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Mechanisms of gluconeogenic activation in the rainbow trout liver Suarez, Raul Kamantigue 1980

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M E C H A N I S M S O F G L U C O N E O G E N I C A C T I V A T I O N I N T H E H A I N B O W T R O U T L I V E S by R A U L K A M A N T I G U E S U A R E Z Bo Sc., A T E N E O D E M A N I L A U N I V E R S I T Y , 1973 M . Sc., U N I V E R S I T Y O F T H E P H I L I P P I N E S , 1976 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O B T H E D E G R E E O F DOCTOR O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( D E P A R T M E N T O F Z O O L O G Y ) WE A C C E P T T H E T H E S I S AS C O N F O R M I N G TO T H E R E Q U I R E D S T A N D A R D THE UNIVERSITY O F BRITISH November 1980 C O L U M B I A © Raul K. Suarez, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Br i t ish Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for f inancial gain shall not be allowed without my written permission. Department of ~^OQ L^O &Y The University of Bri t ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT Experiments were conducted using i s o l a t e d mitochondria, enzyme (pyruvate c a r b o x y l a s e ) , and c e l l s from rainbow t r o u t l i v e r t o study some of the mechanisms which may b e . i n v o l v e d i n the r e g u l a t i o n of gluconeogenesis i n t h i s animal. On the b a s i s of these s t u d i e s , the f o l l o w i n g were found: 1. Mitochondria prepared from rainbow t r o u t l i v e r c o n s i s t e n t l y d i s p l a y high r e s p i r a t o r y c o n t r o l and ADP/O r a t i o s . These appear to possess a complete Krebs c y c l e , s i n c e pyruvate, p a l m i t o y l L - c a r n i t i n e , c i t r a t e , and v a r i o u s Krebs c y c l e i n t e r m e d i a t e s can a l l be o x i d i z e d . Rapid o x i d a t i o n of pyruvate and p a l m i t o y l L - c a r n i t i n e r e q u i r e s the presence of malate. O x i d a t i o n of p a l m i t o y l L - c a r n i t i n e appears to i n h i b i t pyruvate o x i d a t i o n . Malate s t i m u l a t e s a l p h a k e t o g l u t a r a t e o x i d a t i o n w hile a s p a r t a t e i n h i b i t s glutamate o x i d a t i o n , i n d i c a t i n g the presence of malate-a l p h a k e t o g l u t a r a t e and glutamate-aspartate c a r r i e r s . 2. P a l m i t o y l D L - c a r n i t i n e i n h i b i t s 1*C02 pro d u c t i o n from 1-[ 1 * C ] - p y r u v a t e and from 1-[**C ]-alanine by mitochondria from rainbow t r o u t l i v e r . The i n h i b i t o r y e f f e c t occurs i n both r e s p i r a t o r y s t a t e s I I I and IV. F i x a t i o n of H 1 4 C 0 3 _ i n t o a c i d - s t a b l e products by i n t a c t mitochondria r e q u i r e s pyruvate and ATP and i s i n h i b i t e d by sodium a r s e n i t e . T h i s i n h i b i t o r y e f f e c t i s completely a b o l i s h e d by a c e t y l DL-c a r n i t i n e . I t i s proposed t h a t under these c o n d i t i o n s . i i i o x i d a t i o n of p a l m i t o y l D L - c a r n i t i n e r e s u l t s i n i n h i b i t i o n of pyruvate dehydrogenase while o x i d a t i o n of a c e t y l DL-c a r n i t i n e r e s u l t s i n a c t i v a t i o n of pyruvate c a r b o x y l a s e i n i n t a c t rainbow t r o u t l i v e r mitochondria.. 3. Pyruvate carboxylase from rainbow t r o u t l i v e r has a pH optimum of about 8.0, possesses an absolute requirement f o r a c t i v a t i o n by acetylCoA, and p r e f e r s MgATP over other n u c l e o s i d e t r i p h o s p h a t e s . K + causes a decrease i n the apparent Km f o r HC03-. AcetylCoA a c t i v a t i o n shows p o s i t i v e c o o p e r a t i v i t y with Ka = 0.072 mM and nH = 1 r78 at pH 7.7, 2.5 mM f r e e Mg 2 +, 100 mM K+, and s a t u r a t i n g c o n c e n t r a t i o n s of s u b s t r a t e s . A high acetylCoA c o n c e n t r a t i o n causes a decrease i n the apparent Km values f o r MgATP and HC03 -, and a b i p h a s i c double r e c i p r o c a l p l o t with pyruvate as the v a r i e d s u b s t r a t e . MgADP and AMP a r e c o m p e t i t i v e i n h i b i t o r s with r e s p e c t to MgATP. The enzyme shows a "U-type" response to the adenylate energy charge and r e t a i n s c o n s i d e r a b l e a c t i v i t y throughout a wide range of energy charge v a l u e s . I t i s proposed that i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n and the ade n y l a t e energy charge c o n t r o l , t h e r a t e of pyruvate c a r b o x y l a t i o n i n v i v o . 4. I s o l a t e d hepatocytes from rainbow t r o u t l i v e r are capable of net s y n t h e s i s of glucose from CT-[1*C]-lactate. Gluconeogenesis from l a c t a t e i s s t i m u l a t e d by p a l m i t a t e , glucagon, and cAMP, and i n h i b i t e d by 3^-mercaptopicolinic a c i d . Treatment of hepatocytes with glucagon and cAMP at the c o n c e n t r a t i o n s which s t i m u l a t e gluconeogenesis from l a c t a t e r e s u l t s i n i n h i b i t i o n o f pyruvate kinase a c t i v i t y . i v Although maximum enzyme a c t i v i t y i s u n a f f e c t e d , the r a t i o s of a c t i v i t y a t low PEP c o n c e n t r a t i o n t o a c t i v i t y at high PEP c o n c e n t r a t i o n a r e depressed by such treatments. These r e s u l t s c o n firm those of p r e v i o u s s t u d i e s which i n d i c a t e t h a t f a t t y a c i d o x i d a t i o n may s t i m u l a t e gluconeogenesis i n rainbow t r o u t l i v e r . cAMP may a c t as the i n t r a c e l l u l a r messenger of glucagon; gluconeogenic a c t i v a t i o n by t h i s hormone probably i n v o l v e s pyruvate kinase i n a c t i v a t i o n i n v i v o which may occur as a r e s u l t of a cAMP-dependent p h o s p h o r y l a t i o n o f the enzyme. A r e g u l a t o r y scheme i s proposed i n which gluconeogenic a c t i v a t i o n r e s u l t s from i n h i b i t i o n of pyruvate o x i d a t i o n and s t i m u l a t i o n of pyruvate c a r b o x y l a t i o n by f a t t y a c i d o x i d a t i o n and i n h i b i t i o n of f u t i l e c y c l i n g between pyruvate and PEP by glucagon through a cAMP-mediated i n a c t i v a t i o n of pyruvate k i n a s e . V TABLE OF CONTENTS ABSTRACT - .. i i LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS ... . - ..... x i i CHAPTER I . GENERAL INTRODUCTION 1 Pathways Between Glucose And Pyruvate In The V e r t e b r a t e L i v e r 2 Compartmentation And Pathways From Pyruvate To PEP .... 4 The L i m i t a t i o n s Of Eat Biochemistry: Some Known Species D i f f e r e n c e s In Substrate U t i l i z a t i o n And C o n t r o l ... 6 Gluconeogenesis In F i s h : The Problem 9 CHAPTER I I . MATERIALS AND METHODS 25 F i s h 26 Chemicals 26 P r e p a r a t i o n Of Mitochondria 26 Measurement Of Rates Of Substrate O x i d a t i o n .....27 M i t o c h o n d r i a l P r o t e i n Measurement 28 Measurement Of Pyruvate Dehydrogenase A c t i v i t y In I n t a c t Mitochondria 28 Measurement Of Pyruvate Carboxylase A c t i v i t y In I n t a c t Mitochondria ... 30 P a r t i a l P u r i f i c a t i o n Of Pyruvate Carboxylase ..........30 P r o t e i n Measurement 32 Measurement Of Pyruvate Carboxylase A c t i v i t y 32 Determination Of K i n e t i c Constants 32 P r e p a r a t i o n Of I s o l a t e d Hepatocytes 33 v i Measurement Of Rates Of L a c t a t e O x i d a t i o n 34 Measurement Of Rates Of Gluconeogenesis ........... . 35 Demonstration Of Glucagon- And CAMP-induced I n a c t i v a t i o n Of Pyruvate Kinase 36 Measurement Of Pyruvate Kinase A c t i v i t y ............... 36 CHAPTER I I I . PROPERTIES OF RAINBOW TROUT LIVER MITOCHONDRIA .... ...... 38 I n t r o d u c t i o n 39 R e s u l t s . 40 D i s c u s s i o n 43 CHAPTER IV. THE PYRUVATE BRANCH POINT IN FISH' LIVER MITOCHONDRIA:EFFECTS OF ACYLCARNITINE OXIDATION ON PYRUVATE DEHYDROGENASE AND PYRUVATE CARBOXYLASE ACTIVITIES «... 53 I n t r o d u c t i o n 54 R e s u l t s . 55 D i s c u s s i o n 57 CHAPTER V. CATALYTIC AND REGULATORY PROPERTIES OF PYRUVATE CARBOXYLASE FROM RAINBOW TROUT LIVER .. ... 67 I n t r o d u c t i o n 68 R e s u l t s „.... 69 Enzyme P u r i t y And S t a b i l i t y 69 C a t a l y t i c P r o p e r t i e s And Reg u l a t i o n By AcetylCoA ... 70 C o n t r o l By Adenine N u c l e o t i d e s ......... - 73 D i s c u s s i o n 74 CHAPTER VI, CONTROL OF GLUCONEOGENESIS IN ISOLATED HEPATOCYTES FROM RAINBOW TROUT ... 91 I n t r o d u c t i o n 92 v i i R e s u l t s 9H D i s c u s s i o n - 95 CHAPTER V I I . GENERAL DISCUSSION 103 LITERATURE CITED ... 1 . . . . . . . 1 ...... 113 APPENDIX : L i s t Of A b b r e v i a t i o n s , 130 • • i v i i i LIST OF TABLES T a b l e 1-1. Some C l a s s i c a l A c t i v a t o r s And I n h i b i t o r s Of R e g u l a t o r y Enzymes I n G l y c o l y s i s And G l u c o n e o g e n e s i s .. 15 Ta b l e 1.2. I n t r a c e l l u l a r D i s t r i b u t i o n Of PEPCK I n Some V e r t e b r a t e L i v e r s 16 Ta b l e 3.1. R e s p i r a t o r y C o n t r o l (RCR) And ADP/O R a t i o s Of T y p i c a l P r e p a r a t i o n s Of Rainbow T r o u t L i v e r M i t o c h o n d r i a 47 T a b l e 3.2. S t a t e I I I R e s p i r a t o r y R ates O b t a i n e d With V a r i o u s S u b s t r a t e s 48 Ta b l e 3.3. E f f e c t Of P a l m i t o y l L - c a r n i t i n e O x i d a t i o n On The O x i d a t i o n Of P y r u v a t e + M a l a t e 49 Ta b l e 5.1. E f f e c t Of K+ On The K i n e t i c C o n s t a n t s Of T r o u t L i v e r P y r u v a t e C a r b o x y l a s e 78 Tab l e 5.2. N u c l e o s i d e T r i p h o s p h a t e S p e c i f i c i t y Of T r o u t L i v e r P y r u v a t e C a r b o x y l a s e 79 Table 6.1. G l u c o n e o g e n e s i s From U-[ 1*C ] - l a c t a t e 10 1 Tab l e 6.2. E f f e c t s Of Glucagon And CAMP On P y r u v a t e K i n a s e A c t i v i t y I n I s o l a t e d H e p a t o c y t e s 102 i x LIST OF FIGURES Figu r e 1.1. The G l y c o l y t i c And Gluconeogenic Pathways In The V e r t e b r a t e L i v e r ......... 17 Figu r e 1.2. G l y c o l y t i c And Gluconeogenic Reactions I n v o l v e d In F u t i l e C y c l e s In The Pathway Between Glucose And Pyruvate In V e r t e b r a t e L i v e r 18 Fi g u r e 1.3. Proposed Pathway From Pyruvate To PEP In L i v e r s With I n t r a m i t o c h o n d r i a l PEPCK .................. 20 Figure 1.4. Proposed Pathway From Pyruvate To PEP During Gluconeogenesis From L a c t a t e In L i v e r s With Mainly Cytoplasmic PEPCK . . . 21 Fi g u r e 1.5. Proposed Pathway To PEP During Gluconeogenesis From Pyruvate In L i v e r s With Mainly Cytoplasmic PEPCK 22 Figu r e 1.6. Proposed Pathway From Pyruvate To PEP During Gluconeogenesis From A l a n i n e In L i v e r s With Mainly Cytoplasmic PEPCK 23 Fi g u r e 1.7. The Pyruvate Branch P o i n t In T r o u t L i v e r M itochondria In R e l a t i o n To The Gluconeogenic Pathway And The Krebs C y c l e ----- ......... 24 Figure 3.1. O x i d a t i o n Of Various S u b s t r a t e s By Coupled Rainbow Trout L i v e r Mitochondria ....................... 50 Fi g u r e 3.2. Apparent Uncoupling E f f e c t Of MgC12 And I t s R e v e r s a l By Oligomycin .................... 51 Figure 3.3. Dependence Of PalmitoylCoA O x i d a t i o n On The Presence Of C a r n i t i n e 52 F i g u r e 4.1. O x i d a t i o n Of A c e t y l D L - c a r n i t i n e P l u s Malate And P a l m i t o y l D L - c a r n i t i n e P l u s M a l a t e ................ F i g u r e 4.2. E f f e c t Of P a l m i t o y l D L - c a r n i t i n e On MC02 P r o d u c t i o n From 1 - [ 1 * C ] - p y r u v a t e By M i t o c h o n d r i a I n S t a t e I I I F i g u r e 4.3. E f f e c t Of P a l m i t o y l D L - c a r n i t i n e On i*G02 P r o d u c t i o n From 1-[ 1*C ]-pyruvate By M i t o c h o n d r i a . I n S t a t e IV ,. F i g u r e 4.4. E f f e c t Of P a l m i t o y l D L - c a r n i t i n e On **C02 P r o d u c t i o n From 1-[ **C ] - a l a n i n e By M i t o c h o n d r i a I n S t a t e IV i . -F i g u r e 4.5. F i x a t i o n Of H 1*C03~ r By Rainbow T r o u t L i v e r M i t o c h o n d r i a F i g u r e 4.6. E f f e c t s Of Sodium A r s e n i t e And A c e t y l DL-c a r n i t i n e On H 1*C03~ F i x a t i o n F i g u r e 5.1. E f f e c t Of D i v a l e n t C a t i o n s On T r o u t L i v e r P y r u v a t e C a r b o x y l a s e A c t i v i t y F i g u r e 5.2. E f f e c t Of PH On Tr o u t L i v e r P y r u v a t e C a r b o x y l a s e A c t i v i t y -F i g u r e 5.3. AcetylCoA A c t i v a t i o n Of T r o u t L i v e r P y r u v a t e C a r b o x y l a s e . F i g u r e 5.4. Double R e c i p r o c a l P l o t s Of Data I n F i g . 5.3 Showing P o s i t i v e C o o p e r a t i v i t y F i g u r e 5.5. H i l l P l o t s Of Data In F i g . 5.3 I n The Region Of 50% S a t u r a t i o n ..... F i g u r e 5.6. E f f e c t Of A c e t y l C o A C o n c e n t r a t i o n On The K i n e t i c s Of T r o u t L i v e r P y r u v a t e C a r b o x y l a s e With P y r u v a t e As The V a r i e d S u b s t r a t e x i F i g u r e 5.7. E f f e c t Of AcetylCoA C o n c e n t r a t i o n On The K i n e t i c s Of Trout L i v e r Pyruvate Carboxylase With KHC03 As The V a r i e d S u b s t r a t e .... 86 Fig u r e 5.8- E f f e c t Of AcetylCoA C o n c e n t r a t i o n On The K i n e t i c s Of Trout L i v e r Pyruvate Carboxylase With MgATP As The Var i e d Substrate 87 Fi g u r e 5.9. Dixon P l o t Showing Competitive I n h i b i t i o n By MgADP With Respect To MgATP 88 Fig u r e 5.10. Dixon P l o t Showing Competitive I n h i b i t i o n By i " AMP With Respect To MgATP ,. . 89 F i g u r e 5.11. E f e c t Of The Adenylate Energy Charge On Trout L i v e r Pyruvate Carboxylase A c t i v i t y 90 Figu r e 6.1. Glucose S y n t h e s i s From U - [ 1 * C ] - l a c t a t e As A Fun c t i o n Of Time - - - - 99 Fi g u r e 6.2. Rate Of Glucose S y n t h e s i s From D - [ l * C ] -l a c t a t e As A Function Of C e l l Weight 100 Figu r e 7-1. Proposed Pathway For Net Conversion Of Var i o u s Amino A c i d s To Alanine In Salmon White Muscle . 111 Figu r e 7.2.. Proposed Regulatory Mechanisms For I n h i b i t i o n Of Pyruvate Dehydrogenase And Pyruvate Kinase And A c t i v a t i o n Of Pyruvate Carboxylase .................... 112 x i i ACKNOWLEDGEMENTS er Hochachka f o r g i v i n g me the o p p o r t u n i t y to f o r p r o v i d i n g the atmosphere, i d e a s , , and beer which made t h i s p o s s i b l e r members of my r e s e a r c h and examination P. Hahn, D. Holm, D. R a n d a l l , W. M i l s o n , Reeves, J. G o s l i n e , and W. Hoar f o r t h e i r s. to Mike Guppy, B r i a n Murphy, C h r i s French, B a l l a n t y n e , E r i c Shoubridge, and Brian Emmett f t e n endless) d i s c u s s i o n s , advice, and f r i e n d s h i p and h o s p i t a l i t y extended to me by those l i s t e d above, and t h e i r f r i e n d s w i l l always be remembered. The s t u d i e s presented here were funded by grants from the N a t i o n a l Science and Engineering C o u n c i l of Canada to P. W. Hochachka. F i n a n c i a l support i n the form of t e a c h i n g a s s i s t a n s h i p s and summer r e s e a r c h s c h o l a r s h i p s from the U n i v e r s i t y of B r i t i s h Columbia and a p a r t i a l , s c h o l a r s h i p from the Southeast Asian F i s h e r i e s Development Center i s g r a t e f u l l y acknowledged. F i n a l l y , I thank my w i f e . P i n i n g , f o r t y p i n g the t h e s i s and f o r her p a t i e n c e , support and encouragement. I thank Pet do s c i e n c e and s t i m u l a t i o n , funds I thank the committees, Drs. D. Vance, R. h e l p f u l suggestion I am g r a t e f u l Tom Mommsen, Jim f o r c o u n t l e s s (o i n s t r u c t i o n . The Peter and Brenda, 1 CHAPTER I„ GENERAL INTRODUCTION 2 Pathways Between Glucose And Pyruvate In The V e r t e b r a t e L i v e r The v e r t e b r a t e l i v e r i s a b i o c h e m i c a l l y complex organ p o s s e s s i n g both b i o s y n t h e t i c and degradative pathways f o r carbohydrates, f a t s , and amino a c i d s . One of i t s major f u n c t i o n s i s the maintenance of blood glucose c o n c e n t r a t i o n . The pathways f o r glucose breakdown ( g l y c o l y s i s ) and glucose s y n t h e s i s (gluconeogenesis) i n t h i s organ have thus r e c e i v e d c o n s i d e r a b l e a t t e n t i o n and have been the s u b j e c t of r e s e a r c h f o r many years. The two pathways are presented i n F i g . 1.1 i n a manner which emphasizes the flow of carbon through them i n opposite d i r e c t i o n s . Much of our present knowledge of g l y c o l y s i s and gluconeogenesis i n the l i v e r has come from s t u d i e s using r a t s . On the b a s i s of these s t u d i e s , i t i s g e n e r a l l y accepted that many of the enzyme-catalyzed r e a c t i o n s i n g l y c o l y s i s are maintained c l o s e t o e q u i l i b r i u m ( i . e., Kapp = Keg) and are r e a d i l y r e v e r s i b l e i n v i v o (Newsholme and S t a r t , 1973). These r e a c t i o n s are a l s o used i n the g e n e r a t i o n of f l u x i n the reverse or gluconeogenic d i r e c t i o n . However, th e r e i s much evidence which i n d i c a t e s t h a t the g l y c o l y t i c r e a c t i o n s c a t a l y z e d by hexokinase (or g l u c o k i n a s e ) , phosphofructokinase (PFK), and pyruvate kinase (PK) are maintained out of e q u i l i b r i u m ( i . e., Kapp<Keg) and are e s s e n t i a l l y i r r e v e r s i b l e i l l Ziy.o- These r e a c t i o n s are bypassed i n the gluconeogenic pathway by a separate s e t of n o n e g u i l i b r i u m r e a c t i o n s c a t a l y z e d by the enzymes glucose 6-phosphatase (G6Pase) , f r u c t o s e 1,6-diphosphatase (FDPase), pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PEPCK) ( F i g . 1.1) 3 (Newsholme and S t a r t , 1973). The presence of such enzymes which c a t a l y z e opposing n o n e g u i l i b r i u m r e a c t i o n s i n the same c e l l c r e a t e s the p o t e n t i a l f o r f u t i l e c y c l e s ( S c r u t t o n and O t t e r , 1968). In order f o r net f l u x t o occur i n a given d i r e c t i o n , the enzymes c a t a l y z i n g r e a c t i o n s i n t h a t d i r e c t i o n must be a c t i v a t e d while enzymes c a t a l y z i n g r e a c t i o n s i n the r e v e r s e d i r e c t i o n must be i n h i b i t e d . Simultaneous a c t i v i t y r e s u l t s i n f u t i l e c y c l i n g of m e t a b o l i t e s , wastage of energy, and l i t t l e or no net f l u x . Three p o t e n t i a l f u t i l e c y c l e s are apparent i n F i g . 1.1. These are c y c l e s between glucose and G6P, F6P and FDP, and PEP and pyruvate. The r e a c t i o n s i n v o l v e d i n these c y c l e s are presented i n g r e a t e r d e t a i l i n F i g . 