114], [121–122] 3216.73 Competitive Nongonierma & FitzGerald (2013b) !62!Table&2.7&continued Protein (ID)a Sequenceb Locationc IC50 (µM) Mode of inhibition Reference Lactoferrin VR [6–7], [37–38] 826.1 Non-competitive Nongonierma & FitzGerald (2013d) (P24627) WC [8–9], [347–348] 420.0 Non-competitive Nongonierma & FitzGerald (2013a)  WQ [22–23] 120.3 Non-competitive Nongonierma & FitzGerald (2013a)  WR [24–25] 37.8  Non-competitive Nongonierma & FitzGerald (2013a)  AL [42–43], [304–305], [317–318], [391–392], [503–504], [616–617] 882.13 Competitive Nongonierma & FitzGerald (2013b)  EK [51–52], [220–221], [276–277], [521–522] 3216.73 Competitive Nongonierma & FitzGerald (2013b)  VA [77–78], [95–96], [149–150], [206–207],     [256–257], [436–437], [540–541], [591–592] 168.24 Competitive Nongonierma & FitzGerald (2013b)  GL [118–119], [406–407], [445–446], [472–473], [511–512] 2615.03 Competitive Nongonierma & FitzGerald (2013b)  WI [125–126] 138.7 Non-competitive Nongonierma & FitzGerald (2013a)  IP [127–128], [310–311], [469–470] 149.6 Competitive Nongonierma & FitzGerald (2014)  WT [138–139] 482.1 Non-competitive Nongonierma & FitzGerald (2013a)  SL [141–142], [422–423], [450–451] 2517.08 Competitive Nongonierma & FitzGerald (2013b)  YP [166–167] 658.1 Competitive Nongonierma & FitzGerald (2014)  LP [218–219] 712.5 Competitive Nongonierma & FitzGerald (2014)  HL [246–247], [588–589] 143.19 Competitive Nongonierma & FitzGerald (2013b)  WK [268–269] 40.6 Non-competitive Nongonierma & FitzGerald (2013a)  FL [307–308], [686–687] 399.58 Competitive Nongonierma & FitzGerald (2013b)  WS [361–362] 643.5 Non-competitive Nongonierma & FitzGerald (2013a)  WN [448–449], [467–468] 148.5 Non-competitive Nongonierma & FitzGerald (2013a)  WA [560–561] 92.6 Non-competitive Nongonierma & FitzGerald (2013a) β-Lactoglobulin GL [9–10] 2615.03 Competitive Nongonierma & FitzGerald (2013b) (P02754) VA [15–16] 168.24 Competitive Nongonierma & FitzGerald (2013b)  VAGTWY [15–20] 174 ND Uchida et al. (2011)  WY [19–20] 281.0 Non-competitive Nongonierma & FitzGerald (2013a)  SL [21–22], [30–31], [116–117] 2517.08 Competitive Nongonierma & FitzGerald (2013b)  EK [74–75], [134–135] 3216.73 Competitive Nongonierma & FitzGerald, (2013b)  IP [78–79] 149.6 Competitive Nongonierma & FitzGerald (2014)  IPA [78–80] 49 Competitive Tulipano et al. (2011)  IPAVF [78–82] 44.7 ND Silveira et al. (2013)  IPAVFK [78–83] 143.0 ND Silveira et al. (2013)  AL [86–87], [132–133], [139–140], [142–143] 882.13 Competitive Nongonierma & FitzGerald (2013b)  VLVLDTDYK [92–100] 424.4 ND Silveira et al. (2013)  VR [123–124] 826.1 Non-competitive Nongonierma & FitzGerald (2013d) !63!Table&2.7&continued Protein (ID)a Sequenceb Locationc IC50 (µM) Mode of inhibition Reference  TPEVDDEALEK [125–135] 319.5 ND Silveira et al. (2013)  LP [143–144] 712.5 Competitive Nongonierma & FitzGerald (2014)       a!ID!refers!to!the!accession!number!in!the!UniProt!KB!database.!b!Peptide!sequences!shown!in!bold!were!released!and!isolated!from!milk!protein!hydrolysates!or!cheese!extract.!Other!peptides!were!only!chemically!synthesized!based!on!their!occurrence!in!milk!protein!sequences.!c!Numbering!based!on!the!mature!protein!sequence.!d!The!authors!mistakenly!reported!the!β.casein!derived.fragment![63–69]!as!PGPIHNS.!It!should!have!been!PGPIPNS.!!ND,!not!determined.!!! 64!2.4.3.2%In%Vivo%Effect%of%Milk%Derived%Peptides%and%Dairy%Protein%Hydrolysates%on%DPP=IV%Activity%%!Although! a! number! of!milk! protein5derived! peptides! have! been! shown! to! inhibit! the!activity! of! DPP5IV! in# vitro,! their! efficacy! in! human! has! not! yet! been! studied.!Nevertheless,! a! study! by! Gunnarsson! and! colleagues! (2006)! showed! that! the!administration!of!whey!protein! led! to!a!significant!reduction!of!DPP5IV!activity! in! the!proximal!small!intestine!of!mice,!suggesting!that!peptides!released!during!the!digestion!of!whey! protein!may! act! as!DPP5IV! inhibitors.!Moreover,! in! a! recent! investigation! by!Uenishi!et!al.!(2012),!the!oral!administration!of!the!β5casein5derived!DPP5IV!inhibitory!peptide!Leu5Pro5Gln5Asn5Ile5Pro5Pro5Leu!(Table&2.7)!to!rats!in!a!glucose!tolerance!test!was! found! to! lead! to! a! significant! reduction! of! blood! glucose! levels.! It! is! unclear,!however,! whether! the! glucose5lowering! effect! observed! was! directly! caused! by! the!inhibition! of! the! DPP5IV! enzyme.! Similarly,! a! β5lactoglobulin! hydrolysate! displaying!DPP5IV!inhibitory!activity!in#vitro!was!also!reported!to!significantly!lower!blood!glucose!concentrations! in!mice!when!administered!prior! to!a!glucose!challenge! (Uchida!et!al.,!2011).!As!for!the!study!by!Uenishi!et!al.!(2012),!the!exact!mechanism!by!which!the!β5lactoglobulin!hydrolysate!affected!glycemia!regulation!is!unknown.!!In! order! to! exert! their! DPP5IV! inhibitory! activity! in# vivo,! food5derived! peptides!must!retain!their!activity!until!they!reach!the!intestine.!Thus,!the!bioactive!peptides!have!to!either! be! resistant! to! the! action! of! the! gastrointestinal! enzymes! or! be! broken! down!during! digestion! into! peptide! fragments! that! also! possess! DPP5IV! inhibitory! activity.!Investigations!on!the!fate!or!stability!of!peptides!with!DPP5IV!inhibitory!activity!during!gastrointestinal!digestion!are!sparse.!Nongonierma!&!FitzGerald!(2013c)!evaluated!the!DPP5IV! inhibitory! activity! of! a! whey! protein! hydrolysate! before! and! after! simulated!gastrointestinal!digestion!and!found!that!the!digestive!treatment!improved!slightly!the!potency! of! the! hydrolysate! (IC50!values! of! 1.33! and! 1.02!mg/mL!before! and! after! the!simulated! gastrointestinal! digestion,! respectively),! suggesting! that! the! digestive!enzymes!may!be!able!to!release!new!inhibitory!peptides!from!whey!protein.!Similarly,!! 65!the! DPP5IV! inhibitory! activity! of! the! tuna5derived! peptides! Pro5Gly5Val5Gly5Gly5Pro5Leu5Gly5Pro5Ile5Gly5Pro5Cys5Tyr5Glu,!Cys5Ala5Tyr5Gln5Trp5Gln5Arg5Pro5Val5Asp5Arg5Ile5Arg! and! Pro5Ala5Cys5Gly5Gly5Phe5Try5Ile5Ser5Gly5Arg5Pro5Gly! remained! unchanged! or!slightly! improved! following! simulated! gastrointestinal! digestion! (Huang! et! al.,! 2012).!Currently,! the! fate!of! food5derived!DPP5IV! inhibitors!during!gastrointestinal!digestion!has!not!been!investigated!in#vivo.!Clearly,!more!research!is!needed!to!evaluate!the!effects!of!bioactive!peptides!on!DPP5IV!activity!in!humans.!! !! 66!CHAPTER(3:(OBJECTIVES(AND(EXPERIMENTAL(APPROACHES&3.1(OBJECTIVES( As!presented!in!the!previous!section,!the!inhibition!of!the!enzyme!dipeptidyl5peptidase!IV!is!a!promising!approach!for!the!treatment!of!type!2!diabetes.!Recent!findings!in!the!area!of!bioactive!peptides!have!suggested!that!dietary!proteins!could!be!natural!sources!of!DPP5IV!inhibitors.!However,!to!date,!only!a!limited!number!of!peptides!with!DPP5IV!inhibitory!activity!have!been!produced!from!food!proteins,! isolated!and!characterized.!Therefore,!the!overall!objective!of!this!doctoral!thesis!research!was!to!generate!DPP5IV!inhibitory!peptides!from!dietary!proteins!and!study!their!mechanisms!of!action!on!the!enzyme!in#vitro.!The!specific!objectives!of!this!investigation!were!to:!!(1).! Evaluate! the! theoretical! potential! of! dietary! proteins! from! various! food!commodities! to! serve! as! precursors! of! DPP5IV! inhibitory! peptides! using! an! in# silico!approach.!!(2).! Assess! the! DPP5IV! inhibitory! activity! of! dairy! protein! hydrolysates! obtained! by!enzymatic!treatments!using!various!commercially!available!proteases.!!(3).! Isolate,! sequence! and! characterize! the!DPP5IV! inhibitory! peptides! present!within!dairy!protein!hydrolysates.!!(4).! Investigate! the!potential!of! cellulose5bound!peptide!array! to! serve!as!a! screening!tool!to!identify!DPP5IV!inhibitory!peptides.!!(5).! Compare! the! effect! of! protein5derived! peptides! on! the! activity! of! porcine! and!recombinant!human!DPP5IV.!!67!3.2$OVERVIEW$OF$EXPERIMENTAL$APPROACHES$$Figure'3.1'Overview!of!experimental!approaches.!!! Fractionate by successive chromatographic steps the most potent hydrolysates ! Analyze the active fractions by LC-MS/MS for their constituent peptides ! Chemically synthesize the identified peptides and determine their DPP-IV inhibitory activity and mode of action on the enzyme Objective 3 –!Isolate, sequence and characterize the DPP-IV inhibitory peptides present within dairy protein hydrolysates!! Determine the inhibitory activity and mode of action of protein-derived peptides on porcine and recombinant human DPP-IV    Objective 5 –!Compare the effect of protein-derived peptides on the activity of porcine and recombinant human DPP-IV!! Submit dairy ingredients/proteins to enzymatic treatments with digestive enzymes and/or other proteases ! Assess the resulting hydrolysates for their effect on DPP-IV activity Objective 2 –!Assess the DPP-IV inhibitory activity of dairy protein hydrolysates obtained using various commercially available proteases!Objective 1 –!Evaluate the theoretical potential of dietary proteins from various food commodities to serve as precursors of DPP-IV inhibitory peptides using an in silico approach!!! Conduct research of the published literature on non-modified peptide-based   DPP-IV inhibitors ! Search for fragments within protein sequences from animal and plant sources matching those reported to have DPP-IV inhibitory activity !Objective 4 –!Investigate the potential of cellulose-bound peptide arrays to serve as screening tools to identify    DPP-IV inhibitory peptides!! Synthesize on cellulose membranes deca-peptides spanning the α-lactalbumin sequence ! Investigate the interactions (binding and inhibition) between the α-lactalbumin-derived peptides and the DPP-IV enzyme  ! 68!CHAPTER( 4:( EVALUATION( OF( THE( POTENTIAL( OF( DIETARY( PROTEINS( AS(PRECURSORS( OF( DIPEPTIDYL5PEPTIDASE( IV( INHIBITORS( BY( AN( IN# SILICO(APPROACH5(!4.1(SUMMARY(!Synthetic! inhibitors! against! the! enzyme! dipeptidyl7peptidase! (DPP)7IV,! a! serine!protease!implicated!in!the!inactivation!of!incretin!hormones,!are!currently!used!for!the!management! of! type! 2! diabetes.! The! aim! of! the! present! study! was! to! evaluate! the!potential!of!dietary!proteins!from!various!food!commodities!to!serve!as!precursors!of!DPP7IV!inhibitors!by!using!an!in#silico#approach.!A!total!of!2256!fragments!with!peptide!sequences! matching! those! reported! in! the! literature! to! present! DPP7IV! inhibitory!activity!were! found! in! the! 34! proteins! that!were! investigated.! Among! those,! Gly7Ala,!Gly7Pro!and!Pro7Gly!were!the!most!frequently!occurring!sequences.!Caseins!from!cow’s!milk!and!collagens!from!bovine!meat!and!salmon!appeared!to!be!the!richest!potential!sources! of! DPP7IV! inhibitors,! whereas! proteins! from! oat! showed! lower! occurrence!frequency!values.!This!study!suggests!that!dietary!proteins!could!be!used!for!generating!DPP7IV!inhibitory!peptides.!!!!!!!!!!4.2(INTRODUCTION(!Type! 2! diabetes! (T2D)! is! a! chronic! metabolic! disorder! of! increasing! importance!worldwide.! It! is! estimated! that! by!2035! the!number!of! people! affected!with!diabetes!will! reach! 592! million! (International! Diabetes! Federation,! 2013).! One! of! the! new!approaches! in! the! management! of! T2D! consists! of! orally! administered! dipeptidyl!peptidase7IV!(DPP7IV;!EC!3.4.14.5)!inhibitors.!The!enzyme!DPP7IV!is!a!serine!protease!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!5!A!version!of! this!chapter!has!been!published.!Lacroix,! I.M.E,!Li7Chan,!E.C.Y.!Evaluation!of! the!potential!of!dietary!proteins!as!precursors!of!dipeptidyl!peptidase!(DPP)7IV!inhibitors!by!an!in#silico!approach.!J.#Funct.#Foods!2012,!4,!403–422.!! 69!that!preferentially!cleaves!Xaa7proline!or!Xaa7alanine!di7peptides!from!the!N7terminus!of!poly7peptides! (Thoma!et!al.,!2003),!where!Xaa!represents!any!amino!acid!at! the!N7terminus.!This!multifunctional!enzyme,!expressed! in!a!variety!of! cells,!particularly!on!epithelial! tissues,! such!as! liver,!kidney!and! intestine! (Lorey!et!al.,!2003;!Thoma!et!al.,!2003),! is! implicated! in! several! biological! processes,! including! the! inactivation! of! the!incretin!hormones! glucose7dependent! insulinotropic! polypeptide! (GIP)! and! glucagon7like!peptide71! (GLP71).! In! response! to! food!consumption,!GIP!and!GLP71!are! released!and!interact!with!pancreatic!β7cells!via!a!stimulatory!G!protein!causing!the!production!of! cAMP,!which! in! turn! boosts! glucose7dependent! insulin! secretion! (Holst! &! Deacon,!2004).! DPP7IV! inhibitors! exert! their! positive! effect! on! glucose! regulation! by! slowing!down! the!otherwise! rapid! inactivation!of! endogenous!GIP!and!GLP71,! thus!enhancing!insulin!secretion.!!!Numerous! synthetic! DPP7IV! inhibitors! have! been! disclosed! in! the! literature! over! the!last! decade.! These! compounds! can! be! classified! as! either! peptide7derived!peptidomimetics!or!non7peptidomimetic!inhibitors!(Hunziker!et!al.,!2005;!Sebokova!et!al.,!2007).!Those!in!the!former!class!of!DPP7IV!inhibitors,!which!are!designed!to!mimic!the! terminal! di7peptide! that! is! hydrolyzed! by! DPP7IV,! often! present! a! proline! or! a!proline!derived!ring!in!position!P1!and!a!protonated!amino!function!mimicking!the!N7terminus! of! peptide! substrates! (Hunziker! et! al.,! 2005).! Galvus®! (vildagliptin)! from!Novartis! and! Onglyza®! (saxagliptin)! from! Bristol7Meyers! Squibb! are! among! the!peptide7like!inhibitors!currently!approved!for!the!management!of!type!2!diabetes!in!the!European! Union,! Canada! and/or! the! United! States.! Unlike! peptide7derived!peptidomimetic! inhibitors,! which! are! based! on! natural! substrates! of! DPP7IV! as!templates,! most! non7peptide7like! inhibitors! are! the! outcome! of! high7throughput!screening! (Hunziker! et! al.,! 2005).! Xanthine7based,! arylmethylamine7based! and!benzoquinolizine7based! DPP7IV! inhibitors! are! some! of! the! largest! classes! of! non7peptidomimetic! compounds! (Hunziker! et! al.,! 2005;! Sebokova! et! al.,! 2007).! The!heterocyclic! phenethylamine7based! DPP7IV! reversible! inhibitor! Januvia®! (sitagliptin)!from!Merck!is!currently!approved!in!Canada,!the!United!States!and!the!European!Union.!! 70!Studies!conducted!to!understand!the!catalytic!properties!and!functions!of!DPP7IV!have!also!led!to!the!identification!of!a!number!of!di7,!tri7!and!oligo7peptides!presenting!DPP7IV! inhibitory! activity.! Several! of! these! peptides! contain! proline! and/or! hydrophobic!amino!acids!within!their!sequence!(e.g.,!Bella!et!al.,!1982;!Cohen!et!al.,!2004;!Yan!et!al.,!1992)! and! are! competitive! inhibitors! of! DPP7IV! (e.g.,! Harada! et! al.,! 1982;! Stöckel7Maschek!et!al.,!2003).!!!!!Research! on! dietary! proteins! has! shown! that! they! are! precursors! of! a! variety! of!bioactive! peptides! that! have! the! potential! to! improve! human! health! and! prevent!chronic! diseases! by! exerting! positive! effects! on! the! body’s! digestive,! cardiovascular,!immune!and!nervous!systems!(Kamau!et!al.,!2010;!Shahidi!&!Zhong,!2008).!Angiotensin!converting! enzyme! (ACE)7inhibitory! peptides,! used! in! the! treatment! of! hypertension,!are! among! the! food! protein7derived! bioactive! peptides! that! have! been! the! most!intensively!studied!over!the!last!few!decades!(Gu!et!al.,!2011).!ACE7inhibitory!peptides!have!been!successfully!produced!by!enzymatic!hydrolysis!of!various!dietary!proteins,!including!milk!(Guo!et!al.,!2009;!Otte!et!al.,!2007)!and!fish!(Cinq7Mars!&!Li7Chan,!2007;!Nakajima!et!al.,!2009)!proteins.!!On!the!other!hand,!little!attention!has!been!given!to!the!possibility! that! dietary! proteins! may! also! contain! within! their! sequences! fragments!corresponding!to!DPP7IV!inhibitory!peptides!reported!in!the!literature,!and!thus!could!be!used!as!natural!sources!of!DPP7IV!inhibitors!in!the!management!of!T2D.!!!Complementary! to! empirical! methods,! computer7aided# techniques! have! been!demonstrated! to! be! useful! in! evaluating! the! potential! of! proteins! as! precursors! of!bioactive!peptides!with!various!activities!(Cheung!et!al.,!2009;!Dziuba!et!al.,#2009;!Gu!et!al.,! 2011;!Minkiewicz! et! al.,#2011).! Dziuba! et! al.# (2009)! and!Minkiewicz! et! al.# (2011)!have!predicted! the!presence! of!DPP7IV! inhibitors! in! the! sequences! of! cow’s!milk! and!bovine! meat! proteins,! respectively,! using! the! BIOPEP! database.! However,! not! all!peptide! sequences! reported! in! the! literature! to! present! an! inhibitory! activity! against!DPP7IV!are!currently!included!in!the!BIOPEP!database.!Moreover,!no!systematic!in#silico#investigation!comparing!the!ability!of!various!proteins! from!animal!and!plant!sources!as!precursors!of!DPP7IV! inhibitors!has!been!reported.!Therefore,! the!objectives!of! the!! 71!present!study!were!to!consolidate!all! findings!to!date!on!peptide!sequences!that!have!been!shown!to!display!in#vitro!DPP7IV!inhibitory!activity!and!to!evaluate!the!potential!of!dietary! proteins! from! various! food! commodities! to! serve! as! potential! precursors! of!DPP7IV!inhibitory!peptides!using!an!in#silico!approach.!!!4.3(MATERIAL(AND(METHODS(!4.3.1#Proteins#!For! this! investigation,! 9! food! commodities! from! plant! and! animal! sources! and! their!major! proteins!were! selected.! All! protein! sequences!were! obtained! from! the!UniProt!Knowledgebase!of!ExPASy!Proteomics!Server!available!at!http://expasy.org/.!!4.3.2#Peptides#with#DPP:IV#Inhibitory#Activity#!Among!the!numerous!DPP7IV!inhibitors!reported!in!the!literature,!solely!non7modified!peptide7based! DPP7IV! inhibitors! were! considered! in! this! in# silico! analysis.! The!sequences!of!peptide7based!DPP7IV! inhibitors!used! in!this!study,!as!well!as!additional!information! on! the! DPP7IV! inhibitors,! such! as! Ki! and! IC50! values,! were! obtained! by!conducting!research!of!the!published!literature.!!!4.3.3#In#Silico#Analysis#!The!potential!of!various!dietary!proteins! to!serve!as!DPP7IV! inhibitor!precursors!was!predicted! by! searching! for! fragments! within! the! protein! chains! matching! peptide!sequences! reported! in! the! literature! to! present! an! inhibitory! activity! against! the!enzyme! DPP7IV.! Furthermore,! the! potential! of! each! selected! protein! was! quantified!based! on! the! frequency! of! occurrence! of! fragments! matching! peptides! with! DPP7IV!! 72!inhibitory! activity! relative! to! the! length! of! the! protein! chain! using! the! following!equation!as!described!by!Dziuba!et!al.#(2003):!!! ! A!=!a/N!!where! A! is! the! occurrence! frequency,! a! is! the! number! of! peptides! with! DPP7IV!inhibitory!activity!within!the!protein!chain,!and!N!is!the!number!of!amino!acid!residues!in!the!protein!chain.!!!4.4(RESULTS(AND(DISCUSSION((!4.4.1#Peptides#Reported#to#Present#an#Inhibitory#Activity#Against#the#Enzyme#DPP:IV#!Several!di7,!tri7!and!oligo7peptides!presenting!an!inhibitory!activity!against!the!enzyme!DPP7IV! have! been! reported! in! the! literature! (Tables! 4.1! and! 4.2).! The! reported!effectiveness! of! these! peptides! varies! greatly.! However,! it! is! difficult! to! make! direct!comparisons! of! their! absolute! potency.! ! Inhibitor! constant! (Ki)! values! have! been! the!most!commonly!used! indicator!of! the!potency!of! the!peptides!as! inhibitors,!but!a! few!studies!have!presented!DPP7IV!inhibitory!activity!as!IC50!values,!%!inhibition,!%!activity!of!control,!or!%!remaining!enzymatic!activity.!Furthermore,!it!can!be!noted!that!there!is!some!discrepancy!in!the!inhibitory!activity!as!shown!by!the!different!Ki!values!reported!by! different! researchers! for! the! same! peptide! (Table! 4.1).! This! variability! may! be!attributable!to!differences!in!the!experimental!conditions,!such!as!the!type!of!substrate!and!source!of!enzyme,!used!in!the!different!studies!to!assess!the!inhibitory!activity.!!!!! !! 73!Table!4.1!Di7peptides!reported!in!the!literature!to!present!DPP7IV!inhibitory!activity.!! Peptide sequence Reference Ki (mM) DPP-IV activity (%) Ala-Ala Brandt et al. (1995) 0.41a – Yan et al. (1992) 0.378b – Yoshimoto et al. (1978) 3.6c – Ala-Pro Bella et al. (1982) 2.43c – Brandt et al. (1995) 0.2a – Yan et al. (1992) 0.0767b – Arg-Pro Brandt et al. (1995) 0.042a – Asp-Pro Brandt et al. (1995) 0.2a – Gly-Ala Yan et al. (1992) 0.599b – Gly-Phe Brandt et al. (1995) 3.2a – Gly-Pro Brandt et al. (1995) 1.6a – Harada et al. (1982) 13.6b – Yan et al. (1992) 0.0749b – Yoshimoto et al. (1978) 1.83c – His-Ala Bella et al. (1982) 2.35c – Ile-Ala Brandt et al. (1995) 0.01a – Ile-Pro Brandt et al. (1995) 0.0069a – Stöckel-Maschek et al. (2003) Stöckel-Maschek et al. (2003) 0.0149bd 0.0292be – – Ile-Val Brandt et al. (1995) 0.04a – Leu-Ala Bella et al. (1982) 0.5c – Tulipano et al. (2011) – ~ 70 bf Yan et al. (1992); 0.773b – Leu-Leu Bella et al. (1982) 0.34c – Tulipano et al. (2011) – ~ 80bf Leu-Phe Brandt et al. (1995) 0.14a – Leu-Pro Bella et al. (1982) 0.1c – Brandt et al. (1995) 0.07a – Yan et al. (1992) 0.0848b – Lys-Ala Bella et al. (1982) 2.4c – Met-Ala Bella et al. (1982) 0.5c – Met-Pro Bella et al. (1982) 0.45c – Phe-Ala Bella et al. (1982) 1.25c – Phe-Pro Bella et al. (1982) 0.6c – Brandt et al. (1995) 0.047a – Yan et al. (1992) 0.0132b – Pro-Ala Bella et al. (1982) 0.8c – Yan et al. (1992) 0.103b – Yoshimoto et al. (1978) 0.27c – Pro-Gly Brandt et al. (1995) 9.0a – Yan et al. (1992) 0.112b – Pro-Ile Yan et al. (1992) 0.105b – Pro-Leu Yan et al. (1992) 0.102b – Pro-Met Yan et al. (1992) 0.102b – Pro-Phe Yan et al. (1992) 0.0856b – Pro-Pro Harada et al. (1982) 0.25b – ! 74!Table!4.1!continued! Peptide sequence Reference Ki (mM) DPP-IV activity (%) Trp-Pro Stöckel-Maschek et al. (2003) 0.0574bd 0.185be – Tyr-Pro Yan et al. (1992) 0.0853b – Val-Ala Bella et al. (1982) 0.05c – Val-Pro Bella et al. (1982)  0.05c – Brandt et al. (1995) 0.01a – Val-Val Bella et al. (1982) 0.62c – a!Ala7Pro7p7nitroanilide!was!used!as!the!assay!substrate.!b!Gly7Pro7p7nitroanilide!was!used!as!the!assay!substrate.!c!Gly7Pro7β7naphthylamide!was!used!as!the!assay!substrate.!!d!Assay!performed!using!pig!kidney!DPP7IV.!e!Assay!performed!using!human!recombinant!DPP7IV.!!f!Remaining!enzymatic!activity!when!performing!the!assay!in!the!presence!of!100!µM!peptide.!!!75!Table&4.2&Tri'!and!oligo'peptides!reported!in!the!literature!to!present!DPP'IV!inhibitory!activity.&!Peptide sequence Reference Ki (mM) Inhibition (%) Dipeptidase activity         (% of control) IC50 (µM) Ala-Pro-Ala Hanski et al. (1985) – – – – Ala-Pro-Gly Bauvois (1988) – – – ~ 40000a Hanski et al. (1985) – – – – Gly-Pro-Ala Bauvois (1988) – – – ~ 40000a Hanski et al. (1985) – – – – Piazza et al. (1989) – – 9.9b – Ile-Pro-Ala Tulipano et al. (2011) - – – 49a Ile-Pro-Ile  Maruyama et al. (1996) – – – 7.4c Umezawa et al. (1984) 0.0022a – – – Met-Trp-Pro Lorey et al. (2003) 0.0245d – – – Met-Trp-Val Lorey et al. (2003) 0.2d – – – Val-Pro-Leu Hanski et al. (1985) – – – – Maruyama et al. (1996) – – – 47c Piazza et al. (1989) – – 1.9b – Umezawa et al. (1984) 0.0076a – – – Met-Pro-Leu-Pro Maruyama et al. (1996) – – – 270c Pro-Pro-Pro-Pro Harada et al. (1982) 0.6a – – – Tyr-Pro-Trp-Thr Cohen et al. (2004) – ~ 12a – – Met-Pro-Pro-Pro-Leu Maruyama et al. (1996) – – – 500c Met-Pro-Pro-Leu-Pro Maruyama et al. (1996) – – – 930c Tyr-Pro-Trp-Thr-Gln Cohen et al. (2004) – ~ 14a – – Met-Pro-Pro-Pro-Leu-Pro Maruyama et al. (1996) 0.19c – – 490c Val-Ala-Gly-Thr-Trp-Tyr Uchida et al. (2011) – – – 174a Met-Pro-Pro-Pro-Leu-Pro-Ala Maruyama et al. (1996) – – – 1300c Tyr-Pro-Trp-Thr-Gln-Arg-Phe Cohen et al. (2004) – ~ 30a – – Val-Val-Tyr-Pro-Trp-Thr-Gln Cohen et al. (2004) – ~ 8a – – Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg Cohen et al. (2004) – ~ 14a – – Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe Cohen et al. (2004) – ~ 28a – – Met-Trp-Pro-Thr-Ser-Ser-Ser-Thr-Lys Lorey et al. (2003) 0.0159d – – – !76!Table&4.2&continued)Peptide sequence Reference Ki (mM) Inhibition (%) Dipeptidase activity         (% of control) IC50 (µM) Met-Trp-Pro-Asn-Gly-Ser-Ser-Leu-Gly Lorey et al. (2003) 0.00502d – – – Met-Trp-Ile-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.0436d – – – Met-Trp-Pro-Leu-Gly-Pro-Ala-Ser-Ser Lorey et al. (2003) 0.0124d – – – Met-Trp-Pro-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.00212d 0.00170e – – – Met-Lys-Pro-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.0427d – – – Met-Asp-Ile-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 1.75d – – – Met-Asp-Gly-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.487d – – – Trp-Asp-Pro-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.15d – – – Met-Asp-Pro-Val-Asp-Pro-Asn-Ile-Glu Lorey et al. (2003) 0.267d   0.230e  – – – Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe Cohen et al. (2003) – ~ 8a – – Glu-Pro-Pro-Pro-Pro-Glu-Pro-Pro-Pro-Ile Maruyama et al. (1996) – – – 500c Met-Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu Lorey et al. (2003) 0.269d – – – a Gly-Pro-p-nitroanilide was used as the assay substrate. b Gly-Pro-4-methoxy-β-naphthylamide was used as the assay substrate. The activity of the control (i.e. no inhibitor) was considered as 100%.  cArg-Pro-p-nitroanilide was used as the assay substrate. d Ala-Pro-p-nitroanilide was used as the assay substrate. e Gly-Pro-R110-CO-(CH2)4Cl was used as the assay substrate. !! 77!It!is!known!that!di.peptides!of!the!general!structures!Xaa.Pro,!Pro.Xaa!and!Xaa.Ala!are!competitive! inhibitors! of!DPP.IV! (Yan! et#al.,! 1992).! As! shown! in!Tables' 4.1! and!4.2,!most!of!the!inhibitory!peptides!contain!proline!and/or!hydrophobic!amino!acids!within!their! sequence.!The! tri.peptides!diprotin!A! (Ile.Pro.Ile)! and!diprotin!B! (Val.Pro.Leu),!both! containing! a! proline! residue! in! position! P1,! figure! among! the! most! potent! of!peptides! with! DPP.IV! inhibitory! activity.! These! peptides! were! first! reported! by!Umezawa!et!al.!(1984)!to!be!inhibitors!of!DPP.IV,!but!were!later!shown!to!actually!be!substrates!for!the!enzyme!with!low!turnover!rates.!The!apparent!competitive!inhibition!observed!is!believed!to!be!attributable!to!a!kinetic!artifact!resulting!from!the!substrate.like!structure!of!these!compounds!(Rahfeld!et!al.,!1991).!Several!other!inhibition!modes!have!been!reported,!including!non.competitive,!mixed.type!and!irreversible,!suggesting!that!the!enzyme!inhibition!can!occur!by!the!binding!of!the!inhibitor!at!the!active!site!or!outside!the!catalytic!center!of!DPP.IV!(Kühn.Wache!et!al.,!2011;!Lorey!et!al.,!2003).!!!The! substrate! specificity!of!DPP.IV! is!believed! to!primarily! rely!on! the! recognition!of!the! substrate’s!positively! charged!N.terminus!by!a!Glu.Glu!motif!within! the!enzyme’s!active! site! and! on! the! stacking! of! the! amino! acid! residue! in! the! P1! position! in! the!enzyme’s!hydrophobic!pocket!(Engel!et!al.,#2003).!Proline! is! the!preferred!amino!acid!residue!at! the!P1!position,!while! alanine!as!well! as! glycine,!hydroxyproline!and!other!small! uncharged! residues! are! also! accepted! but! with! greatly! reduced! affinity!(Cunningham!&!O’Connor,!1997;!Kühn.Wache!et!al.,#2011).!!In!contrast!to!its!strong!specificity!for!the!residue!at!the!P1!position,!DPP.IV!shows!little!or!no!substrate!specificity!for!residues!in!the!P2!and!P1’!positions,!as!their!side!chains!are!oriented!away!from!the!enzyme!and!do!not!interact!strongly!with!it!(Hunziker!et!al.,!2005).! However,! substrates! bearing! amino! acids! with! bulky! side! chains! in! the! P2!position,! such! as! tryptophan! and! tyrosine,! appear! to! show! enhanced! binding,! while!those!with!acidic!side!chains!in!this!position!have!a!poor!turnover!(Engel!et!al.,!2003).!!Furthermore,!for!hydrolysis!to!occur,!the!third!amino!acid!residue!of!the!substrate!(P1’)!must!not!be!a!proline,!hydroxyproline,!or!N.methyl!glycine,!and!the!P1.P1’!as!well!as!the!P2.P1! bonds! must! be! in! trans.configuration! (Lambeir! et! al.,! 2003).! These! specific!! 78!structural! characteristics! may! be! important! in! determining! whether! a! particular!peptide!sequence!will!be!recognized!by!the!enzyme!as!a!substrate!or!an!inhibitor.!!The!di.peptides!Gly.Gly!(Hanski!et!al.,!1985),!Leu.Val!(Tulipano!et!al.,!2011)!and!the!tri.peptide! Gly.Gly.Gly! (Bauvois,! 1998)! are! some! examples! of! peptides! found! to! not!significantly! affect! DPP.IV! activity.! Interestingly,! the! tri.peptides! Leu.Leu.Phe!(Tulipano! et! al.,! 2011)! and! Pro.Gly.Gly! (Harada! et! al.,! 1982)!were! also! found! to! not!present! any! DPP.IV! inhibitory! activity,! and! this! despite! containing! the! DPP.IV!inhibitory! sequences! Leu.Leu! and! Pro.Gly! at! their! N.terminus.! This! finding! suggests!that!the!amino!acid!at!the!P1’!position!plays!a!role!in!determining!the!inhibitory!activity!of! a! peptide.! Peptides! with! a! protected! and! non.protonated! N.terminus! were! also!investigated! in! some!of! the! studies! reported! in!Table' 4.1' and!4.2,! but! none!of! them!were! found! to!present!DPP.IV! inhibitory! activity! (Bella! et! al.,! 1982;!Yoshimoto! et! al.,!1978).!!!4.4.2$Potential$of$Dietary$Proteins$to$Serve$as$DPP4IV$Inhibitor$Precursors$!The! in# silico# analysis! revealed! that! the! 34! proteins! from! the! 9! food! commodities!investigated! contained! 2255! di.! and! tri.peptides! that! matched! the! sequences! of!peptides!presenting! an! inhibitory! activity! against! the! enzyme!DPP.IV! reported! in! the!literature!(Tables'4.3,'4.4'and!4.5).!On!the!other!hand,!only!one!oligo.peptide!sequence!with!DPP.IV! inhibitory! activity!was! found!within! the! protein! sequences! investigated.!The! fragments! occurring! the! most! frequently! within! the! protein! primary! sequences!were!Gly.Ala,!Gly.Pro!and!Pro.Gly.!!!It!should!be!noted!that!most!of!the!peptides!investigated!in!the!literature!studies!listed!in!Table' 4.1' and!4.2! were! obtained! by! chemical! synthesis,! being! designed!with! the!purpose!of!gaining!knowledge!on!the!enzyme!catalytic!and!binding!properties!as!well!as!on! the! effects! of! structural! characteristics! of! the! peptides! on! their! interactions! with!DPP.IV.! Thus,! particularly! for! the! longer! oligo.peptides,! sequences! of! these! synthetic!! 79!peptides! that! have! been! reported! to! present! DPP.IV! inhibitory! activity!might! not! be!found!to!occur!naturally!in!protein!sequences.!!4.4.2.1%DPP(IV%Inhibitory%Peptides%in%Plant%Proteins%!The! sequences! of! 17! major! proteins! from! 5! plant! commodities! were! investigated!(Table' 4.3).! Barley,! canola,! oat,! soybean! and! wheat! proteins! contained! respectively!161,! 61,! 105,! 248! and! 156! fragments! within! their! sequences! matching! the! peptides!reported!in!the!literature!to!present!DPP.IV!inhibitory!activity.!Among!these!sequences,!Pro.Gly! and! Tyr.Pro! were! the! most! frequently! occurring! sequences! in! wheat! and!barley,!while!Leu.Ala,!Arg.Pro!&!Val.Val!and!Leu.Pro!&!Leu.Leu!were!found!in!higher!number! in! soybean! and! oat,! respectively.! In! order! to! quantify! the! potential! of! the!proteins! to!serve!as!bioactive!peptide!precursors,! the!occurrence! frequency!value!(A)!was!calculated!(Dziuba!et#al.,#2003)!