Medicine, Faculty of
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UBCV
Brownrigg, George
Master of Science - MSc
University of British Columbia
The risk of developing type 2 diabetes (T2D) is ~40% higher in men than in pre-menopausal women. While lifestyle and cultural factors play a role in this male-biased risk of T2D, studies across animal species suggest biological sex contributes significantly to the sex difference in developing T2D. Large-scale gene expression studies suggest sex differences in pancreatic β cells play a role in the differential T2D risk between men and women. Yet, we lack a comprehensive understanding of β cell dysfunction between the sexes in both normal and pathological contexts. Here, we examined scRNA-seq data of human insulin-producing pancreatic β cells from non-diabetic (ND) and T2D men and women, revealing profound sex-specific changes to β cell gene expression in T2D. To gain deeper insight into sex-specific β cell responses in T2D, we sought a detailed understanding of β cell gene expression in normal physiological conditions. Unbiased pathway analysis of our well-powered islet RNAseq dataset from 20-week-old male and female mice with equivalent insulin sensitivity revealed a sex difference in the enrichment of UPR pathway-associated genes under basal conditions. Because female islets had higher expression of genes involved in protein synthesis, folding, and processing compared with males, we hypothesized female islets would be more resilient to acute ER stress induction with thapsigargin. Indeed, we found female islets resolved ER stress-induced protein synthesis repression faster than males and showed less cell death. These differences were significant for β cell function, as female islets maintained better insulin secretion than males in an ER stress context. Given the profound differences that we observed between the sexes in the transcriptional response to thapsigargin and the known links between ER stress and T2D pathogenesis, these findings provide additional insight into potential mechanisms underlying the differential risk of T2D between men and women.
SEX DIFFERENCES IN ISLET STRESS RESPONSES SUPPORT FEMALE BETA CELL RESILIENCE by George Brownrigg B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2022 © George Brownrigg, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Sex differences in islet stress responses support female beta cell resilience submitted by George Brownrigg in partial fulfillment of the requirements for the degree of Master of Science in Cell and Developmental Biology Examining Committee: Dr. Elizabeth J. Rideout, Assistant Professor, Cellular and Physiological Sciences, UBC Co-supervisor Dr. James D. Johnson, Professor, Cellular and Physiological Sciences, UBC Co-supervisor Dr. Angela M. Devlin, Professor, Pediatrics, UBC Supervisory Committee Member Dr. Eric Jan, Professor, Biochemistry, UBC Supervisory Committee Member Dr. Bruce C. Verchere, Professor, Pathology and Laboratory Medicine, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Dan S. Luciani, Associate Professor, Surgery, UBC Supervisory Committee Member iii Abstract The risk of developing type 2 diabetes (T2D) is ~40% higher in men than in pre-menopausal women. While lifestyle and cultural factors play a role in this male-biased risk of T2D, studies across animal species suggest biological sex contributes significantly to the sex difference in developing T2D. Large-scale gene expression studies suggest sex differences in pancreatic β cells play a role in the differential T2D risk between men and women. Yet, we lack a comprehensive understanding of β cell dysfunction between the sexes in both normal and pathological contexts. Here, we examined scRNA-seq data of human insulin-producing pancreatic β cells from non-diabetic (ND) and T2D men and women, revealing profound sex-specific changes to β cell gene expression in T2D. To gain deeper insight into sex-specific β cell responses in T2D, we sought a detailed understanding of β cell gene expression in normal physiological conditions. Unbiased pathway analysis of our well-powered islet RNAseq dataset from 20-week-old male and female mice with equivalent insulin sensitivity revealed a sex difference in the enrichment of UPR pathway-associated genes under basal conditions. Because female islets had higher expression of genes involved in protein synthesis, folding, and processing compared with males, we hypothesized female islets would be more resilient to acute ER stress induction with thapsigargin. Indeed, we found female islets resolved ER stress-induced protein synthesis repression faster than males and showed less cell death. These differences were significant for β cell function, as female islets maintained better insulin secretion than males in an ER stress context. Given the profound differences that we observed between the sexes in the transcriptional response to thapsigargin and the known links between ER stress and T2D pathogenesis, these findings