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The role of mitochondrial (intrinsic) apoptotic pathway in hIAPP mediated β-cell death Wong, Helen Yunshan 2016

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 THE ROLE OF MITOCHONDRIAL (INTRINSIC) APOPTOTIC PATHWAY IN hIAPP-MEDIATED β-CELL DEATH  by  Helen Yunshan Wong  B.Sc., The University of British Columbia, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2016  © Helen Yunshan Wong, 2016 ii  Abstract Type 2 diabetes (T2D) is a complex and heterogeneous metabolic disorder characterized by insulin deficiency due to progressive loss of pancreatic β-cell function/mass and insulin resistance in peripheral tissues.  Islet amyloid is a common pathologic characteristic of the pancreas in patients with T2D which also forms in cultured and transplanted human islets. Intra- and extra-cellular amyloid formation due to aggregation of the β-cell peptide human islet amyloid polypeptides (hIAPP) contributes to progressive β-cell dysfunction and death in T2D and islet grafts.  Our research group previously showed that blocking the amyloid-induced Fas-mediated apoptotic pathway markedly reduces, but does not completely prevent, amyloid toxicity. Since hIAPP aggregates also form intracellularly, we proposed to investigate the potential role of mitochondrial-dependent (intrinsic) apoptotic pathway in amyloid-induced β-cell toxicity. In the present study, we examined if intracellular hIAPP aggregates can initiate the mitochondrial apoptotic pathway and tested if blocking this pathway protects β-cells from toxic hIAPP aggregates. INS-1 transformed rat β-cells and dispersed human islet cells were transduced with an adenovirus that codes for the expression of hIAPP (Ad-prohIAPP; to form intracellular hIAPP aggregates) or non-fibrillogenic rIAPP (Ad-prorIAPP; as control) and cultured with or without the inhibitor of Bax or caspase-9 (to block the intrinsic apoptotic pathway). Adenoviral mediated hIAPP expression and formation of intracellular hIAPP oligomers in both transformed INS-1 β-cells and primary human islet β-cells increased cytochrome c release leading to caspase-9 activation and β-cell apoptosis, both of which were prevented by Bax or caspase-9 inhibitor. We next used mouse islets isolated from CytcKA/KA and BaxBak DKO mice transduced with Ad-prohIAPP, as two models of ex vivo hIAPP aggregation with blocked intrinsic apoptotic pathway.  Blocking the pro-apoptotic function of cytochrome c (CytcKA/KA) or activation of Bax and Bak (BaxBak DKO) in islets, prevented activation of the mitochondrial apoptotic pathway mediated by intracellular hIAPP aggregates and enhanced β-cell survival as compared to wild-type islets despite comparable hIAPP aggregation. These data suggest that: 1) intracellular hIAPP aggregates trigger the mitochondrial apoptotic pathway in islet β-cells; 2) blocking the pro-apoptotic function of cytochrome c or deletion of Bax and Bak protects islet β-cells from hIAPP aggregates.   iii  Preface The content presented in this thesis is the original and unpublished work by the author, Helen Y. Wong. All experiments and data analysis were performed by the author under the supervision of Dr. L. Marzban, with assistance from N. Safikhan and Y.J. Park for animal breeding, PCR genotyping, islet isolation, and optimizing some parts of immunolabeling.   All animal work was conducted in accordance with the guidelines and principles of the laboratory animal care and the ethical protocols which were approved by the Canadian Council on Animal Care and the University of British Columbia’s Animal Policy and Welfare Committee (breeding protocol #A13-0042; experimental protocol #A13-0338). Human islets were provided by the Ike Barber Human Islet Transplant Laboratory (Vancouver, BC, Canada) with approved procedures and guidelines in accordance of the Clinical Research Ethics Board of the University of British Columbia (#H14-00442). The author has completed the Biological Safety Training Course conducted by Risk Management Services of the University of British Columbia (certificate # LB-2012-124863) and ethics training requirements of the Canadian Council on Animal Care (CCAC)/National Institutional Animal User Training (NIAUT) Program (certificate # 5303-12).  iv  Table of Contents  Abstract .......................................................................................................................................... ii	  Preface ........................................................................................................................................... iii	  Table of Contents ......................................................................................................................... iv	  List of Tables .............................................................................................................................. viii	  List of Figures ............................................................................................................................... ix	  List of Abbreviations ................................................................................................................... xi	  Acknowledgements .................................................................................................................... xiv	  Chapter 1: Introduction ................................................................................................................1	  1.1	   Diabetes .............................................................................................................................. 1	  1.1.1	   Diabetes mellitus and classification ............................................................................ 1	  1.1.2	   Type 1 diabetes and islet transplantation .................................................................... 2	  1.1.3	   Type 2 diabetes ........................................................................................................... 4	  1.2	   Islet Amyloid ..................................................................................................................... 7	  1.2.1	   Islet amyloid and islet amyloid polypeptide (IAPP) ................................................... 7	  1.2.2	   IAPP production and storage ...................................................................................... 8	  1.2.3	   Formation of IAPP aggregates .................................................................................... 9	  1.3	   Apoptosis ......................................................................................................................... 12	  1.3.1	   Cell apoptosis signaling pathways ............................................................................ 12	  1.3.2	   Extrinsic apoptotic pathway ...................................................................................... 13	  1.3.3	   Intrinsic apoptotic pathway ....................................................................................... 14	  1.3.4	   Role of the extrinsic apoptotic pathway in β-cell death ............................................ 15	  v  1.3.5	   Role of the intrinsic apoptotic pathway in β-cell death ............................................ 16	  1.4	   Animal Models ................................................................................................................. 18	  1.4.1	   BaxBak double knockout mouse model ................................................................... 18	  1.4.2	   Cytochrome c mutant mouse model (CytcKA/KA) ...................................................... 19	  1.5	   Investigating the role of mitochondrial apoptotic pathway in hIAPP-mediated β-cell death 20	  Chapter 2: Materials and Methods ............................................................................................26	  2.1	   Reagents and materials .................................................................................................... 26	  2.2	   Cell culture ....................................................................................................................... 26	  2.3	   Animals ............................................................................................................................ 26	  2.4	   Human islet isolation and culture .................................................................................... 27	  2.5	   Mouse islet isolation and culture ..................................................................................... 28	  2.6	   Islet dispersion ................................................................................................................. 28	  2.7	   Adenoviral transduction ................................................................................................... 29	  2.8	   Treatment with peptides and inhibitors ............................................................................ 29	  2.9	   Immunolabeling ............................................................................................................... 30	  2.10	   Statistical analysis .......................................................................................................... 31	  Chapter 3: Results ........................................................................................................................35	  3.1	   hIAPP aggregates promote activation of the mitochondrial apoptotic pathway .............. 35	  3.1.1	   Formation of intracellular hIAPP aggregates in β-cells is associated with increased mitochondrial cytochrome c release ..................................................................................... 35	  3.1.2	   Formation of hIAPP aggregates in β-cells results in caspase-9 activation ............... 36	  vi  3.1.3	   Blocking Bax or caspase-9 reduces β-cell apoptosis in Ad-prohIAPP transduced β-cells….. ................................................................................................................................. 37 3.2	   Blocking the mitochondrial apoptotic pathway protects islets from intracellular hIAPP aggregates ................................................................................................................................. 45	  3.2.1	   Blocking the pro-apoptotic function of cytochrome c markedly reduces hIAPP-mediated caspase-9 activation in CytcKA/KA mouse islets during culture ............................. 45	  3.2.2	   Blocking the pro-apoptotic function of cytochrome c markedly reduces β-cell apoptosis  in Ad-prohIAPP-transduced CytcKA/KA islets during culture ............................... 46	  3.2.3	   Reduced β-cell apoptosis in Ad-prohIAPP transduced CytcKA/KA mouse islets was associated with increased islet β- to α-cell ratio ................................................................... 46	  3.2.4	   Loss of Bax and Bak blocks hIAPP-induced caspase-9 activation in islet β-cells during culture ........................................................................................................................ 50	  3.2.5	   Loss of Bax and Bak promotes β-cell survival in Ad-prohIAPP-transduced islets during culture ........................................................................................................................ 50	  3.2.6	   β- to α-cell ratio in Ad-prohIAPP transduced and non-transduced mouse islets with or without Bax and Bak ........................................................................................................ 51	  3.3	   The intrinsic apoptotic pathway may contribute to β-cell apoptosis induced by extracellular hIAPP aggregates ................................................................................................. 54	  3.3.1	   Blocking the pro-apoptotic function of cytochrome c reduces β-cell death in primary islet cells exposed to extracellular hIAPP aggregates ........................................................... 54	  Chapter 4: Discussion ..................................................................................................................56	  4.1	   Formation of hIAPP oligomers in Ad-prohIAPP transduced β-cells .............................. 56	  4.2	   Intracellular hIAPP oligomers induced cytochrome c release ......................................... 57	  vii  4.3	   Intracellular hIAPP oligomers induced caspase-9 activation .......................................... 58	  4.4	   Bax and caspase-9 inhibition protects islet β-cells from apoptosis mediated by intracellular hIAPP aggregates ................................................................................................. 59	  4.5	   The role of pro-apoptotic function of cytochrome c in amyloid-mediated cell death ..... 60	  4.6	   Role of Bax and Bak in amyloid-mediated β-cell death .................................................. 61	  4.7	   Potential crosstalk between the extrinsic and intrinsic apoptotic pathways in amyloid-induced β-cell toxicity .............................................................................................................. 62	  Chapter 5: Conclusion .................................................................................................................64	  5.1	   Conclusions ...................................................................................................................... 64	  5.2	   Future directions .............................................................................................................. 65	  References .....................................................................................................................................67	   viii  List of Tables  Table 1. Culture media and supplements used for islet and cell culture. ...................................... 32 Table 2. List of peptides and inhibitors used for treatment studies. ............................................. 32 Table 3. List of primary antibodies used for immunolabeling studies. ........................................ 33 Table 4. List of secondary antibodies used for immunolabeling studies. ..................................... 34    ix  List of Figures  Figure 1. The main pathological factors and cellular stressors that contribute to the progression of T2D ........................................................................................................................................... 21  Figure 2. Processing of preproIAPP in β-cells ............................................................................ 