(Table'4.6).!Among!the!17!plant!proteins!analyzed,!gamma.hordein.3! from! barley! showed! the! highest! occurrence! frequency! value! (A! =!0.173).!Both!wheat!and!barley,!whose!proteins!showed!occurrence!frequency!values!all!greater!than!0.1,!were!suggested!by!the!in#silico#results!to!be!the!best!potential!sources!of!DPP.IV!inhibitory!peptides!among!the!plant!sources!investigated.!On!the!other!hand,!oat!proteins!were!not!found!to!be!promising!sources!of!DPP.IV!inhibitors.!!! !! 80!Table' 4.3' Location! of! peptide! sequences! of! plant! proteins! with! DPP.IV! inhibition!activity.'Protein source Protein name  and IDa Sequence Locationb Barley B hordein (Q40026) FP [5–6], [10–11], [35–36], [52–53], [58–59], [70–71]  IA [141–142], [237–238], [246–247]  IP [40–41], [110–111]  IV [178–179]  LL [105–106]  LP [96–97], [163–164], [251–252]  MP [137–138], [265–266]  PF [4–5], [9–10], [28–29], [34–35], [51–52], [63–64], [69–70], [75–76]  PI [81–82]  PL [95–96], [260–261]  PP [53–54], [62–63], [74–75], [266–267]  VP [226–227], [259–260]  WP [65–66]  YP [17–18], [24–25], [47–48]  VPL [259–261]         D hordein (Q40054) AA [660–661]  AP [89–90]   FP [145–146], [168–169], [290–291], [616–617]   GA [184–185], [420–421], [429–430], [462–463]   GF [239–240], [487–488], [615–616]   GP [197–198]   IA [220–221], [344–345]   KA [655–656]   LA [659–660], [674–675]   LL [188–189], [675–676]   LP [37–38], [663–664]   PA [664–665]   PF [503–504]   PG [85–86], [132–133], [141–142], [149–150], [155–156], [172–173], [183–184], [191–192], [208–209], [227–228], [233–234], [278–279], [294–295], [328–329], [351–352], [363–364], [469–470], [475–476], [575–576], [609–610], [617–618]   PI [219–220], [243–244]   PL [91–92], [480–481]   PP [90–91]   RP [59–60]   VA [61–62], [653–654]   VP [76–77]   VV [28–29], [66–67]   YP [84–85], [106–107], [140–141], [163–164], [182–183], [218–219], [241–242], [257–258], [285–286], [302–303], [342–343], [383–384], [400–401], [459–460]          Gamma-hordein-3 (P80198) FP [22–23], [55–56], [67–68], [76–77], [88–89], [102–103]  GF [219–220]   HA [204–205]   IV [202–203], [206–207] ! 81!    Table'4.3'continued!Protein source Protein name  and IDa Sequence Locationb   LA [126–127], [187–188]   LF [21–22]   LL [146–147], [158–159]   LP [28–29], [49–50], [61–62], [71–72], [246–247], [264–265]   MA [282–283]   MP [80–81], [92–93], [232–233]   PA [197–198]   PF [33–34], [54–55], [107–108], [278–279]   PG [240–241]   PI [233–234]   PL [27–28], [48–49], [81–82], [93–94], [114–115], [125–126], [157–158]   PM [265–266]   PP [32–33], [273–274]   RP [17–18], [167–168]   VP [156–157], [272–273]   VV [255–256]   YP [43–44], [120–121]   VPL [156–158]         Canola Cruciferin (P11090) AP [35–36]  DP [292–293]   FP [3–4]   GF [137–138], [221–222], [282–283]   GP [256–257], [453–454], [459–460]   HA [34–35]   IA [154–155]   IP [379–380]   IV [174–175]   KA [22–23], [229–230], [462–463]   LA [180–181], [196–197], [227–228], [410–411]   LL [376–377]   LP [57–58], [314–315], [336–337], [420–421]   PA [454–455]   PF [99–100], [193–194], [257–258]   PG [158–159]   PI [315–316]   PL [171–172], [264–265], [421–422]   PP [263–264]   RP [192–193], [262–263], [269–270]   VA [69–70], [160–161]   VP [79–80], [358–359], [385–386]   GPA [453–455]          Napin small chain (P17333) GP [2–3]  IP [6–7]   KA [20–21]   PF [3–4]     ! 82!    Table'4.3'continued'Protein source Protein name  and IDa Sequence Locationb  Napin large chain (P17333) GA [29–30]  GP [83–84]   IP [69–70]   KA [32–33]   LL [7–8]   LP [63–64]   MP [81–82]   PF [76–77]   PG [83–84]   PL [6–7], [20–21]   PP [5–6]   RP [4–5]         Oat Avenin (P27919) FP [35–36]   GF [80–81]   IA [95–96], [119–120]   KA [183–184]   LA [134–135]   LL [57–58]   LP [99–100], [129–130]   MP [61–62]   PI [35–36]   VA [24–25]   WP [38–39]   YP [171–172]          11S Globulin (Q38779) AA [239–240]  AP [77–78], [155–156]   FA [113–114]   FP [94–95], [364–365]   GA [498–499]   GF [88–89], [508–509]   HA [345–346], [403–404]   IA [460–461]   IV [145–146], [157–158], [265–266], [369–370], [428–429]   LA [181–182], [189–190], [206–207], [475–476]   LF [2–3]   LL [180–181], [188–189], [205–206], [228–229], [426–427]   LP [71–72], [141–142], [470–471]   PA [78–79], [98–99], [142–143]   PF [107–108], [387–388]   PG [95–96]   PI [156–157], [264–265], [279–280], [365–366], [471–472]   PL [31–32]   VA [139–140], [158–159]   VP [270–271], [429–430]   VV [138–139], [408–409], [434–435]   APA [77–79]     ! 83!Table'4.3'continued#Protein source Protein name  and IDa Sequence Locationb  12S Globulin (O49257) AA [99–100], [210–211]  AP [77–78], [151–152]   FP [285–286]   GF [88–89], [194–195]   HA [324–325]   IA [135–136], [381–382]   IV [141–142]   KA [445–446]   LA [177–178], [396–397]   LF [2–3]   LL [69–70], [70–71], [83–84], [176–177], [199–200], [347–348]   LP [71–72], [94–95], [137–138], [350–351], [391–392]   PA [98–99], [138–139]   PF [107–108], [308–309]   PG [78–79], [95–96]   PI [286–287]   PL [31–32]   VA [154–155]   VV [153–154], [317–318], [329–330], [355–356]   YP [433–434]   APG [77–79]         Soybean Basic 7S globulin (P13917) AA [83–84]  AP [62–63], [142–143], [164–165], [201–202], [299–300]   FA [285–286], [400–401]   FP [245–246]    GA [194–195], [362–363]   GF [281–282]   GP [129–130], [325–326]   HA [119–120], [163–164]   IV [254–255]   LA [116–117], [172–173], [217–218], [375–376]   LF [138–139], [397–398]   LP [12–13], [151–152], [168–169], [289–290]   PA [82–83]   PF [63–64], [300–301]   PG [87–88], [341–342]   PI [6–7], [101–102], [165–166]   PL [32–33], [130–131], [221–222], [246–247]   RP [86–87]   VA [158–159], [298–299]   VP [36–37], [134–135]   VV [10–11], [371–372]   YP [189–190], [313–314]          Glycinin (P04347) FA [80–81]  FP [82–83]   GA [76–77]   GF [79–80], [207–208]   GP [487–488] ! 84!Table'4.3'continued#Protein source Protein name  and IDa Sequence Locationb   IA [335–336]   IP [125–126], [461–462]   IV [70–71], [237–238]   KA [347–348]   LA [165–166], [213–214]   LL [420–421]   LP [59–60], [63–64], [359–360]   PA [360–361], [427–428]   PG [83–84]   PL [126–127], [146–147], [488–489]   PP [272–273], [299–300]   RP [275–276], [295–296], [302–303], [305–306], [337–338]   VA [142–143], [429–430]   VP [129–130], [423–424]   VV [71–72], [141–142], [372–373], [402–403], [422–423], [441–442]   YP [65–66], [271–272]       Beta-conglycinin, beta-chain (P25974)    AA [319–320]  FP [368–369], [406–407]  GA [257–258], [411–412]  GF [150–151]  GP [404–405]  HA [64–65]  IP [101–102], [125–126], [317–318]  IV [49–50], [185–186], [269–270]  KA [267–268]  LA [123–124], [334–335], [348–349], [366–367]  LF [20–21], [71–72], [171–172]  LL [30–31], [60–61], [70–71], [259–260], [260–261], [378–379]  LP [61–62], [261–262]  PA [102–103], [318–319]  PF [8–9], [214–215], [322–323], [405–406]  PG [95–96], [130–131], [369–370]  PI [222–223]  PL [299–300]  VV [324–325], [355–356]  YP [321–322]  IPA [101–103], [317–319]         Beta-conglycinin, alpha-chain (P13916) DP [350–351]  FA [463–464]  FP [44–45], [91–92], [93–94], [497–498]   GA [386–387]   GF [274–275]   GP [533–534]   HA [188–189]   IP [11–12], [42–43], [249–250], [446–447], [488–489]   IV [195–196], [314–315], [380–381], [398–399]   KA [396–397]     ! 85!    Table'4.3'continued#Protein source Protein name  and IDa Sequence Locationb   LA [247–248], [477–478], [495–496]   LL [182–183], [183–184], [388–389], [389–390], [507–508]   LF [135–136], [144–145], [295–296]   LP [184–185], [390–391]   PA [447–448]   PF [43–44], [92–93], [133–134], [343–344]   PG [28–29], [254–255], [498–499]   PI [41–42], [351–352]   PL [428–429], [534–535]   PP [96–97]   RP [13–14], [15–16], [17–18], [40–41], [46–47], [95–96]   VP [225–226]   VV [233–234], [452–453], [453–454]   WP [63–64]   YP [450–451]   IPA [446–448]          Beta-conglycinin, alpha'-chain (P11827) DP [385–386]  FA [497–498]  FP [41–42], [43–44], [531–532]   GA [420–421]   GF [309–310]   GP [567–568]   HA [204–205], [363–364]   IA [261–262]   IP [12–13], [284–285], [522–523], [480–481]   IV [211–212], [349–350], [432–433]   KA [430–431]   LA [282–283], [511–512], [529–530]   LF [160–161], [330–331], [395–396], [422–423]   LL [199–200], [200–201]   LP [201–202], [424–425]   PA [242–243], [481–482]   PF [40–41], [42–43], [149–150], [378–379]   PG [289–290]   PI [386–387]   PL [462–463], [568–569]   RP [14–15], [16–17], [39–40], [45–46], [86–87]   VP [241–242]   VV [249–250], [268–269], [414–415], [433–434]   YP [484–485]   IPA [480–482]         Wheat Alpha/beta-gliadin (P02863) AP [258–259]  FP [3–4], [37–38], [49–50], [61–62]   HA [177–178]   IA [136–137], [257–258]   IP [124–125], [163–164], [251–252]   LA [238–239] ! 86!    Table'4.3'continued#Protein source Protein name  and IDa Sequence Locationb   LL [151–152]   LP [53–54], [66–67], [229–230], [243–244]   PA [244–245]   PF [36–37], [48–49], [60–61], [73–74], [259–260]   PI [93–94]   PL [23–24], [200–201], [205–206]   PP [38–39], [252–253]   RP [75–76], [212–213]   VP [5–6], [22–23]   VV [129–130], [175–176]   YP [43–44], [80–81], [204–205]   VPL [22–24]          Glutenin, high molecular weight subunit 12 (P08488) AA [608–609]  IP [331–332]  KA [615–616]  LA [30–31], [272–273]   LP [34–35], [623–624]   PA [278–279], [287–288], [302–303], [317–318], [332–333], [347–348], [444–445], [619–620]   PG [81–82], [120–121], [128–129], [134–135], [140–141], [142–143], [155–156], [163–164], [176–177], [221–222], [293–294], [308–309], [314–315], [323–324], [335–336], [338–339], [353–354], [375–376], [381–382], [399–400],[450–451], [457–458], [463–464], [475–476], [490–491], [511–512], [517–518], [550–551], [565–566], [583–584]    PL [86–87]   PP [73–74]   VA [58–59], [63–64], [613–614]   VP [72–73]   VV [25–26], [71–72]   YP [80–81], [107–108], [119–120], [148–149], [169–170], [190–191], [229–230], [256–257], [286–287], [301–302], [316–317], [346–347], [359–360], [407–408], [428–429], [443–444], [483–484], [504–505], [558–559]   IPA [331–333]          Glutenin, low molecular weight subunit 1D1 (P10386) FP [21–22], [36–37], [98–99]   GA [282–283]  IA [242–243]  IP [3–4], [113–114], [169–170]  LA [144–145], [231–232]   LF [26–27], [35–36]   LP [15–16], [63–64], [77–78], [91–92], [166–167], [256–257]   MP [140–141]   PF [48–49], [55–56], [68–69], [82–83], [97–98], [274–275]   PG [4–5]   PI [61–62]   PL [14–15], [25–26], [265–266] ! 87!    Table'4.3'continued#Protein source Protein name  and IDa Sequence Locationb   PP [16–17], [47–48], [54–55], [67–68], [81–82], [92–93]   RP [8–9]   VA [138–139]   VP [264–265], [273–274]   VV [115–116]   VPL [264–266]     a ID refers to the accession number in the UniProt KB database.  b Numbering based on the mature protein sequence. !4.4.2.2%DPP(IV%Inhibitory%Peptides%in%Milk%and%Chicken%Egg%Proteins%!Milk! and! chicken! egg! proteins! are! known! to! be! sources! of! bioactive! peptides! with!various!biological!activities,! including!anti.hypertensive!and!anti.microbial!properties!(Clare!&!Swaisgood,!2000;!Mine!&!Kovacs.Nolan,!2006).!Among!the!three!egg!proteins!studied,! 130! fragments!with!DPP.IV! inhibitory! activity!were! found,!Val.Ala! being! the!sequence! occurring! with! the! highest! frequency! (Table' 4.4).! While! ovalbumin! and!ovotransferrin! showed! similar! occurrence! frequency! values! (0.104! and! 0.111,!respectively),!ovomucoid,!with!an!occurrence!frequency!value!of!0.075,!was!suggested!by! the! in# silico! results! to! be! one! of! the! less! promising! sources! of! DPP.IV! inhibitory!peptides!among!all!proteins!investigated.!!!On!the!other!hand,!211!fragments!reported!to!present!a!DPP.IV!inhibitory!activity!were!found! in! the! six! cow’s!milk! proteins! investigated! (Table' 4.4).! Among! those,! Leu.Leu!and!Val.Pro!were!the!most!frequently!occurring!sequences.!The!in#silico!results!showed!that! beta.casein,! with! an! occurrence! frequency! value! of! 0.249,! had! the! greatest!potential! amongst! milk! proteins! to! serve! as! a! source! of! DPP.IV! inhibitors.! In! a#computer.aided#investigation!of!milk!proteins!as!sources!of!bioactive!peptides,!Dziuba!et!al.!(2009)!also!reported!beta.casein!as!the!most!promising!milk!protein!precursor!of!DPP.IV! inhibitory! peptides.! Conversely,! alpha.lactalbumin!was! found! to! be! a! limited!source! of! peptides!with! DPP.IV! inhibitory! activity.! This!whey! protein,!with! only! five!fragments! within! its! sequence! matching! peptides! known! for! their! DPP.IV! inhibitory!! 88!activity,!had!the!lowest!occurrence!frequency!value!(0.041)!of!all!proteins!investigated!in!this!study.!!!!! !! 89!Table'4.4'Location!of!peptide!sequences!of!chicken!egg!and!cow’s!milk!proteins!with!DPP.IV!inhibitory!activity.''Protein source Protein name and IDa Sequence Locationb Chicken egg Ovalbumin (P01012) AA [5–6], [137–138], [329–330], [351–352] FA [234–235] GA [4–5], [44–45]   GF [64–65]   HA [22–23], [328–329], [331–332]   IA [32–33], [371–372]   IV [178–179]   KA [186–187]   LA [38–39], [101–102]   LF [216–217], [365–366], [377–378]   LL [244–245]   LP [62–63], [114–115], [232–233], [245–246], [282–283]   MA [222–223], [298–299]   MP [196–197]   PF [197–198], [233–234], [363–364]   PG [63–64]   PI [31–32], [112–113], [131–132]   VA [219–220]   VV [56–57], [341–342]   YP [111–112]          Ovotransferrin (P02789) AA [78–79], [248–249]  AP [1–2], [70–71], [186–187], [217–218], [489–490]   DP [177–178], [292–293]   FA [432–433]   GA [136–137], [163–164], [193–194], [526–527]   GF [321–322]   GP [286–287]   HA [250–251], [592–593]   IA [49–50], [77–78]   IP [127–128], [466–467], [508–509]   KA [39–40], [47–48], [269–270], [554–555], [600–601]   LA [69–70], [586–587]   LF [284–285], [299–300], [636–637], [644–645]   LL [132–133], [226–227], [298–299], [568–569], [607–608], [635–636]   MA [411–412]   PA [428–429]   PG [162–163], [288–289], [490–491]   PI [76–77], [128–129]   PM [467–468]   PP [2–3], [287–288], [493–494], [509–510]   RP [427–428], [597–598]   VA [95–96], [152–153], [205–206], [247–248], [253–254], [392–393], [434–435], [436–437], [514–515], [537–538]   VP [161–162], [310–311], [408–409], [589–590]   VV [97–98], [252–253], [363–364], [373–374], [594–595], [595–596]   APG [489–491]   IPI [127–129]     ! 90!    Table'4.4'continued#Protein source Protein name and IDa Sequence Locationb  Ovomucoid (P01005) AA [132–133]  FP [8–9]  GA [116–117]   LA [131–132]   LL [42–43], [107–108]   PI [28–29]   PL [152–153]   PM [67–68]   RP [27–28], [151–152]   VP [66–67]   VV [171–172]   YP [141–142]         Cow’s milk Alpha-S1-casein (P02662) AP [26–27], [176–177] FP [28–29]  GA [162–163]  HA [128–129]   IP [182–183]   IV [71–72], [111–112]   LA [142–143]   LF [149–150]   LL [20–21], [98–99]   LP [11–12]   MP [196–197]   PF [27–28]   PI [5–6], [185–186]   PL [168–169], [197–198]   PM [134–135]   RP [1–2]   VA [25–26]   VP [72–73], [86–87], [106–107], [112–113], [167–168]   YP [146–147], [159–160]   VPL [167–169]          Beta-casein (P02666) AP [103–104]  FA [52–53]  FP [62–63], [111–112], [157–158], [205–206]   GP [64–65], [199–200], [203–204]   IP [66–67], [74–75]   IV [12–13], [208–209]   KA [176–177]   LL [139–140], [191–192]   LP [70–71], [135–136], [137–138], [151–152], [171–172]   MA [102–103]   MP [109–110], [185–186]   PF [51–52], [61–62], [86–87], [110–111], [118–119], [204–205]   PG [9–10], [63–64]   PI [65–66], [186–187], [206–207]     ! 91!Table'4.4'continued#Protein source Protein name and IDa Sequence Locationb   PL [76–77], [136–137], [138–139], [150–151]   PP [75–76], [85–86], [152–153], [158–159]   VP [8–9], [84–85], [173–174], [178–179]   VV [82–83], [83–84]   YP [60–61], [114–115], [180–181]       Kappa-casein (P02668)    AA [65–66]  IA [22–23], [125–126]  IP [26–27], [108–109], [119–120]  LP [56–57]  MA [95–96], [106–107]  PA [64–65], [70–71], [84–85]  PI [8–9], [27–28]  PP [109–110], [156–157]  VA [48–49], [143–144]  VP [83–84]  YP [35–36], [58–59]  IPI [26–28]         Beta-lactoglobulin (P02754) AA [25–26]  AP [37–38]  IA [72–73]  IP [78–79]   IV [2–3]   KA [138–139], [141–142]   LA [22–23], [117–118]   LF [104–105]   LL [31–32], [57–58], [103–104]   LP [143–144]   MA [24–25]   PA [79–80]   PL [38–39]   PM [144–145]   VA [15–16]   IPA [78–80]   VAGTWY [15–20]          Alpha-lactalbumin (P00711) IV [41–42]  KA [108–109]  LA [105–106]   LF [52–53]   LP [23–24]          Lactoferrin (P24627) AA [78–79], [604–605]  AP [1–2], [31–32], [237–238], [492–493], [592–593]   DP [70–71], [496–497]   FA [41–42]     ! 92!Table'4.4'continued Protein source Protein name and IDa Sequence Locationb   GA [30–31], [147–148], [194–195], [202–203], [494–495], [528–529]   GF [306–307]   GP [351–352]   HA [253–254], [595–596]   IA [49–50], [669–670]   IP [127–128], [310–311], [469–470]   IV [281–282], [474–475]   KA [53–54], [221–222], [273–274], [339–340], [441–442]   LA [247–248], [411–412], [434–435], [533–534], [589–590], [648–649]   LF [288–289], [299–300], [617–618], [631–632], [640–641]   LL [229–230], [270–271], [298–299], [371–372], [611–612], [639–640], [680–681]   LP [618–619]   PG [293–294], [493–494]   PL [144–145], [679–680]   PM [128–129], [470–471]   PP [292–293]   RP [75–76], [133–134], [428–429], [654–655]   VA [77–78], [95–96], [149–150], [206–207], [256–257], [436–437], [540–541], [591–592]   VP [158–159], [250–251], [408–409], [516–517]   VV [97–98], [255–256], [345–346], [438–439], [597–598]   YP [166–167]   APG [492–494]  a ID refers to the accession number in the UniProt KB database.  b Numbering based on the mature protein sequence. !%4.4.2.3%DPP(IV%Inhibitory%Peptides%in%Bovine%Meat%and%Salmon%Proteins%!The!four!bovine!and!four!salmon!proteins!investigated!contained!within!their!primary!sequence!685!and!499!fragments,!respectively,!matching!sequences!known!to!present!an!inhibitory!activity!against!the!DPP.IV!enzyme!(Table'4.5).!Among!those,!Gly.Pro!and!Pro.Gly!were! the!most! frequently!occurring! sequences!and!were!mainly! found! in! the!collagen!proteins,!due!to!their!high!contents!of!proline!and!glycine.!!!Collagens!from!bovine!meat!and!salmon,!with!occurrence!frequency!values!of!0.380!and!0.305! respectively,! appeared! to! be! the! best! potential! sources! of! DPP.IV! inhibitory!peptides!among!all!proteins!studied.!A!recent!computational!study!by!Minkiewicz!et!al.!(2011),! in! which! 19! bovine!muscle! proteins! were! investigated! for! their! potential! as!! 93!bioactive! peptide! precursors,! showed! that! collagen! and! elastin! were! the! richest!potential! sources! of! fragments! with! various! biological! functions,! including! DPP.IV!inhibitory! activity.!Moreover,! unlike!most! proteins! investigated! in! the! present! study,!bovine! and! salmon! collagens! contained! relatively! high! numbers! of! the! DPP.IV!inhibitory!tri.peptides!Ala.Pro.Gly!and!Gly.Pro.Ala.!!!It! should! be! noted! that! although! some! proteins! showed! a! high! number! of! DPP.IV!inhibitors! within! their! primary! sequences,! many! shared! overlapping! peptide!sequences.!This!was!particularly!important!in!some!proteins!like!chum!salmon!collagen!in! which! a! high! number! of! peptides,! such! as! Gly.Pro,! Pro.Ala! and! Gly.Pro.Ala,! were!overlapping.! For! example,! Gly.Pro,! Pro.Ala! and! Gly.Pro.Ala! were! located! at! the!positions! [201–202],! [202–203],! [201–203],! respectively,! in! the! collagen! of! chum!salmon.! !! 94!Table'4.5'Location!of!peptide!sequences!of!bovine!meat,!Atlantic!and!chum!salmon!proteins!with!DPP.IV!inhibitory!activity.'Protein source Protein name and IDa Sequence Locationb Bovine meat Myosine-1 (Q9BE40) AA [13–14], [111–112], [387–388], [521–522], [1192–1193], [1193–1194], [1440–1441], [1443–1444], [1545–1546], [1639–1640], [1765–1766]  AP [14–15], [151–152]   DP [42–43], [602–603]   FA [196–197]   FP [78–79], [543–544], [712–713]   GA [67–68], [182–183], [357–358], [428–429], [685–686], [1158–1159]   GF [343–344], [467–468], [711–712], [797–798]   GP [626–627], [635–636]   HA [972–973], [1199–1200], [1334–1335], [1467–1468], [1634–1635]   IA [199–200], [465–466], [992–993], [1519–1520], [1579–1580], [1704–1705], [1915–1916]   IP [677–678], [733–734]   IV [1596–1597]   KA [49–50], [281–282], [386–387], [400–401], [432–433], [545–546], [575–576], [743–744], [769–770], [1000–1001], [1025–1026], [1134–1135], [1243–1244], [1327–1328], [1357–1358], [1366–1367], [1757–1758], [1760–1761]   LA [261–262], [430–431], [520–521], [620–621], [749–750], [784–785], [799–800], [866–867], [901–902], [964–965], [1064–1065], [1103–1104], [1234–1235], [1332–1333], [1396–1397], [1674–1675], [1806–1807]   LF [121–122], [623–624], [653–654], [1484–1485]   LL [229–230], [270–271], [304–305], [398–399], [622–623], [748–749], [772–773], [775–776], [842–843], [1345–1346], [1683–1684], [1709–1710], [1718–1719]   LP [132–133]   MA [7–8], [91–92], [333–334], [852–853], [985–986], [1638–1639], [1768–1769]   PA [100–101], [571–572], [627–628]   PF [31–32]   PG [684–685]   PL [228–229], [603–604], [841–842]   PM [79–80], [530–531]   PP [82–83], [152–153]   VA [40–41], [383–384], [443–444], [1203–1204], [1371–1372]   VP [323–324]   VV [139–140], [608–609]   WP [830–831]   YP [404–405]   GPA [626–628]          Myosin regulatory light chain 12B (A4IF97) AP [75–76], [144–145]  DP [96–97]  FA [21–22], [105–106]  GA [164–165]   GF [45–46] ! 95!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb   GP [77–78]   KA [4–5]   LA [56–57]   LL [124–125]   PG [76–77]   PI [78–79], [145–146]   RP [12–13]   APG [75–77]          Actin, cytoplasmic 1 (P60712) AA [6–7], [230–231]  AP [26–27], [97–98], [108–109], [321–322], [331–332]  FA [21–22]  FP [31–32]   GF [20–21]   GP [366–367]   HA [173–174]   IA [5–6], [309–310], [330–331]   IV [34–35], [75–76], [151–152], [208–209], [369–370]   KA [18–19], [113–114]   LA [180–181], [320–321], [346–347]   LF [261–262]   LL [104–105]   LP [171–172], [242–243]   MA [227–228]   PA [130–131]   PG [307–308]   PI [70–71], [164–165]   PL [109–110]   PP [332–333]   RP [37–38]   VA [96–97], [134–135], [219–220]   VP [163–164]   VV [9–10]   YP [69–70], [306–307]          Collagen alpha-1 (II) chain (P02459) AA [159–160], [225–226], [459–460], [552–553], [576–577], [720–721], [870–871], [1063–1064], [1222–1223]  AP [23–24], [36–37], [189–190], [243–244], [249–250], [306–307], [330–331], [348–349], [399–400], [444–445], [477–478], [510–511], [597–598], [639–640], [681–682], [690–691], [828–829], [885–886], [888–889], [1186–1187], [1285–1286]  DP [366–367], [762–763], [1053–1054], [1120–1121]   FA [654–655], [1042–1043]   FP [96–97], [186–187], [252–253], [336–337], [423–424], [522–523], [717–718], [813–814]                 '! 96!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb   GA [12–13], [35–36], [92–93], [116–117], [122–123], [158–159], [161–162], [188–189], [191–192], [200–201], [206–207], [224–225], [236–237], [242–243], [248–249], [263–264], [299–300], [311–312], [318–319], [329–330], [347–348], [362–363], [377–378], [398–399], [413–414], [428–429], [443–444], [458–459], [476–477], [509–510], [536–537], [551–552], [560–561], [575–576], [596–597], [617–618], [635–636], [638–639], [659–660], [665–666], [680–681], [689–690], [704–705], [707–708], [713–714], [719–720], [749–750], [827–828], [830–831], [860–861], [869–870], [881–882], [884–885], [887–888], [911–912], [923–924], [971–972], [995–996]             GF [5–6], [41–42], [95–96], [185–186], [251–252], [281–282], [335–336], [422–423], [482–483], [521–522], [653–654], [716–717], [812–813], [953–954], [1175–1176]   GP [20–21], [23–24], [26–27], [29–30], [32–33], [38–39], [56–57], [59–60], [62–63], [65–66], [86–87], [89–90], [140–141], [143–144], [155–156], [170–171], [173–174], [176–177], [179–180], [183–184], [197–198], [203–204], [209–210], [221–222], [254–255], [257–258], [260–261], [266–267], [269–270], [287–288], [293–294], [296–297], [302–303], [320–321], [323–324], [344–345], [353–354], [359–360], [389–390], [395–396], [407–408], [410–411], [425–426], [461–462], [464–465], [467–468], [479–480], [488–489], [491–492], [515–516], [548–549], [554–555], [557–558], [563–564], [581–582], [593–594], [608–609], [611–612], [614–615], [626–627], [629–630], [647–648], [650–651], [656–657], [683–684], [686–687], [692–693], [695–696], [701–702], [710–711], [725–726], [734–735], [737–738], [740–741], [746–747], [755–756], [767–768], [770–771], [782–783], [785–786], [788–789], [791–792], [818–819], [836–837], [839–840], [842–843], [848–849], [863–864], [890–891], [896–897], [899–900], [914–915], [917–918], [920–921], [929–930], [932–933], [962–963], [965–966], [974–975], [977–978], [980–981], [983–984], [986–987], [989–990], [1001–1002], [1004–1005], [1007–1008], [1016–1017], [1019–1020], [1025–1026], [1028–1029], [1031–1032], [1034–1035], [1051–1052], [1291–1292], [1301–1302]   IA [246–247], [279–280], [579–580], [1216–1217], [1284–1285]   IP [234–235], [999–1000]   IV [801–802]   KA [9–10], [435–436], [1228–1229]   LA [342–343], [357–358], [795–796], [1185–1186]   LL [1199–1200], [1230–1231]   LP [102–103], [147–148], [375–376], [441–442], [450–451], [486–487], [540–541], [804–805], [816–817], [960–961], [1279–1280]   MA [2–3]   MP [570–571], [927–928]   PA [33–34], [156–157], [171–172], [180–181], [222–223], [294–295], [300–301], [303–304], [321–322], [465–466], [468–469], [555–556], [615–616], [630–631], [651–652], [768–769], [849–850], [897–898], [918–919], [921–922], [975–976], [1017–1018], [1047–1048], [1098–1099]     ! 97!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb   PG [31–32], [37–38], [46–47], [52–53], [64–65], [67–68], [70–71], [79–80], [88–89], [97–98], [100–101], [103–104], [112–113], [124–125], [133–134], [139–140], [148–149], [169–170], [175–176], [184–185], [187–188], [190–191], [214–215], [217–218], [220–221], [229–230], [235–236], [244–245], [250–251], [253–254], [259–260], [277–278], [292–293], [301–302], [316–317], [325–326], [331–332], [337–338], [349–350], [367–368], [370–371], [373–374], [376–377], [385–386], [400–401], [406–407], [409–410], [418–419], [424–425], [433–434], [442–443], [445–446], [451–452], [463–464], [478–479], [487–488], [490–491], [493–494], [499–500], [505–506], [511–512], [523–524], [529–530], [541–542], [544–545], [559–560], [565–566], [571–572], [598–599], [613–614], [628–629], [640–641], [649–650], [658–659], [664–665], [682–683], [691–692], [712–713], [718–719], [727–728], [733–734], [736–737], [739–740], [757–758], [763–764], [772–773], [778–779], [790–791], [805–806], [814–815], [817–818], [823–824], [829–830], [838–839], [844–845], [853–854], [859–860], [865–866], [886–887], [889–890], [892–893], [895–896], [928–929], [961–962], [964–965], [985–986], [1000–1001], [1006–1007], [1021–1022], [1024–1025], [1027–1028], [1030–1031], [1033–1034], [1035–1036]   PI [609–610], [900–901], [1002–1003], [1280–1281]   PL [267–268], [1054–1055]   PM [21–22], [24–25], [57–58], [141–142], [915–916], [1286–1287]   PP [30–31], [63–64], [66–67], [87–88], [174–175], [258–259], [324–325], [408–409], [462–463], [489–490], [492–493], [558–559], [564–565], [612–613], [627–628], [648–649], [657–658], [711–712], [726–727], [735–736], [738–739], [756–757], [771–772], [789–790], [837–838], [843–844], [864–865], [891–892], [963–964], [984–985], [1005–1006], [1020–1021], [1026–1027], [1029–1030], [1032–1033]   RP [369–370], [384–385], [405–406]   VP [504–505], [1050–1051]   YP [111–112], [1144–1145]   APG [36–38], [123–125], [189–191], [243–245], [249–251], [300–302], [330–332], [348–350], [399–401], [444–446], [477–479], [510–512], [597–599], [639–641], [681–683], [690–692], [828–830], [885–887], [888–890]   GPA [32–34], [155–157], [170–172], [179–181], [221–223], [293–295], [302–304], [320–322], [464–466], [467–469], [554–556], [614–616], [629–631], [650–652], [767–769], [848–850], [896–898], [917–919], [920–922], [974–976], [1016–1018]         Atlantic salmon Actin, cytoplasmic 1 (O42161) AA [6–7], [230–231] AP [26–27], [97–98], [108–109], [321–322], [331–332]  FA [21–22]   FP [31–32]   GF [20–21]   GP [366–367]   HA [1173–174]   IA [5–6], [309–310], [330–331] ! 98!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb   IV [34–35], [75–76], [151–152], [208–209], [369–370]   KA [18–19], [113–114]   LA [180–181], [320–321], [346–347]   LF [261–262]   LL [104–105]   LP [171–172], [242–243]   PA [130–131]   PG [307–308]   PI [70–71], [164–165]   PL [109–110]   PP [332–333]   RP [37–38]   VA [96–97], [134–135], [219–220]   VP [163–164]   VV [9–10]   YP [69–70], [306–307]          Myosin regulatory light chain 2 (Q7ZZN0) AA [11–12], [12–13], [143–144]  AP [2–3]  DP [97–98], [110–111]  FP [145–146]   GA [10–11], [95–96]   GF [115–116]   GP [78–79]   IV [102–103]   KA [5–6]   LA [56–57]   LL [125–126]   MA [1–2]   PI [79–80]   PP [146–147]   VA [149–150]          Slow myosin heavy chain (Q2HXU3) AA [12–13], [110–111], [199–200], [207–208]  AP [150–151]  DP [41–42]  FA [195–196]   GA [11–12], [56–57], [181–182]   IA [198–199]   KA [48–49]   LF [102–103], [120–121]   LP [131–132]   MA [6–7], [90–91]   PA [99–100], [224–225]   PF [30–31]   PP [81–82], [151–152]   RP [29–30]   VP [39–40]   VV [138–139]     ! 99!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb Chum salmon Type 1 collagen alpha 2 chain (Q8UUJ4) AA [67–68], [241–242], [301–302], [361–362], [460–461], [622–623], [667–668], [781–782], [931–932], [982–983]  AP [250–251], [310–311], [424–425], [520–521], [541–542], [847–848], [988–989], [1331–1332]  DP [77–78], [337–338]   FA [66–67], [715–716], [1224–1225]   FP [157–158], [247–248], [313–314], [484–485], [778–779]   GA [28–29], [120–121], [153–154], [186–187], [192–193], [195–196], [222–223], [225–226], [240–241], [249–250], [259–260], [279–280], [297–298], [300–301], [309–310], [330–331], [348–349], [360–361], [378–379], [387–388], [391–392], [411–412], [414–415], [423–424], [438–439], [459–460], [504–505], [513–514], [519–520], [540–541], [594–595], [597–598], [621–622], [636–637], [663–664], [666–667], [678–679], [780–781], [846–847], [879–880], [891–892], [930–931], [945–946], [981–982], [984–985], [987–988], [1000–1001], [1062–1063]   GF [102–103], [156–157], [246–247], [312–313], [483–484], [543–544], [714–715], [777–778], [861–862], [933–934], [1162–1163]   GP [25–26], [31–32], [37–38], [40–41], [55–56], [58–59], [79–80], [81–82], [90–91], [93–94], [99–100], [123–124], [148–149], [201–202], [216–217], [219–220], [231–232], [234–235], [237–238], [243–244], [252–253], [264–265], [267–268], [282–283], [285–286], [315–316], [319–320], [321–322], [324–325], [339–340], [363–364], [381–382], [405–406], [417–418], [450–451], [456–457], [468–469], [471–472], [474–475], [486–487], [489–490], [501–502], [511–512], [522–523], [537–538], [549–550], [552–553], [573–574], [585–586], [600–601], [606–607], [627–628], [642–643], [669–670], [672–673], [675–676], [690–691], [693–694], [699–700], [708–709], [711–712], [717–718], [726–727], [732–733], [741–742], [744–745], [747–748], [753–754], [759–760], [762–763], [771–772], [787–788], [789–790], [795–796], [801–802], [807–808], [816–817], [819–820], [828–829], [837–838], [843–844], [852–853], [858–859], [877–878], [888–889], [894–895], [897–898], [924–925], [954–955], [957–958], [972–973], [978–979], [990–991], [993–994], [1023–1024], [1026–1027], [1038–1039], [1041–1042], [1044–1045], [1047–1048], [1050–1051], [1065–1066], [1071–1072], [1077–1078], [1080–1081], [1089–1090], [1092–1093], [1167–1168], [1347–1348]   HA [106–107]   IA [832–833], [1173–1174], [1197–1198], [1330–1331]   IV [619–620]   KA [47–48], [559–560], [1178–1179], [1274–1275]   LA [13–14], [19–20], [1240–1241], [1246–1247]   LL [10–11], [11–12], [12–13], [18–19], [1135–1136], [1245–1246], [1277–1278]   LP [50–51], [163–164], [304–305], [397–398], [442–443], [547–548], [571–572], [865–866], [874–875], [1087–1088], [1325–1326]   MA [1236–1237]   MP [409–410], [565–566], [631–632], [904–905], [1021–1022]     ! 