provide additional insight into potential mechanisms underlying the differential risk of T2D between men and women. iv Lay Summary Type 2 diabetes (T2D) is a disease where your body either does not make enough insulin or does not respond to it effectively. This causes high blood sugar levels in people living with T2D, which is linked with serious health problems. One important risk factor for T2D is whether an individual is male or female. Approximately 40% more men develop T2D than pre-menopausal women. While lifestyle and cultural factors play a role in this male-biased risk, these differences are also present in animal models. This suggests that biological sex contributes to the male-biased T2D risk. Unfortunately, females are underrepresented in research; thus, much remains to be discovered about how diabetes progression is different in males and females. In the present study, we found that male and female insulin-producing beta cells responded differently under normal conditions, and female beta cells were more resilient to cellular stresses. v Preface Unless otherwise specified I designed, performed, and analyzed all experiments presented under the supervision of Dr. Elizabeth J. Rideout and Dr. James D. Johnson. Søs Skovsø performed the in vivo physiology experiments. To analyze the Ins2 gene activity of Ins2GFP/WT β cells, I used custom scripts developed by Jamie Chu. Yi Han Xia performed bioinformatic analysis and data visualization. Betty Hu performed the initial islet isolations. Rachael Bartlett collected the data and organized the sequencing of the initial RNAseq data set. Charlotte Chao created a custom R script to analyze the RNAseq data set on thapsigargin-treated islets. Jiashuo Aaron Zhao compiled human islet perifusion data from the Human Pancreas Analysis Program. The data presented in Chapter 3 is currently in the process of submission to a scientific journal. All animal work conducted was approved by the University of British Columbia Animal Care Committee protocol #A21-0135. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Symbols ...............................................................................................................................x List of Abbreviations ................................................................................................................... xi Acknowledgements .................................................................................................................... xiv Dedication .....................................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Overview of Type 2 Diabetes Pathology ........................................................................ 1 1.1.1 Global Burden of Type 2 Diabetes ............................................................................. 1 1.1.2 Type 2 Diabetes Pathology ......................................................................................... 1 1.2 Diabetes Risk .................................................................................................................. 2 1.2.1 Populations .................................................................................................................. 2 1.2.2 Age .............................................................................................................................. 2 1.2.3 Gender and Lifestyle ................................................................................................... 3 1.2.4 Biological Sex ............................................................................................................. 4 1.2.4.1 Sex Hormones ..................................................................................................... 4 1.2.4.2 Sex Chromosomes .............................................................................................. 6 1.2.5 Male Bias in Diabetes Research and Treatment Outcomes ........................................ 7 1.3 Physiological Mechanisms of Sex Differences in T2D .................................................. 8 1.3.1 Insulin Resistance ....................................................................................................... 8 vii 1.3.2 Glucose Metabolism ................................................................................................... 9 1.4 Pancreatic β cell ............................................................................................................ 12 1.4.1 Sex Hormones Effect on β Cell Function ................................................................. 13 1.4.1.1 Androgens Effect on β Cell Function ............................................................... 13 1.4.1.2 Estrogens Effect on β Cell Function ................................................................. 14 1.4.2 β Cell Transcriptome ................................................................................................ 14 1.4.3 Animal Models of β cell Dysfunction ....................................................................... 