22 Figure 3. Conformational states of hIAPP aggregates at different stages of amyloid formation..23 Figure 4. Proposed mechanisms of hIAPP-induced β-cell death………………………………..24 Figure 5. The intrinsic and extrinsic apoptotic pathways in mammalian cells ............................. 25 Figure 6. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced INS-1 β-cells was associated with mitochondrial cytochrome c release ............................................................. 38 Figure 7. Formation of intracellular hIAPP aggregates co-localized in INS-1 β-cells with mitochondrial cytochrome c release after transduction with Ad-prohIAPP ................................. 39 Figure 8. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced dispersed human islet β-cells was associated with mitochondrial cytochrome c release ............................. 40 Figure 9. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced INS-1 β-cells induced caspase-9 activation. ........................................................................................................ 41 Figure 10. Intracellular hIAPP aggregates formed in Ad-prohIAPP transduced dispersed human β-cells induced caspase-9 activation ............................................................................................. 42 Figure 11. Treatment with Bax or caspase-9 inhibitor reduced β-cell apoptosis in Ad-prohIAPP transduced INS-1 β-cells ............................................................................................................... 43 Figure 12. Treatment with Bax or caspase-9 inhibitors reduced β-cell apoptosis in Ad-prohIAPP transduced dispersed human islet β-cells ...................................................................................... 44 x  Figure 13. Loss of the pro-apoptotic function of cytochrome c in Ad-prohIAPP transduced mouse islet cells prevented hIAPP-mediated caspase-9 activation in β-cells. .............................. 48 Figure 14. Ad-prohIAPP transduced mouse islets lacking the pro-apoptotic function of cytochrome c had reduced level of β-cell apoptosis ..................................................................... 49 Figure 15. β-cell specific deletion of both Bax and Bak in Ad-prohIAPP transduced mouse islet cells prevented caspase-9 activation mediated by hIAPP aggregates. .......................................... 52 Figure 16. β-cell specific deletion of Bax and Bak in Ad-prohIAPP transduced mouse islet cells reduced β-cell apoptosis.. .............................................................................................................. 53 Figure 17. hIAPP-treated islet cells from CytcKA/KA  mice had reduced number of apoptotic β-cells as compared to Cytc+/+ mouse islet cells. ............................................................................. 55     xi  List of Abbreviations Ad-prohIAPP Adenovirus pro-human islet amyloid polypeptide  Ad-prorIAPP Adenovirus pro-rat islet amyloid polypeptide  AIF Apoptosis inducing factor  ANOVA Analysis of variance  Apaf-1 Apoptosis-activating factor 1  ATF3 Activating transcription factor 3  Bad Bcl-2 antagonist of cell death  Bak Bcl-2 homologous antagonist/killer Bax Bcl-2-associated X protein  BaxBak DKO BaxBak double knockout  Bcl-2 B-cell lymphoma 2  BH Bcl-2 Homology BIM Bcl-2-like protein 11  BSA Bovine serum albumin  CAD Caspase-activated DNase  c-FLIP Cellular FLICE-inhibitory protein  CHOP C/EBP homologous protein CPE Carboxypeptidase E Cre-ER Cre recombinase mutant estrogen receptor  CXCL1 Chemokine (C-X-C motif) ligand 1  DISC Death-inducing signaling complex  xii  DMSO Dimethyl sulfoxide  DP5 Death protein 5  ER Endoplasmic reticulum  FA Fas receptor antagonist FADD FAS-associated death domain  FasL Fas ligand FBS Fetal bovine serum  HFD High fat diet hIAPP Human islet amyloid polypeptide IAPP Islet amyloid polypeptide  IEQ Islet equivalent IFN Interferon  IL Interleukin IL-1Ra IL-1 receptor antagonist Mcl-1 Myeloid cell leukemia sequence 1  MOI Multiplicity of infection  MOMP Mitochondrial outer membrane permeabilization  NAC N-acetyl-L-cysteine NFκB Nuclear factor κB  PAM Peptidyl amidating monooxygenase  PBR Peripheral benzodiazepine receptor  PC Prohormone convertase  xiii  PUMA P53 upregulated modulator of apoptosis  RAMP Receptor activity modifying protein  rIAPP Rat islet amyloid polypeptide ROS Reactive oxygen species  SEM Standard error of the mean STS Staurosporine  STZ Streptozotocin sXBP1 Spliced X-box binding protein 1  T1D Type 1 diabetes T2D Type 2 diabetes TNF-α Tumor necrosis factor α  TRADD TNF receptor-associated death domain  TUDCA Taurine-conjugated ursodeoxycholic acid  TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling  UPR Unfolded protein response XIAP X-linked inhibitor of apoptosis protein    xiv  Acknowledgements Being a graduate student is probably the most challenging journey I have ever experienced. I could not have done it without my mentors, family, and friends, who have supported me in the past two years.   I want to offer my enduring gratitude to my mentor Dr. Marzban, for always encouraging me, helping me to grow as a person, and preparing me for the countless challenges that I have or will encounter in life. She was always accessible when I had questions or concerns or needed assistance and even pep talks.  I have to say that I am very privileged to have such a caring mentor. I am also extremely grateful for my supervisory committee members: Dr. Garth Warnock, Dr. Alice Mui, and Dr. Timothy Kieffer. Thank you all for being there by monitoring my progress, teaching me to think critically, and kindly providing me with support and encouragements. I would also like to extend my gratitude to Dr. Kieffer for allowing me to use his fluorescence microscope.    I want to show my sincere gratitude for all the guidance and technical support that I have received during my studies from Nooshin Safikhan, Ingrid Barta, Ali Asadi and Yoo Jin Park. Thank you for being patient with me and taking your time to assist me. I would give special thanks to Crystal Roberson for administrative support, Dr. Ziliang Ao for providing human islets from the Ike Barber Human Transplant Laboratory for our studies, and Dr. Dan Luciani for providing us BaxBak DKO mice.  I am fortunate enough to be part of the Canadian Institutes for Health Research Transplant Research Training Program, which provided me with funding support for my project and educated me on the clinical side of organ transplantation. In addition, I am thankful to the Faculty of Medicine at University of British Columbia for providing financial support through graduate awards.   Finally, I want to thank my family and friends. I would like to thank my mom for always being there and treating every single problem of mine like her own; my dad for making sure that I have food in my tummy; Willy for being a wonderful brother and supporting me when I felt lost; and Grandma and Uncle for being just one phone call away. I have not been the easiest person to put up with so thank you for spoiling me with love and strength. I want to also thank my dearest friends and SK for making me smile and providing moral support when I fall. Last but not least, I would like to express my thanks to Bailey for the non-stop puppy therapy that helps me de-stress.    1 Chapter 1: Introduction 1.1 Diabetes 1.1.1 Diabetes mellitus and classification   Normoglycemia is tightly regulated by clusters of endocrine cells (β, α, δ, F, and ε) in the pancreas known as the islets of Langerhans [1]. β-cells are the predominant islet cell type of the endocrine pancreas that produce, store, and secrete the peptide hormone insulin in response to rise of blood glucose. Insulin has anabolic effects in various target tissues such as reducing hepatic glucose production and promoting glucose uptake in skeletal muscle and adipose tissue [2, 3]. Under pathological conditions, defective insulin secretion and/or inability of peripheral tissues to utilize glucose result in disturbances of carbohydrate, fat, and protein metabolism in the body, leading to a chronic metabolic disorder characterized by hyperglycaemia known as diabetes mellitus [4]. Diabetes consequently causes a deterioration of glucose homeostasis and chronic complications such as neuropathy, blindness, renal failure, and cardiac vascular disease, affecting individuals of all age groups [5]. The growing prevalence of diabetes has become a global health crisis of the 21st century. It is estimated that worldwide there are 415 million adults currently living with diabetes and around 318 million adults at high risk of developing disease due to impaired glucose tolerance [6]. Acute and chronic complications of diabetes result in increased morbidity. More than 673 billion USD was spent in 2015 to treat and prevent diabetes-associated medical problems, which is projected to rise to 802 billion by 2040 [6]. In Canada alone, there were 2,011,347 individuals over the age of 12 living with diabetes in 2014 [7]. By 2020, the prevalence of diabetes is estimated to affect 3.7 million Canadians, costing the Canadian healthcare system $16.9 billion each year [8].   The clinical cases of diabetes are classified under four categories: type 1 diabetes (T1D),  2 type 2 diabetes (T2D), gestational diabetes, and other specific types [9]. T1D accounts for roughly 5-10% of all diagnosed diabetes cases, whereas T2D comprises the majority of the remaining 90% and is rising in prevalence around the world in parallel with increase in obesity [10]. Interestingly, the clinical phenotype and progression of T1D and T2D based on characteristics (age, insulin dependence, onset, ketosis, etc.) can be far less distinct in some patients due to the heterogeneous nature of the disease [11, 12]. For instance, diabetic ketoacidosis, a common manifestation found in untreated T1D, can also be present in some individuals with T2D [12].   1.1.2 Type 1 diabetes and islet transplantation  T1D is an autoimmune disease most commonly found in children and young adolescents where the patients’ antigen-specific T cells selectively destroy β-cells, leading to progressive loss of β-cell mass and hyperglycemia [13]. The pathogenesis of type 1 diabetes involves islet inflammation, infiltration of inflammatory cells, and the creation of autoantibodies specific to islet antigens (e.g. glutamic acid decarboxylase, insulin, and zinc transporter 8), which can be detected in most newly diagnosed patients with T1D (85–90%) [14]. The precise signaling pathway and mechanism of β-cell destruction in T1D are not fully elucidated; however, both genetic and environmental factors are associated with the pathogenesis of T1D. Comparative studies performed on monozygotic twins have reported that defects in T1D risk alleles (e.g. human leukocyte antigen, insulin gene, protein tyrosine phosphatase non-receptor type 22, interleukin (IL)-2 receptor-α, and cytotoxic T lymphocyte-associated protein 4) can genetically predispose individuals and put them at risk of developing the disease [14]. Co-existing environmental factors such as viral [15] or bacterial infection [16], and nutritional factors [17]  3 can trigger T1D onset in these vulnerable individuals. In order to prevent and treat T1D, it is necessary to develop strategies that reverse β-cell destruction, restore glycometabolic control and avoid the risk of microvascular and macrovascular complications [18]. There is no cure for T1D and the current treatment consists of strict diet management and life-long insulin therapy, which consists of multiple daily injections or pump therapy to deliver exogenous insulin [19]. Human insulin and insulin analogues are categorized based on their duration of action, time of onset and peak effects [20]. A common approach in intensive diabetes therapy is to use a basal-bolus insulin regimen with patient-specific adaptations, in which basal insulin is provided by a long/intermediate-acting insulin and bolus insulin is supplied by a short/rapid acting insulin analogue [21]. Instead of subcutaneous injections, insulin also can be administered via an insulin pump (continuous subcutaneous insulin infusion) [22]. Despite the success of hormone replacement therapy, it is still challenging to monitor fluctuations in blood glucose level and to overcome the risk of hypoglycemia associated with intense insulin treatment [13].  Over the past decade, β-cell replacement therapy through islet transplantation has become a feasible clinical approach following success of the Edmonton Protocol [18]. In brief, pancreas is first procured from a heart-beating, brain-dead organ donor and undergoes mechanically enhanced enzymatic digestion in the Ricordi chamber. Next, purified islets are separated from the exocrine and other non-islet tissues using a density gradient and islet yield and quality of the purified islets are assessed [18, 23–25]. Lastly, the resuspended islets are loaded into a gravity-fed transfer bag for intra-portal vein infusion into the liver of selected transplant recipients within 24 hours after isolation. Usually 2 or 3 islet infusions from multiple donors are required for a target islet mass of about 11,000 islet equivalents (IEQ) per  4 kilogram of body weight [18, 23–25]. One IEQ is estimated as an islet with a diameter of 150 µm [26]. The Edmonton Protocol consists of several modifications such as using isolated human islets from multiple donors for intra-portal vein infusion, steroid-free immunosuppression, and improved islet isolation conditions such as omitting the use of xenoproteins [18].  Despite improvements in the procedures, the outcome of islet transplantation is not optimal and currently is limited by the availability of donor pancreatic islets, efficiency of islet isolation, and survival of the grafts after transplantation. Graft survival can be hindered by immune-mediated destruction such as recurrence of autoimmunity [27], graft rejection, as well as side effects associated with long-term immunosuppression [28]. Likewise, a multitude of non-immune factors such as ischemia [29], glucose toxicity [24, 25], islet amyloid [30], stress associated with islet isolation and culture [31], ER and oxidative stress [32], and changes in islet  microenvironment in the engraftment site can also lead to islet injury and loss of β-cells in islet grafts. Ideally, close to 60% of the patients can achieve insulin independence over four years while the rest resume exogenous insulin therapy to restore normoglycemia due to graft loss and injury [33]. Indeed, several studies have suggested that treating freshly isolated islets with assorted therapeutic manipulations (e.g. glucagon-like peptide-1 agonists, anti-inflammatory agents, and pan caspase inhibitors) [18, 28, 34] during isolation or pre-transplant culture can reduce inflammation or prevent loss of β-cell mass and thereby improve clinical outcome [35, 36]. Thus, it is important to understand the mechanisms contributing to early islet graft failure and develop new strategies to maintain long-term function of human islet grafts.  