1.2 t o show t h a t c y c l i n g r e s u l t s only i n net h y d r o l y s i s of ATP (or GTP) . The n o n e g u i l i b r i u m nature of these r e a c t i o n s i n d i c a t e that they may be r a t e - l i m i t i n g i n the g l y c o l y t i c and gluconeogenic pathways ( R o l l e s t o n , 1972). T h i s i s f u r t h e r supported by enzyme measurements which show t h a t these enzymes occur at r e l a t i v e l y low s p e c i f i c a c t i v i t i e s compared to enzymes c a t a l y z i n g r e a c t i o n s c l o s e to e q u i l i b r i u m i n the r a t l i v e r ( S crutton and U t t e r , 1968). Measurements of a c t u a l r a t e s of f l u x i n the g l y c o l y t i c (Hems and Brosnan, 1970) and gluconeogenic (Ross e t a l . , 1967) d i r e c t i o n s i n d i c a t e t h a t these enzymes probably f u n c t i o n at r a t e s w e l l below Vmax under p h y s i o l o g i c a l c o n d i t i o n s . The r e g u l a t o r y nature of these r e a c t i o n s i n . r a t s i s i n d i c a t e d by met a b o l i t e accumulation-d e p l e t i o n p a t t e r n s r e s u l t i n g from g l y c o l y t i c (Hems and Brosnan, 4 1970) and gluconeogenic (Exton and Park, 1966) a c t i v a t i o n , and the, r e g u l a t o r y p r o p e r t i e s of the i s o l a t e d enzymes i n v i t r o (Table 1.1). Although much i s a l r e a d y known about the mechanisms by which r e c i p r o c a l c o n t r o l of g l y c o l y s i s and gluconeogenesis i n the r a t l i v e r i s accomplished, t h i s c o n t i n u e s t o be an area of a c t i v e research,. Compartmentation and Pathways from Pyruvate to PEP Although the o v e r a l l pathway of gluconeogenesis appears to be the same i n a l l v e r t e b r a t e s s t u d i e d so f a r ( F i g . 1-1), there are s i g n i f i c a n t d i f f e r e n c e s i n the pathway between pyruvate and PEP. These are the r e s u l t of the i n t r a m i t o c h o n d r i a l l o c a l i z a t i o n of pyruvate c a r b o x y l a s e and d i f f e r e n c e s i n the compartmentation of PEPCK. Pyruvate c a r b o x y l a s e i s l o c a l i z e d almost e x c l u s i v e l y i n the mitochondria i n l i v e r s of a l l v e r t e b r a t e s examined thus f a r (Scrutton and U t t e r , 1968). The f i r s t step which commits pyruvate d e r i v e d from d i f f e r e n t p r e c u r s o r s e. g., l a c t a t e and a l a n i n e , to entry i n t o the . gluconeogenic pathway t h e r e f o r e occurs i n the mitochondria. However, PEPCK i s l o c a l i z e d mainly i n the cytoplasm of some s p e c i e s and mainly i n the mitochondria of other s p e c i e s . There are a l s o species, i n which the enzyme occurs i n both compartments. Species d i f f e r e n c e s i n the compartmentation of l i v e r PEPCK are presented i n Table 1.2. The o x a l o a c e t a t e generated by the pyruvate c a r b o x y l a t i o n step i s converted to PEP : i n the mitochondria of s p e c i e s with 5 s u b s t a n t i a l amounts of i n t r a m i t o c h o n d r i a l PEPCK. PEP i s then t r a n s p o r t e d out to complete the r e s t of the gluconeogenic pathway (Fig,1.3) (Garber and B a l l a r d , 1969; Garber and Hanson, 1971). In s p e c i e s i n which PEPCK i s mainly c y t o p l a s m i c , such as the r a t ( B a l l a r d and Hanson, 1967), o x a l o a c e t a t e generated by the pyruvate c a r b o x y l a t i o n step must be t r a n s p o r t e d out i n order f o r c o n v e r s i o n to PEP to occur. However, t h i s does not occur to a s i g n i f i c a n t extent because the i n n e r m i t o c h o n d r i a l membrane i s r e l a t i v e l y impermeable to o x a l o a c e t a t e ( C h a p p e l l , 1968). Conversion to other compounds which are capable of e x i t , e. g., a s p a r t a t e or malate, i s t h e r e f o r e necessary (Shrago and Lardy, 1966). In r a t s , gluconeogenesis from l a c t a t e i s i n h i b i t e d by aminooxyacetate (a transaminase i n h i b i t o r ) , but not by D-malate (a malate dehydrogenase i n h i b i t o r ) . On the other hand, gluconeogenesis from pyruvate i s i n h i b i t e d by D-malate but not by aminooxyacetate (Anderson £t a l - , 1971; Rognstad and Katz, 1970). Two separate pathways oc c u r i n g i n r a t l i v e r have thus been proposed. According to t h i s scheme, gluconeogenesis from l a c t a t e occurs v i a the b x a l o a c e t a t e - a s p a r t a t e route ( F i g . 1.4) and gluconeogenesis from pyruvate occurs v i a the oxaloacetate-malate route ( F i g . 1.5). T h i s scheme has been r a t i o n a l i z e d on the b a s i s of the need t o generate NADH r e q u i r e d f o r r e v e r s a l of the g l y c e r a l d e h y d e 3-phosphate dehydrogenase r e a c t i o n i n the cytoplasm d u r i n g gluconeogenesis (Shrago and Lardy, 1966). This occurs,by the t r a n s f e r of reducing e q u i v a l e n t s i n the form of malate from mitochondria to cytoplasm during gluconeogenesis from pyruvate or by r e v e r s a l of the l a c t a t e dehydrogenase 6 r e a c t i o n d u r i n g g l u c o n e o g e n e s i s from l a c t a t e . G l u c o n e o g e n e s i s from a l a n i n e makes use of the MDH r e a c t i o n , t o g e n e r a t e c y t o p l a s m i c r e d u c i n g e q u i v a l e n t s but i s more complex due to c o u p l i n g w i t h urea c y c l e ( F i g . 1.6) ( W i l l i a m s o n e t a l . , 1968). S p e c i e s p o s s e s s i n g high PEPCK a c t i v i t i e s i n both c y t o p l a s m i c and m i t o c h o n d r i a l compartments use both the o x a l o a c e t a t e -p h o s p h o e n o l p y r u v a t e and the o x a l o a c e t a t e - a s p a r t a t e pathways ( A r i n z e e t a l . , 1973). S t u d i e s o f t h e consequences of t h e s e d i f f e r e n c e s i n compartmentation a r e l i m i t e d and have so f a r been c o n f i n e d m a i n l y t o r a t s and guinea p i g s . S i n c e d i f f e r e n c e s i n the r e g u l a t o r y p r o p e r t i e s o f t h e pathway appear t o r e s u l t , a t l e a s t p a r t l y , from d i f f e r e n c e s i n compartmentation ( A r i n z e e t a l . , 1973; S o l i n g and K l e i n e k e , 1976), much can be gained by t h e use o f t h e s p e c i e s as a v a r i a b l e i n s t u d i e s o f the g l u c o n e o g e n i c pathway. The L i m i t a t i o n s of Rat B i o c h e m i s t r y : Some Known S p e c i e s D i f f e r e n c e s i n S u b s t r a t e U t i l i z a t i o n and C o n t r o l I t i s somewhat u n f o r t u n a t e t h a t most s t u d i e s o f metabolism and i t s r e g u l a t i o n a r e conducted u s i n g o n l y a s i n g l e s p e c i e s , the r a t . Hochachka (1975) p o i n t s o u t : "As a f i e l d of s c i e n c e , b i o c h e m i s t r y has never s e r i o u s l y s u b s c r i b e d t o t h e s t r a t e g y of u s i n g organisms as an e x p e r i m e n t a l parameter per s e , f i r s t , f o r th e i d e n t i f i c a t i o n o f unique problems, and second, f o r t h e s o l u t i o n of g e n e r a l ones." A l t h o u g h t h i s has been c o n v e n i e n t i n the f o r m u l a t i o n o f " g e n e r a l models" o f a n i m a l metabolism and 7 i t s r e g u l a t i o n , t h e n e g l e c t o f s p e c i e s d i v e r s i t y s u g g e s t s t h a t the g e n e r a l a p p l i c a b i l i t y of a t l e a s t some.of such models, i s s u s p e c t . T h i s appears t o be t r u e of t h e g l u c o n e o g e n i c pathway. Comparative s t u d i e s , though l i m i t e d i n both number and d e p t h , i n d i c a t e t h a t the mechanisms which r e g u l a t e g l u c o n e o g e n e s i s i n r a t s may not a l l be a p p l i c a b l e t o o t h e r v e r t e b r a t e s . Hanson (1980) has p o i n t e d out the need f o r c o m p a r a t i v e s t u d i e s of t h e c o n t r o l of g l u c o n e o g e n e s i s (as w e l l as o t h e r a s p e c t s of metabolism) i n s u p p o r t o f t h i s s u g g e s t i o n . At l e a s t some o f t h e s p e c i e s d i f f e r e n c e s i n t h e r e g u l a t i o n of t he g l u c o n e o g e n i c pathway may be due t o d i f f e r e n c e s , i n t h e compartmentation o f the enzyme PEPCK ( T a b l e 1-2). The pi g e o n l i v e r , which has a c o m p l e t e l y m i t o c h o n d r i a l PEPCK, i s a b l e t o s y n t h e s i z e g l u c o s e from l a c t a t e , but not from p y r u v a t e - and a l a n i n e ( S o l i n g e t a l . , 1970, 1973). T h i s may be due t o an i n a b i l i t y t o t r a n s p o r t r e d u c i n g e q u i v a l e n t s from t h e m i t o c h o n d r i a t o the c y t o p l a s m which r e s u l t s from t h e i n t r a m i t o c h o n d r i a l s y n t h e s i s o f PEP. The r a t l i v e r , w i t h m a i n l y c y t o p l a s m i c PEPCK, and t h e g u i n e a p i g l i v e r , w i t h both c y t o p l a s m i c and m i t o c h o n d r i a l PEPCK, a r e a b l e t o c o n v e r t a l l t h r e e s u b s t r a t e s t o g l u c o s e ( A r i n z e e t a l . , 1973). F a t t y a c i d o x i d a t i o n i s known t o s t i m u l a t e g l u c o n e o g e n e s i s i n r a t l i v e r ( W i l l i a m s o n , 1967; A r i n z e e t a l . , 1973). T h i s may occ u r by i n h i b i t i o n o f p y r u v a t e o x i d a t i o n ( P o r t e n h a u s e r and Wieland, 1972) and co n c o m i t a n t s t i m u l a t i o n of p y r u v a t e c a r b o x y l a t i o n (Wojtczak e t a l . , 1972) by i n c r e a s e d i n t r a m i t o c h o n d r i a l a c e t y l C o A c o n c e n t r a t i o n . I n c o n t r a s t , f a t t y 8 a c i d o x i d a t i o n i n h i b i t s g l u c o n e o g e n e s i s from l a c t a t e and a l a n i n e i n t h e guin e a p i g l i v e r ( A r i n z e e t a l . , 1973; S o l i n g e t a l - , 1970). T h i s may r e s u l t from an i n c r e a s e d NADH/NAD+ r a t i o i n the m i t o c h o n d r i a l m a t r i x . The i n c r e a s e d r e d u c t i o n o f t h e m i t o c h o n d r i a l redox s t a t e would r e s u l t i n a decrease i n t h e c o n c e n t r a t i o n o f i n t r a m i t o c h o n d r i a l o x a l o a c e t a t e , t h u s i n h i b i t i n g t h e m i t o c h o n d r i a l PEPCK r e a c t i o n (Garber and B a l l a r d , 1970; Garber and Hanson, 1971). Numerous s t u d i e s have been devoted t o t h e hormonal c o n t r o l of g l u c o n e o g e n e s i s i n r a t l i v e r . Glucagon and c a t e c h o l a m i n e s s t i m u l a t e g l u c o n e o g e n e s i s w h i l e i n s u l i n . i n h i b i t s g l u c o n e o g e n e s i s i n t h i s t i s s u e ( P i l k i s e t a l . 1 9 7 8 , Exton e t a l . , 1970). Glucagon a c t i o n i s based m a i n l y upon cAMP-mediated mechanisms (Exton and Park,1968; G a r r i s o n and Haynes, 1973) w h i l e c a t e c h o l a m i n e s t i m u l a t i o n of g l u c o n e o g e n e s i s may not be dependent upon cAMP ( P i l k i s e t a l . , 1978). I n s u l i n appears t o i n h i b i t g l u c o n e o g e n e s i s by c a u s i n g a decrease i n cAMP l e v e l s i n r a t l i v e r (Exton e t a l . , 1970). However, g l u c o n e o g e n e s i s from l a c t a t e i n gui n e a p i g l i v e r i s u n a f f e c t e d by glucagon and cAMP ( S o l i n g e t a l . , 1970). The same t r e a t m e n t s , however, r e s u l t i n s t i m u l a t i o n of g l y c o g e n o l y s i s , u r e o g e n e s i s , and k e t o g e n e s i s , j u s t as i n r a t l i v e r . The c y t o p l a s m i c NADH/NAD+ r a t i o i s h i g h e r i n gu i n e a p i g l i v e r t h a n i n r a t l i v e r (Garber and Hanson, 1971; T i s c h l e r e t a l . , 1977). T h i s may l e a d t o l a r g e d i f f e r e n c e s i n t h e p r o p e r t i e s of p y r i d i n e n u c l e o t i d e - l i n k e d r e a c t i o n s i n v i v o bejtween t h e two a n i m a l s . However, t h e m e t a b o l i c consequences 9 o f t h i s d i f f e r e n c e i n redox s t a t e a r e l a r g e l y unknown (Hanson, 1980) . On t h e bases o f the known s p e c i e s v a r i a t i o n i n PEPCK comp a r t m e n t a t i o n (Table 1.2) and t h e l i m i t e d amount o f i n f o r m a t i o n on s p e c i e s d i f f e r e n c e s i n r e g u l a t i o n p r e s e n t e d above, i t appears t h a t the w i d e s p r e a d use o f t h e r a t i n s t u d i e s of g l u c o n e o g e n e s i s and t h e c o n t i n u e d e x t r a p o l a t i o n from t h e r a t to o t h e r v e r t e b r a t e s i s unwarranted. T h i s i s not w i t h o u t p r a c t i c a l consequences. F o r example, o t h e r a n i m a l s such as t h e guinea p i g may be b e t t e r models o f human g l u c o n e o g e n e s i s i n t h e d i a b e t i c s t a t e than the r a t (Hanson, 1980). G l u c o n e o g e n e s i s i n F i s h : The Problem The c a p a c i t y t o c o n v e r t l a c t a t e t o g l u c o s e appears t o be a common f e a t u r e of v e r t e b r a t e l i v e r s ( P h i l l i p s and H i r d , 1977). L a c t a t e i s an i m p o r t a n t g l u c o n e o g e n i c s u b s t r a t e because i t i s t h e main end product o f a n a e r o b i c g l y c o l y s i s i n v e r t e b r a t e s (Hochachka, 1980). A f t e r s e v e r e e x e r c i s e or e n v i r o n m e n t a l l y imposed a n o x i a , t h e l a c t a t e produced by s k e l e t a l muscle and o t h e r organs i s r e l e a s e d i n t o t h e b l o o d s t r e a m . I t s two major f a t e s a r e o x i d a t i o n t o C02 and H20 v i a t h e Krebs c y c l e and c o n v e r s i o n t o g l u c o s e by the g l u c o n e o g e n i c pathway. The amino a c i d s c o n s t i t u t e a n o ther p o o l o f g l u c o n e o g e n i c s u b s t r a t e s . A l a n i n e i s an i m p o r t a n t g l u c o n e o g e n i c s u b s t r a t e i n the r a t (Ross e t a l . , 1967), g u i n e a p i g ( A r i n z e e t a l . , 1973), and e e l (Hayashi and O o s h i r o , 1979) l i v e r s . G l u c o n e o g e n e s i s from o t h e r 10 amino a c i d s i s known to occur i n the r a t l i v e r (Ross et a l . , 1967) but has not been s y s t e m a t i c a l l y i n v e s t i g a t e d i n other s p e c i e s . Gluconeogenic amino a c i d s may come from the d i e t . In c a r n i v o r o u s animals, d i e t a r y amino a c i d s c o n s t i t u t e a major source o f glucose (Newsholme and S t a r t , 1973). S k e l e t a l muscle i s a source of gluconeogenic amino a c i d s (Goldberg and Chang, 1978; Goldberg e t a l . , 1980) which may be of e s p e c i a l importance during s t a r v a t i o n . Gluconeogenesis from l a c t a t e and amino a c i d s i n f i s h i s of great i n t e r e s t because severe e x e r c i s e r e s u l t s i n accumulation of l a r g e amounts of l a c t a t e i n muscle, much of which i s s l o w l y r e l e a s e d i n t o the bloodstream ( D r i e d z i c and Hochachka, 1978) and because f i s h a re capable of extended p e r i o d s of combined e x e r c i s e and s t a r v a t i o n (Mommsen et a l . , 1980). However, our knowledge of the pathway and r e g u l a t i o n of gluconeogenesis i n f i s h i s l i m i t e d . On the b a s i s of gluconeogenic enzyme content, the l i v e r appears to be the main gluconeogenic organ i n f i s h . The t r o u t l i v e r , f o r example, c o n t a i n s h i g h e r a c t i v i t i e s of pyruvate c a r b o x y l a s e , PEPCK, and FDPase than kidney, g i l l s , and s k e l e t a l (white) muscle (Cowey et a l , , 1977). Gluconeogenesis from l a c t a t e and a number of amino a c i d s has been demonstrated u s i n g hepatocytes from t r o u t (Walton and Cowey, 1979a and b; French et a l . , 1980), Japanese e e l (Hayashi and Ooshiro, 1979), and American e e l (Renaud and Moon, 1979). S t u d i e s of PEPCK compartmentation have shown t h a t the enzyme i s mainly m i t o c h o n d r i a l i n t r o u t l i v e r (Walton and Cowey, 1979b) but i s 11 both c y t o p l a s m i c and m i t o c h o n d r i a l i n the l i v e r of the Japanese e e l (Hayashi and Ooshiro, 1979). The i n t r a m i t o c h o n d r i a l c o n v e r s i o n of pyruvate to PEP by way of the oxaloacetate-PEP route can be i n f e r r e d from the m i t o c h o n d r i a l l o c a l i z a t i o n of PEPCK i n t r o u t l i v e r . On the other hand, t h i s occurs mainly through the o x a l o a c e t a t e - a s p a r t a t e route i n the l i v e r of the Japanese e e l (Hayashi and Ooshiro, 1979). With r e s p e c t to r e g u l a t o r y mechanisms, the f i s h l i v e r i s v i r t u a l l y a black box. The c a p a c i t y f o r c e r t a i n pathways, e. g., glucose o x i d a t i o n (Hochachka, 1968), f a t t y a c i d o x i d a t i o n ( B i l i n s k i and Jonas, 1970), amino a c i d o x i d a t i o n (French et a l , , 1980), gluconeogenesis (Walton, and Cowey, 1979), and f a t t y a c i d s y n t h e s i s (Hazel and S e l l n e r , 1979; Hazel and P r o s s e r , 1979) i s known, but the mechanisms by which f l u x e s are d i r e c t e d and c o n t r o l l e d and the nature of the i n t e r a c t i o n s between pathways are v i r t u a l l y unknown. What determines whether the l i v e r o x i d i z e s or s y n t h e s i z e s g l u c o s e i n f i s h , i . e., whether net f l u x . o c c u r s from glucose to pyruvate or v i c e versa? How does f a t t y a c i d o x i d a t i o n a f f e c t carbohydrate metabolism i n the f i s h l i v e r ? What are the r e g u l a t o r y consequences of d i f f e r e n c e s i n PEPCK compartmentation? To what extent are amino a c i d s used as gluconeogenic s u b s t r a t e s ? What hormones c o n t r o l gluconeogenesis i n f i s h and what are t h e i r mechanisms of a c t i o n ? Host of these areas are unexplored, and one can h a r d l y say that what i s t r u e of the r a t i s a l s o t r u e of f i s h i n the absence of any i n f o r m a t i o n . The nature of the q u e s t i o n s asked above suggests t h a t the 12 problem of gluconeogenic r e g u l a t i o n i n f i s h i s as much a fundamental problem as i t i s a comparative one. The c o n t r o l of gluconeogenesis i n r a t s i s f a r from being a c l o s e d s t o r y ; the l i t e r a t u r e i n t h i s area abounds with r e p o r t s of new mechanisms and c o n t r o v e r s y r e g a r d i n g mechanisms p r e v i o u s l y .proposed. For example, the i d e a t h a t f a t t y a c i d o x i d a t i o n may have a s t i m u l a t o r y e f f e c t on gluconeogenesis i n the r a t i s s t i l l under d i s p u t e . There i s s t i l l much c o n t r o v e r s y r e g a r d i n g whether the phenomenon i s of p h y s i o l o g i c a l importance or not (Exton et a l . , 1970) . C e r t a i n l y , those who are i n t e r e s t e d i n gluconeogenesis and who seek t o apply the August Krogh p r i n c i p l e i n t h e i r work (a sma l l percentage of the human p o p u l a t i o n thus far) would not h e s i t a t e to use f i s h i n s t u d i e s of gluconeogenesis. Members of the f a m i l y Salmonidae (e. g., salmon and trout) are e s p e c i a l l y s u i t a b l e s i n c e they are very a c t i v e swimmers and are capable of accumulating more than 50 mM l a c t a t e i n white muscle d u r i n g bu r s t swimming (Black e t a l . , 1966). C e r t a i n s p e c i e s e, g., sockeye salmon and steel h e a d t r o u t , experience a combination of prolonged e x e r c i s e (with i n t e r m i t t e n t b u r s t swims) and s t a r v a t i o n d u r i n g the spawning m i g r a t i o n ( I d l e r and B i t n e r s , 1958; Mommsen et a l . , 1980). During t h i s p e r i o d , p r o g r e s s i v e breakdown of white muscle p r o t e i n r e s u l t s i n the r e l e a s e of amino a c i d s i n t o the bloodstream (Mommsen e t a l , , 1980), while muscle and l i v e r glycogen and glucose l e v e l s are maintained (C. French and T. Mommsen, p e r s o n a l communication),. T h i s i n d i c a t e s t h a t the gluconeogenic pathway i s c a r e f u l l y 13 c o n t r o l l e d and c r i t i c a l l y important i n t h i s group o f animals. The c a p a c i t y of t r o u t l i v e r t o o x i d i z e l a c t a t e ( B i l i n s k i and Jonas, 1972) and amino a c i d s t h a t give r i s e t o pyruvate ( s e r i n e , g l y c i n e , and alan i n e ) (French e t a l f , 1980) r a i s e s an important q u e s t i o n : How does the gluconeogenic pathway compete f o r s u b s t r a t e with the Krebs c y c l e ? Since l a c t a t e , s e r i n e , g l y c i n e , and a l a n i n e a l l gi v e r i s e t o pyruvate, the common s u b s t r a t e of gluconeogenesis and the Krebs c y c l e , the q u e s t i o n i n more p r e c i s e terms i s , how does pyruvate c a r b o x y l a s e compete f o r s u b s t r a t e with pyruvate dehydrogenase? The s i g n i f i c a n c e of t h i s problem i s made apparent i n F i g . 