16 1.4.4 β Cell Stress Responses ............................................................................................ 17 1.4.4.1 PERK ................................................................................................................ 18 1.4.4.2 ATF6α ............................................................................................................... 19 1.4.4.3 IRE1α ................................................................................................................ 20 1.4.4.4 ER stress-induced apoptosis ............................................................................. 21 1.4.5 Sex Hormones Effects on β Cell Stress Response .................................................... 22 1.4.5.1 Androgens Effect on β Cell Stress Response .................................................... 22 1.4.5.2 Estrogens Effect on β Cell Stress Response ..................................................... 23 1.5 Thesis Investigation ...................................................................................................... 25 Chapter 2: Materials and Methods ............................................................................................26 2.1 Animals ......................................................................................................................... 26 2.2 Islet Isolation, Culture, Dispersion and Treatment. ...................................................... 26 2.3 Analysis of protein synthesis ........................................................................................ 27 2.4 Live Cell Imaging ......................................................................................................... 27 2.5 Western Blot ................................................................................................................. 28 2.6 Islet Secretion and Content ........................................................................................... 29 viii 2.7 In vivo Analysis of Glucose Homeostasis and Insulin Secretion .................................. 30 2.8 Data from the Human Pancreas Analysis Program....................................................... 30 2.9 RNAseq ......................................................................................................................... 30 2.10 Proteomics..................................................................................................................... 31 2.11 Statistical Analysis ........................................................................................................ 32 Chapter 3: Results........................................................................................................................33 3.1 Sex-Specific Transcriptional Profiles in ND and T2D Human Islets ........................... 33 3.2 Sex-Specific Transcriptional Profiles in Mouse Islets .................................................. 34 3.3 Female Islets are More Resilient to ER Stress .............................................................. 35 3.4 Female Islets Retain Greater β Cell Function During ER Stress .................................. 38 3.5 Sex-Specific Transcriptional and Proteomic Profiles during ER Stress ....................... 40 Chapter 4: Discussion ..................................................................................................................60 4.1 Sex Differences in Mouse and Human Gene Expression ............................................. 60 4.2 Sex Differences in ER Stress-Associated Phenotypes .................................................. 61 4.3 Male-Female Differences in β Cell Function ............................................................... 63 4.4 Male-Female Transcriptional Differences in Response to ER Stress ........................... 65 4.5 Overall Significance...................................................................................................... 67 4.6 Limitations .................................................................................................................... 68 4.7 Future Directions .......................................................................................................... 69 References .....................................................................................................................................70 ix List of Figures Figure 1: Transcriptomic analysis of ND and T2D human β cells ............................................... 43 Figure 2: Pathway enrichment analysis of ND and T2D human β cells ....................................... 43 Figure 3: Glucose-stimulated insulin secretion of ND and T2D human β cells ........................... 44 Figure 4: Transcriptomic analysis of mouse islets ........................................................................ 46 Figure 5: ER stress-induced protein synthesis repression in mouse islets .................................... 