1.1.3 Type 2 diabetes  The pathogenesis of T2D is complex and not completely understood. This pathologic  5 condition is associated with both genetic predisposition and acquired risk factors such as aging and obesity. Both insulin resistance in the target peripheral tissues and relative or absolute insulin deficiency due to reduced functional β-cells are characteristic features of T2D [37]. As the condition progresses, β-cell mass and function are further impaired and eventually insulin secretion in response to glucose becomes absent [38].   Insulin resistance occurs when insulin target tissues (muscle, fat, and liver) cannot properly respond to insulin, causing reduction of glucose uptake from the bloodstream and increased glucose output in liver [39]. Obesity is a major risk factor that is closely associated with development insulin resistance in T2D via several proposed mechanisms including alteration in lipid metabolism [40], tissue inflammation, production of abnormal adipokines [41], endoplasmic reticulum (ER) stress [42] and disturbances of gut microbiota [43].     Healthy β-cells can normally adapt and compensate for insulin resistance by increasing insulin secretion and decreasing hepatic insulin clearance to maintain normal glucose tolerance. It has been suggested that β-cell volume is increased by about 50% in healthy people to cope with peripheral insulin resistance [44, 45]. However, when the compensatory pathways in β-cells fail to meet increased insulin demand and lead to β-cell exhaustion, distinct pathologic programs are then initiated to induce β-cell dysfunction and death [46]. Therefore, it appears that progressive decline in functional β-cell mass due to increased β-cell death, rather than reduction in β-cell replication, has a critical role in the pathogenesis in T2D [45].   Often, T2D-associated metabolic overload and insulin resistance can trigger multiple physiologic stressors that can affect β-cell function (Figure 1). For instance, chronic exposure to high levels of glucose can cause β-cell dysfunction, impairment of the ubiquitin/proteasome system, and deterioration of glucose utilization and insulin secretion [47, 48]. β-cells contain low  6 amounts of catalase and superoxide dismutase, which make them more vulnerable to oxidative stress and accumulation of reactive oxygen species (ROS) generated from oxidative glucose metabolism, resulting in subsequent nuclear factor κB (NFκB)-induced β-cell apoptosis [47, 49]. Additionally, growing evidence in literature has shown that elevated glucose induces IL-1β secretion and islet inflammation, which can promote the extrinsic apoptotic pathway in human islet β-cells [50, 51]. Likewise, dyslipidemia and long-term exposure to saturated fatty acids has detrimental effects on islet β-cells such as suppression of insulin biosynthesis [52–54]. In vivo studies have revealed that accumulation of saturated fatty acids such as palmitate can induce inflammation and production of cytokines including IL-1β, IL-6, IL-8, and chemokine (C-X-C motif) ligand 1 (CXCL1), which can further impair β-cell function [55, 56]. Moreover, β-cells become particularly vulnerable to ER stress when insulin biosynthesis is markedly elevated to meet metabolic demand, inducing flux through the rough ER [57]. Other pathologic stimuli such as insulin resistance and accumulation of lipids can further aggravate ER stress, leading to β-cell dysfunction and death [46]. Lastly, islet amyloid is a common pathologic characteristic of the pancreas in T2D that can cause β-cell demise [58]. Similar to high levels of glucose and saturated fatty acids, formation of islet amyloid can promote IL-1β production and cause islet inflammation in T2D [59], the details of which will be discussed in section 1.2.   The majority of patients with T2D can exert glycemic control with lifestyle modifications such as incorporating a balanced diet with high fiber content and low calories, and daily physical activity. In some cases, patients may require pharmacological treatment including but not limited to insulin, metformin, thiazolidinediones, sulfonylureas, and incretin-based therapies [60]. Furthermore, weight-loss strategies such as bariatric surgery also have been reported to have clinical significance in the management of T2D [61–63].  7 1.2 Islet Amyloid  1.2.1 Islet amyloid and islet amyloid polypeptide (IAPP)  Islet amyloid, initially described as “islet hyalinization,” was first discovered in 1901 by two researchers independently in the pancreases from patients with T2D [64, 65]. Close to 90% of patients with T2D have visible amyloid formation in pancreatic tissue post-mortem, a finding that is only reported in a small number of non-diabetic, generally older subjects [66, 67]. Other investigators have shown that islet amyloid in the pancreas of other species vulnerable to T2D such as non-human primates and cats [58, 68–70]. These aggregates are also found in islet grafts during pre-transplant culture or engraftment, which may lead to islet graft failure following islet transplantation [71–75].  In 1987, a polypeptide hormone was isolated and identified by the Westermark and Cooper groups as the principal component of islet amyloid deposits and it was named islet amyloid polypeptide (IAPP) or amylin [68, 76]. IAPP is a neuropancreatic hormone belonging to the calcitonin family of peptides that functions via a complex of the calcitonin receptor and a receptor activity modifying protein (RAMP) [77, 78]. The physiological role of IAPP in humans is not fully understood but animal studies have shown that IAPP has important functions in controlling gastric emptying and satiety, glucose homeostasis, and suppression of glucagon release [79, 80]. Pramlintide, an FDA-approved bioactive analog of endogenous IAPP, has been approved to be administered as an adjunct for insulin treatment in patients with T1D and late stage T2D when IAPP production is minimal to improve glycemic control [81].    8 1.2.2 IAPP production and storage   IAPP is produced in pancreatic β-cells which is co-stored and co-secreted with insulin in response to β-cell secretagogues such as glucose [68, 76, 82]. As shown in Figure 2, IAPP is composed of 37-aa residues, and is first synthesized as an 89-residue preproIAPP molecule. It has a 22-residue signal peptide that leads its transport along the ER to the trans-Golgi network [83, 84]. From there, the signal peptide is cleaved off to form a 67-residue proIAPP. The prohormone then undergoes further cleavage at the dibasic sites near the C-terminus in the Golgi by prohormone convertase (PC) 1/3 and the N-terminus mainly in the secretory granules by PC2 [84]. The remaining C-terminal basic residues are removed by carboxypeptidase E (CPE) and activated peptidyl amidating monooxygenase (PAM) complex assists in the removal of Gly38 and amidation at Tyr37 [79]. Indeed, to obtain complete biological activity, the resultant 37-residue IAPP molecule must have both an intramolecular disulfide bridge between the cysteine residues at positions 2 and 7 and an amidated C-terminus [79]. The biologically active IAPP is co-secreted with insulin in a molar ratio of approximately 1:100 [84].   Mature IAPP molecules are stored with C-peptide in the halo region of the secretory granules in β-cells while insulin hexamers bound to zinc ions occupy the dense core region [85]. Each secretory granule contains about 500 micromolar to several millimolar of IAPP [80]. It has been reported that non-stimulated circulating levels of IAPP are between 3-5 picomolar and can rise up to 20 picomolar with increased blood glucose [86]. IAPP is normally cleared by the kidneys and elevated plasma IAPP levels have been reported in patients with renal failure [87].    9 1.2.3 Formation of IAPP aggregates  Normally, human IAPP (hIAPP) molecules are soluble in the monomeric state. However, these monomers can aggregate into beta-sheet-rich fibrils and form amyloid deposits (Figure 3) [79]. There is a correlation between the amino acid sequence of IAPP and its tendency to form amyloid [88, 89]. hIAPP forms amyloid deposits rapidly in vitro in human islets while islet amyloid is not found in rodent islets. The rat/mouse IAPP sequence contains three unique proline residues at positions 25, 28 and 29, which disrupt formation of the secondary structure and is therefore not amyloidogenic [90]. Likewise, synthetic rodent IAPP (rIAPP) is soluble and does not have cytotoxic effects on β-cells in vitro [89, 91]. As a result, researchers have developed several hIAPP transgenic mouse models to study the mechanism of islet amyloidosis in vivo.  Over-expression of hIAPP in β-cells due to increased insulin demand in peripheral tissues is also a factor that contributes to hIAPP aggregation and amyloid formation [92]. In other words, β-cells in patients with T2D chronically increase the production of (pro)insulin to compensate for their insulin resistance, which leads to a parallel increase in pro-IAPP production [93]. Once protein synthesis exceeds the capacity for protein processing in healthy β-cells, the threshold between normal IAPP expression and formation of misfolded proteins is overcome [94]. At this point, the ubiquitin/proteasome and autophagy/lysosomal systems for protein degradation become dysfunctional and thus, further compromise β-cells from protection against toxic IAPP aggregates [94]. Additionally, β-cell dysfunction and alteration in lipid metabolism can lead to impaired prohIAPP processing and/or trafficking, aggravating the level of islet amyloidogenesis [95, 96]. Interestingly, it has been suggested that hIAPP aggregation is prevented natively in healthy individuals due to environmental characteristics of β-cell granules such as low pH level, high concentrations of insulin, zinc and proinsulin [85, 97, 98].  10  As shown in Figure 3, the kinetics of hIAPP aggregation includes three distinct phases. In the lag phase, monomers undergo conformational changes and form oligomeric nuclei to proceed to an exponential growth phase [80]. Then, fibrils elongate by adding peptides to their ends and secondary nucleation also occurs [80]. Eventually, steady state is reached with soluble peptides at equilibrium with amyloid fibrils [80]. The initial location of amyloid formation is still under investigation; however, it has been proposed that the initial amyloid particle forms intracellularly. These peptide aggregates are then released from the apoptotic cells and act as a seed for progressive formation of extracellular insoluble amyloid deposits [73, 79].  1.1.1 Mechanisms of amyloid-induced cytotoxicity  Several studies have shown that both extracellular hIAPP aggregates and intracellular hIAPP oligomers can lead to deterioration of β-cell mass and function [80, 91, 99–106]. However, the underlying mechanisms of hIAPP-mediated β-cell toxicity have not been completely elucidated. As illustrated in Figure 4, several mechanisms of hIAPP-induced β-cell death have been proposed based on in vitro and in vivo experiments. Recent studies have revealed that hIAPP oligomers are likely the most toxic species to β-cells [99, 107, 108]. Yet, it is challenging to differentiate the cytotoxic effects of oligomers and mature fibrils since different conformation of hIAPP aggregates can co-exist [109–111].   The most commonly hypothesized mechanism of hIAPP-induced β-cell death is through cell membrane permeabilization and disruption of intracellular homeostasis [99, 112–115]. Toxic oligomers can either directly interact with the lipid bilayers or form nonselective ion voltage-dependent channels that results in influx of Ca2+ and Na+ and K+ efflux [112, 113]. In addition, large amyloid deposits in the extracellular space may act as a physical barrier, which results in  11 structural damage in the islets and disruption of blood flow leading to apoptosis [109]. Secondly, hIAPP aggregates can promote production and accumulation of ROS such as hydrogen peroxide (H2O2) and increase oxidative stress [116, 117]. Zraika et al. demonstrated that the antioxidant N-acetyl-L-cysteine (NAC) protected β-cells in the hIAPP transgenic mouse islets [118]. Thirdly, buildup of hIAPP aggregates can lead to ER stress as indicated by upregulation of ER stress genes such as C/EBP homologous protein (CHOP), spliced X-box binding protein 1 (sXBP1) or activating transcription factor 3 (ATF3), promoting defects in the unfolded protein response (UPR)[119–122]. Treatment with chemical chaperones such as taurine-conjugated ursodeoxycholic acid (TUDCA) can reduce ER stress and ameliorate insulin secretion in β-cells overexpressing hIAPP [123]. A recent study has shown that deletion of CHOP, a key mediator of ER stress, delayed β-cell loss and diabetes onset in hIAPP transgenic mice [124]. Additionally, hIAPP aggregates are normally cleared by autophagy and accumulation of hIAPP oligomers can reduce autophagic activity, potentiating the cytotoxic effects of hIAPP oligomers on β-cells [125–127]. Accumulation of hIAPP oligomers can be clearly visualized in islets of hIAPP-expressing mice with β-cell-specific autophagy deficiency, leading to reduction of β-cell mass [128, 129]. Lastly, recent evidence suggests that hIAPP aggregates can activate macrophages and cause local inflammation and cytokine production [130–132]. Depletion of resident macrophages in the islets of transgenic hIAPP-expressing mice achieved by clodronate liposome treatment attenuated gene expression of pro-inflammatory cytokines (IL-1β, IL-1α and tumor necrosis factor α (TNF-α)) and improved glucose tolerance [133]. Furthermore, recent studies have suggested that blocking IL-1 release induced by hIAPP aggregates with IL-1Ra can reduce islet inflammation and improve islet function [131, 134, 135].  