100!Table'4.5'continued#Protein source Protein name and IDa Sequence Locationb   PA [202–203], [217–218], [220–221], [232–233], [235–236], [364–365], [382–383], [418–419], [523–524], [550–551], [553–554], [574–575], [586–587], [601–602], [691–692], [694–695], [709–710], [733–734], [742–743], [745–746], [756–757], [760–761], [790–791], [802–803], [829–830], [889–890], [958–959], [991–992], [1039–1040], [1045–1046], [1078–1079], [1090–1091], [1093–1094], [1145–1146]   PG [51–52], [57–58], [60–61], [78–79], [80–81], [95–96], [98–99], [110–111], [113–114], [131–132], [140–141], [143–144], [149–150], [158–159], [161–162], [164–165], [200–201], [245–246], [248–249], [251–252], [275–276], [290–291], [305–306], [311–312], [314–315], [320–321], [338–339], [353–354], [398–399], [410–411], [425–426], [443–444], [452–453], [470–471], [479–480], [485–486], [490–491], [512–513], [521–522], [542–543], [548–549], [566–567], [572–573], [605–606], [614–615], [628–629], [632–633], [641–642], [674–675], [713–714], [719–720], [764–765], [779–780], [788–789], [797–798], [817–818], [845–846], [848–849], [866–867], [875–876], [878–879], [884–885], [905–906], [926–927], [929–930], [941–942], [944–945], [953–954], [965–966], [989–990], [1022–1023], [1052–1053], [1073–1074], [1082–1083], [1085–1086], [1088–1089]   PI [406–407], [1326–1327]   PL [238–239], [1332–1333]   PM [82–83]   PP [56–57], [59–60], [94–95], [244–245], [451–452], [469–470], [673–674], [712–713], [718–719], [763–764], [796–797], [844–845], [925–926], [1051–1052], [1072–1073], [1081–1082]   RP [139–140], [928–929], [964–965]   VA [53–54], [307–308], [1262–1263]   VV [526–527], [826–827]   APG [250–252], [310–312], [424–426], [520–522], [541–543], [847–849], [987–989]   GPA [201–203], [216–218], [219–221], [231–233], [234–236], [363–365], [381–383], [417–419], [522–524], [549–551], [552–554], [573–575], [585–587], [600–602], [690–692], [693–695], [708–710], [732–734], [741–743], [744–746], [759–761], [789–791], [801–803], [828–830], [888–890], [957–959], [990–992], [1038–1040], [1044–1046], [1077–1079], [1089–1091], [1092–1094]     a ID refers to the accession number in the UniProt KB database.  b Numbering based on the mature protein sequence. ! !! 101!Table' 4.6' Occurrence! frequency! (A)! of! peptides! with! DPP.IV! inhibitory! activity! in!proteins!of!various!food!commodities.!!Protein source Protein name ID numbera Occurrence frequency Ab Barley B hordein Q40026 0.148 D hordein Q40054 0.105 Gamma-hordein-3 P80198 0.173 Canola Cruciferin P11090 0.095 Napin small chain P17333 0.111 Napin large chain P17333 0.151 Oat Avenin P27919 0.075 11S globulin Q38779 0.095 12S globulin Q49257 0.092 Soybean Basic 7S P13917 0.119 Glycinin P04347 0.087 Beta-conglycinin, beta-chain P25974 0.111 Beta-conglycinin, alpha-chain P13916 0.109 Beta-conglycinin, alpha’-chain P11827 0.090 Wheat Alpha/beta-gliadin P02863 0.147 Glutenin, high molecular weight subunit 12 P08488 0.117 Glutenin, low molecular weight subunit 1D1 P10386 0.148 Chicken egg Ovalbumin P01012 0.104 Ovotransferrin P02789 0.111 Ovomucoid P01005 0.075 Cow’s milk Alpha-S1-casein P02662 0.151 Beta-casein P02666 0.249 Kappa-casein P02668 0.130 Beta-lactoglobulin P02754 0.130 Alpha-lactalbumin P00711 0.041 Lactoferrin P24627 0.118 Bovine meat Myosin-1 Q9BE40 0.067 Myosin regulatory light chain 12B A4IF97 0.094 Actin, cytoplasmin 1 P60712 0.117 Collagen alpha-1 (III) chain P02459 0.380 Atlantic salmon Actin, cytoplasmic 1 O42161 0.115 Myosin regulatory light chain 2 Q7ZZN0 0.112 Slow myosin heavy chain Q2HXU3 0.110 Chum salmon Type 1 collagen alpha 2 chain Q8UUJ4 0.305 a ID number refers to the accession number in the UniProt KB database. b A = number of fragments with DPP-IV inhibitory activity in the protein chain/ number of amino acid residues in the polypeptide chain molecule.  !! !! 102!4.5!CONCLUSION!!The!potential!of!34!proteins!from!9!food!commodities!to!serve!as!precursors!of!DPP.IV!inhibitory!peptides!was!assessed!using!an! in#silico! approach.!Caseins! from!cow’s!milk!and! collagens! from! bovine! meat! and! salmon! were! found! to! be! the! best! potential!precursors!of!DPP.IV!inhibitors,!whereas!the!plant!commodity!oat!was!suggested!to!be!the!least!promising!potential!source!of!peptides!with!DPP.IV!inhibitory!activity.!!!Since!the!present!study!relies!entirely!on!the!currently!available!data!on!peptides!with!DPP.IV! inhibitory!activity,! it! is!possible! that! the!dietary!proteins! investigated!contain!within!their!primary!sequences!other! fragments!presenting! inhibitory!activity!against!the!DPP.IV!enzyme,!but! that! these! fragments! are!presently!unknown!or!have!not!yet!been!reported!in!the!literature.!Furthermore,!it!should!be!kept!in!mind!that!the!putative!inhibitor!sequences!must!be!released!from!their!parent!proteins!to!become!active!and!that!consequently,!final!conclusions!on!the!potential!of!dietary!proteins!for!generation!of! DPP.IV! inhibitors! can! only! be! drawn! after! experimentally! assessing! the! release! of!these!bioactive!peptides!upon!in#vitro#or!in#vivo!hydrolysis!or!digestion.!!Moreover,!this!in#silico!approach,!although!providing!useful!information!on!the!potential!of!proteins!to!serve! as! bioactive! peptide! precursors,! does! not! allow! the! identification! of! the! most!potent! proteins! in! terms! of! inhibitory! activity.! The! overall! activity! of! a! protein! will!depend!on!the!potency!of!the!active!peptides!present!within!its!sequence.!This!aspect!could!not,!however,!be!taken!into!account!in!this!study!due!to!the!variability! in!the!Ki!values! reported! by! different! researchers! for! the! same! peptide.! Nevertheless,! the!findings! from! this! analysis! provide! the! basis! in! support! of! further! investigation! to!exploit!dietary!proteins!as!naturally!occurring!materials!for!generation!of!peptides!with!DPP.IV!inhibitory!activity.!!! !! 103!CHAPTER! 5:! DIPEPTIDYL5PEPTIDASE! IV! INHIBITORY! ACTIVITY! OF! DAIRY! PROTEIN!HYDROLYSATES6!!!5.1!SUMMARY!!Dipeptidyl.peptidase! IV! (DPP.IV)! inhibitors! are! promising! new! therapies! for! type! 2!diabetes.! Currently,! these! inhibitors! are! only! available! as! synthetic! drugs.! Therefore,!the!aim!of! the!present! study!was! to! investigate!dairy!protein!hydrolysates!as!natural!sources!of!DPP.IV!inhibitors.!While!the!DPP.IV!inhibitory!activity!of!sodium!caseinate,!skim!milk!powder!and!milk!protein!concentrate!hydrolysates!increased!over!the!course!of!in#vitro!pepsin.pancreatin!digestion,!whey!protein!isolate!(WPI)!hydrolysate!showed!highest! inhibitory! activity! following! peptic! digestion.! Hydrolysates! produced! from!sodium!caseinate!using!11!different!proteases!displayed!higher!inhibitory!activity!than!most!WPI!hydrolysates.!However,!among!all!enzymatic!treatments!investigated,!peptic!digestion! of! WPI! resulted! in! the! greatest! DPP.IV! inhibitory! activity! (IC50! of! 0.075!mg/mL).! Fractionation! of! the! hydrolysates! by! size! generally! improved! the! inhibitory!activity.! This! study! shows! that! peptides! with! DPP.IV! inhibitory! activity! can! be!generated!from!dairy!ingredients.!!5.2!INTRODUCTION!!Type!2!diabetes!(T2D)!is!the!most!prevalent!form!of!diabetes!and!is!one!of!the!fastest!growing! health! concerns!worldwide! (Sebokova! et! al.,! 2007).! Over! the! past! decade,! a!number!of!new!therapies!focusing!on!the!modulation!of!the!incretin!hormones!glucose.dependent! insulinotropic!polypeptide!(GIP)!and!glucagon.like!peptide.1!(GLP.1)!have!been!developed!for!the!management!of!T2D.!Released!following!nutrient!ingestion,!the!gut.derived!hormones!GIP!and!GLP.1!stimulate!glucose.dependent!insulin!secretion!in!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!6!A! version! of! this! chapter! has! been! published.! Lacroix,! I.M.E,! Li.Chan,! E.C.Y.! Dipeptidyl!peptidase.IV!inhibitory!activity!of!dairy!protein!hydrolysates.!Int.#Dairy#J.#2012,!25,!97–102.!! 104!pancreatic! β.cells! (Holst! &! Deacon,! 2004;! Mentlein,! 2005).! However,! incretins! have!very!short!half.lives! in! the!order!of!minutes!due! to!renal!degradation!and!proteolytic!cleavage!by!the!enzyme!dipeptidyl.peptidase!IV!(Mentlein,!2005).!!!In! order! to! extend! the! incretin! effect! and! thus! enhance! insulin! secretion,! two!approaches! can! be! taken:! the! intravenous! administration! of! GLP.1! or! its! protease.resistant!analogs,!or!the!oral!administration!of!DPP.IV!inhibitors!in!order!to!slow!down!the!otherwise!rapid!inactivation!of!endogenous!GIP!and!GLP.1!(Mentlein,!2005).!GLP.1!analogs!offer!the!advantage!of!higher!target!selectivity,!but!suffer!the!disadvantages!of!being! available! only! in! injectable! form! and! of! presenting! undesirable! side! effects!including! vomiting! and! nausea.! On! the! other! hand,! DPP.IV! inhibitors! are! orally!bioavailable!and!have!been! reported! to! cause!only! transient!and!minimal! side!effects!(Mentlein,! 2005).! Several! synthetic! peptide.derived! peptidomimetic! and! non.peptidomimetic! DPP.IV! inhibitors! have! emerged! over! the! past! few! years,! including!Galvus®! (vildagliptin)! from! Novartis! (IC50! =! 3.5! nM),! Onglyza®! (saxagliptin)! from!Bristol.Meyers!Squibb!(IC50!=!0.5!nM)!and!Januvia®!(sitagliptin)!from!Merck!(IC50!=!18!nM)!(Hunziker!et!al.,!2005),!which!have!already!been!approved!for!the!management!of!T2D!in!Canada,!the!European!Union!and/or!the!United!States.!!!In! the! past! two! decades,! extensive! research! in! the! dairy! area! has! shown! that! milk!proteins! are! rich! sources! of! peptides! presenting! a! variety! of! biological! properties.!Peptides! presenting! anti.hypertensive,! anti.oxidative,! anti.bacterial,! anti.cariogenic,!mineral! binding,! opioid! and! immunomodulating! activities! have!been! identified! in! the!sequences!of!proteins! from!both! the!casein!and!whey! fractions!of!milk! (Dziuba!et!al.,#2009;!FitzGerald!&!Murray,!2006;!Hernández.Ledesma!et!al.,!2008;!Korhonen,!2009).!In#vivo#studies!have!shown!that!bioactive!peptides,!such!as!the!opioid!β.casomorphin.11,!can! be! formed! during! digestion! of! milk! proteins! (Segura.Campos! et! al.,! 2011)! and!various! proteases! have! been! successfully! used! to! produce! bioactive! peptides! from!casein!and!whey!proteins!(Korhonen!&!Pihlanto,!2006).!!!!! 105!As!shown!in!the! in#silico#investigation!into!the!potential!of!proteins!from!various!food!commodities! to! serve! as! precursors! of! DPP.IV! inhibitors! presented! in! the! previous!chapter,! milk! proteins! contain! within! their! sequences! fragments! that! match! the!sequences!of!DPP.IV!inhibitory!peptides!reported!in!the!literature!!(Lacroix!&!Li.Chan,!2012;! Chapter! 4).! Moreover,! a! computer.aided! study! using! the! BIOPEP! database! to!evaluate! dairy! proteins! as! sources! of! bioactive!peptides! has! also! shown! that! the! two!biological! properties! occurring!with! the! highest! frequency! in!milk! protein! sequences!were!related!to!anti.hypertensive!and!DPP.IV!inhibitory!activities!(Dziuba!et!al.,!2009).!Despite! the! potential! in! theory! of! milk! proteins! to! serve! as! precursors! of! inhibitory!peptides!against!the!DPP.IV!enzyme,!experimental!investigations!on!the!production!of!DPP.IV! inhibitors! from! milk! or! other! dietary! proteins! are! sparse.! Therefore,! the!objective!of!the!present!study!was!to!investigate!the!DPP.IV!inhibitory!activity!of!dairy!protein! hydrolysates! obtained! by! in# vitro# simulated! digestion! and! by! enzymatic!treatments!using!various!proteases.!!!5.3!MATERIALS!AND!METHODS!#5.3.1$Materials$#Whey! protein! isolate! (WPI! 895,! Fonterra,! New! Zealand)!was! donated! by! Nealanders!International! Inc.! (Delta,! BC,! Canada).! Milk! protein! concentrate! (MPC! 80)! was! from!Vitalus!Nutrition!Inc.!(Abbotsford,!BC,!Canada).!Skim!milk!powder!was!purchased!from!Safeway!(Vancouver,!BC,!Canada).!Sodium!caseinate!(casein!sodium!salt),! thermolysin!(EC! 3.4.24.27,! from!Bacillus# thermoproteolyticus#rokko,! 50.100!units! per!mg!protein),!pepsin! (EC! 3.4.23.1,! from! porcine! gastric! mucosa,! ≥! 2500! units! per! mg! protein),!dipeptidyl.peptidase! IV! (DPP.IV,! EC! 3.4.14.5,! from! porcine! kidney,! ≥10! units! per!mg!protein)! and! Ile.Pro.Ile! (diprotin!A)!were! from!Sigma.Aldrich! (Oakville,!ON,!Canada).!Gly.Pro.p.nitroanilide!(H.Gly.Pro.p?NA⋅HCl)!was!from!Bachem!Americas!(Torrance,!CA,!United!States),!while!2,4,6.trinitrobenzenesulfonic!acid! (TNBS! in!MeOH!solution)!was!! 106!from! Thermo! Scientific! (Rockford,! IL,! United! States).! Protease! N! “Amano”! K! (from!Bacillus# subtilis,#150,000! –! 190,000! units/g),! Protease!A! “Amano”! 2! (from!Aspergillus#oryzae,#20,000!units/g),!Umamizyme!K!(from!Aspergillus#oryzae,!70!units/g)!and!Protin!SD.NY10! (from!Bacillus#subtilis,! ≥!90,000!units/g)!were!donated! !by!Amano!Enzymes!USA!Co.,!Ltd.!(Elgin,!IL,!United!States).!Alcalase®!(from!Bacillus#licheniformis,#2.4!AU/g,!Novozyme!Inc.)!and!Flavourzyme®!(from!Aspergillus#oryzae,!≥!500!units/g,!Novozyme!Inc.)!were! supplied!by!Brenntag!Canada! Inc.! (Langley,!BC,!Canada).! Protamex®! (from!Bacillus,#1.5!AU,!Novozyme!Inc.)!and!bromelain!(from!pineapple!stem,!2000!GDU,!Ultra.Biologics! Inc.)! were! supplied! by! Neova! Technologies! Inc.! (Abbotsford,! BC,! Canada).!Validase®! BNP.L! (from! Bacillus# subtilis# var,! 2000! NPU)! and! Corolase! PP®! (from! pig!pancreas! glands,! ≥! 2500! UHb/g)! were! donated! by! Valley! Research! (South! Bend,! IN,!United!States)!and!AB!Enzymes!(Darmstadt,!Germany),!respectively.!!!5.3.2$In$vitro$Simulated$Gastrointestinal$(GI)$Digestion$of$Dairy$Ingredients$#In#vitro#simulated!GI!digestion!was!performed!in!duplicate!according!to!the!method!of!Garrett!et!al.!(1999).!Sodium!caseinate,!whey!protein!isolate!(WPI),!skim!milk!powder!and!milk!protein! concentrate! (MPC)! solutions! (3%!protein!w/v! in!deionized!distilled!(dd)!H2O),!to!which!0.02%!sodium!azide!was!added!to!prevent!microbial!growth,!were!adjusted!to!pH!2.0!using!6!M!HCl!and!submerged!in!a!water!bath!with!shaker!(Blue!M!Electric!Company;!Blue!Island,! IL,!United!States)!to!bring!up!the!temperature!to!37°C.!Pepsin!(enzyme:substrate!ratio!on!a!w/w!protein!basis!(E/S)!=!4%)!was!added!and!the!solutions! incubated! at! 37°C! with! constant! agitation! for! 1! hour.! The! pH! was! then!adjusted!to!5.3!with!0.9!M!NaHCO3!and!pancreatin!(E/S!=!4%)!was!added.!The!pH!was!further! increased! to! 7.5! with! 1! M! NaOH! and! the! solutions! incubated! at! 37°C! with!constant! agitation! for! 2! hours.! The! digested! dairy! samples! were! then! submerged! in!boiling! water! for! 10! min! to! inactivate! the! enzyme! and! centrifuged! using! a! DuPont!Sorvall! Centrifuge! RD! 5B! (Mandel! Scientific! Co.! Ltd.;! Guelph,! ON,! Canada)! at! room!temperature!for!10!min!at!12,100!g.!The!supernatants!were!collected!and!freeze.dried.!!!!!! 107!5.3.3$Enzymatic$Hydrolysis$of$Sodium$Caseinate$and$Whey$Protein$Isolate$$$#5.3.3.1%Enzyme%Screening%!The! enzymes! investigated! in! this! study! for! production! of! milk! protein! hydrolysates!were!Alcalase,!Protease!A!“Amano”!2,!bromelain,!Corolase!PP,!Flavourzyme,!Protease!N!“Amano”!K,!Protamex,!Protin!SD.NY10,!thermolysin,!Umamizyme!K!and!Validase!BNP.L.!The! activity! of! each! enzyme! was! determined! by! a! colorimetric! assay! using! Folin.Ciocalteu! reagent! as! described! by! Sigma! (Sigma! Quality! Control! Test,! Procedure! for!Enzymatic!Assay!of!Protease,!Casein!as!a!Substrate.!SSCASE01.001,!Revised!04/02/99).!In!this!assay,!one!unit!of!activity!is!represented!by!the!hydrolysis!of!the!substrate!casein!to!produce!color!equivalent!to!1.0!µmole!of!tyrosine!per!minute!at!pH!7.5!and!37°C.!The!concentration! of! each! enzyme! used! for! hydrolysis! in! the! screening! experiment! was!based! on! the! amount! of! enzyme! yielding! 3.6! units! of! activity! in! 30!mL! of! 3%! (w/v)!protein!solution.!This!level!of!activity!corresponds!to!an!enzyme:substrate!ratio!of!3%!(w/w,!protein!basis)!for!the!Flavourzyme!product,!which!falls!within!the!lower!end!of!the! range! recommended! by! the! manufacturer! for! this! enzyme! to! achieve! extensive!hydrolysis!of!proteins.!!Sodium! caseinate! and!WPI! solutions! (30!mL! of! 3!%!w/v! in! ddH2O! containing! 0.02%!sodium!azide,!at!the!unadjusted!pHs!of!6.7!and!6.5,!respectively)!were!pre.incubated!in!a! water! bath! with! shaker! to! the! designated! temperature! for! the! hydrolysis! (55°C,!except!70°C!for!thermolysin)!before!the!addition!of!the!proteases!(3.6!units!of!activity).!The!mixtures!were!then!incubated!with!constant!agitation!for!60!min!and!the!resulting!hydrolysates!assessed!for!their!DPP.IV!inhibitory!activity.!!!!'! %! 108!5.3.3.2%Hydrolysis%Using%Selected%Enzymes%%#!The!peptic! digestion!of!WPI! (described! in! section!5.3.2)! and! the!protease! treatments!(described!in!section!5.3.3.1)!yielding!sodium!caseinate!and!WPI!hydrolysates!with!the!highest! DPP.IV! inhibitory! activity! were! selected! for! further! investigation.! ! For! these!experiments,! the!hydrolysis! conditions!and!enzyme:substrate! ratios!were!modified! in!order!to!optimize!the!conditions!for!enzymatic!activity!and!thus!increase!the!extent!of!the!hydrolysis.!In!particular,!the!pH!and!temperature!conditions!were!selected!based!on!the! optimal! range! for! each! enzyme! as! reported! by! the! manufacturer,! and! the!concentration! of! enzyme! was! chosen! based! on! the! outcome! of! preliminary! trials! in!which! the! extent! of! hydrolysis! of! the! hydrolysates! produced! using! various!concentrations!of!these!enzymes!was!assessed.!!!Sodium! caseinate! hydrolysates!were! prepared! using! thermolysin! (E/S! =! 3%;! pH! 8.0;!70°C;!20!min)!and!bromelain!(E/S!=!5%;!pH!6.7;!45°C;!60!min),!while!WPI!hydrolysates!were!prepared!using!thermolysin!(E/S!=!3%;!pH!8.0;!70°C;!20!min),!Umamizyme!K!(E/S!=!3%;!pH!6.5;!45°C;!60!min)!and!pepsin!(E/S!=!4%;!pH!2.0;!37°C;!60!min).!!!!!!!!5.3.4$Ultrafiltration$of$Sodium$Caseinate$and$Whey$Protein$Isolate$Hydrolysates$$#The! sodium! caseinate! and! WPI! hydrolysates! obtained! by! the! protease! treatments!described!in!section!5.3.3.2!were!centrifuged!at!12,100!g!for!10!min.!The!supernatants!were! collected! and! sequentially! ultrafiltered! using! an! Ultracel! Amicon! ultrafiltration!unit!model!8400!(Millipore!Corporation;!Bedford,!MA,!United!States)!with!membranes!of!molecular!weight!cutoffs!of!10!kDa,!3!kDa!and!1!kDa.!A!pressure!of!50!psi!nitrogen!gas! at! room! temperature! was! applied.! The! ultrafiltered! fractions! were! freeze.dried!prior!to!the!determination!of!DPP.IV!inhibitory!activity.!!! 109!5.3.5$Extent$of$Hydrolysis$##The! extent! of! hydrolysis!was!monitored! by! determining! the! content! of! free! α.amino!groups!measured!in!triplicate!by!the!TNBS!reaction!according!to!the!method!described!by!Lo!&!Li.Chan! (2005),!with! the! following!modifications:! the!digested!aliquots!were!centrifuged! 10! min! at! 10,000! rpm! using! a! VWR! Galaxy! 16! microcentrifuge! (VWR!Scientific!Products;!Bristol,!CT,!United!States)!and!the!supernatants!diluted!50.times!in!ddH2O!prior!to!adding!the!sodium!tetraborate!buffer!and!the!TNBS!reagent.!The!content!of! α.amino! groups!was! expressed! in!mmol! per! g! protein! by! reference! to! a! L.leucine!standard!curve.!Results!were!reported!as!means!±!standard!deviation!(SD).!!5.3.6$DPP4IV$Inhibitory$Activity$Assay$#DPP.IV! inhibitory! activity! was! measured! in! triplicate! according! to! the! method!described!by!Van!Amerongen!et!al.!(2009)!with!some!modifications.!Briefly,!hydrolyzed!samples!were!diluted!in!100!mM!Tris.HCl!buffer!pH!8.0!to!the!desired!concentrations.!In!a!96.well!microplate,!25!µL!of!diluted!sample!was!pre.incubated!with!25!µL!of!Gly.Pro.p.nitroanilide! (1.59!mM)! at! 37°C! for! 10!min,! after! which! 50! µL! of! DPP.IV! (0.01!units/mL)!was!added!and!the!mixture!incubated!at!37°C!for!60!min.!The!reaction!was!stopped!by!addition!of!100!µL!of!1!M!sodium!acetate!buffer!pH!4.0!and!the!absorbance!of!the!samples!measured!at!405!nm!using!a!Labsystems!iEMS!Reader!MF!(Labsystems!Oy;!Helsinki,!Finland).!The!%!DPP.IV!inhibition!was!defined!as!the!percentage!of!DPP.IV! activity! inhibited! by! a! given! concentration! of! hydrolysate! (protein! basis).! Results!were! reported! as! means! ±! SD.! The! concentration! of! the! peptic! WPI! hydrolysate!required!to!cause!a!50%!inhibition!of! the!DPP.IV!activity!(IC50!value)!was!determined!by! measuring! the! %! DPP.IV! inhibition! obtained! using! four! different! hydrolysate!concentrations!ranging! from!0.0625–0.625!mg/mL!(final!assay!concentration,!protein!basis).!The!cubic!regression!equation!generated!by!fitting!the!data!from!the!plot!of!%!DPP.IV!inhibition!versus!the!hydrolysate!concentration!was!then!used!to!calculate!the!IC50.!The!tri.peptide!Ile.Pro.Ile!(diprotin!A)!was!used!as!a!reference!inhibitor.!!! 110!5.3.7$Statistical$Analysis$$$$!Analysis!of!variance!using!general!linear!model!and!pairwise!comparison!with!Tukey’s!method! were! performed! using! Minitab! statistical! software! (version! 16,! Minitab! Inc.,!State!College,!PA,!United!States),! in!order! to! compare! the! extent!of!hydrolysis! and!%!DPP.IV! inhibitory! activity! of! dairy! protein! hydrolysates.! Assays! were! performed! in!triplicate!and!significant!difference!was!established!at!P#<!0.05.!!5.4!RESULTS!AND!DISCUSSION!!5.4.1$ Extent$ of$ Hydrolysis$ and$ DPP4IV$ Inhibitory$ Activity$ of$ Dairy$ Ingredients$ During$ In$Vitro$Digestion$with$Pepsin$and$Pancreatin$#The!progress!of!the!in#vitro!pepsin.pancreatin!digestion!of!sodium!caseinate,!WPI,!skim!milk! powder! and! MPC! was! monitored! by! taking! aliquots! of! each! dairy! sample! at!different!time!intervals.!As!shown!in!Figure'5.1,!the!extent!of!hydrolysis!remained!low!during!the!digestion!with!pepsin!and!increased!most!rapidly!following!the!addition!of!pancreatin.! During! the! peptic! phase! of! the! in# vitro! digestion,! the! enzymatic! activity!tended! to!slow!down!after! the! first!30!min!of!hydrolysis!as!shown!by! the! leveling!off!effect!in!the!content!of!α.amino!groups!generated!from!30!to!60!min.!On!the!other!hand,!the!extent!of!hydrolysis!continued!to! increase!throughout! the!pancreatic!phase!of! the!digestion,!although!not!as!drastically!during!the!last!90!min.!The!complete!hydrolysis!of!casein!and!whey!proteins! to! their!constituent!amino!acids!has!been!reported! to!yield!8.2!and!8.8!mmol!of!amino!acids!per!g!protein,!respectively!(Alder.Nissen,!1986).!Thus,!the! dairy! samples! investigated! were! not! completely! hydrolyzed! during! the! in# vitro!digestion,!and!would!be!expected!to!contain!peptides!as!well!as!free!amino!acids.!!! !! 111!!!!!!!!!!!! !'Figure' 5.1' Extent! of! hydrolysis! (mmol! L.leucine! equiv/g! protein)! of! dairy! protein!hydrolysates!as!a!function!of!time!course!of!digestion.!Sodium!caseinate,!whey!protein!isolate! (WPI),! skim! milk! powder! (SMP),! and! milk! protein! concentrate! (MPC)! (3%)!solutions!were! hydrolysed!with! pepsin! (E/S! =! 4%,! 0–60!min)! and! pancreatin! (E/S! =!4%,!60–180!min).!Values!shown!are!means!and!standard!deviation!(error!bars)!of!three!determinations.''!!'!Among! the! four! dairy! ingredients! investigated,!WPI! consistently! showed! the! highest!extent! of! hydrolysis.! This! finding! is! consistent! with! in# vivo# studies! showing! greater!digestibility! of! the! soluble! components! of!milk! proteins.! By! remaining! soluble! under!gastric!acidic!pH,!whey!proteins!pass!rapidly!through!the!stomach.!On!the!other!hand,!casein!tends!to!clot!or!precipitate!at!low!pH,!which!delays!its!passage!through!the!gut!(Lacroix!et!al.,!2006).!This!phenomenon!was!observed!during!the!peptic!phase!of!the!in#vitro! digestion! of! sodium! caseinate! and! skim!milk! powder.! Conversely,! at! pH! values!closer!to!neutrality,!the!solubility!of!casein!is!increased!and!its!peptide!bonds!are!more!accessible!to!proteolytic!cleavage.!As!a!result,!significantly!higher!extents!of!hydrolysis!were! observed! following! the! addition! of! pancreatin! (Figure' 5.1).! The! extent! of!hydrolysis!of!sodium!caseinate,!however,!remained!lower!than!for!WPI!upon!prolonged!pancreatic!digestion.!This!might!be!attributable!to!the!high!content!of!proline!residues!-0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 0 30 60 90 180 L-leucine equivalents  (mmol per g protein) Digestion duration (min) Sodium caseinate WPI SMP MPC Pepsin Pancreatin ! 112!in!casein.!Because!of! the!cyclic!structure!of! the!amino!acid!proline,! the!peptide!bonds!involving!proline!residues!are!often!resistant!to!proteolytic!action!(Walter!et!al.,!1980).!Thus,! it! is! possible! that! the! enzymes! present! in! the! commercial! preparation! of!pancreatin!were!unable!to!efficiently!hydrolyze!the!sodium!caseinate.!!!Similarly!to!the!extent!of!hydrolysis,!the!inhibitory!activity!against!the!enzyme!DPP.IV!increased!during! the! first!30!min!of!digestion!with!pepsin! (inhibitory!activity!at! time!zero!was!negligible,!data!not!shown),!but!plateaued!during!the!remaining!30!min!of!the!peptic!phase!of! the!digestion!(Figure'5.2).!For!all! four!dairy! ingredients! investigated,!the! DPP.IV! inhibitory! activity! of! the! digests! collected! after! 30!min! of! digestion!with!pepsin!were!not!significantly!different!from!the!inhibitory!activity!after!60!min.!Despite!the!generation!of!additional!free!amino!groups!in!all!samples!following!the!addition!of!pancreatin,! only! sodium! caseinate! showed! a! significant! increase! in!DPP.IV! inhibitory!activity! between!60! and!90!min.!On! the! other! hand,! a! significant! decrease! in!DPP.IV!inhibitory!activity!was!observed!for!WPI!during!that!period,!suggesting!that!the!DPP.IV!inhibitory! peptides! released! during! the! peptic! digestion! of! whey! proteins! might! be!broken! down! by! pancreatin.! Further! digestion! with! pancreatin! did! not! lead! to! any!significant! changes! in! DPP.IV! inhibitory! activity! for! the! dairy! ingredients! with! the!exception! of! WPI,! which! interestingly! showed! a! significant! increase! in! inhibitory!activity!during!the!last!hour!and!a!half!of!the!pancreatic!digestion.!This!improvement!of!inhibitory! activity! suggests! that,! over! time,! pancreatin! is! able! to! release!new!peptide!inhibitors!from!whey!proteins,!thus!compensating!for!the!loss!of!inhibitory!activity!that!may! have! resulted! from! the! cleavage! of! the! inhibitory! peptide! sequences! released!during!the!peptic!digestion.!!!!! 113!!!!!!!!!!!! !Figure'5.2'DPP.IV!inhibitory!activity!of!dairy!protein!hydrolysates!as!a!function!of!time!course!of!digestion.! Sodium!caseinate,!whey!protein! isolate! (WPI),! skim!milk!powder!(SMP),! and! milk! protein! concentrate! (MPC)! (3%)! solutions! were! hydrolysed! with!pepsin!(E/S!=!4%,!0–60!min)!and!pancreatin!(E/S!=!4%,!60–180!min).!The!%!inhibition!of! DPP.IV! activity! was! measured! using! 0.500! mg/mL! of! hydrolysate! (final! assay!concentration,! protein! basis).! Values! shown! are! the! means! and! standard! deviations!(error!bars)!of!three!determinations.'!!!Among!all!four!dairy!ingredients!and!aliquots!collected!during!the!course!of!the!in#vitro#digestion,#the!WPI!digest!obtained!after!60!min!of!hydrolysis!with!pepsin!showed!the!highest!DPP.IV! inhibitory! activity!with! an! IC50!value! of! 0.075!mg/mL.! This! inhibitory!activity!is!about!50!times!lower!than!that!measured!for!the!reference!inhibitor!diprotin!A!(IC50!=!1.6!µg/mL;!data!not!shown).!Since!dairy!protein!hydrolysates!are!composed!of!a!mixture!of!peptides,!only!some!of!which!would!display!DPP.IV!inhibitory!activity,!it!is!not! surprising! that! they! showed! lower! potency! than! the! values! reported! for! isolated!peptides!or!synthetic!drugs.!!!Pepsin! is! known! to! present! a! broad! specificity! and! to! preferentially! cleave! at! the!carboxyl! end! of! hydrophobic! and! aromatic! amino! acid! residues! such! as! leucine,!phenylalanine,! tryptophan! and! tyrosine! (Nelson! &! Cox,! 2008).! This! may! explain! the!strong!increase!in!inhibitory!activity!of!the!dairy!protein!samples!following!the!peptic!0 10 20 30 40 50 60 70 80 30 60 90 180 % DPP-IV inhibition Digestion duration (min) Sodium caseinate WPI SMP MPC Pepsin Pancreatin ! 114!digestion! as! most! peptides! reported! to! present! a! DPP.IV! inhibitory! activity! contain!proline! and/or! hydrophobic! amino! acids! within! their! sequence! (Bella! et! al.,! 1982;!Cohen!et!al.,!2004;!Yan!et!al.,!1992).!!5.4.2$Assessment$of$the$DPP4IV$Inhibitory$Activity$of$Sodium$Caseinate$and$Whey$Protein$Isolate$Hydrolysates$Obtained$Using$Various$Proteases$$!!Further! investigation! was! carried! out! to! evaluate! the! potential! of! casein! and! whey!proteins! to! yield,! upon! enzymatic! treatment! with! various! commercially! available!proteases,! products! with! potent! DPP.IV! inhibitory! activity! that! could! be! used! as!functional! food! ingredients! for! the! management! of! T2D.! ! In! order! to! compare! the!efficacy! of! the! selected! enzymes! to! release! peptides! with! DPP.IV! inhibitory! activity,!hydrolysis!was!carried!out!for!60!min!using!each!enzyme!at!a!concentration!providing!3.6!units!of!activity!in!30!mL!of!3%!(w/v)!sodium!caseinate!and!WPI!solutions!at!their!unadjusted!pH!values!(~!6.7!and!6.5,!respectively).!!!As! shown! in! Figure' 5.3,! the! DPP.IV! inhibitory! activity! of! the! hydrolysates! obtained!after! 60! min! of! hydrolysis! varied! depending! on! the! protease! and! the! type! of! dairy!protein!used.!The!proteases!thermolysin!and!Umamizyme!K!were!found!to!generate!the!WPI!hydrolysates!with! the!highest!DPP.IV! inhibitory!activity! (51!and!47%! inhibition,!respectively)!whereas! the!WPI! hydrolysate! obtained! by! Protease! N! “Amano”! K,!with!14%! inhibition,! showed! the! lowest!DPP.IV! inhibitory! activity.!On! the!other!hand,! the!sodium!caseinate!hydrolysates!presenting! the!highest!DPP.IV! inhibitory!activity!were!obtained!by! the! enzymatic! treatments!with! thermolysin! and!bromelain! (50! and!47%!inhibition,!respectively).!Conversely,!the!sodium!caseinate!hydrolysates!with!the!lowest!DPP.IV! inhibitory! activity! (33%)! were! produced! with! Protease! A! “Amano”! 2! and!Protease!N!“Amano”!K.!!! !! 115!''' !Figure' 5.3' DPP.IV! inhibitory! activity! of! sodium! caseinate! and! whey! protein! isolate!(WPI)! hydrolysates! generated! using! different! enzymes.! Sodium! caseinate! and! whey!protein! isolate! (WPI)! (3%)! solutions! were! hydrolysed! with! 3.6! units! of! Alcalase,!Protease! A! “Amano”! 2,! bromelain,! Corolase! PP,! Flavourzyme,! Protease!N! “Amano”! K,!Protamex,! Protin! SD.NY10,! thermolysin,! Umamizyme! K! or! Validase! BNP.L.! The! %!inhibition! of! DPP.IV! activity!was!measured! using! 0.487!mg/mL! of! hydrolysate! (final!assay!concentration,!protein!basis).! !“ND”!refers!to!“not!determined”!(the!formation!of!clots!during!the!hydrolysis!of!sodium!caseinate!with!Alcalase!prevented!determination!of!DPP.IV!inhibitory!activity!of!the!resulting!hydrolysate).!Values!shown!are!the!means!and!standard!deviations!