48 Figure 6: ER stress-induced suppression of Ins2 gene activity in mouse islets............................ 49 Figure 7: Live-cell imaging cell death assay of ER stress-induced mouse islets ......................... 51 Figure 8: Protein markers of ER stress in ER stress-induced mouse islets .................................. 52 Figure 9: In vitro mouse β-cell function ....................................................................................... 54 Figure 10: In vivo mouse physiology ............................................................................................ 55 Figure 11: Transcriptomic and proteomic analysis of ER stress-induced mouse islets ................ 57 Figure 12: Pathway enrichment analysis of ER stress-induced mouse islet RNAseq data .......... 58 Figure 13: ER Stress Resiliency Model ........................................................................................ 59 x List of Symbols α Alpha β Beta δ Delta xi List of Abbreviations ANOVA Analysis of variance AR Androgen receptor ArKO Aromatase knockout βArKO β cell Aromatase knockout ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 Bak BCL2 antagonist/killer 1 Bax BCL2 associated X, apoptosis regulator BiP Immunoglobulin binding protein CHOP C/EBP homologous protein DAPI 4',6-diamidino-2-phenylindole DIA Data independent acquisition E2 Estradiol eIF2α Eukaryotic initiation factor 2 alpha ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ERα Estrogen receptor alpha ERβ Estrogen receptor beta ERAD Endoplasmic reticulum-associated protein degradation Essrg Estrogen-related receptor gamma FBS Fetal bovine serum GADD34 Growth arrest and DNA damage-inducible protein 34 xii GO Gene ontology GSIS Glucose-stimulated insulin secretion HbA1c Hemoglobin A1c HBSS Hanks balanced salt solution hIAPP Human islet amyloid polypeptide HOMA-IR Homeostasis model assessment of insulin resistance HPAP Human Pancreas Analysis Program IBMX Isobutylmethylxanthine IFG Impaired fasting glucose i.p. Intraperitoneal IRE1 Inositol requiring enzyme 1 IGT Impaired glucose tolerance JNK c-Jun N-terminal kinase KEGG Kyoto Encyclopedia of Genes and Genomes KRB Krebs-Ringer HEPES-buffered solution mRNA Messenger ribonucleic acid ND Non-diabetic OGTT Oral glucose tolerance test OPP O-propargyl-puromycin PBS Phosphate-buffered saline PI Propidium iodide Pparg Proliferator-activated receptor gamma RIDD Regulated Ire1-dependent decay xiii RNA ribonucleic acid RPMI Roswell park memorial institute medium SERCA Sarcoplasmic/endoplasmic reticulum calcium ATPase SEM Standard error of the mean scRNA-seq Single-cell RNA sequencing T1D Type 1 diabetes T2D Type 2 diabetes Tg Thapsigargin UPR Unfolded protein response PERK PKR-like ER kinase Wfs1 Wolframin ER transmembrane glycoprotein XBP1 X-box binding protein 1 XBP1s Spliced X-box binding protein 1 xiv Acknowledgements Graduate school has been a life-changing learning experience, surrounded by incredible mentors and friends. First and foremost, I would like to thank my supervisors Dr. Elizabeth Rideout and Dr. James Johnson who created an environment for me to succeed. Their guidance and unrelenting support helped me to become a better scientist, writer, and communicator. I am immensely grateful to Dr. Elizabeth Rideout for first introducing me to scientific research during my undergraduate degree and continuing to mentor and support me throughout my Master’s. I would also like to extend my deepest gratitude to Dr. James Johnson for allowing me the opportunity to expand my research to mice, lending his expertise and generosity to share resources, support and foster a collaborative environment. I would like to thank Dr. Angela Devlin, Dr. Eric Jan, and Dr. Dan Luciani for participating in my supervisory committee and offering their wisdom, guidance, and valuable feedback. I would also like to acknowledge each member of the Rideout and Johnson labs: thank you for all of your support over the years with experiments, engaging discussions, and lab socials. A special thanks to Jason Millington and Lianna Wat for inspiring my exploration into sex differences research, instilling a passion for research as mentors, and being great friends. I would like to thank my parents for their unwavering support of my professional goals. They have always encouraged me to pursue my passions and continue to support me through life’s many challenges. Lastly, I would like to thank my partner, Samantha, for being by my side through the highs and lows of research and life, always believing in me, and being my best friend. xv Dedication To Sam, for all your love and support.1 Chapter 1: Introduction 1.1 Overview of Type 2 Diabetes Pathology 1.1.1 Global Burden of Type 2 Diabetes Diabetes is a metabolic disorder that affects approximately 537 million people worldwide, a number that is expected to increase to 783 million by 2045 (1). There are two main types of diabetes; type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D accounts for approximately 5-10% of diabetes cases, while type 2 diabetes (T2D) accounts for the majority of diabetes cases. In Canada alone, 9% of the population (3.5 million people) are living with T2D. When considering diagnosed, undiagnosed, and prediabetes combined, the estimated prevalence increases to 26% (10.3 million) (2). These numbers are expected to increase annually with projections for 2031 estimating that 4.5 million Canadians will be diagnosed with T2D and 12.4 million will be living with T2D or prediabetes (2). 