Our research group recently showed that endogenously formed hIAPP aggregates can promote the extrinsic (Fas-dependent) apoptotic  12 pathway, likely by triggering production and release of IL-1β [136] and that blocking IL-1 receptor with Anakinra, a clinically approved IL-1 receptor antagonist (IL-1Ra) for the treatment of the inflammatory disease rheumatoid arthritis, protected β-cells in amyloid forming human islets during culture at elevated glucose [135]. The signaling pathways underlying amyloid-induced β-cell apoptosis will be discussed in section 1.3.  1.3 Apoptosis  1.3.1 Cell apoptosis signaling pathways  Apoptosis or programmed cell death is a highly regulated, energy-dependent process that involves a series of signal cascades. Apoptosis is implicated under both physiologic and pathological conditions [137]. Normally, apoptosis is activated as a homeostatic mechanism to maintain cell population in the body during development and aging [138]. Pathologic apoptosis, occurs in different diseases such as cancers which are associated with reduced rate of apoptosis or diabetes, neurodegenerative diseases, and AIDS which are associated with increased rate of apoptosis [137].   Depending on the type and intensity of stimulus as well as availability of caspases another type of cell death known as necrosis can also occur simultaneously in mammalian cells [139]. Apoptosis can be differentiated from other forms of cell death based on distinct morphological characteristics in cells such as cell shrinkage, pyknosis, membrane blebbing, and DNA fragmentation [138]. Cellular organelles and nuclear fragments are packed into membrane-bound apoptotic bodies that are rapidly engulfed by surrounding phagocytic cells that release cytokines to inhibit an inflammatory response [140, 141]. On the contrary, cells undergoing necrosis display morphological features such enlarged overall size due to dilatation of  13 cytoplasmic organelles and early rupture of plasma membrane, often leading to local inflammation [142].  Apoptosis is activated through two inter-connecting caspase-dependent apoptotic pathways including the extrinsic (or cell death receptor) pathway and the intrinsic (or mitochondrial) pathway in mammalian cells (Figure 5) [143]. Both apoptotic pathways converge at activation of the terminal executioner caspases, which in turn initiates the final events of cell demise including DNA fragmentation, clearance of cytoskeletal and nuclear proteins, formation of cytoplasmic blebs and apoptotic bodies, and expression of ligands to signal uptake by phagocytic cells [137]. There is a third caspase-dependent pathway that involves cytotoxic T-cells named the perforin/granzyme B pathway [144].   1.3.2 Extrinsic apoptotic pathway  As illustrated in Figure 5, the extrinsic or Fas-mediated apoptotic pathway is associated with cell-surface transmembrane death receptors of the TNF receptor gene superfamily that have a cytoplasmic domain known as the “death domain” [145, 146]. The pathway is initiated when extracellular cytokines such as Fas ligand (FasL) and TNF-α bind and activate their corresponding cell-surface death receptors, in this case the Fas receptor (CD95/APO-1) and TNFR1, respectively [147]. This is followed by trimerization of the death receptor and recruitment and binding of adapter proteins such as FAS-associated death domain (FADD) and TNF receptor-associated death domain (TRADD) [148]. FADD then recruits pro-caspase 8 to form the death-inducing signaling complex (DISC), which leads to autocatalytic activation of procaspase-8 [149]. Once caspase-8 activation is triggered, downstream activation of executioner caspases such as caspase-3 takes place. Finally, DNA fragmentation, activation of proteases and  14 cell death occurs. In type II cells such as hepatocytes and pancreatic β-cells, activated caspase-8 can indirectly activate the intrinsic apoptotic pathway via cleavage the pro-apoptotic B-cell lymphoma 2 (Bcl-2) protein Bid, thereby amplifying the death receptor-mediated cell death program (Figure 4) [150]. In addition, cellular FLICE-inhibitory protein (c-FLIP) can bind to FADD and caspase-8 and inhibit the extrinsic apoptotic pathway [151].  1.3.3 Intrinsic apoptotic pathway  The intrinsic (mitochondrial or cytochrome c dependent) apoptotic pathway is regulated by the balance between the pro-apoptotic and the anti-apoptotic members of the Bcl-2 family. As shown in Figure 5, the pathway can be triggered in response to a diverse range of intracellular stimuli such as DNA damage, growth factor withdrawal, toxins, and hypoxia, leading to activation of a group of proteins called Bcl-2 homology (BH) 3-only proteins that have a conserved BH3 domain [152]. Activated BH3-only proteins can bind and inhibit the activity of anti-apoptotic Bcl-2 members, such as Bcl-2 and Bcl-XL, allowing activation of the pro-apoptotic proteins, Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak) [152]. It has been suggested that some BH3-only proteins such as Bcl-2-like protein 11 (BIM) and p53 upregulated modulator of apoptosis (PUMA) may be able to bind directly to Bax and/or Bak and induce activation [152]. Additionally, Bcl-2 antagonist of cell death (Bad) protein from the BH3-only family is an important member of the regulatory process and its phosphorylation state is a critical determinant for cell survival. Under normal circumstances, phosphorylated Bad is bound and sequestered in the cytosol by proteins of the 14-3-3 family [152]. Apoptotic stimuli that result in dephosphorylation of Bad will allow its dissociation from 14-3-3 to bind and block the function of anti-apoptotic proteins [152]. Once Bax and Bak  15 become activated, they translocate to the mitochondria where they oligomerize and induce mitochondrial outer membrane permeabilization (MOMP) [153].   Upon the induction of MOMP, two different groups of apoptotic factors are released from the intermembrane space of the mitochondria into the cytosol [154]. The first group of apoptotic factors released into the cytosol of the cell consists of cytochrome c and Smac/DIABLO that activate the caspase-dependent mitochondrial pathway [155, 156]. As summarized in Figure 5, cytosolic cytochrome c binds to apoptosis-activating factor 1 (Apaf-1) in the presence of ATP and form a complex called the apoptosome [152]. The apoptosome can recruit seven dimers of procaspase-9, leading to proteolytic activation of caspase-9 [155]. This is followed by catalytic maturation of caspase-3 in the execution phase of apoptosis [152]. Smac/DIABLO are released from the mitochondria after MOMP to block X-linked inhibitor of apoptosis protein (XIAP), which is an inhibitor for caspase activity [152]. The second group of mitochondrial apoptotic factors include apoptosis inducing factor (AIF), caspase-activated DNase (CAD), and endonuclease G, which are released much later in the cell [137]. Both AIF and endonuclease G function in a caspase-independent fashion and induce DNA fragmentation and chromatin condensation [157].   1.3.4 Role of the extrinsic apoptotic pathway in β-cell death  Human islets constitutively express Fas ligand but not Fas receptor [51, 158]. Maedler et al. observed that Fas receptor is upregulated in β-cells of patients with T2D and exposure of human islets to elevated glucose ex vivo induced Fas expression and activation of the extrinsic apoptotic pathway [51]. The cytotoxic effects of glucose were inhibited when β-cells were treated with antagonistic Fas antibody (ZB4), indicating that glucose-induced Fas receptor can  16 interact with FasL on neighboring β-cells or other islet and/or non-islet cells thereby activate caspase-8 activation in the extrinsic apoptotic program.   Our research group recently showed that the Fas-mediated apoptotic pathway plays a key role in hIAPP-induced β-cell death. Briefly, we demonstrated the activation of caspase-8 and -3, and apoptosis via Fas upregulation in INS-1 and primary human and mouse islet β-cells following exposure to both exogenously applied and endogenously formed hIAPP aggregates [136, 159]. Furthermore, we demonstrated that blocking caspase-8, Fas or amyloid formation in hIAPP-treated β-cells markedly reduces Fas-mediated apoptosis [136, 159]. Accordingly, deletion of Fas or caspase-8 in hIAPP-expressing mouse islets resulted in greater β-cell area and improved β-cell function as compared with islets expressing hIAPP and Fas or caspase-8 during ex vivo culture [136, 159]. Overall, our lab provided the first evidence to show that endogenously formed hIAPP aggregates can promote the extrinsic (Fas-dependent) apoptotic pathway, likely by triggering production and release of IL-1β as mentioned earlier [135, 136].   1.3.5 Role of the intrinsic apoptotic pathway in β-cell death  Expression and interaction of the various pro-apoptotic and anti-apoptotic Bcl-2 members of the mitochondrial apoptotic pathway remains poorly characterized in β-cells. Several studies have shown that intrinsic apoptotic pathway is involved in β-cell apoptosis upon various types of cellular stress stimuli such as induction of proinflammatory cytokines, hyperglycemia, and high levels of palmitate [147]. For instance, Grunnet et al. reported that proinflammatory cytokines (IL-1β, interferon (IFN)-γ and TNF-α) induced dephosphorylation of Bad via calcineurin, cytochrome c release, and subsequently capase-9 and -3 activation [160]. In addition, blocking Bad Ser136 dephosphorylation or Bax activation protected human islet cells from cytokine  17 toxicity [160]. This finding supports previous data from Rabinovitch et al. that overexpression of anti-apoptotic Bcl-2 protected β-cells from cytokine-mediated β-cell loss and dysfunction [161]. In addition, islets with deletion of the pro-apoptotic BH3-only protein Bid were partially protected from cytokine-mediated β-cell death, supporting the notion that a cross-talk mechanism between the Fas-mediated and mitochondrial-dependent apoptotic pathways may exist in this process [162]. Correspondingly, McKenzie et al. also reported that single Bax or Bak knockout sufficiently protected islet β-cells from cytokine- and FasL-induced cell death in Bax and Bak null mice [162].  Moreover, Federici and colleagues observed that islets cultured in high glucose (16.7 mmol/l) had reduced expression of the anti-apoptotic gene Bcl-XL and overexpression of pro-apoptotic genes such as Bad, Bid, and Bik [163]. Bad and Bid expression was found specifically in human islet β-cells while Bik and Bcl-XL were expressed in other islet cell types, favoring the mitochondrial apoptotic program in β-cells [163]. In another study, McKenzie et al. observed that the intrinsic apoptotic pathway, distinct from the cytokine- or death receptor-mediated apoptosis is a major mediator of glucose induced β-cell death in islets [164]. Finally, ablation of Bax but not Bak protected islet cells from ribose- or glucose-induced cytotoxicity [164]. Taken together, these findings raise the possibility that Bax and Bak have both overlapping and distinct roles in glucotoxicity.  On the other hand, there has been recent evidence showing that the intrinsic apoptotic pathway is directly involved in lipotoxic β-cell death [165]. Biden et al. demonstrated that the protein expression of myeloid cell leukemia sequence 1 (Mcl-1), an anti-apoptotic Bcl-2 protein, was downregulated by palmitate treatment [166]. Reduction of Mcl-1 was associated with Bax translocation to the mitochondria, release of cytochrome c, and activation of the mitochondrial- 18 dependent apoptotic pathway in INS-1E cells and rat pancreatic islets β-cells [166]. In support, Cunha et al. showed that palmitate triggered ER stress and activated the intrinsic apoptotic pathway via BH3-only proteins death protein 5 (DP5) and PUMA [167].  DP5-/- mice were protected from high fat diet–induced β-cell loss and impairment [167].   1.4  Animal Models 1.4.1 BaxBak double knockout mouse model  Bax and Bak are both members of the multi-BH domain Bcl-2 family and have very well-defined pro-apoptotic function in the intrinsic apoptotic pathway [152, 168]. Yet, knowledge on their role(s) in β-cells is limited. Several loss of function studies have been performed to provide insight on the involvement of the two pro-apoptotic proteins in β-cells using various types of stress stimuli. Since we are interested in learning about the consequences of blocking the mitochondrial apoptotic pathway in amyloid-mediated β-cell death rather than the defined individual role(s) of Bax or Bak, we chose to use a mouse model with β-cell specific Bax and Bak double knockout (BaxBak DKO) for our studies.  Bax and Bak are crucial in normal tissue development and homeostasis of cell population. Therefore, global deletion of Bax and Bak has detrimental consequences and more than 90% of Bax-/- Bak-/- mice die perinatally [169–171]. Lindsten et al. also reported that those animals which survived into adulthood displayed several developmental defects such as abnormality in their hematopoietic profile and nervous system, imperforate vaginal canal, interdigital webs, and neurological defects [169]. As detailed in section 2.3, Dr. Luciani generated the BaxBak DKO mouse model with temporal- and β-cell-specific Bax knockout using Cre-lox technology [172]. BaxBak DKO mice were reported to be  19 protected from β-cell apoptosis induced by both staurosporine (STS) and gluco-lipotoxicity [173].   1.4.2 Cytochrome c mutant mouse model (CytcKA/KA)   Cytochrome c in somatic cells is a 13 kDa water-soluble protein located in the inner mitochondrial membrane [174]. Aside from its pro-apoptotic function, cytochrome c normally participates in the electron-transport chain in the absence of cell death stimulus, transferring electrons with its heme group between Complex III and Complex IV [175]. Consequently, knockout of cytochrome c in Cytc-/- null embryos results in early lethality, plausibly due to ineffective oxidative phosphorylation [176].  Hao et al. generated a mouse model that contains a lysine 72 to alanine (K72A) knockin allele in the genomic Cyt c locus, rendering a mutant cytochrome c with intact electron-transport function [177].  Without lysine 72, cytochrome c can no longer bind to Apaf-1, therefore disrupting its pro-apoptotic function [177]. The CytcKA/KA mice were born at a much lower frequency than the expected Mendelian ratio and the majority of surviving animals presented with deformed brain morphology and developmental deficits [177].   