1.7 which shows the pyruvate branch p o i n t i n r e l a t i o n to gluconeogenesis and the Krebs c y c l e . The i n t r a m i t o c h o n d r i a l l o c a l i z a t i o n of PEPCK i n the t r o u t l i v e r and the p o t e n t i a l f o r f u t i l e c y c l i n g between pyruvate and PEP which r e s u l t s from the presence o f . a l l the enzymes i n v o l v e d i n the c y c l e (Cowey et al.,19 77; Somero and Hochachacka, 1968) r a i s e s another s e t of q u e s t i o n s : What determines the f a t e of PEP a f t e r i t i s t r a n s p o r t e d out of the mitochondria i n t r o u t l i v e r ? How i s i t prevented from g e t t i n g r e c o n v e r t e d back to pyruvate by the pyruvate kinase r e a c t i o n ? In more , g e n e r a l terms, how i s carbon flow d i r e c t e d towards g l u c o s e r a t h e r than towards pyruvate? Two s e t s of ob s e r v a t i o n s have been important i n the way I have approached these problems. The f i r s t i s the o b s e r v a t i o n t h a t m itochondria and c e l l s from rainbow t r o u t l i v e r a c t i v e l y 14 o x i d i z e f a t t y a c i d ( B i l i n s k i and Jonas, 1970; French et a l . , 1980) and the o b s e r v a t i o n t h a t m i g r a t i n g salmon ( I d l e r and B i t n e r s , 1958) and t r o u t s u b j e c t e d to combined e x e r c i s e and s t a r v a t i o n (Robinson and and Mead, 1973) m o b i l i z e t h e i r body l i p i d s t o r e s . In the t r o u t , a t r a n s i e n t i n c r e a s e i n blood f r e e f a t t y a c i d c o n c e n t r a t i o n has been observed under these c o n d i t i o n s ( B i l i n s k i and Gardner, 1968). These o b s e r v a t i o n s l e d me t o c o n s i d e r the p o s s i b i l i t y t h a t f a t t y a c i d o x i d a t i o n by the l i v e r may i n h i b i t the o x i d a t i o n of s u b s t r a t e s which give r i s e t o pyruvate, w h i l e . f a v o r i n g the entry of the pyruvate generated i n t o the gluconeogenic pathway. The.experimental demonstration of a c t i v a t i o n of pyruvate c a r b o x y l a t i o n and gluconeogenesis by f a t t y a c i d and the e l u c i d a t i o n of.some of the mechanisms by which these occur c o n s t i t u t e s the major bulk of t h i s work. The second o b s e r v a t i o n i s the s t i m u l a t i o n of gluconeogenesis from l a c t a t e and a l a n i n e i n t r o u t hepatocytes by glucagon (Walton and Cowey, 1979). T h i s . l e d me to examine the p o s s i b i l i t y t h a t f u t i l e c y c l i n g between pyruvate and PEP may be prevented by i n a c t i v a t i o n of the g l y c o l y t i c enzyme pyruvate k i n a s e . T h i s t h e s i s i s t h e r e f o r e concerned with the mechanisms by which entry of carbon i n t o the gluconeogenic pathway and d i r e c t i o n a l i t y of f l u x t o glucose are c o n t r o l l e d i n the t r o u t l i v e r . 15 Table 1.1. Some c l a s s i c a l a c t i v a t o r s and i n h i b i t o r s of E§2^iai2£2 enzymes i n g l y c o l y s i s and g l u c o n e o g e n e s i s J i o d i f i e d from S c r u t t o n and a t t e r x 1968). Enzyme A c t i v a t o r s I n h i b i t o r s H e xokinase G6P P h o s p h o f r u c t o k i n a s e AMP. FDP, P i ATP, c i t r a t e P y r u v a t e k i n a s e FDP ATP P y r u v a t e c a r b o x y l a s e PEP c a r b o x y k i n a s e F r u c t o s e 1,6 d i p h o s p h a t a s e AcetylCoA ADP F e r r o - a c t i v a t o r p r o t e i n ( c y t o p l a s m i c ) AMP 16 Table 1.2. I n t r a c e l l u l a r d i s t r i b u t i o n of PEPCK i n some v e r t e b r a t e l i v e r s . Values are expressed as a percentage of t o t a l enzyme recovered from l i v e r s o f fed animals. Animal Cytoplasm Mitochondria Reference Rat 92.3 4.0 B a l l a r d and Hanson , 1967 Guinea p i g 26.5 73.5 Garber and Hanson, 1971 E e l 57.2 30.9 Hayashi and Ooshiro, 1979 Trout 9.0 91.0 Walton and Cowey, 1979 P l a i c e 3.9 96. 1 Johnston and Moon, 1980 Rabbit 2.7 64i6 Garber and Hanson, 1971 Lamprey 26.5 73. 5 P h i l l i p s and H i r d , 1977 Toad 9.8 9012 P h i l l i p s and H i r d , 1977 L i z a r d 2.0 98.0 P h i l l i p s and H i r d , 1977 Pigeon 10.0 86.0 Gevers, 1967 17 Fi g u r e 1.1. The g l y c o l y t i c and gluconeogenic pathways i n t h e v e r t e b r a t e l i v e r . Curved arrows rep r e s e n t n o n e g u i l i b r i u m r e a c t i o n s c a t a l y z e d by the enzymes l i s t e d i n F i g , . 2. 17a Glucose V F6P, 'FDP G3P«—>DHAP 13DPG 3 PGA 2 PGA •PEP \ Pyruvate Oxaloacetate 18 Figure 1,2. G l y c o l y t i c and gluconeogenic reactions involved i n f u t i l e cycles i n the pathway between cjlucose and pyruvate i n vertebrate l i v e r . 19 F i g . 1.2 A. GLUC0SE-G6P CYCLE Hexokinase ATP + glucose — > ADP + G6P Glucose 6-phosphatase G6P t H20 > glucose + P i Net Result ATP + H20 > A DP + P i B- F6P ZFDP CYCLE • Phosphofructokinase ATP + F6P > ADP + FDP [ F r u c t o s e 1,6 diphospha- FDP + H20 > F6P + Pi tase [ Net Result ATP + H20 > A DP + P i C. PEP-PYRUVATE CYCLE Pyruvate kinase . PEP + ADP — > Pyruvate + ATP Pyruvate c a r b o x y l a s e Pyruvate + C02 + ATP + H2Q > Oxa + ADP + P i PEPCK Oxa + GTP > PEP + C02 + GDP Net R e s u l t GTP + H20 > GDP + P i 20 F i g u r e 1.3. Proposed pathway from pyruvate t o PEP d u r i n g gluconeogenesis from l a c t a t e i n l i v e r s with i n t r a m i t o c h o n d r i a l PEPCK, <giX. guinea p i g f i pigeon^ t r o u t . 20a Lactate \K>NADH Pyruvate cyto mjto CO Py ruvate ATP ->ADP Oxaloacetate Glucose i N A D + N A D + NADH T P E P co2 T P E P GTP GDP 21 F i g u r e 1.4. Proposed pathway from pyruvate t o PEP d u r i n g gluconeogenesis from l a c t a t e i n l i v e r s with mainly c y t o p l a s m i c PEPCK, e.. c j i J L r a t l i v e r . 21a Glucose NAD NADH Lactate ptrp Y N A D + C 0 2 ^ 1 > G D P N A D H Pyruvate GTP >yto OXA<: Glu CXKG i—>> Aspartate 4s mi to- r n Glu oCKG Pyruva te^ O X A — ^ Aspartate ATP ADP 22 F i g u r e 1.5. Proposed pathway t o PEP during gluconeogenesis from pyruvate i n l i v e r s with mainly, c y t o p l a s m i c PEPCK,, e. g., r a t l i v e r . 22a Glucose NAD N A D H /N P E P C 0 2 < J > - * G D P Pyruvate GTP cyto 0XA<r T NADH N A D ' Malate mi to . C ° 2 Pyruvate \ ~ ATP ADP > OXA NADH NAD Ma ate 23 F i g u r e 1.6. Proposed pathway from pyruvate t o PEP d u r i n g a i ^ c o n e o g e n e s i s from a l a n i n e i n l i v e r s with mainly c y t o p l a s m i c PEPCK, e A g i X r a t l i v e r , showing c o u p l i n g with the urea c y c l e . For s i m p l i c i t y , only the pathway i n v o l v i n g i n t r a m i t o c h o n d r i a l glutamate-pyruvate transaminase (GPT) i s shown. 23a cyto Atanjne OXA GTP GDP PEP -> Glucose NADH S- NADH Malate Fum Argsucc Arg Citrulline 4\ Aspartate -> Urea mito Alanine Aspartate v p ^ > Glu ^ Pyruvate > OXA Citrulline As Carbamyl Phosphate 24 F i g u r e 1.7. The p y r u v a t e branch p o i n t i n t r o u t l i v e r l i t o c h o n d r i a i n r e l a t i o n t o t h e g l u c o n e o g e n i c pathway and t h e Krebs c y c l e . 24a Glucose P E P Pyruvate > Pyr / uvate \ AcetylCoA Citrate CHAPTER I I , MATERIALS AND METHODS 26 F i s h Rainbow t r o u t (Salmo g a i r d n e r i Richardson) of both sexes weighing 150-300 grams were obtained from a commercial f i s h farm. These were kept i n aerated running water at 10-15°C and fed d a i l y with 1/4 i n c h Clark f i s h p e l l e t s (Moore-Clark Co., S a l t Lake C i t y , Utah). Chemicals , A c e t y l D L - c a r n i t i n e and bovine glucagon were purchased from Calbiochem, San Diego, CA. 1-[ 1 *C]-pyruvate, U - [ l * C ] -l a c t a t e , and B i o f l u o r were purchased from New England Nuclear, Lachine, Que. 1-[**C ]-alanine was purchased from Amersham, O a k v i l l e , Ont. Hyamine 10-X hydroxide was from BDH Chemicals, Vancouver, B. C. A l l other o r g a n i c m e t a b o l i t e s were o b t a i n e d from Sigma Chemical Co., St. L o u i s , MO. Other chemicals were purchased from v a r i o u s commercial sources and were of a n a l y t i c a l grade. P r e p a r a t i o n of Mitochondria F i s h were k i l l e d by a sharp blow on the head. L i v e r s were g u i c k l y d i s s e c t e d out, f r e e d of g a l l bladders and c o n n e c t i v e t i s s u e , and washed i n c o l d homogenization b u f f e r . T h i s c o n s i s t e d of 10 mM potassium phosphate, 0.25 M sucrose, 0.5 mM EDTA, at pH 7.4. A l l i s o l a t i o n procedures were c a r r i e d out at 27 0-4°C. The l i v e r s were weighed, then minced with ra z o r blades i n homogenization b u f f e r i n a p e t r i d i s h . Another s e r i e s of washings with homogenization b u f f e r was then r e q u i r e d t o remove b i l e and blood. Homogenization was conducted i n about 10 volumes of b u f f e r i n a Potter-Elvehjem homogenizer with a l o o s e - f i t t i n g T e f l o n p e s t l e . Mitochondria were then i s o l a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . The crude homogenate was spun i at 600 x g f o r 10 min. The supernatant from the f i r s t s p i n was decanted and spun a t 9000 x g f o r 10 min. The supernatant from t h i s s p i n was d i s c a r d e d and the p e l l e t was resuspended i n about 10 volumes of b u f f e r by g e n t l e a s p i r a t i o n with a Pasteur p i p e t t e . The dark, pigmented m a t e r i a l at the bottom of the p e l l e t was l e f t u ndisturbed and the r e s t of the resuspended p e l l e t was spun i n another tube at 9000 x g f o r 10 min. M i t o c h o n d r i a l p r e p a r a t i o n s were washed i n t h i s manner 3 times before f i n a l r esuspension i n a s m a l l volume of , homogenization b u f f e r c o n t a i n i n g 1 mg o f . f a t t y a c i d - f r e e bovine serum albumin per ml. F i n a l m i t o c h o n d r i a l p r o t e i n c o n c e n t r a t i o n s of these p r e p a r a t i o n s were g e n e r a l l y between 15-30 mg per ml. P r e p a r a t i o n s were s t a b l e f o r about 8 hours when kept a t i c e temperature. Measurement of Rates of Substrate O x i d a t i o n Oxygen uptake was monitored using a G i l s o n oxygraph with a C l a r k - t y p e 02 e l e c t r o d e . Assays were conducted i n a f i n a l volume of 2 ml i n a water-jacketed c e l l . Temperature was 28 maintained a t 15°C with a constant temperature water bath and c i r c u l a t o r - C e l l contents were mixed with a magnetic s t i r r e r and a T e f l o n - c o v e r e d bar. The assay b u f f e r c o n s i s t e d of 25 mM potassium phosphate, 10 mM T r i s , 100 mM KCL, and 2.7 mg per ml of f a t t y a c i d - f r e e bovine serum albumen, at pH 7.1 (Smith, 1973) . 100 u l of m i t o c h o n d r i a l suspension c o n t a i n i n g between 1.5-3.0 mg m i t o c h o n d r i a l p r o t e i n were used f o r each assay. S u b s t r a t e s were d i s s o l v e d i n assay b u f f e r , n e u t r a l i z e d with KOH, and i n j e c t e d i n t o the c e l l through a s m a l l , removable port using 10 u l Hamilton s y r i n g e s . Rates of 02 consumption, r e s p i r a t o r y c o n t r o l , and ADP/0 r a t i o s were c a l c u l a t e d a c c o r d i n g to Estabrook (1967). R e s p i r a t o r y s t a t e s are d e f i n e d , a c c o r d i n g to Chance and Williams (1956). M i t o c h o n d r i a l P r o t e i n Measurement M i t o c h o n d r i a l p r o t e i n was measured with a b i u r e t method using 10% deoxycholate to s o l u b i l i z e membrane, p r o t e i n s ( G o r n a l l £t a l , , 1948). Bovine serum albumen was used as the standard. Measurement of Pyruvate Dehydrogenase A c t i v i t y i n I n t a c t M i tochondria Assays were conducted i n the same medium used i n the oxygraph study with the a d d i t i o n of 5 mM MgCl2. Pyruvate dehydrogenase a c t i v i t y was assessed by measuring the r a t e of l*C02 pro d u c t i o n from 1-[ 1 * C ] - p y r u v a t e or 1-[ 1*C ] - a l a n i n e . 29 I n c u b a t i o n s were conducted i n 25 ml Erlenmeyer f l a s k s i n a f i n a l volume of 2 ml. F l a s k s were covered with rubber serum caps to which p l a s t i c center w e l l s were attached, The w e l l s c o n t a i n e d 2.4 cm Whatman m i c r o f i b r e f i l t e r paper. Temperature was maintained a t 15°C with a constant temperature water bath and shaker. Reactions were i n i t i a t e d by i n j e c t i o n of 100 u l m i t o c h o n d r i a l suspension, allowed t o proceed with c o n s t a n t shaking, and terminated by i n j e c t i o n of 0.2 ml of 70% p e r c h l o r i c a c i d . C o n t r o l s c o n s i s t e d of f l a s k s i n t o which p e r c h l o r i c a c i d was i n j e c t e d p r i o r to mitochondria and shaken f o r the same l e n g t h of time. Determinations were done i n d u p l i c a t e . Upon t e r m i n a t i o n o f r e a c t i o n s , 0,2 ml of Hyamine hydroxide was i n j e c t e d i n t o the cente r w e l l s and the f l a s k s were shaken f o r 90 min at room temperature. At the end of t h i s p e r i o d , f i l t e r papers were t r a n s f e r r e d to v i a l s c o n t a i n i n g 10 ml of s c i n t i l l a t i o n f l u i d (100 mg POPOP, 2 g PPO, 800 ml to l u e n e , and 200 ml e t h a n o l ) . R a d i o a c t i v i t y was determined with a l i q u i d s c i n t i l l a t i o n counter a t an e f f i c i e n c y of about 80%. Assays conducted with mitochondria i n St a t e I I I r e s p i r a t i o n were conducted under the same c o n d i t i o n s with the a d d i t i o n of 1 u n i t of d i a l y z e d hexokinase, 10 mM glucose, and 0.2 mM ADP. In a d d i t i o n , f l a s k were gassed f o r 10 sec with 02 before capping t o prevent complete deoxygenation d u r i n g the assay. P r e l i m i n a r y experiments i n v o l v i n g measurement of r a t e s of 02 consumption i n d i c a t e d t h a t S t a t e I I I r e s p i r a t i o n was induced and maintained under these c o n d i t i o n s . 30 Measurement of Pyruvate Carboxylase A c t i v i t y , i n I n t a c t Mitochondria Assays were conducted under e s s e n t i a l l y the same c o n d i t i o n s as those used, f o r the measurement of pyruvate dehydrogenase a c t i v i t y . However, i n these experiments, the r a t e o f f i x a t i o n of H1*C03:~ i n t o a c i d - s t a b l e products was measured. Reactions were i n i t i a t e d and , stopped i n s e a l e d f l a s k s as d e s c r i b e d p r e v i o u s l y . A f t e r shaking f o r 90 min at room temperature, p r e c i p i t a t e d p r o t e i n was spun down i n t e s t tubes u s i n g a t a b l e - t o p c e n t r i f u g e . 0.5 ml. a l i g u o t s of supernatant were withdrawn and mixed with 10 ml of B i o f l u o r i n v i a l s f o r l i q u i d s c i n t i l l a t i o n counting. A c i d - s t a b l e r a d i o a c t i v i t y was counted at about 88% e f f i c i e n c y . P a r t i a l P u r i f i c a t i o n of Pyruvate Carboxylase M i t o c h o n d r i a from l i v e r s of 20-30 f i s h were i s o l a t e d and washed 3 times as d e s c r i b e d p r e v i o u s l y . The m i t o c h o n d r i a l p e l l e t was resuspended i n about 10 volumes of b u f f e r c o n s i s t i n g of 50 mM t r i s , 5 mM MgC12, 100 mM KC1, 1mM EDTA, 1 mM DTT, and 2 uM PMSF at pH 7.0, and homogenized with a P o l y t r o n homogenizer at f u l l speed f o r 15 seconds 4 times. The homogenate was spun at 15,000 x g f o r 15 min. The supernatant was decanted and the p e l l e t was resuspended i n 10 volumes of b u f f e r and s o n i c a t e d at maximum output f o r 15 seconds 4 times. The r e s u l t i n g homogenate was spun as b e f o r e and the supernatants were pooled. A l l procedures up t o t h i s p o i n t were 31 c a r r i e d out a t 0-4OC. F u r t h e r t r e a t m e n t s i n v o l v i n g p r e c i p i t a t i o n w i t h ammonium s u l f a t e were conducted a t 10-20°C s i n c e p y r u v a t e c a r b o x y l a s e was found t o be u n s t a b l e a t low tem p e r a t u r e i n the presence o f h i g h ammonium s u l f a t e c o n c e n t r a t i o n s . The p r e p a r a t i o n was brought t o 40% s a t u r a t i o n by slow a d d i t i o n of s o l i d ammonium s u l f a t e and c o n s t a n t m i x i n g w i t h a magnetic s t i r r e r , pH was m a i n t a i n e d between 7.0 and 7.5 by a d d i t i o n o f a s m a l l amount o f s o l i d T r i s base. A f t e r 30 min, t h e p r e p a r a t i o n was spun a t 15,000 x g f o r 15 min a t 10°C, The s u p e r n a t a n t from t h i s s p i n c o n t a i n e d most o f the l a c t a t e dehydrogenase a c t i v i t y and was d i s c a r d e d . The p e l l e t , which c o n t a i n e d a l l o f the p y r u v a t e c a r b o x y l a s e a c t i v i t y , was resuspended i n 30-40 ml of b u f f e r w i t h ammonium s u l f a t e , a t 35% s a t u r a t i o n and s t i r r e d f o r 30 min. C e n t r i f u g a t i o n then r e s u l t e d i n a s u p e r n a t a n t c o n t a i n i n g a l l of the LDH a c t i v i t y l e f t i n the p r e p a r a t i o n and m i n i m a l p y r u v a t e c a r b o x y l a s e a c t i v i t y . T h i s was d i s c a r d e d , l e a v i n g a p e l l e t w i t h e s s e n t i a l l y a l l of t h e p y r u v a t e c a r b o x y l a s e a c t i v i t y . T h i s was resuspended i n 30-40 ml b u f f e r w i t h ammonium s u l f a t e a t 25% s a t u r a t i o n , s t i r r e d f o r 30 min, and spun as b e f o r e . Most of the p y r u v a t e c a r b o x y l a s e a c t i v i t y now remained i n t h e s u p e r n a t a n t , T h i s was c a r e f u l l y decanted and t h e enzyme was p r e c i p i t a t e d by r a i s i n g the ammonium s u l f a t e c o n c e n t r a t i o n t o 50% s a t u r a t i o n , s t i r r i n g f o r 30 min, and s p i n n i n g as p r e v i o u s l y d e s c r i b e d . The f i n a l p e l l e t was resuspended i n a s m a l l volume of 50 mM potassi u m phosphate, 1.5 M s u c r o s e , 1 mM EDTA, 1 mM DTT, and 2 uM PMSF a t pH 7.2 and s t o r e d a t 4<>C. 32 P r o t e i n Measurement P r o t e i n c o n c e n t r a t i o n was measured s p e c t r o p h o t o m e t r i c a l l y using the method of Layne (1957), Measurement of Pyruvate Carboxylase A c t i v i t y Assays were conducted by c o u p l i n g the pyruvate c a r b o x y l a s e r e a c t i o n to malate dehydrogenase and monitoring the decrease i n o p t i c a l d e n s i t y at 340 nm r e s u l t i n g from NADH o x i d a t i o n . Routine assays were conducted u s i n g 50 mM T r i s (pH 7.7), 5 mM pyruvate, 2 mM MgATP, 0.6 mM acetylCoA, 50 mM KHC03, 2.5 mM MgC12, 50 mM KCL, 0.15 mM NADH, and excess d i a l y z e d MDH i n 1 ml c u v e t t e s . Temperature was maintained at 25°C with a water bath, c i r c u l a t o r , and water j a c k e t e d c e l l h o l d e r s . OD was monitored with a Onicara SP 1800 spectrophotometer and c h a r t r e c o r d e r . Reactions were i n i a t e d by a d d i t i o n of 10-20 u l of d i l u t e d enzyme. Determination o f K i n e t i c Constants Apparent Km and K i v a l u e s were determined using Lineweaver-Burk p l o t s of 1 / v e l o c i t y versus 1/substrate c o n c e n t r a t i o n and Dixon p l o t s o