@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Pathology and Laboratory Medicine, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Courtade, Jaques"@en ; dcterms:issued "2016-09-07T02:01:58"@en, "2016"@en ; vivo:relatedDegree "Doctor of Philosophy - PhD"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "Soaring rates of diabetes highlight the importance of controlling this global epidemic, with an estimated 415 million people thought to be living with diabetes in 2015. The defining characteristic of diabetes is elevated fasting blood glucose levels, or hyperglycemia, which if not controlled promotes long-term complications such as neuropathy, kidney failure and damage to blood vessels. Glucose homeostasis is primarily controlled by pancreatic islets, cell clusters that mediate the endocrine functions of the pancreas. To manage circulating glucose concentrations, islet beta cells synthesize proinsulin, a peptide that undergoes proteolytic cleavage to become bioactive. In this dissertation, I examine the processing of the beta-cell protein pro-islet amyloid polypeptide (proIAPP), a propeptide that is cleaved similarly to proinsulin to form the aggregation-prone mature IAPP molecule, and determine whether impairments in the processing of this protein accelerate diabetes development. First, I generated a novel immunoassay to quantify the concentration of IAPP prohormone precursors for the first time in human circulation. Following rigorous validation of this ELISA, I demonstrated that elevated levels of the NH₂-proIAPP₁-₄₈ intermediate form are characteristic of type 1 diabetic recipients of islet transplants, and children with impaired glucose tolerance. Furthermore, I elucidated that this effect was not true for patients with established type 2 diabetes, implicating the peptide intermediate as a biomarker of diabetes onset but not a marker of the diseased state. Following this, I generated a rodent transplant model in which the loss of prohormone convertase 2 (PC2), essential for proIAPP processing in rodents, led to early islet transplant failure. Using a beta cell-specific PC2 null mouse that we generated, I also demonstrated that the loss of this enzyme in beta cells promotes earlier development of diabetes. Lastly, I was successful in establishing an in vitro islet culture model in which the overexpression of a non-cleavable proIAPP substrate leads to increased islet cell death. Altogether, the work in this dissertation highlights the importance of precise prohormone processing in the pancreatic islet, and demonstrates a role for proIAPP processing intermediates as biomarkers of diabetes and contributors to beta-cell dysfunction."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/59064?expand=metadata"@en ; skos:note "IMPAIRED PRO-ISLET AMYLOID POLYPEPTIDE PROCESSING PROMOTES BETA-CELL DYSFUNCTION IN DIABETES AND ISLET TRANSPLANTS by Jaques Courtade B.Sc., The University of British Columbia, 2010 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2016 © Jaques Courtade, 2016 ii Abstract Soaring rates of diabetes worldwide have brought to light the importance of controlling this global epidemic, with an estimated 415 million people thought to be living with diabetes in 2015 and a projected increase to as many as 642 million people living with diabetes in 25 years. The defining characteristic of diabetes is elevated fasting blood glucose levels, or hyperglycemia, which if not controlled promotes long-term complications such as neuropathy, kidney failure and damage to blood vessels. Glucose homeostasis is primarily controlled by pancreatic islets, cell clusters that mediate the endocrine functions of the pancreas. To manage circulating glucose concentrations, islet beta cells synthesize proinsulin, a peptide that undergoes proteolytic cleavage to become bioactive. In this dissertation, I examine the processing of the beta-cell protein pro-islet amyloid polypeptide (proIAPP), a propeptide that is cleaved similarly to proinsulin to form the aggregation-prone mature IAPP molecule, and determine whether impairments in the processing of this protein accelerate diabetes development. First, I generated a novel immunoassay to quantify the concentration of IAPP prohormone precursors for the first time in human circulation. Following rigorous validation of this ELISA, I demonstrated that elevated levels of the NH2-proIAPP1-48 intermediate form are characteristic of type 1 diabetic recipients of islet transplants, and children with impaired glucose tolerance. Furthermore, I elucidated that this effect was not true for patients with established type 2 diabetes, implicating the peptide intermediate as a biomarker of diabetes onset but not a marker of the diseased state. Following this, I generated a rodent transplant model in which the loss of prohormone convertase 2 (PC2), essential for proIAPP processing in rodents, led to early islet transplant failure. Using a beta cell-specific PC2 null mouse that we generated, I also demonstrated that the loss of this iii enzyme in beta cells promotes earlier development of diabetes. Lastly, I was successful in establishing an in vitro islet culture model in which the overexpression of a non-cleavable proIAPP substrate leads to increased islet cell death. Altogether, the work in this dissertation highlights the importance of precise prohormone processing in the pancreatic islet, and demonstrates a role for proIAPP processing intermediates as biomarkers of diabetes and contributors to beta-cell dysfunction. iv Preface We obtained ethics approval from the UBC C&W Research Ethics Board (Ethics Certificates #H06-03112, #H03-70453) for the collection and use of human samples. Chapter 1 in this dissertation is a review of many of the factors that promote diabetes development. This chapter includes a specific focus on the importance of prohormone processing in diabetes and summarizes many of the known biomarkers that predict early diabetes onset. Chapter 2 is currently under revisions for publication in The Journal of Clinical Endocrinology & Metabolism. The initial configuration of the described immunoassay was conceptualized by Dr. Agnieszka Klimek and myself. I designed all of the experiments for this chapter with scientific input from Nirja Patel (ALPCO Diagnostics) and Dr. Phoebe Lu. I conducted the majority of the experiments described in this chapter, with assistance from summer students and co-op students. I also wrote the manuscript and designed all of the figures. Chapter 3 is currently under revisions for publication in Diabetologia. I designed all of the experiments described and maintained all of the mouse colonies. Surgical work was performed by Dr. Derek Dai and Dr. Galina Soukhatcheva. Immunostaining and quantification of tissues were performed with assistance from Evan Wang and Paul Yen. I also wrote the manuscript and designed all of the figures. v Chapter 4 is complementary to Chapter 3, and involves similar analyses in several rodent models of diabetes. I performed all of the animal experiments with surgical and technical assistance from Dr. Derek Dai, Dr. Galina Soukhatcheva and Evan Wang. I worked together with Dr. Paul Orban in generating adenoviruses, adeno-associated viruses and conditional knockout animals. In vitro analyses of culture islets was performed by Paul Yen and myself. In Chapters 2-4, I will use “we” to reflect contributions from co-authors. vi Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Diabetes mellitus ............................................................................................................. 1 1.1.1 Prevalence of type 2 diabetes and its impact .............................................................. 1 1.1.2 Diagnosis and types of diabetes .................................................................................. 3 1.2 Treatments for diabetes ................................................................................................... 4 1.2.1 Metformin ................................................................................................................... 5 1.2.2 Sulfonylureas .............................................................................................................. 6 1.2.3 DPP-IV inhibitors ....................................................................................................... 7 1.2.4 Islet transplantation ..................................................................................................... 9 1.3 Regulation of blood glucose homeostasis ..................................................................... 12 1.3.1 Hormone action ......................................................................................................... 12 1.3.2 Enzymatic processing of prohormones ..................................................................... 14 1.4 Mediators of beta-cell dysfunction ............................................................................... 18 vii 1.4.1 Insulin resistance ....................................................................................................... 18 1.4.2 Glucolipotoxicity ...................................................................................................... 19 1.4.3 Inflammation ............................................................................................................. 20 1.4.4 Impaired proinsulin processing ................................................................................. 21 1.4.5 Islet amyloid formation ............................................................................................. 23 1.4.6 Impaired proIAPP processing ................................................................................... 24 1.5 Biomarkers of diabetes ................................................................................................. 26 1.5.1 Glycosylated hemoglobin A1C ................................................................................. 26 1.5.2 Adiponectin ............................................................................................................... 27 1.5.3 Metabolites ................................................................................................................ 27 1.5.4 Micro RNAs .............................................................................................................. 28 1.5.5 Proinsulin .................................................................................................................. 30 1.6 Summary ....................................................................................................................... 32 Chapter 2: NH2-terminal pro-islet amyloid polypeptide (1-48) is a biomarker of beta-cell dysfunction....................................................................................................................................34 2.1 Introduction ................................................................................................................... 34 2.2 Materials and methods .................................................................................................. 36 2.2.1 Immunoassay reagents .............................................................................................. 36 2.2.2 Animals ..................................................................................................................... 36 2.2.3 Islet isolation ............................................................................................................. 36 2.2.4 Immunostaining and imaging ................................................................................... 37 2.2.5 Preparation of synthetic peptides and labeling of antibodies .................................... 37 2.2.6 Immunoassay protocol .............................................................................................. 38 viii 2.2.7 Plasma collection ...................................................................................................... 38 2.2.8 DPP-IV degradation assay ........................................................................................ 40 2.2.9 Statistical analyses .................................................................................................... 40 2.3 Results ........................................................................................................................... 41 2.3.1 Characterization of proIAPP-specific antibodies in mouse pancreatic islets and human islet grafts .................................................................................................................. 41 2.3.2 Development and cross-reactivity analysis of a human NH2-proIAPP1-48-specific ELISA……. .......................................................................................................................... 43 2.3.3 Determination of NH2-proIAPP1-48 concentrations in plasma from healthy individuals ............................................................................................................................. 45 2.3.4 Children with T1D have an elevated ratio of NH2-proIAPP1-48 to total IAPP.......... 47 2.3.5 NH2-proIAPP1-48 levels are elevated in children with impaired glucose tolerance .. 49 2.3.6 NH2-proIAPP1-48 concentrations and ratios of NH2-proIAPP1-48 to total IAPP species are elevated in recipients of human islet transplants but not in patients with T2D .............. 51 2.4 Discussion ..................................................................................................................... 53 Chapter 3: Loss of prohormone convertase 2 promotes beta-cell dysfunction in a rodent transplant model of human pro-islet amyloid polypeptide expression ...................................57 3.1 Introduction ................................................................................................................... 57 3.2 Materials and methods .................................................................................................. 58 3.2.1 Mice .......................................................................................................................... 58 3.2.2 Islet isolation and transplantation ............................................................................. 59 3.2.3 Blood glucose monitoring ......................................................................................... 59 3.2.4 Glucose and insulin tolerance tests ........................................................................... 60 ix 3.2.5 Plasma proinsulin, human proIAPP and IAPP measurements.................................. 60 3.2.6 Immunofluorescent staining...................................................................................... 60 3.2.7 Quantification of islet cell areas and islet cell death ................................................. 61 3.2.8 RNA extraction of isolated islets and islet grafts...................................................... 62 3.2.9 Statistical analyses .................................................................................................... 63 3.3 Results ........................................................................................................................... 63 3.3.1 Lack of PC2 promotes mild hypoglycemia and improved glucose tolerance .......... 63 3.3.2 hproIAPPTg/0PC2-/- mice have abnormal islet morphology and reduced amyloid formation ............................................................................................................................... 65 3.3.3 Loss of PC2 and human proIAPP overexpression lead to rapid failure of islet transplants ............................................................................................................................. 68 3.3.4 Elevated proinsulin and human proIAPP levels lead to early islet graft failure ....... 72 3.3.5 Altered gene expression in human proIAPPTg/0PC2-/- islet grafts ............................. 74 3.4 Discussion ..................................................................................................................... 76 Chapter 4: Loss of prohormone convertase 2 promotes beta-cell dysfunction in a rodent type 2 diabetic model of human pro-islet amyloid polypeptide overexpression ....................79 4.1 Introduction ................................................................................................................... 79 4.2 Materials and methods .................................................................................................. 80 4.2.1 Mice .......................................................................................................................... 80 4.2.2 Development of Pcsk2-floxed animals ..................................................................... 81 4.2.3 Generation of adeno-associated viral (AAV) vectors ............................................... 82 4.2.4 Generation of adenoviral vectors .............................................................................. 83 4.2.5 Islet isolation and adenoviral infection ..................................................................... 84 x 4.2.6 Pancreatic duct injection ........................................................................................... 84 4.2.7 Blood glucose monitoring ......................................................................................... 84 4.2.8 Glucose and insulin tolerance tests ........................................................................... 85 4.2.9 Plasma human NH2-proIAPP1-48 and human mature IAPP levels ............................ 85 4.2.10 Western blot for proIAPP and proIAPP-derived species ...................................... 85 4.2.11 Immunofluorescent staining.................................................................................. 86 4.2.12 Determination of alpha to beta cell ratios and %TUNEL-positive islet cells ....... 87 4.2.13 Statistical analyses ................................................................................................ 87 4.3 Results ........................................................................................................................... 87 4.3.1 Alpha cell rescue of PC2 expression normalizes glycemia and islet architecture in PC2 null animals ................................................................................................................... 87 4.3.2 Restoration of PC2 in alpha cells of hproIAPPTg/0PC2-/- animals does not lead to early diabetes development ................................................................................................... 91 4.3.3 Generation of a mouse model with specific deletion of PC2 in pancreatic beta cells…. .................................................................................................................................. 93 4.3.4 Short-term loss of PC2 expression in human proIAPP-expressing animals is not associated with early beta-cell dysfunction .......................................................................... 95 4.3.5 Tissue-specific deletion of PC2 in human proIAPP-expressing PdxCreER mice .... 97 4.3.6 Loss of PC2 in beta cells of human proIAPP-expressing animals leads to early diabetes development ............................................................................................................ 99 4.3.7 Generation of an uncleavable NH2-proIAPP1-48 substrate ...................................... 101 4.3.8 Impaired processing of human proIAPP in vitro promotes islet cell death ............ 103 4.4 Discussion ................................................................................................................... 106 xi Chapter 5: Conclusion ...............................................................................................................110 Bibliography ...............................................................................................................................119 xii List of Tables Table 2.1 Cross-reactivity of IAPP-targeted and NH2-proIAPP1-48-targeted ELISAs with proIAPP-derived synthetic peptides. ............................................................................................ 46 Table 2.2 NH2-proIAPP1-48-specific ELISA limit of detection as determined with three independent operators over two days. ........................................................................................... 46 Table 2.3 Determined concentrations of low, medium and high NH2-proIAPP1-48 peptide spikes by three independent operators over two days. ............................................................................. 46 Table 2.4 Inter-assay CV and intra-assay CV values for the NH2-proIAPP1-48-specific ELISA as determined with three independent operators over two days. ....................................................... 46 Table 2.5 Linearity analysis for NH2-proIAPP1-48 detection in human plasma. ........................... 47 Table 2.6 Spike and recovery analysis in healthy human plasma for the NH2-proIAPP1-48-specific ELISA in the presence of DPP-IV inhibitor. ................................................................................ 47 Table 2.7 Clinical characteristics of healthy children, and children with impaired glucose tolerance, T2D and T1D. .............................................................................................................. 48 Table 2.8 Clinical characteristics of healthy individuals, T1D recipients of islet transplants and T2D adults. .................................................................................................................................... 51 Table 3.1 Primers used for RT-qPCR ........................................................................................... 62 Table 4.1 Determination of adenoviral titres by GFP fluorescence and qPCR ............................ 83 xiii List of Figures Figure 1.1 Amino acid sequence variation between mouse and human mature IAPP. ................ 11 Figure 1.2 Processing of proinsulin to insulin and C-peptide. ...................................................... 15 Figure 1.3 Processing of proIAPP1-67 to mature IAPP in rodent pancreatic beta cells. ................ 17 Figure 2.1 Identification of IAPP precursors in mouse pancreatic islets and islet grafts. ............ 42 Figure 2.2. Development of an immunoassay specific for human NH2-proIAPP. ....................... 44 Figure 2.3. The ratio of NH2-proIAPP1-48 to total IAPP is elevated in T1D................................. 48 Figure 2.4. NH2-proIAPP1-48 levels are elevated in impaired glucose tolerance. ......................... 50 Figure 2.5. NH2-proIAPP1-48 and ratio of NH2-proIAPP1-48 to total IAPP are elevated in T1D recipients of islet transplants but not T2D. ................................................................................... 52 Figure 3.1. Hypoglycemia and improved glucose tolerance in PC2 deficient mice. .................... 64 Figure 3.2. Islet histology in mice expressing human proIAPP and lacking PC2. ....................... 66 Figure 3.3. Early islet graft failure in recipients of transplanted islets expressing human proIAPP and lacking PC2. ........................................................................................................................... 70 Figure 3.4. Elevated proinsulin and human proIAPP levels in recipients of transplanted islets expressing human proIAPP and lacking PC2. .............................................................................. 73 Figure 3.5. Gene expression in transplanted islets expressing human proIAPP and lacking PC2........................................................................................................................................................ 75 Figure 4.1. Rescue of alpha-cell PC2 expression in global PC2 null mice restores normoglycemia and islet architecture. .................................................................................................................... 89 Figure 4.2. Loss of beta-cell PC2 is not associated with early diabetes development in a rodent model of human proIAPP overexpression. ................................................................................... 92 xiv Figure 4.3. Generation of PdxCreER/Pcsk2 conditional knockout mice...................................... 94 Figure 4.4. Transient loss of PC2 in mouse pancreatic beta cells overexpressing human proIAPP does not further compromise beta-cell function. .......................................................................... 96 Figure 4.5. Generation of a Cre-inducible, human proIAPP-expressing Pcsk2 conditional knockout mouse. ........................................................................................................................... 98 Figure 4.6. Permanent loss of PC2 in mouse pancreatic beta cells expressing human proIAPP lead to compromised beta-cell function. ..................................................................................... 100 Figure 4.7. Mutation of KRK to KHK in the human proIAPP amino acid sequence leads to impaired processing of human NH2-proIAPP1-48. ....................................................................... 102 Figure 4.8. Impaired cleavage at the human proIAPP NH2-terminal end promotes islet cell death but not changes in expression of processing genes and ER stress genes. ................................... 