Despite low survival rate in CytcKA/KA mice, Choi et al. demonstrated that blocking the pro-apoptotic function of cytochrome c did not affect glucose homeostasis and β-cell mass under basal conditions [178]. Interestingly, CytcKA/KA mice were not protected from streptozotocin (STZ)-induced diabetes as seen with loss of β-cell area comparable with their wild-type littermates [178]. Moreover, abolishing the pro-apoptotic function of cytochrome c failed to preserve β-cells from c-Myc-induced β-cell death [178]. In the present study, we investigated the potential role of pro-apoptotic function of cytochrome c in hIAPP-induced β-cell apoptosis using CytcKA/KA mutant mouse model.   20  1.5 Investigating the role of mitochondrial apoptotic pathway in hIAPP-mediated β-cell death   Previously, our research group has demonstrated that the Fas-mediated apoptotic pathway plays a key role in hIAPP-induced β-cell death [159]. Yet, blocking the amyloid-induced Fas-mediated apoptotic pathway markedly reduced β-cell death but not to basal level. This suggests a potential role for the mitochondrial apoptotic pathway in amyloid toxicity since this pathway can be activated either directly by intracellular cellular stress stimuli or indirectly via caspase-8-mediated cleavage of Bid in β-cells.   In the present study, we hypothesized that hIAPP aggregates promote activation of the mitochondrial apoptotic pathway and blocking the key mediators of this pathway can protect β-cells from amyloid toxicity, thereby enhancing β-cell survival.  To test this hypothesis, we addressed the following two aims: Aim 1: To investigate if hIAPP aggregates can activate the mitochondrial apoptotic pathway in islet β-cells;  Aim 2: To test if blocking the mitochondrial apoptotic pathway can reduce amyloid-induced β-cell toxicity.   Analyzing the role of mitochondrial apoptotic pathway in hIAPP cytotoxicity enhances our knowledge on the pathogenesis of T2D and may provide valuable insight into novel therapeutic interventions to prevent β-cell death in T2D.  21         Figure 1. The main pathological factors and cellular stressors that contribute to the progression of T2D. T2D is associated with both genetic pre-disposition and environmental risk factors (e.g. excessive nutrition, obesity, lack of physical activity and aging) that can promote insulin resistance in peripheral tissues. β-cells are constantly exposed to physiologic stressors associated with T2D (e.g. hyperglycemia, hyperlipidemia, oxidative stress, inflammation, ER stress and islet amyloid formation) that impair functional β-cells, leading to hyperglycemia and development of T2D.   22     Figure 2. Processing of preproIAPP in β-cells. IAPP is first synthesized as an 89-residue preproIAPP molecule. The signal peptide is cleaved off in the Golgi to form a 67-residue proIAPP. The prohormone undergoes further cleavage at the dibasic sites near the C-terminus in the Golgi by prohormone convertase (PC) 1/3, and the N-terminus mainly in the secretory granules by PC2. The remaining C-terminal basic residues are removed by carboxypeptidase E (CPE). Activated peptidyl amidating monooxygenase (PAM) complex assists in the removal of Gly38 and amidation at Tyr37. The biologically active form of IAPP has an intramolecular disulfide bridge between the cysteine residues at positions 2 and 7 and an amidated C-terminus (adapted from [79]).   23       Figure 3. Conformational states of hIAPP aggregates at different stages of amyloid formation. The kinetics of hIAPP aggregation contains three distinct phases. In the lag phase, monomers undergo conformational changes and form oligomeric nuclei to proceed to an exponential growth phase. Next, fibrils elongate by addition of peptides to their ends and secondary nucleation also occurs. Eventually, steady state is reached with soluble peptides at equilibrium with amyloid fibrils (adapted from [80, 109]).   24     Figure 4. Proposed mechanisms of hIAPP-induced β-cell death. hIAPP can form toxic oligomers and induce cytotoxicity in β-cells via several proposed mechanisms including cell membrane permeabilization and disruption of intracellular homeostasis, generation of reactive oxygen species, endoplasmic reticulum stress, mitochondrial dysfunction, defects in autophagy, local inflammation and cytokine production (adapted from [103]).    25  Figure 5. The intrinsic and extrinsic apoptotic pathways in mammalian cells.  (i) The extrinsic (Fas-mediated) pathway is initiated when FasL or TNF-α bind and activate their cell-surface death receptors. This is followed by recruitment of FADD and pro-caspase 8 to form DISC, which leads to autocatalytic activation of procaspase-8. Once caspase-8 is activated, it can directly activate executioner caspases such as caspase-3 in type I cells. In type II cells, activated caspase-8 can also initiate the intrinsic pathway by cleavage of Bid.   (ii) The intrinsic (mitochondrial dependent) apoptotic pathway is regulated by the balance between the pro-apoptotic and anti-apoptotic members of the Bcl-2 family. This pathway is triggered upon activation of the multi-domain pro-apoptotic proteins, Bax and Bak, which form homo-oligomeric pores that cause mitochondrial outer membrane permeabilization (MOMP) and release of apoptotic factors such as cytochrome c. Cytosolic cytochrome c subsequently binds to apoptosis-activating factor 1 in the presence of ATP to form a protein complex called apoptosome that recruits and activates caspase-9. Activated caspase-9 then cleaves and activates executioner caspases, leading to apoptosis. Smac is also released from the mitochondria after MOMP to block XIAP, an inhibitor for caspase activity (adapted from [205]). Black arrows, activation. Red lines, inhibition.   26 Chapter 2: Materials and Methods 2.1 Reagents and materials  Trypsin-EDTA, RPMI-1640, fetal bovine serum (FBS), bovine serum albumin (BSA), Tryphan Blue, Avertin, glutamax, MEM sodium pyruvate, penicillin, streptomycin, gentamycin, and cell dissociation buffer were purchased from Invitrogen Canada Inc. (Burlington, ON, CA). 2-mercaptoethanol, HEPES buffer, poly-L-lysine, Triton X-100, dithizone, collagenase (Type XI), DNase 1, Sigmacote™ siliconizing reagent, Thioflavine S, dimethyl sulfoxide (DMSO) and Bisbenzimide Hoechst were from Sigma-Aldrich (Oakville, ON, CA). CMRL 1066 and Ham’s-F10 culture media were from Mediatech (Herndon, VA, USA). In Situ Cell Death Detection Kit, TMR red was from Roche Diagnostics (Laval, QC, Canada). Fluorescence mounting media with or without DAPI ((4', 6-diamidino-2-phenylindole) was from Dako (Carpinteria, CA, USA).   2.2 Cell culture   The transformed rat β-cell line, INS-1 (832/13), was kindly provided by Dr. C. Newgard (Duke University Medical Center, NC, USA). Cells were cultured in RPMI-1640 medium supplemented with FBS, penicillin, streptomycin, 2-mercaptoethanol, glutamax, MEM sodium pyruvate, and HEPES (as detailed in Table 1) at 37°C (95% humidity, 5% CO2). Cells were seeded into 8-well chamber slides at 140,000 cells per well.   2.3 Animals  Mice carrying a mutant cytochrome c (Cytc KA/KA) were generated by Hao et al. and provided by Dr. M. Woo (University of Toronto, ON, CA). In this mouse model, the pro-apoptotic function of cytochrome c is abolished due to the lysine 72 to alanine (KA) mutation in  27 genomic Cyt c [177]. The BaxBak wild-type and BaxBak double knockout (BaxBak DKO) mice were generated and provided by Dr. D. Luciani (University of British Columbia, BC, CA) [172].  In brief, Bak-/-:Baxflox/flox mice (Jax stock number 006329; B6:129 genetic background; The Jackson Laboratory) were bred with Pdx1-CreER mice (CD-1 genetic background; provided by Dr. Doug Melton, Harvard University, MA, USA) to generate Bak-/-:Baxflox/flox:Pdx1-CreER and Bak-/-:Baxflox/flox animals. Ablation of Bax was achieved by intra-peritoneal injection of tamoxifen (3mg/40 g body weight) for five consecutive days and the resulting BaxBak DKO mice had temporal- and β-cell-specific Bax knockout using Cre-lox technology [172]. All animals were cared and treated with standard protocols in compliance with the Canadian Council on Animal Care guidelines and studies were approved by the University of British Columbia Animal Care Committee.   2.4 Human islet isolation and culture   Human pancreatic islets provided by the Ike Barber Clinical Human Islet Transplant Laboratory (Vancouver, BC, CA) were from cadaveric organ donors and were isolated in compliance with guidelines of the Clinical Research Ethics Board of the University of British Columbia. The details on processing of human pancreata are reported previously [179]. Human islets were hand-picked and islet purity was assessed by dithizone staining (purity >70%). Islets were then cultured overnight (37°C, 95% humidity, 5% CO2) in CMRL media supplemented with FBS, penicillin, streptomycin, and gentamicin (as detailed in Table 1) before islet dispersion.    28 2.5  Mouse islet isolation and culture   Mouse pancreatic islets were isolated as described before with some modifications [180]. Animals were anaesthetised with Avertin (0.25 mg/g body weight, i.p.) and euthanized by cervical dislocation. Mouse pancreata were perfused with ice-cold collagenase (1000 U/ml) dissolved in calcium-free Hank’s buffer via the common bile duct. The pancreata were then harvested and incubated with collagenase/Hanks’ solution (1,000 U/ml) in a shaker water bath (14 min, 37°C, 120 rpm). This was followed by 2 min of gentle shaking and digestion was then terminated by addition of ice-cold Hanks’ solution with 1 mmol/l CaCl2. Next, digested tissues were rinsed and resuspended in completed Ham’s-F10 medium (refer to Table 1), and filtered through a 70 µm nylon mesh cell strainer (BD Biosciences, Oakville, ON, CA). Mouse islets were hand-picked and cultured in supplemented Ham’s-F10 medium (shown in Table 1) for 7 days (37°C, 95% humidity, 5% CO2) and the culture medium was changed every 2 days.  2.6 Islet dispersion   Isolated human or mouse islets were cultured overnight to allow them to recover from the isolation process and islets were dispersed as previously described with some modifications [180]. Briefly, 300-350 islets were dissociated into single cells in 190 µl cell dissociation buffer by gently pipetting up and down for 1 min. Cells were then incubated in 37°C water bath undisturbed for 1 min. This was followed by addition of 2 µl trypsin-EDTA (0.25%) and 1 µl DNase I (0.4 mg/ml). Dissociation was continued with 1 min pipetting up and down and 1 min rest, which were repeated for a total of 5 min. 200µl medium (CMRL for human islets; Ham’s-F10 for mouse islets) was then added and cells were centrifuged at 1500-2000 RPM for 5 min at room temperature. Cell pellets were resuspended in 400 µl culture medium and the number of  29 live/dead cells was assessed with Trypan Blue. Counted cells (150,000-180,000 per well) were seeded in poly-L-lysine-coated chamber slides and cultured as detailed for each study (see Table 1).   2.7 Adenoviral transduction   Recombinant adenoviruses expressing human and rat pre-proIAPP (Ad-prohIAPP and Ad-prorIAPP, respectively) were generated by Dr. C. Rhodes (University of Chicago, IL, USA) and details were described previously [181]. In brief, the complementary cDNA construct encoding the full-length human or rat pre-proIAPP were sub-cloned into pAd/cytomegalovirus/DEST adenovirus vector (Invitrogen, Carlsbad, CA) and a high-titer recombinant virus was generated.  Purified Pre-proIAPP is produced and processed normally in β-cells using the host cell’s machinery for protein synthesis [181]. INS-1 β-cells or dispersed islet cells were cultured in chamber slides for 48 h before adenoviral transduction. Cells were transduced with Ad-prohIAPP or Ad-prorIAPP at multiplicity of infection (MOI: 10) in culture medium for 2 h, followed by 40 h incubation with fresh medium for recovery. Isolated mouse islets were cultured in Hams-F10 (16.7 mM glucose) and transduced with Ad-prohIAPP (MOI: 10) for 16 h. Time points were pre-determined based on preliminary studies from our lab.   2.8 Treatment with peptides and inhibitors  Aliquots of lyophilised peptide were prepared using synthetic hIAPP or rIAPP peptide as described before [180]. hIAPP or rIAPP solutions were freshly made for each study in culture medium and added promptly to cells at a final concentration of 10 µmol/l. The caspase-9 inhibitor and Bax inhibitor were diluted in culture medium and added to cells 1 h before IAPP  30 peptide treatment or 2 h after adenoviral transduction as detailed in figure legends. The final concentration and supplier details of the peptides and inhibitors used in these studies are summarized in Table 2.  2.9 Immunolabeling   INS-1 β-cells and primary islet cells cultured in 8-well chamber slides were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5 % Triton X-100 in PBS for 5 min. Paraffin-embedded mouse islet sections (5 µm) were deparaffinized with xylene and rehydrated with ethanol, followed by antigen retrieval with citrate buffer in a steamer. Cells and islet sections were blocked with 2% normal goat and/or donkey serum (Vector Laboratories, Burlingame, CA, USA) for 30 and 45 minutes, respectively. This was followed by immunolabeling with primary antibodies listed in Table 3 and the slides were incubated overnight at 4ºC. The next day, cells and islet sections were washed with PBS and incubated with the species-corresponding secondary antibodies (Table 4) for 1 h at room temperature. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect β-cell apoptosis in cells/islet sections by incubation with TUNEL reaction mixture (1:20) for 30 min at 37°C following immunolabeling for insulin. Amyloid formation was detected with thioflavin S staining (0.5%; 5 min; room temperature). For quantification studies, cells/islet sections were counterstained with the nuclear dye DAPI or Hoechst. Fluorescent signals were captured using an Axiovert 200 (Zeiss, Toronto, CA) inverted fluorescence microscope and Openlab 3.0 software (Improvision, Lexington, MA, USA).    31 2.10 Statistical analysis  Statistical analysis was performed in Microsoft Excel and GraphPad Prism 6 (GraphPad Software Inc.) and data are presented as mean +/− SEM (standard error of the mean).  Data were analyzed by one-way analysis of variance (ANOVA) with Newman-Keuls multiple comparison test. A p value of <0.05 was considered significant.                     32 Table 1. Culture media and supplements used for islet and cell culture.      Table 2. List of peptides and inhibitors used for treatment studies.   