1.1.2 Type 2 Diabetes Pathology T2D is a multifactorial disease; however, it is well established that insulin resistance and hyperinsulinemia play critical roles in the progression of diabetes pathology. Insulin resistance results in elevated insulin production to compensate for decreased insulin sensitivity to maintain glucose homeostasis (3, 4). Hyperinsulinemia, excess circulating insulin, can both result from insulin resistance and play a causal role in the development of insulin resistance (3, 5). Both insulin hypersecretion and insulin resistance occur during the progression of T2D and can consequently lead to exhaustion of insulin-producing β cells (3). Without pharmacological intervention, β cells become unable to sustain adequate insulin secretion to maintain euglycemia; consequently, blood sugar levels rise leading to a clinical diabetes diagnosis accompanied by a 2 decrease in β cell mass and/or a decline in β cell function (6, 7). Therefore, T2D is characterized by chronically elevated blood glucose levels, insulin resistance, and β cell failure. 1.2 Diabetes Risk 1.2.1 Populations It is well established that in Canada and globally, the prevalence of T2D is higher in men than women (8–15). Of note, there are regions in which the prevalence of T2D is reversed. For instance, in South Africa, the Caribbean, and South Asia, the prevalence of T2D is higher in women than men (15, 16). In addition, different populations are at a higher risk of developing T2D. In Canada, Indigenous populations have a higher prevalence of T2D than the general population, and it is more prevalent among Indigenous women than Indigenous men (17, 18). 1.2.2 Age The risk of developing T2D increases with age and the prevalence of T2D varies among different age categories. The SEARCH study found that in youth (< age 10-19 years; n=2846) more girls were diagnosed with T2D than boys (19). Studies on diabetes prevalence—the majority of which have been performed on adults—have found that the prevalence of T2D is higher in adult men than in adult women (9–15). The male-biased prevalence tends to disappear with age after menopause (8), yet the T2D prevalence in men and women has been found to peak at different ages: 65-69 in men and 70-79 in women (8). In some countries, a higher prevalence of T2D has been found in older men. For instance, a population-based prospective study in Sweden comprising participants in the Healthy Aging Initiative project (age=70, n=1393) found a higher prevalence of T2D in older men (14.6%) than in older women (9.1%) (20). 3 1.2.3 Gender and Lifestyle Gender differences in men and women arise from socio-cultural factors including, but not limited to, differences in nutrition, behaviour, stress, environment, and lifestyle (21, 22). Socio-cultural factors may differentially impact the risk of developing T2D in men and women. However, these differences have been challenging to isolate (23). Furthermore, women have a higher prevalence of healthcare-seeking behaviour for both physical and mental health concerns (24, 25). Studies on psychosocial factors in individuals with T2D have found that men display a greater number of factors associated with treatment success, including a lower prevalence of depression and anxiety, and a more active problem-solving approach (26, 27). In contrast, women have lower adherence to medication and worse glycemic control. These outcomes in women have been attributed to lower satisfaction with social support, reduced quality of life, lower sexual desire, and a higher prevalence of negative emotions despite greater knowledge of diabetes management (26, 28). Another explanation for lower medication adherence may be the higher prevalence of hypoglycemia in T2D women treated with insulin (29), as hypoglycemia itself is associated with a reduced quality of life (30). Low physical activity is a well-known risk factor for the development of T2D (3) and many randomized control trials have been conducted to determine the impact of lifestyle interventions. These trials found that dietary changes and regular exercise delayed or prevented the progression from prediabetes to diabetes (31–33). While increasing physical activity improved glucose tolerance, long-term adherence to lifestyle interventions remains a challenge. A comprehensive review of lifestyle intervention found the beneficial effect of reducing diabetes incidence was reduced after lifestyle interventions stopped (34). However, long-term follow-up 4 studies found that diabetes incidence remained lower