104 xv List of Abbreviations AAV Adeno-associated virus CMV Cytomegalovirus CPE Carboxypeptidase E Cre Causes recombination CreER Causes recombination estrogen receptor CV Coefficient of variation DPP-IV Dipeptidyl peptidase IV FRT Flippase recognition target Flp Flippase GAPDH Glyceraldehyde phosphate dehydrogenase GFP Green fluorescent protein GIP Glucose-dependent insulinotropic peptide GLP-1 Glucagon-like peptide 1 GLUT Glucose transporter GRPP Glucagon-related polypeptide GSIS Glucose-stimulated insulin secretion HbA1c Hemoglobin A1c IAPP Islet amyloid polypeptide IFG Impaired fasting glucose IGT Impaired glucose tolerance IPGTT Intraperitoneal glucose tolerance test xvi ITT Insulin tolerance test LoxP Locus of crossing (x) over, P1 NOD Non-obese diabetic PAM Peptidylglycine alpha-amidating monooxygenase PC2 Prohormone convertase 2 PC1/3 Prohormone convertase 1/3 Pdx1 Pancreatic and duodenal homeobox 1 ProIAPP Pro-islet amyloid polypeptide RGP Rat glucagon promoter RIP Rat insulin promoter qPCR Quantitative real-time polymerase chain reaction T1D Type 1 diabetes T2D Type 2 diabetes xvii Acknowledgements I would like to thank everyone that has helped me throughout my PhD studies. I feel lucky to have you all in my life and would never have gotten this far without your continued support. Firstly, I would like to thank my supervisor, Dr. Bruce Verchere, for accepting me as a co-op student (with very limited lab experience) back in 2008. You have always allowed me to pursue my own ideas and always reminded me to think about the “big picture”. I am grateful to have worked in your lab and to have you as a mentor. Also, a sincere thank you to Dr. Paul Orban who taught me everything I ever needed to know about molecular biology and encouraged me to consider every single possibility, no matter how improbable. Thank you also to my committee members, Dr. Angela Devlin, Dr. Francis Lynn, Dr. Cheryl Wellington and Dr. Dina Panagiotopoulos. Your continued support at my committee meetings taught me to put my work in perspective and helped me to stay on track. Also, thank you to Dr. Pritchard for not only being supportive during my PhD, but also for encouraging me to think about the world following graduation. Finally, thank you to the funding agencies, CIHR and JDRF, for supporting the majority of the research presented in this dissertation. I would also like to thank all of the Verchere lab members, past and present, for your constant support throughout the years. I have learned so much from all of you, especially Derek and Galina for teaching me even the most difficult animal surgeries, and Agnes for providing me with a strong fundamental understanding of how an ELISA works. Thank you all for giving me advice and keeping me sane when I struggled with experiments. xviii Finally, I would like to thank my parents and grandma for all of their support and encouragement from elementary school to PhD. You provided me the opportunity to pursue any career of my choosing and gave me unwavering support in all of my decisions. Also, I would like to thank my sister Francesca for all her advice throughout the years and for always being there. Lastly, a big thank you to my wife, Phoebe, for listening to my endless complaints about experiments and helping me edit my thesis. The most memorable moment of my PhD was meeting you, and I look forward to enjoying a long future together. xix Dedication I dedicate this thesis to my parents for always being supportive and for encouraging me to pursue my dreams. 1 Chapter 1: Introduction 1.1 Diabetes mellitus 1.1.1 Prevalence of type 2 diabetes and its impact The burden of diabetes mellitus is shared worldwide and has significant impact on both economies and public health. Complications from diabetes accounted for close to 4.6 million deaths worldwide in 2011 [1], highlighting the importance of reducing disease incidence. According to the Global Status Report on noncommunicable diseases in 2014, obesity was marked as one of the biggest risk factors for diabetes development, present in 11% of men and in 15% of women aged 18 years or older [1]. In that same year, the prevalence of diabetes was estimated to be close to 9% globally [1], a significant increase from 8.3% (approximately 382 million people) calculated in 2013 [2]. It is currently thought that the number of individuals affected will rise to 592 million people in 20 years [2]. Close to 3.4 million Canadians were living with diabetes in 2015 [3]. This number equates to about 9.3% of the total population [3]. Most concerning however, is that 5.7 million individuals over the age of 20 are thought have prediabetes [3]. Furthermore, it is estimated that by 2025 these percentages will rise to 12.1% for diabetes and 23.2% for prediabetes [3]. Left untreated or without intervention, prediabetes promotes the development of diabetes and many of the complications associated with the disease. In addition to complications, the financial out-of-pocket costs of diabetes medications equate to 3% of the average Canadian income [4]. As many as 57% of Canadians reported that they were not able to meet the financial cost of diabetes treatments, linking income status as a determinant of disease severity [4]. The statistics above 2 illustrate the need for improved diagnostic tools and earlier intervention to prevent the transition from prediabetes to diabetes, which may indirectly relieve some of the costs associated with diabetes treatments. Individuals afflicted with diabetes are at increased risk for kidney and cardiovascular problems. In 2008-2009, diabetic patients were reported to have a 3-fold higher chance of being hospitalized for heart disease, and a 12-fold higher chance of being hospitalized for symptoms of renal disease [5]. Close to 50% of patients with diabetes display signs of kidney damage throughout their lifetime and often go on to develop renal failure [6], requiring either dialysis or a kidney transplant. Another long-term complication of diabetes is neuropathy or nerve damage, a condition common to 60-70% of patients [7]. Peripheral neuropathy as a result of diabetes accounts for 70% of all foot or leg amputations in Canada [8]. Autonomic neuropathy affects internal organs and was demonstrated in one study to be present in approximately half of newly-diagnosed type 2 diabetic patients after 1.3 years [9]. Diabetes has been identified as a global crisis and many governments have implemented programs to combat this growing epidemic. Provinces across Canada have allocated public funds to cover the cost of insulin pumps, reducing the number of diabetes-related complications compared to daily insulin injections [10], while other programs have offset some of the costs associated with blood glucose monitoring to relieve financial burden [8]. Aside from these initiatives, the Canadian federal government also endorsed programs aimed at increasing diabetes awareness [8] and promoting healthier weights in young children [8]. Altogether, it is 3 clear that combatting the diabetes epidemic requires a co-operative effort from the government, researchers, physicians and individuals. 1.1.2 Diagnosis and types of diabetes The major hallmark of diabetes is chronic high blood sugar, or hyperglycemia, resulting from the inability to maintain blood glucose homeostasis [8]. The Canadian Diabetes Association (CDA) suggests that healthy fasting blood glucose levels are in the range of 4.0 to 6.0 mM, while at-risk individuals with impaired fasting glucose are in the 6.1 mM to 6.9 mM fasting glucose range [8]. Patients are also considered to be at risk if their blood glucose concentrations fall within the range of 7.8 mM to 11.0 mM following a two-hour glucose tolerance test, in which patients consume a 75 g bolus of glucose. Patients are diagnosed with diabetes if they have a fasting blood glucose level greater than or equal to 7.0 mM or a value greater than 11.0 mM glucose following an oral glucose tolerance test [8]. As an indicator of glycemic control over a period of three months, physicians also assess percent circulating hemoglobin A1c (HbA1c) levels. An HbA1c value greater than 6.5% is sufficient to diagnose diabetes, although caution is warranted in patients that recently experienced significant blood loss [11] or have a pre-existing condition that affects normal red blood cell turnover [11]. Historically, diabetes has been characterized as type 1 diabetes (T1D) or type 2 diabetes (T2D). In T1D, formerly referred to as juvenile diabetes, autoimmune-mediated destruction of insulin-producing islet beta cells results in chronic hyperglycemia [8]. The peak age of diagnosis for T1D is around 14 years of age, although cases have been reported in individuals older than 40 years of age [8]. T2D, on the other hand, is frequently diagnosed in older individuals, although 4 the number of younger individuals being diagnosed with T2D is increasing yearly [8]. Unlike T1D, patients with T2D, previous referred to as non-insulin dependent diabetes, develop insulin resistance [12]. During insulin resistance, tissues such as adipose, muscle and liver experience reduced glucose absorption and are unable to efficiently clear glucose from circulation [12]. More than 98% of diabetes cases are classified as T1D or T2D [13], while some of the remaining individuals develop chronic hyperglycemia as a result of a single gene mutation. For these monogenic disorders, close to 20 genes have been identified [13]. Mutations in the HNF1A and GCK genes are commonly associated with the development of mature onset diabetes of the young (MODY), while mutations in the KCNJ11, ABCC8, or INS genes can lead to neonatal diabetes [13]. Neonatal diabetes persists throughout the lifetime of the individual and is concomitant with reduced weight and growth rates, and in certain cases neurological problems [14]. Finally, the CDA reports that approximately 3-20% of pregnant women develop a form of diabetes referred to as gestational diabetes [15]. Similar to patients with T2D, women with gestational diabetes develop insulin resistance [15]. This loss of insulin sensitivity may be due to elevated concentrations of placental lactogens, such as cortisol and progesterone, which have been demonstrated to interfere with the binding of insulin to its receptor [16]. Although gestational diabetes can be managed effectively, left undiagnosed, the disease can lead to a difficult delivery and a higher risk that the mother or child will develop T2D later in life [17]. 1.2 Treatments for diabetes A number of oral anti-hyperglycemic medications are being used for the treatment of type 2 diabetes. For the purpose of brevity, this dissertation will only discuss the clinical implications of several of these treatments. Two newer treatments for type 2 diabetes, glucagon-like peptide-1 5 (GLP-1) agonists and sodium/glucose cotransporter 2 (SGLT2) inhibitors, will not be discussed in detail but are worth highlighting. GLP-1 receptor agonists potentiate insulin secretion by enhancing binding of GLP-1 to the GLP-1 receptor expressed on the surface of beta cells [18]. SGLT2 inhibitors work by preventing glucose re-absorption in the kidney, thus lowering the concentration of glucose in the plasma but also leading to reductions in body weight and blood pressure [19]. The treatments that will be described in more detail include metformin, sulfonylureas, DPPIV inhibitors and islet transplantation. 1.2.1 Metformin Since the discovery of insulin in 1921, a number of drug treatments and therapies have been developed for the management of T2D. Many of these medications work to enhance beta-cell secretion while others act outside of the pancreas to lower glucose production. The first choice among the oral anti-hyperglycemic medications prescribed for the management of T2D is metformin. Metformin is well-known for being low-risk and having fewer side effects compared to other oral anti-hyperglycemic medications available [20]. Although the exact mechanism by which this drug acts is still not completely understood, the drug’s primary mode of action is to suppress gluconeogenesis and glycogenolysis in the liver [20]. The mechanism of metformin may involve a direct effect on AMPK activation or the elevation of AMP/ATP ratios in the liver, leading to reduced gluconeogenesis [21, 22]. The potent therapeutic effects of metformin are closely reflected by changes in HbA1c levels. As part of the United Kingdom Prospective Diabetes Study (UKPDS), T2D patients given metformin monotherapy responded positively, displaying an HbA1c level of 7.1% compared to 7.8% in the obese control group after three years [23]. Similarly, a randomized 29-week trial of 289 patients undergoing metformin 6 monotherapy reported an HbA1c level of 7.1% in the experimental group versus a value of 8.6% in the placebo group [24]. While the effects of metformin on glucose metabolism have been well tested, several other studies have focused on the ability to reduce cancer risk in patients with T2D. A 10 year study revealed a 7.3% cancer incidence in T2D patients taking metformin compared to 11.6% in T2D patients that were not on metformin medication [25]. A similar risk reduction has been identified both in pancreatic adenocarcinoma [26] and prostate cancer [27]. It has been suggested that these effects may be an indirect result of reductions in hyperinsulinemia during diabetes, which leads to inhibition of mammalian target of rapamycin (mTOR) [28]. Despite many of the positive aspects of this drug, side effects have been reported. The most common side effects include nausea and diarrhea in approximately 30% of patients within two weeks of use [29], with symptoms persisting in about 5-10% of patients. One study also identified a 19% drop in serum vitamin B12 levels in patients taking metformin [30], which itself has been demonstrated as a risk for peripheral neuropathy [31]. As a whole, metformin persists as one of the most cost-effective and low-risk drugs available for the management of T2D, provided that physicians administer cautious doses to reduce unwanted side effects. 1.2.2 Sulfonylureas Prior to the use of metformin, the most highly prescribed class of drugs given to patients were sulfonylureas. This class of drug was available at low cost and demonstrated effective glycemic control in individuals diagnosed with T2D [20]. This class of drug consists of glyburide, glipizide and glimepiride, which mechanistically all act to bind the sulfonylurea receptor 1 (SUR1) complex in pancreatic beta cells [32]. Binding to the SUR1 receptor results in the closure of Kir6.1/2 channels, followed immediately by the influx of calcium ions and the release 7 of insulin from secretory granules [33, 34]. With respect to HbA1c levels, an average 1.5% drop is expected in newly diagnosed T2D patients [35], and approximately half of patients are expected to reach an HbA1c level lower than 7% during monotherapy [36]. A comparison between sulfonylureas and metformin in the UKPDS (United Kingdom Prospective Diabetes Study) revealed that failure rates were similar among both drugs, whereas the ADOPT (A Diabetes Outcome Progression Trial) study reported a 34% rate of failure for glyburide versus a 21% rate for metformin after a five year period [37]. Because of these conflicting reports, sulfonylureas are not the first choice of treatment due to risk of hypoglycemia. Hypoglycemia occurs in as many as 5% of glyburide users in the first month following usage compared to 1.7% of patients on glimepiride [35]. In addition, glyburide is not recommended for individuals with kidney disease since the risk of severe hypoglycemia in these patients is increased [20]. Effects of sulfonylureas on cardiovascular function have also been investigated. For instance, glyburide use promotes closure of myocardial SUR2 channels [38], encouraging increased infarct size and increased risk of heart failure [39, 40]. Interestingly, these effects were not reproducible with glimepiride or glipizide, implying that these drugs may have a reduced affinity for SUR2 channels in the heart compared to glyburide [41]. Due to many of the risks associated with sulfonylurea use, this family of drugs is often prescribed as second-line therapy. 1.2.3 DPP-IV inhibitors More recently, drug inhibitors of DPP-IV have been utilized in clinical trials. DPP-IV is a specialized protease that acts on the incretin hormones GLP-1 and glucose-dependent insulinotropic peptide (GIP) [42]. Both GLP-1 and GIP are released by the gut following a meal and work to amplify the insulin secretory response [43, 44]. Because DPP-IV-mediated cleavage 8 of GLP-1 and GIP results in a loss of their respective bioactivities, inhibition of DPP-IV increases the half-life of these incretins in circulation, thus allowing them to further enhance insulin secretion [42]. One clinical trial utilizing a DPP-IV inhibitor (NVP DPP728) reported a statistically significant drop in HbA1c levels from 7.4% to 6.9% over the course of four weeks in T2D patients [45]. The same report also saw a decrease in postprandial glucose levels and reduced insulin levels over a 24 hour period [45]. Two additional studies in obese T2D patients also demonstrated that use of the DPP-IV inhibitors vidagliptin or sitagliptin led to reductions in endogenous glucose production and suppression of plasma glucagon levels [46, 47]. In combination with metformin, vidagliptin resulted in decreased HbA1c levels over a span of 12 weeks and improved glycemic control over a period of 52 weeks compared to patients on metformin alone [46]. Although GLP-1 related therapies have displayed great promise for the management of glycemia in diabetes, treatment with these drugs may also be linked to pancreatitis and pancreatic cancer [48]. A report published in 2011 examined the number of pancreatitis and pancreatic cancer events in patients administered sitagliptin [49]. Cases from 2006 onwards reported increased pancreatitis risk at an odds ratio of 6.74 for sitagliptin, while an odds ratio of 2.72 was reported for the development of pancreatic cancer [50]. These associated risks may be linked to impaired immune function, which has been demonstrated in rodents administered DPP-IV inhibitors [51]. Altogether, DPP-IV inhibitors have demonstrated improved glycemic control in patients with T2D, but a closer examination of potential side effects is warranted. 9 1.2.4 Islet transplantation Many of the available diabetes therapies are designed to enhance beta-cell function. Because people with T1D have a limited source of beta cells, many of the oral anti-hyperglycemic drugs prescribed for T2D patients are ineffective. Pancreatic islet transplant was proposed as a treatment for T1D in the 1970’s [52], although the first successful islet transplant took place in 1989 [53]. In an islet transplant, healthy pancreatic islet cells are isolated from a cadaveric donor and infused into the hepatic portal vein of a T1D individual, allowing for the newly-transplanted islets to engraft in the liver. From here, these islets are able to release insulin into circulation in response to high glucose. A major advancement in the field of islet transplantation was the establishment of the Edmonton protocol, which included a shorter islet culture period prior to transplant, an increase in the number of islet equivalents given to each individual and the utilization of steroid-free immunosuppression [54]. Using these improved parameters, close to 10% out of a total of 47 islet transplant patients remained insulin independent 5 years post-transplant [55]. From 2007 to 2010, further improvements to the transplant protocol have increased the rate of insulin independence (three years post-transplant) to 44%, compared to 27% in previous years [56]. In spite of these early successes, there are still a number of factors that limit the survival of pancreatic islet grafts. Currently, the majority of healthy islets are isolated from cadaveric donors and in recent years, xenogenic islets from pigs [57]. The number of islet transplants per year is limited by the number of available organ donors, with a patient often requiring more than one donor in order to achieve glycemic control [58]. In addition, islets from non-beating heart donors suffer from increased ischemic damage compared to islets from brain-dead donors, further limiting the pool of 10 available human islets [59]. As an alternative to human islets, researchers have considered the use of islets derived from animals. Pigs are excellent candidates based mainly on the structural and physiological similarities between pig islets and human islets [60]. One of the biggest obstacles to using pig islets is a more intense immune response associated with xenotransplantation relative to allotransplantation [58]. To circumvent this issue, many investigations are employing the use of encapsulation devices that allow for free gas exchange but restrict access of immune cells to the enveloped islets [61]. Apart from animal-derived islets, a potentially unlimited source of insulin-producing cells may come from embryonic stem (ES) cells. With respect to ES cells, significant advances have been made in the last decade towards manipulating these cells to secrete insulin in response to glucose. Most notably, stem cell-derived insulin-producing cells normalize glycemia in rodent models following transplant and secrete insulin following glucose challenge, although these cells adopt an altered secretory profile in comparison to healthy human islets [62]. Regardless of the cell source, successful engraftment is one of the challenges associated with the infusion of beta cells. In rodents, it takes approximately 14 days following an islet transplant for islets to re-establish intra-graft blood perfusion [63]. During this time, there is a loss of transplanted islet cell mass and a prolonged period of hypoxia [63]. The islet vascularization process in mice is largely dependent on the recruitment of endothelial cells from the recipient to establish a rich blood vessel network [64]. The overexpression of vascular endothelial growth factor (VEGF) in islets prior to transplant is able to amplify the revascularization process [64, 65], offering a potential solution to the engraftment problem. This process can be further accelerated by co-transplantation of islets with vascular endothelial cells [66]. 11 An equally important but less appreciated contributor to islet graft failure is the deposition of islet amyloid plaques. Amyloid plaques are present in approximately 90% of T2D individuals but have also been identified in T1D recipients of islet transplants [67–69]. Deposits of amyloid arise from the misfolding of islet amyloid polypeptide (IAPP), a 37-amino acid hormone co-secreted with insulin from beta cells. Interestingly, mouse IAPP is incapable of aggregation due to several amino acid differences compared to the human IAPP sequence (Figure 1.1). Transplantation of human IAPP-expressing transgenic mouse islets promotes rapid islet failure in comparison to the transplantation of wild-type mouse islets [70]. We [71] and others [70, 72] have speculated that increased beta-cell stress during transplantation drives IAPP aggregation resulting in the deposition of amyloid plaques. In agreement with this hypothesis, an islet graft isolated from an islet transplant that was insulin independent for 13 years yielded nearly no detectable amyloid, indicating that healthy beta cells are not prone to IAPP-induced aggregation [73]. From these data, it is apparent that a more comprehensive understanding of the key players involved in amyloid formation is necessary in order to elucidate the mechanism that links amyloid deposition to declining islet graft function. Figure 1.1 Amino acid sequence variation between mouse and human mature IAPP. Three proline residues within the amyloidogenic region (NFGAILSS) of mature IAPP prevent rat or mouse IAPP from forming amyloid deposits. 