(error!bars)!of!three!determinations.!'!!For! most! of! the! enzymatic! treatments! in! this! study,! sodium! caseinate! hydrolysates!displayed!significantly!higher!DPP.IV!inhibitory!activity!than!WPI!hydrolysates!(Figure'5.3).!The!ability!of!casein!to!be!a!precursor!of!peptides!with!DPP.IV!inhibitory!activity!was!also!suggested! from!the!results!of! the! in#silico!analysis!presented! in! the!previous!chapter!(Lacroix!&!Li.Chan,!2012;!Chapter!4).!!The!authors!found!that!β.casein!contains!within! its! sequence! 52! fragments! reported! in! the! literature! to! present! DPP.IV!inhibitory!activity.!Moreover,!among!all!major!dairy!proteins!investigated,!β.casein!was!shown! to! have! the! highest! occurrence! frequency! value,!which! is! an! indication! of! the!potential! of! a! protein! to! serve! as! a! precursor! of! bioactive! peptides! based! on! the!0 10 20 30 40 50 60 Alcalase A"Amano"2 Bromelain Corolase PP Flavourzyme N"Amano"K Protamex Protin SD-NY10 Thermolysin Umamizyme K Validase BNP-L % DPP-IV inhibition Protease WPI Sodium caseinate ND ! 116!occurrence!of! fragments!matching!peptides!with!DPP.IV! inhibitory!activity!relative! to!the!length!of!the!protein!chain.!Similar!findings!were!also!reported!in!a!study!by!Dziuba!et! al.! (2009),! which! investigated! the! theoretical! release! by! proteolytic! cleavage! of!bioactive! peptides! from! milk! proteins.! Their! results! suggested! that! bromelain! and!thermolysin! could!have! the!ability! to! release!peptides!with!DPP.IV! inhibitory!activity!from!milk!proteins.!In!fact,!the!enzyme!thermolysin!has!already!been!successfully!used!to! produce! bioactive! peptides,! including! angiotensin! I! converting! enzyme! (ACE)!inhibitory!peptides,!from!various!milk!proteins!(Hernández.Ledesma!et!al.,!2002;!Otte!et!al.,!2007).!!!5.4.3$ Fractionation$ of$ Sodium$ Caseinate$ and$WPI$ Hydrolysates$ Showing$ Potent$ DPP4IV$Inhibitory$Activity$$!Fractionation! by! molecular! size! of! sodium! caseinate! and! WPI! hydrolysates! was!performed!in!order!to!evaluate!the!feasibility!of!generating!hydrolysate!fractions!with!more! potent! DPP.IV! inhibitory! activity.! Having! yielded! hydrolyates! with! the! highest!DPP.IV! inhibitory! activity,! the! peptic! digestion! of! WPI! as! well! as! the! enzymatic!treatments! of!WPI! with! thermolysin! &! Umamizyme! K! and! of! sodium! caseinate! with!bromelain!&!thermolysin!were!selected!for!this!investigation.!!As!shown!in!Table'5.1,!smaller!peptides!were!not!always!the!major!contributors!to!the!DPP.IV!inhibition!effect.!In!fact,!only!the!sodium!caseinate!hydrolysate!with!bromelain!and!the!WPI!hydrolysate!with!thermolysin!showed!<!1!kDa!fractions!with!significantly!greater! DPP.IV! inhibitory! activity! compared! to! the! unfractionated! hydrolysates.!Conversely,! the! inhibitory! activity! of! the! <! 1! kDa! fraction! of! the! WPI! hydrolysate!produced!by! pepsin!was! reduced! compared! to! the! higher!molecular!weight! fractions!and! the! unfractionated! hydrolysate.! As! for! the! WPI! hydrolysate! obtained! with!Umamizyme!K,!only!the!1.3!kDa!fraction!was!found!to!present!a!significantly!improved!inhibitory!activity!compared!to!the!unfractionated!hydrolysate.!On!the!other!hand,!the!! 117!ultrafiltration!of!sodium!caseinate!hydrolysate!with! thermolysin!did!not!generate!any!fraction!with!significantly!higher!DPP.IV!inhibitory!activity.!!!Table' 5.1' DPP.IV! inhibitory! activity! of! sodium! caseinate! and! whey! protein! isolate!hydrolysates! and! their! fractions! obtained! by! ultrafiltration.! Sodium! caseinate! (3%)!solutions! were! hydrolysed! with! bromelain! (E/S! =! 5%;! pH! 6.7;! 45°C;! 60! min)! and!thermolysin! (E/S! =! 3%;! pH! 8.0;! 70°C;! 20!min).!Whey! protein! isolate! (3%)! solutions!were!hydrolysed!with!pepsin!(E/S!=!4%;!pH!2.0;!37°C;!60!min),!thermolysin!(E/S!=!3%;!pH!8.0;!70°C;!20!min)!and!Umamizyme!K!(E/S!=!3%;!pH!6.5;!45°C;!60!min).!!Fraction DPP.IV!inhibition!(%)!Sodium!caseinate! Whey!protein!isolate!Bromelaina! Thermolysina! Pepsinb! Thermolysina! Umamizyme!Ka!Hydrolysate!before!ultrafiltration! 61! 53! 78! 63! 61!Ultrafiltration!fraction! ! ! ! ! !>!10!kDa! 49! 50! 78! 60! 58!3.10!kDa! 60! 50! 82! 63! 63!1.3!kDa! 70! 52! 83! 62! 68!<!1!kDa! 74! 58! 63! 73! 64!a % DPP-IV inhibition was measured at a final concentration of 0.475 mg/mL (protein basis) b % DPP-IV inhibition was measured at a final concentration of 0.375 mg/mL (protein basis) !!Since!most!peptides!reported!in!the!literature!as!presenting!DPP.IV!inhibitory!activity!are! di.! and! tri.peptides! (Bella! et! al.! 1982;! Yan! et! al.,! 1992;! Cohen! et! al.,! 2004),! it! is!surprising!that!some!of!the! lower!molecular!weight!fractions!of!sodium!caseinate!and!WPI!hydrolysates!did!not!show!improved!DPP.IV!inhibitory!activity.!DPP.IV!is!known!to! exert! its! catalytic! activity! on! peptides! that! vary! in! length! and! amino! acid!composition.!In#vitro#kinetic!studies!have!revealed!that!the!substrate!specificity!of!DPP.IV! is!not!only!determined!by! the! amino!acid! sequence! surrounding! the! scissile!bond,!but! also! by! the! substrate’s! specific! structural! characteristics! (Lambeir! et! al.,! 2003).!Moreover,! the!peptide! length!of! the!substrate! (up! to!about!100!amino!acids)!was!not!found!to!be!linked!to!the!kinetic!constant!(Lambeir!et!al.,!2003).!It!is!possible!that!some!of! the! higher! molecular! weight! peptides! released! during! enzymatic! hydrolysis! of!sodium!caseinate!and!WPI!have!structural!features!recognized!by!DPP.IV!and!thus!act!as!substrates! for! the!enzyme.! If! this! is! the!case,! the!observed! inhibition!by!the!higher!molecular!weight!fractions!of!sodium!caseinate!and!WPI!hydrolysates!may!be!a!kinetic!artifact! resulting! from! the! substrate.like! structure! of! the! peptides! present! in! these!! 118!fractions.! In! fact,! two!of! the!most!potent!peptides!with!DPP.IV! inhibitory!activity! that!have! been! reported! in! the! literature,! the! tri.peptides! diprotin! A! (Ile.Pro.Ile)! and!diprotin! B! (Val.Pro.Leu),! have! been! shown! to! actually! be! substrates! for! the! enzyme!with! low! turnover! rates! (Rahfeld! et! al.,! 1991).! The! apparent! competitive! inhibition!observed!by!these!peptides!is!believed!to!be!attributable!to!their!substrate.like!feature!as!both!compounds!contain!a!proline!residue!in!the!P1!position!(Rahfeld!et!al.,!1991).!!''! !! 119!5.5!CONCLUSION!'The!results!from!this!study!showed!that!peptides!with!DPP.IV!inhibitory!activity!can!be!generated!by!in#vitro!pepsin.pancreatin!digestion!and!by!enzymatic!treatments!of!dairy!proteins.! Products! obtained! by! hydrolysis! of! sodium! caseinate! using! various!commercially! available! proteases! were! found! to! generally! display! greater! DPP.IV!inhibitory!activity! than! those!obtained!using!WPI.!With! the!exception!of! the!WPI!and!sodium!caseinate!hydrolysates!obtained!respectively!with!pepsin!and!thermolysin,!the!fractionation! by! size! of! WPI! and! sodium! caseinate! hydrolysates! led! to! some!improvement! of! their! inhibitory! activity.! These! findings! provide! the! basis! to! support!the!potential!of!dairy!proteins!to!serve!as!natural!precursors!of!peptides!with!DPP.IV!inhibitory!activity.!Although!these!DPP.IV!inhibitory!peptides!are!not!as!potent!as!the!synthetic!drugs,!and!thus!are!not!meant!to!replace!them,!they!could!be!used!as!a!natural!complementary! approach! in! the! management! of! T2D.! Further# research! is,! however,!needed!to!confirm!the#in#vivo#release!of!peptides!with!DPP.IV!inhibitory!activity!during!digestion! of! dairy! proteins.! Additional! investigations! are! also! needed! to! isolate! and!sequence! the! potent! DPP.IV! inhibitory! peptides! from! dairy! protein! hydrolysates,! to!assess!their!modes!of!inhibition!as!well!as!their!efficacy!in#vivo.!!!!! !! 120!CHAPTER! 6:! ! INHIBITION! OF! DIPEPTIDYL5PEPTIDASE! IV! AND! α5GLUCOSIDASE!ACTIVITY!BY!PEPSIN5TREATED!WHEY!PROTEINS7!!6.1!SUMMARY!!Inhibitors! of! the! enzymes! dipeptidyl! peptidase! (DPP).IV! and! α.glucosidase! are! two!classes!of!pharmacotherapeutical!agents!used! for! the! treatment!of! type!2!diabetes.! In!the! present! study,! whey! protein! isolate! (WPI),!α.lactalbumin,! β.lactoglobulin,! serum!albumin! and! lactoferrin! hydrolysates! obtained! by! peptic! digestion!were! investigated!for! their!potential! to!serve!as!natural! sources!of!DPP.IV!and!α.glucosidase! inhibitors.!While! inhibition! of!DPP.IV! activity!was! observed! in! all! pepsin.treated!whey!proteins!studied,!the!α.lactalbumin!hydrolysate!showed!the!greatest!potency!with!an!IC50!value!of!0.036!mg/mL.!Conversely,!only!WPI,!β.lactoglobulin!and!α.lactalbumin!hydrolysates!displayed! some! inhibitory! activity! against! α.glucosidase.! This! study! suggests! that!peptides! generated! from!whey!proteins!may!have!dual! beneficial! effects! on! glycemia!regulation!and!could!be!used!as!functional!food!ingredients!for!the!management!of!type!2!diabetes.!!6.2!INTRODUCTION!!Type! 2! diabetes! represents! one! of! the!most! challenging! health! problems! of! the! 21st!century.!Both!the!prevalence!and!incidence!of!this!chronic!and!multifactorial!disorder,!which!is!characterized!by!defects!in!insulin!secretion!and/or!action!(Schmidt!&!Hickey,!2009),! are! increasing! at! an! alarming! rate! worldwide! (International! Diabetes!Federation,! 2013).! When! not! properly! treated,! type! 2! diabetes! can! lead! to! serious!complications,!including!cardiovascular!disorders,!blindness!and!renal!failure!(Inzucchi!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!7!A! version! of! this! chapter! has! been! published.! Lacroix,! I.M.E,! Li.Chan,! E.C.Y.! Inhibition! of!dipeptidyl!peptidase!(DPP).IV!and!α.glucosidase!activities!by!pepsin.treated!whey!proteins.! J.#Agric.#Food#Chem.#2013,!61,!7500–7506.!! 121!et! al.,! 2012).! Therefore,! effective! diabetes! management! strategies! are! of! primary!importance.!!!Twelve!classes!of!glucose.lowering!agents!are!currently!available!for!the!management!of! diabetes.! Of! these,! dipeptidyl.peptidase! IV! (DPP.IV;! EC! 3.4.14.5)! inhibitors! are!among! the! newest! medications! that! have! been! introduced! to! the! type! 2! diabetes!pharmacopeia!(Bennett!et!al.,!2011;!Tahrani!et!al.,!2011).!!!Recent! studies! in! the! area! of! bioactive! peptides! have! suggested! that! dietary! proteins!could! be! natural! precursors! of! DPP.IV! inhibitors.! An! in# silico# analysis! revealed! that!peptide! sequences! matching! those! reported! in! the! literature! to! present! inhibitory!activity!against!DPP.IV!are!contained!within!the!primary!sequences!of!various!proteins!from!plant! and! animal! sources,! including! dairy! and! fish! proteins! (Lacroix!&! Li.Chan,!2012a;! Chapter! 4).!Moreover,! hydrolysates! and! peptides! from! a! number! of! proteins,!including! those! from!egg!(Van!Amerongen,!2009),! fish!(Huang!et!al.,!2012;!Li.Chan!et!al.,! 2012),! amaranth! (Velarde.Salcedo! et! al.,! 2013),! rice! bran! (Hatanaka! et! al.,! 2012),!corn!(Mochida!et!al.,!2010)!and!milk!(Tulipano!et!al.,!2011;!Uchida!et!al.,!2011;!Tulipano!et!al.,!2012;!Uenishi!et!al.,!2012;!Lacroix!&!Li.Chan,!2012b;!Nongonierma!&!FitzGerald,!2013b),! have! been! found! to! be! able! to! inhibit! the! activity! of! DPP.IV.! Among! those!proteins,! dairy! proteins! seem! to! be! a! particularly! interesting! source! of! DPP.IV!inhibitors,! peptides! derived! from! the!whey! and! casein! fractions! of!milk! having! been!shown!to!display!DPP.IV!inhibitory!activity!in#vitro#(Tulipano!et!al.!2011;!Uchida!et!al.,!2011;! Tulipano! et! al.,! 2012;! Uenishi! et! al.,! 2012;! Lacroix! &! Li.Chan,! 2012b;!Nongonierma!&!FitzGerald,!2013b)!and!to!reduce!blood!glucose!levels!in#vivo#(Uchida!et!al.,! 2011;! Uenishi! et! al.,! 2012).! Although! β.casein! (Uenishi! et! al.,! 2012)! and! β.lactoglobulin!(Tulipano!et!al.,!2011;!Uchida!et!al.,!2011;!Tulipano!et!al.,!2012)!have!been!investigated!as!precursors!of!peptides!with!DPP.IV!inhibitory!activity,!little!is!known!on!the!potential!of!other!individual!milk!proteins!to!serve!as!sources!of!DPP.IV!inhibitors.!!!!! 122!Another! therapeutic! approach! in! the!management! of! type! 2! diabetes! consists! in! the!suppression! of! the! digestion/absorption! of! glucose! in! the! gut! by! the! inhibition! of!carbohydrate.hydrolyzing!enzymes!such!as!α.glucosidase!(Lebovitz,!1997).!In!the!past!decade,! numerous! studies! have! been! carried! out! to! identify! dietary! products! or!constituents!that!may!serve!as!natural!sources!of!inhibitors!of!these!enzymes.!A!variety!of! fruits! and! vegetables,! including! blueberry! (McDougall! et! al.,! 2005),! strawberry!(McDougall!et!al.,!2005;!da!Silva!Pinto!et!al.,!2008)!broccoli!sprouts!(McCue!et!al.,!2005)!and!green!pepper!(McCue!et!al.,!2005),!have!been!shown!to!display!α.amylase!and/or!α.glucosidase! inhibitory! activity.! Polyphenolic! compounds,! such! as! flavonoids! and!tannins,! in! these! foods! have! been! suggested! to! be! responsible! for! the! observed!inhibitory! activity! (Kim! et! al.,! 2000;! McDougall! et! al.,! 2005;! Tadera! et! al.,! 2006;!Gonçalves!et!al.,!2011;!Etxeberria!et!al.,!2012).!In!addition!to!food!commodities!of!plant!origin,! peptides!derived! from!sardine!muscle! (Matsui! et! al.,! 1996)! and!more! recently!egg!white!protein!(Yu!et!al.,!2011)!have!also!been!reported!to!suppress!the!activity!of!α.glucosidase.! To! the! author’s! knowledge,! the!α.glucosidase! inhibitory! activity! of! dairy!protein!hydrolysates!has!not!been!reported.!!!!The! consumption! of! dairy! proteins,! particularly! whey! protein,! has! been! shown! in! a!number!of! intervention!studies!to!have!beneficial!effects!on!glucose!metabolism!(Graf!et! al.,! 2011;! Jakubowicz! &! Froy,! 2013).! The! addition! of! whey! protein! to! high!carbohydrate! containing!meals!has!been! found! to! lead! to! a! reduction! in!postprandial!blood!glucose! levels!and!an! increase! in!plasma! insulin!concentrations! in!both!healthy!and!type!2!diabetes!individuals!(Graf!et!al.,!2011;!Jakubowicz!&!Froy,!2013).!The!anti.diabetic! properties! of! whey! protein! have! been! suggested! to! be! attributable! to! its!content! of! bioactive! peptides! which,! following! their! release! during! gastrointestinal!digestion,! could! stimulate! the! secretion! of! gut! derived! hormones! and/or! inhibit!enzymes!involved!in!glycemia!homeostasis!(Graf!et!al.,!2011;!Jakubowicz!&!Froy,!2013).!Little! is! known,! however,! on! the! specific! whey! proteins! that! these! peptides! are!originated!from.!!! 123!In!a!previous!investigation!to!assess!whether!inhibition!of!the!DPP.IV!enzyme!may!be!one!of!the!potential!mechanisms!contributing!to!the!reported!benefits!of!whey!protein!on! regulating! glucose! metabolism,! it! was! found! that,! among! several! enzymatic!treatments!using!various!proteases,!the!peptic!digestion!of!whey!protein!isolate!(WPI)!generated! the! hydrolysate!with! the! greatest!DPP.IV! inhibitory! activity! (Lacroix!&! Li.Chan,! 2012b;! Chapter! 5).! The! specific! protein! constituents! in! the! peptic! WPI!hydrolysate! responsible! for! the! observed! inhibitory! activity! were,! however,! not!identified.! Also! unknown! is! the! ability! of! whey! protein! hydrolysates! to! inhibit! other!enzymes,! such! as!α.glucosidase,!which! are! involved! in! glycemia! regulation.!Thus,! the!objective! of! the! present! study! was! to! investigate! the! DPP.IV! and! α.glucosidase!inhibitory! activities! of! protein! hydrolysates! obtained! by! peptic! digestion! of!WPI! and!four!major!protein!constituents!of!whey,!namely!β.lactoglobulin,!α.lactalbumin,!bovine!serum!albumin!and!lactoferrin.!!!!'6.3!MATERIALS!AND!METHODS!'6.3.1$Materials$$!Pepsin! (EC! 3.4.23.1,! from! porcine! gastric! mucosa,! ≥! 2500! units! per! mg! protein),!dipeptidyl.peptidase! IV! (DPP.IV,! EC!3.4.14.5,! from!porcine!kidney,! ≥!10!units! per!mg!protein),!α.glucosidase!in!the!form!of!rat!intestine!acetone!powder,!acarbose,!maltose,!Ile.Pro.Ile! (diprotin! A),! glucose! assay! kit! (o.dianisidine! reagent! and! glucose!oxidase/peroxidase!reagent),!α.lactalbumin!from!bovine!milk!(≥!85%!protein),!albumin!from! bovine! serum! (≥! 96%! protein)! and! β.lactoglobulin! from! bovine! milk! (≥! 90%!protein)! were! purchased! from! Sigma.Aldrich! (Oakville,! ON,! Canada).!Trinitrobenzenesulfonic! acid! (TNBS,! in! methanolic! solution)! was! from! Thermo!Scientific!(Rockford,! IL,!United!States).!Whey!protein! isolate!(WPI!895,!Fonterra,!New!Zealand;! 92.4%! protein)! was! donated! by! Nealanders! International! Inc.! (Delta,! BC,!Canada)!and!lactoferrin!from!bovine!milk!was!supplied!by!DMV!International!(Fraser,!! 124!NY,!United!States;!80!%!protein).!Gly.Pro.p.nitroanilide!(H.Gly.Pro.pNA⋅HCl)!was!from!Bachem! Americas! (Torrance,! CA,! United! States).! Ultralow! Range! Molecular! Weight!Marker! (1.0–26.6! kDa)! was! from! Sigma.Aldrich! (Oakville,! ON,! Canada)! while!BioReagents! EZ.RunTM! Rec! Protein! Ladder! (10–200! kDa)! was! from! Fisher! Scientific!(Fair!Lawn,!NJ,!United!States).!Polyacrylamide!pre.cast!gels! (gradient!10–15!and!high!density),!PhastGel!SDS!buffer!strips,!PhastGel!Blue!R!tablets!and!PlusOne!silver!staining!kit!were!purchased!from!GE!Healthcare!(Uppsala,!Sweden).!!!6.3.2$Peptic$Hydrolysis$of$Whey$Proteins$!Whey! protein! isolate! (WPI),! α.lactalbumin,! β.lactoglobulin,! lactoferrin! and! bovine!serum!albumin!solutions!(3%!protein,!w/v,! in!deionized!distilled!(dd)!H2O!containing!0.02%! sodium! azide)!were! adjusted! to! pH! 2.0! using! 6! M!HCl! and! pre.incubated! in! a!water! bath!with! shaker! (Blue!M! Electric! Company;! Blue! Island,! IL,! United! States)! to!bring! up! the! temperature! to! 37°C.! The! pepsin! enzyme! was! added! (4%!enzyme:substrate! ratio! on! a!w/w! protein! basis)! and! the! solutions! incubated! at! 37°C!under! constant! agitation! for! 60! min.! The! hydrolysed! whey! proteins! were! then!centrifuged!using!a!DuPont!Sorvall!Centrifuge!RD!5B!(Mandel!Scientific!Co.!Ltd.;!Guelph,!ON,! Canada)! at! room! temperature! for! 10! min! at! 12,100! g.! The! supernatants! were!collected,!freeze.dried!and!stored!at!.18°C!until!further!analysis.!!6.3.3$Extent$of$Hydrolysis$'The!extent!of!hydrolysis!of!the!hydrolysed!whey!protein!samples!was!assessed!by!the!determination!of!the!content!of!free!α.amino!groups!measured!in!triplicate!by!the!TNBS!reaction! as! described! in! Chapter! 5,! section! 5.3.5! (Lacroix! &! Li.Chan,! 2012b).! The!α.amino! group! content! was! expressed! in! mmol! per! gram! protein! by! reference! to! a!L.leucine!standard!curve.!!!! 125!6.3.4$SDS4PAGE$$!The! un.hydrolysed! and! hydrolysed! whey! proteins! were! characterized! by! sodium!dodecyl!sulfate.polyacrylamide!gel!electrophoresis!(SDS.PAGE).!The!samples!(80!µL!of!0.1–1!mg/mL!solutions!in!10!mM!Tris.HCl,!1!mM!EDTA!pH!8.0)!were!combined!with!20!µL!10%!SDS,!2!µL!of!2.mercaptoethanol,!and!1!µL!of!bromophenol!blue!(1%,!w/v),!to!yield!final!concentrations!of!0.078!mg/mL!for!the!unhydrolysed!whey!protein!samples,!0.39! mg/mL! for! whey! protein! isolate! and! β.lactoglobulin! hydrolysates,! and! 0.78!mg/mL! for! lactoferrin,! α.lactalbumin! and! bovine! serum! albumin! hydrolysates.! ! The!mixtures!were!submerged!in!boiling!water!for!20!min,!centrifuged!at!14,000!rpm!for!5!min! using! a! VWR! Galaxy! 16! microcentrifuge! (VWR! Scientific! Products;! Bristol,! CT,!United! States)! and! 1! µL! of! the! supernatants! was! loaded! onto! the! gels.! The!electrophoresis! was! performed! on! a! PhastSystem! (Pharmacia! Biotech;! Uppsala,!Sweden)!using!PhastGel!gradient!10–15!and!PhastGel!high!density!pre.cast!gels!for!the!un.hydrolysed! proteins! and! hydrolysates,! respectively.! The! un.hydrolysed! whey!proteins!were!revealed!by!silver!staining!while!the!hydrolysate!samples!were!detected!using! Coomassie! blue! stain! followed! by! silver! stain.! ! Stainings! were! performed!according! to! the!manufacturer’s! instructions.!Molecular!weight!markers!ranging! from!10–200! kDa! and! 1.0–26.6! kDa! were! run! with! the! un.hydrolysed! and! hydrolysed!samples,!respectively.!!6.3.5$Determination$of$DPP4IV$Inhibitory$Activity$!The!effect!of!the!hydrolysed!whey!protein!samples!on!DPP.IV!activity!was!determined!using! a! modified! version! of! the! DPP.IV! assay! described! in! Chapter! 5,! section! 5.3.6!(Lacroix!&!Li.Chan,!2012b).!The!samples!were!reconstituted!in!100!mM!Tris.HCl!buffer!pH! 8.0! to! concentrations! ranging! from! 0.0125! mg/mL! to! 1.25! mg/mL! (final! assay!concentrations).!In!a!96.well!microplate,!25!µL!of!test!sample!was!pre.incubated!with!25!µL!of!substrate!Gly.Pro.p.nitroanilide!(12!mM)!at!37°C!for!10!min,!after!which!50!µL!of!DPP.IV! (0.02!units/mL)!was! added!and! the!mixture! incubated!at!37°C! for!30!min.!! 126!The! enzymatic! reaction!was! terminated!by! addition!of! 100!µL!of! 1!M! sodium!acetate!buffer!pH!4.0!and!the!absorbance!of! the!released!p.nitroanilide!was!measured!at!405!nm!using!a!Labsystems!iEMS!Reader!MF!(Labsystems!Oy;!Helsinki,!Finland).!Each!test!sample!was!analyzed!in!triplicate,!and!the!absorbance!values!were!corrected!for!sample!blanks! in! which! DPP.IV! was! replaced! with! Tris.HCl! buffer! (100! mM,! pH! 8.0).! The!positive! control! (DPP.IV! activity! with! no! inhibitor)! and! negative! control! (no! DPP.IV!activity)! were! prepared! by! using! Tris.HCl! buffer! (100! mM,! pH! 8.0)! in! place! of! the!sample,! and! in! place! of! the! sample! and! DPP.IV! solution,! respectively.! The!%!DPP.IV!inhibition!was!calculated!as!follows:!!%!DPP.IV! inhibition!=!100!×{1.(A405!test!sample.A405!test!sample!blank)/(A405!positive!control.A405!negative!control)}.!!The!IC50!values!(concentrations!of!hydrolysate!required!to!cause!a!50%!inhibition!of!the!enzyme! activity)!were! determined! from! the! cubic! regression! equations! generated! by!fitting!the!data!from!the!plot!of!%!DPP.IV!inhibition!against!hydrolysate!concentrations!ranging!from!1.25–0.0125!mg/mL!(final!assay!concentration).!The!tri.peptide!diprotin!A!(Ile.Pro.Ile)!was!used!as!a!reference!inhibitor.!!!6.3.6$Extraction$of$Rat$Intestinal$α4Glucosidase$'α.Glucosidase!was!extracted!from!rat!intestine!acetone!powder!as!described!by!Liu!et!al.!(2011).!Briefly,!0.5!g!of!rat!intestine!acetone!powder!was!suspended!in!20!mL!of!0.1!M!sodium!phosphate!buffer!pH!6.9!and!stirred!for!30!min!at!4°C.!The!mixture!was!then!centrifuged! at! 2,000! g! at! 4°C! for! 10! min,! the! supernatant! was! collected! and! kept!at!.18°C!for!further!use.!!! $! 127!6.3.7$Determination$of$α4Glucosidase$Inhibitory$Activity$'The! inhibitory! activity! of! the! hydrolysed! whey! protein! samples! was! assessed! by!comparing! the! amount! of! glucose! released! by! the! α.glucosidase! enzyme! from! its!natural!substrate,!maltose,! in!the!presence!and!absence!of!hydrolysed!samples!(Liu!et!al.,!2011).!!The!hydrolysed!whey!protein!samples!were!diluted!in!0.1!M!sodium!phosphate!buffer!pH! 6.9! to! the! desired! concentrations.! The! sample! solutions! (150! µL)! were! pre.incubated!with!150!µL!of!rat!α.glucosidase!solution!(obtained!as!described!above!and!diluted! 40! times!with! 0.1!M! sodium!phosphate! buffer! pH! 6.9)! for! 5!min! at! 37°C! in! a!shaker! incubator! (New! Brunswick! Scientific;! Enfield,! CT,! United! States)! prior! to! the!addition! of! 300! µL! of! 20! mg/mL! maltose! solution! (in! ddH2O).! The! mixtures! were!incubated!at!37°C!with!agitation!for!6!min!and!then!submerged!in!boiling!water!for!4!min! to! stop! the! enzymatic! reaction.!Aliquots! of! the! inactivated! suspensions! (100!µL)!were! incubated! with! 200! µL! of! glucose! assay! reagent! (prepared! according! to! the!supplier’s!instructions)!at!37°C!for!30!min.!The!reaction!was!then!stopped!by!addition!of!200!µL!of!6!M!sulfuric!acid!and! the!mixtures!were!centrifuged! for!5!min!at!14,000!rpm.! The! supernatants! (200! µL)! were! pipetted! into! a! 96.well! microplate! and! the!absorbance! of! the! samples!measured! at! 540! nm.! The! positive! control! (α.glucosidase!activity!with!no! inhibitor)!was!prepared!by!using!150!µL!of! 0.1!M! sodium!phosphate!buffer! pH! 6.9! in! place! of! the! sample! solution.! Each! test! sample! was! analyzed! in!triplicate.! The! concentrations! of! glucose! produced! from! the! substrate! by! the! α.glucosidase!enzyme!were!determined!from!a!glucose!calibration!curve!prepared!using!the!glucose!assay!kit.!The!%!α.glucosidase!inhibition!was!calculated!as!follows:!!%!α.glucosidase!inhibition!=!100!×!{1.([glucose]S!/[glucose]B)}!!where![glucose]S!and![glucose]B!represent!the!concentration!of!glucose!produced!in!the!presence!and!absence!of!test!sample,!respectively.!!!! 128!The! IC50! values! were! determined! from! the! cubic! regression! equations! obtained! by!fitting! the! data! from! the! plot! of! the! %! α.glucosidase! inhibition! against! hydrolysate!concentrations!ranging!from!2.5–7.5!mg/mL!(final!assay!concentration).!Acarbose!was!used!as!a!reference!inhibitor.!!6.3.8$Statistical$Analysis$$!To!compare!the!extents!of!hydrolysis!and!DPP.IV!or!α.glucosidase!inhibitory!activities!of!the!hydrolysed!whey!protein!samples,!one.way!analysis!of!variance!using!the!general!linear! model! and! pairwise! comparison! with! Tukey’s! method! were! performed! using!Minitab!statistical!software!(version!16,!Minitab!Inc.,!State!College,!PA,!United!States).!All! experiments!were! conducted! at! least! in! triplicate.!Data! are! expressed! as!means!±!standard!deviation!(SD)!and!significant!difference!was!established!at!P!<!0.05.!!!6.4!RESULTS!AND!DISCUSSION!'6.4.1$Extent$of$Hydrolysis$of$Pepsin4Treated$Whey$Proteins$!The!extent!to!which!the!whey!proteins!were!hydrolysed!by!pepsin!during!the!enzymatic!hydrolysis! treatment!was!evaluated.!As!shown! in!Figure' 6.1,! the!extent!of!hydrolysis!differed! significantly! between! the! pepsin.treated! whey! proteins.! The! complete!hydrolysis!of!whey!proteins!to!their!constituent!amino!acids!has!been!reported!to!yield!8.8!mmol!of!amino!acids!per!g!protein!(Alder.Nissen,!1986).!With!extent!of!hydrolysis!values!all! lower! than!1.0!mmol! L.leucine!equivalents!per!g!protein,!none!of! the!whey!proteins!were!completely!hydrolysed!by!the!peptic!treatment!used!in!this!study.!Among!the!four!individual!proteins!investigated,!the!serum!albumin!hydrolysate!displayed!the!highest! content! of! α.amino! groups,! while! the! lowest! content! was! observed! for! the!hydrolysate!of!β.lactoglobulin.!!! 129!!!!!!!!!!!!!! !Figure' 6.1' Extent! of! hydrolysis! (mmol! L.leucine! equiv/g! protein)! of! whey! protein!hydrolysates!obtained!by!peptic!treatment.!Each!bar!represents!the!mean!and!standard!deviation!of! three!determinations.!Bars! labeled!with!different! letters! are! significantly!different! (P! <! 0.05).! α.la,! α.lactalbumin;! β.lg,! β.lactoglobulin;! Lf,! lactoferrin;! BSA,!bovine!serum!albumin;!WPI,!whey!protein!isolate.''!!!Moreover,! the! SDS.PAGE! of! the! pepsin.treated! β.lactoglobulin! (Figure' 6.2B' lane! 3)!revealed!that! it!contained!intact!proteins!and!fewer! low!molecular!weight!bands!than!the!pepsin.treated!lactoferrin,!α.lactalbumin!and!serum!albumin!samples!(Figure'6.2B'lanes! 2,! 4! and! 6! respectively).! The! resistance! of! native! β.lactoglobulin! to! pepsin!digestion!has!been!previously!reported!and!suggested!to!be!caused!by!the!folded!calyx!structure!of!this!protein,!making!target!peptide!bonds!unavailable!to!proteolytic!action!(Reddy!et!al.,!1988).!Although!mainly!composed!of!β.lactoglobulin!(Figure'6.2A),!whey!protein!isolate!(WPI)!had!an!extent!of!hydrolysis!almost!two!times!higher!(Figure'6.1).!It!is!possible!that!the!process!used!to!produce!the!WPI!may!have!caused!changes!to!the!proteins’! structure! and! consequently! affected! their! susceptibility! to! proteolytic!cleavage.!!!!0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 α-la β-lg Lf BSA WPI L-leucine equivalent  (mmol per g protein) Whey protein hydrolysate a b c d e ! 130!!! !'Figure' 6.2' SDS.PAGE! electrophoregrams! of! whey! protein! samples! (A)! and! their!hydrolysates! after! peptic! treatments! (B).! Lanes:! 1,! molecular! weight! markers;! 2,!lactoferrin;! 3,! β.lactoglobulin;! 4,! α.lactalbumin;! 5,! whey! protein! isolate;! 6,! bovine!serum!albumin.''6.4.2$DPP4IV$Inhibitory$Activity$of$Pepsin4Treated$Whey$Proteins$$!The! DPP.IV! inhibitory! activity! of! the! whey! protein! hydrolysates! obtained! by! peptic!treatment!was!investigated.!As!shown!in!Figure'6.3,!when!present!at!a!concentration!of!0.500!mg/mL,! all!whey! protein! hydrolysates! obtained! after! 60!min! of! digestion!with!pepsin!were!able!to!cause!the!inhibition!of!the!DPP.IV!enzyme.!The!α.lactalbumin!and!WPI!hydrolysates,!with!91!and!82%!inhibition!respectively,!showed!the!highest!DPP.IV!inhibitory! activity,! while! the! pepsin.treated! β.lactoglobulin! sample! was! the! least!effective! at! inhibiting! the! activity! of! the! enzyme! (28%! inhibition).! Un.hydrolysed!proteins,!on!the!other!hand,!displayed!no!inhibitory!activity!(data!not!shown).!The!IC50!value!measured!for!the!α.lactalbumin!hydrolysate!(0.036!mg/mL),!was!found!to!be!of!the!same!order!of!magnitude!as!the!IC50!of!WPI!hydrolysate,!but!about!10!times!lower!than! that!observed! for! the!serum!albumin!and! lactoferrin!hydrolysates!while! roughly!35!times!lower!than!that!for!the!β.lactoglobulin!hydrolysate!(Table'6.1).!The!relatively!low!inhibitory!activity!of! the!hydrolysed!β.lactoglobulin!sample!compared!to!the!WPI! 2 1 4 5 6 10 15 20 25 30 40 50 60 70 85 100 120 150 200 3 MW (kDa) 2 1 4 5 6 3 MW (kDa) 26.6 17.0 14.2 6.5 3.5 1.0 A B  2 1 4 5 6 10 15 20 25 30 40 50 60 70 85 100 120 150 200 3 MW (kDa) 2 1 4 5 6 3 MW (kDa) 26.6 17.0 14.2 6.5 3.5 1.0 A B A B ! 131!hydrolysate! is! surprising! since! β.lactoglobulin! is! the!major! protein! fraction! in! whey!(Figure'6.2).!!!!!!!!!!!!!!!!!!! !!Figure'6.3'DPP.IV!inhibitory!activity!of!whey!protein!hydrolysates!obtained!by!peptic!treatment.! The! percent! inhibition! of! DPP.IV! was! measured! using! 0.500! mg/mL! of!hydrolysate! (final! assay! concentration,! protein! basis).! Each! bar! represents! the!mean!and!standard!deviation!of!three!determinations.!Bars!labeled!with!different!letters!are!significantly! different! (P! <! 0.05).! α.la,! α.lactalbumin;! β.lg,! β.lactoglobulin;! Lf,!lactoferrin;!BSA,!bovine!serum!albumin;!WPI,!whey!protein!isolate.'!!!!!!!!!!!!!' '0 10 20 30 40 50 60 70 80 90 100 α-la β-lg Lf BSA WPI % DPP-IV inhibition Whey protein hydrolysate a b c d e ! 132!Table'6.1'DPP.IV!and!α.glucosidase!inhibitory!activities!of!hydrolyzed!whey!proteins!obtained!by!peptic!treatment.'Hydrolysate IC50 a (mg/mL) DPP-IV α-glucosidase α-Lactalbumin 0.036 ± 0.002 c ND β-Lactoglobulin 1.279 ± 0.100 a 3.5 ± 0.4 a Lactoferrin 0.379 ± 0.035 b ND Bovine serum albumin 0.513 ± 0.056 b ND Whey protein isolate 0.075 ± 0.006 c 4.5 ± 0.6 a Diprotin Ab 0.0016 ± 0.0003 NA Acarboseb NA 2.8 x 10-4 ± 0.3 x 10-4 a IC50 values are reported as the means and standard deviations from triplicate determinations and expressed at final assay concentration. Within the same column, figures with different lower case letters are significantly different (P < 0.05). NA, not applicable; ND, not determined.  