Cell/tissue type Culture media Supplements INS-1 (832/13) cells RPMI-1640  (containing 11.1 mmol/l glucose) • 10% (vol./vol.) FBS • 2 mmol/l Glutamax 1 • 1 mmol/l MEM sodium pyruvate  • 50 U/ml penicillin • 50 µg/ml streptomycin • 50 µmol/l 2-mercaptoethanol • 1% HEPES Dispersed human islet cells CMRL  (containing 5.5 mmol/l glucose) • 10% FBS • 50 U/ml penicillin • 50 µg/ml streptomycin • 50 µg/ml gentamicin • 1% Glutamax Dispersed mouse islet cells Ham’s-F10  (containing 10 mmol/l glucose) • 0.5% BSA • 50 U/ml penicillin • 50 µg/ml streptomycin • 50 µg/ml gentamicin Mouse islets Ham’s-F10  (containing 10 mmol/l glucose)  • 0.5% BSA • Final [glucose]: 16.7 mmol/l  • 50 U/ml penicillin • 50 µg/ml streptomycin • 50 µg/ml gentamicin Peptides and inhibitors Abbreviation Concentration Supplier Synthetic human islet amyloid poly peptide hIAPP 10 µmol/l Bachem  (Torrance, CA, USA) Synthetic rat islet amyloid polypeptide rIAPP 10 µmol/l Bachem  (Torrance, CA, USA) Caspase-9 inhibitor  z-LEHD-FMK; CASP9 inh 100 µmol/l Bachem  (Torrance, CA, USA)  Bax Inhibitor VPMLK; Bax inh or BI 100 µmol/l Bachem  (Torrance, CA, USA)  33 Table 3. List of primary antibodies used for immunolabeling studies.        1°Antibody Species Cross-Reactivity Supplier  Catalogue Number  Host Species  Dilution ATP synthase β antibody (monoclonal) Human, mouse, rat Sigma-Aldrich (Oakville, ON, CA) A9728 Mouse Cell: 1:1000  Cleaved caspase-9 (Asp330) antibody (polyclonal) Human Cell Signaling (Pickering, ON, CA) 9501 Rabbit Cell: 1:75 Cleaved caspase-9 (Asp353) antibody (polyclonal) Mouse Cell Signaling (Pickering, ON, CA) 9509 Rabbit Islet: 1:100 Cleaved caspase-9 (Asp353) antibody (polyclonal) Rat Cell Signaling (Pickering, ON, CA) 9507 Rabbit Cell: 1:200 Cytochrome c antibody (monoclonal) Human, mouse, rat Cell Signaling (Pickering, ON, CA) 12963 Mouse Cell: 1:250 Islet: 1:250 Glucagon antibody (polyclonal) Human, mouse, rat Dako (Carpinteria, CA, USA) A056501 Rabbit Islet: 1:750 Insulin antibody (polyclonal) Human, mouse, rat Dako (Carpinteria, CA, USA) A056401 Guinea Pig Cell: 1:400 Islet: 1:1000 Oligomer A11 antibody (polyclonal) Human, mouse, rat Invitrogen (Burlington, ON, CA) AHB0052 Rabbit Cell: 1:400 Islet: 1:400   34 Table 4. List of secondary antibodies used for immunolabeling studies.     Type of staining 2°Antibody (dilution) Supplier Catalogue Number Insulin/Glucagon Texas red-conjugated goat α guinea pig IgG (1:1000) Jackson Laboratories (WestGrove, PA, USA) 106-076-003 Alexa Fluor 488-conjugated goat α rabbit IgG (1:750) Invitrogen (Burlington, ON, CA) A-11034 Insulin/Cytochrome c Texas red-conjugated goat α guinea pig IgG (1:400) Jackson Laboratories (WestGrove, PA, USA) 106-076-003 Alexa Fluor 488-conjugated goat α mouse IgG (1:500) Invitrogen (Burlington, ON, CA) A-11029 Insulin/A11 (oligomer) Alexa Fluor 488-conjugated goat α guinea pig IgG (1:400) Invitrogen (Burlington, ON, CA) A-11073 Texas red-conjugated donkey α rabbit IgG (1:400) Jackson Laboratories (WestGrove, PA, USA) 711-075-152 Insulin/Cleaved caspase-9 Texas red-conjugated goat α guinea pig IgG (1:400) Jackson Laboratories (WestGrove, PA, USA) 106-076-003 Alexa Fluor 488-conjugated goat α rabbit IgG (1:200) Invitrogen (Burlington, ON, CA) A-11034 Insulin/Cytochrome c/Cleaved caspase-9 AMCA-conjugated donkey α guinea pig IgG (1:100) Jackson Laboratories (WestGrove, PA, USA) 706-155-148 Alexa Fluor 488-conjugated goat α mouse IgG (1:500) Invitrogen (Burlington, ON, CA) A-11029 Texas red-conjugated donkey α rabbit IgG (1:75) Jackson Laboratories (WestGrove, PA, USA) 711-075-152 Insulin/Cytochrome c/A11 (oligomer) AMCA-conjugated donkey α guinea pig IgG (1:200) Jackson Laboratories (WestGrove, PA, USA) 706-155-148 Alexa Fluor 488-conjugated goat α mouse IgG (1:500) Invitrogen (Burlington, ON, CA) A-11029 Texas red-conjugated donkey α rabbit IgG (1:400) Jackson Laboratories (WestGrove, PA, USA) 711-075-152  35 Chapter 3: Results 3.1 hIAPP aggregates promote activation of the mitochondrial apoptotic pathway  3.1.1 Formation of intracellular hIAPP aggregates in β-cells is associated with increased mitochondrial cytochrome c release  We first tested whether formation of intracellular hIAPP aggregates in INS-1 β-cells transduced with an adenovirus that codes for the expression of hIAPP (Ad-prohIAPP), can induce activation of the mitochondrial apoptotic pathway. INS-1 β-cells transduced with an adenovirus that codes for expression of non-fibrillogenic rat IAPP was used as a control for any potential adverse effects of adenoviral transduction on β-cells. In contrast to control and rIAPP-expressing INS-1 β-cells, Ad-prohIAPP transduced β-cells formed intracellular oligomers as detected by immunolabeling for insulin and A11 (to detect hIAPP oligomers) 42 hours after adenoviral transduction (Figure 6a). Treatment with Bax inhibitor did not have any detectable effect on the formation of hIAPP oligomers.   Intracellular hIAPP aggregation in transduced INS-1 β-cells was associated with the release of mitochondrial cytochrome c as assessed by the presence of diffuse homogeneous cytochrome c in the cytoplasm observed by immunolabeling (Figure 6b). On the other hand, cytochrome c staining appeared in a punctate pattern where intact nuclei were found in the control and rIAPP-expressing β-cells, as normally seen in healthy cells.   Importantly, hIAPP-induced cytochrome c release was alleviated in Ad-prohIAPP-transduced β-cells treated with Bax inhibitor (Figure 6b). The proportion of β-cells with cytochrome c release from the mitochondria was significantly higher in hIAPP-expressing INS-1 β-cells than both control cells and those transduced to express non-fibrillogenic rIAPP and was markedly reduced in Ad-hIAPP transduced cells treated with Bax inhibitor (Figure 6c).   36  Furthermore, there was a close correlation between the presence of hIAPP oligomers and release of cytochrome c in INS-1 β-cells and cytochrome c release was evident in the majority of A11-positive cells (Figure 7). Taken together, these findings suggest that hIAPP aggregates can mediate the release of mitochondrial cytochrome c.   To validate our findings in transformed INS-1 β-cells using primary islet β-cells, same experiments were performed in dispersed human islet cells. Similar to what we observed in INS-1 β-cells, human islet β-cells transduced with Ad-prohIAPP formed oligomeric hIAPP aggregates (Figure 8a), which was associated with release of cytochrome c from the mitochondria (Figure 8b).     3.1.2 Formation of hIAPP aggregates in β-cells results in caspase-9 activation   Next, we explored the role of caspase-9, a downstream mediator of cytosolic cytochrome c in the mitochondrial apoptotic pathway, in hIAPP-expressing INS-1 β-cells. Cytochrome c release in Ad-prohIAPP transduced INS-1 β-cells preceded activation of caspase-9 and apoptosis. As seen in Figures 9a and 9b, hIAPP-expressing INS-1 β-cells had a significantly higher proportion of cleaved caspase-9 positive cells as compared to control β-cells. Moreover, hIAPP-expressing β-cells treated with Bax inhibitor had comparable levels of caspase-9 activation with non-transduced control INS-1 β-cells (Figures 9a-b). Similarly, dispersed human islet β-cells transduced with Ad-prohIAPP had a marked elevation in the active form of caspase-9 and Bax inhibitor effectively blocked caspase-9 activation in those cells (Figures 10a-b).  The proportion of cleaved caspase-9 positive primary human islet β-cells transduced with Ad-prorIAPP was similar to non-transduced control β-cells.    37 3.1.3 Blocking Bax or caspase-9 reduces β-cell apoptosis in Ad-prohIAPP transduced β-cells  To examine if blocking the mitochondrial-mediated apoptotic pathway by inhibition of Bax or caspase-9 can reduce β-cell apoptosis in Ad-prohAPP transduced INS-1 β-cells, the proportion of TUNEL-positive (apoptotic) β-cells was quantified.  As shown in Figures 11a-b, Ad-prohIAPP transduction in INS-1 β-cells aggravated apoptosis.  Blocking the activation of either Bax or caspase-9, two crucial mediators of the mitochondrial apoptotic pathway, alleviated amyloid-induced β-cell apoptosis in Ad-prohIAPP transduced INS-1 β-cells. Similarly, in Ad-prohIAPP-transduced dispersed human islet cells, inhibition of Bax or caspase-9 markedly reduced hIAPP-induced β-cell apoptosis (Figures 12a-b).             38    Figure 6. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced INS-1 β-cells was associated with mitochondrial cytochrome c release. (a) Immunolabeling of INS-1 β-cells for insulin (green) and hIAPP oligomers (A11; red) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with or without Bax inhibitor (BI). (b) Immunolabeling of INS-1 β-cells for cytochrome c (green) and nuclear staining (Hoechst; blue). Scale bar=25 µm.	   (c) The percentage of INS-1 β-cells with cytochrome c release. Quantification was performed by manual counting of β-cells with cytochrome c release (verified with ATP synthase β; mitochondrial marker) from a minimum of five microscopic fields each containing 300-400 INS-1 β-cells. Results are expressed as means +/− SEM of three independent studies (N=3). (*p<0.05, one-way ANOVA).  a	  b	  Control Ad-rIAPP Ad-hIAPP Ad-hIAPP+BI02468% cells with cytochrome c release**c	   39    Figure 7. Formation of intracellular hIAPP aggregates co-localized in INS-1 β-cells with mitochondrial cytochrome c release after transduction with Ad-prohIAPP.   Immunolabeling of INS-1 β-cells for cytochrome c (green), hIAPP oligomers (A11; red) and nuclear staining (blue) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with or without Bax inhibitor (BI). Scale bar=25 µm. Micrographs represent three independent studies (N=3).  40                    Figure 8. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced dispersed human islet β-cells was associated with mitochondrial cytochrome c release. (a) Immunolabeling of dispersed human islet cells for insulin (green) and hIAPP oligomers (A11; red) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with or without Bax inhibitor (BI). (b) Immunolabeling of dispersed human islet cells for cytochrome c (green), insulin (red) and nuclear staining (DAPI; blue). Scale bar=25 µm. The micrographs represent three independent studies (N=3) performed on human islet preparations from three donors.  a	  b	   41   Figure 9. Formation of intracellular hIAPP aggregates in Ad-prohIAPP transduced INS-1 β-cells induced caspase-9 activation. (a) Immunolabeling of INS-1 β-cells for insulin (red) and cleaved caspase-9 (green) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with or without Bax inhibitor (BI). (b) The percentage of INS-1 β-cells positive for cleaved caspase-9 was quantified by manual counting of double insulin and cleaved caspase-9 positive cells in a minimum of five microscopic fields each containing 300-400 INS-1 β-cells. Results are expressed as means +/− SEM of three independent studies (N=3). (*p<0.05, one-way ANOVA).  b	  a	  Control Ad-rIAPP Ad-hIAPP Ad-hIAPP+BI01234% Cleaved CASP-9-positive β-cells * * 42                    Figure 10. Intracellular hIAPP aggregates formed in Ad-prohIAPP transduced dispersed human β-cells induced caspase-9 activation. (a) Immunolabeling of dispersed human islet cells for insulin (red) and cleaved caspase-9 (green) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with or without Bax inhibitor (BI). Scale bar=25 µm. (b) The percentage of dispersed human β-cells positive for cleaved caspase-9 was quantified by manual counting of double insulin and cleaved caspase-9 positive cells in a minimum of five microscopic fields each containing 100-150 β-cells from two independent studies (N=2) performed on human islet preparations from two donors.   a	  bControl Ad-rIAPP Ad-hIAPP Ad-hIAPP+BI012345% Cleaved CASP-9-positive β cells 43   Figure 11. Treatment with Bax or caspase-9 inhibitor reduced β-cell apoptosis in Ad-prohIAPP transduced INS-1 β-cells. (a) Immunolabeling of INS-1 β-cells for insulin (green) and TUNEL (red) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with Bax or caspase-9 inhibitor. (b) The percentage of TUNEL-positive INS-1 β-cells was quantified by manual counting of insulin- and TUNEL-positive cells from a minimum of five microscopic fields each containing 300-400 INS-1 β-cells. Results are expressed as means +/− SEM of three independent studies (N=3) and treatments were conducted in duplicates. (*p<0.05, vs control; #p<0.05, vs Ad-hIAPP-transduced; one-way ANOVA).  b	  a	  Control Ad-rIAPP Ad-hIAPP Ad-hIAPP+Bax inh Ad-hIAPP+CASP9 Inh051015TUNEL-positive β-cells(fold increase over control) *# # 44    Figure 12. Treatment with Bax or caspase-9 inhibitors reduced β-cell apoptosis in Ad-prohIAPP transduced dispersed human islet β-cells. (a) Immunolabeling of dispersed human islet cells for insulin (green) and TUNEL (red) after 2 hours of adenoviral transduction (MOI= 10) and 40 hours of incubation with Bax or caspase-9 inhibitor. Scale bar=25 µm. (b) The percentage of dispersed human β-cells positive for TUNEL was quantified by manual counting of insulin- and TUNEL-positive islet β-cells from a minimum of five microscopic fields each containing 100-150 β-cells in two independent studies (N=2) performed on human islet preparations from two donors.   b	  a	  Control Ad-rIAPP Ad-hIAPP Ad-hIAPP+Bax inh Ad-hIAPP+CASP9 Inh051015TUNEL-positive β-cells(fold increase over control)  45 3.2 Blocking the mitochondrial apoptotic pathway protects islets from intracellular hIAPP aggregates   We used islets from two transgenic mouse models, CytcKA/KA and BaxBak DKO mice to examine if blocking the mitochondrial apoptotic pathway can reduce β-cell toxicity induced by intracellular hIAPP aggregates and enhance β-cell survival. As detailed in section 1.4, homozygous cytochrome c mutant (CytcKA/KA) mice have disrupted pro-apoptotic function but normal electron-transport function [177]. Also, BaxBak DKO mice have global Bak and β-cell specific Bax deletion, which results in blocking activation of the intrinsic apoptotic pathway [172]. Freshly isolated islets from sex- and age-matched CytcKA/KA and BaxBak DKO mice and their wild-type littermates were transduced with Ad-prohIAPP and cultured to form intracellular hIAPP aggregates.  Previous studies from our group have shown that adenoviral transduction at the MOI range used for these studies does not have any significant impact on β-cell survival.   