12 1.3 Regulation of blood glucose homeostasis 1.3.1 Hormone action A hormone is a biologically active molecule released into circulation that acts distally to its origin to stimulate a target tissue. The lowering of blood glucose levels following a meal is a highly-coordinated process requiring the action of various pancreas and gut-derived hormones. Food is broken down into glucose and other nutrients, which are absorbed in the intestine and enter the circulation. High circulating glucose concentration is the main stimulus for insulin release from the beta cell. In humans, glucose is taken up by islet beta cells, driving oxidative phosphorylation and resulting in increased levels of cytosolic ATP [74]. Increased ATP results in an influx of calcium ions and the exocytosis of insulin-containing secretory granules [74]. In addition to insulin, pancreatic beta cells also co-secrete IAPP in response to high blood glucose levels [75]. IAPP is a regulator of gastric emptying and hunger satiety [76], but is most notorious for its ability to form amyloid deposits in T2D [77]. IAPP is able to bind the calcitonin receptor upon co-expression of RAMP1 or RAMP 3 in the brain [78]. Other studies suggest that this hormone acts in autocrine fashion to inhibit the secretion of insulin [79, 80]. Endogenous IAPP production has also been demonstrated to inhibit glucagon secretion from pancreatic alpha cells [81]. Insulin and IAPP secretion from beta cells is significantly amplified in response to incretins, such as GLP-1 and GIP, which are gut hormones that increase insulin levels [82]. GLP-1 and GIP are expressed from intestinal L cells and K cells, respectively, and released when these cells are exposed to protein, carbohydrates or lipids [83, 84]. Both these hormones amplify insulin secretion through a similar mechanism. Binding of GLP-1 to the GLP-1 receptor and GIP to the 13 GIP receptor results in increased cAMP levels, and the potentiation of insulin release in a glucose-dependent manner [85, 86]. Conversely, GLP-1 also suppresses glucagon production [87] and both incretin hormones demonstrate proliferative and anti-apoptotic effects on beta cells [88, 89]. Following its exit from the beta cell, insulin stimulates peripheral tissues such as adipocytes, skeletal muscles cells and hepatocytes to absorb glucose from the blood stream, thus allowing for glucose to be stored or utilized in these tissues. Insulin activates the insulin receptor, which phosphorylates insulin receptor substrate-1 (IRS-1). Activation of IRS-1 leads to downstream activation of Akt [90] and increased translocation of GLUT4 to the cell membrane in adipocytes and muscle cells, allowing for glucose uptake [91, 92]. Over-secretion of insulin can lead to excessive clearance of glucose from the circulation and result in hypoglycemia. The first line of defense against hypoglycemia is the release of glucagon from pancreatic alpha cells. Glucagon entry into the liver is facilitated by the glucagon receptor, which activates the protein kinase A (PKA) signaling cascade [93, 94]. PKA acts on a number of protein targets such as CREB, CRTC2 and inositol-1,4,5-trisphophate receptors to enhance gluconeogenesis [94, 95]. Similarly, phosphorylation of glycogen synthase and glycogen phosphorylase through the action of PKA promotes glycogenolysis [94, 96]. Both gluconeogenesis and glycogenolysis work together to augment blood glucose concentrations. 14 1.3.2 Enzymatic processing of prohormones Many hormones are initially synthesized as non-bioactive form that requires several post-translational modifications before activation. Each of the hormones described in Section 1.1.3 is initially derived from a prohormone precursor. Proteolytic cleavage of prohormones is accomplished by a family of proteases called prohormone convertases (PCs) [97]. The prohormone convertase family is composed of nine genes, PCSK1 through PCSK9, some of which are ubiquitously expressed and others that are tissue-specific [97, 98]. A PC is capable of cleaving a peptide substrate provided that it carries two consecutive basic amino acid residues (namely arginine and lysine residues), although the efficiency of cleavage is strongly dictated by the substrate’s tertiary structure [97, 98]. In certain cases, such as for proglucagon, a substrate may differentially processed depending on the particular PC enzyme expressed within the cell type. Insulin and C-peptide are initially derived from a preproinsulin precursor containing a signal peptide sequence (Figure 1.2), which is removed to generate proinsulin [99]. In rodents, proinsulin is first cleaved in the trans-Golgi network by PC1/3 to generate the des 31,32 split-proinsulin intermediate [100]. The intermediate proinsulin species is then processed at the C-terminal end by PC2 in the secretory granule to liberate mature insulin and C-peptide [101]. In both PC-mediated steps, carboxypeptidase E (CPE) is involved in the removal of the paired basic residues [102]. Interestingly, rodents lacking PC2 generate mature insulin due to compensatory action by PC1/3 [101]. In humans, recent data suggest an absence of beta-cell PC2 following the transplant of human islets into athymic nude mice [103], implying that proinsulin may be completely processed by PC1/3. Conversion of proinsulin to mature insulin is vital to its 15 biological action. Proinsulin carries only 10% of the total biological activity of insulin [104]. With respect to circulating half-life, insulin has an average half-life of less than 4.3 min [105] whereas proinsulin has a half-life of approximately 25.6 min in circulation [105]. Figure 1.2 Processing of proinsulin to insulin and C-peptide. Proinsulin is cleaved at the NH2-terminal side by the enzyme PC1/3 to generate the des-31,32 proinsulin. This process is followed by the removal of basic residues RR by CPE. The des-31,32 proinsulin is then cleaved at the COOH-terminal side by PC2 to generated mature insulin and C-peptide, which are produced in equimolar amounts. 16 Similar to the conversion of proinsulin to insulin, proIAPP is processed in parallel to generate the mature IAPP protein (Figure 1.1). In mice, the proIAPP1-67 precursor is cleaved by PC1/3 to generate NH2-proIAPP1-48 [106] and subsequently cleaved by PC2 in the secretory granule to generate mature IAPP [107]. CPE is also involved in the removal of paired basic residues following cleavage [108]. Unlike insulin, IAPP is a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), which removes the C-terminal glycine residue and amidates the neighbouring tyrosine [109]. By mass spectrometry, O-linked glycosylation modifications were suggested to be present on threonines 6 and 9 of the mature IAPP sequence [110] although the importance of this modification is not well understood. In rodents, PC2 is absolutely essential for the complete maturation of IAPP [101, 111]. Overexpression of human proIAPP in rat pituitary GH3 cells, which express low levels of PC1/3, results in the production of mature human IAPP [112], implying that PC2 may not be absolutely essential for human proIAPP processing in humans. Whether the NH2-proIAPP1-48 and proIAPP1-67 precursors have biological activity has not been determined but both intermediates are capable of fibril formation in vitro [113, 114]. Similarly, the relative half-lives of these species have not been assessed relative to mature IAPP. A previous study in our laboratory, however, suggests that these intermediates are potential substrates for DPP-IV [115]. 17 Figure 1.3 Processing of proIAPP1-67 to mature IAPP in rodent pancreatic beta cells. ProIAPP1-67 is cleaved by PC1/3 at the C-terminus to generate NH2-proIAPP1-48. This intermediate is then cleaved by PC2 to generate the mature IAPP peptide. CPE removes paired basic residues following proteolytic cleavage by PCs and PAM removes the C-terminal glycine and amidates the adjacent tyrosine residue. 18 Pancreatic alpha cells mainly express PC2 [116] but are also thought to express PC1/3 under conditions of stress [117]. In both rodents and humans, the combined action of PC2 and CPE is required for conversion of proglucagon to glucagon, generating several side-products including glicentin-related polypeptide (GRPP), intervening peptide 1 (IP-1) and MPGF (major proglucagon fragment) [118, 119]. Apart from alpha cells, proglucagon is also synthesized in the brain [120, 121] and intestinal L-cells [121]. L-cells express PC1/3 rather than PC2, and do not generate mature glucagon [122]. Processing by PC1/3 and CPE in L-cells generates GLP-1, GLP-2, IP-2, GRPP and oxyntomodulin [122, 123]. Of these products, GLP-1 is the most well-characterized. Similar to IAPP, GLP-1 also undergoes an amidation modification at the C-terminal end, which appears to enhance its survival in plasma but is not essential for the potentiation of insulin secretion [124]. Additionally, DPP-IV-mediated cleavage of GLP-1 limits its circulating half-life to approximately two minutes [124, 125]. Processing of proGIP to GIP is completely dependent on the action of PC1/3 in vivo, while PC2 is capable of proGIP cleavage but not expressed in intestinal K cells [126]. 1.4 Mediators of beta-cell dysfunction 1.4.1 Insulin resistance While hormones are the main regulators of blood glucose homeostasis, there are a number of non-hormonal factors that promote hormone dysregulation and the development of diabetes. One factor is resistance to insulin, which develops in several tissues responsible for glucose absorption. During insulin resistance, the beta cell compensates by increasing insulin output in an attempt to balance reduced glucose uptake by muscle, liver and fat [12]. Aside from increased beta-cell stress, insulin resistance shares a strong positive correlation with several metabolic 19 disorders such as high blood pressure, dyslipidemia and atherosclerosis [127]. Several studies have revealed that lack of exercise and excess weight are two of the strongest predictors of insulin resistance [128, 129]. In patients with impaired glucose tolerance and T2D, as little as 30-60 minutes of aerobic exercise was sufficient to see a significant decline in blood glucose levels [130]. Acute muscle contractions stimulate glucose uptake through increased translocation of GLUT4 to the plasma membrane and increased expression of IRS-1 [130, 131]. Chronic exercise on the other hand, also upregulates GLUT4 protein levels and enhances expression of genes involved mitochondrial biogenesis [131]. Aside from exercise, diet can have profound effects on insulin sensitivity. Patients administered a Mediterranean diet, fortified with monounsaturated fatty acids, have increased glucose uptake in peripheral monocytes in the basal state and following insulin stimulation [132]. Increased insulin sensitivity was also accompanied by decreased plasma glucose and cholesterol levels [132]. An increased ratio of monounsaturated to unsaturated fatty acids in addition to higher dietary fiber in these patients is thought to be responsible for decreased insulin resistance and enhancements in beta-cell secretory capacity [133]. 1.4.2 Glucolipotoxicity Another factor implicated in diabetes development is glucolipotoxicity, which refers to the combined effect of high glucose and excess fatty acid consumption [134]. In particular, islets cultured in high glucose in the presence of palmitate have decreased PDX1 and MAFA expression, two transcription factors that bind to the insulin promoter and enhance its transcription [135]. Palmitate also induces cleavage of the ATF6 unfolded protein response protein, which decreases ER load in the beta cell via inhibition of insulin gene expression [136]. 20 Along the same lines, accumulation of saturated fatty acids depletes ER calcium stores, a process that promotes JNK-mediated cell death [137] and degradation of CPE protein [138]. Palmitate induces loss of CPE in rodent islets, potentially contributing to impaired proinsulin processing and enhances constitutive secretion of proinsulin [139], which may further promote beta-cell dysfunction. Another link by which glucolipotoxocity compromises beta-cell function is through activation of the protein kinase C (PKC)-ε isoform [140]. Inhibition of PKCε in ob/ob mice potentiates insulin secretion, and mice with a global knockout of this isoform are protected against fatty acid-induced reductions in insulin secretion [140]. Lastly, rodents with beta cell-specific loss of the ABCA1 cholesterol transporter results in the accumulation of cholesterol within the islet, impairing both glucose tolerance and glucose-stimulated insulin secretion [141]. This experimental model suggests that cholesterol accumulation, either through reduced function of ABCA1 or genetic loss of ABCA1, is a potential contributor to diabetes development in humans. 1.4.3 Inflammation In the past few years, inflammation has gained more attention as an important contributor to the development of T2D and affects tissue such as adipose, liver, muscle and pancreas [142]. The key mediators of inflammation in these tissues are macrophages, which can be broadly classified as M1- or M2-like [143]. In obese rodent models, macrophages undergo a phenotypic switch from M2 macrophages that secrete the anti-inflammatory IL-10 cytokine to M1 macrophages that secrete pro-inflammatory cytokines such as IL-1β, TNF-α and IL-6 [144]. A switch to an M1-like phenotype has a negative impact on both adipogenesis and insulin signaling within adipose tissue [144]. Conversely, resident Kupffer cells in the liver that become M2-skewed are 21 able to reverse insulin resistance and delay the progression of non-alcoholic steatohepatitis (NASH) [145]. Macrophages are less prevalent in skeletal muscle tissue relative to adipose and hepatocytes [146]. Regardless of this, evidence also suggests that M1-like macrophages in adipose tissue within muscle promote local insulin resistance through the release of pro-inflammatory cytokines [146]. In islet macrophages, signaling through toll-like receptor (TLR) 2 and TLR4, along with activation of the NLRP3 inflammasome work together to trigger inflammation in response to a particular insult [147]. Molecules such as saturated fatty acids are recognized by TLRs, leading to NFκB production and the release of IL-6, TNFα and pro-IL-1β [148, 149]. At the same time, activation of NLRP3 inflammasome stimulates caspase-1 activation, resulting in the cleavage of pro-IL-1β to active IL-1β [148, 149]. Inhibition of IL-1β signaling with anakinra, an IL-1β receptor antagonist, improves glycemic and beta-cell secretory capacity in T2D patients, demonstrating the importance of islet inflammation in diabetes development [150]. Previous work in our laboratory revealed that aggregates of IAPP are capable of activating a TLR2/6 heterodimer in addition to the NLRP3 inflammasome, leading to release of pro-inflammatory cytokines including IL-1β [151, 152]. These inflammatory pathways promote beta-cell stress and dysfunction, possibly resulting in increased secretion of IAPP species prone to aggregation, which would work further to amplify the inflammatory response. 1.4.4 Impaired proinsulin processing As discussed in Section 1.3.2, conversion of prohormones into their active biological forms is essential to proper blood glucose regulation. Most studies focusing on the importance of prohormone processing in diabetes have been limited to rodent models. The generation of a PC2 global knockout mouse in 1997 led to a number of insights into the function of this prohormone 22 convertase in the islet [101, 111]. In the absence of PC2, intact proinsulin and des 31,32 split-proinsulin levels are highly elevated, while mature insulin levels are not severely affected [101, 111]. In contrast, PC1/3 knockout mice have an 85% reduction in mature insulin levels relative to wild type controls and an overabundance of proinsulin intermediates [97, 153]. Interestingly, even with hyperproinsulinemia and reduced levels of active GLP-1 in these animals, glucose tolerance is not impaired [97, 153]. In humans, mutations in PC2 have never been documented and only two cases of PC1/3 mutations have been documented in the literature. In one of these case studies, absence of PC1/3 protein was linked to high plasma proinsulin levels, severe reduction in mature insulin and impaired glucose tolerance [154]. Disproportionate secretion of proinsulin has also been documented in T2D [155, 156], early T1D [157] and recipients of islet transplants [155], and will be examined more closely in Section 1.5.5. Equally as important as PC enzymes in the islet is the production of CPE. Mice lacking CPE activity due to a S202P mutation are viable but have hyperproinsulinemia [158]. In agreement with this effect, complete deletion of CPE in rodents manifests as 50-100 fold higher intact proinsulin concentrations, severe obesity and glucose intolerance [159]. CPE knockout mice are also insulin resistant [159] and have elevated levels of a C-terminally-extended mature insulin peptide [158]. In humans, CPE polymorphisms result in decreased enzyme activity and may be linked to hyperproinsulinemia in diabetes [160]. In addition, a homozygous CPE mutation in humans closely resembles the phenotype of CPE knockout mice, resulting in characteristics such as obesity, neurological defects and impaired glucose tolerance [161]. 23 1.4.5 Islet amyloid formation At autopsy, approximately 90% of patients with T2D have visible amyloid plaques within pancreatic islets [77]. Amyloid typically consists of an aggregated protein bound to components of the cell basement membrane [162–164]. Islet amyloid, in addition to IAPP, is also composed of serum amyloid P, the heparan sulfate proteoglycan perlecan and apolipoprotein E [162–164]. Amyloid deposits are not a characteristic of patients with T1D, likely due to these patients having not only fewer beta cells, but also reduced beta-cell secretory capacity. T1D recipients of islet transplants, however, accumulate amyloid plaques under conditions of beta-cell stress [71, 72]. In a Japanese population, an S20G IAPP mutation has been linked to increased risk of type 2 diabetes development, and may be a result of differences in the aggregation rate of the mutant peptide versus the canonical peptide [165]. As described in Section 1.2.4, mouse IAPP does not aggregate due to proline substitutions within the characteristic amyloidogenic region of the human sequence (Figure 1.1). For this reason, transgenic mouse and rat models of human proIAPP overexpression have been used to examine the effects of IAPP aggregation on pancreatic beta cells. Mice with homozygous overexpression of human proIAPP become diabetic soon after weaning and experience beta-cell apoptosis between 5-10 weeks of age [166]. Similarly, rats with heterozygous overexpression of human proIAPP become diabetic between 5-10 months [167]. Not surprisingly, pancreas isolated from both these models exhibit a significant decrease in beta-cell mass, likely a result of IAPP aggregation-induced toxicity [166–168]. Culture of wild type rodent islets with synthetic IAPP induces beta-cell dysfunction and inflammation, whereas incubation with pre-formed amyloid fibrils has no effect [152]. This observation suggests the possibility that beta-cell cytotoxicity is mediated by an intermediate 24 oligomeric species rather than the mature fibrils. Bone marrow-derived macrophages exposed to IAPP aggregates (but not monomeric or fibrillar IAPP) secrete high levels of IL-1β [149, 152] leading to the activation of inflammatory pathways. The precise molecular structure of the elusive cytotoxic intermediate has not been identified; however, an antibody raised against Aβ oligomeric species (an amyloid-forming protein in the brain) displays immunoreactivity for aggregated human IAPP [169]. Immunostaining with this antibody suggests that oligomeric IAPP species may form intracellularly [170]. Several mechanisms as to how IAPP aggregates induce beta-cell apoptosis have been proposed. One possibility is that IAPP prefibrillar aggregates may interact with the ER membrane and promote the leakage of calcium ions [171]. This in turn, promotes the leakage of cytochrome C through the mitochondria and downstream activation of caspase 3 [171], while the depletion of Ca2+ in the ER promotes protein misfolding and ER stress. As evidence for this, human proIAPP-overexpressing rats stain positive for both caspase 12 and C/EBP homologous protein (CHOP), which are downstream signaling molecules of apoptosis during ER stress [172]. A second model proposes that IAPP fibrillary structures introduce pores into lipid membranes which lead to uncontrolled ion flux, generation of reactive oxygen species (ROS) and subsequent beta-cell death [173]. 1.4.6 Impaired proIAPP processing The mechanisms that initiate amyloid formation and the key players involved in this process have not been fully characterized. Immunoreactivity for proIAPP1-67 and NH2-proIAPP1-48 within amyloid deposits suggests that these prohormones contribute to the amyloid formation process 25 [174, 175]. In vitro, synthetic peptides of these intermediate are both capable of forming aggregate structures [113, 176], and also in the presence of artificial anionic membranes [114]. The reason for this is not understood, but may be partly due to the positive-charged N-terminal present on the precursors but not on the mature peptide [177]. This N-terminal region has been identified as a heparin-binding domain, and may interact closely with basement membrane heparan sulfate proteoglycan perlecan [177]. Binding of these prohormone intermediates to the cell basement membrane may initiate a nidus for amyloid fibril formation, resulting in localized plaque formation [176, 177]. A suitable animal model to examine the cytotoxic impact of proIAPP1-67 and NH2-proIAPP1-48 in vivo has not been generated, although several inferences can be made based on in vitro evidence. PC-expressing cell lines transfected with a human proIAPP-expressing vector revealed that enhanced processing of human proIAPP reduces amyloid formation and beta-cell apoptosis [178], implying that rapid processing of IAPP precursors is important in preventing amyloid plaque deposition and generation of toxic prefibrillar species. In human proIAPP-expressing rodent islets, PC2 overexpression using an adenovirus also resulted in reduced amyloid severity and fewer TUNEL-positive beta cells [112]. Based on these results, it is difficult to conclude whether it is the absence of partially processed proIAPP that prolongs beta-cell survival or if increased prohormone processing efficiency (through PC2 overexpression) reduces beta-cell death. Apart from proteolytic cleavage, proIAPP also undergoes C-terminal amidation prior to secretion from the beta cell. Amidation may be important for the physiological function of IAPP [179], and it has also been demonstrated that amidated synthetic IAPP aggregates more rapidly than the synthetic non-amidated species [180], although this hypothesis has not been evaluated in vivo. 26 1.5 Biomarkers of diabetes 1.5.1 Glycosylated hemoglobin A1C Considering that T2D is such a rapidly growing epidemic, identification of biomarkers that predict early disease onset may significantly reduce disease incidence. A biomarker is defined by the US Food and Drug Administration as any measurable diagnosis indicator that is used to assess the risk or presence of disease [181]. Molecules that are upregulated or downregulated during the course of diabetes are numerous but identification of molecules that foreshadow diabetes onset is a challenging endeavor. For many years, HbA1c levels have been the gold standard in measuring glycemic control over a period of 2-3 months, the average lifespan of a red blood cell [182, 183]. The Canadian Diabetes Association currently defines prediabetes as HbA1c levels ranging from 6.0% to 6.4%, although a recent study in 2011 suggested 5.6% as a more appropriate cut-off for determining if an individual is at risk for the development of T2D [184]. In a separate investigation, an HbA1c level greater than 5.6% was the best predictor for a plasma glucose level greater than 7.8 mM following a two-hour glucose tolerance test [185]. Higher HbA1c levels are also closely correlated with changes in lipid profile. A comparison of patients with HbA1c levels greater than 7% versus patients with HbA1c levels less than 7% revealed increased triacylglycerol levels, increased total cholesterol concentrations and an elevated ratio of LDL-C to HDL-C [186]. The same study was also successful in using elevated HbA1c levels as predictors of cardiovascular risk [186]. With respect to efficacy, conditions affecting red cell turn-over rate, such as anemia or an overactive spleen, as well as over-ingestion of salicylates can impact the accuracy of HbA1c results [187, 188]. 27 1.5.2 Adiponectin In recent years, the hormone adiponectin has been evaluated as a potential biomarker for diabetes. Tissue biopsies in human patients suggest that abdominal and gluteal subcutaneous adipose tissue are the main sources of circulating adiponectin [189]. Adiponectin has functions in fatty acid oxidation and glucose regulation, and is inversely correlated with weight gain [190]. Concentrations of adiponectin were found to be significantly higher in plasma from T2D individuals, and generally higher in women compared to men [191]. More importantly, one study indicated that prediabetic patients with impaired glucose tolerance had significantly lower adiponectin levels compared to age-matched healthy individuals [192]. This report also uncovered a strong association of reduced adiponectin levels with higher fasting glucose levels, increased insulin levels and increased insulin resistance [192]. Another investigation utilizing a larger cohort of individuals was able to reproduce these observations in women but only saw a trend towards decreased adiponectin levels in men [193]. With respect to exercise intervention, aerobic exercise in postmenopausal women led to increased circulating adiponectin levels, although this study did not measure parameters for glycemic status [193]. Additionally, an increase in adiponectin concentrations from 6.9 to 8.5 µg/mL following one week of acute exercise in this study correlated with increased insulin sensitivity in men [193]. 