f 1 / v e l o c i t y versus i n h i b i t o r c o n c e n t r a t i o n , r e s p e c t i v e l y . Ka and n values were determined with H i l l p l o t s . K i n e t i c c o n s t a n t s were g e n e r a l l y r e p r o d u c i b l e to w i t h i n ±10% except f o r the apparent Km f o r HC03- which 33 v a r i e d maximally by ±20%. P r e p a r a t i o n of I s o l a t e d Hepatocytes The method i s based on that d e s c r i b e d by Walton and Cowey (1979) and used i n our l a b o r a t o r y with s l i g h t , m o d i f i c a t i o n (French e t a l . , 1980). For each p r e p a r a t i o n , a f i s h was a n a e s t h e s i z e d i n water c o n t a i n i n g MS 222 (1: 2000 w/v) and i n j e c t e d with 250 u n i t s of sodium h e p a r i n ( i n 0.1 ml 0.9% NaCl) v i a the c a u d a l v e i n . The f i s h was allowed t o recover i n water to allow the heparin to c i r c u l a t e . I t was r e a n a e s t h e s i z e d and a m i d v e n t r a l i n c i s i o n was made to expose the v i s c e r a . A cannula (PE 50) was i n s e r t e d i n t o the p o s t e r i o r i n t e s t i n a l v e i n and secured with a l i g a t u r e around the i n t e s t i n e . I n i t i a l p e r f u s i o n was conducted f o r 10 min with a p e r i s t a l t i c pump using a medium c o n s i s t i n g of g l u c o s e - and C a 2 + - f r e e Hanks medium (Hanks and Wallace, 1949) with 25 mM NaHC03, 10 mM HEPES, and gassed with 95% 02:5% C02 to a f i n a l pH of 7,4. The bulbus a r t e r i o s u s was c u t upon i n i t i a t i o n of p e r f u s i o n and the l i v e r was g e n t l y massaged t o c l e a r i t of blood. I f w e l l p e r f u s e d , the l i v e r turned yellowish-brown,and showed no s i g n s of b l o o d . A f t e r 10 min, the p e r f u s i o n medium, was changed to one with the same composition plus 30 mg c o l l a g e n a s e and 20 mg h y a l u r o n i d a s e i n 50 ml. P e r f u s i o n s were c a r r i e d out with a flow r a t e of 0.5 ml per min. A f t e r 45 min of p e r f u s i o n , the l i v e r was d i s s e c t e d out and the g a l l bladder was removed. The l i v e r was washed i n 2 changes o f p e r f u s i o n medium and minced 34 with r a z o r blades. The l i v e r fragments were then t r a n s f e r r e d to a f l a s k c o n t a i n i n g 10 ml of the enzyme-containing p e r f u s i o n medium. The f l a s k was shaken at 2 c y c l e s per sec f o r 5-10 min at 15°C. The contents were, then passed through a nylon mesh (mesh s i z e of 253 um). Large p i e c e s of l i v e r caught by the mesh were g e n t l y pressed through with a f i n g e r and washed through with enzyme-free p e r f u s i o n medium. The f i l t r a t e was passed through a second mesh (mesh s i z e o f 73 um). The r e s u l t i n g 40-80 ml of c e l l suspension was spun a t 50 x g f o r 2 min at 10°C. The c e l l p e l l e t was resuspended i n 40 ml of i n c u b a t i o n medium. T h i s was the same as the p e r f u s i o n medium except f o r the absence of enzymes and contained, i n a d d i t i o n , 1 mM CaC12 and 1% f a t t y a c i d f r e e bovine serum albumen. The suspension was spun again and the p e l l e t washed a second time. A f t e r f i n a l c e n t r i f u g a t i o n , the p e l l e t was resuspended i n i n c u b a t i o n medium f o r use i n experiments. C e l l weight was determined as d e s c r i b e d p r e v i o u s l y (Walton and Cowey, 1979). Y i e l d s of 30-40% of t o t a l l i v e r weight were u s u a l l y obtained. Measurement of Rates of L a c t a t e O x i d a t i o n I n c u b a t i o n s were done i n 25 ml f l a s k s at 15°C i n a constant temperature water bath and shaker. F l a s k s c o n t a i n i n g 0.9 ml of i n c u b a t i o n medium and n o n - r a d i o a c t i v e m e t a b o l i t e s were gassed with 95% 02:5% C02 f o r 30 sec and covered with rubber serum caps with p l a s t i c c e n t e r wel l s c o n t a i n i n g 2.4 cm Whatman m i c r o f i b r e f i l t e r paper. A f t e r shaking f o r 15 min to 35 allow e q u i l i b r a t i o n , 1 ml of c e l l suspension was added to g i v e a volume of 1.9 ml per f l a s k . A f t e r g a s s i n g f o r another 30 sec and a l l o w i n g 15 min f o r e q u i l i b r a t i o n , 0.1 ml of i n c u b a t i o n medium c o n t a i n i n g a mixture of [ 1*C ] - l a b e l l e d and n o n - l a b e l l e d s u b s t r a t e was i n j e c t e d i n t o each f l a s k . Reactions were allowed to proceed with constant shaking and terminated by i n j e c t i o n of 0.2 ml of 70% p e r c h l o r i c a c i d . Zero-time c o n t r o l s were f l a s k s to which p e r c h l o r i c a c i d was added a f t e r the c e l l s , p r i o r to i n j e c t i o n of r a d i o a c t i v e s u b s t r a t e . R a d i o a c t i v e C02 was trapped with 0.2 ml of Hyamine 10-X hydroxide and counted as d e s c r i b e d p r e v i o u s l y . Measurement of Rates of Gluconeogenesis A f t e r i n j e c t i o n of p e r c h l o r i c a c i d and shaking f o r 1 hr, f l a s k contents were t r a n s f e r r e d to small, t e s t tubes and p r e c i p i t a t e d p r o t e i n was spun down with a t a b l e - t o p c e n t r i f u g e . The supernatants were c o o l e d i n i c e and n e u t r a l i z e d with 3 M K2C03 i n 0.5 M t r i e t h a n o l a m i n e base. P r e c i p i t a t e d KC104 was spun down and l a b e l l e d glucose was i s o l a t e d from the n e u t r a l i z e d supernatants u s i n g the method of Walton and Cowey (1979). T h i s was done by shaking 0.5 ml a l i q u o t s with 4.5 ml of 1 M glucose and 1.5 gm of Amberlite MB3 mixed bed r e s i n i n 25 ml f l a s k s f o r 1.5 hr. 2 ml a l i q u o t s were withdrawn and spun. 1 ml of supernatant was mixed with 10 ml Aquasol i n g l a s s counting v i a l s and counted a t more than 90% e f f i c i e n c y i n a l i q u i d s c i n t i l l a t i o n counter. The procedure d e s c r i b e d was 36 found to remove more than 99% of excess r a d i o a c t i v e s u b s t r a t e . Demonstration of Glucagon- and cAMP-induced I n a c t i v a t i o n of Pyruyate Kinase Hepatocytes were prepared and incubated as d e s c r i b e d p r e v i o u s l y . A f t e r 1 hr of i n c u b a t i o n , the suspensions were t r a n s f e r r e d to c o l d 15 ml Corex c e n t r i f u g e tubes and homogenized with a P o l y t r o n homogenizer. This was done at f u l l speed f o r 15 sec. P r e l i m i n a r y experiments i n d i c a t e d t h a t f u r t h e r homogenization does not i n c r e a s e the amount of pyruvate kinase r e l e a s e d . The homogenates were spun a t 12,000 x g f o r 5 min a t 4°C. The supernatants were used f o r assays without f u r t h e r treatment. Measurement of Pyruvate Kinase A c t i v i t y Assays were conducted by c o u p l i n g the pyruvate k i n a s e r e a c t i o n t o LDH and monitoring the decrease i n o p t i c a l d e n s i t y at 340 nm r e s u l t i n g from NADH o x i d a t i o n . T h i s was done i n 1 ml cu v e t t e s at 25°C using a Onicam SP 1800 spectrophotometer and r e c o r d e r . Temperature was maintained with a constant temperature bath, c i r c u l a t o r , and water-jacketed c u v e t t e -h o l d e r s . Routine assays were done at s a t u r a t i n g (5 mM) and no n - s a t u r a t i n g (0.1 mM) c o n c e n t r a t i o n s of phosphoenolpyruvate i n 50 mM HEPES with 5 mM MgC12, 100 mM KCl, 1.5 mM ADP, 0.15 mM NADH, and excess LDH at pH 7.0. Reactions were i n i t i a t e d by a d d i t i o n o f 50 u l o f enzyme e x t r a c t . 38 CHAPTER I I I . PROPERTIES OF RAINBOW TROUT LIVER MITOCHONDRIA 39 Ifi P r o d u c t i o n The l i v e r i s probably the most m e t a b o l i c a l l y v e r s a t i l e organ i n the v e r t e b r a t e body. In mammals,,it i s known to have the c a p a c i t y f o r both the o x i d a t i o n and s y n t h e s i s of numerous me t a b o l i t e s and i s the organ mainly r e s p o n s i b l e f o r the r e g u l a t i o n of blood glucose c o n c e n t r a t i o n (Newsholme and S t a r t , 1973). There i s c o n s i d e r a b l e evidence that the main pathways of carbohydrate and f a t metabolism are present i n the l i v e r of t e l e o s t f i s h as w e l l . The f i s h l i v e r has been shown to have the c a p a c i t y f o r glucose o x i d a t i o n (Hochachka, 1968) as w e l l as gluconeogenesis (Walton and Cowey, 1979; Hayashi and Ooshiro, 1979; Renaud and Moon, 1980). I s o l a t e d f i s h hepatocytes are capable of f a t t y a c i d s y n t h e s i s (Hazel and S e l l n e r , 1979) and amino a c i d o x i d a t i o n (French e t a l . , 1980) and mitochondria i s o l a t e d from f i s h l i v e r a c t i v e l y o x i d i z e f a t t y a c i d s (Brown and Tappel, 1959; B i l i n s k i and Jonas, 1970),. S i n c e these pathways i n c l u d e r e a c t i o n s c a t a l y z e d by i n t r a m i t o c h o n d r i a l enzymes and r e g u i r e t r a n s p o r t a c r o s s m i t o c h o n d r i a l membranes, s t u d i e s of f i s h l i v e r mitochondria are of c o n s i d e r a b l e i n t e r e s t . However, i n f o r m a t i o n on m i t o c h o n d r i a l metabolism i n f i s h i s fragmentary. Although a few s t u d i e s have been conducted on l i v e r mitochondria of a number of s p e c i e s i n c l u d i n g carp (Brown and Tappel, 1959; Gumbmann and Tappel, 1962), e e l (Wodtke, 1974; Moon and O u e l l e t , 1979), and rainbow t r o u t (Smith, 1973), the r e s u l t s p r o v i d e a very incomplete p i c t u r e and the p r o p e r i e s observed appear to vary g r e a t l y between s p e c i e s . There i s , t h e r e f o r e , a 40 need f o r more d e t a i l e d and comparative s t u d i e s . My i n t e n t i o n t o study the i n t r a m i t o c h o n d r i a l r e a c t i o n s of gluconeogenesis i n rainbow t r o u t l i v e r and p o s s i b l e i n t e r a c t i o n s between these r e a c t i o n s and other pathways made necessary a study of the p r o p e r t i e s of mitochondria from t h i s t i s s u e . These p r o p e r t i e s i n d i c a t e the presence of a complete Krebs c y c l e , i n h i b i t i o n of pyruvate o x i d a t i o n by f a t t y a c i d , and c a r r i e r - m e d i a t e d exchange between glutamate and a s p a r t a t e and between malate and a l p h a k e t o g l u t a r a t e . The r e s u l t s are compared with those obtained with l i v e r mitochondria from other s p e c i e s . R e s u l t s The i s o l a t i o n procedure and assay c o n d i t i o n s c o n s i s t e n t l y y i e l d e d w e l l coupled mitochondria as judged by r e s p i r a t o r y c o n t r o l and ADP/0 r a t i o s with glutamate, s u c c i n a t e , or pyruvate + malate as s u b s t r a t e s . F i g , 3.1 shows t y p i c a l r e s u l t s obtained with the ADP pulse method. Pulses o f ADP i n i t i a t e dramatic i n c r e a s e s i n the r a t e of oxygen consumption (State III) f o l l o w e d by a r e t u r n t o a low r a t e upon d e p l e t i o n of ADP (State IV) (Chance and W i l l i a m s , 1956). R e s p i r a t o r y c o n t r o l and ADP/O r a t i o s obtained with these s u b s t r a t e s are g i v e n i n Table 3.1. Coupled mitochondria o x i d i z e exogenous extremely low r a t e . A d d i t i o n of NADH i n the NADH absence at an of other 41 s u b s t r a t e s r e s u l t s i n no i n c r e a s e i n the r a t e of oxygen consumption, A subsequent p u l s e of ADP has no e f f e c t ( F i g . 3.1d). P a r a l l e l experiments i n v o l v i n g s p e c t r o p h o t o m e t r i c measurement of the r a t e of o x i d a t i o n o f exogenous NADH by these mitochondria y i e l d r a t e s o f about 5 nanomoles per min per mg p r o t e i n . The e f f e c t of Mg 2 + was examined due to i t s apparent uncoupling e f f e c t on rainbow t r o u t l i v e r mitochondria (Smith, 1973). F i g . 3.2 shows t h a t with pyruvate + malate as s u b s t r a t e s , MgC12 causes an i n c r e a s e i n the S t a t e IV r e s p i r a t o r y r a t e . However, a d d i t i o n of o l i g o m y c i n , an i n h i b i t o r of ATPase ( S l a t t e r and Ter Welle, 1969) , r e s u l t s i n r e v e r s a l of t h i s e f f e c t , Subseguent uncoupling of the p r e p a r a t i o n w i t h . t r i f l u o r o m e t h o x y c a r b o n y l c y a n i d e p h e n y l h y d r a z o n e (FCCP) r e s u l t s i n a maximal r a t e of r e s p i r a t i o n u n t i l the medium i s deplete d of oxygen. T h i s i n d i c a t e s t h a t Mg 2 + does not uncouple the mitochondria, but merely s t i m u l a t e s ATPase a c t i v i t y i n the p r e p a r a t i o n s . Using the ADP pulse method, the r a t e s of o x i d a t i o n o f a number of Krebs c y c l e i n t e r m e d i a t e s , glutamate, pyruvate, and p a l m i t o y l L - c a r n i t i n e s i n g l y and i n combination, were compared (Table 3.2). Pyruvate, p a l m i t o y l L - c a r n i t i n e , and malate are j o x i d i z e d at r e l a t i v e l y low r a t e s . When malate i s used i n combination with pyruvate p a l m i t o y l L - c a r n i t i n e , a " s p a r k i n g " e f f e c t i s observed. The r a t e s of o x i d a t i o n f a r exceed the sum of the r a t e s obtained with pyruvate alone and malate alone, and the sum of the r a t e s with p a l m i t o y l L - c a r n i t i n e alone and 42 malate alon e . PalmitoylCoA o x i d a t i o n r e q u i r e s the presence of c a r n i t i n e ( F i g . 3.3). T h i s i n d i c a t e s t h a t a s . i n mammals, mitochondria from rainbow t r o u t l i v e r are impermeable t o acylCoA e s t e r s and t h a t a c a r n i t i n e a c y l t r a n s f e r a s e system i s i n v o l v e d i n f a t t y a c i d o x i d a t i o n . These r e s u l t s agree with those obtained i n p r e v i o u s . s t u d i e s ( B i l i n s k i and Jonas, 1970). Glutamate, s u c c i n a t e , and i s o c i t r a t e are o x i d i z e d at high r a t e s . C i t r a t e i s o x i d i z e d at about h a l f the r a t e obtained with i s o c i t r a t e and glutamate. The a d d i t i o n of malate with c i t r a t e r e s u l t s i n only a s m a l l i n c r e a s e i n the r a t e , while a d d i t i o n of malate with i s o c i t r a t e has no e f f e c t on the r a t e of i s o c i t r a t e o x i d a t i o n . The h i g h e r r a t e of o x i d a t i o n o f pyruvate + malate than p a l m i t o y l L - c a r n i t i n e + malate at s a t u r a t i n g c o n c e n t r a t i o n s i s noteworthy s i n c e r a t l i v e r mitochondria o x i d i z e a c y l c a r n i t i n e e s t e r s , i n c l u d i n g p a l m i t o y l L - c a r n i t i n e , . i n , p r e f e r e n c e to pyruvate (Bremer, 1966). I have taken advantage of t h i s to demonstrate an apparent i n h i b i t i o n of pyruvate o x i d a t i o n by p a l m i t o y l L - c a r n i t i n e (Table 3.3). Under the c o n d i t i o n s used i n t h i s p a r t i c u l a r study, mitochondria o x i d i z e p a l m i t o y l L-c a r n i t i n e + malate at about 55% of the r a t e with pyruvate + malate. I f p a l m i t o y l L - c a r n i t i n e o x i d a t i o n r e s u l t s i n i n h i b i t i o n of pyruvate o x i d a t i o n , the r a t e obtained with pyruvate + malate + p a l m i t o y l L - c a r n i t i n e should be lower than t h a t with pyruvate + malate, and should approach, i f not e q u a l , 43 t h a t with p a l m i t o y l L - c a r n i t i n e + malate. The r e s u l t s show that t h i s i s indeed the case. S u c c i n a t e o x i d a t i o n , however, i s h a r d l y a f f e c t e d by p a l m i t o y l L - c a r n i t i n e , A l p h a k e t o g l u t a r a t e i s a l s o o x i d i z e d r e l a t i v e l y s l o w l y . A d d i t i o n of malate g r e a t l y s t i m u l a t e s a l p h a k e t o g l u t a r a t e o x i d a t i o n , suggesting t h a t a m a l a t e - a l p h a k e t o g l u t a r a t e c a r r i e r i s present as i n the mitochondria from mammalian heart and l i v e r (Williamson e t a l . , 1973; Cederbaum et a l . , 1973). Glutamate and a s p a r t a t e a r e thought to compete f o r the same s i t e on the glutamate-aspartate c a r r i e r (LaNoue et a l . , 1979). A s p a r t a t e i n h i b i t s glutamate o x i d a t i o n by mitochondria from t r o u t l i v e r (Table 3.2), s u g g e s t i n g the presence of t h i s c a r r i e r as w e l l . These o b s e r v a t i o n s i n d i c a t e t h a t the malate-a s p a r t a t e s h u t t l e may operate i n f i s h l i v e r mitochondria as i n the mitochondria of mammalian l i v e r (Cederbaum et a l . , 1973). D i s c u s s i o n The rainbow t r o u t l i v e r appears to be s i m i l a r t o other v e r t e b r a t e l i v e r s i n using f a t as one of i t s main energy sources ( P h i l l i p s and H i r d , 1977). Mitochondria from rainbow t r o u t l i v e r , however, o x i d i z e pyruvate at a higher r a t e than p a l m i t o y l L - c a r n i t i n e i n the presence of a m e t a b o l i t e sparker (Table 3,2) i n c o n t r a s t to r a t l i v e r mitochondria which p r e f e r a c y l c a r n i t i n e e s t e r s (Bremer, 1966). The mechanism by which the high r a t e of pyruvate o x i d a t i o n i s brought under c o n t r o l i s of i n t e r e s t and i s the s u b j e c t of the next c h a p t e r . However, 44 the present r e s u l t s i n d i c a t e t h a t p a l m i t o y l L - c a r n i t i n e o x i d a t i o n r e s u l t s i n i n h i b i t i o n of the o x i d a t i o n of pyruvate (Table 3.3). Rates of pyruvate and p a l m i t o y l L - c a r n i t i n e o x i d a t i o n i n the absence of malate are low probably because a low i n t r a m i t o c h o n d r i a l o x a l o a c e t a t e c o n c e n t r a t i o n i s l i m i t i n g to the c i t r a t e synthase r e a c t i o n . Malate i s o x i d i z e d at a low r a t e probably because of the unfavourable e g u i l i b r i u m constant of the malate dehydrogenase r e a c t i o n (Kun, 1963). Many of t h e . p r o p e r t i e s observed d i f f e r g r e a t l y with those of l i v e r mitochondria from other s p e c i e s of f i s h . For example, s t u d i e s on carp l i v e r mitochondria (Gumbmann and Tappel, 1962) showed t h a t s u c c i n a t e and a l p h a k e t o g l u t a r a t e are r a p i d l y o x i d i z e d at s i m i l a r r a t e s . T h i s i s i n c o n t r a s t with my r e s u l t s which show t h a t while s u c c i n a t e i s o x i d i z e d r a p i d l y , a l p h a k e t o g l u t a r a t e o x i d a t i o n i s probably l i m i t e d by malate a v a i l a b i l i t y to the m a l a t e - a l p h a k e t o g l u t a r a t e c a r r i e r (Table 3.2). I t i s p o s s i b l e t h a t our p r e p a r a t i o n s c o n t a i n e d lower l e v e l s of endogenous malate than t h e i r s . T h i s i s suggested a l s o by the high degree to which malate s t i m u l a t e s pyruvate and p a l m i t o y l L - c a r n i t i n e o x i d a t i o n i n my experiments. Carp l i v e r mitochondria a l s o o x i d i z e c i t r a t e at a high r a t e , suggesting a high degree of p e r m e a b i l i t y to t h i s metabolite and a high l e v e l of a c o n i t a s e a c t i v i t y . Rainbow t r o u t l i v e r mitochondria o x i d i z e c i t r a t e r e l a t i v e l y s l o w l y , and the r a t e of o x i d a t i o n i s only s l i g h t l y i n c r e a s e d by the a d d i t i o n of malate (Table 3.2). The t r i c a r b o x y l a t e c a r r i e r i n mammalian mitochondria t r a n s p o r t s c i t r a t e , c i s - a c o n i t a t e , and i s o c i t r a t e i n t o the m i t o c h o n d r i a l 45 matrix i n exchange f o r malate ( C h a p p e l l , 1964; 1968). The l a c k of s t i m u l a t i o n by malate of i s o c i t r a t e o x i d a t i o n i n my system may be due to r a p i d s y n t h e s i s of malate from i s o c i t r a t e . E e l l i v e r mitochondria do not o x i d i z e pyruvate and c i t r a t e at a l l and do not c o n t a i n the enzyme a c o n i t a s e (Moon and O u e l l e t , 1979). However, i s o c i t r a t e , a l p h a k e t o g l u t a r a t e , s u c c i n a t e , and malate are a l l o x i d i z e d a t high r a t e s , A scheme i n which c i t r a t e i s t r a n s p o r t e d i n t o the cytoplasm, converted to i s o c i t r a t e and a l p h a k e t o g l u t a r a t e , and then r e t r a n s p o r t e d i n t o the mitochondria as such t o complete the Krebs c y c l e has been proposed. Mitochondria from rainbow t r o u t l i v e r appear to have a complete Krebs c y c l e s i n c e pyruvate, p a l m i t o y l L-c a r n i t i n e ; c i t r a t e , and a l l the Krebs c y c l e i n t e r m e d i a t e s t e s t e d can a l l be o x i d i z e d . The malate-aspartate s h u t t l e i n r a t l i v e r i s thought to p a r t i c i p a t e i n the t r a n s p o r t of carbon out of mitochondria during gluconeogenesis and i n the t r a n s f e r of r e d u c i n g e q u i v a l e n t s between c y t o s o l and mitochondria (Cederbaum e t a l . , 1973). Hayashi and Ooshiro (1977; 1979) have proposed, on the b a s i s of s t u d i e s i n v o l v i n g the use of aminooxyacetate and D-malate, t h a t gluconeogenesis i n e e l l i v e r occurs p r i m a r i l y through the o x a l o a c e t a t e - a s p a r t a t e pathway and s e c o n d a r i l y through the oxaloacetate-phosphoenolpyruvate pathway. In rainbow t r o u t l i v e r , phosphoenolpyruvate carboxykinase i s a m i t o c h o n d r i a l enzyme ( P h i l l i p s and H i r d , 1977; Walton and Cowey, 1979b). T h i s means t h a t phosphoenolpyruvate, r a t h e r than malate or a s p a r t a t e , may be the m e t a b o l i t e t r a n s p o r t e d out 46 t o s e r v e as t h e p r e c u r s o r f o r g l u c o s e . I f a m a l a t e - a s p a r t a t e c y c l e does f u n c t i o n as such i n rainbow t r o u t l i v e r , i t s p r i m a r y f u n c t i o n may be t h e t r a n s f e r o f r e d u c i n g e q u i v a l e n t s g e n e r a t e d by a e r o b i c g l y c o l y s i s i n t o the m i t o c h o n d r i a . The ease w i t h which m i t o c h o n d r i a o f good q u a l i t y can be p r e p a r e d from rainbow t r o u t l i v e r and t h e p r o p e r t i e s r e p o r t e d here i n d i c a t e t h a t t h i s system i s i d e a l f o r t h e s t u d y o f the Krebs c y c l e and i t s i n t e g r a t i o n w i t h o t h e r m e t a b o l i c pathways i n f i s h . 