3.2.1 Blocking the pro-apoptotic function of cytochrome c markedly reduces hIAPP-mediated caspase-9 activation in CytcKA/KA mouse islets during culture  Transduction with Ad-prohIAPP led to formation of comparable levels of hIAPP aggregates in the wild-type (Cytc+/+) and CytcKA/KA islets during 7-day culture as shown by thioflavin S staining (Figure 13a). Amyloid formation was associated with an increase in the number of active caspase-9 positive β-cells in 7-day cultured wild-type islets as shown in Figures 13a-b. On the contrary, activation of caspase-9 was mostly absent in CytcKA/KA islet β-cells (Figures 13a,b).  Thioflavin S staining was also observed in non β-cells within islets since Ad-prohIAPP does not target β-cells exclusively (Figure 13a). However, very low number of islet non-β-cells with active caspase-9 were present which is in line with previous studies from our  46 group that have shown islet α-cells are more resistant than β-cells to amyloid toxicity and hIAPP [180].  3.2.2 Blocking the pro-apoptotic function of cytochrome c markedly reduces β-cell apoptosis  in Ad-prohIAPP-transduced CytcKA/KA islets during culture  Paraffin-embedded islet sections were immunolabeled for insulin and TUNEL to assess β-cell apoptosis. Pre-culture islets from CytcKA/KA and Cytc+/+ mice had comparable levels of basal β-cell apoptosis but the proportion of apoptotic β-cells markedly increased during 7-day culture. Islets from CytcKA/KA mice had lower number of apoptotic β-cells than wild-type islets after 7-day culture but this difference did not reach the level of significance (Figure 14a, b).   Amyloid formation in Ad-prohIAPP transduced islets from both CytcKA/KA and Cytc+/+ mice resulted in a marked increase in the proportion of apoptotic β-cells as compared to non-transduced islets cultured under the same conditions (Figures 14a, b). Interestingly, the proportion of apoptotic β-cells was markedly lower in Ad-prohIAPP transduced islets from CytcKA/KA mice lacking the pro-apoptotic function of cytochrome c as compared to wild-type Cytc+/+ islets (Figures 14a, b).    3.2.3 Reduced β-cell apoptosis in Ad-prohIAPP transduced CytcKA/KA mouse islets was associated with increased islet β- to α-cell ratio  Islet β- to α-cell ratio was assessed in freshly isolated and cultured Cytc+/+ and CytcKA/KA mouse islets by quantitative immunolabeling for insulin and glucagon. Islet culture resulted in progressive loss of islet cells mainly β-cells. Therefore, β- to α-cell ratio was markedly lower in 7-day cultured islets as compared to pre-culture islets. Following 7-day culture, Ad-prohIAPP  47 transduced CytcKA/KA islets had a higher β- to α-cell ratio as compared to transduced islets from their wild-type littermates which correlated with lower β-cell apoptosis observed in cultured CytcKA/KA islets (Figure 14a,c).                       48         Figure 13. Loss of the pro-apoptotic function of cytochrome c in Ad-prohIAPP transduced mouse islet cells prevented hIAPP-mediated caspase-9 activation in β-cells. (a) Paraffin-embedded sections of cultured islets (non-transduced and transduced with Ad-prohIAPP (MOI=10) for 16 hours) from CytcKA/KA and Cytc+/+ mice were double immunolabeled for insulin (green), cleaved caspase-9 (red) and thioflavin S (Thio S; blue)). (b) Quantification of mouse β-cells positive for cleaved caspase-9 was performed by manual counting of cells positive for insulin and cleaved caspase-9 from each islet in total of at least 20 islets per condition using pooled animals (3 mice per genotype). Results are expressed as means +/− SEM (*p<0.05, one-way ANOVA).  b	  a	  +/+d7 controlKA/KAd7 control+/+d7 +Ad-hKA/KAd7 +Ad-h01234% Cleaved Casp9-positive islet β-cells * 49   Figure 14. Ad-prohIAPP transduced mouse islets lacking the pro-apoptotic function of cytochrome c had reduced level of β-cell apoptosis. (a) Paraffin-embedded sections of cultured islets (non-transduced and transduced with Ad-prohIAPP (MOI=10) for 16 hours) from CytcKA/KA and Cytc+/+ mice were double immunolabeled for insulin (green)/TUNEL (red) and insulin (red)/glucagon (green). (b) Quantification of the proportion of islet β-cells positive for TUNEL and (c) β- to α-cell ratio was performed by manual counting of cells with insulin and TUNEL staining or insulin- and glucagon- positive cells from each islet in a total of about 50 islets per condition using pooled animals (3 mice per genotype). Results are expressed as means +/− SEM (*p<0.05, one-way ANOVA).  b	  c	  a	  +/+ KA/KA +/+ KA/KA +/+ KA/KA02468Islet β- to α-cell ratio *d7	controld0 d7	+Ad-h+/+ KA/KA +/+ KA/KA +/+ KA/KA0246810TUNEL-positive β-cells (%) d7	controld0 d7	+Ad-h* 50 3.2.4 Loss of Bax and Bak blocks hIAPP-induced caspase-9 activation in islet β-cells during culture   Next, as an independent approach, we used islets from mice lacking Bax and Bak, to examine the role of mitochondrial apoptotic pathway in mediating β-cell death induced by intracellular hIAPP aggregates formed in Ad-prohIAPP-transduced islets. After 7 days, Ad-prohIAPP-transduced islets from both BaxBak wild-type and BaxBak DKO mice formed comparable amyloid levels as detected by thioflavin S (Figure 15a). Amyloid (thioflavin S)-positive islets showed significantly greater activation of caspase-9 in BaxBak wild-type islets after transduction with Ad-prohIAPP when compared with BaxBak DKO islets (shown in Figure 15a, b). A low number of active caspase-9 positive β-cells were also found in BaxBak wild-type islets without detectable amyloid formation (Figure 15a). The proportion of cleaved caspase-9 positive β-cells was greatly reduced in BaxBak DKO islets despite formation of hIAPP aggregates (Figure 15b).   3.2.5 Loss of Bax and Bak promotes β-cell survival in Ad-prohIAPP-transduced islets during culture  Formation of hIAPP aggregates and increased caspase-9 activation in Ad-prohIAPP transduced BaxBak wild-type islets was associated with significant higher β-cell apoptosis than transduced BaxBak DKO mouse islets expressing hIAPP (Figures 16a, c). Ad-prohIAPP-transduced BaxBak DKO islets had almost comparable number of apoptotic β-cells with non-transduced control islets. Basal cell death in BaxBak DKO islets after 7-day culture was markedly lower than BaxBak wild-type islets (Figures 16a, c).  51 3.2.6 β- to α-cell ratio in Ad-prohIAPP transduced and non-transduced mouse islets with or without Bax and Bak   Similar to Cyto c mouse islet studies in section 3.2.3, islet β- to α-cell ratio was assessed in cultured and freshly isolated islets from transduced and non-transduced wild-type and BaxBak DKO mice with quantitative double insulin and glucagon immunolabeling.  Consistent with our findings in the last section, islet β- to α-cell ratio was lower after 7-day culture as compared to freshly isolated islets. The β- to α-cell ratio was not significantly different between Ad-prohIAPP transduced wild-type and BaxBak DKO mouse islets, although Ad-prohIAPP transduced wild-type islets had somewhat lower β- to α-cell ratio than Ad-prohIAPP transduced BaxBak islets (Figure 16b). This observation may be partially due to the limited number of animals we had available for this study.     .     52  Figure 15. Global Bak and β-cell specific Bax deletion in Ad-prohIAPP transduced mouse islet cells prevented caspase-9 activation mediated by hIAPP aggregates. (a) Paraffin-embedded sections of cultured islets (non-transduced and transduced with Ad-prohIAPP (MOI=10) for 16 hours) from BaxBak DKO mice and their wild-type littermates were double immunolabeled for insulin (green), cleaved caspase-9 (red) and thioflavin S (Thio S; blue)). (b) Quantification of mouse β-cells positive for cleaved caspase-9 was performed by manual counting of cells positive for insulin and cleaved caspase-9 from each islet in a total of at least 20 islets per condition from pooled islets (5 mice per genotype). Results are expressed as means +/− SEM (*p<0.05, one-way ANOVA). b	  a	  WT d7 controlKOd7 controlWT d7 +Ad-hKOd7 +Ad-h01234% Cleaved Casp9-positive islet β-cells * 53     Figure 16. Global Bak and β-cell specific Bax deletion in Ad-prohIAPP transduced mouse islet cells reduced β-cell apoptosis. (a) Paraffin-embedded sections of cultured islets (non-transduced and transduced with Ad-prohIAPP (MOI=10) for 16 hours) from BaxBak DKO and their wild-type littermates were double immunolabeled for insulin (green)/TUNEL (red) and insulin (red)/glucagon (green). (b) The proportion of islet β-cells positive for TUNEL and (c) islet β- to α-cell ratio were quantified by manual counting of insulin positive cells with TUNEL staining or insulin- and glucagon- positive cells from each islet in a total of about 50 islets per condition using pooled islets (5 mice per genotype). Results are expressed as means +/− SEM (*p<0.05, one-way ANOVA).   b	   c	  a	  WT KO WT KO WT KO02468Islet β- to α-cell ratio d7	controld0 d7	+Ad-hWT KO WT KO WT KO0246810TUNEL-positive β-cells (%) d7	controld0 d7	+Ad-h*** 54 3.3 The intrinsic apoptotic pathway may contribute to β-cell apoptosis induced by extracellular hIAPP aggregates  3.3.1 Blocking the pro-apoptotic function of cytochrome c reduces β-cell death in primary islet cells exposed to extracellular hIAPP aggregates  Our research group has recently shown that the Fas-mediated apoptotic pathway plays a key role in mediating β-cell apoptosis by extracellular hIAPP aggregates [159]. As described earlier, activation of the Fas-mediated apoptotic pathway may lead to activation of the mitochondrial apoptotic pathway. Thus, to investigate the potential indirect role of intrinsic apoptotic pathway in β-cell death induced by extracellular hIAPP aggregates, we cultured dispersed islet cells from CytcKA/KA and Cytc+/+ mice with synthetic hIAPP (to form extracellular hIAPP aggregates) or non-fibrillogenic rIAPP (control). As presented in Figures 17a-b, hIAPP-treated CytcKA/KA islet cells had lower number of apoptotic β-cells than Cytc+/+ islet cells cultured in the presence of synthetic hIAPP. The observations from this preliminary study suggest that the mitochondrial apoptotic pathway, in addition to its role in mediating β-cell death induced by intracellular hIAPP aggregates, may also indirectly play a role in β-cell death caused by extracellular hIAPP aggregates.  Further studies with higher number of independent experiments are required to examine the potential indirect role of the cytochrome c apoptotic pathway in extracellular amyloid β-cell toxicity.    55   Figure 17. hIAPP-treated islet cells from CytcKA/KA  mice had reduced number of apoptotic β-cells as compared to Cytc+/+ mouse islet cells. (a) Dispersed islet cells from CytcKA/KA and Cytc+/+ mouse islets were treated with 10 µmol/l hIAPP or rIAPP (as control) for 24 hours and immunolabeled for insulin (green) and TUNEL (red). (b) The proportion of dispersed mouse β-cells positive for TUNEL was quantified by manual counting of insulin positive cells with TUNEL staining from a minimum of five microscopic fields each containing 100-150 β-cells from two independent studies (N=2) performed on pooled islets from animals.    b 01234TUNEL-positive islet beta cells (fold increase over control)        Control         + rIAPP         + hIAPPCytc +/+Cytc KA/KAa  56 Chapter 4: Discussion  4.1 Formation of hIAPP oligomers in Ad-prohIAPP transduced β-cells  Growing evidence suggests that β-cell death is associated with formation of toxic hIAPP oligomeric species that are predominantly intracellular when amyloid deposition is minimal [45, 112, 182]. Oligomers are defined as the intermediates of multiple monomeric hIAPP units during fibril formation that can co-exist with other forms of aggregates [109–111]. Soluble oligomers can disrupt membranes of the secretory pathway [99, 183], which may subsequently lead to Ca2+ influx into the cytoplasm, a pro-apoptotic signal of the intrinsic apoptotic pathway [184–186]. In this thesis project, we tested if intracellular hIAPP aggregates can activate the mitochondrial-dependent apoptotic pathway. To test this hypothesis, we used an adenoviral approach to express fibrillogenic hIAPP in transformed and primary islet β-cells. The MOI of Ad-prohIAPP was optimized to express hIAPP within physiological range in β-cells. Cells were transduced at MOI of 10 to ensure optimal hIAPP expression without any significant adverse effects associated with adenoviral transduction. The presence of hIAPP oligomers was detected by double immunolabeling of cells or paraffin-embedded islet sections for insulin and A11 (an antibody that detects oligomer form of peptides) in Ad-prohIAPP transduced β-cells. We chose immunolabeling approach for detection of hIAPP oligomers in our studies because binding of the antibody is conformation-dependent and hIAPP oligomers can be denatured and therefore unlikely to be accurately detected by Western blot. The oligomer antibody (A11) may not exclusively bind to hIAPP oligomers and has reactivity against amyloid β as well [187] which typically is not present in  β-cells. Therefore, oligomers detected in our experimental models, are mainly hIAPP aggregates. We also used non-transduced or Ad-prorIAPP-transduced cells (which express non-fibrillogenic rIAPP) as controls to detect any potential adverse effects of adenoviral  57 transduction or overexpression of peptides on β-cells, respectively.   We first used dispersed islet cells and transformed INS-1 β-cells to ensure sufficient penetration of peptide inhibitors into β-cells since it may be difficult for synthetic peptides to penetrate the outer membrane of intact islets [109]. Previously, Zhao et al. examined formation of hIAPP oligomers in islet sections from both subjects with or without T2D using the A11 antibody by immunolabeling [188]. They detected large curvilinear hIAPP aggregates (oligomers) mostly confined in islet β-cells of patients with T2D which had similar morphology as described in studies on amyloid β in Alzheimer's disease [189, 190] and transgenic mouse islets overexpressing hIAPP [191]. Similarly, we found that β-cells transduced with Ad-prohIAPP formed hIAPP oligomers in the cytosol of cells with similar morphologic characteristics observed in T2D islet β-cells. The majority of hIAPP oligomers detected by immunolabeling resembled larger, more pronounced shapes rather than small punctate dots and were found mainly in insulin-positive cells.  We also found that addition of Bax inhibitor to cell culture did not interfere with oligomer formation. These studies validated our immunolabeling technique for oligomer-specific immunoreactivity in Ad-prohIAPP transduced β-cells.   