1.5.3 Metabolites Because diabetes induces drastic changes in metabolism, it is not unusual that circulating metabolites may be strong indicators of glycemic status. Flow injection analysis tandem mass spectrometry was utilized in a metabolomics screen to identify metabolites associated with the onset of T2D in a middle-aged population [194]. The study revealed that serum hexose and 28 phenylalanine, as well as several diacyl-phosphatidylcholines were associated with an increased risk of T2D [194]. Conversely, glycine, sphingomyelin C16:1, lysophosphatidylcholine C18:2, as well as several acyl-alkyl-phosphatidylcholines, were associated with a decreased risk of T2D [194]. Complementary to this study, aromatic amino acids, including phenylalanine, as well as branched chain amino acids (BCAAs) were reported to be increased in T2D patients as part of the Framingham heart study [195]. Increases in phenylalanine concentrations support insulin secretory pathways during insulin resistance, while an upregulation of BCAA production increases substrate availability for hepatic gluconeogenesis [196, 197]. In addition, reduced glycine levels may reflect increased gluconeogenesis [198]. Apart from amino acids, low levels of long chain phosphatidylcholine derivatives have been observed in obese patients and patients with insulin resistance [194], and are inversely correlated to triglyceride concentrations in plasma. This observation may account for why the absence of these molecules in circulation promotes T2D development [194]. In contrast, phosphatidylcholine molecules with fewer carbons and fewer double bonds are associated with increased T2D risk [194]. 1.5.4 Micro RNAs The first microRNA, or miRNA, was identified in C. elegans [199] in the early 2000’s. miRNAs silence gene expression by targeting a specific mRNA molecule, either by cleaving the mRNA, shortening the polyA tail of the mRNA or by reducing the translation efficiency of the mRNA [200]. Dysregulation of specific miRNAs, similar to alterations in protein levels, contribute to disease development. Specific miRNAs have been shown to be elevated in diseases such as cancer [201], atherosclerosis [202] and diabetes [203]. miR-375, for instance, is not only imperative for the maintenance of normal alpha-cell and beta-cell mass, but is also significantly 29 upregulated in NOD mice two weeks prior to onset and in mice treated with streptozotocin prior to hyperglycemia [204]. A later paper examining plasma from patients with T2D versus individuals with normal glucose tolerance revealed an upregulation of circulating miR-375, along with microRNAs miR-101 and miR-802 [205]. In agreement with this, another investigation was able to replicate elevated levels of miR-375 in newly-diagnosed T2D individuals but not in prediabetic patients [206], which suggests that this molecule is a late-stage marker of T2D and may reflect changes in beta-cell mass or beta-cell death. Unlike miR-375 which is expressed in islets, the endothelial-specific miR-126 was also identified as an indicator of T2D in humans [207]. A separate investigation found levels of this microRNA to be specifically down-regulated in patients with impaired glucose tolerance (IGT) or impaired fasting glucose (IFG) relative to healthy controls [208], while patients with established T2D presented with even lower concentrations. Biologically, miR-126 is involved in a number of processes such as angiogenesis, vascular growth and wound repair [207]. Because elevated glucose levels contribute to vascular endothelium growth factor (VEGF) resistance and dysfunction of endothelial cells [207], miR126 may act as a both a marker of glycemic and cardiovascular status. Two other microRNAs, miR-192 and miR-193b, are also found in higher concentrations in the plasma of individuals with IGT and IFG [209]. Of note, this correlation was even stronger in patients with fatty liver, a risk factor for T2D [209]. A striking finding from this study was that both microRNAs were not differentially regulated between T2D patients and healthy controls, signifying that upregulation of these microRNAs is specific to the prediabetic state [209]. 30 Accordingly, exercise intervention in the prediabetic patients led to reduced fasting glycemia accompanied by a reduction in circulating miR-192 and miR-193b levels [209]. In humans, miR-192 is abundant in liver and pancreatic beta cells, and its levels correlate strongly with triacylglycerol levels and the fatty liver index [210]. miR-193b on the other hand has been shown to be important for brown adipocyte development [210] and may have a role in modulating inflammation [211]. In combination, these two microRNAs may not only be useful as predictors of diabetes onset, but may also be a reflection of liver dysfunction and status of inflammation. 1.5.5 Proinsulin Many factors affect insulin gene expression and protein production, and ultimately, lead to changes in blood glucose homeostasis. The amount of DNA methylation at specific gene promoters has been linked to various diseases such as cancer [212], neurodevelopment disorders [213] and viral-induced diseases [214]. The methylation of cytosine residues in gene promoters is an important regulator of gene expression. Although many of the transcription factors that bind the preproinsulin promoter have been deciphered, the methylation pattern at this promoter was not elucidated until 2009 [215]. This study found that an important step in the differentiation of immature beta cells to mature beta cells was the demethylation of -182 and-414 nucleotides. identity [215]. Recently, the percent methylation of several sites within the preproinsulin promoter has been suggested as a potential marker of beta-cell dysfunction [216]. An increase in the ratio of unmethylated to methylated DNA was reported as early as 24 hours following the administration of streptozotocin, while NOD mice had similar elevations at 14 weeks of age, just prior to the onset of diabetes [216]. Increased absolute levels of unmethylated and methylated DNA at position -69 bp has also been correlated with recent onset of T1D, implying an increase 31 in circulating preproinsulin DNA in these individuals [217]. These studies associate methylation at the insulin promoter as a potential biomarker of diabetes but warrants further characterization before it can be used as a diagnostic tool in the clinic. In Section 1.3.2, it was highlighted that proinsulin has only 10% of the bioreactivity compared to insulin, illustrating the importance of post-translational processing of this molecule. Mechanisms implicated during prediabetes, such as ER stress and abnormally-high insulin secretion, have been proposed to be involved in early release of proinsulin from the beta cell before it can be fully processed [218]. For this reason, parameters such as total proinsulin concentrations and the ratio of proinsulin to insulin (or C-peptide) have been identified as potential biomarkers for diabetes. Various groups have reported an increased ratio of proinsulin to insulin or proinsulin to C-peptide in T2D [155, 156], in addition to prediabetic patients with impaired glucose tolerance [219]. An increase in total proinsulin levels, including intact proinsulin and des 31,32 split-proinsulin, is suggestive of reduced PC1/3 or CPE activity within beta cells [220]. Measurements of proinsulin and insulin following acute insulin secretion in humans also led to elevated proinsulin to insulin ratios in both healthy individuals and T2D patients [156]. This investigation concluded that increased demand for insulin, regardless of glycemic status, is sufficient to increase circulating proinsulin levels, and that the increased ratio is mainly a result of exaggerated beta-cell secretion. Similar to pre-T2D, proinsulin concentrations and proinsulin to C-peptide ratios are heightened in auto-antibody positive patients prior to the onset of T1D [221]. T1D recipients of islet transplants, as well as patients with pancreatitis that received an islet autotransplant also have elevated proinsulin to C-peptide ratios, suggesting that enhanced 32 beta-cell secretory stress following islet transplant is mirrored by increased circulating proinsulin [155]. Preceding the development of T1D, at-risk individuals test positive for at least one autoantibody against either insulin [222], GAD [223], IA-2 [223, 224] or ZnT8 [225]. In addition, proinsulin-targeted autoantibodies have been demonstrated to be more closely associated with the development of T1D compared to insulin-targeted autoantibodies [226]. A study from 2013 introduced a more robust and sensitive chemiluminescent ELISA for the detection of proinsulin autoantibodies [227]. Not only was this platform successful in predicting T1D in 46 out of 47 children, the authors also demonstrated a correlation between age of onset and mean proinsulin autoantibody concentrations [227]. 1.6 Summary Many factors are involved in determining whether an individual will develop diabetes mellitus. Enzymatic processing of prohormones is essential for proper glucose homeostasis. As such, impairments in the processing of prohormones such as proIAPP may be detrimental to beta-cell health. Discovery and validation of biomarkers not only adds to our understanding of disease, but is also required to identify at-risk individuals and administer early intervention. In this dissertation, I hypothesized that impaired human proIAPP processing is a contributor to beta-cell dysfunction in diabetes and islet transplantation. I evaluated the beta-cell prohormone, pro-islet amyloid polypeptide, as a biomarker for diabetes. At the same time, I developed several animal models of impaired human proIAPP processing and examined the rate at which these animals developed diabetes, along with parameters associated with the regulation of blood glucose 33 homeostasis. In Chapter 2, I measured the circulating concentrations of IAPP and its precursors in several patient populations, and discuss the relevance of partially processed proIAPP intermediates as markers of beta-cell dysfunction. In Chapter 3, I generated an animal transplant model to investigate the role of proIAPP processing intermediates in beta-cell failure and make inferences as to how these intermediates may be linked to the generation of toxic prefibrillar aggregates in islets. In Chapter 4, I describe the phenotype of two generated mouse models that express human proIAPP and lack PC2 in beta cells to recapitulate the findings from Chapter 3. Furthermore, I examine whether inability to cleave human proIAPP in an in vitro model accelerates beta-cell apoptosis. In Chapter 5, I highlight the findings from Chapters 2-4 and discuss how the research presented in this dissertation increases our current understanding of diabetes. I also compare my findings with previous reports in the literature and describe future investigations that will enhance our knowledge of dysfunctional prohormone processing in the diabetic or prediabetic state. 34 Chapter 2: NH2-terminal pro-islet amyloid polypeptide (1-48) is a biomarker of beta-cell dysfunction 2.1 Introduction The discovery and validation of biomarkers of beta cell death and dysfunction could allow for earlier prediction of type 1 and type 2 diabetes onset. Historically, both fasting blood glucose and HbA1c levels have been utilized as measures of glycemic control. More recently, molecules such as adiponectin [191], microRNAs [203], amino acids [195] and proinsulin [219] have surfaced as potential markers of prediabetes. Therapeutic and exercise interventions at early stages of disease could significantly improve prognosis [228]. For this reason, the utilization of multiple biomarkers to more accurately predict disease and elucidation of earlier biomarkers holds significant promise for reducing the burden of diabetes. Islet amyloid polypeptide (IAPP) is a hormone that is co-secreted with insulin from pancreatic beta cells [75]. Physiologically, this molecule likely functions as a regulator of satiety and gastric emptying [76]. Under conditions of beta-cell stress, IAPP aggregates to form insoluble amyloid plaques that accumulate within pancreatic islets of patients with T2D [77]. Data suggest that these deposits also contain immunoreactivity for the IAPP precursors, proIAPP1-67 and NH2-proIAPP1-48. Both these intermediates display rapid aggregation in the presence of anionic lipid membranes [114] and may potentiate beta-cell failure similar to the mature species [174, 175]. Hormones in the islet are derived from inactive prohormone forms. In rodents, preproIAPP1-89 undergoes a series of proteolytic cleavages and post-translational modifications before becoming 35 the biologically active IAPP species (Figure 1.1). Following the removal of the signal sequence, proIAPP1-67 is cleaved at the C-terminus by PC1/3 to generate NH2-proIAPP1-48 [106]. This intermediate is then cleaved in the secretory granule by PC2 to generate mature IAPP [107]. Interestingly, proIAPP processing closely resembles the pathway for proteolytic conversion of proinsulin to insulin and C-peptide, involving the concerted actions of PC1/3 and PC2. An increased ratio of proinsulin to C-peptide has been demonstrated in serum from T2D patients [155, 156] and in T1D recipients of islet transplants [155], suggesting that increased circulating proinsulin levels are reflective of beta-cell dysfunction. Like impairments in proinsulin processing, impairments in proIAPP processing may be a potential contributor to diabetes development and to the early failure of islet transplantation [112]. Based on the parallel processing pathway for proinsulin and proIAPP, we sought to determine if IAPP precursor intermediates may be a marker of beta-cell dysfunction and may complement proinsulin as a valuable biomarker for diabetes. To test this hypothesis, we developed a sandwich-type immunoassay capable of specifically detecting NH2-proIAPP1-48 with minimal cross-reactivity to other IAPP species. We assessed NH2-proIAPP1-48 levels and the ratio of NH2-proIAPP1-48 to total IAPP in subjects with impaired glucose tolerance, T1D, T2D and T1D recipients of islet transplants. 36 2.2 Materials and methods 2.2.1 Immunoassay reagents Monoclonal mouse antibodies, F002, F064 and F025, raised against human proIAPP-derived peptides were provided by MedImmune (Gaithersburg, Maryland). Electrode-plated 96-well multi-array plates, secondary antibody MSD-SULFO-labeling reagent and Read Buffer T (4x) detection reagent were purchased from Mesoscale Discovery (Rockville, USA). Heterophilic antibody-blocking reagent HBR-1 was purchased from Scantibodies (Santee, USA). 2.2.2 Animals Male and female C57BL/6J, FVB/N and FVB/N-Tg(Ins2-IAPP)RHFSoel/J (hproIAPPTg/0) were purchased from The Jackson Laboratory (Bar Harbor, ME). B6;129-Pcsk2tm1Dfs/J (PC2-/-) mice were generated as described previously [17]. All animals were maintained according to the Canadian Council on Animal Care guidelines and experiments were approved by the University of British Columbia Committee on Animal Care. 2.2.3 Islet isolation C57BL/6 and PC2-/- mice were sacrificed at 12 weeks of age by cervical dislocation following surgical plane anesthesia. Digestion of islets was performed by perfusing the pancreas through the common bile duct with 0.84 mg/mL of Collagenase Type XI (1200 CDU/mg, Sigma, Canada), incubating at 37°C and shaking the pancreas tissue gently for 5 min. Islet samples were washed in 1x HBSS (containing 1 mM CaCl2), passed through a 70 μm cell strainer and transferred to RPMI 1640 supplemented with 10% FBS. 37 2.2.4 Immunostaining and imaging Pancreata from PC2-/- and littermate controls, with or without expression of human proIAPP, were harvested at 12 weeks of age and fixed with 4% paraformaldehyde overnight at 4°C prior to paraffin embedding. Embedded pancreas were cut into 5 µm sections and dried overnight, then rehydrated through a series of xylene, 95% ethanol and 70% ethanol washes. Antigen retrieval of hydrated tissues was performed with 10 mM citrate pH6 buffer for 10 minutes in a microwave under high power. Sections were incubated with primary antibodies, F002, F064 or F025 each at a 1/200 dilution, and with a secondary goat anti-mouse Alexa Fluor 594 antibody (1:400, Molecular Probes, Canada). Serial sections were also stained for 2 minutes with 0.5% thioflavin S for detection of amyloid deposits and washed with 70% ethanol prior to the addition of mounting media (Vector laboratories, USA). 2.2.5 Preparation of synthetic peptides and labeling of antibodies Synthetic forms of proIAPP1-67 and NH2-proIAPP1-48 peptides were generated by Bio-Synthesis (Lewisville, USA). These peptides were dissolved in trifluoroacetic acid (TFA) overnight at room temperature, lyophilized and frozen at -80°C to maintain peptides in monomeric form. Synthetic, amidated mature IAPP was purchased from Bachem (Torrance, USA), dissolved in hexafluoroisopropanol (HFIP), lyophilized as above and stored at -80°C prior to reconstitution. F064 and F025 monoclonal antibodies were labeled using MSD-Sulfo-labeling reagent as per the manufacturer’s instructions (Mesoscale, USA). 38 2.2.6 Immunoassay protocol Bare, high affinity MSD electrode plates (Mesoscale, USA) were coated with F002 monoclonal antibody (1:250 dilution) and incubated overnight at 4°C. Plates were blocked at room temperature with 2% BSA before the addition of samples. For the generation of standard curves and cross-reactivity studies, synthetic peptides were diluted in assay buffer, Diluent 41 (Mesoscale, USA), supplemented with a 1 in 50 dilution of HBR-1 (Scantibodies, USA) and prepared at concentrations ranging from 0 to 100 pM. Plasma from humans and rodents were diluted two-fold in assay buffer to counteract matrix effects. Secondary labeled antibodies (F064 or F025, 1:2000 dilution) were incubated for one hour at room temperature. Chemiluminescent signals were generated by covering wells with Read Buffer 2x and reading plates on the Sector Imager 2400 (Mesoscale, USA). Spike and recovery analysis of NH2-proIAPP1-48 peptide was performed by spiking the peptide (at three concentrations) into three healthy individual plasma samples and compared to samples from the same individual without spiking. Intra- and inter-assay coefficients of variation were determined by three independent operators over two days (three plates each day). Linearity was tested by diluting a spiked plasma sample of known concentration two-fold and determining percent recovery. 2.2.7 Plasma collection The Clinical Research Ethics Board at the University of British Columbia, the Research Review Committees at BC Children’s Hospital and Vancouver General Hospital and the University of Cincinnati Institutional Review Board approved the study on human islet transplant recipients. Written consent was obtained from each individual or from parents prior to blood collection. 39 Islet transplant patients were recruited from the Vancouver Islet Transplant Program at Vancouver General Hospital. All of these patients were on tacrolimus and mycophenolate mofetil with the exception of one individual that received tacrolimus and sirolimus. These twelve individuals were taking the following medication: pioglitazone and exenatide (7 patients), pioglitazone (3) or exenatide (2). Of these patients, 7 were on insulin therapy while 5 were insulin independent. Healthy age-matched individuals were used as controls. In addition, we also recruited 15 patients with type 2 diabetes that were not on insulin medication and were reported to have an increased proinsulin to total insulin ratio. These patients tended to be older (70.1 ± 1.2 years), have higher fasting blood glucose (7.0 ± 0.3 mmol/L) and increased HbA1c (6.4 ± 0.2%) relative to healthy controls (Table 2.8). T2D individuals were taking the following medications: metformin (8 patients), glyburide (5), gliclazide (2) or no medication (5). We also collected plasma from children from 9-16 years old that were classified as healthy (n = 29), impaired glucose tolerance (n = 20), T2D (n = 4) or T1D (n = 10). Average fasting glycemia was 5.3 ± 0.1 mmol/L for healthy individuals, 5.7 ± 0.1 mmol/L for IGT patients, and 7.7 ± 0.7 mmol/L for T2D patients (Table 2.7). Two-hour blood glucose levels following a 75 g bolus of glucose were 8.9 ± 0.3 mmol/L in the IGT group (Table 2.7). Fasting blood glucose levels were not measured in patients with type 1 diabetes and HbA1c% levels were not available. For all measurements and validation tests, blood was collected in the presence or absence of a protease inhibitor cocktail and with a 5 µM final concentration of dipeptidyl peptidase IV (DPP-40 IV) inhibitor (Sigma, Canada). Blood was immediately spun at 2000 x g at 4°C and the plasma layer stored at -80°C before hormone measurements. 2.2.8 DPP-IV degradation assay Plasma from a healthy individual was collected in the absence of protease inhibitors and spiked with a 10 pM concentration of NH2-proIAPP1-48. Six separate samples were incubated for 0, 1, 2, 3, 8 or 24 hours in the presence of DPP-IV inhibitor at 37°C. Each collected sample was diluted into assay buffer and frozen prior to analysis. 2.2.9 Statistical analyses Data are expressed as mean ± SEM. Data from study participants were tested for normality using the D’Agostino and Pearson omnibus normality test. Differences between healthy and patient groups were determined using Student’s t-test or by one-way ANOVA (with Tukey’s post hoc test for multiple comparisons) for normally distributed data. For non-normally distributed data, we utilized the Kruskal-Wallis test followed by Dunn’s post-hoc test for multiple comparisons. Statistical analyses were performed using Graphpad Prism 5.0. Values were considered significant at P < 0.05. The inter- and intra-assay CV was compared using the same samples across six different plates (three per day) by three independent operators and calculated as the standard deviation divided by the mean. 41 2.3 Results 2.3.1 Characterization of proIAPP-specific antibodies in mouse pancreatic islets and human islet grafts In order to generate an immunoassay specific to human IAPP precursors, we first tested whether our candidate antibodies were capable of detecting human proIAPP-derived peptides (Figure 2.1A). Our analysis of mouse pancreatic islets with or without expression of human proIAPP determined that F002 and F064 antibodies are specific to human proIAPP, whereas the F025 antibody does not discriminate between mouse and human forms (Figure 2.1B). It was unclear from the previous literature whether the NH2-proIAPP1-48 could be amidated prior to PC2-mediated cleavage in rodents. To test this, we immunostained islets from PC2 deficient animals that cannot generate mature IAPP [103]. Positive staining with the amidation-specific F025 antibody in PC2 knockout mice implies that amidated NH2-proIAPP1-48 exists in pancreatic islets (Figure 2.1C). To confirm that IAPP precursors have a role in amyloid plaque deposition, we co-stained amyloid-containing human islet grafts with thioflavin S (specific to amyloid fibrils) and F064 (specific to IAPP precursors). Overlap of these two stains only suggests that IAPP precursor species are constituents of islet amyloid since the image was not generated on a confocal microscope (Figure 2.1D). 42 Figure 2.1 Identification of IAPP precursors in mouse pancreatic islets and islet grafts. A) Binding sites for human proIAPP-specific antibodies on NH2-proIAPP1-48. F064 antibody binds the NH2-terminal portion of NH2-proIAPP1-48 but not mature IAPP. F002 antibody binds to mature IAPP and its precursors. F025 antibody binds to amidated species of mature IAPP and NH2-proIAPP1-48. B) F025, F002 and F064-positive staining (red) in isolated C57BL/6 mouse islets co-stained with insulin (green, top panels) or glucagon (green, bottom panels). WT represents a section of wild-type mouse pancreas and Tg a section of pancreas from a hproIAPPTg/0 mouse. C) F025 (green) staining and glucagon (red) staining in PC2-/- mouse islets. D) Co-localization of F064-positive staining (red) with amyloid (pseudo-coloured blue for clarity) in a human islet graft. White scale bars represent a distance of 100 µm. 43 2.3.2 Development and cross-reactivity analysis of a human NH2-proIAPP1-48-specific ELISA We utilized a configuration wherein F002 was used as a capture antibody and F064 as a detection antibody (Figure 2.2A). In order to generate ELISA standard curves and determine relative cross-reactivities between the different proIAPP intermediates, we used synthetic proIAPP1-67, NH2-proIAPP1-48 and mature IAPP peptides as standards (Figure 2.2B). Using this antibody pair, cross-reactivity of intact proIAPP1-67 was 15.6 ± 0.9% (relative to NH2-proIAPP1-48) whereas mature IAPP was undetectable, as expected (Figures 2.2C, Table 1), indicating that this ELISA is relatively specific for NH2-proIAPP1-48. To evaluate whether the ELISA was capable of detecting NH2-proIAPP1-48 from human islets, we ran serial dilutions of human islet lysates. Plotting the concentration of NH2-proIAPP1-48 versus total protein revealed a linear relationship, suggesting that the F002/F064 antibody arrangement was indeed measuring this intermediate species (Figure 2.2D). Notably, we also tested the configuration of an existing ELISA for the detection of mature IAPP by using F002 as a capture antibody and F025 as a detection antibody on the MesoScale Discovery platform. Comparable to the commercial kit, we determined that the mature IAPP ELISA has a cross-reactivity of 0% with proIAPP1-67 and less than 20% with NH2-proIAPP1-48 relative to mature IAPP (Table 2.1). This analysis allowed us to correct values for mature IAPP by subtracting the contribution of the NH2-proIAPP1-48 intermediate. 