47 Table 3.1. R e s p i r a t o r y c o n t r o l (RCR) and ADP/O r a t i o s of i i f i i c a l p r e p a r a t i o n s of rainbow t r o u t l i v e r m i tochondria. Values were c a l c u l a t e d a c c o r d i n g t o Estabrook (1967). n= the number of m i t o c h o n d r i a l p r e p a r a t i o n s used. Values presented are averages and ranges obtained with these p r e p a r a t i o n s . State I I I r e s p i r a t i o n was induced with a 0.4 umole pulse of ADP. Mitochondria were allowed to r e t u r n to State IV and given another p u l s e of ADP as i l l u s t r a t e d i n F i g . 3.1. S u b s t r a t e s RCR ADPJLO 5 mM Glutamate 5 mM S u c c i n a t e 5 mM Pyruvate + 3 mM Malate 3 2 5.0 (4.0-6.7) 3.7 (3.1-4.2) 4.8 (4.7-4.9) 2.6 (2.5-2.7) 1.81 (1.7-1.9) 2.5 (2.4-2.6) 48 Table 3,2. S t a t e I I I r e s p i r a t o r y r a t e s obtained with v a r i o u s s u b s t r a t e s . Two pulses of 0.4 umole ADP were i n j e c t e d as d e s c r i b e d i n Table 1. n= number.of m i t o c h o n d r i a l p r e p a r a t i o n used. Values presented are averages and ranges o b t a i n e d with these p r e p a r a t i o n s , Rates were c a l c u l a t e d according t o Estabrook (1967) and are expressed i n nanoatoms 0 per min per mg m i t o c h o n d r i a l p r o t e i n (where 1 nanoatom 0 = 0.5 nanomole 02). S u b s t r a t e s n State I I I Rate 5 mM Pyruvate 3 8.8 (8 . 3 - 9.1) 50 mM P a l m i t o y l L - c a r n i t i n e 3 4.6 (4 . 2 - 4.9) 3 mM Mai ate 3 8.7 (8 . o- 9.4) 5 mM Pyruvate + 3 mm Malate 4 50.8 ( 46. 8-55. 5) 50 + uM P a l m i t o y l L - c a r n i t i n e 3 mM Malate 4 27.1 ( 23. 2-31. 5) 5 mM S u c c i n a t e 2 64.3 ( 59. 8-68. 8) 5 mM Glutamate 3 31.9 ( 28. 8-33. 5 > 5 mM Glutamate + 5 mM A s p a r t a t e 2 20. 1 ( 17. 6-22. 5) 5 mM Glutamate + 3 mM Malate 2 35.4 ( 30. 2-40. 5) 5 mm C i t r a t e 3 13.4 ( 11. 3-15. 2) 5 mM C i t r a t e + 3 mM Malate 3 16.7 ( 13. 3-20. 2) 5 mM I s o c i t r a t e 2 31.3 ( 30. 1-32. 5) 5 mM I s o c i t r a t e + 3 mM Malate 2 31.3 ( 29. 1-33. 5) 5 mm I s o c i t r a t e + 5 mM C i t r a t e 2 33.1 ( 30. 0-36. 2) 5 mm A l p h a k e t o g l u t a r a t e 3 18.0 ( 17. 7-18. 3) 5 3 mM mM A l p h a k e t o g l u t a r a t e + Malate 3 47.2 ( 43. 8-50. 6) 49 Table 3.3. E f f e c t o f p a l m i t o y l L - c a r n i t i n e o x i d a t i o n on the o x i d a t i o n of p_y.ruvate + malate. Experiments and c a l c u l a t i o n s were a l l done as d e s c r i b e d p r e v i o u s l y . n = the number of m i t o c h o n d r i a l p r e p a r a t i o n s used. Values presented are averages and ranges. Rates are expressed i n nanoatoms 0 per min per mg m i t o c h o n d r i a l p r o t e i n . S u b s t r a t e s n State of I I I Rate % of C o n t r o l 1 mM Pyruvate + 1 mM Malate 3 38. 1 (36.4-39.3) 100 50 uM P a l m i t o y l L-c a r n i t i n e + 1 mM Malate 3 20.8 (19.0-23.6) 54.6 1 mM Pyruvate + 1 mM Malate + 50 uM P a l m i t o y l L - c a r n i t i n e 3 26. 4 (21.0-29.8) 69.3 5 mM S u c c i n a t e + 1 mM Malate 2 62.4 (59.7-65.1) 100 5 mM S u c c i n a t e + 1 mM Malate + 50 uM P a l m i t o y l L - c a r n i t i n e 2 61.0 (54.2-67.8) 97.8 50 Figure 3.1. O x i d a t i o n of v a r i o u s s u b s t r a t e s by coupled rainbow t r o u t l i v e r mitochondria. 1.9 6 mg m i t o c h o n d r i a l p r o t e i n was i n j e c t e d where i n d i c a t e d . Substrates i n j e c t e d were (a) 10 moles pyruvate and 6 umoles malate, (b) 10 umoles s u c c i n a t e , (c) 10 umoles glutamate, (d) 2 umoles NADH. State I I I r e s p i r a t i o n was i n i t i a t e d by i n j e c t i o n of 0.4 umole ADP. 51 Figu r e 3.2. Apparent uncoupling e f f e c t of MqCl2 and i t s r e v e r s a l by qligomycin. 2.11 mg m i t o c h o n d r i a l p r o t e i n , 10 umoles pyruvate, and 6 umoles malate were i n j e c t e d where shown. State I I I r e s p i r a t i o n was i n i t i a t e d with 0.4 umole ADP. Upon r e t u r n t o S t a t e IV r e s p i r a t i o n , 10 umoles MgC12 was i n j e c t e d . Oligomycin and FCCP were d i s s o l v e d i n ethanol and i n j e c t e d t o give f i n a l c o n c e n t r a t i o n s of 5 ug per ml and 0.4 uM, r e s p e c t i v e l y . 500 nanoatoms 0 • mitochondria - pyruvate J— malate /—ADP ' —MgCI2 - oligomycin ,—FCCP 52 Fi g u r e 3.3. Dependence of palrgitoy ICoA o x i d a t i o n on the presence of c a r n i t i n e . The assay medium contained palmitoylCoA at a f i n a l c o n c e n t r a t i o n of 50 uM. 2.11 mg m i t o c h o n d r i a l p r o t e i n , 6 umoles malate, 0.4 umole ADP, and 10 umoles D L - c a r n i t i n e were i n j e c t e d where shown. 300 nanoatoms 0 I — i on o Q O •< o o >l I — mitochondria — malate — ADP • DL-carni t ine — ADP 53 CHAPTER IV. THE PYRUVATE BRANCH POINT IN FISH LIVER MITOCHONDRIA: EFFECTS OF ACYLCARNITINE OXIDATION ON, PYRUVATE DEHYDROGENASE AND PYRUVATE CARBOXYLASE A C T I V I T I E S 54 I n t r o d u c t i o n A number of s t u d i e s i n r e c e n t years have shown t h a t the l i v e r i s a gluconeogenic organ i n t e l e o s t f i s h . Gluconeogenesis from pyruvate or p r e c u r s o r s . o f pyruvate, e, g,, l a c t a t e and a l a n i n e , has been demonstrated using rainbow t r o u t l i v e r s l i c e s ( P h i l l i p s and H i r d , 1977; Cowey et a l , , 1977), i s o l a t e d p e r f u s e d e e l l i v e r (Hayashi and Ooshiro, 1975; 1977), and hepatocytes from rainbow t r o u t (Walton and Cowey, 1979; French et a l , , 1980) and e e l (Hayashi and Ooshiro, 1979; Renaud and Moon, 1980). Glucagon s t i m u l a t e s gluconeogenesis from pyruvate, l a c t a t e , and a l a n i n e i n hepatocytes from rainbow t r o u t (Walton and Cowey, 1979a) while i n s u l i n i n h i b i t s the process i n i n t a c t f i s h (Cowey et a l , , 1977). T h i s i n f o r m a t i o n and the o b s e r v a t i o n t h a t mitochondria from rainbow t r o u t l i v e r o x i d i z e pyruvate (in presence of malate) at a high r a t e (Chapter I I I ) prompted me to examine the r e g u l a t i o n of pyruvate metabolism i n t h i s system. Since pyruvate c a r b o x y l a s e i s a m i t o c h o n d r i a l enzyme i n rainbow t r o u t l i v e r ( P h i l l i p s and H i r d , 1977; Walton and Cowey, 1979b), c o m p e t i t i o n between t h i s enzyme and pyruvate dehydrogenase would determine whether pyruvate i s o x i d i z e d or channeled i n t o the gluconeogenic pathway. S t u d i e s with r a t and chicken l i v e r m itochondria have shown that o x i d a t i o n of a c y l c a r n i t i n e e s t e r s r e s u l t s i n i n h i b i t i o n of pyruvate dehydrogenase (Jagow et al.,1968; Batenburg and Olson, 1976) and a c t i v a t i o n of pyruvate c a r b o x y l a s e ( B a r r i t t e t a l - , 1976; Wojtczak et a l . , 1972). Since rainbow t r o u t l i v e r mitochondria a c t i v e l y o x i d i z e f a t t y 55 a c i d s ( B i l i n s k i and Jonas, 1970), I t r i e d to determine whether s i m i l a r i n t e r a c t i o n s between f a t t y a c i d o x i d a t i o n and pyruvate metabolism occur i n t h i s system as w e l l . Here, I present evidence t h a t o x i d a t i o n of p a l m i t o y l D L - c a r n i t i n e r e s u l t s i n i n h i b i t i o n of pyruvate dehydrogenase and t h a t o x i d a t i o n of a c e t y l D L - c a r n i t i n e i n the presence of pyruvate, MgATP, and sodium a r s e n i t e r e s u l t s i n a c t i v a t i o n of pyruvate c a r b o x y l a s e i n i n t a c t mitochondria from rainbow t r o u t l i v e r . R e s u l t s F i g . 4.1 shows oxygraph t r a c e s of m i t o c h o n d r i a l r e s p i r a t i o n with a c e t y l D L - c a r n i t i n e + malate and p a l m i t o y l DL-c a r n i t i n e + malate as s u b s t r a t e s . P r e l i m i n a r y s t u d i e s showed t h a t the s u b s t r a t e c o n c e n t r a t i o n s used are s a t u r a t i n g and t h a t malate i s r e q u i r e d t o "spark" the Krebs c y c l e (Chapter I I I ) . Pulses of ADP induce r a p i d r a t e s of 02 uptake (State I I I ) f o l l o w e d by a r e t u r n to a low r a t e upon d e p l e t i o n of ADP ( S t a t e IV). The e f f e c t of p a l m i t o y l D L - c a r n i t i n e on the r a t e of X*C02 production from 1-[ X*C]-pyruvate by mitochondria i n State I I I i s shown i n F i g . 4.2- The r a t e s of **C02 production are roughly l i n e a r , and p a l m i t o y l D L - c a r n i t i n e i n h i b i t s the process by about 70%. Since p a l m i t o y l D L - c a r n i t i n e may i n h i b i t 1-[i*C ]-pyruvate o x i d a t i o n by i n h i b i t i n g the adenine n u c l e o t i d e t r a n s l o c a s e (thus slowing down the r a t e of r e s p i r a t i o n ) , or by i n h i b i t i n g pyruvate t r a n s p o r t i n t o the mitochondria. 56 experiments were conducted to r u l e out these p o s s i b i l i t i e s . That the i n h i b i t o r y e f f e c t of p a l m i t o y l D L - c a r n i t i n e cannot be accounted f o r by i n h i b i t i o n of the adenine n u c l e o t i d e t r a n s l o c a s e i s shown by the r e s u l t s i n F i g . 4.3. Although the a b s o l u t e r a t e s of **C02 pr o d u c t i o n by both c o n t r o l and t r e a t e d p r e p a r a t i o n s i n S t a t e IV are much lower than the r a t e s obtained i n S t a t e I I I , the degree of i n h i b i t i o n remains about the same. The p o s s i b i l i t y t h a t i n h i b i t i o n of pyruvate t r a n s p o r t may account f o r the r e s u l t s was i n v e s t i g a t e d by p r o v i d i n g mitochondria with 1-[i*C ]-alanine + a l p h a k e t o g l u t a r a t e , thus a l l o w i n g them to generate 1-[**C]-pyruvate with i n t r a m i t o c h o n d r i a l a l a n i n e aminotransferase. The r e s u l t s again show t h a t J*C02 production i s i n h i b i t e d by p a l m i t o y l DL-c a r n i t i n e ( F i g - 4.4). Thus, o x i d a t i o n of p a l m i t o y l DL-c a r n i t i n e r e s u l t s i n i n h i b i t i o n of pyruvate dehydrogenase i n i n t a c t mitochondria from rainbow t r o u t l i v e r . A c e t y l DL-c a r n i t i n e , however, has a s t i m u l a t o r y e f f e c t on 1*C02 prod u c t i o n from 1 - [ 1 * C ] - a l a n i n e . P r e p a r a t i o n s of rainbow t r o u t l i v e r mitochondria c o n t a i n o l i g o m y c i n - s e n s i t i v e ATPase a c t i v i t y (Chapter I I I ) , Since experiments to demonstrate pyruvate carboxylase a c t i v i t y i n i n t a c t mitochondria r e q u i r e d the use of ATP, oligomycin was used i n a l l the experiments to prevent h y d r o l y s i s t o ADP and P i and r e s u l t i n g changes i n the i n t r a m i t o c h o n d r i a l ATP/ADP r a t i o . The r e s u l t s i n F i g . 4.5 show t h a t mitochondria i n c o r p o r a t e H.i*C03- i n t o a c i d - s t a b l e products at a very low r a t e i n the absence of pyruvate. Pyruvate s t i m u l a t e s the process and 57 MgATP, a c o s u b s t r a t e of the pyruvate c a r b o x y l a s e r e a c t i o n , allows H 1*G03 - f i x a t i o n to occur a t a high, l i n e a r r a t e . Experiments in.which a c e t y l D L - c a r n i t i n e was added with MgATP showed no f u r t h e r i n c r e a s e . i n the r a t e . The reason for. t h i s i s probably t h a t pyruvate dehydrogenase, under these c o n d i t i o n s , i s s u f f i c i e n t l y a c t i v e to maintain i n t r a m i t o c h o n d r i a l acetylCoA at a c o n c e n t r a t i o n high enough to f u l l y a c t i v a t e pyruvate c a r b o x y l a s e . The evidence f o r t h i s i s t h a t i n h i b i t i o n of pyruvate dehydrogenase with sodium a r s e n i t e r e s u l t s i n i n h i b i t i o n of H 1 4C03- f i x a t i o n ( F i g . 4.6) furthermore, a d d i t i o n of a c e t y l D L - c a r n i t i n e with sodium a r s e n i t e completely o v e r r i d e s the i n h i b i t o r y e f f e c t on H 1*C03~ f i x a t i o n . T h i s probably occurs through r e s t o r a t i o n , of a high i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n and r e s u l t i n g a c t i v a t i o n of pyruvate c a r b o x y l a s e . These r e s u l t s i n d i c a t e t h a t pyruvate , carboxylase i s modulated by changes i n the c o n c e n t r a t i o n of i n t r a m i t o c h o n d r i a l acetylCoA, D i s c u s s i o n T e l e o s t f i s h are w e l l known f o r t h e i r a b i l i t y to s u r v i v e long p e r i o d s of s t a r v a t i o n . Under these c o n d i t i o n s , they m o b i l i z e t h e i r body l i p i d s t o r e s ( I d l e r and B i t n e r s , 1958; Nagai and Ikeda, 1971; Robinson and Mead, 1973), r a i s e blood f r e e f a t t y a c i d l e v e l s ( B i l i n s k i and Gardner, 1968), and probably a c t i v a t e l i v e r . g l u c o n e o g e n e s i s (Hayashi and Ooshiro, 1977; French e t a l . , 1980). S i n c e the f i s h l i v e r appears to 58 use f a t as one of i t s main energy source ( P h i l l i p s and H i r d , 1977), i t i s tempting t o s p e c u l a t e t h a t an,increased r a t e of f a t t y a c i d o x i d a t i o n may c o n t r i b u t e to i n c r e a s e the r a t e of gluconeogenesis i n f i s h as i n mammals (Williamson, 1967). I t i s c l e a r from the present s t u d i e s t h a t f a t t y a c i d o x i d a t i o n may have an important r o l e i n the c o n t r o l of gluconeogenesis i n f i s h by i t s e f f e c t on c o m p e t i t i o n at the pyruvate branch poi n t i n l i v e r mitochondria. O x i d a t i o n of p a l m i t o y l D L - c a r n i t i n e r e s u l t s i n i n h i b i t i o n of pyruvate o x i d a t i o n by a d i r e c t i n h i b i t o r y e f f e c t on pyruvate dehydrogenase ( F i g s . 4.2, 4.3, and 4.4). Studi e s of the i n h i b i t o r y e f f e c t s o f f a t t y a c i d s and t h e i r c a r n i t i n e e s t e r s on pyruvate dehydrogenase i n i n t a c t mitochondria have thus f a r been c o n f i n e d t o mammalian h e a r t (Olson e t a l . , 1978; Hansford, 1976; 1977; Hansford and Cohen, 1978) and l i v e r (Jagow e t a l . , l 1968; Portenhauser and Wieland, 1972; Walajtys-Rode, 1976; Batenburg and Olson, 1976). As f a r as I am aware, t h i s r e p r e s e n t s the f i r s t demonstration of such an e f f e c t i n l i v e r m itochondria from f i s h . H**C03- f i x a t i o n by i s o l a t e d t r o u t l i v e r mitochondria i s dependent upon the presence of both pyruvate and MgATP ( F i g . 4.5). These s u b s t r a t e requirements, the i n h i b i t i o n of H 1*C03~ f i x a t i o n by sodium a r s e n i t e , and the r e v e r s a l of t h i s i n h i b i t o r y e f f e c t by a c e t y l D L - c a r n i t i n e ( F i g . 4.6) i n d i c a t e t h a t the method i s a v a l i d measure of pyruvate c a r b o x y l a s e a c t i v i t y i n i n t a c t mitochondria and suggest t h a t the a c t i v i t y of t h i s enzyme i s modulated by changes i n the 59 i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n . S i m i l a r experiments using p a l m i t o y l D L - c a r n i t i n e were not p o s s i b l e because sodium a r s e n i t e i n h i b i t s B - o x i d a t i o n (Rein et a l . , 1979). S t u d i e s on chicken l i v e r mitochondria using methods s i m i l a r to mine have y i e l d e d e s s e n t i a l l y the same r e s u l t s ( B a r r i t t et a l . , 1976). S t u d i e s on r a b b i t heart mitochondria r e v e a l t h a t a c e t y l c a r n i t i n e i s more e f f e c t i v e than p a l m i t o y l c a r n i t i n e i n causing m i t o c h o n d r i a l NADH/NAD+ and acetylCoA/CoASH r a t i o s to r i s e (Olson et a l . , 1978). Since i n c r e a s e d NADH/NAD+, acetylCoA/CoASH, and ATP/ADP r a t i o s are a l l known to i n h i b i t pyruvate dehydrogenase a c t i v i t y by f a v o r i n g p h o s p h o r y l a t i o n o f the enzyme and by d i r e c t feedback i n h i b i t i o n ( H a l a j t y s et a l . , 1974; P e t i t et a l . , 1975; Batenburg and Olson, 1976; Hansford and Cohen, 1978), i t was q u i t e s u r p r i s i n g t h a t a c e t y l DL-j c a r n i t i n e s l i g h t l y s t i m u l a t e d 1*C02 production;from 1 - [ 1 4 C ] -a l a n i n e i n my p r e p a r a t i o n ( F i g . 4.3). Olson e t a l . (1978) found t h a t a c e t y l c a r n i t i n e i s much l e s s e f f e c t i v e than p a l m i t o y l c a r n i t i n e i n b r i n g i n g about i n h i b i t i o n of pyruvate o x i d a t i o n and i n h i b i t i o n of pyruvate dehydrogenase a c t i v i t y i n i n t a c t r a b b i t heart mitochondria. I t i s p o s s i b l e t h a t other f actors besides NADH/NAD+, acetylCoA/CoASH, and ATP/ADP r a t i o s are of importance (Olson e t a l . , 1978),. Further s t u d i e s are re q u i r e d t o determine whether such f a c t o r s can account f o r the opp o s i t e e f f e c t s of a c e t y l D L - c a r n i t i n e on pyruvate dehydrogenase a c t i v i t y i n t r o u t . l i v e r mitochondria. A more d e t a i l e d e l u c i d a t i o n of the nature of the enzyme-60 m e t a b o l i t e i n t e r a c t i o n s which r e s u l t i n m o d u l a t i o n of p y r u v a t e c a r b o x y l a s e and p y r u v a t e dehydrogenase a c t i v i t i e s i n my system w i l l r e q u i r e measurements o f i n t r a m i t o c h o n d r i a l m e t a b o l i t e c o n c e n t r a t i o n s under d i f f e r e n t c o n d i t i o n s , and k i n e t i c s t u d i e s on t h e i s o l a t e d enzymes. 