in intervention groups (35–37). Gender differences exist in exercise habits. Globally, men are more physically active than women (15, 38, 39). Despite this difference, a large meta-analysis of all randomized control trials that included sex-specific data on lifestyle interventions found that lifestyle interventions had similar effectiveness in men and women with prediabetes (40). Therefore, other aspects of male-female differences need to be explored such as the higher incidence of T2D in adult men. 1.2.4 Biological Sex While some of the differential T2D risk is explained by gender, one overlooked factor that affects T2D risk is biological sex (41, 42). Biological sex is known to influence the differential T2D risk since the male bias in diabetes phenotypes persists in animal species (43–51). 1.2.4.1 Sex Hormones Clinical and experimental studies have identified a role for sex hormones in regulating T2D risk. The primary sex hormones of males and females are testosterone and estradiol, respectively (52). After puberty, the actions of estrogens and androgens contribute to male-female differences in physiology and metabolism (53). Insight into the role of sex hormones in T2D risk factors and T2D incidence has been elucidated by studies in humans and mice. In humans, studies on menopause and menopausal hormone replacement therapy have provided insight into the protective role of estrogen in T2D. The EPIC-InterAct prospective study (n=8099) found that early menopause, before 40 years of age, was associated with a greater risk of developing T2D compared with women who underwent menopause between 50-54 years of age (54). Furthermore, meta-analyses of hormone-replacement therapy trials in 5 postmenopausal women found that hormone-replacement therapy reduced the incidence of T2D (55, 56). Additionally, estrogen and progestin hormone therapy reduced the incidence of T2D in postmenopausal women compared to the placebo group by 20% in a one-year follow-up (57). Overall the consensus is that estrogen, endogenous or exogenous, reduces the development of obesity and metabolic diseases, including T2D (55, 58). In mice, many elegant studies have demonstrated that estrogens play an important protective role in female mice (59–67). For instance, ovariectomies performed on control and diabetic mice to study estrogen deficiency, revealed that ovariectomies led to worsened metabolic profiles including an increase in fasting blood glucose, serum insulin, and insulin resistance (68, 69). Supplementing ovariectomized mice with estradiol (E2), reduced the incidence of diabetes (68). Conversely, supraphysiological levels of E2 have been implicated in insulin resistance and may even increase the risk of developing T2D (70, 71). Functional estrogen is also important for males. Aromatase, an enzyme responsible for converting androgens into estrogens, is necessary to maintain normal physiology in both males and females (72). Males with loss-of-function mutations in the aromatase gene developed insulin resistance along with other metabolic abnormalities which were alleviated with low-dose conjugated estrogen replacement therapy (73, 74). Therefore, physiological levels of estrogen and progesterone are important in both sexes. Beyond estrogen, the maintenance of appropriate levels of testosterone is important in both sexes. Men with prostate cancer treated with androgen deprivation therapy were rendered hypogonadal and became more insulin resistant (75, 76). Men receiving androgen deprivation therapy had a higher incidence of developing T2D on average than the general population (75, 77). A cross-sectional study in men showed that independent of obesity and metabolic syndrome, 6 prediabetes was associated with low testosterone levels (<300 ng/dL) (78). Testosterone supplementation in men with hypogonadism and prediabetes prevented the progression to T2D and improved glucose metabolism (79). Testosterone imbalances not only affect men, as women with high testosterone levels and transgender women are at a higher risk of developing T2D (80, 81). These observations provide evidence that an appropriate physiological range of sex hormones is important in maintaining normal physiology in both sexes; however, these whole-body studies do not address how sex hormones affect physiological mechanisms. 1.2.4.2 Sex Chromosomes In addition to sex hormones, sex chromosomes also play a role. The effect of sex chromosomes can be described as the direct effect of genes on the X and Y chromosomes that produce differences in the cellular function of XX female and XY male cells (53). Individuals with a sex chromosome complement, other than XX or XY, impact the risk of developing T2D. Turner syndrome is caused by a complete or partial loss of an X chromosome, and only affects females. People living with Turner syndrome are at a higher risk of developing T2D and the majority of these individuals experience abnormal glucose metabolism including impaired glucose tolerance, hyperinsulinemia, and insulin resistance (82). Klinefelter syndrome is a genetic condition where males are born with an additional X chromosome. People living with Klinefelter syndrome are also at a higher risk of developing insulin resistance and diabetes. Interestingly, unlike Turner syndrome where the incidence of T1D is increased to a greater extent than the incidence of T2D, the majority of diabetic cases in Klinefelter syndrome were T2D (82, 83). 