44 Figure 2.2. Development of an immunoassay specific for human NH2-proIAPP. A) Sandwich-immunoassay for detection of human IAPP precursors. F002 was used as capture antibody and F064 as detection antibody labeled with an MSD-Sulfo tag. B) Amino acid sequence of human synthetic peptides proIAPP1-67, NH2-proIAPP1-48 and mature IAPP. PC cleavage sites are indicated in red and the amyloidogenic region indicated in green. C) Standard curves for synthetic proIAPP1-67, NH2-proIAPP1-48 and mature IAPP using F002 as capture and F064 as detection. D) Detection of NH2-proIAPP1-48 in serial dilutions of healthy human islet lysate. E) Percent recovery of spiked NH2-proIAPP1-48-spiked plasma samples from three healthy individuals with or without DPP-IV inhibitor. DDPIV-mediated degradation experiment was performed with three technical replicates per time point. 45 2.3.3 Determination of NH2-proIAPP1-48 concentrations in plasma from healthy individuals Prior to testing our antibody configuration for the detection of NH2-proIAPP1-48 in human plasma, we analyzed the lower limit of detection for the assay. We determined the limit of detection of the immunoassay to be 0.18 ± 0.06 pM, as determined by three independent operators with synthetic NH2-proIAPP1-48 peptide (Table 2.2). In the same experiment, plasma samples spiked with known concentrations of NH2-proIAPP1-48 were run on two consecutive days to assess reproducibility (Table 2.3). Inter- and intra-assay CV values were <12% and <11% (Table 2.4), respectively, and within recommended guidelines for immunoassay validation [229]. To assess linearity, we performed two-fold serial dilutions with a sample containing a known concentration of NH2-proIAPP1-48. This analysis revealed that as low as a 4-fold dilution of plasma was appropriate for measurement of this intermediate (Table 2.5). To assess potential loss of NH2-proIAPP1-48 in human plasma samples, we performed spike and recovery experiments in the presence of protease inhibitors. At 37°C, NH2-proIAPP1-48 levels were reduced to below 60% in the absence of any protease inhibitor, while treatment of plasma samples from the same individual with a dipeptidyl peptidase IV (DPP-IV) inhibitor was able to completely preserve the stability of the NH2-proIAPP1-48 peptide for at least three hours (Figure 2.2E). Treatment of samples with DDPIV inhibitor resulted in close to 100% percent recovery of the protein intermediate (Table 2.6). 46 Table 2.1 Cross-reactivity of IAPP-targeted and NH2-proIAPP1-48-targeted ELISAs with proIAPP-derived synthetic peptides. Table 2.2 NH2-proIAPP1-48-specific ELISA limit of detection as determined with three independent operators over two days. Table 2.3 Determined concentrations of low, medium and high NH2-proIAPP1-48 peptide spikes by three independent operators over two days. Table 2.4 Inter-assay CV and intra-assay CV values for the NH2-proIAPP1-48-specific ELISA as determined with three independent operators over two days. 47 Table 2.5 Linearity analysis for NH2-proIAPP1-48 detection in human plasma. Table 2.6 Spike and recovery analysis in healthy human plasma for the NH2-proIAPP1-48-specific ELISA in the presence of DPP-IV inhibitor. 2.3.4 Children with T1D have an elevated ratio of NH2-proIAPP1-48 to total IAPP We measured NH2-proIAPP1-48 and IAPP levels in a cohort of children with established T1D (for greater than six months) (Table 2.7). As expected, these patients had reduced plasma concentrations of both mature IAPP (Figure 3A) and NH2-proIAPP1-48 (Figure 3B) compared to healthy age-matched controls, although this difference was much less for NH2-proIAPP1-48. Thus, the ratio of NH2-proIAPP1-48 relative to total IAPP species was significantly higher in patients with T1D (61.3 ± 6.9%) than in non-diabetic healthy children (17.6 ± 2.0%) (Figure 3C). These data suggest that T1D may be characterized by secretion of NH2-proIAPP1-48 before it can be properly processed into mature IAPP. 48 Table 2.7 Clinical characteristics of healthy children, and children with impaired glucose tolerance, T2D and T1D. Figure 2.3. The ratio of NH2-proIAPP1-48 to total IAPP is elevated in T1D. A) Mature IAPP concentrations, B) NH2-proIAPP1-48 concentrations and C) ratio of NH2-proIAPP1-48 over total IAPP ratios in healthy children and children with T1D. Data are expressed as mean + SEM of 10-28 individuals per group. *P < 0.05, ***P < 0.001. 49 2.3.5 NH2-proIAPP1-48 levels are elevated in children with impaired glucose tolerance We also examined circulating NH2-proIAPP1-48 levels in children at risk for development of T2D. In this cohort, patient samples were segregated based on the 2-hour blood glucose level following an oral glucose tolerance test. A 2-hour blood glucose level less than 7.8 mM was deemed healthy, ≥ 7.8 to 11.0 mM was classified as impaired glucose tolerance (IGT) and ≥11.1 mM was characterized as T2D (Table 2.7). At these different stages of disease progression, we found a trend towards increased mature IAPP levels in children with IGT and significantly elevated levels of mature IAPP in T2D children compared to healthy controls (Figure 2.4A). Interestingly, NH2-proIAPP1-48 levels displayed a very similar trend compared to the mature peptide (Figure 2.4B), and this resulted in a ratio of NH2-proIAPP1-48 to total IAPP species that was similar among all the groups (Figure 2.4C). 50 Figure 2.4. NH2-proIAPP1-48 levels are elevated in impaired glucose tolerance. A) Mature IAPP concentrations, B) NH2-proIAPP1-48 concentrations and C) NH2-proIAPP1-48 over total IAPP ratios in healthy children, children with impaired glucose tolerance (IGT) and children with T2D. Data are expressed as mean + SEM of 10-28 individuals per group. 51 2.3.6 NH2-proIAPP1-48 concentrations and ratios of NH2-proIAPP1-48 to total IAPP species are elevated in recipients of human islet transplants but not in patients with T2D We next examined two adult populations that either had T2D or were given an islet transplant for the treatment of T1D. Previously, both these cohorts have been demonstrated to have an increased ratio of total proinsulin to C-peptide [155]. Characteristics for both these patient populations are described in Table 2.8. T1D recipients of islet transplants had normal levels of circulating mature IAPP and (Figure 4.5A) significantly higher levels of NH2-proIAPP1-48 (Figure 4.5B). Thus, compared to healthy controls, transplant recipients have an increased ratio of NH2-proIAPP1-48 to total IAPP species (Figure 4.5C). Under the conditions of islet transplantation, elevated NH2-proIAPP1-48 may be reflective of the secretory stress imposed on the donor islet beta cells. Interestingly, patients with T2D (on average longer than 9.8 years) had normal physiological levels of NH2-proIAPP1-48, implying that NH2-proIAPP1-48 may only act as a biomarker of an early beta-cell stress, but not once the disease is well-established (Figure 4.5A). Despite having normal NH2-proIAPP1-48 levels, T2D patients had increased total proinsulin levels (Figure 4.5D). Table 2.8 Clinical characteristics of healthy individuals, T1D recipients of islet transplants and T2D adults. 52 Figure 2.5. NH2-proIAPP1-48 and ratio of NH2-proIAPP1-48 to total IAPP are elevated in T1D recipients of islet transplants but not T2D. Plasma concentrations of A) mature IAPP, B) NH2-proIAPP1-48, and C) the ratio of NH2-proIAPP1-48 to total IAPP ratio. D) Plasma total proinsulin concentrations in healthy individuals, islet allostranplant recipients (Allo Tx) and type 2 diabetic patients (T2D). Data are expressed as mean + SEM of 10-15 individuals per group. *P < 0.05, **P < 0.01, ***P < 0.001. 53 2.4 Discussion In these studies, we used a new ELISA to measure circulating levels of the IAPP precursor intermediate species NH2-proIAPP1-48, created by PC1/3 cleavage of proIAPP1-67, in humans for the first time. Findings with this ELISA revealed that normal circulating concentrations of NH2-proIAPP1-48 are in the low picomolar range (0.5-2.0 pM), with an average of 1.5 ± 0.2 pM in healthy individuals. The ratio of NH2-proIAPP1-48 to total IAPP in circulation was higher than reported values for the ratio of proinsulin to insulin [230, 231]. Measurement of this intermediate in circulation allowed us to establish the peptide as a potential marker of beta cell dysfunction and diabetes. Using our ELISA, we were able to evaluate NH2-proIAPP1-48 as a marker of beta-cell function in several patient populations, including children with impaired glucose tolerance, T2D and T1D, and adults with T2D and T1D recipients of islet transplants. Patients with T1D have a drastic reduction in the number of beta cells compared to healthy individuals and secrete low levels of insulin and C-peptide. In our study, plasma samples from patients with T1D similarly had reduced levels of both NH2-proIAPP1-48 and mature IAPP, but the ratio of the NH2-proIAPP1-48 intermediate to mature IAPP in T1D patients was approximately 3-fold higher than in normal individuals, suggesting that these individuals have profound defects in their beta-cell prohormone processing machinery. In agreement with our findings, prediabetic NOD mice have an elevated circulating proinsulin to insulin ratio prior to disease onset [232]. Beta cells in NOD mice and T1D individuals may secrete increased proportions of these prohormone precursors due to several different mechanisms, including changes in PC enzyme expression or activity, or increased beta cell secretory stress in these individuals in the face of reduced beta cell mass. Whether IAPP precursors levels are elevated in pre-T1D has not been 54 determined; a recent study, however, described a dramatic rise in circulating IAPP levels in a population of recent-onset T1D patients days 1 to 3 after diagnosis, suggesting that the precursor peptides may also be measurable in these individuals [233]. Due to the limited availability of healthy pancreatic islets, T1D patients often require multiple islet donors and sometimes receive a suboptimal mass of islet tissue. Three years post-transplant, only about 44% of patients remain insulin independent [56]. Transplanted human islets can develop extensive amyloid deposition [69, 71], associated with graft dysfunction, and this may be attributed to beta-cells in these individuals undergoing increased secretory stress. Analysis of pancreatic tissue in an individual that remained insulin independent for 13 years revealed no traces of islet amyloid [73], consistent with the notion that beta-cell dysfunction in islets transplants promotes amyloid development and/or that IAPP aggregation contributes to graft failure. Since NH2-proIAPP1-48 appears to be a component of islet amyloid [174], it is plausible that the elevated levels of this intermediate form in the circulation of islet transplant recipients contributes to its aggregation and amyloid formation. Under these circumstances, the measurement of IAPP and its precursor species in circulation may mirror the progression of amyloidogenesis at the level of the pancreatic islet. Because it is difficult to evaluate whether an islet transplant will fail based on parameters such as the age of the donor, age of the recipients and quality of the islet preparation, having improved biomarkers of impending graft failure would be of considerable value. The elevated proportions of NH2-proIAPP1-48 in islet transplant recipients and T1D patients suggests it is worth further investigation as a biomarker of beta cell dysfunction prior to graft failure or diabetes onset. 55 Based on elevated proinsulin to C-peptide ratios in patients with T2D [155, 156], we expected to see elevated circulating NH2-proIAPP1-48 concentrations. To our surprise, T2D adults and healthy controls had comparable circulating levels of NH2-proIAPP1-48 and similar ratios of NH2-proIAPP1-48 to total IAPP species. It is possible that IAPP precursors are secreted at higher concentrations earlier during disease progression. It is also possible that patients with T2D were on medications that improved beta cell function, possibly correcting prohormone processing defects, including metformin which many of our patients were being treated with and which has been shown to reduce circulating proinsulin to insulin ratios [234]. Finally, since our ELISA detects almost exclusively NH2-proIAPP1-48 and not intact proIAPP1-67, we cannot rule out the possibility that the intact precursor is elevated in T2D. Elevated proIAPP1-67 levels would be indicative of a processing impairment at the C-terminal end of the peptide, possibly at the level of PC1/3 or CPE, and could reflect a separate stage of the disease. To determine whether NH2-proIAPP1-48 is elevated earlier in T2D progression, we measured the propeptide in children with impaired glucose tolerance that had not yet progressed to T2D and found they had elevated NH2-proIAPP1-48 levels in circulation compared to age-matched controls. These data suggest that prohormone processing defects may be more detectable earlier in disease. Further studies are needed to assess the possibility that NH2-proIAPP1-48 or other prohormone forms have biomarker potential in T2D prediction. Elevated circulating proinsulin levels have been studied as a marker of beta cell dysfunction in diabetes, and are indicative of prohormone processing impairments in the beta cell. Here, we demonstrate that, NH2-proIAPP1-48, a precursor form of another beta-cell hormone IAPP, is 56 detectable in the circulation of humans and is disproportionately elevated relative to mature IAPP in some conditions of beta cell stress and dysfunction, including T1D, impaired glucose tolerance (in adolescents), and in islet transplant recipients. Our finding that changes in NH2-proIAPP1-48 levels do not necessarily correlate with proinsulin levels and suggests possible differences in their processing during disease. This raises the possibility that measurement of IAPP precursors may complement those of proinsulin and provide additional insight into beta cell function in disease states. 57 Chapter 3: Loss of prohormone convertase 2 promotes beta-cell dysfunction in a rodent transplant model of human pro-islet amyloid polypeptide expression 3.1 Introduction Islet amyloid is a pathological lesion identified in approximately 90% of T2D islets post-mortem [77, 170]. The major component of amyloid is IAPP, a self-aggregating hormone co-secreted with insulin from beta cells [75]. Misfolded IAPP monomers assemble into oligomeric intermediates, which further interact to generate insoluble amyloid fibers containing heparan sulfate proteoglycans, serum amyloid P and apolipoprotein E [162, 164, 177]. It has been previously demonstrated that prefibrillar IAPP species that arise during the transition from monomer to amyloid fibril elicit beta-cell dysfunction and apoptosis [169, 170], as well as provoking islet inflammation [151, 152]. We [152], and others [70, 168] have proposed that cytotoxic IAPP aggregates mediate beta-cell death in T2D and in transplanted human islets, although the precise mechanism of this cytotoxic effect is poorly understood. Adequate processing of pancreatic prohormones is vital to the maintenance of blood glucose homeostasis. Processing of proinsulin is impaired both in T2D [156] and T1D recipients of islet transplants [155], resulting in elevated circulating ratios of proinsulin to C-peptide. In rodents, the proinsulin processing involves cleavage by prohormone convertase (PC) 1/3 and PC2 in order to generate mature insulin [100, 153]. Interestingly, absence of PC2 is partially compensated by the action of PC1/3 [111]. Despite this, mice with global PC2 deficiency exhibit 58 high proinsulin levels in circulation [101]. Similarly, processing of proIAPP in mice requires sequential action by PC1/3 and PC2 to generate mature IAPP [106, 107], though it is unclear whether impaired proIAPP processing plays a role in the onset of diabetes. Studies aimed at determining the effect of impaired prohormone processing have been limited by the lack of a suitable mouse model, since global loss of prohormone convertase enzymes results in developmental abnormalities [153, 154] and, in the case of PC2, hypoglycemia [111]. We have previously demonstrated that in culture, human proIAPP-overexpressing islets lacking PC2 develop increased amyloid deposition and beta-cell apoptosis compared to control islets with normal PC2 expression [112]. Based on this evidence, we hypothesize that elevated prohormone levels, namely proinsulin and proIAPP, promote beta-cell dysfunction in vivo. To test our hypothesis, we transplanted islets from mice with beta cell expression of human proIAPP but lacking PC2 and assessed graft function, survival, and amyloid formation. 3.2 Materials and methods 3.2.1 Mice NOD.CB17-Prkdcscid/J (NOD/SCID), C57BL/6J, FVB/N and FVB/N-Tg(Ins2-IAPP)RHFSoel/J were purchased from the Jackson Laboratory (Bar Harbor, ME). B6;129-Pcsk2tm1Dfs/J mice were generated as described previously [17]. All animals were housed in the animal facility of the Child and Family Research Institute. PC2 null (B6;129-Pcsk2tm1Dfs/J) mice were crossed to human proIAPP transgenic (FVB/N-Tg(Ins2-IAPP)RHFSoel/J) to generate hproIAPPTg/0PC2+/- animals. These animals were then crossed with PC2-/- mice to generate hybrid FVB;B6 hproIAPPTg/0PC2-/- donors and control littermates (hproIAPPTg/0PC2+/-, hproIAPP0/0PC2-/- and 59 hproIAPP0/0PC2+/-). For transplant experiments, we isolated islets from 16-week-old donor mice that were fed a 9% fat diet (Research diets, NJ, USA) from weaning. All animals were maintained according to the Canadian Council on Animal Care guidelines and the experiments were approved by the University of British Columbia Committee on Animal Care. 3.2.2 Islet isolation and transplantation Islet donors were sacrificed at 16 weeks of age by cervical dislocation following anesthesia. Digestion of islets was performed by injection through the common bile duct of 0.84 mg/mL of collagenase Type XI (1200 CDU/mg, Sigma), followed by incubation at 37°C and shaking of the pancreas tissue gently for 5 min. Islet samples were washed in HBSS (containing 1 mM CaCl2), passed through a 70 μm cell strainer and transferred to RPMI 1640 supplemented with 10% FBS. Approximately 150 islets were manually picked and allowed to recover overnight at 37°C. NOD/SCID recipients were administered a single 180 mg/kg body weight dose of streptozotocin (STZ; Sigma) and utilized for transplant if their blood glucose measurements were greater than 20 mM glucose. NOD/SCID mice were transplanted with 150 islets into the left renal subcapsular space 4 days after STZ administration. Animals in which blood glucose levels did not normalize (<8 mM glucose) in the first week following transplant were removed from the study. 3.2.3 Blood glucose monitoring Blood glucose was measured via tail bleeds in islet donors prior to islet isolation. Blood glucose was monitored weekly in islet transplant recipients. Islet graft failure was identified when fasting blood glucose levels were above 16.9 mM, at which point mice were sacrificed. 60 3.2.4 Glucose and insulin tolerance tests To assess glucose tolerance, mice were fasted for 6 h and administered glucose via intraperitoneal (i.p.) injection (1.0 g/kg body weight). Glucose was measured by glucometer from tail bleeds taken at 0, 15, 30, 60 and 120 min following glucose administration. To assess insulin tolerance, mice were fasted for 6 hours and administered insulin (Novo Nordisk, Canada) i.p. at a dose of 0.7 units/kg. Glucose measurements were obtained at 0, 15, 30, 60 and 120 min. Mice were excluded from the experiment if blood glucose levels dropped below 1.8 mM, in which case they were administered glucose. 3.2.5 Plasma proinsulin, human proIAPP and IAPP measurements At 6 weeks post-transplant, blood was collected via the saphenous vein in recipient mice. Plasma samples were isolated by centrifuging blood samples at 2000 x g at 4 degrees and stored at -80 degrees prior to analysis. Mouse total proinsulin was measured using the Mercodia rat/mouse proinsulin ELISA (Uppsala, Sweden). Human proIAPP and IAPP levels were measured using an in-house ELISA (see Chapter 2) utilizing F002 antibody (MedImmune, USA) as a capture antibody, and either F064 antibody (Medimmune, USA) for detection of human proIAPP or F025 (Medimmune, USA) antibody for detection of mature human IAPP. 3.2.6 Immunofluorescent staining Kidneys containing islet grafts were removed at 6 weeks post-transplant and fixed overnight in 4% paraformaldehyde at four degrees. Kidneys were placed in 70% ethanol prior to paraffin wax embedding. Tissues were sectioned at a thickness of 5 μm. Pancreas from mice which were age-61 matched to 16 week old islet donor mice, and islet graft sections were dehydrated through a series of xylene/ethanol washes and blocked in 4% goat serum for one hour. Following blocking, some sections were stained with polyclonal guinea pig anti-insulin antibody (1:200, DAKO, Canada) and polyclonal rabbit anti-glucagon antibody (1:200, AbCam) for hour at room temperature. Sections were then incubated for one hour with the secondary antibodies Alexa 594 goat anti-guinea pig (1:200, Invitrogen) and Alexa 488 goat anti-rabbit (1:200, Invitrogen). For amyloid staining, sections were placed in 0.5% thioflavin S for 2 min following staining with secondary antibodies. Slides were washed with two rounds of 70% ethanol and water prior to mounting. Images were taken on an Olympus BX61 fluorescent microscope at 20x magnification. Cell death was measured via TUNEL staining using the in situ cell death detection kit (Roche, USA). 3.2.7 Quantification of islet cell areas and islet cell death Quantitative image analysis to determine graft beta cell and alpha cell area, TUNEL positive cells and amyloid area was performed using Image-Pro Plus Analyzer V6 (Olympus). Beta-cell area was determined as the insulin positive area divided by the combined insulin and glucagon-positive area. Amyloid area was calculated as the thioflavin S positive area divided by the total graft area. %TUNEL-positive beta cell was calculated as the number of TUNEL-positive insulin cells divided by the total insulin-positive cells x 100. For characterization of insulin and amyloid area in pancreatic sections, 20 islets were counted per section. Percent amyloid severity was calculated as the total amyloid area divided by the total islet area x 100. Percent amyloid prevalence was calculated as the number of islets containing amyloid divided by the total number of islets in the section x 100. 62 3.2.8 RNA extraction of isolated islets and islet grafts Islet and islet grafts were washed in PBS prior to RNA extraction. Cells were homogenized in lysis buffer and RNA extracted according to the Purelink RNA Micro kit (Invitrogen, Canada). RNA was converted to cDNA using Superscript Vilo (Invitrogen, Canada). Quantitative PCR was performed with SYBR green master mix and with the primers listed in Table 3.1. Table 3.1 Primers used for RT-qPCR 63 3.2.9 Statistical analyses Data are expressed as the mean ± SEM. Differences between two groups were determine using Student’s t-test and differences between several groups were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Statistical analyses were performed using Graphpad Prism 5.0. Values were considered significant if p < 0.05. 3.3 Results 3.3.1 Lack of PC2 promotes mild hypoglycemia and improved glucose tolerance To test whether elevated prohormone levels promote beta cell failure in a human proIAPP overexpression model, we generated mice expressing human proIAPP and lacking PC2 (hproIAPPTg/0PC2-/-), along with control mice with human proIAPP and one copy of PC2 (hproIAPPTg/0PC2+/-) (Figure 3.1A). We also generated additional control groups of mice that do not express human proIAPP but lack PC2 (hproIAPP0/0PC2-/-) as well as mice with that do not express human proIAPP and PC2 (hproIAPP0/0PC2+/-) (Figure 3.1A). Haploinsufficient PC2+/- mice were used instead of PC2+/+ mice for ease of breeding. We first determined whether global PC2 deficiency had an impact on blood glucose homeostasis, with or without expression of the human proIAPP transgene. There was not statistical significance between body weights of mice with or without PC2 expression, although a trend towards decreased body weight was observed in hproIAPP0/0PC2-/- mice versus hproIAPP0/0PC2+/- mice, (Figure 3.1B), in agreement with a previous study that reported modest reduction in body weight in PC2 deficient animals fed normal chow [111]. None of the four genotypes were hyperglycemic, although mice without PC2 expression demonstrated mild hypoglycemia (Figure 3.1C), likely related to their inability to produce mature glucagon [111]. Glucose tolerance was also improved in PC2 deficient animals 64 (Figure 3.1C), as previously reported. We observed only mild glucose tolerance between hproIAPPTg/0PC2+/- and hproIAPP0/0PC2+/- mice at 16 weeks of age, demonstrating that expression of the human proIAPP transgene promotes early beta-cell dysfunction in our model. Figure 3.1. Hypoglycemia and improved glucose tolerance in PC2 deficient mice. A) Diagram of breeding scheme used to generate study animals. B) Body weight of donors at 16 weeks of age. C) Intraperitoneal glucose tolerance test (IPGTT) in 16-week-old donor animals. Data are expressed as mean ± SEM of 4-6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- donor mice. #P < 0.05 represents a comparison between hproIAPPTg/0PC2+/- and hproIAPP0/0PC2+/- donors. 