61 F i g u r e 4.1. O x i d a t i o n of a c e t y l D L - c a r n i t i n e p l u s malate and p a l m i t o y l D L - c a r n i t i n e p l u s malate. . In (a) , 4 mM a c e t y l D L - c a r n i t i n e was present i n the assay b u f f e r . 3.46 mg m i t o c h o n d r i a l p r o t e i n and 6 umoles malate were i n j e c t e d where i n d i c a t e d . In (b), 2.11 mg m i t o c h o n d r i a l p r o t e i n , 0. 1 umole p a l m i t o y l D L - c a r n i t i n e , and 6 umoles malate were i n j e c t e d where i n d i c a t e d . State I I I r e s p i r a t i o n was i n i t i a t e d by i n j e c t i o n of 0.4 umole p u l s e s of ADP. 61a 62 Figure 4.2. E f f e c t of p a l m i t o y l D L - c a r n i t i n e on MC02 production from 1-:[,* *CJ-pyruvate by mitochondria i n State III.. Reactions were i n i t i a t e d by i n j e c t i o n of 3.000 mg m i t o c h o n d r i a l p r o t e i n . Substrates i n (o) were 1 uCi 1-[**C]-pyruvate, 1 mM pyruvate, and 3 mM malate. (A) con t a i n e d the same s u b s t r a t e s plus 50 uM p a l m i t o y l D L - c a r n i t i n e . P o i n t s r e p r e s e n t the averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l values d i f f e r e d from the averages by a maximum of 13%. 62a = 300 r minutes 63 F i g u r e 4.3- E f f e c t of p a l m i t o y l D L - c a r n i t i n e on 1»C02 production from 1-L 1*C ]-pyruvate by mitochondria i n State IV. Reactions were i n i t i a t e d by i n j e c t i o n of 3.25 mg m i t o c h o n d r i a l p r o t e i n . Assay c o n d i t i o n s were, the same as those d e s c r i b e d i n F i g . 2 except f o r the absence of components r e q u i r e d to maintain State I I I r e s p i r a t i o n . (o) con t a i n e d 1 uCi 1 - [ 1 *C ]-pyruvate, 1 mM pyruvate, and 3 mM malate. (A) c o n t a i n e d the same s u b s t r a t e s p l u s 50 uM p a l m i t o y l DL-c a r n i t i n e . P o i n t s represent the averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l values d i f f e r e d from the averages by a maximum of 2%. nanomoles C0 2 per mg mitochondrial protein 64 Figure 4.4. E f f e c t o f p a l m i t o y l D L - c a r n i t i n e on 1, 4C02 production from J-££*CJ-alanine by mitochondria i n State IV^ Reac t i o n s were i n i t i a t e d by i n j e c t i o n of 3.07 mg m i t o c h o n d r i a l p r o t e i n . Substrates i n (o) were 0.5 uCi 1-[**C]-a l a n i n e , 5 mM a l a n i n e , and 5 mM a l p h a k e t o g l u t a r a t e . (n) contained 4 mM a c e t y l D L - c a r n i t i n e and ( A ) contained 50 uM p a l m i t o y l D L - c a r n i t i n e i n a d d i t i o n t o these s u b s t r a t e s . P o i n t s r e p r e s e n t the averages of d u p l i c a t e determinations. I n d i v i d u a l values d i f f e r e d from the averages by a maximum of 3%. nanomoles UC0 2 per mg mitochondrial protein — ' — » N-> N J C O ( j j cn o cn o cn o cn 65 Fi g u r e 4.5. F i x a t i o n of H * 4C03- by rainbow t r o u t l i v e r m itochondria. Assays were conducted i n the presence of 5 ug oli g o m y c i n per ml. Reactions were i n i t i a t e d by i n j e c t i o n of 2.47 mg m i t o c h o n d r i a l p r o t e i n - Substrates were 1 uCi NaH**C03 and 12 mM NaHC03 i n (o) , 1 uCi NaHi*C03, 12 mM NaHC03, and 5 mM pyruvate i n (A), and 1 uCi NaH l 4C03, 5 mM pyruvate, and 5 mM MgATP i n (n). P o i n t s r e p r e s e n t the averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l values d i f f e r e d from the averages by a maximum of 3%. 65a 7.0 r minutes 66 F i g u r e 4.6. E f f e c t s of sodium a r s e n i t e and a c e t y l D L r c a r n i t i n e on Hi*C03- f i x a t i o n . Assays were conducted i n the presence o f 5 ug oli g o m y c i n per ml. Reactions were i n i t i a t e d by i n j e c t i o n of 2.74 mg m i t o c h o n d r i a l p r o t e i n . Substrates were 1 uCi NaHi*C03, 5 mM pyruvate, and 5 mM MgATP (control) i n (o) , c o n t r o l plus 0 . 5 mM sodium a r s e n i t e i n (A), and c o n t r o l p l u s 0 . 5 mM sodium a r s e n i t e and 4 mM a c e t y l D L - c a r n i t i n e i n (n). Po i n t s r e p r e s e n t the averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l v a l u e s d i f f e r e d from the averages by a maximum of 6%. 66a 11.0 r 5 10 15 20 minutes 67 C H A P T E R V . C A T A L Y T I C A N D R E G U L A T O R Y P R O P E R T I E S O F P Y R U V A T E C A R B O X Y L A S E F R O M R A I N B O W T R O U T L I V E R 68 I n t r o d u c t i o n The l i v e r of t e l e o s t f i s h has a high c a p a c i t y f o r gluconeogenesis using l a c t a t e and a l a n i n e as s u b s t r a t e s (Walton and Cowey, 1979; Hayashi and Ooshiro, 1979).. Gluconeogenesis from these p r e c u r s o r s may be an important process c o n t r i b u t i n g to the a b i l i t y of these animals to s u r v i v e a combination of prolonged s t a r v a t i o n and e x e r c i s e as, f o r example, d u r i n g spawning.migration (Mommsen e t a l . , 1980). The f i r s t enzymatic step which commits pyruvate d e r i v e d from l a c t a t e , a l a n i n e , and other p r e c u r s o r s i n t o the gluconeogenic pathway i s c a t a l y z e d by pyruvate c a r b o x y l a s e (pyruvate: C02 l i g a s e (ADP) , E. C. 6.4.1.1) i n the r e a c t i o n : Pyruvate + HC03- + ATP > o x a l o a c e t a t e + ADP + P i I have p r e v i o u s l y shown t h a t mitochondria i s o l a t e d from rainbow t r o u t l i v e r f i x H 1 4C03~ i n t o a c i d - s t a b l e product(s) by a r e a c t i o n dependent upon pyruvate and ATP (Chapter I V ) . The process i s i n h i b i t e d ' ( i n d i r e c t l y ) by sodium a r s e n i t e and the i n h i b i t i o n i s reversed by acetylcarnitine™ P r e l i m i n a r y experiments with pyruvate carboxylase i n s o n i c a t e d m i t o c h o n d r i a l e x t r a c t s i n d i c a t e d a requirement f o r a c t i v a t i o n by acetylCoA. These r e s u l t s suggested t h a t the r a t e o f pyruvate c a r b o x y l a t i o n may be c o n t r o l l e d by the i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n . I t was t h e r e f o r e of i n t e r e s t to examine, some of the k i n e t i c p r o p e r t i e s of 69 pyruvate c a r b o x y l a s e from rainbow t r o u t l i v e r . Here, k i n e t i c p r o p e r t i e s are d e s c r i b e d which i n d i c a t e t h a t acetylCoA and the adenine n u c l e o t i d e s may be i n v o l v e d i n the c o n t r o l of pyruvate c a r b o x y l a t i o n i n v i v o . R e s u l t s Enzyme P u r i t y and S t a b i l i t y The procedure d e s c r i b e d r e s u l t e d i n enzyme p u r i f i e d 6- to 9 - f o l d with y i e l d s of 40 t o 70% with r e s p e c t to the crude m i t o c h o n d r i a l homogenate. The maximum s p e c i f i c a c t i v i t y obtained was 0.49 umole m i n - 1 mg p r o t e i n - 1 . Pyruvate c a r b o x y l a s e prepared and s t o r e d as d e s c r i b e d was u n s t a b l e . About 50% of the a c t i v i t y was l o s t i n the f i r s t week of s t o r a g e and an a d d i t i o n a l 10% was l o s t during each succeeding week. However, k i n e t i c parameters remained unchanged f o r at l e a s t 3 weeks. Attempts to f u r t h e r p u r i f y the enzyme by g e l f i l t r a t i o n with Sephadex G-200 r e s u l t e d i n l a r g e l o s s e s of a c t i v i t y . The p r e p a r a t i o n s were s u b s t a n t i a l l y f r e e of enzymes which . c o u l d i n t e r f e r e with the k i n e t i c s t u d i e s r e p o r t e d here. 70 C a t a l y t i c P r o p e r t i e s and Re g u l a t i o n by AcetylCoA Trout l i v e r pyruvate c a r b o x y l a s e r e q u i r e s a d i v a l e n t c a t i o n f o r a c t i v i t y ( F i g . 5.1). The s a t u r a t i o n curve o b t a i n e d i with MgC12 with ATP c o n c e n t r a t i o n f i x e d at 2 mM shows maximum v e l o c i t y a t 2.5 mM and p r o g r e s s i v e i n h i b i t i o n as the c o n c e n t r a t i o n i s i n c r e a s e d f u r t h e r . A higher maximum v e l o c i t y i s o b t a i n e d with MgC12 than MnC12. I t appears t h a t l i t t l e , i f any, Mg 2 + i n excess of the c o n c e n t r a t i o n necessary t o complex ATP i s r e q u i r e d by the t r o u t l i v e r enzyme. T h i s property i s u n l i k e t h a t o f other pyruvate c a r b o x y l a s e s which r e q u i r e much higher c o n c e n t r a t i o n s of f r e e Mg 2 + f o r maximum a c t i v i t y (Keech and U t t e r , 1963; McClure et a l , , 1971). Mn2+ i s an e f f e c t i v e i n h i b i t o r at c o n c e n t r a t i o n s higher than 1 mM. The s p e c i f i c i t y of the t r o u t l i v e r enzyme f o r MgATP i s shown i n Tabl e 5.1. Of the other n u c l e o s i d e t r i p h o s p h a t e s t e s t e d , only MgUTP i s used to a c o n s i d e r a b l e e x t e n t . The enzyme has a pH optimum of 8.0. A c t i v i t y decreases r a p i d l y as pH i s dropped below 7.6 or r a i s e d above 8.0 ( F i g . 5.2). K+ g r e a t l y a f f e c t s the k i n e t i c p r o p e r t i e s of the enzymes from r a t (McClure et a l . , 1971) and chicken l i v e r (Barden and S c r u t t o n , 1974). When the apparent Km values of the t r o u t l i v e r enzyme f o r the d i f f e r e n t s u b s t r a t e s are measured i n the presence of 100 mM K +, a l a r g e e f f e c t i s seen on the apparent Km f o r HC03- (Table 5.2). In the presence of 100 mM K+, t h i s drops 6 - f o l d t o a value w i t h i n the range of p h y s i o l o g i c a l HC03 -71 c o n c e n t r a t i o n s i n t r o u t blood (Randall and Cameron, 1973). The apparent Km values f o r the other s u b s t r a t e s are much lower and are only s l i g h t l y a f f e c t e d by K+. Unless otherwise i n d i c a t e d , the remainder of the k i n e t i c s t u d i e s r e p o r t e d here were done under c o n d i t i o n s intended to approximate the environment of .the enzyme i n v i v o , i . e. , pH 7.7, 2-5 mM f r e e Mg+, and 100 mM K+ (Schulman e t . , 1979; T i s c h l e r et a l . , 1977; Reinhart and Lardy, 1980). Trout l i v e r pyruvate c a r b o x y l a s e shows an a b s o l u t e requirement f o r acetylCoA f o r a c t i v i t y ( F i g . 5.3). In t h i s r e s p e c t , i t i s s i m i l a r to the enzyme from c h i c k e n l i v e r ( S c r u t t o n and U t t e r , 1967). In c o n t r a s t , the rat, l i v e r enzyme r e t a i n s some a c t i v i t y i n the absence of acetylCoA (Scrutton and White, 1961) . As with other pyruvate c a r b o x y l a s e s , the s a t u r a t i o n curves obtained with acetylCoA are s i g m o i d a l . P o s i t i v e c o o p e r a t i v i t y i s i n d i c a t e d by upward concave double r e c i p r o c a l p l o t s ( F i g . 5.4) and H i l l p l o t s ( F i g , 5.5) of the data showing n. >1 i n the region o f 50% s a t u r a t i o n . Under n e a r - p h y s i o l o g i c a l c o n d i t i o n s , the enzyme from t r o u t l i v e r has a high e r Ka and a lower,nH f o r acetylCoA than pyruvate c a r b o x y l a s e s from other animals ( S c r u t t o n and U t t e r , 1967; S c r u t t o n , 1974; Rowan e t a l . , 1978), su g g e s t i n g a lower a f f i n i t y f o r acetylCoA and a lower degree of c o o p e r a t i v i t y . Pyruvate c o n c e n t r a t i o n has a smal l e f f e c t on the Ka f o r acetylCoA. The Ka i n c r e a s e s s l i g h t l y when pyruvate c o n c e n t r a t i o n i s low ( F i g . 5.5). 72 Pyruvate c a r b o x y l a s e s from mammalian l i v e r , kidney, and b r a i n (McClure e t a l . , 1971; Ashman et a l . , 1972; Mahan et a l . , 1975) and from c h i c k e n l i v e r (Scrutton e t a l . , 1965) show b i p h a s i c double r e c i p r o c a l p l o t s with pyruvate as the v a r i e d s u b s t r a t e . At s a t u r a t i n g acetylCoA c o n c e n t r a t i o n , a b i p h a s i c p l o t i s a l s o obtained with the enzyme from t r o u t l i v e r ( F i g . 5.6). However, while p l o t s of data obtained with the mammalian and a v i a n enzyme r e v e a l "breaks" at about 0.2 mM pyruvate or hi g h e r , my p l o t s show such d i s c o n t i n u i t i e s at lower c o n c e n t r a t i o n s . The n o n - l i n e a r i t y i s not seen i n experiments conducted with 0.1 mM acetylCoA. E x t r a p o l a t i o n to h o r i z o n t a l i n t e r c e p t s g i v e s a low apparent Km (low pyruvate c o n c e n t r a t i o n range) corresponding to the pyruvate I and pyruvate I I v a l u e s r e p o r t e d by other authors (McClure et a l . , 1971; Mahan et a l . , 1975).. AcetylCoA c o n c e n t r a t i o n a l s o has profound e f f e c t s on the apparent Km valu e s f o r the other s u b s t r a t e s . Lowering the acetylCoA c o n c e n t r a t i o n from 0.6 mM to 0.1 mM r e s u l t s i n a 6-f o l d i n c r e a s e i n the apparent Km f o r HCOS^- ( F i g . 5.7) and a 2 . 5 - f o l d i n c r e a s e i n the apparent Km f o r MgATP ( F i g . 5.8). Using the r a t l i v e r enzyme and, conseguently, a much lower range of acetylCoA c o n c e n t r a t i o n s , S c r u t t o n and White (1972) found t h a t the apparent Km values f o r MgATP and pyruvate are independent of acetylCoA c o n c e n t r a t i o n . However, the apparent Km f o r HC03 - was found to decrease with i n c r e a s i n g acetylCoA c o n c e n t r a t i o n . 73 C o n t r o l by Adenine N u c l e o t i d e s MgADP and AMP are co m p e t i t i v e i n h i b i t o r s with r e s p e c t to MgATP, with K i valu e s of 0.16 mM and 0.88 mM, r e s p e c t i v e l y ( F i g s . 5.9 and 5.10). The k i n e t i c constants f o r MgATP, MgADP, and AMP are much lower than the c o n c e n t r a t i o n s of these m e t a b o l i t e s i n the m i t o c h o n d r i a l matrix of r a t l i v e r ( S i e s s et a l . , 1977). Although s i m i l a r data are not a v a i l a b l e f o r t r o u t l i v e r mitochondria, i t i s probable t h a t m i t o c h o n d r i a l adenylate c o n c e n t r a t i o n s would be s i m i l a r l y h i gh. T h i s l e d me t o examine the e f f e c t of adenylate c o n c e n t r a t i o n r a t i o s , . e x p r e s s e d as the energy charge (Atkinson, 1968) on enzyme a c t i v i t y . F i g . 5.11 shows the response o f the enzyme to energy charge and i t s a c t i v i t y a t MgATP c o n c e n t r a t i o n s corresponding, to v a r i o u s energy charges i n the absence of other a d e n y l a t e s . The energy charge response i s u n l i k e that of many other enzymes with "U-type" response curves (Atkinson and Walton, 1967; Atkinson amd F a l l , 1967; Klungsoyr e t a l . , 1968). The t y p i c a l response of t h i s type shows very low a c t i v i t y i n the low energy charge range and a r a p i d i n c r e a s e as the high end of the range i s reached. Trout l i v e r pyruvate carboxylase, however, r e t a i n s s i g n i f i c a n t a c t i v i t y even i n the low range of energy charge v a l u e s , . Op t o about an energy charge of 0.5, a c t i v i t y i n c r e a s e s i n d i r e c t p r o p o r t i o n to energy charge. Above t h i s v a l u e , a gradual i n c r e a s e i n the steepness o f the response curve i s observed ( F i g . 5.11). 74 D i s c u s s i o n Pyruvate carboxylase has been i m p l i c a t e d i n a vide v a r i e t y of metabolic processes i n d i f f e r e n t t i s s u e s i n c l u d i n g s t e r o i d o g e n e s i s , l i p o g e n e s i s , glutamate and tf-amino b u t y r a t e s y n t h e s i s , augmentation of the Krebs c y c l e , and gluconeogenesis (Scruttonr 1978). While the c a t a l y t i c p r o p e r t i e s of the enzymes from numerous, sources are remarkably s i m i l a r ( S c r u t t o n and Young, 1972), the r e g u l a t o r y p r o p e r t i e s d i f f e r i n ways which are thought to r e f l e c t the p h y s i o l o g i c a l f u n c t i o n s of the enzymes (Scrutt o n , 1978). Numerous s t u d i e s i n v o l v i n g the measurement of changes i n metabolite c o n c e n t r a t i o n s i n v i v o i n response to v a r i o u s treatments, measurement of r a t e s of pyruvate c a r b o x y l a t i o n by i n t a c t mitochondria, and i n v i t r o k i n e t i c s i n d i c a t e t h a t the enzyme i s i n v o l v e d i n the c o n t r o l of gluconeogenesis i n v e r t e b r a t e l i v e r s ( S c r u t t o n and O t t e r , 1968; B a r r i t t e t a l . , 1976). Only i n r e c e n t years have s t u d i e s been conducted on the gluconeogenic pathway and i t s r e g u l a t i o n i n f i s h . Such s t u d i e s are of i n t e r e s t s i n c e t e l e o s t f i s h are w e l l adapted to s u r v i v e prolonged s t a r v a t i o n , o f t e n combined with, e x e r c i s e d u r i n g m i g r a t i o n (Mommsen et a l . , 1980). There i s evidence to suggest that the gluconeogenic pathway may be important under these c o n d i t i o n s . For example, r e c e n t s t u d i e s have shown t h a t gluconeogenesis i s a c t i v a t e d i n i s o l a t e d , perfused l i v e r s and hepatocytes from s t a r v e d e e l s (Hayashi and Ooshiro, 1977; Renaud and Moon, 1980) and i s o l a t e d hepatocytes from s t a r v e d t r o u t (French e t a l , , 1980) , and. i s probably maintained 75 throughout the spawning migrat i o n of salmon (Mommsen et a l . , 1980). The r e s u l t s presented here s u b s t a n t i a t e those of p r e v i o u s s t u d i e s i n d i c a t i n g c o n t r o l of pyruvate c a r b o x y l a s e i n t r o u t l i v e r by i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n (Chapter I V ) . Trout l i v e r pyruvate carboxylase i s completely i n a c t i v e when assayed without acetylCoA i n the presence of s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n s . The e f f e c t s of acetylCoA on the k i n e t i c p r o p e r t i e s of the enzyme with pyruvate, MgATP, and HC03 - as the v a r i e d s u b s t r a t e s (Figs- 5.6, 5.7 and 5.8), and the e f f e c t of pyruvate c o n c e n t r a t i o n on the k i n e t i c s with acetylCoA v a r i e d ( F i g . 