b Diprotin A and acarbose were used as reference inhibitors for DPP-IV and α-glucosidase, respectively. !!To! assess! whether! the! low! DPP.IV! inhibitory! activity! of! the! pepsin.treated! β.lactoglobulin!could!be!the!result!of!the!resistance!of!this!protein!to!being!hydrolysed,!a!3%! β.lactoglobulin! solution! was! heated! at! 90°C! for! 15! min! prior! to! performing! the!hydrolysis! treatment!with!pepsin.!Heating!β.lactoglobulin!at!80.90°C!has!been!shown!to! cause! structural! changes! that! improve! the! susceptibility! of! its! peptide! bonds! to!peptic!digestion!(Reddy!et!al.,!1988).!The!heating!pre.treatment!did!increase!the!extent!of! hydrolysis! of! the! β.lactoglobulin! hydrolysates! to! 0.75! mmol! L.leucine! equiv/g!protein,!but!had!no!significant!effect!on!the!DPP.IV!inhibitory!activity!(data!not!shown).!The!IC50!value!for!the!pepsin.treated!β.lactoglobulin!found!in!this!study!is!of!the!same!order! as! the! value! reported!by!Uchida! et! al.! (2011)! for! a!β.lactoglobulin! hydrolysate!obtained!by!enzymatic!hydrolysis!with!the!digestive!enzyme!trypsin!(IC50!=!210!µM!or!~! 3.9!mg/mL).! The! authors! isolated! and! identified! the! hexa.peptide!Val.Ala.Gly.Thr.Trp.Tyr!(IC50!=!174!µM!or!~!0.12!mg/mL)!as!being!the!active!component!responsible!for!the!inhibitory!activity!of!the!tryptic!hydrolysate!(Uchida!et!al.,!2011).!!!!!Peptides! with! different! biological! properties,! including! opioid! and! angiotensin!I.converting! enzyme! (ACE)! inhibitory! activities,! have! been! found! in! α.lactalbumin!hydrolysates! obtained! by! peptic! digestion! (Pihlanto.Leppälä,! 2001).! However,! to! the!author’s! knowledge,! the! generation! of! peptides! with! DPP.IV! inhibitory! activity! from!! 133!α.lactalbumin!has!never!been!reported.!!α.Lactalbumin!is!the!second!main!constituent!of!WPI,!accounting!for!about!14%!of!its!total!protein!content!(Figure'6.2A).!The!similar!inhibitory! activity! observed! for!α.lactalbumin! and!WPI!hydrolysates! suggests! that!α.lactalbumin.derived! peptides!might! be! among! the! active! components! responsible! for!the!inhibition!of!DPP.IV!activity!observed!with!the!WPI!hydrolysate.!!!The! hydrolysates! obtained! by! the! peptic! digestion! of! lactoferrin! and! serum! albumin!showed! similar! DPP.IV! inhibitory! activity! (Table' 6.1).! A! lactoferrin! hydrolysate! of!comparable! potency! (IC50! =! 1.088!mg/mL),! obtained! using! a! food! grade! commercial!gastro.intestinal!preparation,!was!also!recently!reported!in!a!study!by!Nongonierma!&!FitzGerald!(2013b).!!!!!6.4.3$α4Glucosidase$Inhibitory$Activity$of$Pepsin4Treated$Whey$Proteins$!The! whey! protein! hydrolysates! obtained! by! peptic! digestion! were! also! assessed! for!their!inhibitory!activity!against!the!α.glucosidase!enzyme.!Most!in#vitro!studies!on!the!α.glucosidase! inhibitory! activity! of! natural! compounds! have! used! microbial! α.glucosidase! (from!Baker’s! yeast)! (Matsui! et! al.,! 1996).!However,! the!magnitude! of!α.glucosidase! inhibition! has! been! reported! to! be! greatly! affected! by! the! origin! of! the!enzyme.!The!synthetic!α.glucosidase!inhibitors!voglibose!and!acarbose!were!shown!in!a!study!by!Oki!et!al.#(1999)!to!strongly!inhibit!the!activity!of!mammalian!α.glucosidase,!but! to!have! little! or!no! effect! on!baker’s! yeast!α.glucosidase.!On! the!other!hand,! (+).catechin!as!well!as!some!food!products,!such!as!yogurt,!chicken!essence!and!fish!sauce,!showed!inhibitory!activity!against!yeast!α.glucosidase!but!not!against!the!mammalian!enzyme!(Oki!et!al.,!1999).!It!has!been!suggested!that!altered!binding!of!the!inhibitors!to!the!active!site,!which!may!arise!from!structural!differences!among!the!enzymes,!could!be! responsible! for! these! conflicting! findings! (Oki! et! al.,! 1999;! Kim! et! al.,! 2011b).! In!order!for!the!investigation!of!the!α.glucosidase!inhibitory!activity!of!the!whey!protein!hydrolysates! to! have! better! relevance! to! human! health,! mammalian! (rat)! intestinal!! 134!α.glucosidase! was! used! in! the! present! study.! Moreover,! maltose! was! chosen! as! the!substrate! for! the!enzyme!assay! in! this!study!since! it! is! the!major!digestive!product!of!starch!in!the!small!intestine!(Tadera!et!al.,!2006).!!The!inhibition!of!α.glucosidase!activity!in!the!presence!of!whey!protein!hydrolysates!at!2.50! mg/mL! was! investigated.! Of! the! five! samples,! β.lactoglobulin! and! WPI!hydrolysates!displayed!the!highest!α.glucosidase!inhibitory!activity,!with!33!and!36%!inhibition,! respectively! (Figure' 6.4).! The!α.lactalbumin! hydrolysate!was! also! able! to!inhibit! the! activity! of!α.glucosidase!but! not! as! effectively! (24%! inhibition),!while! the!hydrolysed! lactoferrin!and!serum!albumin!samples!had! low! inhibitory!activity! (5!and!6%! inhibition,! respectively)! (Figure' 6.4).! None! of! the! un.hydrolysed! proteins! were!able! to! inhibit! the! α.glucosidase! enzyme! (data! not! shown).! The! IC50! values! of! the!hydrolysed!β.lactoglobulin! and!WPI! samples! (3.5! and!4.5!mg/mL,! respectively)!were!not! found! to!be!significantly!different! (Table' 6.1).! Similarly! to! the!results! for!DPP.IV!inhibitory!activity!of!the!hydrolysates!relative!to!the!reference!inhibitor!diprotin!A,!the!α.glucosidase! inhibitory! activity! of! the! whey! protein! hydrolysates! was! significantly!lower! than! that! measured! for! the! reference! inhibitor! acarbose! (IC50! =! 0.28! µg/mL).!Since! the! protein! hydrolysates! are! composed! of! a!mixture! of! peptides,! only! some! of!which! would! display! inhibitory! activity,! their! lower! potency! compared! to! isolated!peptides!or!synthetic!drugs!is!not!surprising.!!!!!!!!!!!!!!!! 135!!!!!!!!!!!!!!!!!! !Figure'6.4'α.Glucosidase!inhibitory!activity!of!whey!protein!hydrolysates!obtained!by!peptic! treatment.! The! percent! inhibition! of! α.glucosidase! was! measured! using! 2.50!mg/mL!of! hydrolysate! (final! assay! concentration,! protein!basis).! Each!bar! represents!the!mean!and!standard!deviation!of! three!determinations.!Bars! labeled!with!different!letters!are!significantly!different!(P!<!0.05).!α.la,!α.lactalbumin;!β.lg,!β.lactoglobulin;!Lf,!lactoferrin;!BSA,!bovine!serum!albumin;!WPI,!whey!protein!isolate.'!!!Althought! the! majority! of! synthetic! α.glucosidase! inhibitors,! such! as! acarbose! and!voglibose,!are!sugars!or!derivatives!of!sugar!moieties!(Bharatham!et!al.,!2008),!most!α.glucosidase! inhibitory!compounds!reported!to!be!derived! from!natural!sources!are!of!phenolic!nature,!including!flavonoids!and!tannins!(Matsui!et!al.,!2001;!Gonçalves!et!al.,!2011;!You!et!al.,!2012).!On!the!other!hand,!a!few!peptides!isolated!from!food!proteins!of!animal!origin!have!also!been!reported!to!inhibit!the!activity!of!α.glucosidase!(Matsui!et!al.,!1996;!Matsui!et!al.,!1999;!Yu!et!al.,!2011).! In!a!survey!of! the! in#vitro#α.glucosidase!inhibitory!activity!of!different! food!commodities,!Matsui!et!al.! (1996)! showed! that!an!alkaline! protease! hydrolysate! from! sardine! muscle! was! able! to! inhibit! the!carbohydrate.hydrolyzing! enzyme! with! an! IC50! value! of! 48.7! mg/mL.! Upon! further!fractionation!of!the!sardine!muscle!hydrolysate,!the!authors!isolated!and!identified!the!α.glucosidase! inhibitory!peptides!Val.Trp!(IC50!=!22.6!mM!or!~!6.9!mg/mL)!and!Tyr.Tyr.Pro.Leu!(IC50!=!3.7!mM!or!~!2.1!mg/mL)!(Matsui!et!al.,!1999).!More!recently,! the!hexa.peptide! Arg.Val.Pro.Ser.Leu.Met! (IC50!=! 23.07! µM! or! ~! 0.016!mg/mL)! and! the!0 5 10 15 20 25 30 35 40 45 α-la β-lg Lf BSA WPI % α-glucosidase inhibition Whey protein hydrolysate a a b b c c ! 136!penta.peptide!Thr.Pro.Ser.Pro.Arg!(IC50!=!40.02!µM!or!~!0.022!mg/mL),!derived!from!egg!white!protein,!were!reported!to!possess!α.glucosidase!inhibitory!activity!(Yu!et!al.,!2011).!To!the!best!of!the!author’s!knowledge,!the!present!study!is!the!first!to!report!the!generation! of! α.glucosidase! inhibitory! activity! by! proteolysis! of! whey! proteins.! The!exact!mechanism! by!which! peptides! could! inhibit! α.glucosidase! activity! is! unknown,!but! it! has! been! suggested! that! non.saccharide! compounds!may! exert! their! inhibitory!activity!by!binding!to!the!enzyme’s!active!site!via!hydrophobic!interactions!(Bharatham!et!al.,!2008).!!!6.5!CONCLUSION!!Results!from!the!present!study!demonstrated!that!peptides!with!inhibitory!properties!against!DPP.IV!and!α.glucosidase!activities!can!be!generated!from!the!peptic!digestion!of!whey!proteins.!While!WPI,!α.lactalbumin!and!β.lactoglobulin!hydrolysates!displayed!both!DPP.IV!and!α.glucosidase!inhibitory!activities,!products!obtained!by!hydrolysis!of!lactoferrin! and! serum! albumin! were! only! able! to! inhibit! the! DPP.IV! enzyme.! These!findings! suggest! that! whey! protein! hydrolysates! with! DPP.IV! and! α.glucosidase!inhibitory! activities! may! have! the! potential! to! improve! blood! glucose! regulation! by!means!of!their!ability!to!slow!down!both!the!inactivation!of!the!incretin!hormones!and!the! intestinal! digestion! of! carbohydrates.! Additional! investigations! to! isolate! and!sequence!the!bioactive!peptides!present!within!dairy!protein!hydrolysates!would!allow!a!better!understanding!of!their!modes!of!action!on!DPP.IV!and!α.glucosidase!activities.!Furthermore,! clinical! studies! are! also! needed! to! confirm! the! efficacy! of! these! whey!protein.derived!peptides!in!humans.!!!!! 137!CHAPTER!7:! ISOLATION!AND!CHARACTERIZATION!OF!PEPTIDES!WITH!DIPEPTIDYL5PEPTIDASE! IV! INHIBITORY! ACTIVITY! FROM! PEPSIN5TREATED! BOVINE! WHEY!PROTEINS8!!7.1!SUMMARY!!Inhibition!of! the! enzyme!dipeptidyl.peptidase! IV! is! one!of! the! strategies!used! for! the!treatment!of!type!2!diabetes.!In!the!present!study,!pepsin.treated!whey!protein!isolate!(WPI)! and! α.lactalbumin! displaying! DPP.IV! inhibitory! activity! were! fractionated! by!successive!chromatographic!steps!and!the!resulting!active!fractions!analyzed!for!their!constituent! peptides! by! liquid! chromatography–electrospray! ionization.tandem!mass!spectrometry.!Among!the!identified!sequences,!24!peptides!derived!from!α.lactalbumin!and! 11! from! β.lactoglobulin! were! synthesized! and! their! effects! on! DPP.IV! activity!assessed.!The!most!potent! fragments,!LKPTPEGDL!and!LKPTPEGDLEIL!(IC50!=!45!and!57! µM,! respectively),! were! found! to! inhibit! DPP.IV! in! an! un.competitive! manner.!Although!several!of!the!peptides!tested!showed!some!inhibitory!activity,!only!two!were!as! effective! as! the! un.fractionated!WPI! hydrolysate! and! none! were! as! potent! as! the!un.fractionated!α.lactalbumin!hydrolysate.!The!peptides’!structural!features,!including!length!and!amino!acid!composition,!were!found!to!impact!their!inhibitory!activity.!This!study! provides! new! insights! on! the! active! components! responsible! for! the! DPP.IV!inhibitory!activity!of!pepsin.treated!whey!proteins.!!!!7.2!INTRODUCTION!!!It! is! recognized! that! diet! plays! a! major! role! in! the! incidence! and! management! of!diabetes.!Over!the!past!decade,!a!number!of!interventional!studies!have!shown!that!the!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!8!A! version! of! this! chapter! has! been! published.! Lacroix,! I.M.E.,! Li.Chan,! E.C.Y.! Isolation! and!characterization! of! peptides! with! dipeptidyl! peptidase.IV! inhibitory! activity! from! pepsin.treated!bovine!whey!proteins.!Peptides!2014,!54,!39–48.!! 138!consumption!of!whey!protein!has!a!beneficial!effect!on!glycemia!regulation!(Akhavan!et!al.,!2010;!Jakubowicz!&!Froy,!2013).!Pre.meal!consumption!of!whey!or!the!addition!of!whey! protein! to!meals! high! in! carbohydrate! has! been! found! to! lead! to! a! decrease! in!postprandial!blood!glucose! levels!and!an! increase! in!plasma! insulin!concentrations! in!healthy! individuals! and! type! 2! diabetic! patients! (Frid! et! al.,! 2005;! Ma! et! al.,! 2009;!Petersen! et! al.,! 2009;! Akhavan! et! al.,! 2010).! However,! how! whey! protein! exerts! its!antidiabetic! properties! is! not! fully! understood.! One! of! the! possible! mechanisms!suggested!by!recent! findings! is! that! the!release!of!bioactive!peptides!and!amino!acids!during!gastrointestinal!digestion!could!promote!the!secretion!of!the!incretin!hormones!and/or!cause!the!inhibition!of!dipeptidyl.peptidase!IV!(Jakubowicz!&!Froy,!2013).!!!Although!the!beneficial!effect!of!whey!protein! intake!on!glucose!metabolism!has!been!recognized!for!a!number!of!years!and!extensive!research!on!synthetic!DPP.IV!inhibitors!has!been!carried!out!for!more!than!a!decade,! it! is!only!recently!that!whey!protein!has!been! identified! as! a! precursor! of! DPP.IV! inhibitory! peptides.! An! in# silico! analysis!showed! that! several! dietary! proteins,! including! the! whey! proteins! α.lactalbumin,!β.lactoglobulin! and! lactoferrin,! contain! fragments! nascent! within! their! primary!structure! that! correspond! to! the! sequences! of! synthetic! peptides! reported! in! the!literature! to!display!DPP.IV! inhibitory!activity! (Lacroix!&!Li.Chan,!2012a;!Chapter!4).!Moreover,! whey! protein! hydrolysates! produced! using! digestive! enzymes,! such! as!pepsin!and! trypsin,!have!been!recently! found! to! inhibit! the!activity!of!DPP.IV! in#vitro!(Uchida!et!al.,!2011;!Lacroix!&!Li.Chan,!2012b;!Lacroix!&!Li.Chan,!2013;!Nongonierma!&! FitzGerald,! 2013b;! Nongonierma! &! FitzGerald,! 2013c;! Silveira! et! al.,! 2013).! A!β.lactoglobulin! hydrolysate! prepared! by! tryptic! treatment! was! also! reported! to!significantly! lower! blood! glucose! levels! in! mice! when! administered! before! an! oral!glucose! tolerance! test! (Uchida! et! al.,! 2011).! The! hexa.peptide! VAGTWY!was! isolated!from!the!hydrolysate!and!suggested!to!be!the!main!active!sequence!responsible!for!the!DPP.IV! inhibitory! activity! of! the! trypsin.treated!β.lactoglobulin! (Uchida! et! al.,! 2011).!Similarly,! Silveira! et! al.! (2013)! isolated! and! identified! the!DPP.IV! inhibitory! peptides!TPEVDDEALEK,! IPAVF,! IPAVFK! and! VLVLDTDYK! from! a! trypsin.hydrolysed! whey!! 139!protein! concentrate! rich! in!β.lactoglobulin.! In! the! past! few! years,! a! number! of! other!peptide! fragments! found! within! the! sequences! of! whey! proteins! have! also! been!chemically!synthesized!and!studied!for!their!effect!on!DPP.IV!activity!(Tulipano!et!al.,!2011;! Nongonierma! &! FitzGerald,! 2013b;! Nongonierma! &! FitzGerald,! 2013c).! These!peptides,!however,!were!not!actually!identified!in!whey!protein!hydrolysates,!but!only!hypothesized!to!be!among!the!active!components!responsible!for!the!DPP.IV!inhibitory!activity!of!whey.!Thus,!despite! the!knowledge! that!peptide! fragments!associated!with!DPP.IV!inhibitory!activity!are!contained!within!the!sequences!of!whey!proteins!and!that!DPP.IV!inhibitors!can!be!released!from!whey!protein!by!enzymatic!hydrolysis,!to!date!only! a! few! studies! have! isolated! and! identified! DPP.IV! inhibitory! peptides! in! whey!protein!hydrolysates.!!!!A! previous! investigation! on! various! whey! protein! hydrolysates! produced! by! pepsin!digestion! revealed! that! the! hydrolysates! of! whey! protein! isolate! (WPI)! and!α.lactalbumin! were! the! most! potent! at! inhibiting! the! DPP.IV! enzyme! (Lacroix! &! Li.Chan,!2013;!Chapter!6).!The!specific!peptides!responsible!for!the!observed!inhibition!of!DPP.IV!were,!however,!unknown.!Therefore,!the!objective!of!the!present!study!was!to!isolate!and!sequence!the!bioactive!peptides!present!within!the!WPI!and!α.lactalbumin!peptic!hydrolysates!and!to!determine!their!modes!of!action!on!DPP.IV!activity.!!!7.3!MATERIALS!AND!METHODS!!!7.3.1$Materials$'Pepsin! (EC! 3.4.23.1,! from! porcine! gastric! mucosa,! ≥2500! units! per! mg! protein),!dipeptidyl.peptidase! IV! (DPP.IV,! EC! 3.4.14.5,! from! porcine! kidney,! ≥10! units! per!mg!protein),!α.lactalbumin!from!bovine!milk!(Product!#!L6010,!≥85%!protein),!lysozyme,!HPLC! peptide! standard! mixture! and! Ile.Pro.Ile! (diprotin! A)! were! purchased! from!Sigma.Aldrich! (Oakville,! ON,! Canada).! Antifreeze! protein! type! 1!was! donated! by! A/F!! 140!Protein!Canada!(St! Johns,!NF,!Canada).!Whey!protein! isolate!(WPI!895,!Fonterra,!New!Zealand;! 92.4%! protein)! was! donated! by! Caldic! (Delta,! BC,! Canada).!Gly.Pro.p.nitroanilide!(H.Gly.Pro.pNA⋅HCl)!was!from!Bachem!Americas!(Torrance,!CA,!United!States).!All!other!chemicals!used!were!of!analytical!grade.!!7.3.2$Preparation$of$Whey$Protein$Hydrolysates$!WPI!and!α.lactalbumin!hydrolysates!were!prepared!by!digestion!with!pepsin!using!a!4%! (w/w)! enzyme/substrate! ratio! and! incubating! at! pH!2.0! and!37°C! for! 60!min,! as!previously!described!in!Chapter!6,!section!6.3.2!(Lacroix!&!Li.Chan,!2013).!!7.3.3$Isolation$of$DPP4IV$Inhibitory$Peptides$From$Whey$Protein$Hydrolysates$!WPI! and!α.lactalbumin! hydrolysates! were! sequentially! fractionated! using! successive!chromatographic! techniques! to! obtain! fractions! enriched! in! peptides! with! DPP.IV!inhibitory!activity.!The!peptides!in!the!WPI!hydrolysate!were!separated!on!the!basis!of!charge,!size!and!polarity,!whereas!those!in!the!α.lactalbumin!hydrolysate!were!isolated!on!the!basis!of!size!and!polarity.!!!7.3.3.1%Cation(Exchange%Chromatography%!The!WPI! hydrolysate!was! first! fractionated! by! cation.exchange! chromatography.! The!freeze.dried!hydrolysate!was!dissolved!in!20!mM!sodium!acetate!buffer!pH!4.0!(starting!buffer)! to! a! concentration! of! 30!mg/mL! (solid! basis)! and! 500! µL! was! loaded! into! a!Mono! STM! 5/50! GL! cation.exchange! column! (5! x! 50! mm;! GE! Healthcare,! Uppsala,!Sweden)!pre.equilibrated!with!the!starting!buffer!and!connected!to!a!fast!protein!liquid!chromatography! (FPLC)! system! (GE! Healthcare,! Uppsala,! Sweden).! Unbound! protein!material!was!washed!from!the!column!with!the!starting!buffer!for!10!min!at!a!flow!rate!of!2!mL/min.!The!adsorbed!protein!material!was! then!eluted!with! the!starting!buffer!! 141!containing!increasing!NaCl!concentrations!at!a!flow!rate!of!2!mL/min!according!to!the!following!sequential!gradients:!(1)!0–0.10!M!NaCl!over!5!min,!(2)!0.10!M!NaCl!for!8!min,!(3)!0.10–0.20!M!NaCl!over!5!min,!(3)!0.20!M!NaCl!for!8!min,!(4)!0.20–0.30!M!NaCl!over!5!min,!(5)!0.30!M!NaCl!for!8!min,!(6)!0.30–0.40!M!NaCl!over!5!min,!(7)!0.40!M!NaCl!for!8!min,! (8)!0.40–1.00!M!NaCl!over!5!min,! (9)!1.00!M!NaCl! for!5!min.!Fractions!of!1.4!mL!were! collected! and! the! absorbance!was!monitored! at! 214! and!280!nm.!The! fractions!eluted!within!each!peak!were!pooled,!desalted!(Micro!Acilyzer!S1,!Aston!Corporation,!Japan),! freeze.dried! and! assessed! for! their!DPP.IV! inhibitory! activity.! The! separation!was!repeated!multiple! times! to!obtain!enough!sample! for! the!subsequent!purification!steps!and!analysis.!!!!!7.3.3.2%Size(Exclusion%Chromatography%!The! pooled! fraction! collected! from! cation.exchange! chromatography! of! WPI!hydrolysate! displaying! the! highest! DPP.IV! inhibitory! activity!was! further! purified! by!size.exclusion! chromatography.! !The! fraction!was!dissolved! in!20!mM!Tris.HCl!buffer!pH!8.0!to!a!concentration!of!20!mg/mL,!500!µL!was!loaded!into!a!SuperdexTM!Peptide!10/300!GL!column!(10!x!300–310!mm;!GE!Healthcare,!Uppsala,!Sweden)!connected!to!a!FPLC!system!(GE!Healthcare,!Uppasala,!Sweden),!and!eluted!with!20!mM!Tris.HCl!buffer!pH!8.0!at!a!flow!rate!of!0.2!mL/min.!Fractions!of!0.4!mL!were!collected!and!the!peaks!observed!by!monitoring!the!absorbance!at!214!and!280!nm!were!pooled,!freeze.dried!and!assessed!for!their!DPP.IV!inhibitory!activity.!The!separation!was!repeated!multiple!times! to! obtain! enough! sample! for! the! subsequent! purification! steps! and! analysis.!Lysozyme!(MW!14300!Da),!the!antifreeze!protein!(MW!3230!Da)!as!well!as!the!peptides!DRVYIHPF!(MW!1046.2!Da),!YGGFM!(MW!573.7!Da),!YGGFL!(MW!555.6!Da),!VYV!(MW!379.5!Da)!and!GY!(MW!238.2!Da)!from!the!HPLC!peptide!mixture!standard!were!used!as!molecular!weight!standards.!!! 142!The!α.lactalbumin!hydrolysate!was!also! submitted! to! size.exclusion!chromatography.!The!hydrolysate!was!dissolved!in!20!mM!Tris.HCl!buffer!pH!8.0!to!a!concentration!of!20!mg/mL!and!fractionated!as!described!above.!!!!!!7.3.3.3%Reversed(Phase%High%Performance%Liquid%Chromatography%(RP(HPLC)%!The! peptide! fractions! with! the! highest! DPP.IV! inhibitory! activity! from! the! size.exclusion! chromatography! were! separated! by! RP.HPLC! using! an! Agilent! 1100! HPLC!system! (Agilent! Technologies,! Mississauga,! ON,! Canada).! The! freeze.dried! fractions!were!dissolved!in!0.1%!(v/v)!TFA!in!H2O!to!a!concentration!of!2!mg/mL!and!90!µL!was!injected! into! a! Phenomenex! JupiterTM! C12! Proteo! 90! Å! (250! x! 4.6! mm,! 4! µ;!Phenomenex®,!Torrance,! CA,!United! States)!HPLC! column!at! 40°C.!The! samples!were!eluted!using!a!linear!gradient!of!acetonitrile!(8.0–34.2%!(v/v)!in!35!min)!and!0.1%!TFA!(v/v)! in! H2O! at! a! flow! rate! of! 1! mL/min.! Fractions! were! collected! based! on! the!absorbance!at!214!and!280!nm.!The!procedure!was!repeated!multiple!times!to!obtain!enough! sample! for! the! subsequent! analysis.! After! removal! by! evaporation! of! the!acetonitrile! under! a! stream! of! nitrogen,! the! fractions! were! freeze.dried! (Labconco!Corporation,! Kansas! City,!MO,! United! States)! and! assessed! for! their! effect! on! DPP.IV!activity.!!!!7.3.4$ Identification$ of$ Peptide$ Sequences$ by$ Liquid$ Chromatography4Electrospray$IonisationTtandem$Mass$Spectrometry$(LC–ESI4MS/MS)$'The!purified!peptide!fractions!displaying!the!greatest!inhibitory!activity!against!DPP.IV!were!analyzed!at! the!Fred!Hutchinson!Cancer!Research!Center! (Seattle,!WA,!USA)! for!their! amino! acid! sequences! by! LC–ESI.MS/MS! using! an! Easy.nLC! system! (Thermo!Scientific,!Waltham,!MA,!USA)!coupled!to!a!hybrid!Orbital!Elite!ETD!mass!spectrometer!(Thermo! Scientific,! Waltham,! MA,! USA).! The! samples! were! solubilized! in! 2%! (v/v)!acetonitrile! in! H2O! containing! 0.1%! (v/v)! formic! acid! and! 6! µL! was! injected! onto! a!reversed.phase! trap! column! (100! µm! x! 20! mm)! packed! with! C18! reversed.phase!! 143!material! (Magic! C18AQ,! 5! µm,! 200! Å,! Michrom! Bioresources,! Auburn,! CA,! USA)! and!separated!on!a!reversed.phase!column!(75µm!x!250!µm)!packed!with!Magic!C18AQ!(5!µm,! 100! Å,! Michrom! Bioresources,! Auburn,! CA,! USA)! using! a! linear! gradient! of!acetonitrile!(2–35%!(v/v)!in!60!min)!and!0.1%!(v/v)!formic!acid!in!H2O!at!a!flow!rate!of!400!nL/min.!The!eluate!from!the!column!was!directly!injected!into!the!MS!system.!!!A! spray! voltage! of! 2000V!was! applied! to! the! electrospray! tip! and! the! Orbitrap! Elite!instrument! was! operated! in! the! data.dependent! mode! (Yi! et! al.,! 2003),! switching!automatically!between!MS!survey!scans!in!the!Orbitrap!with!MS/MS!spectra!acquisition!acquired!with!collision!induced!dissociation!(CID)!detected!in!the!linear!ion!trap,!high.energy! collision! dissociation! (HCD)! detected! in! the! Orbitrap! and! electron! transfer!dissociation! (ETD)!detected! in! the!Orbitrap.!The! three!most! intense! ions!observed! in!the! Fourier.transform! full! scan! were! sequentially! isolated! in! the! ion! trap! and!fragmented!by!CID!(35%!normalized!collision!energy),!HCD!(35%!normalized!collision!energy)!and!ETD!(100!ms!activation!time).!Selected!ions!were!dynamically!excluded!for!30! sec.! !The! tandem!MS!data!were!analyzed!using! the!Proteome!Discoverer! software!(Thermo!Scientific,!Waltham,!MA,!USA)! and! the! amino!acid! sequence!of! each!peptide!was! identified! by! comparison! with! identified! peptide! sequences! from! Bos# taurus#(UniProt!database).!!!7.3.5$Peptide$Synthesis$'Peptides! identified! by! LC–ESI.MS/MS! were! synthesized! and! purified! by! CanPeptide!(Pointe.Claire,! QC,! Canada)! and! GL! Biochem! (Shanghai,! China).! The! purity! of! the!peptides!was!≥!95%.!!7.3.6$DPP4IV$inhibitory$Activity$Assay$!The!effect!of!the!purified!peptide!fractions!and!synthesized!peptides!on!the!activity!of!DPP.IV!was!determined!using!the!chromogenic!substrate!Gly.Pro.p.NA!as!described!in!! 144!Chapter! 6,! section! 6.3.5! (Lacroix! &! Li.Chan,! 2013).! Briefly,! peptide! fractions! and!synthesized!peptides!were!dissolved!to!the!desired!concentrations!in!100!mM!Tris.HCl,!pH! 8.0,! buffer.! For! the! synthesized! peptides! that! were! not! soluble! in! buffer! alone,!acetonitrile!(up!to!30%!(v/v)!in!100!mM!Tris.HCl,!pH!8.0)!was!also!added.!In!a!96.well!microplate,!25!µL!of!sample!was!pre.incubated!with!25!µL!of!substrate!(12!mM)!at!37°C!for!10!min,!whereupon!50!µL!of!DPP.IV!(0.02!units/mL)!was!added!and!the!enzymatic!reaction!carried!out!at!37°C!for!30!min.!The!reaction!was!then!terminated!by!addition!of! 100! µL! of! 1! M! sodium! acetate! buffer,! pH! 4.0,! and! the! absorbance! of! the! released!p.nitroaniline! was! measured! at! 405! nm! using! a! Labsystems! iEMS! Reader! MF!(Labsystems!Oy,!Helsinki,!Finland).!!!The!concentrations!of!peptide! fractions!and!synthesized!peptides! required! to! cause!a!50%!inhibition!of!the!enzyme!activity!(IC50)!were!determined!from!the!cubic!regression!equations! generated! by! fitting! the! data! from! the! plot! of! percent! DPP.IV! inhibition!against!test!sample!concentrations.!The!IC50!value!and!mode!of!inhibition!of!a!reference!inhibitor,!the!tri.peptide!Ile.Pro.Ile!(diprotin!A),!were!also!determined!for!comparison.!!!!7.3.7$Determination$of$the$Modes$of$Inhibition$'The! most! potent! synthesized! peptides! were! studied! for! their! modes! of! action! on!DPP.IV.!The!initial!rate!of!hydrolysis!of!the!synthetic!substrate!Gly.Pro.p.NA!by!DPP.IV!was!measured!using!substrate!concentrations!ranging!from!38.5!to!385!µM!(final!assay!concentrations)! in! the! absence! and! presence! of! different! concentrations! of! the!inhibitors!(synthesized!peptides).!The!substrate!(25!µL),!with!or!without!inhibitor!(25!µL),!was!pre.incubated!at!37°C!for!10!min!and!the!reaction!was!started!by!addition!of!the!DPP.IV!enzyme!(50!µL!of!a!0.02!units/mL!solution).!The!absorbance!of!the!released!p.NA!was!monitored!at!37°C!and!405!nm!using!a!Tecan! Infinite®!200!Pro!microplate!reader!(ESBE!Scientific,!Markham,!ON,!Canada).!When!no!inhibitor!was!added,!25!µL!of!100!mM!Tris.HCl,!pH!8.0,!buffer!was!used.!!! 145!The! modes! of! inhibition! of! the! synthesized! peptides! were! determined! by! fitting! the!initial! velocity! versus! substrate! concentration! data! to! nonlinear! regression! models!using!the!GraphPad!Prism!software!(version!5,!GraphPad!Software,!San!Diego,!CA,!USA).!Lineweaver.Burk!plots!of!DPP.IV!activity! in! the!absence!and!presence!of! synthesized!peptides!were!created!using!the!aforementioned!software!to!help!visualize!the!modes!of!inhibition.!!!!'7.3.8$Statistical$Analysis$!Significant!differences!between!the!DPP.IV! inhibitory!activity!of! the!purified! fractions!and!synthesized!peptides!were!established!by!performing!one.way!analysis!of!variance!using!the!general!linear!model!and!pairwise!comparison!with!Tukey’s!method!(Minitab!Statistical! Software! Version! 16,!Minitab! Inc.,! State! College,! PA,! USA).! All! assays!were!performed!at!least!in!triplicate!and!significant!difference!was!established!at!P#<!0.05.!!!7.4!RESULTS!'7.4.1$Fractionation$of$Pepsin4Treated$Whey$Proteins$with$DPP4IV$Inhibitory$Activity$$'WPI! and! α.lactalbumin! hydrolysates! obtained! by! peptic! treatment,! and! displaying!DPP.IV!inhibitory!activity,!were!fractionated!by!successive!chromatographic!steps!and!the!resulting!fractions!assessed!for!the!effect!on!DPP.IV!activity.!!!7.4.1.1%Fractionation%of%Pepsin(Treated%Whey%Protein%Isolate%!A!WPI!hydrolysate!obtained!by!digestion!with!pepsin!was!first!fractionated!by!cation.exchange!chromatography!and!the!resulting!pooled!fractions!were!evaluated!for!their!DPP.IV!inhibitory!activity.!As!shown!in!Figure'7.1A,!seven!fractions!were!obtained!and!! 146!all!were!able! to! cause! the! inhibition!of!DPP.IV!activity.!The!WPI.4,!WPI.5!and!WPI.7!fractions,! with! 53,! 57! and! 53%! inhibition,! respectively,! were! found! to! be! the! most!effective! at! inhibiting! the! enzyme.! Their! potency! was! similar! to! that! of! the! un.fractionated!WPI! (55%! inhibition,! data! not! shown).! Conversely,! fraction!WPI.2,!with!18%! inhibition,! was! the! least! effective! at! inhibiting! DPP.IV! activity! (Figure' 7.1B).!Displaying! the! greatest! DPP.IV! inhibitory! activity! and! having! the! highest! peptide!content,! the! WPI.5! fraction! was! selected! to! undergo! further! purification! by! size.exclusion!chromatography.!!Fractionation! by! size! of! the! WPI.5! fraction! revealed! a! relatively! simple! molecular!weight! distribution! profile! with! only! two! main! peaks! (Figure' 7.2A).! The! WPI.5b!fraction,! comprised! of! peptides!with!molecular!weights! around! 1350!Da,! displayed! a!significantly! greater! DPP.IV! inhibitory! activity! than! the! WPI.5a! fraction! containing!larger!peptides!(47!and!15%!inhibition,!respectively)!(Figure'7.2B).!!!!!The!WPI.5b! fraction! from!size!exclusion!chromatography!was! further! fractionated!by!RP.HPLC.! As! shown! by! the! elution! profile! (Figure' 7.3A),! the! fraction! contained!primarily! non.polar! peptides.! Of! the! six! fractions! collected,!WPI.5b.3! and!WPI.5b.4,!which!eluted!at!about!27.28!minutes,!displayed!the!highest!DPP.IV! inhibitory!activity!(23!and!46%! inhibition,! respectively).!The!WPI.5b.6! fraction!was! also! able! to! inhibit!the! DPP.IV! enzyme,! but! not! as! effectively! (20%! inhibition),! whereas! the! WPI.5b.1,!WPI.5b.2!and!WPI.5b.5!had!low!or!no!inhibitory!activity!(Figure'7.3B).!!!!147!!!!!!!!!!!!! !!!Figure'7.1'Elution!profile!(A)!and!DPP7IV!inhibitory!activity!(B)!of!whey!protein!isolate!(WPI)!hydrolysate!fractions!obtained!by!cation7exchange!FPLC.!The!percent! inhibition!of!DPP7IV!activity!was!determined!using!0.094!mg/mL!sample!(final!assay!concentration).!Each!bar!represents!the!mean!and!standard!deviation!of!three!determinations.'!0 10 20 30 40 50 60 70 WPI-1 WPI-2 WPI-3 WPI-4 WPI-5 WPI-6 WPI-7 % DPP-IV inhibition Fraction !-0.2 0 0.2 0.4 0.6 0.8 1 1.2 0 10 20 30 40 50 60 70 80 90 Absorbance at 280 nm Elution time (min) WPI-1 WPI-3 WPI-2 WPI-4 WPI-5 WPI-6 WPI-7 ! ! ! !! !A B !148!!!!!!!!!!!! !Figure' 7.2' Elution! profile! (A)! and! DPP7IV! inhibitory! activity! (B)! of!WPI75! fractions! obtained! by! size7exclusion! FPLC.! The!percent! inhibition! of!DPP7IV! activity!was! determined! using! 0.094!mg/mL! and! 0.024!mg/mL! (final! assay! concentration)! of!fraction!WPI75a!and!WPI75b,!respectively.!Each!bar!represents!the!mean!and!standard!deviation!of!three!determinations.'!!!!!!'0 10 20 30 40 50 60 WPI-5a WPI-5b % DPP-IV inhibition Fraction !0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 90 100 Absorbance at 280 nm Elution time (min) WPI-5a WPI-5b 3800 Da 1350 Da A B !149!'''''''''''''' '!!Figure'7.3'Elution!profile!(A)!and!DPP7IV!inhibitory!activity!(B)!of!WPI75b!fractions!obtained!by!reversed7phase!HPLC.!The!percent!inhibition!of!DPP7IV!activity!was!determined!using!0.063!mg/mL!(final!assay!concentration).!Each!bar!represents!the!mean!and!standard!deviation!of!three!determinations.!!!mAU min WPI-5b-1 WPI-5b-2 WPI-5b-3 WPI-5b-4 WPI-5b-5 WPI-5b-6 -400    -200 5 10 15 20 25 30 40 0 0 200 400 60 800 1000 1200 1400 0 10 20 30 40 50 60 WPI-5b-1 WPI-5b-2 WPI-5b-3 WPI-5b-4 WPI-5b-5 WPI-5b-6 % DPP-IV inhibition Fraction A B ! 150!7.4.1.2&Fractionation&of&Pepsin4Treated&α4Lactalbumin&!An!