65 3.3.2 hproIAPPTg/0PC2-/- mice have abnormal islet morphology and reduced amyloid formation Prior to determining whether hproIAPPTg/0PC2-/- mice had an increased amyloid deposition compared to hproIAPPTg/0PC2+/- mice, we thought to first measure the proportion of beta cells to alpha cells in these animals. Mice lacking PC2 had an increased proportion of alpha cells, such that the ratio of beta to alpha cells was ~ 1:1 (Figure 3.2A, B), as reported previously [111]. Staining of pancreas sections from hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- 16-week-old mice with thioflavin S revealed small, punctate amyloid deposits in islets of hIAPPTg/0PC2+/- mice but not in those of hproIAPPTg/0PC2-/- mice (Figure 3.2C, D, E). The finding of no detectable amyloid in mice expressing human proIAPP and lacking PC2 is likely due to these animals being mildly hypoglycemic, resulting in a low drive for proIAPP synthesis [235]. 66 Figure 3.2. Islet histology in mice expressing human proIAPP and lacking PC2. A) Insulin and glucagon staining in donors animals. B) Quantification of beta cell area to total alpha cell plus beta cell area. C) Amyloid staining in donor islets. White arrows denote indicate the location of amyloid deposits. D) Quantification of amyloid severity as determined by amyloid area relative to total islet graft area. E) Quantification of amyloid prevalence as determined by the number of islets containing amyloid relative to the total number of islets. Data are expressed as mean ± SEM of 4-6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- donor mice. White scale bars represent a distance of 100 µm. 67 Figure 3.2 (continued). Islet histology in mice expressing human proIAPP and lacking PC2. A) Insulin and glucagon staining in donors animals. B) Quantification of beta cell area to total alpha cell plus beta cell area. C) Amyloid staining in donor islets. White arrows denote indicate the location of amyloid deposits. D) Quantification of amyloid severity as determined by amyloid area relative to total islet graft area. E) Quantification of amyloid prevalence as determined by the number of islets containing amyloid relative to the total number of islets. Data are expressed as mean ± SEM of 4-6 mice per group. White scale bars represent a distance of 100 µm. 68 3.3.3 Loss of PC2 and human proIAPP overexpression lead to rapid failure of islet transplants To test in vivo whether loss of PC2 promotes amyloid formation and beta-cell dysfunction under normoglycemic conditions, we transplanted islets from these mice into diabetic recipients. We used streptozotocin (STZ)-induced diabetic NOD/SCID recipients, enabling us to assess primary graft failure in the absence of allograft rejection. Following transplantation of the donor islets, non-fasting blood glucose levels steadily increased in mice transplanted with hproIAPPTg/0PC2-/- islets (Figure 3.3A). By contrast, we observed no changes in glycemia in recipients of hproIAPPTg/0PC2+/-, hproIAPP0/0PC2-/- or hproIAPP0/0PC2+/- islet grafts. In a cohort of islet graft recipients followed for 16 weeks, we observed that 15 out of 16 recipients of hproIAPP0/0PC2-/- islet transplants became hyperglycemic (blood glucose > 16.9 mM), whereas all recipients of control islet grafts remained normoglycemic (<16.9 mM fasting glucose) (Figure 3.3B). These results support that the loss of PC2 promotes beta-cell failure in a model of human proIAPP overexpression. Next, we examined whether islet graft failure was accompanied by increased amyloid formation and beta-cell apoptosis. TUNEL staining of islets grafts six weeks post-transplant revealed no significant differences among any of the groups (Figure 3.3C, D). Additionally, we calculated beta-cell area in grafts as a proportion of total islet (alpha plus beta cell) area. Interestingly, hproIAPPTg/0PC2-/- islet transplants had a ~10% reduction in beta cell area compared to hproIAPPTg/0PC2+/- grafts (Figure 3.3E, F). The proportion of beta to alpha cells in hproIAPP0/0PC2-/- islet grafts appeared similar to other control groups by six weeks post-transplant (Figure 3.3F), in contrast to the morphology of these islets prior to transplant when the proportion of alpha cells was markedly greater. Thus, the alpha cell hyperplasia associated with PC2 deficiency is normalized following transplantation of islets into a recipient with normal 69 glucagon production. We then determined whether the observed reductions in beta cell mass were associated with increased amyloid formation. Interestingly, thioflavin S staining revealed that hproIAPPTg/0PC2-/- islet grafts had a similar degree of amyloid deposition compared to hproIAPPTg/0PC2+/- islet grafts (Figure 3.3G, H). It should be noted, however, that hproIAPPTg/0PC2+/- mice, unlike hproIAPPTg/0PC2-/- mice, had detectable amyloid in their islets prior to transplant, which may have initiated further IAPP aggregation post-transplant. 70 Figure 3.3. Early islet graft failure in recipients of transplanted islets expressing human proIAPP and lacking PC2. A) Weekly non-fasting blood glucose measurements in transplant recipients. B) Survival graph displaying % of recipients with blood glucose levels below 16.9 mM glucose. C) Histological staining (insulin in red, TUNEL in green, nuclei in blue) and D) quantification showing the extent of TUNEL positivity in 6-week-old-recipients. E) Histological staining and F) quantification of insulin-positive area relative to graft area. G) Histological staining of amyloid deposits and H) quantification of amyloid severity calculated as amyloid area relative to total graft area. Data are expressed as mean ± SEM of 5-13 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- graft recipients. White scale bars represent a distance of 100 µm. 71 Figure 3.3 (continued). Early islet graft failure in recipients of transplanted islets expressing human proIAPP and lacking PC2. A) Weekly non-fasting blood glucose measurements in transplant recipients. B) Survival graph displaying % of recipients with blood glucose levels below 16.9 mM glucose. C) Histological staining and D) quantification showing the extent of TUNEL positivity in 6-week-old-recipients. E) Histological staining (insulin in red, glucagon in green) and F) quantification of insulin-positive area relative to graft area. G) Histological staining of amyloid deposits (insulin in red, amyloid in green) and H) quantification of amyloid severity calculated as amyloid area relative to total graft area. Data are expressed as mean ± SEM of 5-13 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- graft recipients. White scale bars represent a distance of 100 µm. 72 3.3.4 Elevated proinsulin and human proIAPP levels lead to early islet graft failure To better characterize the effect of PC2 loss in prohormone processing in our models, we measured plasma concentrations of mouse proinsulin, human proIAPP and human mature IAPP. As expected, at 6 weeks post-transplant, recipients of grafts lacking PC2 had elevated circulating proinsulin levels (Figure 3.4A). Inability to measure mature insulin levels in our model was limited by the high cross-reactivity of existing mouse insulin ELISAs with partially processed proinsulin species. For this reason, we evaluated mouse C-peptide concentrations in animal recipients, which were similar among all groups (Figure 3.4B). Interestingly, human NH2-proIAPP1-48 levels were elevated in mice that received hproIAPPTg/0PC2-/- islets compared to those who received hproIAPPTg/0PC2+/- islets (Figure 3.4C), while only a trend towards increased human mature IAPP levels was observed (p = 0.054)(Figure 3.4D). Since hproIAPP0/0PC2-/- islet grafts did not fail during the 16 week study period, our data suggests that elevated proinsulin levels are not sufficient to induce early beta-cell failure. Our data suggests that high NH2-proIAPP1-48 concentrations in hproIAPPTg/0PC2-/- islet grafts led to rapid transplant failure. Additionally, we performed insulin tolerance tests in recipients 6 weeks post-transplant to ascertain whether transplant failure might be attributed to changes in insulin sensitivity. Compared to mice with hproIAPPTg/0PC2+/- grafts, recipients with hproIAPPTg/0PC2-/- grafts had significantly higher blood glucose levels during the first 30 minutes of the test (Figure 3.4E) and a greater change in blood glucose levels at 60 and 120 minutes (Figure 3.4F) compared to mice with hproIAPPTg/0PC2+/- grafts. These data imply that animals with graft failure were, if anything, were more insulin sensitive compared to recipients of hproIAPPTg/0PC2+/- islet grafts and that hyperglycemia was not a result of insulin resistance in peripheral tissues but rather defects in the islet graft itself. 73 Figure 3.4. Elevated proinsulin and human proIAPP levels in recipients of transplanted islets expressing human proIAPP and lacking PC2. A) Total plasma proinsulin, B) C-peptide, C) NH2-proIAPP1-48 and D) mature IAPP concentrations in recipients 6 weeks post-transplant. E) Insulin tolerance tests in recipients 6 weeks post-transplant represented as blood glucose concentration and C) change in glucose concentration. Data are expressed as mean ± SEM of 4-9 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- graft recipients. 74 3.3.5 Altered gene expression in human proIAPPTg/0PC2-/- islet grafts To delineate a possible mechanism as to why hproIAPPTg/0PC2-/- islet grafts failed earlier than controls, RNA was isolated from these grafts 6 weeks post-transplant for qPCR analysis. There was a tendency towards decreased expression of two genes important for differentiated beta-cell function, Ins2 and Pdx1, in animals with hproIAPPTg/0PC2-/- grafts (Figure 3.5A). Our own previous data demonstrated that human IAPP aggregates induce islet inflammation via TLR signaling in macrophages [151]. Islet transplants from hproIAPPTg/0PC2-/- donors displayed a trend towards increased ITGAM expression compared to the other groups (Figure 3.5B); however, no trend was apparent for the expression of IL1b, Nlrp3 or Tlr2 genes. Lastly, because PC2 deficiency in human proIAPP-expressing animals led to islet graft failure, we performed gene expression analysis on several other prohormone processing genes. There was a trend towards decreased expression of Pcsk1 and Pcsk1n, necessary for cleavage the C-terminal end of proIAPP, in animals with hproIAPPTg/0PC2-/- islet grafts (Figure 3.5C), which may impact the C-terminal processing of human proIAPP. Additionally, we identified a trend towards the downregulation of Sgne1, which codes for the PC2 chaperone 7B2, and Cpe, responsible for removal of paired basic residues following PC-mediated cleavage, in these same animals (Figure 3.5C). These trends suggest that impaired prohormone processing may be a characteristic of declining islet graft function but require further validation at the protein level. 75 Figure 3.5. Gene expression in transplanted islets expressing human proIAPP and lacking PC2. A) qPCR analysis of proinflammatory genes. B) qPCR analysis of prohormone processing genes. C) qPCR analysis of beta-cell survival markers. Data are expressed as mean ± SEM of 5-6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Unless otherwise indicated by, statistical comparisons were done between hproIAPPTg/0PC2-/- and hproIAPPTg/0PC2+/- graft recipients. 76 3.4 Discussion Identification of factors that limit beta-cell viability is vital to the understanding of diabetes development. Here, we demonstrate that dysregulation of prohormone processing can lead to premature beta-cell failure in an islet transplant model. Although the degree of amyloid deposition was similar between mice with or without expression of PC2, the loss of this enzyme led to a significant reduction in beta cell mass. The data suggest a model in which elevated proinsulin and human proIAPP levels promote beta-cell failure or may reduce beta-cell proliferation. The toxic species associated with islet amyloid polypeptide aggregation remains elusive, although several studies reveal that both soluble monomers and fully formed fibrils do not impact beta-cell survival [151, 170]. Here, we examined whether overexpression of human proIAPP in a model lacking PC2 production exacerbated beta-cell failure. Surprisingly, even with rapid islet graft failure and reduced beta cell mass, we were unable to detect increases in the levels of amyloid deposition in contrast to a previous in vitro study [112]. Mature amyloid fibrils may be less deleterious to beta cells than pre-fibrillar species [152], and improvements in glucose tolerance do not always correlate with reduced amyloid formation [152]. From these data, it is plausible that undetected oligomeric proIAPP aggregates are present in PC2 deficient, human proIAPP-expressing islet grafts, and are inducing beta-cell dysfunction. Although the nature of IAPP precursor aggregation has not been studied as extensively as for mature IAPP, various reports indicate that these species aggregate in the presence of negatively-charged artificial membranes [176]. It has been reported that the positively-charged N-terminus 77 of unprocessed proIAPP interacts with heparan, possibly enabling attachment to heparan sulphate proteoglycans on islet basement membranes and the formation of a nidus for amyloid development [177]. Whether this process impacts other signaling pathways or is a contributor to beta-cell apoptosis has not been closely examined. Although preliminary, our gene expression analysis suggests that human proIAPP aggregate species may compromise beta-cell survival, prohormone processing and induce inflammatory pathways, although a more thorough analysis at the protein level is necessary to elucidate a concrete mechanism. A previous transplant model revealed that human proIAPP expression led to early graft failure as early as two weeks following transplantation of a sub-optimal mass of 100 islets [70]. Because our transplant studies had the additional stress of impaired human proIAPP and proinsulin processing, we chose to transplant a greater islet mass (150 islets). Notably, islets from PC2 deficient mice have a relative increase in alpha cells (~1:1 ratio of beta cells to alpha cells) compared to wild-type islets. Despite this abnormal islet morphology, transplanted islets lacking PC2 and without human proIAPP expression remained functional throughout the 16 weeks post-transplant, and histological analysis at six weeks post-transplant revealed similar beta and alpha cell proportions to hproIAPP0/0PC2+/- islet grafts. Loss of glucagon signaling in glucagon receptor knockout animals also results in altered islet morphology and alpha cell hyperplasia, suggesting the importance of glucagon in maintaining islet architecture [236]. It would therefore be interesting to analyze in our model how early PC2 null islets undergo a normalization in islet architecture following transplant into glucagon-producing animals. 78 Whether elevated proIAPP levels are characteristic of diabetes development has not been determined; however it has been proposed that elevated proinsulin to insulin ratios in circulation may be a marker of T1D [157], T2D [155, 156] and islet transplant failure [155]. In this study, we present tor the first time a model in which increased levels of proinsulin and proIAPP exacerbate islet transplant failure, and we speculate may contribute to diabetes as well. Altogether, these findings implicate IAPP precursors as cytotoxic species in the beta cell and as potential circulating biomarkers of early beta-cell dysfunction. 79 Chapter 4: Loss of prohormone convertase 2 promotes beta-cell dysfunction in a rodent type 2 diabetic model of human pro-islet amyloid polypeptide overexpression 4.1 Introduction Patients with diabetes suffer from chronically elevated blood sugar levels. Regulation of blood glucose levels is controlled by pancreatic islet hormones, and changes in the levels of these hormones results in conditions such as hyperglycemia or hypoglycemia. Insulin secreted from islet beta cells acts on peripheral tissues to promote glucose uptake while glucagon secreted from islet alpha cells stimulates gluconeogenesis in the liver to counteract insulin action. Understanding the factors that lead to alterations in insulin and glucagon production is crucial for the management of diabetes. A second beta-cell protein, IAPP, has been suggested to have a role in insulin and glucagon secretion [79, 81]. Apart from this, IAPP is also involved in inducing hunger satiety [75] but it is more commonly acknowledged as a protein that forms insoluble amyloid deposits in T2D pancreatic islets [70, 168, 170]. The process of amyloid formation, but not the mature IAPP fibrils themselves, has been shown to impair beta-cell function and promote beta-cell apoptosis [169, 170]. The precise mechanism of this beta-cell failure has not been fully elucidated but has been suggested to involve membrane damage [170], inflammation [151] and activation of pro-apoptotic pathways [173]. In addition, defects in the production of mature IAPP are also thought to compromise beta-cell survival [112]. 80 Insulin, glucagon and IAPP are derived from proinsulin [107], proglucagon [118] and proIAPP [106], respectively, prohormones that carry only a fraction of the biological activity of their mature forms. As an example, proinsulin has been demonstrated to be only 10% as bioactive relative to mature insulin [104] and differential processing of proglucagon can result in either the counterregulation of insulin or enhancements in insulin output [42, 94]. The biological activities of IAPP precursors proIAPP1-67 and NH2-proIAPP1-48 have not been examined, but these peptides have been shown to form amyloid deposits in pancreatic islets [72, 174]. Furthermore, evidence from Chapter 2 suggests that elevated levels of NH2-proIAPP1-48 reflect beta-cell dysfunction in diabetes and islet transplants. In Chapter 3, we demonstrated that the loss of PC2 in a transplant model of human proIAPP overexpression led to early transplant failure, suggesting that IAPP precursor accumulation contributes to beta cell failure likely through the generation of toxic prefibrillar aggregates. We wanted to test whether accumulation of these precursors promotes beta cell dysfunction in a rodent model of diabetes and in isolated islets. We hypothesized that IAPP precursor accumulation in these models would compromise beta-cell function and exacerbate beta-cell death. 4.2 Materials and methods 4.2.1 Mice C57BL/6J, FVB/N and FVB/N-Tg(Ins2-IAPP)RHFSoel/J were purchased from the Jackson Laboratory (Bar Harbor, ME). B6;129-Pcsk2tm1Dfs/J mice were generated as described previously [17]. All animals were housed in the animal facility of the Child and Family Research Institute. 81 PC2 deficient (B6;129-Pcsk2tm1Dfs/J) mice were crossed to human proIAPP transgenic (FVB/N-Tg(Ins2-IAPP)RHFSoel/J) to generate hproIAPPTg/0PC2+/- animals and controls. These animals were then crossed with PC2-/- mice to generate hybrid FVB;B6 hproIAPPTg/0PC2-/-. hproIAPPTg/0PC2+/- mice were also crossed to PdxCreER and Pcsk2-floxed animals to generate CreERTg/0hproIAPPTg/0PC2loxP/- mice and controls. At 8 weeks of age, mice were placed onto a high fat diet (Research Diets, USA) and monitored for 16 weeks. All animals were maintained according to the Canadian Council on Animal Care guidelines and the experiments were approved by the University of British Columbia Committee on Animal Care. 4.2.2 Development of Pcsk2-floxed animals To generate a vector for recombination into the Pcsk2 mouse locus, we began with the pFlrt4 plasmid containing a pre-existing PGK-neomycin cassette with flanking FRT sites (FRT-PGK-Neo-FRT) and two loxP sites containing a multiple cloning region between them. Upstream of FRT-PGK-Neo-FRT region we cloned a short arm of homology from Pcsk2 containing 146 bp of the 3’ end of intron 2, exon 3 and 2550 bp of the 5’ end of intron 3. Between the loxP sites, we cloned in 99 bp of the 3’ end of intron 3, exon 4 and 91 bp of the 5’ end of intron 4. Downstream of the second loxP site, we cloned a long arm of homology from Pcsk2 containing 6129 bp of the 3’ end of intron 4, exon 5 and 3584 bp of the 5’ end of intron 5. Lastly, downstream of the long homology arm we added a thymidylate kinase cassette to select against non-specific recombination using ganciclovir, generating the final vector, pFlrt4Pcsk2ExtTK. pFlrt4Pcsk2ExtTK was electroporated into mouse C57Bl/6 embryonic stem cells and individual stem cell clones were grown on plates containing neomycin and ganciclovir (positive and negative selection). To verify we had correct recombination, we performed a Southern blot using 82 the DIG-High Prime DNA Labeling and Detection Kit II (Sigma, Canada). Briefly, suspect ES cell genomic DNA was isolated with the Invitrogen DNAseI isolation kit. digested with BglII and run overnight on a DNA agarose electrophoresis gel. BglII-digested DNA was transferred onto a positively-charged membrane via capillary transfer overnight. We utilized a probe just 5’ of the short homology arm to detect an expected 6.0 kb fragment derived from the conditional allele (the wild type allele generated a 7.9 kb band by Southern blot). 4.2.3 Generation of adeno-associated viral (AAV) vectors We used the pK9-CMV-mFIX vector and pDSAAV-RIP-GFP vector (Children’s Hospital of Philadelphia viral vector core; Philadelphia, PA), to generated single-stranded AAV6 vectors and double stranded AAV6 vectors, respectively. To generate our pAAV6-RGP-PC2 vector, we removed the CMV promoter and mouse factor IX sequence (mFIX) from the pK9-CMV-mFIX vector and substituted it with 1298 bp of the rat glucagon promoter (RGP). Downstream of RGP we cloned the 418 bp of the human beta-globin intron 3 sequence and mouse Pcsk2 cDNA derived from C57Bl/6 genomic DNA. pAAV6-RGP-Cre was generated by removing the Pcsk2 coding sequence and replacing it with the Cre coding sequence. Generation of pAAV6-RIP-Cre was performed by replacing the GFP sequence from pDSAAV-RIP-GFP with the Cre coding sequence. Plasmid pAAV6-RGP-PC2 and pAAV6-RIP-Cre were used to generate the AAV vectors AAV6-RGP-PC2 and AAV6-RIP-Cre by 2x CsCl gradient ultracentrifugation (CHOP). Plasmid pAAV6-RGP-Cre was used to generate the AAV vector AAV6-RGP-Cre (SignaGen Laboratories, USA). 83 4.2.4 Generation of adenoviral vectors To generate adenoviruses expressing human proIAPP and human proIAPP-derived mutants, we utilized the pAdTrack system of vectors (AddGene, USA) [237]. Briefly, we inserted the human proIAPP cDNA sequence into pAdTrack-CMV to generate pAdTrack-CMV-hproIAPP. Downstream of the human proIAPP cDNA, we cloned an internal ribosome entry site (IRES) linked to the GFP coding sequence. pAdTrack-CMV-hproIAPP(KKK), pAdTrack-CMV-hproIAPP(KHK) and pAdTrack-CMV-hproIAPP(KQK) were derived from pAdTrack-CMV-hproIAPP using site-directed mutagenesis. pAdTrack vectors were recombined with pAdEasy vectors and transfected into 293T cells. Following viral amplification, viruses were purified via anion-exchange purification (Sartorius, USA). Titres of adenoviruses were performed by both GFP fluorescence in 293A cells and by qPCR using primers against the GFP coding sequence, and reported as pfu/mL (Table 4.1). Table 4.1 Determination of adenoviral titres by GFP fluorescence and qPCR 84 4.2.5 Islet isolation and adenoviral infection Mouse islet donors were sacrificed at 16 weeks of age by cervical dislocation following anesthesia. Digestion of islets was performed by injection through the common bile duct of 0.84 mg/mL of collagenase Type XI (1200 CDU/mg, Sigma), followed by incubation at 37°C and shaking of the pancreas tissue gently for 5 min. Islet samples were washed in HBSS (containing 1 mM CaCl2), passed through a 70 μm cell strainer and transferred to RPMI 1640 supplemented with 10% FBS. For adenovirus experiments islets were infected at an MOI of 200 (assuming 1000 cells per islet) directly after isolation and allowed to recover overnight at 37°C. 4.2.6 Pancreatic duct injection Animals at 8 weeks of age were anesthetized with isoflurane to reach surgical plane anaesthesia. A vertical incision was made along the abdomen and the skin held open using a small retractor. A straight clamp was placed at the sphincter leading from the common bile duct to the intestine and a second angled clamp around the common bile duct adjacent to the gall bladder. A single injection of 100 µL of AAV virus containing green dye was administered into the pancreatic duct. Success of the surgery was evaluated by spreading of dye into the pancreas and no signs of bleeding near the intestine. Animals were closely monitored for three consecutive days to confirm that sutures remained intact. 