5.5) i n d i c a t e i n t e r a c t i o n s between c a t a l y t i c and a l l o s t e r i c s i t e s . These r e s u l t s suggest a r e g u l a t o r y mechanism i n which pyruvate c o n c e n t r a t i o n a f f e c t s acetylCoA b i n d i n g and a c t i v a t i o n of the enzyme while acetylCoA c o n c e n t r a t i o n c o n t r o l s s u b s t r a t e b i n d i n g and c a t a l y t i c e f f i c i e n c y . Thus, an i n c r e a s e i n i n t r a m i t o c h o n d r i a l pyruvate c o n c e n t r a t i o n which may r e s u l t from a hormone-induced i n c r e a s e i n pyruvate t r a n s p o r t (Titheradge and Coore, 1976a and b) may lower the Ka f o r acetylCoA and f a c i l i t a t e a c t i v a t i o n . C o n v e r s e l y , an i n c r e a s e i n i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n r e s u l t i n g from hormonal e f f e c t s ( S i e s s et a l . , 1977) and/or f a t t y a c i d o x i d a t i o n (Williamson, 1967; Batenburg and Olson, 1976) would r e s u l t i n an i n c r e a s e i n the a f f i n i t y f o r s u b s t r a t e s and an i n c r e a s e i n c a t a l y t i c e f f i c i e n c y . During s t a r v a t i o n , f i s h m o b i l i z e body f a t ( I d l e r and B i t n e r s , 1958; Nagai and Ikeda, 1971; Robinson and Mead, 1973) and r a i s e blood 76 f r e e f a t t y a c i d l e v e l s ( B i l i n s k i and Gardner, 1968). I t i s p o s s i b l e t h a t an i n c r e a s e d r a t e of h e p a t i c f a t t y , a c i d o x i d a t i o n may c o n t r i b u t e to i n c r e a s e the r a t e of gluconeogenesis i n f i s h by a c t i v a t i o n of pyruvate c a r b o x y l a s e v i a the mechanisms d e s c r i b e d . Since the probable i n t r a m i t o c h o n d r i a l c o n c e n t r a t i o n of ATP i s high (Siess et a l . , 1977) and the apparent Km of the enzyme f o r MgATP i s low, the enzyme may be fa c e d with a s a t u r a t i n g c o n c e n t r a t i o n of MgATP i n v i v o under most p h y s i o l o g i c a l c o n d i t i o n s . S i m i l a r l y , the K i value s f o r MgADP and AMP are lower than measured i n t r a m i t o c h o n d r i a l c o n c e n t r a t i o n s ( S i e s s e t a l . , 1977). Atkinson has proposed t h a t i n such a s i t u a t i o n , enzymes may be re g u l a t e d not by m e t a b o l i t e c o n c e n t r a t i o n s per se but by met a b o l i t e , c o n c e n t r a t i o n r a t i o s (Atkinson, 1968; Atkinson e t a l . , 1975). In the case o f t r o u t l i v e r pyruvate c a r b o x y l a s e , both MgADP and AMP are c o m p e t i t i v e i n h i b i t o r s with r e s p e c t to MgATP ( F i g s . . 5.9 and 5.10). The ade n y l a t e energy Charge may thus be a r e g u l a t o r y parameter to, which the enzyme responds. The "O-type" response curve obtained ( F i g . 5.11) i s a t y p i c a l of other enzymes which have been found t o respond i n t h i s manner. I f the Ki f o r MgADP and the apparent Km f o r MgATP are assumed t o approximate the d i s s o c i a t i o n c o n s t a n t s f o r these m e t a b o l i t e s , Kd (ADP)/Kd (ATP) , the r a t i o between the d i s s o c i a t i o n c o n s t a n t s , i s approximately 2. I t has been p r e d i c t e d t h a t the steepness of the "U-type", response curve i n c r e a s e s as Kd (ADP)/Kd (ATP) decreases (Atkinson, 1968). S i n c e the s t e e p e r response curves g e n e r a l l y observed are obtained 77 with enzymes f o r which Kd(ADP)/Kd(ATP)<<1, the behavior of t r o u t l i v e r pyruvate, carboxylase i s not unexpected. The i n c r e a s e i n i n t r a m i t o c h o n d r i a l energy s t a t e which may occur under c o n d i t o n s of i n c r e a s e d gluconeogenesis (Siess e t a l . , 1977; T i t h e r a d g e et a l - , 1979) may thus a c t i v a t e pyruvate c a r b o x y l a s e i n s p i t e of con s t a n t s a t u r a t i o n of the adenylate i b i n d i n g s i t e . 78 Table 5.1. E f f e c t o f K+ on the k i n e t i c c o n s t a n t s of t r o u t l i v e r pyruvate c a r b o x y l a s e . Assays were conducted with s a t u r a t i n g c o n c e n t r a t i o n s of acetylCoA and c o s u b s t r a t e s as i n d i c a t e d i n M a t e r i a l s and Methods. In experiments with [ K + ] = 0 r NaHC03 was used i n s t e a d of KHC03. Apparent Km v a l u e s are given i n m i l l i m o l a r c o n c e n t r a t i o n s . K i n e t i c Constant M e t a b o l i t e Pyruvate I Pyruvate I I MgATP HC03-AcetylCoA I_K±J = 0 0.038 0.066 0.108 19,71 0.089 (nH=1.70) fK+ 1 = JOOmM 0.042 0.109 0.083 3. 20 0.072 (nH=1.78) 79 Table 5.2. N u c l e o s i d e t r i p h o s p h a t e s p e c i f i c i t y P.f t r o u t l i v e r pyruvate c a r b o x y l a s e . N u c l e o s i d e t r i p h o s p h a t e s were added as Mg2 + s a l t s a t 5mM c o n c e n t r a t i o n s . Other assay c o n d i t i o n s were as d e s c r i b e d i n M a t e r i a l s and Methods. Nucleoside Triphosphate % of C o n t r o l Rate ATP (Control) 100 GTP 2.5 DTP 23.7 ITP 2.5 CTP 2.5 80 Figure 5.1. E f f e c t o f d i v a l e n t c a t i o n s on t r o u t l i v e r pyruvate car b o x y l a s e a c t i v i t y . Assays were conducted as d e s c r i b e d i n M a t e r i a l s and Methods except t h a t MgCl2 and MnC12 c o n c e n t r a t i o n s were v a r i e d as i n d i c a t e d with ATP c o n c e n t r a t i o n f i x e d at 2 mM. v = (A OD/sec) x 10 5. 80a 0 1 2 3 4 5 6 7 8 81 F i g u r e 5.2. E f f e c t of p_H on t r o u t l i v e r pyruvate c a r b o x y l a s e a c t i v i t y . Assays were conducted under c o n d i t i o n s d e s c r i b e d i n M a t e r i a l s and Methods. 82 F i g u r e 5.3. A c e t y l C o A a c t i v a t i o n o f t r o u t l i v e r p y r u v a t e c a r b o x y l a s e . A ssays were conducted under c o n d i t i o n s d e s c r i b e d i n M a t e r i a l s and Methods. P y r u v a t e c o n c e n t r a t i o n was 5 mM i n (o) and 0.05 mM i n (A). V= (AOD/sec) x 10*. [AcetylCoA] mM 83 F i g u r e 5.4. Double r e c i p r o c a l p l o t s of data i n Fig,.. 5.3 showing p o s i t i v e c o o p e r a t i v i t y . Pyruvate c o n c e n t r a t i o n was 5 mM i n (o) and 0.05 mM i n (A). 83a 0 1 0 2 0 3 0 4 0 5 0 1 [AcetylCoA] mM 84 F i g u r e 5.5. H i l l p l o t s of data i n Fig.. 5.3 i n the r e g i o n of 50% s a t u r a t i o n . Pyruvate c o n c e n t r a t i o n was 5 mM i n (o) , g i v i n g Ka = .072 mM and nH = 1.78, and 0.05 mM i n (A), g i v i n g Ka = .084 mM and nH = 1. 50. 84a :12 HL1 -U) log [ Acetyl CoA ] 85 F i g u r e 5-6. E f f e c t of acetylCoA c o n c e n t r a t i o n on the k i n e t i c s of t r o u t l i v e r pyruvate c a r b o x y l a s e with pyruvate as the v a r i e d s u b s t r a t e . Double r e c i p r o c a l p l o t with 0.6 mM acetylCoA (o) g i v e s apparent Km v a l u e s of Pyruvate I = .042 and Pyruvate I I = .109 mM. With 0.1 mM acetylCoA (A), the apparent Km f o r pyruvate i s .096 mM. Other c o n d i t i o n s were as d e s c r i b e d i n M a t e r i a l s and Methods. 85a 40 r [Pyruvate] mM 86 F i g u r e 5.7- E f f e c t of acetylCoA c o n c e n t r a t i o n on the k i n e t i c s of t r o u t l i v e r pyruvate carboxylase with KHC03 as the v a r i e d s u b s t r a t e . Double r e c i p r o c a l p l o t s with 0.6 mM (o) and 0.1 mM ( A ) acetylCoA g i v e apparent Km values f o r HC03- of 3.20 mM and 19.32 mMr r e s p e c t i v e l y . Other c o n d i t i o n s were as d e s c r i b e d i n M a t e r i a l s and Methods. 150 r [ KHCO3] mM 87 F i g u r e 5.8- E f f e c t of acetylCoA c o n c e n t r a t i o n on the k i n e t i c s of t r o u t l i v e r pyruvate c a r b o x y l a s e with MgATP as the > ! .> : • v a r i e d s u b s t r a t e . Double r e c i p r o c a l p l o t s with 0.6 mM (o) and 0.1 mM (A) acetylCoA g i v e apparent Km values f o r MgATP of .083 mM and .210 mM, r e s p e c t i v e l y . Other c o n d i t i o n s were as. d e s c r i b e d i n M a t e r i a l s and Methods, [MgATP] mM 88 Fi g u r e 5.9- Dixon p l o t showing c o m p e t i t i v e i n h i b i t i o n by_ M3ADP with r e s p e c t t o MgATP. MgATP c o n c e n t r a t i o n s (mM) were v a r i e d as i n d i c a t e d . Other 1 c o n d i t i o n s were as d e s c r i b e d i n M a t e r i a l s and Methods. Ki (MgADP) = 0.16 mM. 88a 18r [MgADP] mM 89 Figu r e 5,10. Dixon p l o t showing, c o m p e t i t i v e i n h i b i t i o n by_ AMP with r e s p e c t t o MgATP. MgATP c o n c e n t r a t i o n s (mM) were v a r i e d as i n d i c a t e d . Other c o n d i t i o n s were as d e s c r i b e d i n M a t e r i a l s , a n d Methods. Ki (AMP) = 0.88 mM. 89a 2h -1.0 -0.5 0 0.5 1.0 1.5 2.0 2.5 [AMP]mM 90 Figu r e 5 . 1 1 - E f f e c t of the ade n y l a t e energy charge on t r o u t l i v e r pyruvate c a r b o x y l a s e a c t i v i t y . C o n c e n t r a t i o n s of ATP, ADP, and AMP at d i f f e r e n t energy charge v a l u e s were c a l c u l a t e d assuming an e q u i l i b r i u m c o n s t a n t f o r the adenylate kinase r e a c t i o n of 0.8 and a t o t a l adenine n u c l e o t i d e pool of 5 mM. The curve l a b e l l e d MgATP alone shows a c t i v i t y at MgATP c o n c e n t r a t i o n s c o r r e s p o n d i n g to d i f f e r e n t energy charges i n the absence of ADP and AMP. Mg 2 + c o n c e n t r a t i o n was maintained at 2.5 mM i n excess of [ATP] + [ADP], Other c o n d i t i o n s were , as d e s c r i b e d i n M a t e r i a l s and Methods. 90a 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 Energy Charge 91 CHAPTER V I . CONTROL OF GLUCONEOGENESIS IN ISOLATED HEPATOCYTES FEOM RAINBOW TROUT 92 I n t r o d u c t i o n The method developed by Berry and F r i e n d (1969) f o r the i s o l a t i o n of hepatocytes from r a t l i v e r has r e c e n t l y been used with s l i g h t m o d i f i c a t i o n f o r the i s o l a t i o n of l i v e r c e l l s from f i s h (Hayashi and Ooshiro, 1978; Renaud and Moon, 1980; Walton and Cowey, 1979; Hazel and P r o s s e r , 1979). T h i s has made p o s s i b l e experiments which have shown t h a t f i s h hepatocytes are capable of gluconeogenesis from p r e c u r s o r s of pyruvate, e. g., l a c t a t e and a l a n i n e , and t h a t t h i s process i s s t i m u l a t e d by glucagon (Hayashi and Ooshiro, 1979;. Renaud and Moon, 1979; Walton and Cowey, 1979). S t u d i e s using i s o l a t e d t r o u t l i v e r m itochondria (Chapter IV) i n d i c a t e t h a t entry of pyruvate i n t o the gluconeogenic pathway i s c o n t r o l l e d by the r e l a t i v e a c t i v i t i e s of pyruvate carboxylase and pyruvate dehydrogenase. I t was t h e r e f o r e proposed t h a t i n h i b i t i o n of pyruvate dehydrogenase and concomitant * a c t i v a t i o n of pyruvate c a r b o x y l a s e may be i n v o l v e d i n the a c t i v a t i o n of gluconeogenesis from p r e c u r s o r s of pyruvate. T h i s may occur, at l e a s t p a r t l y , as the r e s u l t of an i n c r e a s e i n i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n d u r i n g f a t t y a c i d o x i d a t i o n (Chapters IV and V) . T h i s was c o n s i d e r e d t o be a p l a u s i b l e mechanism of gluconeogenic a c t i v a t i o n s i n c e t r o u t l i v e r mitochondria ( B i l i n s k i and Jonas, 1970) and c e l l s (French et a l . , 1980) o x i d i z e f a t t y a c i d s and because f a t s are depleted f o r use as o x i d a t i v e s u b s t r a t e during the spawning m i g r a t i o n of salmon ( I d l e r and B i t n e r s , 1958) and as a r e s u l t of prolonged e x e r c i s e combined with s t a r v a t i o n i n t r o u t (Robinson and Mead, 93 1973) . Pyruvate carboxylase and PEPCK are both completely i n t r a m i t o c h o n d r i a l enzymes i n rainbow t r o u t l i v e r (Walton and cowey, 1979). T h i s i s t r u e of PEPCK i n both f e d and s t a r v e d s t a t e s (T. Mommsen, personal communication). Net c o n v e r s i o n of pyruvate to PEP t h e r e f o r e occurs i n the mitochondria, and PEP must be t r a n s p o r t e d out t o complete the pathway. The presence of the g l y c o l y t i c enzyme pyruvate kinase i n the cytoplasm (Somero and Hochachka, 1968) makes . p o s s i b l e the r e c o n v e r s i o n of PEP back t o . p y r u v a t e , r e s u l t i n g i n a f u t i l e c y c l e ( Scrutton and U t t e r , 1968) . I t was t h e r e f o r e d e s i r a b l e t o determine the e f f e c t of f a t t y a c i d on the r a t e of gluconeogenesis from a p r e c u r s o r of pyruvate ( l a c t a t e ) u s ing i n t a c t c e l l s from t r o u t l i v e r . Since glucagon has been found to a c t i v a t e gluconeogenesis from l a c t a t e and a l a n i n e i n t r o u t hepatocytes (Walton and Cowey, 1979), i t was a l s o of i n t e r e s t t o determine whether glucagon and i t s i n t r a c e l l u l a r messenger, cAMP ( P i l k i s e t a l . , 1978), c o u l d i n h i b i t r e c o n v e r s i o n of PEP to pyruvate. In t h i s chapter, I present r e s u l t s which show t h a t p a l m i t a t e , glucagon, and cAMP s t i m u l a t e gluconeogenesis from l a c t a t e i n hepatocytes from rainbow t r o u t . R e s u l t s are a l s o shown which suggest t h a t glucagon may induce a cAMP-raediated i n a c t i v a t i o n of pyruvate kinase i n v i v o . 94 R e s u l t s When viewed under the l i g h t microscope, the c e l l p r e p a r a t i o n s appeared homogeneous, contained few red blood c e l l s (<10%), and excluded Trypan blue s t a i n . Glucose s y n t h e s i s from U - [ 1 4 C l a c t a t e o c c u r r e d at a constant r a t e f o r up to 4 hours a f t e r an i n i t i a l l a g ( F i g , 6,1). The r a t e was d i r e c t l y p r o p o r t i o n a l to c e l l weioght up t o about 100 mg ( F i g . 6.2). The c e l l p r e p a r a t i o n s appeared to be v i a b l e on the bases of these c r i t e r i a . The r a t e s of gluconeogenesis obtained (Table 6,1) are i n the same range as those measured by French et a l . (1980) i n our l a b o r a t o r y , but are much lower than the r a t e s r e p o r t e d by Walton and Cowey (1979). The e f f e c t s o f v a r i o u s treatments on 0 - [ 1 4 C ] - l a c t a t e o x i d a t i o n and co n v e r s i o n t o glucose are presented i n Table 6.1. Glucagon and p a l m i t a t e s t i m u l a t e gluconeogenesis from l a c t a t e . T h e i r e f f e c t s , at the c o n c e n t r a t i o n s used i n these experiments, are not a d d i t i v e . cAMP s t i m u l a t e s gluconeogenesis, while glucose has no e f f e c t . 3 - m e r c a p t o p i i c o l i n i c a c i d (3MPA) , a s p e c i f i c i n h i b i t o r of PEPCK, i n h i b i t s gluconeogenesis markedly. Treatments which s t i m u l a t e gluconeogenesis from l a c t a t e , with the e x c e p t i o n of cAMP, a l s o i n c r e a s e the r a t e of l a c t a t e o x i d a t i o n . 3MPA, however, i n h i b i t s l a c t a t e o x i d a t i o n . The e f f e c t s of glucagon and cAMP on the a c t i v i t y of pyruvate kinase i n i s o l a t e d t r o u t hepatocytes a r e presented i n Table 6.2. Although the maximum enzyme a c t i v i t i e s are u n a f f e c t e d by these treatments, the r a t i o s o f a c t i v i t y at low 95 PEP c o n c e n t r a t i o n to a c t i v i t y at high PEP c o n c e n t r a t i o n are depressed. T h i s i n d i c a t e s t h a t pyruvate kinase i s a t a r g e t of glucagon a c t i o n i n the t r o u t l i v e r and that i n a c t i v a t i o n of t h i s enzyme may occur as the r e s u l t of a cAMP-mediated process. D i s c u s s i o n The p a l m i t a t e s t i m u l a t i o n of gluconeogenesis from l a c t a t e i n i s o l a t e d rainbow t r o u t hepatocytes i s c o n s i s t e n t with p r e v i o u s data i n d i c a t i n g i n h i b i t i o n of pyruvate o x i d a t i o n and s t i m u l a t i o n of pyruvate c a r b o x y l a t i o n by f a t t y a c i d o x i d a t i o n (Chapters IV and V). I t i s p a r t i c u l a r l y , i n t e r e s t i n g s i n c e f a t t y a c i d i n h i b i t s gluconeogenesis from l a c t a t e and a l a n i n e i n guinea p i g l i v e r ( S o l i n g e t a l . , 1970; A r i n z e et a l . , 1973) but s t i m u l a t e s gluconeogenesis from these s u b s t a t e s i n r a t l i v e r (Williamson, 1967; A r i n z e e t a l , , 1973). PEPCK i s mainly c y t o p l a s m i c i n r a t l i v e r ( B a l l a r d and Hanson, 1967). In t h i s t i s s u e , net f l u x from pyruvate t o PEP occurs through both the o x a l o a c e t a t e - a s p a r t a t e and oxaloacetate-malate routes (Shrago and Lardy, 1966). However, i n guinea p i g l i v e r , a c o n s i d e r a b l e amount of PEPCK i s m i t o c h o n d r i a l (Garber and Hanson, 1971) and the oxaloacetate-PEP route accounts f o r a s i g n i f i c a n t amount of the f l u x from pyruvate t o PEP (Garber and B a l l a r d , 1970; Garber and Hanson, 1971). In t h i s t i s s u e , the i n h i b i t o r y e f f e c t of f a t t y a c i d s on gluconeogenesis i s thought to be due t o an i n c r e a s e i n the m i t o c h o n d r i a l NADH/NAD+ r a t i o . The i n c r e a s e d r e d u c t i o n of the m i t o c h o n d r i a l redox s t a t e i s thought to s h i f t 96 the malate dehydrogenase r e a c t i o n f a r t h e r i n the d i r e c t i o n of malate, r e s u l t i n g i n d e p l e t i o n of i n t r a m i t o c h o n d r i a l o x a l o a c e t a t e and consequent i n h i b i t i o n of the PEPCK r e a c t i o n ( S o l i n g et a l . , 1970; A r i n z e e t a l . , 1973),. An assumption which appears i n h e r e n t i n t h i s proposed mechanism o f i n h i b i t i o n i s the n e a r - e q u i l i b r i u m nature of the m i t o c h o n d r i a l malate dehydrogenase r e a c t i o n . However, T i s c h l e r et a l . (1977) haye found t h a t t h i s r e a c t i o n i s f a r from e q u i l i b r i u m i n r a t l i v e r mitochondria. The s t i m u l a t o r y e f f e c t of p a l m i t a t e on gluconeogenesis i n t r o u t l i v e r hepatocytes i n d i c a t e s t h a t under c o n d i t i o n s of net gluconeogenic f l u x , f a t t y a c i d o x i d a t i o n does not r e s u l t i n a decrease i n i n t r a m i t o c h o n d r i a l o x a l o a c e t a t e c o n c e n t r a t i o n u n l i k e i n the guinea pig l i v e r . The o b s e r v a t i o n t h a t treatments which s t i m u l a t e gluconeogenesis from l a c t a t e a l s o s t i m u l a t e i*C02 r e l e a s e must be i n t e r p r e t e d with c a u t i o n . The p o s s i b i l i t y of entry of l a b e l i n t o the Krebs c y c l e by more than one route and randomization of l a b e l among Krebs c y c l e i n t e r m e d i a t e s make the method an i m p r e c i s e measure of the r a t e of net s u b s t r a t e o x i d a t i o n . Thus, although i t i s p o s s i b l e that the apparent i n c r e a s e i n l a c t a t e o x i d a t i o n may r e f l e c t a h i g h e r energy requirement r e s u l t i n g from i n c r e a s e d r a t e s of gluconeogenesis, other methods must be a p p l i e d to determine the r a t e of f l u x through the pyruvate dehydrogenase r e a c t i o n under, these d i f f e r e n t c o n d i t i o n s . 97 S i n c e most e a r l y s t u d i e s of the hormonal c o n t r o l of carbohydrate metabolism i n f i s h merely i n v o l v e d measurement of blood glucose c o n c e n t r a t i o n s , i t was not p o s s i b l e , on the bases of such s t u d i e s , t o d i s t i n g u i s h between hormonal e f f e c t s on g l y c o g e n o l y s i s , g l y c o l y s i s , and gluconeogenesis. Unequivocal demonstration of hormonal e f f e c t s on gluconeogenesis per se are t h e r e f o r e of i n t e r e s t . S t i m u l a t i o n by glucagon of gluconeogenesis from l a c t a t e and a l a n i n e i n i s o l a t e d hepatocytes from rainbow t r o u t was f i r s t r e p o r t e d by Walton and Cowey (1979). Both glucagon and d i b u t y r y l cAMP s t i m u l a t e gluconeogenesis from l a c t a t e i n i s o l a t e d hepatocytes from American e e l (Renaud and Moon, 1980). In the present study, the s t i m u l a t o r y e f f e c t of glucagon on gluconeogenesis from l a c t a t e i n t r o u t hepatocytes i s confirmed. cAMP a l s o s t i m u l a t e s gluconeogenesis, i n d i c a t i n g t h a t i t may f u n c t i o n as the i n t r a c e l l u l a r messenger of glucagon a c t i o n i n f i s h as i n mammals. The i n h i b i t i o n of pyruvate k i n a s e a c t i v i t y i n t r o u t hepatocytes r e s u l t i n g from treatment with glucagon and cAMP i s probably the consequence of c o v a l e n t m o d i f i c a t i o n of the enzyme. The l a c k of e f f e c t on maximum v e l o c i t y and the de p r e s s i o n of a c t i v i t y a t low PEP c o n c e n t r a t i o n suggest t h a t t h i s m o d i f i c a t i o n r e s u l t s i n a decrease i n . a f f i n i t y f o r PEP. Thus, i n . t h e i n t a c t animal, glucagon may cause an i n c r e a s e i n l i v e r cAMP l e v e l s which, i n t u r n , may s t i m u l a t e a p r o t e i n kinase c a t a l y z e d p h o s p h o r y l a t i o n of pyruvate kinase. T h i s r e s u l t s i n markedly decreased a c t i v i t y at the PEP c o n c e n t r a t i o n s found i n v i v o . Such a mechanism has been found to operate i n r a t l i v e r (Riou e t a l . , 1976; 1978) and may serve 98 to prevent f u t i l e c y c l i n g between pyruvate and PEP d u r i n g gluconeogenesis i n f i s h as w e l l as i n mammals. 