α'lactalbumin!hydrolysate!produced!by!peptic!digestion!was!also!fractionated!by!successive!chromatography!steps.!Unlike!the!WPI,!which!was!composed!primarily!of!α'lactalbumin,! β'lactoglobulin! and! bovine! serum! albumin! (14,! 69! and! 3.3%,!respectively),! the! α'lactalbumin! product! consisted! of! almost! exclusively! the! α'lactalbumin! protein! and! some! trace! of! β'lactoglobulin! (Lacroix! &! Li'Chan,! 2013;!Chapter!6).!Therefore,!the!α'lactalbumin!hydrolysate!was!expected!to!contain!a!less!complex! peptide! mixture! than! the! WPI! hydrolysate.! For! this! reason,! the! pepsin'treated!α'lactalbumin!was!fractionated!only!on!the!basis!of!size!and!polarity.!!!!As!shown!in!Figure'7.4A,!the!α'lactalbumin!hydrolysate!contained!peptides!with!a!broad! spectrum! of!molecular!weights,! ranging! from! around! 2610!Da! to! less! than!250! Da.! Among! the! seven! fractions! collected,! α'la'1! and! α'la'2,! with! molecular!weights!of!about!2610!and!1410!Da!and!31!and!78%!inhibition,!respectively,!were!shown!to!be!the!most!effective!at!inhibiting!the!activity!of!the!enzyme.!The!potency!of! the! α'la'2! fraction! was! similar! to! that! of! the! un'fractionated! α'lactalbumin!hydrolysate!(70%!inhibition,!data!not!shown).!On!the!other!hand,!the!five!remaining!fractions!had!weak!or!no!effect!on!DPP'IV!activity!(Figure'7.4B).!!!Displaying! the! highest! DPP'IV! inhibitory! activity,! the! α'la'2! fraction! was! further!purified!by!RP'HPLC.!Among!the!seven! fractions!collected!(Figure' 7.5A),! the!non'polar! fractions!α'la'2e!and!α'la'2f,!eluting!at!27'28!minutes,!were! found!to!be!the!most!potent!(81!and!82%!inhibition,!respectively).!The!enzyme!was!also! inhibited!by!the!α'la'2c,!α'la'2d!and!α'la'2g!fractions,!but!to!a!lesser!extent.!The!more!polar!fractions! α'la'2a! and! α'la'2b! had! low! inhibitory! activity! (6! and! 5%! inhibition,!respectively)!(Figure'7.5B).!!!!!151!!!!!!!!!!!!!!! !!Figure'7.4'Elution!profile!(A)!and!DPP6IV!inhibitory!activity!(B)!of!α6lactalbumin!(α6la)!hydrolysate!fractions!obtained!by!size6exclusion!FPLC.!The!percent!inhibition!of!DPP6IV!activity!was!determined!using!0.125!mg/mL!(final!assay!concentration).!Each!bar!represents!the!mean!and!standard!deviation!of!three!determinations.'!!!!! !             !0 0.5 1 1.5 2 0 20 40 60 80 100 120 140 160 180 Absorbance at 280 nm Elution time (min) α-la-5 A 2610 Da 1410 Da 362 Da 248 Da α-la-1 α-la-2 α-la-3 α-la-4 α-la-7 α-la-6 α-la-5 0 10 20 30 40 50 60 70 80 90 α-la-1 α-la-2 α-la-3 α-la-4 α-la-5 α-la-6 α-la-7 % DPP-IV inhibition Fraction A B !152!!!!!Figure'7.5'Elution!profile!(A)!and!DPP6IV!inhibitory!activity!(B)!of!α6la62!fractions!obtained!by!reversed6phase!HPLC.!The!percent!inhibition!of!DPP6IV!activity!was!determined!using!0.063!mg/mL!(final!assay!concentration).!Each!bar!represents!the!mean!and!standard!deviation!of!three!determinations.!!   !5 10 15 20 25 30 0 min 250 500 750 1000 1250 1500 1750 mAU 0 α-la-2a α-la-2g α-la-2d α-la-2f α-la-2e α-la-2c α-la-2b 0 10 20 30 40 50 60 70 80 90 α-la-2a α-la-2b α-la-2c α-la-2d α-la-2e α-la-2f α-la-2g % DPP-IV inhibition Fraction A B ! 153!7.4.1.3& DPP)IV& Inhibitory& Activity& of& Fractions& Isolated& from& Pepsin)Treated& WPI& and& α)Lactalbumin&!The!potency!of!the!most!active!α4la!and!WPI!fractions!from!RP4HPLC!were!determined!and! compared! to! their! respective!un4fractionated!hydrolysate! (Table& 7.1).! ! Fractions!α4la42e!and!α4la42f,!with!IC50!values!of!0.019!and!0.016!mg/mL!respectively,!were!both!more!effective!at!inhibiting!the!DPP4IV!enzyme!than!the!un4fractionated!α4lactalbumin!hydrolysate!(IC50!=!0.036!mg/mL).!Conversely,!neither!of!the!WPI!hydrolysate!fractions!obtained!from!successive!chromatographic!separation!was!more!potent!than!the!whole!hydrolysate! (IC50! =! 0.075!mg/mL! for! the! whole! hydrolysate! versus! 0.216! and! 0.081!mg/mL!for!the!WPI45b43!and!WPI45b44!fractions,!respectively).!!!!!!!Table& 7.1&Dipeptidyl4peptidase! IV! inhibitory! activity! of!whey! protein! protein! isolate!(WPI)! and! α4lactalbumin! (α4la)! hydrolysates! obtained! by! peptic! treatment! and! the!most!active!RP4HPLC!fractions!isolated!from!them.&&Hydrolysate or fraction IC50 (mg/mL)a WPI hydrolysateb 0.075 ± 0.006 y WPI-5b-3 0.216 ± 0.038 z WPI-5b-4 0.081 ± 0.011 y α-lactalbumin hydrolysateb 0.036 ± 0.002 x α-la-2e 0.019 ± 0.001 w α-la-2f 0.016 ± 0.001 w a IC50 values are reported as the mean and standard deviations from triplicate determinations and expressed as final assay concentration. Values with different lower case letters are significantly different (P < 0.05). b IC50 values of the hydrolysates as reported in Chapter 6 and Lacroix & Li-Chan (2013).  !7.4.2% Identification%of% the%Peptide%Sequences%Present% in% the%Purified%Fractions%WPI:5b:3,%WPI:5b:4,%α:la:2e%and%α:la:2f%by%LC–ESI:MS/MS%!Being! the! most! effective! at! inhibiting! the! DPP4IV! enzyme,! the! fractions! WPI45b43,!WPI45b44,! α4la42e! and! α4la42f! from! RP4HPLC! were! analyzed! by! LC–ESI4MS/MS! to!identify!their!constituent!peptides.!The!147!unique!peptide!sequences!identified,!which!ranged! from!5! to! 17! amino! acids! in! length,! are! presented! in! the!Appendix!A! (Table&A.1).!!! 154!The!sequences!of!90!peptides!derived!from!β4lactoglobulin!and!19!from!α4lactalbumin!were! identified! in! the! WPI45b43! fraction,! while! 74! peptides! were! identified! in! the!WPI45b44!fraction,!65!of!these!deriving!from!β4lactoglobulin!and!the!remaining!9!from!α4lactalbumin.!Among! the! sequences! identified,!36!were! found! in!both!WPI! fractions.!On! the! other! hand,! 22! peptide! sequences! were! identified! in! the! α4la! fractions.! The!α4la42e! fraction!was! found! to! contain!2!peptides!derived! from!β4lactoglobulin!and!18!from! α4lactalbumin,! whereas! 1! peptide! derived! from! β4lactoglobulin! and! 8! from!α4lactalbumin!were! identified! in! the!α4la42f! fraction.! Seven! of! these! sequences! were!common! to! both! α4la! fractions.! Several! of! the! α4lactalbumin4! and! β4lactoglobulin4derived!peptide!sequences!identified!were!overlapping.!!!7.4.3%DPP:IV%Inhibitory%Activity%of%Synthesized%Peptides%!All!22!peptide!sequences!identified!in!the!α4la!fractions!as!well!as!13!of!the!sequences!identified! in! the! WPI! fractions! were! synthesized! and! evaluated! for! their! DPP4IV!inhibitory! activity! (Table& 7.2).! The! peptides!were! selected! based! on! their! structural!properties,! in!order! to! investigate! the!effect!of! length!and!amino!acid!composition!on!DPP4IV! activity.! The! location! of! the! selected! peptides! within! the! α4lactalbumin! and!β4lactoglobulin!sequences!is!presented!in!Figure&7.6.!!!!!!!!!!!! 155!!&Figure& 7.6& Mature! amino! acid! sequences! of! bovine! (A)! α4lactalbumin! (UniProt! KB!database! accession! number! P00711)! and! (B)! β4lactoglobulin! (UniProt! KB! database!accession!number!P02754).!Peptide!sequences!identified!in!the!α4lactalbumin!and!WPI!hydrolysate!fractions!that!were!selected!for!synthesis!are!underlined.&&!!10 20 30 40 50 60 70 80 90 110 100 120 1 1 10 20 30 40 50 60 70 80 90 100 110 120 130 150 160   A EQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPH SSNICNISCDKFLDDDLTDDIMCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL  B LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKK IIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKA LPMHIRLSFNPTQLEEQCHI  140 !156!Table&7.2&Dipeptidyl-peptidase!IV!inhibitory!activity!and!mode!of!inhibition!of!synthesized!peptides.&!Protein of origin Peptide sequence MW pI Fraction(s) % DPP-IV inhibitiona IC50 (µM)b Mode of inhibition α-lactalbumin WLAHKAL   838.02 8.76 WPI-5b-3 33  d 286  cd Non-competitive WLAHKALCSEKLDQ 1641.91 6.74 α-la-2e; α-la-2f; WPI-5b-3; WPI-5b-4 44  bc 141  b Un-competitive LAHKALCSEKL 1212.47 8.21 α-la-2e; α-la-2f; WPI-5b-3; WPI-5b-4 41  bcd 165  b Competitive LAHKALCSEKLDQ 1455.69 6.74 α-la-2e; α-la-2f; WPI-5b-3; WPI-5b-4 22  ef ND ND AHKALCSEKLDQ 1342.53 6.78 α-la-2e; WPI-5b-3 9  hijk ND ND KALCSEKLDQ 1134.31 6.06 α-la-2e 14  fgh ND ND ALCSEKLDQ 1006.14 4.37 α-la-2e 9  hijk ND ND LCSEKLDQ   935.06 4.37 α-la-2e 46  b 186  b Non-competitive TKCEVF   725.86 5.66 WPI-5b-3; WPI-5b-4 23 e ND ND TKCEVFRE 1011.16 5.81 α-la-2e; WPI-5b-3; WPI-5b-4 41  bcd 166  b Un-competitive RELKDLKG   958.13 8.59 WPI-5b-3; WPI-5b-4 -3  m ND ND RELKDLKGY 1121.30 8.50 α-la-2e; α-la-2f; WPI-5b-3; WPI-5b-4  2  klm ND ND RELKDLKGYG 1178.35 8.50 α-la-2e  3  jklm ND ND RELKDLKGYGG 1235.41 8.50 α-la-2e; α-la-2f; WPI-5b-3  9  hijk ND ND RELKDLKGYGGVS 1421.62 8.50 α-la-2e; α-la-2f  7  hijk ND ND RELKDLKGYGGVSL 1534.78 8.50 α-la-2e 11  hij ND ND TFHTSGYDTQA 1227.25 5.05 α-la-2e  6  hijklm ND ND AIVQNNDSTE 1090.11 3.67 α-la-2e; α-la-2f; WPI-5b-3  5  ijklm ND ND IVQNNDSTE 1019.03 3.67 α-la-2f 13  ghi ND ND IVQNNDSTEY 1182.21 3.67 α-la-2e 23 e ND ND IVQNNDSTEYGLF 1499.60 3.67 α-la-2e 37  cd 337  d Non-competitive CVKKIL   702.95 9.31 WPI-5b-3; WPI-5b-4 -2  m ND ND CVKKILD   818.04 8.20 WPI-5b-3; WPI-5b-4   1  klm ND ND ILDKVGINY 1034.22 5.83 α-la-2e 34 d 263  c Competitive β-lactoglobulin LIVTQTMKG   990.23 8.75 WPI-5b-3; WPI-5b-4 10  hijk ND ND  LIVTQTMKGLD 1218.47 5.84 WPI-5b-3; WPI-5b-4   9  hijk ND ND  LIVTQTMKGLDIQ 1459.76 5.84 WPI-5b-3; WPI-5b-4   8  hijk ND ND  IVTQTMKGLD 1105.31 5.84 α-la-2e; WPI-5b-3; WPI-5b-4 22  ef ND ND  IVTQTMKGLDIQ 1346.60 5.84 WPI-5b-3; WPI-5b-4 20  efg ND ND  TMKGLDIQ   905.08 5.50 WPI-5b-3; WPI-5b-4 -1  lm ND ND  ELKPTPEGD   985.06 4.14 WPI-5b-3; WPI-5b-4  3  jklm ND ND !157!         Table&7.2&continued)Protein of origin Peptide sequence MW pI Fraction(s) % DPP-IV inhibitiona IC50 (µM)b Mode of inhibition  LKPTPEGDL   969.10 4.37 α-la-2e; WPI-5b-4 73 a   45  a Un-competitive  LKPTPEGDLEIL 1324.54 4.14 WPI-5b-4 67  a   57  a Un-competitive  IPAVFKIDA   973.18 5.84 WPI-5b-4 38  bcd 191  b Competitive  VRTPEVDD   929.98 4.03 α-la-2f; WPI-5b-3; WPI-5b-4 20  efg ND ND diprotin Ac IPI   341.45 5.52 NA       NA 4.7 Competitive a The percent DPP-IV inhibition values are reported as the mean from triplicate determinations and measured using 125 µM of peptides (final assay concentration). Percent inhibition values not sharing any common lower case letters are significantly different (P < 0.05). b IC50 values are reported as the mean from triplicate determinations and expressed as final assay concentration. Values not sharing any common lower case letters are significantly different (P < 0.05). c The inhibitory activity and mode of inhibition of diprotin A were included as references. ND, not determined; NA, not applicable. !! 158!The!β(lactoglobulin(derived!peptides!LKPTPEGDL!and!LKPTPEGDLEIL,!with!IC50!values!of! 45! and! 57! µM,! respectively,! were! found! to! be! the!most! effective! at! inhibiting! the!DPP(IV! enzyme.! These! peptides! were,! however,! about! 10! times! less! potent! than! the!reference! inhibitor!diprotin!A!(4.7!µM).!Although!not!as!effective,! the!β(lactoglobulin(derived! ennea(peptide! IPAVFKIDA! was! also! able! to! inhibit! the! enzyme.! With! the!exception! of! the! TMKGLDIQ! peptide,!which! did! not! inhibit! DPP(IV,! the! other! peptide!sequences!derived!from!β(lactoglobulin!tested!had!low!inhibitory!activity.!!!Among! the! α(lactalbumin(derived! peptides,! WLAHKALCSEKLDQ,! LAHKALCSEKL,!TKCEVFRE! and! LCSEKLDQ! showed! the! highest! inhibitory! activity! (IC50! values! =! 141,!165,! 166! and! 186! µM,! respectively).! The! peptides! ILDKVGINY,! WLAHKAL! and!IVQNNDSTEYGLF!were!also!able!to!inhibit!the!DPP(IV!enzyme,!albeit!to!a!lesser!extent.!Conversely,!the!hexa(peptide!CVKKIL!and!the!octa(peptide!RELKDLKG!had!no!effect!on!the!activity!of!the!enzyme.!The!remaining!15!α(lactalbumin(derived!peptides!tested!had!only!limited!inhibitory!activity.!!!7.4.4$Modes$of$Inhibition$of$Synthesized$Peptides$$!The!synthesized!peptides!found!to!cause!the!greatest!inhibition!of!DPP(IV!activity!were!further! investigated! to! determine! their! modes! of! action! on! the! enzyme.! Similarly! to!diprotin! A,! the! peptides! IPAVFKIDA,! LAHKALCSEKL! and! ILDKVHINY! were! shown! to!inhibit! the! DPP(IV! enzyme! in! a! competitive!manner,!whereas! LCSEKLDQ,!WLAHKAL,!and! IVQNNDSTEYGLF! behaved! as! non(competitive! inhibitors! and! LKPTPEGDL,!LKPTPEGDLEIL,! WLAHKALCSEKLDQ! and! TKCEVFRE! exhibited! un(competitive!behavior.! The! Lineweaver(Burk! plots! for! peptides! LAHKALCSEKL,! LKPTPEGDL! and!LCSEKLDQ,! which! were! representative! of! the! competitive,! un(competitive! and! non(competitive!modes!of!inhibition!exhibited!by!the!peptides!in!this!study,!are!presented!in!Figure'7.7.!!!!! 159!!!''' '''Figure' 7.7' Lineweaver(Burk! plots! of! DPP(IV! activity! in! the! presence! of! (A)! peptide!LAHKALCSEKL!(250–1250!µM,!final!assay!concentration),!(B)!peptide!LKPTPEGDL!(63–250!µL,!final!assay!concentration)!and!(C)!peptide!LCSEKLDQ!(188–750!µM,!final!assay!concentration).!!Plots!show!results!from!triplicate!determinations.!'''! !!-0.01 0.00 0.01 0.02 0.031000200030001250 µM750 µM250 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.01 0.00 0.01 0.02 0.031000200030001250 µM750 µM250 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.01 0.00 0.01 0.02 0.031000200030001250 µM750 µM250 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.01 0.00 0.01 0.02 0.031000200030001250 µ750 µ250 µ0 µ1/[ ly-Pro-pNA] (1/µ )1/V 0(1/Abs per min)1/V 0(1/Abs per min)!-0.03 -0.02 -0.01 0.00 0.01 0.02 0.03100200300400500250 µM125 µM63 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.03 -0.02 -0.01 0.00 0.01 0.02 0.03100200300400500250 µM125 µM63 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.03 -0.02 -0.01 0.00 0.01 0.02 0.03100200300400500250 µM125 µM63 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)1/V 0(1/Abs per min)1/V 0(1/Abs per min)!-0.01 0.00 0.01 0.02 0.03500100015002000750 µM375 µM188 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)-0.01 0.00 0. 1 0. 2 0. 3500100015002000750 µM375 µM188 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)- . . . . ./[ l - r - ] ( / )1/V 0(1/Abs per min)-0.01 0. 0 0. 1 0. 2 0. 3500100015002000750 µM375 µM188 µM0 µM1/[Gly-Pro-pNA] (1/µM)1/V 0(1/Abs per min)1/V 0(1/Abs per min)A B C ! 160!7.5!DISCUSSION!!!While! the! fractionation! of! the! α(lactalbumin! hydrolysate! led! to! the! generation! of!peptide! fractions! with! more! potent! DPP(IV! inhibitory! activity! than! the! starting!hydrolysate! (Table' 7.1),! none! of! the! individual! peptides! identified! in! α(la(5e! and!α(la(5f! (Table' 7.2)! were! as! potent! as! the! whole! fractions.! Conversely,! the! peptide!fractions! obtained! from! the! successive! chromatographic! separation! of! the! WPI!hydrolysate! by! charge,! size! and! polarity! were! not! significantly! more! effective! at!inhibiting! the! DPP(IV! enzyme! than! the! crude! hydrolysate! (Table' 7.1).! Among! the!peptide!sequences!tested,!only!LKPTPEGDL!and!LKPTPEGDLEIL,!both!identified!in!the!WPI(5b(4! fraction,!were!more! effective! at! inhibiting! DPP(IV! than! the! un(fractionated!hydrolysate!(Table'7.2).!!!In! a! previous! investigation! on! the! DPP(IV! inhibitory! activity! of! pepsin(treated! whey!proteins,! including! α(lactalbumin,! β(lactoglobulin,! bovine! serum! albumin,! lactoferrin!and! WPI,! it! was! found! that! the! peptic! hydrolysis! of! β(lactoglobulin! was! the! least!effective! at! inhibiting! the! activity! of! DPP(IV,! whereas! the! α(lactalbumin! and! WPI!hydrolysates! caused! the! greatest! inhibition! of! the! enzyme! (Lacroix! &! Li(Chan,! 2013;!Chapter! 6).! Alpha(lactalbumin! being! the! second! main! constituent! of! WPI,! it! was!hypothesized! that! α(lactalbumin(derived! peptides! might! be! among! the! active!components! responsible! for! the! enzymatic! inhibition! observed! with! the! WPI!hydrolysate.! Although! some! of! the! α(lactalbumin(derived! peptides! identified! in! the!WPI(5b(3!and!WPI(5b(4!fractions!were!able!to! inhibit!the!activity!of!DPP(IV,!the!most!potent! peptides,! LKPTPEGDL! and! LKPTPEGDLEIL,! were! derived! from! the!β(lactoglobulin!protein.!This!discrepancy!may!be!due!to!distinct!β(lactoglobulin(derived!peptides! being! generated! by! the! pepsin! treatment! of! WPI! and! β(lactoglobulin.!Alterations! in! protein! molecular! conformation! arising! from! the! different! processes!involved! in! production! of! β(lactoglobulin! and!WPI,! as! well! as! the! presence! of! other!whey!proteins!in!the!WPI,!may!have!affected!the!accessibility!and!susceptibility!of!the!peptide! bonds! in! β(lactoglobulin! to! peptic! digestion! and,! consequently,! the! peptides!! 161!released.! Therefore,! it! is! possible! that! more! potent! and/or! a! greater! number! of! β(lactoglobulin(derived!peptides!were!generated!from!the!WPI!than!from!β(lactoglobulin.!!!!!The!most!potent!peptides! identified!both!have!a!proline! residue!at! the! third!position!(P1#),!a!structural! feature!found!in!several!peptides!reported!to! inhibit!DPP(IV!activity!(Lacroix!&!Li(Chan,!2012a;!Chapter!4).!While!proline!at!the!second!position!from!the!N(terminal! (P1)" of! the! peptide! is! believed! to! be! the! preferred! amino! acid! residue! for!DPP(IV!activity,!substrates!with!proline,!hydroxyproline,!or!N(methyl!glycine!at!the!P1#!position! cannot! be! hydrolyzed! by! DPP(IV! (Lambeir! et! al.,! 2003;! Lorey! et! al.,! 2003).!Among! all! peptides! identified! in! the! WPI! and! α(la! fractions,! only! the! sequences!LPMHIRL,! IPAVFKID! and! IPAVFKIDA! had! a! proline! residue! at! their! P1!position.! The!shorter!fragments!IPA!(Hernández(Ledesma!et!al.,!2008),!IPAVF!and!IPAVFK!(Silveira!et!al.,!2013)!have!also! recently!been! identified! in!β(lactoglobulin!hydrolysates.!Although!these! peptides! all! have! the! same! three! first! amino! acid! residues! at! their! N(terminal,!IPAVF! and! IPAF! (IC50! =!49! and!44.7!µM,! respectively)!were! found! to!be!more!potent!DPP(IV! inhibitors! than! the! longer!peptides! IPAVFK!and! IPAVFKIDA! (IC50!=!143.0!and!191! µM,! respectively).! The! tri(peptide! diprotin! A! (IPI),!which! also! contains! a! proline!residue! at! its! P1! position,! figures! among! the! most! potent! peptides! with! DPP(IV!inhibitory!activity!reported!in!the!literature.!Despite!its!apparent!competitive!behavior,!it!was!found!to!actually!be!a!substrate!for!the!DPP(IV!enzyme!with!a!low!turnover!rate!(Rahfeld!et!al.,!1991).! It! is!possible! that,!similarly! to!diprotin!A,!whey!protein(derived!peptides!bearing!a!proline!residue!at!their!P1!position,!such!as!IPAVFKIDA,!may!also!act!as!substrates!for!the!enzyme!and!that!their!apparent!inhibitory!activity!may!be!a!kinetic!artifact!resulting!from!their!substrate(like!structure.!!!Although!the!amino!acid!residues!surrounding!the!scissile!bond!may!play!a!role!in!the!inhibitory!activity!of!a!peptide,!other!structural!characteristics,!such!as!the!length!and!amino!acid!composition,!have!also!been!suggested!to!have!an!influence!(Kühn(Wache!et!al.,!2011).!Peptides!varying!primarily!from!two!to!thirteen!amino!acids! in! length!have!been!reported!in!the!literature!to!display!DPP(IV!inhibitory!activity!(Lacroix!&!Li(Chan,!! 162!2012a;!Chapter!4).!In!the!present!study,!the!most!potent!peptides!identified!contained!seven! to! fourteen!amino!acid! residues.! In!order! to! further! assess!how!peptide! length!affects!DPP(IV!activity,! sets!of!successively! longer!peptides!sharing! the!same! first! few!amino! acids! at! the! N(terminal! were! selected! among! the! α(lactalbumin(! and!β(lactoglobulin(derived!fragments!identified!in!the!WPI!and!α(la!fractions!(Table'7.2).!The!addition!of!the!residues!Arg!and!Glu!at!the!C(terminal!of!the!peptide!TKCEVF!led!to!a! significant! increase! in! the! DPP(IV! inhibitory! activity.! Similarly,! the! addition! of! the!residues!Tyr,!Gly,!Leu!and!Phe!to!the!peptide!IVQNNDSTE!and!the!residues!Cys,!Ser,!Glu,!Lys,! Leu,! Asp,! Gln! to! the! fragment! WLAHKAL! significantly! augmented! the! DPP(IV!inhibitory!activity.!An! increase! in! length!did!not,!however,!always! result! in! inhibitory!peptides!with!greater!potency.!The!hendeca(peptide!LAHKALCSEKL!was!more!effective!at!inhibiting!the!DPP(IV!enzyme!than!the!triskaideca(peptide!LAHKALCSEKLDQ.!On!the!other!hand,!no!significant!effects!on!DPP(IV!activity!were!observed!by!adding!the!amino!acid!residues!Tyr,!Gly,!Gly,!Val,!Ser!and!Leu!to!the!fragment!RELKDLKG!or!the!residues!Leu,! Asp,! Ile! and! Gln! to! the! peptide! LIVTQTMKG.! Similarly,! the! peptide! sequences!CVKKIL! and! CVKKILD! as!well! as! IVTQTMKGLD! and! IVTQTMKGLDIQ! did! not! differ! in!their!effect!on!DPP(IV.!!!In!a!similar!way,!the!net!charge!of!the!peptides!did!not!seem!to!have!a!predictable!effect!on! their! potency.! Peptides! with! identical! isoelectric! point! (pI),! such! as! the! ennea(peptides! LKPTPEGDL! and! ALCSEKLDQ,! were! found! to! display! significantly! different!DPP(IV! inhibitory! activity! (73! and! 9%,! respectively)! (Table' 7.2).! The! polarity! of! the!peptides!also!did!not!appear!to!have!a!predictable!impact!on!their!ability!to!inhibit!DPP(IV.!Although!all! sequences!shown! in!Table' 7.2!were! identified! in! the!more!non(polar!fractions! eluted! from!RP(HPLC,! not! all!were! found! to! be! active.! Thus,! the! ability! of! a!peptide! to! inhibit! the! DPP(IV! enzyme! seems! to! be! predominantly! determined! by! its!specific!amino!acid!sequence.!!The!peptides!displaying!the!highest!DPP(IV!inhibitory!activity!showed!different!modes!of!inhibition!(Figure'7.7)!suggesting!that!they!may!inhibit!DPP(IV!by!targeting!different!! 163!domains!of!the!enzyme.!The!peptides!that!exhibit!a!competitive!mode!of!action!inhibit!the! enzyme!by!binding! to! and!blocking! its! active! site!whereas! peptides! acting! as! un(competitive! inhibitors!affect!enzymatic!activity!by!binding!outside!the!catalytic!center!of! the! enzyme(substrate! complex! and! slowing! down! the! catalysis.! Similarly,! peptides!acting!as!non(competitive!inhibitors!bind!outside!the!catalytic!center,!but!can!do!so!on!either! the!enzyme!or! the!enzyme(substrate!complex.!Since! the!peptides! identified!are!acting!on!different!sites!of!the!enzyme,!it!is!possible!that,!when!present!in!combination,!they!operate!synergistically!by!targeting!different!domains!of!the!enzyme,!thus!causing!a!greater!inhibition!than!the!individual!peptides!acting!alone.!!!!!!7.6!CONCLUSION!'Findings!from!this!study!showed!that!the!hydrolysis!of!whey!proteins!by!the!digestive!enzyme! pepsin! generated! α(lactalbumin(! and! β(lactoglobulin(derived! peptides! with!inhibitory! activity! against! the! enzyme! DPP(IV.! These! active! peptides! were! found! to!inhibit! DPP(IV! activity! in! either! a! competitive,! un(competitive! or! non(competitive!manner,!indicating!that!they!may!exert!their!inhibitory!activity!by!binding!to!the!active!site!or!outside!the!catalytic!center!of!the!enzyme.!The!individual!whey!protein(derived!peptides! generally! showed!a! lower!potency! than! the!hydrolysates! and! fractions! from!which!they!were!isolated,!suggesting!that!they!may!act!in!synergy!with!one!another.!!!Although!it!is!not!possible!to!conclude!from!these!results!that!the!glucose!lowering!and!insulinotropic!properties!of!whey!protein!are!linked!to!its!content!of!DPP(IV!inhibitory!peptides,!the!present!findings!do!suggest!that!the!enzymatic!digestion!of!whey!protein!can! generate! peptides! with! inhibitory! activity! against! the! DPP(IV! enzyme! that! could!help!improve!blood!glucose!regulation.!Additional!investigation!is,!however,!needed!to!evaluate!the!efficacy!of!whey!protein(derived!DPP(IV!inhibitors!in!humans.!!!!!!!!! !! 164!CHAPTER! 8:! PEPTIDE! ARRAY!ON! CELLULOSE! SUPPORT!—!A! SCREENING! TOOL! TO!IDENTIFY!PEPTIDES!WITH!DIPEPTIDYL;PEPTIDASE! IV! INHIBITORY!ACTIVITY!WITHIN!THE!SEQUENCE!OF!α;LACTALBUMIN9!!8.1!SUMMARY!!The! inhibition! of! the! enzyme! dipeptidyl(peptidase! IV! (DPP(IV)! is! an! effective!pharmacotherapeutic!approach!for!the!management!of!type!2!diabetes.!Recent!findings!have! suggested! that! dietary! proteins,! including! bovine! α(lactalbumin,! could! be!precursors!of!peptides!able!to!inhibit!DPP(IV.!However,!information!on!the!location!of!active! peptide! sequences! within! the! proteins! is! far! from! being! comprehensive.!Moreover,! the! traditional! approach! to! identify! bioactive! peptides! from! foods! can! be!tedious! and! long.! Therefore,! the! objective! of! this! study!was! to! use! peptide! arrays! to!screen!α(lactalbumin(derived!peptides!for!their!interaction!with!DPP(IV.!Deca(peptides!spanning!the!entire!α(lactalbumin!sequence!with!a!frame!shift!of!1!amino!acid!between!successive! sequences! were! synthesized! on! cellulose! membranes! using! “SPOT”!technology!and!their!binding!to!and!inhibition!of!DPP(IV!were!studied.!Among!the!114!α(lactalbumin(derived!decamers!investigated,!the!peptides!60WCKDDQNPHS69!(αKi!=!76!µM),! 105LAHKALCSEK114! (Ki! =! 217! µM)! and! 110LCSEKLDQWL119! (Ki! =! 217! µM)! were!among!the!strongest!DPP(IV! inhibitors.!While! the!SPOT(!and!traditionally(synthesized!peptides! showed! consistent! trends! in! DPP(IV! inhibitory! activity,! the! cellulose(bound!peptides’!binding!behavior!was!not!correlated!to!their!ability!to!inhibit!the!enzyme.!This!research! showed! for! the! first! time! that! peptide! arrays! are! useful! screening! tools! to!identify!DPP(IV!inhibitory!peptides!from!dietary!proteins.!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!9!A!version!of! this!chapter!has!been!published.!Lacroix,! I.M.E.,!Li(Chan,!E.C.Y.!Peptide!array!on!cellulose! support! —! a! screening! tool! to! identify! peptides! with! dipeptidyl(peptidase! IV!inhibitory!activity!within!the!sequence!of!α(lactalbumin.!Int."J."Mol."Sci."2014,!15,!20846–20858.!! 165!8.2!INTRODUCTION!!Dietary! proteins! are! known! to! be! instrumental! in! a! wide! range! of! nutritional! and!biological! processes! (Jahan(Mihan! et! al.,! 2011).!Over! the! last! two!decades,! a! growing!body! of! research! has! shown! that! they! can! be! precursors! of! an! array! of! biologically!active! peptides! that! have! the! potential! to! improve! human! health! (Shahidi! &! Zhong,!2008).!!Even!though!bioactive!peptides!have!been!found!encrypted!in!the!sequence!of!a!number! of! proteins! from! both! plant! and! animal! sources,!milk! proteins! are! currently!considered! the! most! important! precursors! of! peptides! with! biological! activities!(Korhonen,!2009).!In!addition!to!containing!protein!fragments!with!anti(hypertensive,!anti(bacterial,! anti(cariogenic,! anti(oxidative,! mineral! binding,! opioid! and!immunomodulating! activities! (Hartmann!&!Meisel,! 2007;! Jauregi,! 2008;!Kamau! et! al.,!2010),! dietary! proteins,! particularly!whey! proteins,! have! recently! been! found! to! also!contain! within! their! sequence! peptides! able! to! inhibit! the! activity! of! the! enzyme!dipeptidyl(peptidase! IV!(DPP(IV)! (Dziuba!et!al.,!2009;!Tulipano!et!al.,!2011;!Uchida!et!al.,! 2011;! Uenishi! et! al.,! 2012;! Nongonierma!&! FitzGerald,! 2013b;! Lacroix! &! Li(Chan,!2012a;!Lacroix!&!Li(Chan,!2012b;!Lacroix!&!Li(Chan!2013,! Lacroix!&!Li(Chan!2014c).!The! DPP(IV! enzyme! is! known! to! inactivate! the! incretins! glucose(dependent!insulinotropic!polypeptide!(GIP)!and!glucagon(like!peptide(1!(GLP(1),!two!gut!derived(hormones! that! play! crucial! roles! in! glucose! regulation! by! stimulating! pancreatic!glucose(dependent!insulin,!suppressing!glucagon!release,!promoting!β(cell!proliferation!and! survival,! retarding! gastric! emptying! and! modulating! appetite! (Lim! &! Brubaker,!2006;! Phillips! &! Prins,! 2011).! Prolonging! the! half(lives! of! the! incretin! hormones! by!administration! of! orally! available! DPP(IV! inhibitors! such! as! the! peptidomimetic!compounds!sitagliptin,!vildagliptin!and!saxagliptin,!is!currently!a!promising!strategy!for!the!management!of! type!2!diabetes! (Deacon,!2011).! !Although!peptides!derived! from!dietary!proteins!have!not!yet!been!shown!to!prevent!the!degradation!of!the!incretins!in"vivo,! the!discovery!of! their!ability! to! inhibit! the!activity!of! the!DPP(IV!enzyme! in"vitro"has!triggered!great!interest!in!the!bioactive!peptide!research!area.!!! 166!The! traditional! approach! to! study! bioactive! peptides! from! dietary! proteins! typically!involves!a!number!of!steps,!such!as!hydrolysis!of!the!proteins!by!enzymatic!treatment,!isolation!of!the!active!peptides,!identification!of!the!peptides’!amino!acid!sequence!and!finally! chemical! synthesis! of! the! identified! peptides! for! validation! of! their! biological!activity! (Khaldi,! 2012;!Udenigwe,!2014).!This!methodology!has! recently!been!used! to!identify! peptides! with! DPP(IV! inhibitory! activity! from! casein! (Uenishi! et! al.,! 2012),!whey!(Lacroix!&!Li(Chan,!2014c),!fish!(Li(Chan!et!al.,!2012;!Huang!et!al.,!2012)!and!rice!bran!(Hatanaka!et!al.,!2012)!proteins.!However,!this!empirical!way!of!studying!bioactive!peptides!is!rather!tedious!and!presents!a!number!of!limitations.!It!is!technically!nearly!impossible! to!characterize!all!bioactive!peptides!present!within!a!protein!hydrolysate!and! only! those! that! are! released! from! the! parent! protein! during! the! enzymatic!treatment! can! be! identified! by! this! approach.! Another! investigation! strategy! that! has!been!successfully!used!to!identify!bioactive!peptides!consists!of!chemically!synthesizing!amino! acid! fragments! found! within! dietary! proteins! based! on! their! structural!properties!and!similarities!with!peptides!previously!reported!to!have!known!activities!(Udenigwe,!2014).!Yet,!synthesizing!and!screening!a!large!number!of!peptides!using!the!traditional!methods! for!peptide!synthesis!can!be!expensive!and! time!consuming,! thus!limiting!the!applicability!of!this!approach!(Hilpert!et!al.,!2007).!!!First! introduced! more! than! two! decades! ago,! peptide! array! technology! has! been!developed!as!a!complementary!method!to!the!traditional!solid!phase!peptide!synthesis!to! allow! the! parallel! production! of! hundreds! to! thousands! of! peptides! (Hilpert! et! al.,!2007).!Cellulose(bound!peptide!arrays,!which!are!cellulose!membranes!on!which!small!amounts! of! peptides! are!built,! have!been!used! as! screening! tools! for! a!wide! range!of!applications,! including! the! study!of! peptide(antibody,! peptide(receptor,! peptide(metal!ion!and!peptide(enzyme!interactions.!In!addition,!peptide!arrays!can!also!be!utilized!in!assays!requiring!soluble!peptides!by!cleaving!them!off!the!membrane!(Min!&!Mrksich,!2004;! Hilpert! et! al.,! 2007;! Volkmer! et! al.,! 2012).! Despite! the! numerous! possible!applications!of!peptide!arrays,!to!the!author’!knowledge,!this!approach!has!never!been!used!to!identify!bioactive!peptides,!such!as!DPP(IV!inhibitors,!from!dietary!proteins.!!! 167!The!objective!of! this!study!was!to!evaluate! the!potential!of!peptide!arrays! to!serve!as!screening! tools! to! identify! DPP(IV! inhibitory! peptides.! Using! SPOT! technology,! deca(peptides!spanning!the!entire!sequence!of!α(lactalbumin,!a!protein!previously!found!to!contain! within! its! primary! sequence! fragments! able! to! inhibit! the! activity! of! DPP(IV!(Lacroix!&!Li(Chan,!2013;!Lacroix!&!Li(Chan,!2014c;!Chapter!6!&!7),!were!synthesized!on! cellulose! membranes! and! their! binding! to! and! inhibition! of! DPP(IV! were!investigated.!!!!!!!!!!!!8.3!MATERIALS!AND!METHODS!"8.3.1$Materials$"Recombinant!human!dipeptidyl(peptidase!IV!