4.2.7 Blood glucose monitoring Blood glucose was measured via tail bleeds in experimental animals. Blood glucose was monitored weekly in all transgenic animals and animals infected with AAV vectors. Diabetes 85 was identified when fasting blood glucose levels were above 16.9 mM, at which point mice were sacrificed. 4.2.8 Glucose and insulin tolerance tests To assess glucose tolerance, mice were fasted for 6 h and administered glucose i.p. (0.3 g/kg body weight). Glucose was measured by glucometer from tail bleeds taken at 0, 15, 30, 60 and 120 min following glucose administration. To assess insulin tolerance, mice were fasted for 6 hours and administered insulin (Novo Nordisk, Canada) i.p. at a dose of 0.4 units/kg. Glucose measurements were obtained at 0, 15, 30, 60 and 120 min. Mice were excluded from the experiment for humane reasons if blood glucose levels dropped below 1.8 mM. 4.2.9 Plasma human NH2-proIAPP1-48 and human mature IAPP levels At 6 weeks post-transplant, blood was collected via the saphenous vein in recipient mice. Plasma samples were isolated by centrifuging blood samples at 2000 x g at 4°C and stored at -80°C prior to analysis. Human NH2proIAPP1-48 and mature human IAPP levels were determined using the ELISAs described in Chapter 2. 4.2.10 Western blot for proIAPP and proIAPP-derived species Islet grafts were removed from the kidney at 6 weeks post-transplant and lysed in RIPA lysis buffer containing 1 mM PMSF, 25 µg/mL aprotinin, 3.3 µg/mL pepstatin and 20 µg/mL leupeptin. After 25 minutes on ice, the lysates were centrifuged at 14 000 x g and the supernatant collected for western blot. Samples were resolved on a 15% Tris-tricine gel and transferred onto a nitrocellulose membrane for one hour at 100 volts via wet transfer. Overnight blocking with 86 2% BSA in tris-buffered saline was followed by a one hour incubation with a polyclonal rabbit anti-mouse IAPP antibody (1:1000, Bachem, USA) or monoclonal mouse anti-human IAPP antibody (F002) (1:1000, Medimmune) and a one hour incubation with a goat anti-rabbit or goat anti-mouse IR Dye 680RD antibody (1:10 000, Licor). Relative band densities were determined using Image Pro Analyzer Plus V6.0 (Olympus). 4.2.11 Immunofluorescent staining Tissues were sectioned at a thickness of 5 μm. Pancreas from mice between 12 to 24 weeks of age were dehydrated through a series of xylene/ethanol washes and blocked in 4% goat serum for one hour. Following blocking, some sections were stained with polyclonal guinea pig anti-insulin antibody (1:200, DAKO, Canada) and polyclonal rabbit anti-glucagon antibody (1:200 AbCam, USA) paired with either rabbit anti-PC2 antibody (1:200) (Thermo, Canada) or rabbit anti-PC2 antibody specific to exon 3 (Abcam, USA) for one hour at room temperature. Sections were then incubated for one hour with the secondary antibodies Alexa 594 goat anti-guinea pig (1:200, Invitrogen) and Alexa 488 goat anti-rabbit (1:200, Invitrogen). GFP staining in islets was done using rabbit anti-GFP antibody (1:1000 Abcam, USA) followed by a secondary Alexa 488 goat-anti rabbit antibody (ThermoFisher, Canada?). TUNEL staining was performed after GFP staining using the Roche TMR green cell death detection kit (Roche, USA). Slides were washed with two rounds of 70% ethanol and water prior to mounting. Images were taken on an Olympus BX61 fluorescent microscope at 20x magnification. 87 4.2.12 Determination of alpha to beta cell ratios and %TUNEL-positive islet cells All area measurements were performed using Image-Pro Plus Analyzer V6 (Olympus). For characterization of insulin and glucagon area in pancreatic sections, a minimum of 20 islets were counted per section. Alpha to beta cell ratios were determined by divided the glucagon-positive area by the insulin-positive area. Percent islet cell death was calculated as the TUNEL-positive area divided by the GFP-positive area. 4.2.13 Statistical analyses Data are expressed as mean ± SEM. Differences between two groups were determined using Student’s t-test and differences between several groups were analyzed by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Statistical analyses were performed using Graphpad Prism 5.0. Values were considered significant if p < 0.05. 4.3 Results 4.3.1 Alpha cell rescue of PC2 expression normalizes glycemia and islet architecture in PC2 null animals In order to examine the impact of impaired NH2-terminal processing of human proIAPP, we crossed mice with global PC2 deficiency with human proIAPP expressing mice to generate hproIAPPTg/0PC2-/- offspring. We suggested in Chapter 3 that these animals did not develop amyloid deposits due to chronic hypoglycemia and a reduced drive for IAPP secretion. For this reason, we tested whether specific overexpression of PC2 in the alpha cells of PC2 global knockout mice would normalize glycemia and proIAPP biosynthesis, creating an environment permissive for amyloid formation. Following pancreatic duct delivery of an AAV6-RGP-PC2 88 virus expressing PC2 under the control of the rat glucagon promoter, PC2 deficient mice became normoglycemic by one week post-infection (Figure 4.1A). This effect was likely linked to the re-establishment of proglucagon processing in islet alpha cells, because we found mature glucagon produced in isolated islets from animals infected with the AAV6-RGP-PC2 vector (Figure 4.1B). Immunostaining of pancreas from PC2-/- animals transduced with AAV6-RGP-PC2 revealed that PC2 expression was present in alpha cells (Figure 4.1C). We further examined the specificity of the rat glucagon promoter by utilizing ROSAMT/MG animals, a mouse model that expresses GFP in Cre-expressing cells. Infection of these animals with an AAV6-RGP-Cre virus resulted in several beta cells expressing both insulin and GFP (Figure 4.1D). Despite PC2 expression in beta cells of PC2-/- mice, production of mature mouse IAPP was not restored (Figure 4.1E). To further confirm the production of mature glucagon in this model, we also quantified the proportion of alpha cells to beta cells in PC2-/- mice treated with AAV6-RGP-PC2 or no virus. The analysis revealed that while uninfected PC2-/- mice had a 1:1 alpha to beta cell ratio, infection with AAV6-RGP-PC2 restored this proportion to near wild-type levels (Figure 4.1F, G). In summary, by rescuing alpha cell PC2 expression in PC2 deficient mice, we were successful in generating a mouse model lacking PC2 in beta cells without the presence of chronic hypoglycaemia. 89 Figure 4.1. Rescue of alpha-cell PC2 expression in global PC2 null mice restores normoglycemia and islet architecture. A) Weekly blood glucose measurements in PC2-/- and PC2+/- animals. At 7 weeks, PC2-/- mice were infected via pancreatic duct injection of AAV6-RGP-PC2 and PC2+/- mice were infected with an AAV6-RIP-Empty vector control. B) Western blot in islet lysates from wild type mice (lane 1), and PC2-/- mice infected with 1 x 1011 vg and 1 x 1012 vg of AAV6-RGP-PC2 virus (lanes 2 and 3, respectively). GAPDH was used as a loading control. C) PC2 (red) and glucagon (blue) levels in AAV6-RGP-PC2-infected PC2-/- mouse 4 weeks post-infection. D) Glucagon (red) and GFP (green) staining in pancreatic islets of RosaMT/MG rodents two weeks after infection with AAV6-RGP-Cre. E) Western blot showing proglucagon and mature glucagon levels in islet lysates from wild type animals (lane 1), PC2-/- mice (lane 2) and PC2-/- rodents infected with 1 x 1011 vg and 1 x 1012 vg of AAV6-RGP-PC2 virus (lanes 3 and 4, respectively). F) Insulin (red) and glucagon (green) staining in pancreas from PC2+/-, PC2 null animals and PC2-/- mice infected with AAV6-RGP-PC2 16 weeks post-infection. G) Quantification of alpha to beta cell ratios in wild type animals, and PC2-/- mice 16 weeks post-infection. Data are expressed as mean ± SEM of 3-6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. White scale bars represent a distance of 100 µm. 90 Figure 4.1 (continued). Rescue of alpha-cell PC2 expression in global PC2 null mice restores normoglycemia and islet architecture. A) Weekly blood glucose measurements in PC2-/- and PC2+/- animals. At 7 weeks, PC2-/- mice were infected via pancreatic duct injection of AAV6-RGP-PC2 and PC2+/- mice were infected with an AAV6-RIP-Empty vector control. B) Western blot in islet lysates from wild type mice (lane 1), and PC2-/- mice infected with 1 x 1011 vg and 1 x 1012 vg of AAV6-RGP-PC2 virus (lanes 2 and 3, respectively). GAPDH was used as a loading control. C) PC2 (red) and glucagon (blue) levels in AAV6-RGP-PC2-infected PC2-/- mouse 4 weeks post-infection. D) Glucagon (red) and GFP (green) staining in pancreatic islets of RosaMT/MG rodents two weeks after infection with AAV6-RGP-Cre. E) Western blot showing proglucagon and mature glucagon levels in islet lysates from wild type animals (lane 1), PC2-/- mice (lane 2) and PC2-/- rodents infected with 1 x 1011 vg and 1 x 1012 vg of AAV6-RGP-PC2 virus (lanes 3 and 4, respectively). F) Insulin (red) and glucagon (green) staining in pancreas from PC2+/-, PC2 null animals and PC2-/- mice infected with AAV6-RGP-PC2 16 weeks post-infection. G) Quantification of alpha to beta cell ratios in wild type animals, and PC2-/- mice 16 weeks post-infection. Data are expressed as mean ± SEM of 3-6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. White scale bars represent a distance of 100 µm. 91 4.3.2 Restoration of PC2 in alpha cells of hproIAPPTg/0PC2-/- animals does not lead to early diabetes development We next tested whether human proIAPP overexpression in PC2 deficient animals with viral rescue of alpha cell PC2 expression (hproIAPPTg/0PC2-/- + AAV6-RGP-PC2) led to diabetes development and amyloid formation. Monitoring of blood glucose for 16 weeks post-infection did not reveal any significant differences in blood glucose levels among any of our mouse cohorts (Figure 4.2A), although there was a trend towards increased glycemia in hproIAPPTg/0PC2-/- mice infected with AAV6-RGP-PC2. Interestingly, expression of PC2 in alpha cells of PC2 deficient animals, irrespective of human proIAPP overexpression, led to normal glucose tolerance curves during an IPGTT (Figure 4.2B). Expression of human proIAPP overexpression alone also appeared to be associated with impaired glucose intolerance (Figure 4.2B). At 16 weeks post-infection, however, none of the animals became diabetic (Figure 4.2C). These data suggest that in this model, loss of beta-cell PC2 in hproIAPPTg/0 animals is not sufficient to induce diabetes over a 16-week time period. 92 Figure 4.2. Loss of beta-cell PC2 is not associated with early diabetes development in a rodent model of human proIAPP overexpression. A) Weekly blood glucose measurements in hproIAPPTg/0PC2-/- mice and controls (hproIAPPTg/0PC2+/-, hproIAPP0/0PC2-/- and hproIAPP0/0PC2+/-). Mice were infected with AAV6-RGP-or an AAV6-RIP-Empty vector control at time zero. B) IPGTT results 4 weeks post-infection. C) Percent diabetic free animals within a period of 16 weeks post-infection. Data are expressed as mean ± SEM of 3-6 mice per group. 93 4.3.3 Generation of a mouse model with specific deletion of PC2 in pancreatic beta cells In the previous model, we found that the AAV6-RGP-PC2 virus had limited specificity for alpha cells, resulting in some PC2 expression in pancreatic beta cells. We therefore generated a Pcsk2-floxed animal to selectively remove PC2 from all pancreatic beta cells without affecting PC2 levels in other cells, including alpha cells. We developed a plasmid construct with homology to the Pcsk2 locus in order to introduce loxP sites around exon 4 (Figure 4.3A). We predicted, based on the PC2 coding sequence, that the loss of exon 4 would result in a truncated peptide lacking proteolytic activity. For specific recombination at the Pcsk2 locus in C57Bl/6 stem cells, we generated a plasmid containing homology arms upstream and downstream of exon 4 (Figure 4.3A). Between these homology arms, we cloned a PGK-neomycin cassette flanked by FRT sites and flanked exon 4 with loxP sites (Figure 4.3A). Single chromosome site-specific integration of this vector into the Pcsk2 locus was confirmed by the presence of 6.0 kb band detected by Southern blot (Figure 4.3B). Animals containing the conditional knockout allele were crossed to Flp recombinase mice in order to remove the FRT-flanked neomycin cassette (Figure 4.3C). Next, in order to specifically remove PC2 from beta cells, we generated an AAV6-RIP-Cre vector to preferentially express Cre in beta cells. Infection of homozygous PC2-floxed mice revealed loss of PC2 in pancreatic beta cells but not alpha cells three weeks post-infection (Figure 4.3D). Loss of mature mouse IAPP production in islets from these animals was consistent with PC2 loss in beta cells and led to accumulation of mouse proIAPP1-67 and NH2-proIAPP1-48 intermediates (Figure 4.3E). Surprisingly, we determined that as early as 12 weeks post-infection, PC2-floxed homozygous animals infected with AAV6-RIP-Cre had regained PC2 94 expression in the majority of beta cells (data not shown), suggesting only a transient loss of PC2 expression, possibly reflecting a survival advantage of PC2 expressing beta cells. Figure 4.3. Generation of PdxCreER/Pcsk2 conditional knockout mice. A) Diagram of strategy for generating a Pcsk2 conditional knockout allele. Exons are symbolized by red squares and homology arms are in gray. Also shown are FRT sites, loxP sites, a neomycin cassette and a thymidylate kinase cassette. BglII restriction sites are shown for Southern blot analysis using a DNA probe (shown in blue). B) Southern blot of recombined ES cell genomic DNA (lane 1), wild type DNA (lane 2) and negative control DNA (lane 3). BglII-mediated cleavage of the wild type Pcsk2 allele is expected at 7.9 kb whereas cleavage of the conditional Pcsk2 allele generates a 6.0 kb band. C) Diagram of the Pcsk2 locus in heterozygous PC2-floxed mice, displaying loxP sites flanking exon 4 and a residual FRT site from removal of the neomycin cassette. D) PC2 (green) and glucagon (red) staining in homozygous PC2-floxed mice infected with AAV6-RIP-Empty (top panels) or AAV6-RIP-Cre (lower panels). E) Western blot for mouse proIAPP species in homozygous PC2-floxed mice infected with either AAV6-RIP-Empty (lane 1) or AAV6-RIP-Cre (lane 2). White scale bars represent a distance of 100 µm. 95 4.3.4 Short-term loss of PC2 expression in human proIAPP-expressing animals is not associated with early beta-cell dysfunction In order to evaluate the effect of beta-cell PC2 loss in a model of human proIAPP overexpression, we crossed Pcsk2 floxed animals to human proIAPP transgenic mice and infected their offspring with AAV6-RIP-Cre (hproIAPPTg/0PC2loxP/- + AAV6-RIP-Cre). The impact of successful PC2 deletion in beta cells was confirmed by measuring plasma proinsulin levels, which were elevated at 6 weeks post-infection relative to animals infected with null virus (Figure 4.4A). hproIAPPTg/0PC2loxP/+ and hproIAPPTg/0PC2loxP/- animals infected with either AAV6-RIP-Cre or AAV6-RIP-Empty shared similar glucose tolerance curves at 4 weeks and 8 weeks post-infection (Figure 4.4B). However, only groups infected with AAV6-RIP-Cre, regardless of beta-cell PC2 expression, became diabetic over a period of 16 weeks following infection (Figure 4.4C). These findings suggest that infection with AAV6-RIP-Cre alone in human proIAPP-expressing animals causes beta cell dysfunction and early diabetes development, possibly a result of Cre overexpression in beta cells. Lastly, histological data revealed that the beta cell and alpha cell proportions were similar among all animals, suggesting that the apparent hyperglycemia in this model was likely related to beta-cell dysfunction and not beta-cell death (Figure 4.4D). 96 Figure 4.4. Transient loss of PC2 in mouse pancreatic beta cells overexpressing human proIAPP does not further compromise beta-cell function. A) Fasted plasma proinsulin levels in homozygous PC2-floxed animals infected with AAV6-RIP-Empty or AAV6-RIP-Cre. B) IPGTT at 4 and 8 weeks post-infection in human proIAPP-expressing PC2-floxed animals. C) Percent diabetes-free animals remaining within 16 weeks post-infection. D) Quantification of percent beta-cell area relative to total alpha-cell and beta-cell area in human proIAPP-overexpressing PC2-floxed mice. Data are expressed as mean ± SEM of 3-13 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. 97 4.3.5 Tissue-specific deletion of PC2 in human proIAPP-expressing PdxCreER mice In the AAV6-RIP-Cre infection model, we were limited by transient knockout of PC2 in beta cells, likely due to AAV-mediated Cre expression under the rat insulin promoter. To bypass this issue, we crossed human proIAPP-expressing PC2-floxed mice with the PdxCreER inducible mice to generate CreERTg/0hproIAPPTg/0PC2loxP/- animals and controls (Figure 4.5A). To verify beta cell-specific deletion of PC2, CreERTg/0hproIAPPTg/0PC2loxP/- mice were treated with tamoxifen or corn oil. Tamoxifen was dissolved at a concentration of 4 mg/mL in corn oil and was administered via oral garage on days 1, 3 and 5. Animals were sacrificed 20 weeks post-tamoxifen injection. Staining of pancreas from these mice with PC2, insulin and glucagon antibodies resulted in selective knockout of PC2 in beta cells of CreERTg/0hproIAPPTg/0PC2loxP/- mice but not controls lacking CreER expression (Figure 4.5B). These results demonstrate that we were successful in inducing long-term PC2 removal with single administration of tamoxifen. 98 Figure 4.5. Generation of a Cre-inducible, human proIAPP-expressing Pcsk2 conditional knockout mouse. A) Breeding scheme for the generation of Cre-inducible, human proIAPP-expressing Pcsk2 conditional knockout animals and controls. B) Insulin (green), glucagon (green) and PC2 (red) immunostaining in CreERTg/0hproIAPPTg/0PC2loxP/- animals treated with or without tamoxifen. C) Plasma measurements of human NH2proIAPP1-48 levels and D) human mature IAPP levels in CreERTg/0hproIAPPTg/0PC2loxP/- animals treated with or without tamoxifen. N = 5-9 mice per group. White scale bars represent a distance of 100 µm. 99 4.3.6 Loss of PC2 in beta cells of human proIAPP-expressing animals leads to early diabetes development We first analyzed the effects of Cre overexpression, human proIAPP overexpression and tamoxifen administration alone on glucose tolerance. Glucose tolerance curves in CreER0/0hproIAPP0/0PC2loxP/- and CreERTg/0hproIAPP0/0PC2loxP/- mice were not significantly different, indicating that transgenic CreER overexpression alone does not affect glucose homeostasis (Figure 4.6A), in contrast to viral overexpression. As we expected, animals that varied only in the expression of human proIAPP (CreER0/0hproIAPP0/0PC2loxP/- versus CreER0/0hproIAPPTg/0PC2loxP/-) exhibited changes in glucose tolerance, likely due to human proIAPP misfolding and aggregation (Figure 4.6B). Third, we identified that treatment with tamoxifen versus corn oil in our animal models led to a modest improvement in glucose tolerance (Figure 4.6C). For these reasons, we chose to compare CreER0/0hproIAPPTg/0PC2loxP/- and CreERTg/0hproIAPPTg/0PC2loxP/- mice, both treated with tamoxifen. By this comparison, Cre-mediated removal of PC2 in beta cells led to a trend towards impaired glucose tolerance compared to the tamoxifen-treated control (Figure 4.6D). Over a period of 16 weeks, several CreERTg/0hproIAPPTg/0PC2loxp/- mice treated with tamoxifen developed overt diabetes (fasting blood glucose >16.9 mM) while CreER0/0hproIAPPTg/0PC2loxp/- mice treated with tamoxifen remained normoglycemic (Figure 4.6E). These findings are in agreement with our findings from the islet transplant model in Chapter 3, in which loss of beta-cell PC2 in transplanted islets with human proIAPP-overexpression leads to impaired beta-cell function and graft failure. 100 Figure 4.6. Permanent loss of PC2 in mouse pancreatic beta cells expressing human proIAPP lead to compromised beta-cell function. A) IPGTT at 20 weeks in CreER0/0hproIAPP0/0PC2loxP/- versus CreERTg/0hproIAPP0/0PC2loxP/- mice not treated with tamoxifen; B) CreER0/0hproIAPP0/0PC2loxP/- versus CreER0/0hproIAPPTg/0PC2loxP/- mice not treated with tamoxifen; C) comparison of CreER0/0hproIAPP0/0PC2loxP/- mice with or without tamoxifen, and CreERTg/0hproIAPP0/0PC2loxP/- mice with or without tamoxifen; and D) CreER0/0hproIAPPTg/0PC2loxP/- versus CreERTg/0hproIAPPTg/0PC2loxP/- mice treated with tamoxifen. E) Survival curve showing percent diabetes-free CreER0/0hproIAPPTg/0PC2loxP/- and CreERTg/0hproIAPPTg/0PC2loxP/- animals treated with tamoxifen. # denotes the comparison between CreER0/0hproIAPP0/0PC2loxP/- tamoxifen-treated versus corn oil-treated animals. Data are expressed as mean ± SEM of 5-9 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05. 101 4.3.7 Generation of an uncleavable NH2-proIAPP1-48 substrate To test the cytotoxic effect of the NH2-proIAPP1-48 intermediate alone, which in Chapter 2 we demonstrated is upregulated during beta-cell stress, we sought to generate a human proIAPP substrate that cannot be cleaved at the NH2-terminal end. We generated adenoviruses expressing human proIAPP with four cleavage sites mutations, replacing the wild type KRK sequence with either KKK, KHK and KQK (Figures 4.7A, 7B). KR is the sequence required for PC2-medicated cleavage of NH2-proIAPP1-48 in rodents. Infection of wild type C57Bl/6 islets at an MOI of 200 resulted in approximately 10% infection of islets as evidenced by GFP positivity (Figure 4.7C). To verify similar levels of protein overexpression among the different adenoviral constructs, we quantified titres by both qPCR and GFP fluorescence (Figure 4.7C). Next, to determine whether any of our candidate constructs expressed a non-cleavable form of human NH2-proIAPP1-48, we ran ELISAs specific to human mature IAPP and human NH2proIAPP1-48 on infected wild type islet lysates isolated from human proIAPP-overexpressing rodents. Infection with Ad-CMV-hIAPP(KKK) did not lead to impaired processing, suggesting that replacement of arginine with lysine does not affect enzymatic cleavage at this site (Figure 4.7D). Infection with Ad-CMV-hIAPP(KHK) and Ad-CMV-hIAPP(KQK), however, did result in a markedly elevated ratio of NH2proIAPP1-48 to mature IAPP (Figure 4.7D). We chose to use the Ad-CMV-hIAPP(KHK) mutant for the remainder of the study to retain the overall positive charge at the NH2-terminal domain. 102 Figure 4.7. Mutation of KRK to KHK in the human proIAPP amino acid sequence leads to impaired processing of human NH2-proIAPP1-48. A) Diagram showing four prohormone convertase cleavage site mutations at the NH2-terminal domain of human proIAPP (amino acids 1-19). B) Control adenovirus construct expressing human proIAPP under the CMV promoter (left) and C) infected versus non-infected wild type C57Bl/6 islets two days post-infection. D) Measurement of the NH2proIAPP1-48 over mature IAPP ratio in islet cell lysates infected with Ad-CMV-hIAPP, Ad-CMV-hIAPP(KHK), Ad-CMV-hIAPP(KKK) and Ad-CMV-hIAPP(KQK). White scale bars represent a distance of 100 µm. 103 4.3.8 Impaired processing of human proIAPP in vitro promotes islet cell death To determine if there were any compensatory effects resulting from lack of NH2-terminal cleavage of human proIAPP, we measured prohormone processing gene transcripts after 10-day islet culture. The strongest trends observed were for the expression of Pcsk1 and its chaperone Pcsk1n (Figure 4.8A), whose upregulation may suggest a build-up of PC1/3 and ProSAAS levels in attempt to cleave the NH2-proIAPP1-48(KHK) intermediate. Overexpression of human proIAPP(KHK) did not promote changes in the expression of ER stress genes (Figure 4.8B), indicating that overexpression of human proIAPP(KHK) did not induce ER stress-related pro-apoptotic pathways. Lastly, to test whether impaired human proIAPP processing led to increased islet cell death, we measured the number of TUNEL positive cells after a 10 day infection in low glucose and high glucose culture. Infection of wild type C57Bl/6 islets with Ad-CMV-hIAPP(KHK) led to an increase in the number of TUNEL-positive islet cells under low glucose conditions, suggesting that accumulation of human NH2proIAPP1-48(KHK) intermediates may be more potent in inducing beta-cell death relative to the mature species (Figures 4.8C, D). Interestingly, high glucose culture conditions resulted in a similar percentage of TUNEL-positive cells in islets overexpressing human proIAPP versus human proIAPP(KHK) (Figure 4.8E). 