99 F i g u r e 6 . 1 . Glucose s y n t h e s i s from 0 ~ L 1 * c ] ~ l a c t a t e as a f u n c t i o n of time. Values p l o t t e d are averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l v a l u e s d i f f e r e d from the averages by a maximum of 10%. . 100 F i g u r e 6.2. gate of glucose s y n t h e s i s from U-[£ 4CJ-lactate as a f u n c t i o n of c e l l weight. Values p l o t t e d are averages of d u p l i c a t e d e t e r m i n a t i o n s . I n d i v i d u a l values d i f f e r e d from the averages by a maximum of 10%. 101 Table 6.1 Gluconeogenesis from U - [ 1 ^ C l ^ l a c t a t e . I n c u b a t i o n s were done i n d u p l i c a t e . F l a s k s contained 30-50 mg of hepatocytes and were incubated f o r 3 hours. Rates are presented as umoles l a c t a t e o x i d i z e d to C02 or converted to glucose per gram of c e l l s per hour. Values are means ±S.E. n = number of hepatocyte p r e p a r a t i o n s used. Treatment n C02 Glucose c o n t r o l 6 2.97 + .10 2.40 + .23 10 uM glucagon 3 3.38 + .09 4.20 ± .03 0.25 mM p a l m i t a t e 3 3.48 ± .01 3.73 ± .12 10 uM glucagon + 0.25 mM p a l m i t a t e 3 3.86 ± .08 4.25 ± .06 0.1 mM cAMP 3 2.78 ± .17 3.24 + .06 5 mM gl u c o s e 3 2.78 + .09 2.56 ± .01 0.2 mM 3MPA 3 1. 74 + .12 0.35 + .07 102 Table 6,2- E f f e c t s of glucagon and cAMP on pyruvate k i n a s e a c t i v i t y i n i s o l a t e d hepatocytes. F l a s k s contained 50-100 mg of hepatocytes and were inc u b a t e d f o r 1 hour. A c t i v i t y r a t i o s = a c t i v i t y at 0-1 mM P E P / a c t i v i t y at 5 mM PEP. Maximum a c t i v i t i e s are expressed as umoles per gram of hepatocytes per min. Values are means ±S.E. n = number of hepatocyte p r e p a r a t i o n s used-Treatment c o n t r o l 10 uM glucagon 0.1 mM cAMP £ A c t i v i t y R a t i o 3 .347 + ,049 3 .157 + .022 3 .170 + .011 Maximum A c t i v i t y 10.87 ± 1.40 11.88 ± 2.79 10.70 + 1.74 CHAPTER V I I . GENERAL DISCUSSION 104 The a d a p t i v e s i g n i f i c a n c e of t h e observed d i f f e r e n c e s i n l i v e r PEPCK compartmentation (Table 1-2) i s u n c l e a r . The i n t r a m i t o c h o n d r i a l l o c a l i z a t i o n of both p y r u v a t e c a r b o x y l a s e and PEPCK i n t h e l i v e r of some a n i m a l s (e., g., pi g e o n and t r o u t ) a p p e a r s advantageous f o r r e g u l a t o r y r e a s o n s s i n c e t h e r e a c t i o n s t h a t make PEP from p y r u v a t e and t h a t which makes p y r u v a t e from PEP ( i . e., p y r u v a t e k i n a s e ) occur, i n d i f f e r e n t c e l l u l a r compartments. Such a m e t a b o l i c o r g a n i z a t i o n appears i d e a l l y s u i t e d f o r g l u c o n e o g e n e s i s from l a c t a t e and may be of g r e a t i m p o r t a n c e f o r C o r i c y c l e a c t i v i t y i n h i g h l y g l y c o l y t i c a n i m a l s . However, t h e l i m i t e d i n f o r m a t i o n a v a i l a b l e s u g g e s t s t h a t t h i s may l e a d t o i n a b i l i t y t o u t i l i z e p y r u v a t e p r e c u r s o r s o t h e r t h a n l a c t a t e a t a h i g h r a t e ( S o l i n g e t a l . , 1970, 1973; French e t a l . , 1980). T h i s may r e s u l t from an i n a b i l i t y t o s u p p l y c y t o p l a s m i c r e d u c i n g e g u i v a l e n t s f o r the r e v e r s a l o f g l y c o l y s i s . C l e a r l y , the p o s s i b i l i t y of i n d u c t i o n of c y t o p l a s m i c PEPCK under c o n d i t i o n s which f a v o r g l u c o n e o g e n e s i s from such s u b s t r a t e s i s worthy of f u r t h e r i n v e s t i g a t i o n . A n i m a l s which p o s s e s s c y t o p l a s m i c PEPCK (e. g., r a t s ) and PEPCK i n both c y t o p l a s m and m i t o c h o n d r i a (e. g,, gui n e a p i g s ) i n t h e l i v e r appear t o be more v e r s a t i l e i n t h e i r a b i l i t y t o use p y r u v a t e p r e c u r s o r s as g l u c o n e o g e n i c s u b s t r a t e s . However, more complex r e g u l a t o r y problems, p a r t i c u l a r l y w i t h r e s p e c t t o c o n t r o l of the pyruvate-PEP c y c l e and c o n t r o l of t h e t r a n s f e r o f r e d u c i n g e q u i v a l e n t s from m i t o c h o n d r i a t o c y t o p l a s m , must be d e a l t w i t h i n such systems. I n t e l e o s t f i s h e s , s u b s t r a t e a v a i l a b i l i t y may be i m p o r t a n t 105 i n the c o n t r o l of h e p a t i c gluconeogenesis. S a t u r a t i o n curves of gluconeogenic r a t e i n i s o l a t e d hepatocytes as a f u n c t i o n of l a c t a t e and pyruvate c o n c e n t r a t i o n are roughly h y p e r b o l i c , with maximum v e l o c i t y reached at more than 10 mM s u b s t r a t e (Walton and Cowey, 1979). White muscle l a c t a t e c o n c e n t r a t i o n i n t r o u t can i n c r e a s e t o more than 50 mM as a r e s u l t of burs t swimming (Black e t a l . , 1962) while blood l a c t a t e c o n c e n t r a t i o n can i n c r e a s e to about 10 mM during r e c o v e r y ( D r i e d z i c and Kic e n i u k , 1976). Thus, the pathway i s able t o respond t o changes i n s u b s t r a t e c o n c e n t r a t i o n under d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . Amino a c i d s are important gluconeogenic s u b s t r a t e s d u r i n g s t a r v a t i o n i n f i s h . In nature, s t a r v a t i o n i s o f t e n accompanied by e x e r c i s e as, f o r example, d u r i n g the spawning migrat i o n of salmonid f i s h . Onder these c o n d i t i o n s , . s k e l e t a l muscle.in the salmon undergoes p r o g r e s s i v e p r o t e o l y s i s , r e s u l t i n g i n r e l e a s e of amino, a c i d s i n t o the bloodstream ( I d l e r and B i t n e r s , 1958; Mommsen e t a l . , 1980). An e x t e n s i v e study o f t i s s u e enzyme p r o f i l e s and amino a c i d c o n c e n t r a t i o n s i n muscle and blood l e d Mommsen e t a l , (1980) t o propose t h a t net con v e r s i o n of d i f f e r e n t amino a c i d s t o a l a n i n e occurs i n salmon white muscle, The pathway proposed i s presented i n F i g - 7-1. The a l a n i n e r e l e a s e d i n t o the bloodstream may be used as o x i d a t i v e and gluconeogenic s u b s t r a t e by the l i v e r and other organs. Rainbow t r o u t are c a r n i v o r o u s f i s h and amino a c i d s from the d i e t c o n s t i t u t e an important source of gluconeogenic s u b s t r a t e s . , Cowey et a l . (1977) found t h a t gluconeogenic 106 enzyme a c t i v i t i e s are higher i n l i v e r s o f t r o u t fed a high p r o t e i n d i e t than i n t r o u t f e d a high carbohydrate d i e t . Furthermore, gluconeogenesis from U - [ 1 * C ] - a l a n i n e i s reduced as a r e s u l t of fe e d i n g with a high carbohydrate d i e t (Cowey e t a l . , 1977). The importance of c o n t r o l at the pyruvate branch p o i n t i s ev i d e n t from the f a c t t h a t l a c t a t e and a number o f amino a c i d s , e. g., a l a n i n e , s e r i n e , g l y c i n e , e nter both o x i d a t i v e and gluconeogenic pathways as pyruvate ( F i g . 1-7). Pyruvate c a r b o x y l a s e , the enzyme which c a t a l y z e s the r e a c t i o n i n v o l v e d i n pyruvate entry i n t o the gluconeogenic pathway, i s a c t i v a t e d by acetylCoA (McClure and Lardy, 1971). Pyruvate dehydrogenase, which c a t a l y z e s pyruvate c o n v e r s i o n t o acetylCoA, i s i n h i b i t e d by d i r e c t feedback i n h i b i t i o n by NADH and acetylCoA (Hansford and Cohen, 1978) and by p h o s p h o r y l a t i o n r e s u l t i n g from i n c r e a s e d NADH/NAD+, acetylCoA/CoA, and ATP/ADP r a t i o s ( P e t t i t et a l . , 1975; Hansford, 1976). Changes i n m i t o c h o n d r i a l metabolite c o n c e n t r a t i o n s which would be expected to a c t i v a t e pyruvate c a r b o x y l a s e and i n h i b i t pyruvate dehydrogenase are known to occur as a r e s u l t of f a t t y a c i d o x i d a t i o n (Williamson, 1967) and glucagon treatment ( S i e s s e t a l . , 1977) i n r a t l i v e r . A c cumulation-depletion p a t t e r n s of metabolic i n t e r m e d i a t e s i n the gluconeogenic pathway r e s u l t i n g from treatment with f a t t y a c i d , glucagon, and cAMP i n d i c a t e a c t i v a t i o n of pyruvate c a r b o x y l a t i o n i n v i v o (Williamson, 1967; Exton and Park, 1968). The p h y s i o l o g i c a l s i g n i f i c a n c e o f gluconeogenic 107 s t i m u l a t i o n by f a t t y a c i d s i n r a t l i v e r has been questioned mainly on the grounds t h a t glucagon-stimulated l i p o l y s i s and the conseguent i n c r e a s e i n the r a t e of f a t t y a c i d o x i d a t i o n may not completely account f o r the s t i m u l a t i o n of .gluconeogenesis by glucagon (Exton et a l . , 1970). In guinea p i g s , f a t t y a c i d i n h i b i t s gluconeogenesis from l a c t a t e and a l a n i n e (Arinze et a l . , 1973; S o l i n g et a l . , 1970), p o s s i b l y by i n c r e a s i n g m i t o c h o n d r i a l NADH/NADt r a t i o , and s h i f t i n g the malate dehydrogenase e q u i l i b r i u m i n f a v o r of malate. This, i s thought to r e s u l t i n d e p l e t i o n of i n t r a m i t o c h o n d r i a l o x a l o a c e t a t e and i n h i b i t i o n of the PEPCK r e a c t i o n (Garber and B a l l a r d , 1970; Garber and Hanson, 1971). I t appears, however, that the f a t t y a c i d o x i d a t i o n may be of p h y s i o l o g i c a l importance i n the s t i m u l a t i o n of h e p a t i c gluconeogenesis i n t r o u t . P a l m i t a t e , at a c o n c e n t r a t i o n w i t h i n the range measured i n t r o u t blood ( B i l i n s k i and Gardner, 1968) s t i m u l a t e s gluconeogenesis from l a c t a t e i n i s o l a t e d t r o u t hepatocytes (Chapter V I ) . Since the pyruvate c a r b o x y l a s e r e a c t i o n i s an o b l i g a t o r y part of the pathway, an i n c r e a s e d r a t e of f l u x through t h i s r e a c t i o n must have occurred as a r e s u l t of f a t t y a c i d o x i d a t i o n . However, i t i s not known whether the pyruvate dehydrogenase r e a c t i o n i s i n h i b i t e d under these c o n d i t i o n s . T h i s may occur i n the i n t a c t l i v e r , s i n c e o x i d a t i o n of p a l m i t o y l c a r n i t i n e r e s u l t s i n i n h i b i t i o n of pyruvate dehydrogenase a c t i v i t y i n i s o l a t e d t r o u t l i v e r m i tochondria (Chapters I I I and I V ) . On the other hand, a c t i v a t i o n of the pyruvate c a r b o x y l a s e r e a c t i o n i n i s o l a t e d 108 mitochondria by a c e t y l c a r n i t i n e (Chapter IV) and a c t i v a t i o n of the i s o l a t e d enzyme by acetylCoA (Chapter V) i n d i c a t e t h a t f a t t y a c i d o x i d a t i o n may i n c r e a s e the r a t e of pyruvate c a r b o x y l a t i o n i n v i v o by causing an i n c r e a s e i n i n t r a m i t o c h o n d r i a l acetylCoA c o n c e n t r a t i o n . That f a t t y a c i d o x i d a t i o n may be i n v o l v e d i n the r e c i p r o c a l c o n t r o l of the enzymes at the pyruvate branch p o i n t i n t r o u t l i v e r i s a l s o suggested by other l i n e s of evidence. S t a r v a t i o n r e s u l t s i n d e p l e t i o n of body l i p i d s t o r e s i n salmon ( I d l e r and B i t n e r s , 1958) and t r o u t (Robinson and Head, 1973) . In the l a t t e r , a t r a n s i e n t i n c r e a s e i n blood f r e e f a t t y a c i d c o n c e n t r a t i o n i s a l s o observed under these c o n d i t i o n s ( B i l i n s k i and Gardner, 1968). Hepatocytes i s o l a t e d from s t a r v e d , e x e r c i s e d t r o u t d i s p l a y an i n c r e a s e d c a p a c i t y f o r f r e e f a t t y a c i d o x i d a t i o n (French et a l . , 1980), Gluconeogenesis from pyruvate p r e c u r s o r s i s a c t i v a t e d as a r e s u l t of s t a r v a t i o n i n a number of s p e c i e s of f i s h . S t a r v a t i o n of American and Japanese e e l s r e s u l t s i n i n c r e a s e d r a t e s of gluconeogenesis from l a c t a t e and a l a n i n e i n i s o l a t e d hepatocytes (Renaud and Moon, 1980; Hayashi and Ooshiro, 1977). Combined e x e r c i s e and s t a r v a t i o n r e s u l t s i n s i g n i f i c a n t l y i n c r e a s e d r a t e s of gluconeogenesis from s e r i n e and decreased r a t e s of o x i d a t i o n of l a c t a t e and a l a n i n e i n rainbow t r o u t hepatocytes (French e t a l . , 1980). Metabolism occurs i n a hormonal m i l i e u which i s i n v o l v e d i n the r e g u l a t i o n of d i r e c t i o n s and r a t e s of f l u x .through pathways. In t e l e o s t f i s h , glucagon and i n s u l i n may be i n v o l v e d i n the c o n t r o l of blood glucose c o n c e n t r a t i o n by 109 r e g u l a t i n g h e p a t i c gluconeogenesis (Cowey et a l . , 1977; Walton and Cowey, 1979; Renaud and Moon, 1980). The s t i m u l a t i o n of gluconeogenesis from l a c t a t e (Chapter VI) and a l a n i n e (Walton and Cowey, 1979) i n i s o l a t e d t r o u t hepatocytes by glucagon suggests t h a t t h i s hormone may be i n v o l v e d i n gluconeogenic a c t i v a t i o n i n v i v o . T h i s s t i m u l a t o r y e f f e c t occurs with a concomitant i n c r e a s e i n the r a t e of f l u x through the pyruvate c a r b o x y l a s e r e a c t i o n s i n c e t h i s i s an e a r l y and o b l i g a t o r y step i n the pathway. The mechanisms by which hormones a c t i v a t e h e p a t i c pyruvate c a r b o x y l a t i o n i n any v e r t e b r a t e l i v e r are not known with c e r t a i n t y . However, re c e n t s t u d i e s with r a t s i n d i c a t e t h a t hormonal s t i m u l a t i o n of net f l u x i n the d i r e c t i o n of glucose s y n t h e s i s may be f a c i l i t a t e d by i n a c t i v a t i o n of r e g u l a t o r y enzymes i n the g l y c o l y t i c pathway. Evidence from r a d i o i s o t o p e experiments r e v e a l t h a t f l u x through the phosphofructokinase (Rognstad and Katz, 1976; 1980) and pyruvate k i n a s e r e a c t i o n s (Rognstad, 1976; Rognstad and Katz, 1977) are i n h i b i t e d i n v i v o by glucagon. T h i s has been s u b s t a n t i a t e d by experiments which have shown that glucagon and i t s i n t r a c e l l u l a r messenger, cAMP, induce i n h i b i t i o n of phosphofructokinase (Castano et a l . , 1979; Kagimoto. and Uyeda, 1979,1980) and pyruvate kinase (Riou e t a l . , 1976; 1978) by a p r o t e i n kinase c a t a l y z e d p h o s p h o r y l a t i o n of these enzymes. I t appears that a s i m i l a r cAMP-mediated i n a c t i v a t i o n of pyruvate kinase may be one of the mechanisms by which glucagon s t i m u l a t e s gluconeogenesis i n t r o u t (Chapter V I ) . Although no evidence of concomitant i n a c t i v a t i o n of phosphofructokinase 110 and/or a c t i v a t i o n of f r u c t o s e 1,6-diphosphatase as a r e s u l t of glucagon treatment are a v a i l a b l e , these may a l s o occur during hormonal s t i m u l a t i o n of gluconeogenesis. The t r o u t l i v e r i s l e s s of a black box now than when I s t a r t e d my s t u d i e s . Gluconeogenesis i s a major, f u n c t i o n of t h i s organ, and much evidence i n d i c a t e s t h a t a c t i v a t i o n of the process may be of great importance under c e r t a i n p h y s i o l o g i c a l c o n d i t i o n s . T h i s may occur, a t l e a s t p a r t l y , as a r e s u l t of the mechanisms I have proposed ( F i g . 7.2). V e r t e b r a t e gluconeogenesis i s c o n t r o l l e d by many processes a c t i n g i n c o n c e r t . S u b s t r a t e a v a i l a b i l i t y , i n t e r a c t i o n s with other pathways, hormonal changes r e s u l t i n g i n a l l o s t e r i c c o n t r o l or cov a l e n t m o d i f i c a t i o n o f r e g u l a t o r y enzymes, and a l t e r a t i o n s i n the r a t e of met a b o l i t e t r a n s p o r t are a l l i n v o l v e d i n the r e g u l a t i o n of h e p a t i c glucose metabolism. These may be more f r u i t f u l l y s t u d i e d and more meaningful g e n e r a l i z a t i o n s may r e s u l t from a comparative approach u s i n g animals capable of both common and uncommon metabolic f e a t s . 111 F i g u r e 7.1. Proposed pathway f o r net c o n v e r s i o n of v a r i o u s amino a c i d s to a l a n i n e i n salmon white muscle _[from Mommsen e t a l i X . 1980) . Threonine Cysteine Cystine Glycine Serine Malic Enzyme Malate Fumarate Phenylalanine \ ^ Tyrosine Succinyl - CoA / V Isoleucine Valine Methionine Pyruvate i Glutamate ' 2-0xoglutarate V > Alanir GPT Asparagine Aspartate Oxaloacetate 2-0xoglutarate i i i I Acetyl - CoA Glutamate \ Glutamine Arginine Proline Histidine 112 Figure 7.2. Proposed regulatory mechanisms for i n h i b i t i o n of pyruvate dehydrogenase and pyruvate kinase and acti v a t i o n of pyruvate carboxylase during Sliiconeogenic activation i n rainbow trout l i v e r . 112a ; Glucagon , Glucose I I Protein ^ © cAMP<-—ATP t kinase uvate P E P 4 OXA Pyruvate^_^-ATP e ) < r ^ NADH* % - W e t y l C o A Citrate .fatty acid oxidation 113 LITERATURE CITED Anderson, J . H., N i c k l a s , W. . J . , Blank, B., E e f i n o , C., and Willi a m s o n , J . R. 1971. T r a n s f e r of carbon and hydrogen a c r o s s the m i t o c h o n d r i a l membrane i n the c o n t r o l of gluconeogenesis. 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FEBS L e t t s . 129 28: 253-25 8. 130 APPENDIX LIST OF ABBREVIATIONS AcetylCoA - acetyl-S-coenzyme A AMP, ADP, ATP - adenosine 5'-mono, d i , t r i p h o s p h a t e Asp - a s p a r t a t e CAMP - c y c l i c 3* 5'-adenosine monophosphate CoA - coenzyme A DHAP - dihydroxyacetone phosphate 1,3 DPG - 1,3-diphosphoglycerate DTT - d i t h i o t h r e i t o l EDTA - ethylenediamine t e t r a a c e t i c a c i d FCCP - tri f l u o r o m e t h o x y c a r b o n y l c y a n i d e p h e n y l h y d r a z o n e FDP - f r u c t o s e diphosphate FDPase - f r u c t o s e 1,6 diphosphatase F6P - f r u c t o s e 6-phosphate Glu - glutamate G3P - g l y c e r a l d e h y d e 3-phosphate G6P - glucose 6-phosphate G6Pase - glucose 6-phosphatase GTP - guanosine t r i p h o s p h a t e Kapp - mass a c t i o n r a t i o Keg ^ e q u i l i b r i u m constant MPA - 3 - m e r a c p t o p i c o l i n i c a c i d NAD+(H) - n i c o t i n a m i d e adenine d i n u c l e o t i d e (reduced) 0. D- - o p t i c a l d e n s i t y Oxa - o x a l o a c e t a t e PDH - pyruvate dehydrogenase PEP - phosphoenolpyruvate PEPCK - phosphoenolpyruvate carboxykinase PFK - phosphofructokinase 132 2PGA - 2-phosphoglycerate 3PGA - 3-phosphoglycerate P i - i n o r g a n i c phosphate PMSF - p h e n y l m e t h y l s u l f o n y l f l u o r i d e 

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