7 1.2.5 Male Bias in Diabetes Research and Treatment Outcomes Many preclinical researchers do not include female rodents in studies and consequently are underrepresented in research. One common misconception is that the female reproductive cycle results in increased variability compared to males (84). However, a meta-analysis of mice used in research studies found that females had similar or less data variability than males (85). While there has been an increase in studies using both males and females, unfortunately, many of these studies fail to analyze their data by sex and others continue to use a single-sex (86–88). Consequently, this can lead to findings that are only relevant to a single-sex since there are fundamental differences in the regulation of metabolic homeostasis in males and females (89–95). These male-female differences in metabolic regulation can impact the development of T2D and the response to pharmacological intervention. In fact, in adults after treatment with lifestyle intervention, metformin or sulfonylurea, women experienced a greater reduction in body weight than men. In contrast, men had a greater reduction of Hba1c levels than women following lifestyle intervention and metformin (96). In addition, thiazolidinediones (insulin sensitizers) have been shown to have a stronger effect in women than in men (97). Importantly, the side effects of diabetes drugs differ in men and women. For instance, metformin treatment had a beneficial impact on heart metabolism in women, shown by a reduction in fatty acid utilization and increased glucose uptake by the heart, whereas it had the opposite effect in men (98). To have a complete understanding of metabolic disease, including diabetes pathology, it is critical to study both sexes. Furthermore, prospective studies are needed to investigate sex differences in diabetes treatments. 8 1.3 Physiological Mechanisms of Sex Differences in T2D 1.3.1 Insulin Resistance While it is well established that there are sex differences in T2D incidence and treatment outcomes, a lot remains to be uncovered to identify underlying causes for these male-female differences. One mechanism that has been proposed is insulin resistance. Insulin resistance is a condition in which muscle, liver, and adipose tissues are unable to respond to circulating insulin (3), as a result, blood glucose levels increase. Therefore, insulin resistance is one factor that contributes to diabetes. Insulin resistance in adipocytes results in a decreased uptake of circulating lipids and an increased breakdown of triglycerides. This results in elevated circulating free fatty acids which can further increase insulin resistance (99, 100). Another factor associated with higher insulin resistance is a greater body fat to lean mass ratio (101–104). Overall, increased lipid accumulation in skeletal muscle and liver, a greater adipose mass, and increased circulating free fatty acids can all promote insulin resistance (53, 105). While these factors are well defined, less is known about sex differences in insulin resistance. What is known is that despite women possessing a greater disposition of factors known to promote insulin resistance, women are more or as insulin sensitive as men (53, 106). Studies encompassing a large European population have found that among healthy individuals, women have higher insulin sensitivity and lower fasting insulin than men (106, 107). In addition, data from human studies are consistent with rodent data that showed female mice have greater insulin sensitivity than male mice (108, 109). Moreover, across dozens of strains of mice fed a high-fat diet, female mice had lower levels of HOMA-IR (Homeostasis model assessment of insulin resistance, an estimate of insulin resistance), indicating female mice were less insulin resistant than male mice (110). Interestingly, in both a regular and a high-fat diet, gonadectomies decreased HOMA-IR in male 9 mice but increased HOMA-IR in female mice. This indicates gonadal hormones impact insulin resistance and suggests estrogen may be insulin-sensitizing in females which could partly explain the lower HOMA-IR in females across the many strains of mice (110). In humans, sex differences in insulin action change throughout puberty, a key developmental stage where changes in sex hormone levels occur (111). During puberty, a transient increase in insulin resistance, fasting glucose and fasting insulin occurs (111–113). While the increase in fasting glucose follows a similar trajectory in both males and females, the peak increase in fasting insulin