104 Figure 4.8. Impaired cleavage at the human proIAPP NH2-terminal end promotes islet cell death but not changes in expression of processing genes and ER stress genes. A) Measurement of prohormone processing transcripts (relative to untreated controls) in wild type C57Bl/6 islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK). B) Measurement of ER stress transcripts (relative to untreated controls) in islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK). C) Insulin (green) and TUNEL (red) staining and D) %TUNEL-positive islet cell quantification in wild type C57Bl/6 islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK) in 11.1 mM glucose culture and E) 22.2 mM glucose culture. Data are expressed as mean ± SEM of n = 4-6 biological replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001, ##p < 0.05. * represents significance compared to untreated and # represents significance compared to rIAPP control. White scale bars represent a distance of 100 µm. 105 Figure 4.8 (continued). Impaired cleavage at the human proIAPP NH2-terminal end promotes islet cell death but not changes in expression of processing genes and ER stress genes. A) Measurement of prohormone processing transcripts (relative to untreated controls) in wild type C57Bl/6 islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK). B) Measurement of ER stress transcripts (relative to untreated controls) in islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK). C) Insulin (green) and TUNEL (red) staining and D) %TUNEL-positive islet cell quantification in wild type C57Bl/6 islets infected with Ad-CMV-rIAPP, Ad-CMV-hIAPP and Ad-CMV-hIAPP(KHK) in 11.1 mM glucose culture and E) 22.2 mM glucose culture. Data are expressed as mean ± SEM of n = 4-6 biological replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001, ##p < 0.05. * represents significance compared to untreated and # represents significance compared to rIAPP control. White scale bars represent a distance of 100 µm. 106 4.4 Discussion Prohormone processing in the pancreatic islet is essential to the maintenance of blood glucose homeostasis by hormones such as insulin, glucagon and IAPP. In accordance with Chapter 3, the studies presented in this chapter suggest that elevated IAPP precursor levels predispose the beta cell to early failure. Equally as important, we demonstrated that accumulation of NH2-proIAPP1-48 in the absence of mature IAPP may have an even more profound impact on the survival of pancreatic islets. Altogether, the models summarized in this chapter replicate an environment in which prohormone processing defects, either via the loss of PC2 in beta cells or the overexpression of uncleavable human NH2-proIAPP1-48 in islets, compromise beta-cell function. To explore the importance of PC2 in regulating amyloid deposition by human IAPP and its precursor species, we crossed PC2 global knockout animals to human proIAPP-overexpressing animals. To overcome the limitation of chronic hypoglycemia and provoke amyloid formation, we chose to restore glycemic balance via rat glucagon promoter-mediated PC2 overexpression in pancreatic alpha cells. Previous characterization of the rat glucagon promoter suggests that regulatory elements greater than 2.0 kb upstream of the transcriptional start site regulate preproglucagon expression in intestinal cells [238], while elements ranging from -1 kb to 0 kb are imperative for alpha cell-specific expression of the preproglucagon gene [239]. To retain alpha cell-specific expression of PC2, we cloned the 1089 bp region upstream of the transcriptional start site of the preproglucagon gene. We found that this segment of the promoter indeed led to transgene overexpression in alpha cells, but also resulted in protein overexpression in some beta cells. These results imply that although elements within 1 kb of the transcriptional start site are sufficient for preproglucagon expression in alpha cells, it is likely that regulatory 107 sequences further upstream are necessary for preventing preproglucagon expression in pancreatic beta cells. Restoration of glucagon levels by micro-osmotic pump in PC2 global knockout mice leads to the complete restoration of islet architecture [240]. Similar disruptions on islet morphology have been noted in glucagon receptor knockout mice [236], which would imply that it is not lack of glucagon that leads to abnormal alpha to beta cell ratios, but rather reduced glucagon signaling that plays an important role. In agreement with this, we found that alpha-cell rescue of PC2 in PC2 deficient animals reinitiates the production of mature glucagon, restoring both islet architecture and normoglycemia. Although the impairment of proglucagon processing is not a mechanism implicated in diabetes development, and may in fact be a potential therapeutic approach to treating diabetes, this example clearly indicates how impaired prohormone processing can have drastic effects in the regulation of glucose levels and the relative proportions of islet cell types. Along the same lines, early T2D is characterized by beta-cell hyperplasia due to a need for increased insulin production [127] and obese rodents have abnormally-large pancreatic islets [241]. A potentially important addition to our study would be to examine whether there is a relationship between impairments in proinsulin processing and beta-cell hyperplasia in these individuals. Beta cell-specific knockout of PC2 utilizing an AAV6-RIP-Cre vector resulted in transient loss of PC2 expression and recovery of the protein as early as 12 weeks post-infection. Previous studies in our laboratory and by other groups have utilized beta cell-specific AAV vectors at similar titres, and demonstrated long-term expression in beta cells [242]. On the other hand, RIP-108 Cre mice develop glucose intolerance even in the absence of loxP-flanked genes, and it has been suggested that overexpression of Cre in beta cells impairs beta-cell physiology [243]. These observations allude to the idea that the absence of PC2-null beta cells 12 weeks post-infection was likely related to Cre overexpression (possibly in combination with human proIAPP transgene expression) rather than AAV toxicity. Conversely, the expression of Cre under the Pdx1 promoter in our tamoxifen-inducible model was successful in permanently deleting PC2 from pancreatic beta cells. Even though relative to RIP-Cre mice the PdxCreER model has reduced levels of Cre protein in beta cells, few copies of Cre are required for recombination to occur. Utilization of the PdxCreER-inducible model to permanently remove PC2 from beta cells, as opposed to the RIP-Cre model that allowed for only short-term loss of PC2, allowed us to see a progressive decline in beta-cell function in the presence of human proIAPP overexpression. Many of the results presented in this chapter and Chapter 3 focused on a model with elevated NH2-proIAPP1-48 levels and parallel increases in mature IAPP levels as well, making it difficult to isolate the toxic effects of IAPP precursors alone. In order to focus more specifically on the relative toxicity of NH2-proIAPP1-48, we generated a human proIAPP(KHK) substrate that remained cleavable at the C-terminal domain but was uncleavable at the N-terminal domain. Based on the amyloidogenic sequence of human proIAPP, which is situated at amino acids 33-41, we predicted that the R10H mutation would not affect the peptide’s ability to aggregate but would retain the overall charge. In this respect, it was not surprising that overexpression of human proIAPP(KHK) yielded a toxic effect on beta cells in the absence of mature IAPP. Studies on the folding of NH2-proIAPP1-48 suggest that aggregation of this peptide may be impacted in the presence of negatively-charged membranes [176], owing partly to its overall 109 positive charge at the N-terminus that interacts with heparan [177]. Although our results are preliminary, it suggests that NH2-proIAPP1-48 is a potent mediator of beta-cell dysfunction. We propose in a future investigation to study this effect in vivo using two knock-in rodent models, one expressing full-length human proIAPP under the mouse IAPP promoter, and a second expressing the mutant human proIAPP(KHK) sequence under the same promoter. This method would ensure similar levels of human proIAPP expression and allow us to isolate the effect of NH2-proIAPP1-48 overproduction alone. It is clear that proteolytic processing of prohormones is essential to the regulation of blood glucose homeostasis and that changes in processing patterns have an impact on islet morphology and beta-cell health. Altogether, this chapter has examined multiple in vivo models wherein the loss of PC2 expression in human proIAPP-expressing rodents may promote diabetes onset, likely due to modulations in prohormone processing activity within the beta cell. In addition, we have defined an extreme case in which the complete loss of mature IAPP in vitro, and accumulation of NH2-proIAPP1-48, results in increased islet cell apoptosis. This work implicates IAPP precursors as important constituents of amyloid plaques and mediators of beta-cell stress, and defines these molecules as potential therapeutic targets for the treatment of diabetes. 110 Chapter 5: Conclusion The rising epidemic of diabetes worldwide reinforces the importance of understanding the factors that promote beta-cell dysfunction. Identification of these factors has offered insight into potential biomarkers of early diabetes development, such as proinsulin to C-peptide ratios, and allowed for the development of novel drugs targeted against chronic hyperglycemia. Cell-based therapies such as islet transplantation or infusion of stem-cell derived beta cells hold promise for a cure, but may still be limited by mechanisms such as inflammation and islet amyloid formation. Even though mechanistic models of IAPP-induced amyloid formation have been suggested, it remains unclear as to the precise cytotoxic species that threatens beta-cell viability. Furthermore, there may be key players contributing to amyloid formation in vivo that have not been considered. This dissertation examines the role of IAPP precursors both as markers of early diabetes development and as contributors to amyloid-induced beta-cell dysfunction. I examined increased secretion of the proIAPP processing intermediate, NH2-proIAPP1-48, as a marker of declining beta-cell function, and demonstrated this phenomenon in patients at risk for the development of diabetes but not in patients with established T2D. Furthermore, we found that patients with established T1D had low circulating levels of mature IAPP and NH2-proIAPP1-48 but a significant increase in the ratio of NH2-proIAPP1-48 to total IAPP species. I also developed and characterized several mouse models lacking PC2 and determined that absence of this enzyme in the beta cell exacerbates diabetes development in the presence of human proIAPP overexpression. 111 We identified the IAPP precursor intermediate, NH2-proIAPP1-48 as a potential biomarker of diabetes development. Based on western blot evidence that beta-cell stressors such as high glucose [244] and ER stress [172] result in abnormal ratios of IAPP precursors to mature IAPP, one interpretation of the data in Chapter 2 is that a higher circulating concentration of this intermediate may be related to compromised beta-cell function. Because various stressors are involved in prediabetes, we measured NH2-proIAPP1-48 concentrations in children with impaired glucose tolerance and T1D recipients of islet transplants. Considering that impaired glucose tolerance often develops into T2D and that only 44% of islet transplant patients remain insulin dependent after three years [56], it was perhaps not surprising that NH2-proIAPP1-48 levels in both groups were increased compared to age-matched healthy controls. The ratio of NH2-proIAPP1-48 to total IAPP species was not elevated in children with impaired glucose tolerance, suggesting that the prohormone processing machinery was still intact in these individuals whereas the opposite may be true in islet transplant recipients. Alternatively, we speculate that patients with impaired glucose tolerance could have increased levels of circulating proIAPP1-67, which was not fully detectable with our ELISA. Measurement of this precursor species would offer more information about processing at the C-terminal domain, similar to how levels of NH2-proIAPP1-48 describe processing at the NH2-terminal domain. Higher levels of circulating NH2-proIAPP1-48 may also be exaggerated by a slower clearance of proIAPP intermediates relative to mature IAPP, although the half-lives of IAPP precursor species have not been determined. On the other hand, I demonstrated that 37°C incubation of synthetic NH2-proIAPP1-48 in human plasma supplemented with DPP-IV inhibitor resulted in increased detectability of the peptide for up to 4 hours, whereas the sample without inhibitor showed a significant decline in immunoreactivity in less than one hour. Based on this experiment, it is tempting to speculate that 112 IAPP precursor intermediates may be rapidly cleaved by DPP-IV, preventing their detection by our ELISA. This mechanism would suggest that the circulating concentration of detectable NH2-proIAPP1-48 is solely dependent on beta-cell secretory activity. With respect to proteolytic degradation, other enzymes such as insulin degrading enzyme (IDE) and metalloprotease 9 have been shown to cleave mature IAPP [245, 246], although the affinity of these enzymes for proIAPP intermediates, and whether or not these enzymes are active in circulation has not been described. Various reports in the literature define prediabetes as patients with impaired glucose tolerance, impaired fasting glucose or both. For certain biomarkers, such as microRNAs (discussed in Chapter 1), concentrations were higher in patients with impaired glucose tolerance but not impaired fasting glucose, suggesting that these two phenomena may define two distinct stages of early diabetes development. Due to limited sample availability, we were only able to measure NH2-proIAPP1-48 in children with impaired glucose tolerance but not in children with impaired fasting glucose. Similar to what has been published for proinsulin [219], elevated NH2-proIAPP1-48 was a characteristic of impaired glucose tolerance. One study published that insulin resistance occurs in patients with impaired glucose tolerance but not in patients with only impaired fasting glucose [247]. Because insulin resistance stimulates increased insulin secretion, and by extension increased IAPP secretion, it is not surprising that NH2-proIAPP1-48 and mature IAPP levels are elevated in children with impaired glucose tolerance. Nonetheless, it would most certainly be interesting to measure NH2-proIAPP1-48 levels in patients with impaired fasting glucose, in which diabetes may be further advanced. A second comparison we would like to address in the future is whether NH2-proIAPP1-48 concentrations are more highly elevated in adults with impaired 113 glucose tolerance versus children with impaired glucose tolerance. Because amyloid deposition accumulates over many years in adults with T2D, and because this thesis and others [72, 174] have shown that amyloid is composed of partially processed proIAPP intermediates, it is plausible that the duration of beta-cell dysfunction may dictate the degree of proIAPP intermediate accumulation. Lastly, the antibodies we currently have at our disposal are not able to selectively measure proIAPP1-67 versus NH2-proIAPP1-48, and the ELISA we developed only had 15% cross-reactivity with this species relative to the NH2-proIAPP1-48 form. Although there is very little literature available on the function and amyloidogenicity of proIAPP1-67, we suspect that levels of this precursor may be similarly elevated in the circulation of patients at risk for diabetes. Prior to complete maturation, IAPP undergoes several post-translational modifications such as disulfide bond formation, proteolytic cleavage, possible glycosylation [110] and C-terminal amidation [124]. Chapter 2 discusses the N-terminally-extended NH2-proIAPP1-48 intermediate, which is derived from proIAPP1-67 by proteolytic cleavage, as a marker of beta-cell dysfunction. Presumably, impairments in other post-translational-modifications may also play a role in the development of diabetes. The function of glycosylation at threonine residues 6 and 9 of the mature IAPP sequence has not been identified and 25-45% of secreted mature IAPP is thought to be glycosylated in circulation [248]. These values were estimated based on the subtracted values of two ELISAs designed to detect either total mature IAPP levels or only non-glycosylated IAPP levels. Interestingly, plasma from patients with T2D did not show increased glycosylation relative to healthy controls [248]. At the same time, data from Chapter 2 suggests that NH2-proIAPP1-48 levels are also not elevated during T2D. Measurement of glycosylated proIAPP 114 species during different stages of disease, such as T1D and islet transplants, is warranted. Apart from glycosylation, the enzyme PAM involved in C-terminal amidation of IAPP has been identified as a risk allele in persons with T2D [249], suggesting that C-terminal amidation of IAPP may be affected during diabetes. Unlike GLP-1 in which amidation at the C-terminus is imperative to its function as an insulin secretagogue [124], there is limited evidence as to whether amidation of IAPP is important for its physiological function. The first paper that sequenced the IAPP gene also tested its function in isolated rat soleus muscle, indicating that amidation of IAPP is required for glycogenolysis [250]. Since this investigation, IAPP has been demonstrated to have roles in hunger satiety and gastic emptying, and perhaps in the inhibition of insulin, glucagon and somatostatin secretion [79, 81]. The importance of amidation in these processes has not been elucidated, although a non-amylodogenic analogue of IAPP, pramlintide, is manufactured with an amidated C-terminus and utilized as weight-loss drug [251]. With respect to amyloid formation, an in vitro study reports that amidated synthetic IAPP aggregates more rapidly relative to the non-amidated synthetic species [180]. Although the results in this dissertation do not examine the importance of amidation for amyloid fibril formation, it was found that intermediate NH2-proIAPP1-48 can exist in an amidated state, implying that this process can occur before or at the same time as N-terminal proteolytic cleavage. Because of this, elevated levels of non-amidated proIAPP species may also a represent a state of impaired prohormone processing, and slower aggregation of these species may result in an abundance of cytotoxic prefibrillar species. Development of an ELISA to detect non-amidated proIAPP species may also hold value as a marker of beta-cell health, similarly to the one that was developed for detection of NH2-proIAPP1-48. 115 The appearance of amyloid-like protein aggregates is a pathological feature of many diseases, and is strongly associated with cell degeneration both in the brain and in the islet. Various groups have demonstrated that the degree of fibril formation does not always correlate with the extent of cell death, and cytotoxicity is likely mediated through a prefibrillar intermediate species. In vitro aggregation of proIAPP1-67 and NH2-proIAPP1-48 has been shown to occur, but whether either of these processes promotes beta-cell dysfunction and death has not been demonstrated. In the transplant model we developed, it is difficult to discern whether IAPP precursors also transition through a cytotoxic intermediate prior to their incorporation into amyloid deposits, mainly because PC2 deficiency did not reduce the formation of mature human IAPP. For similar reasons, we were also unable to test this in our human proIAPP-expressing Pcsk2-floxed mouse. Using adenoviruses expressing human proIAPP or the non-cleavage human proIAPP(KHK) substrate, we were able to directly show that precursor proIAPP(KHK) species were more potent in their ability to induce beta-cell apoptosis under low glucose conditions, and elicited similar levels of beta-cell apoptosis under high glucose conditions compared to the cleavable wild type human proIAPP species. Relating this finding to our data from Chapter 2 and to other studies that demonstrate impaired human proIAPP processing is a characteristic of unhealthy beta cells, it is apparent that increased production and secretion of human IAPP precursors is a major factor that promotes beta-cell dysfunction. In order to make a stronger case for this effect in vivo, it would be imperative to study the non-cleavable human proIAPP(KHK) substrate using a knock-in mouse model, against a separate knock-in mouse model with comparative expression of wild type human proIAPP. 116 The processing pathway for the conversion of proIAPP to mature IAPP was initially elucidated in rodents through various studies, and involves proteolytic cleavage by PC1/3, PC2 and CPE. Global PC2 deficient animals, for instance, are completely devoid of mature IAPP but still make mature insulin due to partial compensation by PC1/3. Presumably, since proIAPP processing is more greatly affected than proinsulin processing, this model appears an attractive candidate to isolate the effect of impaired proIAPP processing. I found, in agreement with another group [103], that healthy human islets express PC2 mainly in alpha cells and not in beta cells. In Chapter 3, nevertheless, I found that islet grafts expressing human proIAPP and lacking PC2 function failed more rapidly compared to islet grafts expressing human proIAPP and normal PC2 protein levels. In vitro cleavage of human proIAPP by recombinant mouse PC2 in Chinese hamster ovary (CHO) cell lines has been demonstrated [112], and I verified by ELISA that mature human IAPP is detectable in mice lacking PC2. These findings imply that PC2 may not be essential for the cleavage of human, in contrast to mouse proIAPP, but is certainly capable of N-terminal cleavage of the peptide. Animals with islet grafts lacking PC2 had higher human proIAPP and human IAPP levels, but the ratio of NH2-proIAPP1-48 to total IAPP was not affected, which mirror our findings from Chapter 2 in children with impaired glucose tolerance. These data support that having an accumulation of NH2-proIAPP1-48, irrespective of processing efficiency, is associated with and may contribute to beta-cell dysfunction. In Chapter 4, we observed, that PC2 deficient animals have an equal ratio of alpha to beta cells and irregular islet architecture. This effect is complete reversed when mice are fitted with a micro-osmotic pump delivering controlled doses of glucagon [240]. I demonstrated that rescue of PC2 in the pancreatic alpha cells of PC2 global knockout animals restores islet architecture and 117 reverses the chronic hypoglycemic phenotype, further verifying the importance of the glucagon signaling pathway in the maintenance of normal islet architecture. In the presence of human proIAPP, we expected the animals with alpha-cell PC2 rescue would develop diabetes earlier than controls due to the recovery of glucagon levels and normoglycemia. Although we did not see any signs of uncontrolled glycemia during the 16 weeks post-infection, we suspect that over a longer period of time, the rescue of PC2 in alpha cells may create the appropriate environment for increased secretion of proIAPP-derived species and increased amyloidogenesis. Loss or reduced activity of prohormone processing genes in humans results in changes in proinsulin processing and abnormalities in blood glucose homeostasis. PC1/3 deficiency, for instance, promotes impaired glucose tolerance and significant reductions in insulin levels [154], and the R293W mutation in the CPE gene leads to earlier onset of T2D [160]. Although the contribution of amyloid deposition to these phenotypes has never been examined, changes in peptide processing have been shown to affect amyloidogenicity of other aggregate proteins. The loss of β-secretase (or BACE1), an enzyme important for the conversion of amyloid precursor protein (APP) to Aβ in the brain, promotes a reduction in amyloid plaque severity [252]. In contrast, our findings demonstrate that mutation of the N-terminal human proIAPP cleavage site increased beta-cell apoptosis under low glucose conditions relative to the wild type human proIAPP peptide. Differences between these amyloidogenic peptides, such as the aggregation potential of their processing intermediates, may account for this observation. In vitro incubation of synthetic NH2-proIAPP1-48 in the presence of membranes aggregates more slowly relative to mature IAPP [176], similar to how the aggregation of CT99, the processing intermediate prior to β-secretase cleavage, remains membrane bound, possibly limiting its ability to aggregate [253]. 118 In agreement with our findings, impairments in the processing of α-synuclein characterize both Parkinson disease and dementia with Lewy bodies [254]. The A53T and A30P point mutations in the α-synuclein gene have been linked to early onset of Parkinson disease but fibrillize at different rates relative to the wild type form [255]. In summary, there are many factors that impact beta-cell health and alter the regulation of blood glucose homeostasis. Impairments in the processing of prohormones in the islet are tightly linked to beta-cell dysfunction, and partially processed intermediates may be present at higher circulating concentrations at various stages of diabetes development. 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"@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2016-09"@en ; edm:isShownAt "10.14288/1.0314119"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Pathology and Laboratory Medicine"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "Attribution-NonCommercial-NoDerivatives 4.0 International"@* ; ns0:rightsURI "http://creativecommons.org/licenses/by-nc-nd/4.0/"@* ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Impaired pro-islet amyloid polypeptide processing promotes beta-cell dysfunction in diabetes and islet transplants"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/59064"@en .