(DPP(IV,!EC!3.4.14.5,!expressed!in!HEK293!human! cells,! >! 95%! purity,! activity! >! 5500! pmoles! Gly(Pro(7(amido(4(methylcoumarin/min/µg)! was! purchased! from! Creative! BioMart! (Shirley,! NY,! United!States)! while! polyclonal! goat! DPP(IV! antibody! and! polyclonal! donkey! anti(goat!secondary! antibody! conjugated! to! horseradish! peroxidase! were! obtained! from! R&D!Systems!(Minneapolis,!MN,!United!States).!PierceTM!ECL!western!blotting!substrate!and!protein(free! (TBS)! blocking! buffer!were! from!Thermo! Scientific! (Rockford,! IL,! United!States).! Gly(Pro(p(nitroanilide! (H(Gly(Pro(p(NA⋅HCl)! was! purchased! from! Bachem!Americas! (Torrance,! CA,! United! States).! Synthesized! peptides! (≥! 95%! purity)! were!prepared!and!purified!by!GL!Biochem!(Shanghai)!Ltd!(Shanghai,!China).!!8.3.2$Peptide$Array$Synthesis$!Peptide! arrays! of! deca(peptides! spanning! the! entire! α(lactalbumin! sequence! with! a!frame!shift!of!one!amino!acid!between!successive!sequences!were!prepared!by!Kinexus!Bioinformatics! Corporation! (Vancouver,! BC,! Canada).! The! peptides! were! synthesized!from!the!C(terminal!amino!acid!using! the!SPOT! technology!consisting!of! the!stepwise!! 168!coupling!of! the!Fmoc(protected!amino!acids!and!cleavage!of! the!side!chain!protection!groups.! The! α(lactalbumin(derived! peptides! were! synthesized! on! trioxatridecanedi(amine! (TOTD)! and! β(alanine(modified! Whatman! 540! cellulose! membranes! for! the!binding!and!inhibition!assays,!respectively.!For!the!inhibition!assay,!the!peptides!were!synthesized! in! large!dots! (5–7!mm! in!diameter)! and! the! quality! of! the! synthesis!was!verified!by!HPLC!analysis!of!quality!control!spots!included!on!the!membranes.!!Along! with! the! 114! deca(peptides! derived! from! the! α(lactalbumin! sequence,! seven!additional!peptides!were!included!on!the!arrays!(Appendix'B,'Table'B.2).!Each!array!was!prepared!in!duplicate.!!8.3.3$Probing$of$the$Membrane@Bound$Peptide$Arrays$!The! membranes! were! first! blocked! with! protein(free! (Tris(buffered! saline! (TBS))!blocking!buffer!containing!0.05%!Tween(20!for!4!hours!at!room!temperature!and!then!incubated!with!DPP(IV!(10!µg/mL!in!blocking!buffer)!overnight!at!4˚C.!The!membranes!were!washed!with!TBS!(50!mM!Tris,!136!mM!NaCl,!0.05%!Tween(20,!pH!8.0)!four!times!for! 15! min! each,! after! which! they! were! incubated! with! the! polyclonal! goat! DPP(IV!antibody!(0.2!µg/mL)!for!eight!hours!at!4˚C.!The!membranes!were!once!again!washed!with!TBS!four!times!for!15!min!each!prior!to!being!incubated!with!anti(goat!secondary!antibody! conjugated! to! horseradish! peroxidase! (0.1! µg/mL)! overnight! at! 4˚C.! Finally,!the!membranes!were!washed! four!more! times! for! 15!min! each;! then! a! thin! layer! of!chemiluminescence! detection! mixture,! prepared! according! to! the! manufacturer’s!instructions,!was!applied!for!1!min!and!the!luminescence!generated!from!the!spots!on!the!membranes!with!bound!DPP(IV!was!measured!using!a!ChemiDocTM!MP!system!(Bio(Rad!Laboratories!(Canada)!Ltd,!Mississauga,!ON,!Canada).!!!!!!! 169!8.3.4$DPP@IV$Inhibition$Assay$on$SPOT@Synthesized$Peptides$!In!order!to!determine!the!effect!of!the!SPOT(synthesized!peptides!on!the!activity!of!the!DPP(IV! enzyme,! the! peptides! were! first! cleaved! from! the! cellulose! support! by!incubating! the!membranes!with!gaseous!ammonia!overnight.! Peptide! spots! (5!mm! in!diameter)! were! then! punched! out! using! a! single(hole! punch! and! transferred! into!microcentrifuge!tubes!containing!150!µL!of!acetonitrile!(50%!in!100!mM!Tris(HCl!buffer!pH!8.0).!The!peptide!solution!(50!µL)!was!pipetted!into!a!96(well!microplate!with!35!µL!of! the! chromogenic! substrate! Gly(Pro(p(NA! (0.29!mM! in! 100!mM!Tris(HCl! buffer! pH!8.0).!The!mixture!was!pre(incubated! for!10!min!at!37! ˚C,!whereupon!15!µL!of!DPP(IV!(25!mU/mL)!was!added!and!the!enzymatic!reaction!was!carried!out!for!30!minutes!at!37! ˚C.!The!reaction!was! then! terminated!by!addition!of!100!µL!of!1!M!sodium!acetate!buffer!pH!4.0!and! the!absorbance!of! the!released!p(nitroaniline!was!measured!at!405!nm!using!a!Tecan!Infinite®!200!Pro!microplate!reader!(ESBE!Scientific,!Markham,!ON,!Canada).!Positive!and!negative!controls!(DPP(IV!activity!with!no!inhibitor!and!no!DPP(IV! activity,! respectively)! were! prepared! using! punched! peptide(free! cellulose! spots!from! the! same! membrane! used! for! peptide! synthesis,! and! adding! Tris(HCl! buffer! in!place!of!the!sample!and!in!place!of!the!sample!and!enzyme!solution,!respectively.!!!One!unit!of!DPP(IV!was!defined!as!the!concentration!of!enzyme!that!releases!1!µmole!per!minute!of!p(nitroaniline!from!Gly(Pro(p(NA!(100!µM)!at!37!˚C!and!pH!8.0.!!!8.3.5$DPP@IV$Inhibition$Assay$on$Traditionally@Synthesized$Peptides$!A!number!of!SPOT(synthesized!peptides!that!either!showed!no/low!DPP(IV!inhibitory!activity!or!that!were!found!to!be!potent!inhibitors!were!obtained!by!the!traditional!solid!phase!peptide!synthesis!to!confirm!their!effect!on!the!enzyme.!!The!peptides!were!dissolved!in!a!mixture!of!acetonitrile!and!H2O!and!further!diluted!in!100!mM!Tris(HCl!pH!8.0!buffer!to!the!desired!concentrations.! In!a!96(well!microplate,!! 170!25!µL!of!sample!were!pre(incubated!with!25!µL!of!100!mM!Tris(HCl!pH!8.0!buffer!and!35!µL!of!Gly(Pro(p(NA!(0.29!mM!in!100!mM!Tris(HCl!buffer!pH!8.0)!at!37!˚C!for!10!min.!!The! enzymatic! reaction! was! started! by! addition! of! 15! µL! of! DPP(IV! solution! (25!mU/mL)!and!carried!out!at!37˚C!for!30!min.!Sodium!acetate!buffer!(1!M,!pH!4.0,!100!µL)!was!added!to!stop!the!reaction!and!the!absorbance!of! the!released!p(nitroaniline!was!measure!at!405!nm!using!a!Tecan!Infinite®!200!Pro!microplate!reader.!The!positive!and!negative!controls!were!prepared!by!using!Tris(HCl!buffer!(100!mM,!pH!8.0)!in!place!of!the!sample!and!in!place!of!the!sample!and!enzyme!solution,!respectively.!!8.3.6$Determination$of$the$Mode$of$Inhibition$and$the$Inhibition$Constant$(Ki)$!The! traditionally(synthesized!peptides! showing! the!highest! inhibitory! activity! against!DPP(IV! were! further! studied! for! their! mode! of! action! on! the! enzyme! as! previously!described! in! Chapter! 7,! section! 7.4.4! (Lacroix! &! Li(Chan,! 2014a)! with! the! following!modifications:!the!substrate!(25!µL)!was!combined!with!100!mM!Tris(HCl!buffer!pH!8.0!(35!µL)!and!the!sample!(25!µL)!and!pre(incubated!for!10!min!at!37!˚C,!after!which!the!enzymatic! reaction! was! started! by! addition! of! the! DPP(IV! enzyme! (15! µL! of! a! 25!mU/mL!solution).!!!The!mode! of! inhibition! and!Ki!values! of! the! inhibitory! peptides! were! determined! by!fitting! the! initial! velocity! versus! substrate! concentration!data! to!nonlinear! regression!models!using!the!GraphPad!Prism!software!(version!6,!GraphPad!Software,!San!Diego,!CA,!United!States).!The!Ki!values!are!expressed!as!final!assay!concentrations.!!! !! 171!8.4!RESULTS!!8.4.1$Binding$of$DPP@IV$to$Deca@Peptides$on$the$Array$$'The!interaction!between!the!DPP(IV!enzyme!and!deca(peptides!spanning!the!entire!α(lactalbumin!sequence!(Appendix'B,!Table'B.1)!was!first!determined!by!immunoassay!and!visualized!using!an!enhanced!chemiluminescence!substrate!(Figure'8.1).!As!shown!in!Figure'8.2,!the!probing!of!the!peptide!array!with!DPP(IV!revealed!that!a!number!of!α(lactalbumin(derived!peptides!are!able!to!interact!with!the!enzyme!(dark!spots!on!the!array).!Since!every!consecutive!spot!on!the!membrane!differs!by!only!one!amino!acid,!the!presence!of!consecutive!dark!spots!indicates!that!some!regions!of!the!α(lactalbumin!molecule! such! as! 1EQLTKCEVFRELK13! (spots! A1–A4),! 45NDSTEYGLFQINNKIWCK62!(spots!E1–E9)!and!89IMCVKKILDKVGINYWLAHKALCSEKL115!(spots!I1–J7)!were!able!to!bind! to! DPP(IV! while! others! like! 61CKDDQNPHSSNICN74! (spots! F6–F10)! and!68HSSNICNISCDKFLD82!(spots!G2–G7)!did!not!seem!to!interact!with!the!enzyme.!!In!addition!to!the!114!α(lactalbumin(derived!deca(peptides,!seven!additional!decamers!were! synthesized!on! the!membrane! (spots!K5–K11,!Appendix' B,!Tables' B.1'&!B.2).!These! peptides! were! selected! based! on! their! known! effect! on! DPP(IV! activity,! their!similarity!to!peptides!previously!shown!to!have!inhibitory!activity!or!because!they!are!derived!from!natural!substrates!for!the!enzyme!(Appendix'B,'Table'B.2).!Among!these,!only! five!were! found! to! bind! to! DPP(IV,! namely! the!β(lactoglobulin(derived! peptides!IVTQTMKGLD! and! LKPTPEGDLE,! and! the! peptides! HSQGTFTSDY,! YAEGTFISDY! and!HAEGTFTSDY!derived!from!glucagon,!GIP!and!GLP(1,!respectively.!!!!!!!!!! 172!!!!!! !''Figure'8.1'Schematic!representation!of!peptide!array!synthesis,!binding!and!inhibition!experiments.!Binding!of!α(lactalbumin(derived!decamers!to!the!dipeptidyl(peptidase!IV!(DPP(IV)! enzyme! was! investigated! directly! on! the! cellulose! membrane,! whereas! the!inhibition! of! DPP(IV! was! measured! using! a! colorimetric! assay! with! the! cleaved!peptides.'!!!!!!!!!!!!!! ! !!!! 173!!!!!!!!!!!!!!!!!!!!!!!!! !Figure' 8.2' Binding! of! DPP(IV! to! deca(peptides! derived! from! α(lactalbumin! (A1–K4,!Table'B.1,!Appendix'B)!and!from!other!protein!(K5–K11,!Appendix'B,!Tables'B.1'&!B.2).! The! binding! is! inversely! correlated! to! the! light! signal,! the! darkest! spots!representing!peptides!on!which!more!enzyme!was!bound.'!!!8.4.2$DPP@IV$Inhibitory$Activity$of$SPOT@Synthesized$Deca@Peptides$'In! order! to! determine! whether! the! binding! of! DPP(IV! to! the! α(lactalbumin(derived!peptides!is!indicative!of!their!ability!to!inhibit!the!activity!of!the!enzyme,!the!cellulose(bound! peptides! were! cleaved! off! from! the! membrane! and! their! DPP(IV! inhibitory!activity! was! assessed! (Figure' 8.1).! ! Among! the! 114! deca(peptides! derived! from! α(lactalbumin,!33!were!able!to!decrease!DPP(IV!activity!by!at!least!20!%!(Table'8.1).!The!regions! of! the! protein! comprising! the! amino! acids! 84DLTDDIMCVKKIL96! (spots! H7–H10),! 97DKVGINYWLAHKA109! (spots! I9–J1)!and!105LAHKALCSEKLDQWLC120! (spots! J6–K1)! appeared! to! be! particularly! effective! at! inhibiting! DPP(IV.! Amid! the! seven!additional!decamers!not!derived!from!α(lactalbumin,!the!sequences!IVTQTMKGLD!and!HSQGTFTSDY,!previously!found!to!have!some!DPP(IV!inhibitory!activity!(Kühn(Wache!! 174!et! al.,! 2011;! Lacroix! &! Li(Chan,! 2014d)! showed! 16! and! 15%! inhibition,! respectively.!With!the!exception!of!GLP(1(1(10),!all!other!non(α(lactalbumin(derived!peptides!caused!more!than!23%!inhibition!of!the!enzyme’s!activity.!!!!!!!Table' 8.1' Percent! inhibition! of! DPP(IV! activity! caused! by! SPOT(synthesized! deca(peptides!derived!from!α(lactalbumin!(A1–K4,!Appendix'B,!Table'B.1)!and!from!other!proteins!(K5–K11,!Appendix'B,!Tables'B.1'&!B.2).!Values!equal!to!or!greater!than!20%!inhibition!are!highlighted!in!gray.!Percent!inhibition!values!obtained!for!the!decamers!not! derived! from!α(lactalbumin! are! shown! in! red! font.! The! data! are! reported! as! the!averages!of!four!determinations!obtained!from!two!separate!inhibition!experiments.'!  1 2 3 4 5 6 7 8 9 10 11 A 11 16 20 20 17 16 18 14 17 14 8 B 11 11 13 13 11 17 15 15 15 15 9 C 26 14 18 26 16 10 5 13 12 11 14 D 11 9 11 14 14 14 11 15 13 14 11 E 14 14 13 9 10 11 9 13 20 6 21 F 22 17 19 21 29 15 16 15 15 8 15 G 11 9 16 19 25 31 17 19 18 21 22 H 19 18 18 21 14 19 25 20 20 20 15 I 17 19 26 18 17 20 19 16 22 22 20 J 25 18 15 20 16 27 21 20 24 21 25 K 22 19 24 19 16 23 25 15 24 19 31 !!!Interestingly,!the!binding!of!the!deca(peptides!to!the!DPP(IV!enzyme!was!not!correlated!to! their! inhibitory! activity! (Figure' 8.3).! In! fact,! the!peptide! sequences!displaying! the!strongest! binding! to! DPP(IV,! VKKILDKVGI! and! KKILDKVGIN,! caused! less! than! 20%!reduction!of!the!enzyme’s!activity,!whereas!the!most!potent!decamers,!WCKDDQNPHS,!ICNISCDKFL!and!YPSKPDNPGE,!showed!moderate!or!no!binding!to!the!enzyme.!!!! !! 175!!!!!!!!!!!!!!!!!!! !Figure' 8.3' Correlation! between! binding! of! DPP(IV! to! deca(peptides! on! the! array!(indicated! by! luminescence! intensity)! and! inhibition! of! the! enzyme! activity! by! the!peptides.'!8.4.3$Validation$of$the$DPP@IV$Inhibitory$Activity$of$the$Deca@Peptides$!Being!built!on!the!membrane!in!small!amounts,!the!SPOT(synthesized!peptides!cannot!easily! be! purified! or! quantified.! Therefore,! to! validate! the! DPP(IV! inhibitory! activity!results,! a! number! of! deca(peptides! found! to! cause! high! and! low! inhibition! of! DPP(IV!activity!were!obtained!by!traditional!solid!phase!peptide!synthesis!and!tested!for!their!effect!on!DPP(IV!activity!(Figure'8.4).!!!!!!!!! !!Figure' 8.4' Mature! amino! acid! sequence! of! α(lactalbumin! (UniProt! KB! database!accession! number! P00711).! Deca(peptides! underlined! were! chemically! synthesized!according! to! the! traditional!method!of! solid!phase!peptide! synthesis! to! validate! their!effect!on!DPP(IV!activity.'0 3000 6000 9000 12000 15000 18000 0 10 20 30 40 Luminescence intensity (RLU) % DPP-IV inhibition ! 176!!The! peptides! obtained! by! the! two! synthesis! methods! displayed! consistent! trends! in!their! DPP(IV! inhibitory! activity! (Table' 8.2).! The! deca(peptides! WCKDDQNPHS! and!IPAVFKIDAL,!with!51!and!48!%!inhibition!respectively,!were!the!most!potent!inhibitors.!Conversely,! the! α(lactalbumin(derived! peptides! VFRELKDLKG,! FHTSGYDTQA,!TSGYDTQAIV,! DTQAIVQNND,! CKDDQNPHSS! and! QNPHSSNICN! did! not! significantly!affect!the!activity!of!the!enzyme.!!!!!'Table' 8.2' Dipeptidyl(peptidase! IV! inhibitory! activity! of! SPOT(! and! traditionally(synthesized!deca(peptides.''Categorya Position Sequence pI % DPP-IV inhibition SPOT-synthesizedb Traditionally- synthesizedcd High inhibitory activity, strong binding C1 LPEWVCTTFH 5.24  26 26 J6 LAHKALCSEK 8.21  27 31 K6 LKPTPEGDLE 4.14 23 24 High inhibitory activity, moderate/no binding  F5 WCKDDQNPHS 5.21  29 51 J11 LCSEKLDQWL 4.37 25 25 K7 IPAVFKIDAL 5.84 25 48 K11 YPSKPDNPGE 4.37 31 25 Low inhibitory activity, strong binding A1 EQLTKCEVFR 6.23 11 10 A8 VFRELKDLKG 8.58 14 3 B6 GYGGVSLPEW 4.00 17 8 C9 FHTSGYDTQA 5.08 12 2 Low inhibitory activity, moderate/no binding C11 TSGYDTQAIV 3.80 14 -1 D4 DTQAIVQNND 3.56 14 2 F6 CKDDQNPHSS 5.21 15 -3 F10 QNPHSSNICN 6.73 8 2 a High inhibitory activity refers to peptides able to cause more than 20% decrease in DPP-IV activity whereas strong binding refers to peptide spots with a relative luminescence intensity > 4000. b Values shown are averages of four determinations obtained from two separate inhibition experiments.  c Values shown are averages of three determinations.  d Assay performed using 250 µM of peptides (final assay concentration). !!!!!! 177!8.4.4$Modes$of$Inhibition$of$Deca@Peptides$!The! deca(peptides! shown! to! cause! the! greatest! inhibition! of! DPP(IV! activity! were!further!studied!to!determine!their! inhibition!constant!(Ki)!and! identify!their!modes!of!action! on! the! DPP(IV! enzyme.! ! The! sequences! LPEWVCTTFH,! LAHKALCSEK,!IPAVFKIDAL!and!YPSKPDNPGE!were!found!to!inhibit!DPP(IV!in!a!competitive!manner,!whereas! WCKDDQNPHS! behaved! as! an! un(competitive! inhibitor! and! the! decamers!LKPTPEGDLE! and! LCSEKLDQWL! exhibited! mixed! inhibition! (Table' 8.3! and! Figure'8.5).! The! α(lactalbumin(derived! peptide! WCKDDQNPHS! and! the! β(lactoglobulin(derived!sequence!IPAVFKIDAL,!with!αKi!and!Ki!values!of!76!and!156!µM,!respectively,!were! shown! to! have! the! lowest! inhibition! constants.! ! Conversely,! the! deca(peptide!LKPTPEGDLE!was! found! to! be! the! least! potent! inhibitor! (Ki! value! =! 729! µM)! (Table'8.3).!!!!Table'8.3'Inhibition!constant!(Ki)!and!mode!of!inhibition!of!traditionally!synthesized!deca(peptides.!Sequence Ki (µM)a Mode of inhibition LPEWVCTTFH 300 ± 16 Competitive LAHKALCSEK 217 ± 9 Competitive LKPTPEGDLE 729 ± 128 Mixed WCKDDQNPHS   76 ± 5 Un-competitive LCSEKLDQWL 217 ± 15 Mixed IPAVFKIDAL 156 ± 11 Competitive YPSKPDNPGE 262 ± 17 Competitive a The Ki (or αKi in the case of un-competitive inhibition) values are reported as the averages and standard errors from triplicate determinations. !!178!!!!Figure! 8.5! Lineweaver-Burk! plots! of! DPP-IV! activity! in! the! absence! and! presence! of! peptides.! (A)! LPEWVCTTFH,! (B)!LAHKALCSEK,!(C)!LKPTPEGDLE,!(D)!WCKDDQNPHS,!(E)!LCSEKLDQWL,!(F)!IPAVFKIDAL,!(G)!YPSKPDNPGE. !-0.02 -0.01 0.00 0.01 0.02 0.031002003004005001/[Gly-Pro-pNA] (1/µM)1/V0(1/Abs per min)375 µM0 µM1250 µM750 µMC -0.01 0.00 0.01 0.02 0.032004006008001/[Gly-Pro-pNA] (1/µM)1/V0(1/Abs per min)1250 µM750 µM375 µM0 µMA E -0.01 0.00 0.01 0.02 0.032004006008001/[Gly-Pro-pNA] (1/µM)1/V0(1/Abs per min)1125 µM250 µM0 µMB -0.01 0.00 0.01 0.02 0.032004006008001/[Gly-Pro-pNA] (1/µM)1/V0(1/Abs per min)1250 µM750 µM375 µM0 µMG F D ! 179!8.5$DISCUSSION$!The!DPP*IV! inhibitory! activity! of! a! peptide! has! been! suggested! to! be! influenced! by! a!number!of!factors,! including!its! length,!charge!and!amino!acid!composition!(Lan!et!al.,!2014).!However,! to! date,! it! is! still! unclear!which!of! these! structural! or! compositional!characteristics! play! a! predominant! role.! In! the! present! study,! the! net! charge! of! the!peptides!did!not!seem!to!have!a!predictable!effect!on!their!potency!nor!their!ability!to!bind!to!the!enzyme.!Peptides!with!similar!isoelectric!point!(pI),!such!as!LPEWVCTTFH!and! CKDDQNPHSS,! were! found! to! display! significantly! different! DPP*IV! inhibitory!activity! and! binding! (Table! 8.2! and! Figure! 8.2).! Similarly,! since! all! sequences!investigated!in!this!study!were!composed!of!10!amino!acids,!the!length!of!the!peptides!could! not! be! considered! a! factor! influencing! their! potency! and! binding! behavior.!Therefore,! the! specific! amino! acid! sequences! of! the! peptides,! rather! than! other!structural!properties,!appear!to!be!the!predominant!element!determining!their!ability!to!inhibit!DPP*IV.!!The! interactions!between!an!enzyme!and! ligands! that!may!be!potential! inhibitors!can!be! investigated! by! binding! and! inhibition! assays.! In! the! present! study,! these! two!approaches!were!used!to!study!the!interaction!between!the!enzyme!DPP*IV!and!deca*peptides! derived! from! α*lactalbumin,! a! protein! known! to! be! a! precursor! of! DPP*IV!inhibitory!peptides! (Lacroix!&!Li*Chan,!2013;!Lacroix!&!Li*Chan,!2014c;!Chapters!6!&!7).!Even!though!some!peptides!showed!both!binding!to!and!inhibitory!activity!against!DPP*IV,! overall! the! results! obtained! from! these! two! assays! were! not! found! to! be!correlated.!This!discrepancy!may!be!explained!by!a!number!of!factors!including!the!fact!that! binding! of! the!α*lactalbumin*derived! peptides! to! DPP*IV!may! occur! at! different!domains!of!the!enzyme.!Therefore,!peptides!that!showed!strong!binding!but!low!DPP*IV!inhibitory! activity,! such! as! EQLTKCEVFR,! VFRELKDLKG,! GYGGVSLPEW! and!FHTSGYDTQA,! may! bind! outside! the! enzyme’s! catalytic! center! without! affecting! its!activity.!Interestingly,!some!of!the!most!potent!deca*peptides,!including!WCKDDQNPHS,!LCSEKLDQWL,! IPAVFKIDAL! and! YPSKPDNPGE! (Table! 8.2),! displayed! no! or! only!! 180!moderate! interaction! with! DPP*IV! based! on! the! binding! assay! (Figure! 8.2).!Investigation!on!the!mode!of!inhibition!of!these!peptides!revealed!that!WCKDDQNPHS!and! LCSEKLDQWL! exhibited! an! un*competitive! and! a! mixed! mode! of! action,!respectively,! suggesting! that! they!may! affect! enzymatic! activity! by!binding!not! to! the!enzyme!itself,!but!rather!to!the!enzyme*substrate!complex!(Lan!et!al.,!2014).!The!deca*peptides! IPAVFKIDAL! and! YPSKPDNPGE,! on! the! other! hand,! were! found! to! act! as!competitive! inhibitors!and! therefore!would!be!expected! to! interact!with!DPP*IV!since!they!reduce!the!enzyme’s!activity!by!binding!to!and!blocking!its!active!site!(Table!8.3!and!Figure!8.5).!Since!the!binding!assay!is!performed!directly!on!the!bound!peptides,!the!peptide!density! in! the! spots!may! limit! their! accessibility! to! the! enzyme.! It! is! also!possible!that!the!peptides!are!able!to!interact!with!DPP*IV,!but!that!the!binding!leads!to!a! change! in! the! enzyme’s! conformational! structure! preventing! its! recognition! by! the!primary! antibody!and! therefore! its!detection.!Moreover,! because! each!peptide!on! the!array! is! synthesized! with! a! different! yield,! depending! on! the! amino! acid! sequence,!hydrophobicity!and!conformation!(Katz!et!al.,!2011),!peptides!able!to!interact!with!DPP*IV,!but!that!are!present!in!lower!concentrations!on!the!membrane,!may!show!no!or!only!moderate!binding!to!the!enzyme.!Hence,!information!on!the!ability!of!a!peptide!to!bind!to!DPP*IV!cannot!be!used!to!predict!its!ability!to!inhibit!the!enzyme.!!The!SPOT*!and! traditionally*synthesized!decamers!showed!a!consistent! trend! in! their!DPP*IV!inhibitory!activity,!thus!confirming!that!peptide!array!technology!can!be!used!to!identify!DPP*IV!inhibitory!peptides.!Since!the!yield!and!purity!of!peptides!on!the!array!are!unknown,! this!approach! is!only!semi*quantitative!(Volkmer!et!al.,!2012),!allowing!the!comparison!of! the!relative!potency!of!peptides,!but!not! the!determination!of! their!absolute!DPP*IV! inhibitory!activity.!Nevertheless,!peptide!arrays!could!be!useful! tools!to!screen!a!large!number!of!peptides!for!their!DPP*IV!inhibitory!activity!and!to!identify!in! the! primary! sequence! of! a! protein! the! areas! that! can! inhibit! the! enzyme.! By! then!exploring!where!in!the!tertiary!structure!of!the!protein!these!areas!are!located,!it!would!be! possible! to! know! whether! the! active! peptides! are! exposed! on! the! surface! of! the!molecule,! and! thus! prone! to! be! cleaved! by! treatment! with! proteolytic! enzymes,! or!buried!within! the! interior! of! the!molecule! and! likely! needing! to! be! exposed! prior! to!! 181!being!released!from!the!protein.!Ultimately,!this!knowledge!could!help!identify!the!best!treatments!to!generate!DPP*IV!inhibitory!peptides!from!dietary!proteins.! 8.6$CONCLUSION$!Findings!from!this!study!showed!that!peptide!arrays!on!cellulose!support!can!be!used!to!investigate!protein*derived!peptides! for! their! interaction!with! the!enzyme!DPP*IV.!By!allowing! the! parallel! synthesis! of! a! large! number! of! peptides! in! small! amounts,! this!method!makes!possible!the!screening!of!entire!protein!sequences!for!active!peptides!in!a!timely!and!cost!effective!manner.!!!!Since! results!obtained!using!peptide!array! technology!are!only! semi*quantitative,! any!information!gained!must!be!confirmed!by!other!methods.!Nevertheless,!peptide!arrays!are! useful! screening! tools! for! investigating! biologically! active! peptides! and! could! be!used!to!facilitate!structure*function!studies,!and!complement!or!support!the!traditional!methods!currently!used!to!identify!peptides!with!DPP*IV!inhibitory!activity.!!!!! !  182 CHAPTER 9: COMPARISON OF THE SUSCEPTIBILITY OF PORCINE AND HUMAN DIPEPTIDYL-PEPTIDASE IV TO INHIBITION BY PROTEIN-DERIVED PEPTIDES10  9.1 SUMMARY  The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only 6 caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (DKi or Ki = 11.5, 13.4, 13.3 µM and 31.4, 28.2, 75.0 µM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme.                                                             10 A version of this chapter has been accepted for publication. Lacroix, I.M.E., Li-Chan, E.C.Y. Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides. Peptides 2015, http://dx.doi.org/10.1016/j.peptides.2015.03.016    ! 183!9.2$INTRODUCTION$!Dipeptidyl*peptidase!IV!is!a!ubiquitously!distributed!110!kDa!glycoprotein!belonging!to!the! prolyl! oligopeptidase! family,! a! group! of! enzymes! that! preferentially! cleaves! N*terminal!dipeptides!from!substrates!containing!proline!or!alanine!at!their!penultimate!position! (Thoma! et! al.,! 2003).! Existing! mainly! as! a! membrane*anchored! cell*surface!enzyme,!but!also!found!in!soluble!form,!DPP*IV!is!implicated!in!a!number!of!biological!processes!by!acting!as!both!a!regulatory!protease!and!a!binding!protein!(Zhong!et!al.,!2013;! Filippatos! et! al.,! 2014).! Although! the! enzyme! exerts! its! action! on! a! number! of!molecules,! including!chemokines,!neuropeptides!and!regulatory!peptides!(Chen,!2006;!Stulc!&!Sedo,!2010),!DPP*IV! is!most!widely!known!for! its!catalytic!activity!against!the!incretins! glucose*dependent! insulinotropic! polypeptide! (GIP)! and! glucagon*like!peptide*1! (GLP*1),! two! gut*derived! hormones! that! play! a! pivotal! role! in! glycemic!regulation.!The!finding!that!the!DPP*IV!enzyme!is!responsible!for!more!than!95%!of!the!inactivation!of!GLP*1!has! attracted! considerable! research! interest! in! the! inhibition!of!the!enzyme!as!a!strategy!for!the!management!of!type!2!diabetes!(Thoma!et!al.,!2003).!As!a! result,! extensive!work! has! been! done! over! the! last! decade! to! develop! and! identify!molecules!able! to! inhibit! the!activity!of!DPP*IV!and! therefore! increase! the!circulatory!levels! of! intact! incretins.!This! growing!body!of! research!has! led! to! the!discovery!of! a!variety! of! compounds,! including! peptidomimetics,! xanthine*based,! arylmethylamine*based!and!benzoquinolizine*based!DPP*IV! inhibitors! (Hunziker!et!al.,!2005).!Of! these,!several!have!been!approaved!for!the!management!of!type!2!diabetes!(Filippatos!et!al.,!2014).!!!More! recently,! there! has! been! a! great! interest! in! investigating! naturally! occurring!materials,!particularly!dietary!proteins,!as!sources!of!DPP*IV!inhibitors.!In!the!last!few!years,!proteins!from!a!variety!of!food!commodities,!including!milk!(Uchida!et!al.,!2011;!Uenishi! et! al.,! 2012;! Lacroix! &! Li*Chan,! 2012b,! 2013,! 2014c,d;! Nongonierma! &!FitzGerald,!2013a,b,d,!2014;!Silveira!et!al.,!2013),!fish!(Huang!et!al.,!2012;!Li*Chan!et!al.,!2012),! egg! (Van!Amerongen!et!al.,!20109),!dry*cured!ham!(Gallego!et!al.,!2014),!pork!! 184!(Hsu!et!al.,!2013),! rice!bran! (Hatanaka!et!al.,!2012),!amaranth! (Velarde*Salcedo!et!al.,!2013),!corn!(Mochida!et!al.,!2010)!and!azuki!beans!(Tominaga!et!al.,!2012)!have!been!reported!in!the!literature!to!be!precursors!of!peptides!able!to!inhibit!the!activity!of!the!DPP*IV! enzyme! (Table! 9.1).! These! peptides,! although! not! as! potent! as! the! synthetic!drugs,! have! triggered! interest! as! a! possible! complementary! approach! to!pharmacotherapy!in!the!management!of!type!2!diabetes.!!!!!!Table! 9.1!Dietary! proteins! reported! to! be! precursors! of! peptides! able! to! inhibit! the!activity!of!porcine!or!human!DPP*IV.!Protein source DPP-IV source used to assess inhibitory activity Reference Amaranth Porcine Velarde-Salcedo et al. (2013) Azuki bean Human Tominaga et al. (2012) Chicken egg Porcine Van Amerongen et al. (2009) Corn Human Mochida et al. (2010) Cow’s milk Porcine Lacroix & Li-Chan (2012b, 2013, 2014c); Nongonierma & FitzGerald (2013a, 2013b, 2013d, 2014) Human Uchida et al (2011); Uenishi et al. (2012); Silveira et al. (2013); Lacroix & Li-Chan (2014d) Porcine skin gelatin Porcine Hsu et al. (2013) Rice bran Human Hatanaka et al. (2012) Salmon skin gelatin Porcine Li-Chan et al. (2012) Spanish dry-cured ham Porcine Gallego et al. (2014) Tuna cooking juice Porcine Huang et al. (2012) !!Most! studies! that! have! investigated! the! in$ vitro! effect! of! synthetic! molecules! or!compounds! of! natural! origin! on! DPP*IV! have! been! conducted! using! the! human! or!porcine!enzyme.!Although!the!enzyme!sequence!is!highly!conserved!among!mammalian!species,! the! human! and! porcine! DPP*IV! enzymes! are! not! identical,! their! overall!sequence!identity!being!88%!(Bär!et!al.,!2003;!Kühn*Wache!et!al.,!2005)!(Figure!9.1).!To!date,! studies! comparing! the!kinetic!parameters! and! inhibition!patterns!of! the! two!enzymes! are! sparse! and! have! given! contradictory! results.! Findings! from! Stöckel*Maschek!et!al.!(2003)!suggested!that!porcine!DPP*IV!might!be!slightly!more!susceptible!to!inhibition!by!di*peptides!than!the!human!enzyme.!With!the!exception!of!the!inhibitor!Trp*Pyrr,!which!was!found!to!act!on!porcine!and!human!DPP*IV!as!a!linear!mixed*type!and! a! competitive! inhibitor! respectively,! the! authors! found! that! the! same! mode! of!inhibition!was!displayed!on!the!two!species.!Conversely,!Bär!et!al.!(2003)!reported!that!! 185!both!enzymes!were!inhibited!similarly!by!di*peptide!analogue!inhibitors,!but!observed!differences! in! their!binding!affinity! to!natural! substrates.!Thus,!despite! some! findings!suggesting! that! porcine! and! human! DPP*IV! might! differ! in! their! affinity! to! natural!substrates!and!susceptibility!to!short!modified!peptide*based!inhibitors,!to!the!author’s!knowledge,!the!effect!of!non*modified!protein*derived!peptides!on!the!activity!of!DPP*IV!from!both!species!has!never!been!compared.!!Therefore! the! objective! of! this! research!was! to! determine! and! compare! the! effect! of!protein*derived!peptides!of!various!length!and!amino!acid!composition!on!the!activity!of!both!porcine!and!human!DPP*IV!and!to!determine!their!mechanism!of!action!on!the!enzymes.!!!!!!!!!!!!!!!!!!!!!!!! 186!!!!! !!Figure! 9.1! Sequence! alignment! of! human! (UniProt! KB! database! accession! number!P27487)!and!porcine!(UniProt!KB!database!accession!number!P22411)!DPP*IV.!Amino!acids! that!differ!between!the!two!species!are!shown!bigger,!bold!red! font!while! those!representing!the!potential!glycosylation!sites!are!underlined.!The!amino!acids!making!up!the!catalytic!triad!are!highlighted!in!blue.!Adapted!from!Bär!et!al.!(2003)!and!Engel!et!al.!(2003).!!!!!!!Figure'1'' MKTPWKVLLG LLGAAALVTI ITVPVVLLNK GTDDATADSR KTYTLTDYLK NTYRLKLYSL MKTPWKVLLG LLGIAALVTV ITVPVVLLNK GTDDAAADSR RTYTLTDYLK STFRVKFYTL  RWISDHEYLY KQENNILVFN AEYGNSSVFL ENSTFDEFGH SINDYSISPD GQFILLEYNY QWISDHEYLY KQENNILLFN AEYGNSSIFL ENSTFDELGY STNDYSVSPD RQFILFEYNY  VKQWRHSYTA SYDIYDLNKR QLITEERIPN NTQWVTWSPV GHKLAYVWNN DIYVKIEPNL VKQWRHSYTA SYDIYDLNKR QLITEERIPN NTQWITWSPV GHKLAYVWNN DIYVKNEPNL  PSYRITWTGK EDIIYNGITD WVYEEEVFSA YSALWWSPNG TFLAYAQFND TEVPLIEYSF SSQRITWTGK ENVIYNGVTD WVYEEEVFSA YSALWWSPNG TFLAYAQFND TEVPLIEYSF  YSDESLQYPK TVRVPYPKAG AVNPTVKFFV VNTDSLSSVT NATSIQITAP ASMLIGDHYL YSDESLQYPK TVRIPYPKAG AENPTVKFFV VDTRTLSPNA SVTSYQIVPP ASVLIGDHYL  CDVTWATQER ISLQWLRRIQ NYSVMDICDY DESSGRWNCL VARQHIEMST TGWVGRFRPS CGVTWVTEER ISLQWIRRAQ NYSIIDICDY DESTGRWISS VARQHIEIST TGWVGRFRPA  EPHFTLDGNS FYKIISNEEG YRHICYFQID KKDCTFITKG TWEVIGIEAL TSDYLYYISN EPHFTSDGNS FYKIISNEEG YKHICHFQTD KSNCTFITKG AWEVIGIEAL TSDYLYYISN  EYKGMPGGRN LYKIQLSDYT KVTCLSCELN PERCQYYSVS FSKEAKYYQL RCSGPGLPLY EHKGMPGGRN LYRIQLNDYT KVTCLSCELN PERCQYYSAS FSNKAKYYQL RCFGPGLPLY  TLHSSVNDKG LRVLEDNSAL DKMLQNVQMP SKKLDFIILN ETKFWYQMIL PPHFDKSKKY TLHSSSSDKE LRVLEDNSAL DKMLQDVQMP SKKLDVINLH GTKFWYQMIL PPHFDKSKKY  PLLLDVYAGP CSQKADTVFR LNWATYLAST ENIIVASFDG RGSGYQGDKI MHAINRRLGT PLLIEVYAGP CSQKVDTVFR LSWATYLAST ENIIVASFDG RGSGYQGDKI MHAINRRLGT  FEVEDQIEAA RQFSKMGFVD NKRIAIWGWS YGGYVTSMVL GSGSGVFKCG IAVAPVSRWE FEVEDQIEAT RQFSKMGFVD DKRIAIWGWS YGGYVTSMVL GAGSGVFKCG IAVAPVSKWE  YYDSVYTERY MGLPTPEDNL DHYRNSTVMS RAENFKQVEY LLIHGTADDN VHFQQSAQIS YYDSVYTERY MGLPTPEDNL DYYRNSTVMS RAENFKQVEY LLIHGTADDN VHFQQSAQLS  KALVDVGVDF QAMWYTDEDH GIASSTAHQH IYTHMSHFIK QCFSLP KALVDAGVDF QTMWYTDEDH GIASNMAHQH IYTHMSHFLK QCFSLP!60 60 120 120 240 240 300 300 360 360 420 420 480 480 540 540 600 600 660 660 720 720 180 180 Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine Human Porcine 766 766 ! 