4.2 Intracellular hIAPP oligomers induced cytochrome c release    As discussed earlier, cytochrome c release is a crucial hallmark of activation of the intrinsic apoptotic pathway.  We identified release of mitochondrial cytochrome c by diffuse homogeneous immunolabeling of cytochrome c in the cytosol of hIAPP-expressing INS-1 β-cells or dispersed primary human islet β-cells, 42 hours post-transduction, the time point at which β-cell apoptosis was clearly observed. There was a higher number of INS-1 β-cells and human islet β-cells with cytochrome c release present after Ad-prohIAPP transduction, which was alleviated  58 with a Bax inhibitor in both transformed and primary islet β-cells. We observed a close correlation between oligomer formation and diffused cytochrome c immunostaining in the cytosol. The majority of A11-positive cells showed diffuse cytochrome c pattern. The pattern of hIAPP-mediated cytochrome c release was congruent with cytochrome c release from INS-1 β-cells treated with other β-cell apoptotic factors such as elevated glucose [164] and palmitate [167]. Taken together, these findings suggest that intracellular hIAPP oligomers can mediate cytochrome c release in both transformed and primary islet β-cells and that hIAPP-induced cytochrome c release from the mitochondria was blocked by preventing permeabilization of the outer mitochondrial membrane and release of cytochrome c by the Bax inhibitor.   Interestingly, recent studies have demonstrated that cytochrome c release is also a crucial event that connects mitochondrial dysfunction and activation of caspases in amyloid β-induced neuronal apoptosis in Alzheimer’s disease [192–194]. Kim et al. proposed that amyloid β oligomers could directly interact with Bax pores on the mitochondrial membrane to provoke cytochrome c release in vitro [193]. However, the mechanisms underlying the interaction between hIAPP oligomers and the pro-apoptotic members of the Bcl-2 family have yet to be determined. Accordingly, inhibitors of cytochrome c release such as methazolamide and carbonic anhydrase inhibitor have been identified in Huntington’s disease using in vitro assays which may have potential benefits in β-cells that are vulnerable to hIAPP oligomers [195].   4.3  Intracellular hIAPP oligomers induced caspase-9 activation    Typically after cytochrome c release, immediate activation of caspase-9, a key mediator of the intrinsic pathway, can be detected in cells undergoing apoptosis. After formation of the apoptosome, the initiator procaspase-9, is recruited and undergoes proteolysis after clustering to  59 form mature caspase-9, which then promotes activation of the downstream cascade of events in the intrinsic apoptotic pathway [137]. Therefore, we examined if cytochrome c release mediated by formation of intrinsic hIAPP aggregates in Ad-prohIAPP transduced islet β-cells can promote caspase-9 activation. We found that the number of cleaved caspase-9 positive β-cells was significantly higher in Ad-prohIAPP transduced INS-1 β-cells, as compared to non-transduced control cells. A similar pattern was observed in Ad-prohIAPP transduced dispersed primary human islet β-cells. Furthermore, hIAPP-mediated caspase-9 activation was prevented by treatment with the Bax inhibitor.  These studies suggest that intracellular hIAPP aggregates can promote cytochrome c release leading to activation of caspase-9.   In a similar manner, studies performed on Alzheimer's disease have demonstrated that formation of amyloid β oligomers leads to caspase-9 activation and subsequent destruction of neural cells [196, 197]. Therefore, it appears that hIAPP and amyloid β may activate the same apoptotic signaling pathways. In summary, our studies suggest that intracellular hIAPP aggregates can act as a cell death signal to trigger the mitochondrial apoptotic pathway via activation of caspase-9. However, we cannot exclude the potential indirect activation of this pathway via activated caspase-8 due to Fas upregulation and activation of the Fas-mediated apoptotic pathway initiated by formation of extracellular hIAPP aggregates.    4.4 Bax and caspase-9 inhibition protects islet β-cells from apoptosis mediated by intracellular hIAPP aggregates  Our studies demonstrated that key components of the intrinsic apoptotic pathway are involved in oligomeric hIAPP-mediated apoptotic β-cell death. In our complementary studies, we applied irreversible cell permeable inhibitors of Bax and caspase-9 to examine if inhibition of  60 the mitochondrial apoptotic pathway can prevent β-cell death induced by hIAPP aggregates in Ad-prohIAPP transduced β-cells. Treatment with Bax or caspase-9 inhibitor protected β-cells from intracellular hIAPP aggregates manifested as a marked reduction in the number of β-cells undergoing apoptosis as compared to Ad-prohIAPP transduced cells without inhibitor treatment. This suggests that the intrinsic apoptotic pathway plays a significant role in mediating β-cell death by formation of intracellular hIAPP oligomers. Since small oligomeric hIAPP is hypothesized to be the most toxic conformation to β-cells [99], targeting the specific apoptotic pathway initiated  by the hIAPP oligomers may provide a new strategy to protect β-cells from amyloid toxicity.  4.5 The role of pro-apoptotic function of cytochrome c in amyloid-mediated cell death  In our CytcKA/KA mouse model, caspase-9 activation was blocked due to the mutation of cytochrome c. The intrinsic apoptotic pathway is known to be involved in β-cell apoptosis upon various types of cellular stress stimuli such as hyperglycemia, hyperlipidemia, and proinflammatory cytokines [147, 160, 164, 166, 167, 198]. Accordingly, CytcKA/KA mice were shown to be resistant to apoptotic stimuli such as ultraviolet irradiation, serum withdrawal, and STS [176].    We found a markedly lower number of apoptotic β-cells in Ad-prohIAPP transduced CytcKA/KA mouse islets that formed hIAPP aggregates as compared to islets from their wild-type littermates following 7-day culture. This correlated with an increase in islet β- to α-cell ratio during culture. Interestingly, we further observed that some β-cells positive for cleaved caspase-9 staining were not in proximity to thioflavin S- stained islet areas. This observation is in line with our findings in single β-cells showed that hIAPP oligomers, which cannot be detected with  61 thioflavin S, likely contribute to this process.   Our studies further suggest that blocking the pro-apoptotic function of cytochrome c may provide a new approach to protect islet β-cells in conditions associated with islet amyloid formation such as pre-transplant islet culture. Importantly, Choi et al. [178] has demonstrated that blocking the pro-apoptotic function of cytochrome c does not affect glucose homeostasis and β-cell mass in CytcKA/KA mice under basal conditions. Therefore, it seems feasible to block the pro-apoptotic function of cytochrome c during pre-transplant islet culture without any significant adverse effects on islet function.    4.6 Role of Bax and Bak in amyloid-mediated β-cell death  To further investigate the role of intrinsic apoptotic signaling pathway in amyloid associated β-cell toxicity, we used BaxBak DKO mice as another experimental model in which the intrinsic apoptotic pathway is blocked. Our data suggests that, similar to what we observed in CytcKA/KA mice, blocking the intrinsic apoptotic pathway in BaxBak DKO mouse islets protected β-cells from intracellular hIAPP aggregates and significantly reduced both hIAPP-induced caspase-9 activation and β-cell death. These studies demonstrate that blocking the intrinsic pathway by genetic manipulation of pro-apoptotic Bax and Bak leads to enhanced β-cell survival in the presence of hIAPP aggregates. Intriguingly, BaxBak DKO islets despite formation of intracellular hIAPP aggregates had much lower β-cell apoptosis which was almost comparable to non-transduced islets from these mice. BaxBak DKO mice were also reported to be protected from STS and gluco-lipotoxicity [173]. Therefore, blocking Bax and Bak can protect islet β-cells from both glucose toxicity and amyloid toxicity.    Interestingly, β-cell apoptosis was even lower in Ad-prohIAPP transduced BaxBak DKO  62 mouse islets than Ad-prohIAPP transduced CytcKA/KA mouse islets shown earlier. Normally, Bax and Bak can compensate for each other. Therefore, loss of both Bax and Bak in β-cells is expected to completely prevent activation of the mitochondrial apoptotic pathway without a compensatory mechanism. Accordingly, it has been shown in mouse embryonic fibroblasts that cells with either Bax or Bak ablation are still sensitive to apoptotic cell death mediated by BH3-only proteins [199, 200] whereas mouse embryonic fibroblasts with BaxBak DKO are impervious to apoptotic stimuli [201].   Finally, in our studies, we did not see any significant difference between β- to α-cell ratio in islets from BaxBak DKO mice and wild-type islets with or without hIAPP expression, which might be potentially due to low number of mice in these studies. Also, it should be noted that changes in β- to α-cell ratio may not be significant in islets when the rate of β-cell apoptosis is not drastically increased.    4.7 Potential crosstalk between the extrinsic and intrinsic apoptotic pathways in amyloid-induced β-cell toxicity   Extracellular hIAPP aggregates upregulate Fas expression via IL-1β signaling in human islets, triggering caspase-8 activation and the extrinsic apoptotic pathway in β-cells [135, 136, 159]. Activation of caspase-8 may in turn promote the mitochondrial apoptotic pathway. As shown earlier, islet β-cells from CytcKA/KA mice cultured with synthetic hIAPP had a lower β-cell apoptosis than Cytc+/+ islet cells cultured at the same condition. This suggests that the pro-apoptotic function of cytochrome c, in addition to its role in mediating β-cell apoptosis by intracellular hIAPP aggregates, may also play a role in β-cell toxicity mediated by extracellular hIAPP aggregates. In line with this notion, it has been suggested that type II cells are more  63 resistant to Fas-induced apoptosis unless the caspase cascade is amplified via cleavage of Bid by caspase-8 and subsequent activation of the intrinsic pathway of cell death [202]. Moreover, a study from Li and colleagues has shown that hIAPP aggregates stimulate cleavage of Bid by activated caspase-8 in INS-1 β-cells [121]. Taken together, these findings suggest that formation of islet amyloid and islet inflammation promote production of IL-1β [130–134], leading to Fas upregulation and activation of the Fas-mediated apoptotic pathway which then indirectly activates the mitochondrial apoptotic program via active caspase-8. Thus, extracellular hIAPP aggregates may activate the intrinsic apoptotic pathway via the extrinsic apoptotic pathway and inhibition of both pathways yields maximal protection on β-cell survival. Further mechanistic experiments are required to test the role of cytochrome c in extracellular amyloid toxicity.        64 Chapter 5: Conclusion 5.1 Conclusions   The objective of our study was to identify the role of the intrinsic mitochondrial apoptotic pathway in amyloid-mediated β-cell death.  We used INS-1 β-cells and dispersed human islet β-cells transduced with Ad-prohIAPP as two experimental models of intracellular hIAPP aggregation. Our data suggests that small hIAPP aggregates (oligomers) trigger the mitochondrial-dependent apoptotic pathway in both transformed and primary islet β-cells as detected by key events such as release of cytochrome c, activation of caspase-9 and β-cell apoptosis, all of which are prevented by peptide inhibitors to halt activation of the intrinsic apoptotic pathway.   Next, We further demonstrated that Ad-prohIAPP-transduced mouse islets from CytcKA/KA and BaxBak DKO mice, two animal models in which the intrinsic apoptotic pathway is blocked, had better β-cell survival than islets from their wild-type littermates, despite forming comparable hIAPP aggregates. Therefore, it appears that blocking the pro-apoptotic function of cytochrome c or deletion of Bax and Bak both can protect β-cells from hIAPP-induced cytotoxicity.  Finally, lower β-cell death in islet cells from CytcKA/KA than Cytc+/+ mice following treatment with synthetic hIAPP suggests that potential activation of the intrinsic apoptotic pathway may also play a role in β-cell death mediated by extracellular hIAPP aggregates by amplifying the Fas-mediated apoptotic signaling. Two limitations of this project include the small sample size (transgenic mouse models) available to perform experiments and lack of in vivo studies.   Overall, the proof of concept studies presented in this thesis project have enhanced our knowledge on the pathological mechanisms of T2D in the context of islet amyloid formation.  65 Therefore, these findings may have potential therapeutic application in T2D. Furthermore, current work may provide new insight into development of new strategies to protect islet grafts and thereby improve outcome of clinical islet transplantation.   5.2 Future directions  Accumulating evidence suggests that T2D is a protein misfolding disease in which amyloid formation occurs in the majority of patients [103]. In the current study, we have examined the role of intrinsic (mitochondrial) apoptotic pathway in amyloid toxicity using in vitro and ex vivo experimental models.  The next steps for this project include: 1.  To examine the activation of intrinsic apoptotic program in vivo in a transgenic mouse model that expresses hIAPP. This can be achieved by crossbreeding homozygous hIAPP transgenic mice such as FVB-Tg(IAPP)6Jdm/Tg(IAPP)6Jdm [182] with CytcKA/KA  or BaxBak DKO mice to generate a mouse model with β-cell specific  hIAPP expression similar to that found  in human subjects with T2D but lacking activation of the intrinsic apoptotic pathway.  2. The relationship between the extrinsic and intrinsic apoptotic pathways in β-cell death mediated by hIAPP aggregates has not been fully studied. It is important to distinguish the individual and combined roles of the two cell death pathways and unravel the redundancy of the mitochondrial-dependent pathway in extracellular and intracellular hIAPP-mediated cytotoxicity.  3. It has been reported that IL-1β plays a key role in amyloid-induced β-cell deterioration [130–134] and that IL-1β expression can trigger the intrinsic apoptotic pathway under diabetogenic milieu [160].  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