187!9.3$MATERIALS$AND$METHODS$!9.3.1%Materials%!Porcine! DPP*IV! (purified! from! porcine! kidney,! >! 90%! purity,! activity! ≥! 35! units/mg!protein;!one!unit!being!defined!as! the!amount!of!enzyme! that!hydrolyzes!1.0!µmol!of!Gly*Pro*7*amido*4*methylcoumarin/min! at! 37°C,! pH! 8.5)! was! obtained! from! EMD!Millipore! (Darmstadt,! Germany)! while! recombinant! human! DPP*IV! (expressed! in!HEK293,! >95%! purity,! activity! >! 5500! pmole! Gly*Pro*7*amido*4*methyl*coumarin/min/µg)!was!purchased! from!Creative!BioMart! (Shirley,!NY,!United!States).!The! chromogenic! substrate!Gly*Pro*p*nitroanilide! (H*Gly*Pro*p*NA·HCl)!was!obtained!from!Bachem!Americas!(Torrance,!CA,!United!States).!The!synthesized!peptides!(≥!95%!purity)!were!prepared!and!purified!by!GL!Biochem! (Shanghai)!Ltd! (Shanghai,! China).!Whey! protein! isolate! (WPI! 895,! Fonterra,! NZ,! 92.4%! protein)!was! donated! by! Caldic!(Delta,!BC,!Canada)!whereas!α*lactalbumin!from!bovine!milk!(≥!85%!protein),!diprotin!A!(Ile*Pro*Ile)!and!pepsin!(from!porcine!gastric!mucosa,!≥!2500!units/mg!protein;!one!unit!being!defined!as!the!amount!of!enzyme!that!produces!a!ΔA280!of!0.001!per!min!at!pH! 2.0! and! 37°C,!measured! as! TCA*soluble! products! using! hemoglobin! as! substrate)!were!purchased!from!Sigma*Aldrich!(Oakville,!ON,!Canada).!!9.3.2%Preparation%of%Whey%Protein%Hydrolysates%!Whey! protein! isolate! (WPI)! and! α*lactalbumin! hydrolysates! were! prepared! by!enzymatic!treatment!of!the!proteins!with!pepsin!(4%!(w/w)!enzyme/substrate!ratio)!at!37°C!and!pH!2.0!for!60!min!as!described!in!Chapter!6,!section!6.3.2!(Lacroix!&!Li*Chan,!2013).!!! %! 188!9.3.3%Porcine%(p)%DPP=IV%Inhibition%Assay%!The! effect! of! whey! protein! hydrolysates! and! synthesized! peptides! on! the! activity! of!pDPP*IV! was! assessed! using! the! chromogenic! substrate! Gly*Pro*p*NA! as! previously!described! in! Chapter! 7,! section! 7.3.6! (Lacroix! &! Li*Chan,! 2014c).! Briefly,! the!hydrolysates!were! dissolved! in! 100!mM!Tris*HCl! buffer! pH! 8.0!while! the! synthesized!peptides!were!solubilized!in!a!mixture!of!acetonitrile!and!water,!then!further!diluted!in!100!mM!Tris*HCl!buffer!pH!8.0! to! the!desired!concentrations.! In!a!96*well!microplate,!25!µL!of!sample!were!combined!with!25!µL!of!Gly*Pro*p*NA!(12!mM!in!100!mM!Tris*HCl!buffer!pH!8.0)!and! the!mixture!was!pre*incubated! for!10!min!at!37°C.!The!enzymatic!reaction!was! then! started! by! addition! of! 50! µL! of! pDPP*IV! (0.02! unit/mL! in! 100!mM!Tris*HCl!buffer!pH!8.0)!and!carried!out!at!37°C! for!30!min!whereupon!100!µL!of!1!M!sodium!acetate!buffer!pH!4.0!was!added! to! inactivate! the!enzyme.!The!absorbance!of!the!released!p*nitroaniline!was!measured!at!405!nm!using!a!Labsystems!iEMS!Reader!MF!(Labsystems!Oy).!The!positive!and!negative!control,!corresponding!to!the!activity!of!DPP*IV!in!the!absence!of!inhibitor!and!the!absence!of!DPP*IV!activity,!respectively,!were!prepared!by!using!Tris*HCl!buffer!(100!mM,!pH!8.0)!in!lieu!of!the!sample!and!in!lieu!of!the!sample!and!enzyme!solution,!respectively.!!One!unit!of!pDPP*IV!was!defined!as!the!concentration!of!enzyme!that!produced!1!µmole!per!min!of!p*nitroaniline!from!Gly*Pro*p*NA!at!pH!8.0!and!37°C.!!9.3.4%Recombinant%Human%(rh)%DPP=IV%Inhibition%Assay%%!The!effect!of!the!whey!protein!hydrolysates!and!synthesized!peptides!on!the!activity!of!rhDPP*IV!was!determined!using!the!chromogenic!substrate!Gly*Pro*p*NA!according!to!the!method!described!in!the!Enzo®!Life!Sciences!DPPIV!Drug!Discovery!Kit!Instruction!Manual! (BML*AK499)! with! some! modifications.! The! samples! (25! µL)! were! pre*incubated!with!25!µL!of!100!mM!Tris*HCl!buffer!pH!8.0!and!35!µL!of!Gly*Pro*p*NA!(0.29!mM!in!Tris*HCl!buffer!pH!8.0)!for!10!min!at!37°C,!thereupon!15!µL!of!rhDPP*IV!enzyme!(25!mU/mL!in!100!mM!Tris*HCl!pH!8.0)!was!added.!The!enzymatic!reaction!was!carried!! 189!out! at! 37°C! for! 30!min! after!which! 100! µL! of! 1! M! sodium! acetate! buffer! pH! 4.0!was!added!to!inactivate!the!enzyme!(Chapter!8,!Lacroix!&!Li*Chan,!2014d).!The!absorbance!of!the!released!p*nitroaniline!was!measured!at!405!nm!using!a!Labsystems!iEMS!Reader!MF!and!the!positive!and!negative!controls!were!prepared!as!described!for!the!porcine!enzyme!(section!9.3.3).!!!As!for!pDPP*IV,!one!unit!of!rhDPP*IV!was!defined!as!the!concentration!of!enzyme!that!released!1!µmol!per!min!of!p*nitroaniline!from!Gly*Pro*p*NA!at!pH!8.0!and!37°C.!!9.3.5%Determination%of%the%Mode%of%Inhibition%and%the%Inhibition%Constant%(Ki)%!The! whey! protein! hydrolysates! and! the! synthesized! peptides! found! to! cause! the!strongest! inhibition!of!both!pDPP*IV!and!rhDPP*IV!were!further! investigated!for!their!mode! of! action! on! the! enzymes.! The! initial! rate! of! hydrolysis! of! the! chromogenic!substrate! Gly*Pro*p*NA! by! pDPP*IV! and! rhDPP*IV! was! measured! using! substrate!concentrations!ranging!from!38.5!to!385!µM!(final!assay!concentrations)!as!detailed!in!Chapter!7,!section!7.3.7!(Lacroix!&!Li*Chan,!2014c)!for!pDPP*IV!and!Chapter!8,!section!8.3.6!(Lacroix!&!Li*Chan,!2014d)!for!rhDPP*IV.!The!Michaelis*Menten!constants!(Km)!for!pDPP*IV! and! rhDPP*IV! were! 185.7! ±! 31.4! µM! and! 173.8! ±! 17.1! µM! (n! =! 23),!respectively.!!The!Ki!values!and!mode!of!inhibition!of!the!hydrolysates!and!synthesized!peptides!were!determined! by! fitting! the! initial! velocity! data! versus! the! substrate! concentrations! to!nonlinear! regression! models! using! the! graphing! and! statistical! software! GraphPad!Prism! (version! 6).! The! Ki! values! are! expressed! as! final! assay! concentrations.!Lineweaver*Burk!plots!of!pDPP*IV!and!rhDPP*IV!activities!in!the!presence!and!absence!of!inhibitors!were!drawn!using!the!aforementioned!software!in!order!to!help!visualize!the!modes!of!inhibition!determined!by!nonlinear!regression.!!! %! 190!9.3.6%Statistical%Analysis%!One*way!analysis!of!variance!using!the!general!linear!model!and!pairwise!comparison!with! Tukey’s! method! (Minitab! Statistical! Software! Version! 17,! Minitab! Inc.,! State!College,! PA,! USA)! were! used! to! compare! the! inhibitory! activity! of! the! synthesized!peptides! on! porcine! and! recombinant! human! DPP*IV.! All! assays! were! performed! at!least!in!triplicate!and!significant!difference!was!established!at!P!<!0.05.!!9.4$RESULTS$!9.4.1% Effect%of% Synthesized%Peptides%on% the%Activity%of%Porcine%and%Recombinant%Human%DPP=IV%!A!number!of!peptides!(Appendix!C,!Table!C.1)!derived!from!dietary!proteins!or!from!natural! substrates! of! the! DPP*IV! enzyme! that! have! previously! been! investigated! for!their! effects! on! either! the! porcine! or! human! DPP*IV! were! synthesized! and! their!inhibitory!activity!against!both!species!was!determined.!!!As!shown!in!Figure!9.2,!pDPP*IV!and!rhDPP*IV!differ!in!their!susceptibility!to!inhibition!by!peptides,!the!porcine!enzyme!being!generally!more!easily!inhibited!than!the!rhDPP*IV.!Among! the!62! synthesized!peptides! investigated,!37! caused!a! significantly!greater!inhibition!of!pDPP*IV,!while!only!six!were!significantly!more!effective!at!inhibiting!the!recombinant!human!enzyme.!Peptides!that!showed!low!or!no!inhibitory!activity!against!pDPP*IV!were!also!not!effective!at!inhibiting!rhDPP*IV.!Conversely,!a!number!of!peptide!sequences,! such!as!FHTSGYDTQA,! IVQNNDSTEYGLF!and!NNDSTEYGLF,!were! found! to!inhibit!pDPP*IV!but!had!marginal!effects!on!the!activity!of!rhDPP*IV.!The!di*peptide!WR!and!deca*peptide!WCKDDQNPHS,!with!92%!and!94%!inhibition!respectively,!were!the!most! effective! at! inhibiting! the! activity! of! pDPP*IV,!whereas! the!penta*peptide! IPIQY,!with!89%!inhibition,!was!the!most!potent!inhibitor!of!rhDPP*IV.!!!191!!!!!Figure! 9.2! Percent! inhibition! of! porcine! (p)! and! recombinant! human! (rh)! DPP7IV! activities! by! synthesized! peptides.! The!percent! inhibition!was!measured!using!250!µM!of!synthesized!peptides!(final!assay!concentration).!Each!bar!represents!the!mean!and!standard!deviation!(n!=!3).!Bars!labeled!with!the!symbols!*!and![*]!indicate!peptides!found!to!cause!a!significantly!greater! inhibition! of! pDPP7IV! and! rhDPP7IV! respectively! (P" <! 0.05).! The! origin! of! the! peptide! sequences! is! presented! in!Appendix!C,!Table!C.1.!!0"10"20"30"40"50"60"70"80"90"100"EQLTKCEVFR"TKCEVF"VFRELKDLKG"RELKDLKG"RELKDLKGY"RELKDLKGYG"RELKDLKGYGGVS"RELKDLKGYGGVSL"GYGGVSLPEW"LPEWVCTTFH"WV"TFHTSGYDTQA"FHTSGYDTQA"TSGYDTQAIV"DTQAIVQNND"AIVQNNDSTE"IVQNNDSTE"IVQNNDSTEY"IVQNNDSTEYGLF"NNDSTEYGLF"WCKDDQNPHS"CKDDQNPHSS"QNPHSSNICN"CDKFLDDDLT"LDDDLTDDIM"CVKKIL"CVKKILD"CVKKILDKVG"ILDKVGINY"DKVGINYWLA"GINYWLAHKA"NYWLAHKALC"YWLAHKALCS" WL"WLAHKAL"LAHKALCSEK"LAHKALCSEKLDQ"AHKALCSEKLDQ"KALCSEKLDQ"ALCSEKLDQ"LCSEKLDQWL"LIVTQTMK"LIVTQTMKG"LIVTQTMKGLD"IVTQTMKGLD"TMKGLDIQ"ELKPTPEGD"LKPTPEGDL"LKPTPEGDLE"LKPTPEGDLEIL"IPAVFKIDA"IPAVFKIDAL"VRTPEVDD"IPIQY"LPYPY"FLQP" FP" WR"HAEGTFTSDY"YPSKPDNPGE"GPAE"GPGA"%"inhibiEon"PepEde"sequence"pDPP$IV'rhDPP$IV'**************** [*]'************ [*]'******[*]'[*]'***[*]'[*]'! 192!Since!different!enzyme!and!substrate!concentrations!were!used!to!assay!the!activity!of!the! two! enzymes,! the! percent! inhibition! values! measured! only! provide! qualitative!information! on! the! susceptibility! of! porcine! and! human! DPP?IV! to! inhibition! by! the!peptides.!In!order!to!directly!compare!the!potency!of!the!peptides!on!both!species,!the!inhibition!constants!(Ki),!which!are!independent!of!assay!conditions,!were!determined.!!9.4.2%Modes%of%Inhibition%and%Inhibition%Constants%of%the%Synthesized%Peptides%Displaying%the%Strongest%Inhibitory%Activity%against%Porcine%and%Recombinant%Human%DPPDIV%!The! synthesized!peptides! found! to! cause! the! strongest! inhibition!of!both!porcine!and!recombinant! human! DPP?IV! activities! were! further! investigated! to! determine! their!inhibition!constants!(Ki)!and!identify!their!mechanism!of!action!on!the!enzymes.!!!The! peptides!were! found! to! inhibit! the! DPP?IV! enzyme! in! either! a! competitive,! non?competitive,!un?competitive!or!mixed?type!manner!(Table&9.2!and!Appendix&C,&Figure&C.1).! Among! the! 21! synthesized! peptides! studied,! 6! sequences!were! found! to! act! on!pDPP?IV!differently! than!on!rhDPP?IV.!While! the!peptides! IPAVFKIDAL,!LAHKALCSEK!and!YPSKPDNPGE!inhibited!rhDPP?IV!in!a!competitive!manner,!the!former!behaved!on!pDPP?IV!as!a!mixed!inhibitor!while!the!latter!two!acted!as!non?competitive!inhibitors.!On!the!other!hand,!the!β?lactoglobulin?derived!peptides!LKPTPEGDL,!LKPTPEGDLE!and!LKPTPEGDLEIL! acted! as! un?competitive! inhibitors! against! pDPP?IV,! but! the! first! two!were! found! to! inhibit! rhDPP?IV! in! a! mixed! manner! whereas! the! third! was! a! non?competitive!inhibitor.!!!With! the! exception! of! the! decamers! LAHKALCSEK! and! YPSKPDNPGE,! which! showed!similar! inhibition! constants! for! the! two! enzymes,! the!Ki!values! of! the! peptides! were!lower! for! pDPP?IV! than! for! rhDPP?IV.! Against! pDPP?IV,! the! peptides!WR,! IPIQY! and!WCKDDQNPHS,! with!αKi! and!Ki! values! of! 11.5,! 13.4! and! 13.3! µM! respectively,! were!found!to!have!the!lowest!inhibition!constants,!albeit!about!five!times!higher!than!that!of!the! reference! inhibitor! diprotin!A! (IPI;!Ki! =! 2.6! µM).! Similarly,! the!peptide! sequences!! 193!WR! and! IPIQY!were! found! to! be! the!most! potent! inhibitors! of! rhDPP?IV! (αKi! and!Ki!values!of!31.4!and!28.2!µM,!respectively),!but!were!also!less!effective!than!diprotin!A!(Ki!=!5.3!µM).!For!both!species,!the!di?peptide!FP!had!the!highest!inhibition!constant!among!all!21!peptides! tested! in! this!study!(Ki! values!of!548.8!and!990.5!µM!for!pDPP?IV!and!rhDPP?IV,!respectively).!!Table&9.2& Inhibition!constant!(Ki)!and!mode!of! inhibition!of!synthesized!peptides!and!whey!protein!hydrolysates!on!porcine!(p)!and!recombinant!human!(rh)!DPP?IV.!The!Ki!(or!αKi! in!the!case!of!un?competitive!inhibition)!values!are!reported!as!the!means!and!standard!errors!(n!=!3).&Peptide/ hydrolysate Ki (µM) Mode of inhibition pDPP-IV rhDPP-IV pDPP-IV rhDPP-IV HAEGTFTSDY 355.5  ± 17.5  445.4 ± 16.0  Competitive Competitive IPAVFKIDA 92.0 ± 3.1  200.9 ± 15.1  Competitive Competitive IPAVFKIDAL 107.2 ± 6.5 159.9 ± 11.6 Mixed Competitive LAHKALCSEK 223.9 ± 7.3  216.9 ± 9.1 Non-competitive Competitive LCSEKLDQWL 82.0 ± 5.0  217.1 ± 14.6 Mixed Mixed LKPTPEGDL 81.9 ± 3.1 489.9 ± 28.8  Un-competitive Mixed LKPTPEGDLE 94.0 ± 4.5  729.1 ± 127.6 Un-competitive Mixed LKPTPEGDLEIL 142.6 ± 7.5 715.5 ± 14.4  Un-competitive Non-Competitive LPEWVCTTFH 75.2 ± 3.6 300.0 ± 16.0 Competitive Competitive WCKDDQNPHS 13.3 ± 0.7  75.0 ± 5.4 Un-competitive Un-competitive YPSKPDNPGE 271.5 ± 6.2  261.6 ± 16.5 Non-competitive Competitive FLQP 120.9 ± 4.0 295.5 ± 6.0 Competitive Competitive GPAE 69.4 ± 1.4 185.9 ± 5.0 Competitive Competitive GPGA 154.9 ± 4.3 177.2 ± 7.2 Competitive Competitive IPIQY 13.4 ± 0.3 28.2 ± 0.6 Competitive Competitive LPYPY 47.0 ± 1.8  74.0 ± 2.1  Competitive Competitive FP 548.8 ± 13.4  990.5 ± 27.5 Non-competitive Non-competitive WL 200.2 ± 12.2 770.5 ± 52.9  Mixed Mixed WR 11.5 ± 0.9 31.4 ± 1.4 Un-competitive Un-competitive WV 137.9 ± 37.7 377.1 ± 59.5 Mixed Mixed IPI (diprotin A) 2.6 ± 0.1 5.3 ± 0.1   Competitive Competitive α-la hydrolysatea 137.2 ± 13.3 942 ± 145   Mixed Mixed WPI hydrolysatea 471.6 ± 99.9 1568 ± 193 Mixed Mixed a Ki values are expressed in µg/mL &&&&! 194!9.4.3%Modes%of%Inhibition%and%Inhibition%Constants%of%Whey%protein%Hydrolysates%on%Porcine%and%Recombinant%Human%DPPDIV%!The! peptic! hydrolysates! of! α?lactalbumin! (α?la)! and! whey! protein! isolate! (WPI)!proteins,!previously!found!to!be!potent!inhibitors!of!pDPP?IV!(Lacroix!&!Li?Chan,!2013;!Chapter!6),!were!further!investigated!in!the!present!study!to!determine!their!inhibition!constants!and!mode!of!action!on!both!enzymes.!!!The!α?la!and!WPI!hydrolysates!both! inhibited!pDPP?IV!and!rhDPP?IV! in!a!mixed?type!manner! (Table& 2& and!Appendix& C,& Figure& C1).! The! porcine! enzyme,! however,! was!much!more!sensitive!to!inhibition!by!the!hydrolysates!than!the!human!enzyme!(Table&2).!!9.5$DISCUSSION$&The! sequence! of! the! DPP?IV! enzyme! is! known! to! be! highly! conserved! among!mammalian! species.! Comparison! of! the! amino! acid! sequences! of! porcine! and! human!DPP?IV! (Figure& 9.1)! revealed!92%! identity!within! the! catalytic! centre! and!an!overall!identity!of!88%!between! the! two! species! (Bär! et! al.,! 2003;!Kühn?Wache!et! al.,! 2005).!Human! and! porcine! DPP?IV! have! nine! and! ten! potential! N?glycosylation! sites!respectively,! seven!of! these!being! conserved!between! the! two! enzymes! (Figure& 9.1).!DPP?IV! from! both! species! has! been!widely! used! to! gain! knowledge! on! the! enzymes’!catalytic!and!binding!properties!and!to!screen!compounds!for!their!inhibitory!activity.!However,! the! impact! of! the! enzymes’! structural! differences! on! their! susceptibility! to!inhibition!is!mostly!unknown.!!In!the!present!study,!we!found!that!the!enzymes!differed!in!their!inhibition!patterns,!porcine!DPP?IV!being!generally!more!strongly!inhibited!by!peptides!derived!from!dietary!proteins!than!the!human!enzyme.!In!a!study!by!Bär!et!al.!(2003),!porcine!and!human!DPP?IV!were!found!to!be!inhibited!similarly!by!the!analogue!inhibitors! Ile?Thia! and! Ile?Cyano?Pyrr,! but! displayed! different! binding! affinity! for! the!incretin! hormone! GLP?17?36! and! the! enzyme! adenosine! deaminase! (ADA),! the! latter!! 195!having!a!greater!affinity!for!the!human!enzyme,!while!the!former!bound!more!strongly!to! porcine! DPP?IV.! The! authors! suggested! that! these! differences! may! result! from!secondary! interactions!between! longer!molecules!and! the!enzymes’!propeller!domain!(amino!acids!55–497)!which!features!a!lower!identity!(85%)!between!the!two!species.!Similarly,!in!the!present!study,!it!is!possible!that!the!variations!in!the!inhibitory!activity!of!the!peptides!observed!between!the!two!enzymes!may!have!resulted!from!the!binding!of!the!inhibitors!with!the!less!conserved!regions!of!the!enzymes.!!!Several!of!the!peptides!displaying!the!greatest!potency!against!porcine!and!human!DPP?IV! showed! different! modes! of! inhibition! (Table& 2! and! Appendix& C,& Figure& C.1),!suggesting! that! they!exert! their! inhibitory!effect!by! targeting!different!domains!of! the!enzymes.! ! Interestingly,! all! short! (≤! 5! amino! acid! residues)! peptides! investigated!exhibited! the! same! inhibition!mechanism!on!both! enzymes,!while! some!of! the! longer!peptides!acted!differently!on!the!two!species.!!!!Peptides!containing!a!Trp!amino!acid!at!their!N?terminal!(WCKDDQNPHS,!WL,!WR,!WV)!were!found!to!behave!on!porcine!and!human!DPP?IV!as!un?competitive!or!mixed?type!inhibitors.! A! few! studies! have! also! reported! that! peptides! or! peptide! derivatives!containing!a! tryptophan!residue!at! their!P2!position!showed!un?competitive!or!mixed!type! inhibition! toward! the! DPP?IV! enzyme! (Stöckel?Maschek! et! al.,! 2003;! Lan! et! al.,!2014),!suggesting!that!the!compounds!reduce!the!enzymatic!activity!by!binding!outside!of!the!active!site!of!the!free!enzyme!or!to!the!enzyme?substrate!complex.!On!the!other!hand,! with! the! exception! of! IPAVFKIDAL! and! YPSKPDNPGE! found! to! act! on! porcine!DPP?IV! as! mixed! and! non?competitive! inhibitors,! respectively,! all! peptides! having! a!proline! residue! at! their! P1! position! behaved! as! competitive! inhibitors! against! both!human! and! porcine! DPP?IV.! The! DPP?IV! enzyme! is! known! to! preferentially! act! on!substrates! containing! a! proline! residue! at! their! penultimate! amino! acid! position!(Lambeir!et!al.,!2003).!Therefore,!it!is!possible!that,!similarly!to!diprotin!A!(IPI),!a!well!known!DPP?IV!inhibitor!that!is!in!fact!a!substrate!with!a!low!turnover!rate!(Rahfeld!et!al.,!1991),!the!whey!protein?derived!peptides!bearing!the!amino!acid!proline!at!their!P1!! 196!position! may! act! as! substrates! for! the! enzyme,! their! apparent! competitive! behavior!being!a!kinetic!artifact!resulting!from!their!substrate?like!structure.!!!In!previous! investigations! (Chapters!7!&!8;!Lacroix!&!Li?Chan,!2014c,d),! it!was! found!that! the! specific! amino! acid! sequence! of! a! peptide! was! the! predominant! factor!determining! its! ability! to! inhibit! the! activity! of! DPP?IV,! rather! than! other! structural!features!such!as!its!length!or!charge.!The!peptides’!amino!acid!sequence!also!appeared!to!be!the!primary!factor!influencing!their!potency!against!human!DPP?IV.!!!9.6$CONCLUSION$&Evidence!from!the!present!study!showed!that!porcine!and!human!DPP?IV!differ!in!their!susceptibility!to!inhibition!by!protein?derived!peptides.!!In!general,!the!porcine!enzyme!was! inhibited! more! strongly! than! the! human! enzyme,! therefore! suggesting! that! the!usage! of! porcine! DPP?IV! to! assess! the! inhibitory! effect! of! peptides! may! lead! to! an!overestimation!of!their!potency!or!effectiveness!on!human!DPP?IV.!!!The!reasons!behind!the!different!inhibition!patterns!observed!between!the!two!species!are!unknown,!but!could!be!suggested!to!result!from!the!binding!of!the!inhibitors!with!regions!of!the!enzymes!that!are!less!conserved.!Additional!investigations!are!needed!to!identify!the!interaction!sites!between!the!inhibitory!peptides!and!the!DPP?IV!enzymes.!!!! !! 197!$CHAPTER$10:$CONCLUSION$!10.1$MAIN$FINDINGS$$!Dipeptidyl?peptidase! IV! inhibitors! are! a! class! of! glucose?lowering! agents! with!established! efficacy! in! individuals! with! type! 2! diabetes! (Filippatos! et! al.,! 2014).!Currently,! these! inhibitors! are! only! available! as! synthetic! drugs.! Over! the! past! few!decades,!a!growing!body!of!scientific!research!has!shown!that!dietary!proteins!can!be!used!to!generate!peptides!with!a!variety!of!biological!activities!that!have!the!potential!to!improve!human!health!(Shahidi!&!Zhong,!2008;!Kamau!et!al.,!2010).!However,!until!now,!little!attention!had!been!given!to!the!possibility!that!proteins!may!contain!within!their!sequences!peptides!able!to!inhibit!the!activity!of!the!DPP?IV!enzyme.!Hence,!this!study!sought!to!gain!a!better!understanding!of!the!potential!of!dietary!proteins!to!serve!as!natural!sources!of!DPP?IV!inhibitors!that!could!potentially!be!used!as!functional!food!ingredients!for!the!regulation!of!blood!glucose!levels.!!The!theoretical!ability!of!a!dietary!proteins!to!serve!as!precursors!of!DPP?IV!inhibitory!peptide! was! first! assessed! using! an! in% silico% analysis.! Peptides! reported! to! have!inhibitory! activity! against! the! DPP?IV! enzyme! were! observed! in! the! sequences! of! a!variety! of! proteins! from! both! plant! and! animal! sources.! Among! these,! caseins! from!cow’s!milk!and!collagen!from!bovine!meat!and!salmon!were!found!to!have!the!highest!occurrence! frequency! of! peptide! fragments! with! known! DPP?IV! inhibitory! activity!within!their!primary!sequence,!therefore!appearing!to!be!the!richest!potential!sources!of!DPP?IV!inhibitors.!!!To! be! precursors! of! DPP?IV! inhibitors,! proteins! must! not! only! contain! within! their!sequences!active!peptides,!but!it!must!be!possible!to!release!these!fragments!from!the!parent!proteins.!To!confirm!the!finding!from!the!in%silico!analysis!that!milk!proteins!can!be! used! to! generate! peptides!with! DPP?IV! inhibitory! activity,! dairy! ingredients!were!submitted!to!enzymatic!treatments!with!digestive!enzymes!and!commercially!available!! 198!proteases.!Although!a!number!of!proteins!were!suggested!by!the!in%silico!analysis!to!be!potential! sources! of! DPP?IV! inhibitors,! dairy! proteins! were! chosen! because! of! their!known!beneficial!effects!on!blood!glucose!regulation!as!presented!in!Chapter!2.!!!Peptides!with!DPP?IV!inhibitory!activity!were!successfully!generated!from!a!number!of!dairy!proteins!and!dairy!ingredients.!Even!though!caseins!were!proposed!by!the!in%silico!analysis!to!be!the!most!promising!sources!of!DPP?IV!inhibitors!among!all!the!cow’s!milk!proteins!investigated,!the!most!potent!hydrolysates!were!obtained!from!the!enzymatic!treatments!of!proteins!found!in!whey.!This!discrepancy!could!be!explained!by!the!fact!that! the! in% silico! approach! relies! entirely! on! the! currently! available! data! on! peptides!with! DPP?IV! inhibitory! activity! and! does! not! take! into! account! the! potency! of! the!peptides! nor! their! ability! to! be! released! from! the! proteins.! The! most! active!hydrolysates,! the! peptic! digests! of! whey! protein! isolate! and! α?lactalbumin,! were!fractionated! by! successive! chromatographic! steps! and! the! active! fractions! were!analyzed!by!liquid!chromatography?electrospray!ionization?tandem!mass!spectrometry!to! identify! their! constituent! peptides.! While! a! number! of! peptides! of! various!effectiveness! and!modes! of! action!were! identified,! their! potency!was! generally! lower!than!the!hydrolysates!and!fractions!from!which!they!were!isolated,!therefore!suggesting!that!they!may!act!in!synergy!with!one!another.!The!specific!amino!acid!sequence!of!the!peptides! appeared! to! be! the! predominant! structural! feature! determining! their!inhibitory! activity.! Albeit! the! majority! of! DPP?IV! inhibitory! peptides! that! have! been!reported!in!the!literature!are!short!(Tables!2.7,!4.1!&!4.2),!most!of!the!active!peptides!identified!in!this!study,!including!some!of!the!most!potent,!contained!seven!to!fourteen!amino!acid!residues.!!!!Peptide! arrays! on! cellulose! support! were! investigated! as! an! alternative! strategy! to!identify!DPP?IV! inhibitory!peptides!within! the! sequence!of! the!α?lactalbumin!protein.!Despite! having! been! introduced! more! than! two! decades! ago! and! having! been!successfully! used! for! a! wide! range! of! applications,! this! technology! has! never! been!employed!to!identify!bioactive!peptides!from!dietary!proteins.!Deca?peptides!spanning!! 199!the! complete! sequence! of! α?lactalbumin! were! synthesized! on! cellulose! membranes!using!SPOT!technology!and!their!binding!to!and!inhibition!of!DPP?IV!was!study.!SPOT?synthesized!peptides!were! found! to!display!a! similar! trend! in! their!DPP?IV! inhibitory!activity!to!that!of!the!peptides!obtained!by!the!traditional!solid!phase!synthesis!method.!The!peptides’!binding!behavior,!on!the!other!hand,!was!not!shown!to!be!correlated!to!their!ability!to!inhibit!the!enzyme.!By!allowing!the!parallel!synthesis!of!a!large!number!of!peptides! in! small!amount,!peptide!arrays!enable! the! investigation!of!entire!protein!sequences! and! can! complement! the! traditional! methods! currently! used! to! identify!bioactive!peptides!such!as!DPP?IV!inhibitors.!!!Most!studies!on!the!in%vitro!effects!of!DPP?IV!inhibitors,!including!synthetic!compounds!or!molecules! of! natural! origin,! have!been! conducted!using!human!or!porcine!DPP?IV.!However,! the!question!of!whether!the!two!enzymes!display!the!same!susceptibility!to!inhibition!has!not!been!clearly!addressed.!The!present!investigation!into!the!effects!of!protein?derived!peptides!on!the!activity!of!porcine!and!human!DPP?IV!showed!that!the!two!species,!although!structurally!alike,!differ! in! their! inhibition!patterns,! the!porcine!enzyme! being! generally! inhibited! more! strongly! than! the! human! DPP?IV.! While! the!mode! of! inhibition! by! short! peptides! (≤! 5! amino! acid! residues)!was! similar! for! both!species,!some!of!the!longer!fragments!were!found!to!act!differently!on!the!enzymes.!The!reasons! behind! the! different! inhibition! patterns! observed! are! unknown,! but! could!possibly!result!from!the!binding!of!the!inhibitory!peptides!with!less!conserved!regions!of!the!enzymes.!Still,!these!findings!suggest!that!human!DPP?IV!should!be!employed!in!future!research!to!assess!the! inhibitory!activity!of!peptides!since!the!usage!of!porcine!DPP?IV!may!result!in!an!overestimation!of!their!potency!or!effectiveness!on!the!human!enzyme.!!10.2$FUTURE$AREAS$OF$RESEARCH$$!The!individual!whey!protein?derived!peptides!identified!in!this!study!generally!showed!a!lower!potency!than!the!hydrolysates!or!fractions!from!which!they!were!isolated.!This!! 200!finding! raises! the!question!as! to!whether! the!peptides,!when!present! in! combination,!may! act! synergistically! with! one! another.! Further! work! to! determine! the! relative!amounts! of! the! most! potent! peptides! within! the! active! hydrolysate! fractions! would!allow!to!create,!using!the!individual!synthesized!peptides,!peptide!mixtures!mimicking!the!composition!of!the!active!fragments!present!in!these!fractions.!By!then!comparing!the!effect!of!these!mixtures!on!the!activity!of!DPP?IV!to!that!of!each!individual!peptide,!it!would!be!possible!to!determine!whether!they!exert!a!synergistic!effect!on!the!enzyme.!!!The!different!inhibition!patterns!observed!in!the!present!research!between!porcine!and!human!DPP?IV!were!suggested!to!result!from!the!binding!of!the!inhibitory!peptides!to!less! conserved! regions! of! the! enzymes.! Additional! investigations! using! a! molecular!docking!approach!could!provide!useful!information!on!the!interaction!sites!between!the!enzymes! and! the! inhibitors.! Moreover,! computational! techniques! could! also! help!determine!whether!the!length!of!the!peptides!affects!its!binding!location!on!the!enzyme.!!Over!the!last!few!years,!a!number!of!protein?derived!peptides!and!protein!hydrolysates!have! been! shown! to! be! able! to! inhibit! the! activity! of! DPP?IV! in! in% vitro! assays! using!synthetic! substrates.! However,! the! ability! of! these! bioactive! peptides! to! prevent! the!degradation! of! biological! substrates,! such! as! the! incretin! hormones,! by! DPP?IV! is!currently! unknown.! This! could! be! assessed! by! incubating! the! DPP?IV! enzyme! in! the!presence!of!GLP?1!or!GIP!and!the!inhibitory!peptides!and!monitoring!the!hydrolysis!by!DPP?IV!of! the! incretins! into! their! shorter,! inactive! forms! (GLP?19?36!and!GIP3?42)!using!mass!spectrometry.!!!!!!Further!research!is!also!needed!to!investigate!the!effects!of!the!protein?derived!DPP?IV!inhibitors!on!the!activity!of! the!other!members!of! the!DPP?IV!family.!As!mentioned!in!Chapter! 2,! DPP?IV! belongs! to! a! family! of! structurally! related! prolyl! oligopeptidases,!which!includes!the!enzymatically!active!members!FAP,!DPP?8!and!DPP?9.!!Some!studies!(Lankas!et!al.,!2005;!Mentlein,!2005),!but!not!all!(Burkey!et!al.,!2008;!Wu!et!al.,!2009),!have!suggested!that!the!inhibition!of!DPP?8!and!DPP?9!can!result! in!the!occurrence!of!thrombocytopenia,! reticulocytopeia,! multiorgan! histopathological! changes,! enlarged!! 201!spleen!and!mortality! (Lankas!et! al.,! 2005),!whereas! the! inhibition!of!FAP!could!affect!wound! healing! (Mentlein,! 2005).! Hence,! to! avoid! potential! adverse! events,! DPP?IV!inhibitors! should! only! affect! the! activity! of! the! DPP?IV! enzyme.! All! gliptins! currently!approved!for!the!management!of!type!2!diabetes!have!been!reported!to!have!some!level!of!selectivity!for!DPP?IV!over!the!other!related!enzymes,!with!sitagliptin!and!alogliptin!being! the! most! selective! inhibitors! (Filippatos! et! al.,! 2014;! Patel! &! Gate,! 2014).! The!target! selectivity! of! the! protein?derived! DPP?IV! inhibitors,! on! the! other! hand,! is!unknown!and!needs!to!be!examined.!!!In! order! to! exert! their! effect! in% vivo,! the! DPP?IV! inhibitory! peptides! have! to! remain!intact! until! they! reach! the! intestine.! Additional! work! is! therefore! required! to! assess!their! fate! during! the! digestion! process.! This! could! be! achieved! by! submitting! the!peptides! through! in% vitro% digestion! using! a! dynamic! digestion! model,! such! as! the!computer?controlled! multi?compartmental! dynamic! model! “TIM?1”,! which! has! been!successfully! used! to! study! the! digestibility,! availability! or! absorption! of! a! number! of!compounds! including!proteins,!vitamins,!minerals!and!nutraceuticals!(Framroze!et!al.,!2014).! Moreover,! to! prevent! the! inactivation! of! the! incretin! hormones,! inhibitory!peptides!have!to!be!absorbed!in!the!lumen!and!reach!the!endothelium!of!the!capillary!bed!within!the!intestinal!wall!where!the!DPP?IV!enzyme!can!be!found!in!close!proximity!to! the!cells!secreting! the! incretins!(Deacon,!2004).! It! is!unknown!whether! the!DPP?IV!inhibitory! peptides! identified! in! this! study! or! those! that! have! been! reported! in! the!literature! can! be! absorbed! in! the! lumen.! ! The! cellular! permeability! of! the! DPP?IV!inhibitory!peptides!and!hydrolysates!with!DPP?IV! inhibitory!activity! could!be! studied!using!the!Caco?2!cell!model.!This!approach!has!been!employed!in!a!number!of!studies!to!predict!the!in%vivo!permeability!of!bioactive!peptides!(Segura?Campos!et!al.,!2011).!!The! effectiveness! of! bioactive! peptides! on! DPP?IV! activity! in! humans! is! currently!unknown.!The!few!studies!that!have!investigated!the!effect!of!peptides!or!hydrolysates!with! DPP?IV! inhibitory! activity! in! rodents! have! suggested! that! the! administration! of!these!DPP?IV!inhibitors! led!to!a!reduction!in!blood!glucose!levels!(Uchida!et!al.,!2011;!Uenishi! et! al.,! 2012).! However,! it! is! unclear! whether! the! glucose?lowering! effect!! 202!observed! was! directly! caused! by! the! inhibition! of! DPP?IV.! 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