and insulin resistance is greater in females than in males (112, 114). In both sexes, fasting insulin and insulin resistance plateau at equivalent levels by approximately 16 years of age (112, 115). In addition, studies in healthy young women found that insulin resistance also fluctuates throughout the menstrual cycle (116, 117) and during pregnancy (118, 119). Therefore, changes in sex hormone levels may be an important component in differences in insulin sensitivity. In addition to sex hormones' impact on insulin sensitivity, sex chromosomes can also influence insulin sensitivity. The four-core genotype mouse model enables studies to distinguish between the effects of sex hormones and sex chromosomes. Data from gonadectomized male and female mice on a high-fat diet revealed a 2-fold increase in fasting insulin levels and HOMA-IR in XX male and XX female mice but not in XY mice. This demonstrates the sex chromosome complement affects insulin resistance (120). 1.3.2 Glucose Metabolism Glucose metabolism involves multiple processes to maintain appropriate levels of blood glucose through the storage and breakdown of glucose and glycogen. During fasted states, glycogen is 10 broken down and released as glucose as energy for the cells. After a meal, insulin is released and glucose is taken up by fat and muscle cells, thereby lowering blood glucose levels. In people with prediabetes, insufficient insulin is released to compensate for the degree of insulin resistance and consequently, blood glucose levels remain elevated. Therefore, insulin resistance is a major driving factor in the development of impaired glucose metabolism which is a risk factor for progressing to overt diabetes (23). Abnormal glucose metabolism is revealed by impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) (121–123). Fasting glucose mostly reflects endogenous glucose production, whereas glucose levels following an oral glucose tolerance test (OGTT) reflect the increase in blood glucose after a glucose challenge (124). Interestingly, the prevalence of IFG and IGT differ in men and women. IFG is more prevalent in men, while IGT is more prevalent in women (121, 125). Data from the World Health Organization showed that the global prevalence of IGT is higher in women than in men from ages 20 to 75 (125). Furthermore, a comprehensive meta-analysis encompassing multiple populations and ages (n=51,520), found that IFG was much more common in men, while IGT was slightly more common in women (121). Specifically, from the DECODE (n=26190; age 17-92) and DECODA studies (n=16,931; age 30-89) the prevalence of IFG was approximately 1.5-3 times higher in men than women across all age groups except for European populations between the ages of 70-79 and Asian populations between the ages of 80-89. The prevalence of IGT was higher in women than men except for European populations over the age of 80 and Asian populations over the age of 60 (121). While the reasons for this sex difference are unknown, researchers have suggested potential explanations to explain the sex bias (23, 53). 75 grams of glucose is given during a standard OGTT. Therefore, one proposed explanation is that healthy women have a prolonged gut glucose absorption compared to men; thus, the longer gut glucose 11 half-life in women may explain the higher glucose concentrations at the end of an OGTT (124). Another explanation for the higher prevalence of IGT in women is that they ingest more glucose per kg of lean mass than males (126). Indeed, when individuals were administered a glucose dose proportional to their body weight and insulin action was normalized to lean muscle mass, insulin action was found to be greater in women than men. However, when lean mass was not taken into account, the opposite conclusion was reached due to the lower proportion of insulin-sensitive muscle in women than in men (126). In postmenopausal women without diabetes, randomized control trials have found that hormone replacement therapy either decreased or had no effect on fasting glucose or insulin levels (55, 127–129). A randomized control trial of postmenopausal women with coronary disease and varying degrees of glucose tolerance (no diabetes, IGT, T2D) were treated with placebo or hormone therapy. The investigators found a significant worsening of fasting glucose levels in the placebo group compared to the hormone therapy group (130). Interestingly, several studies on the effect of hormone therapy on glucose metabolism also found hormone therapy increased post-challenge glucose concentrations (130–132), a similar phenotype observed in healthy women without diabetes (124), which may indicate hormone therapy delayed glucose clearance. A large study on individuals with different degrees of impaired glucose tolerance across a broad age range (n=1572) found that women have better glucose metabolism than men; however, the sex advantage in normal glucose tolerance disappeared with age (107). Additionally, females' sex advantage also decreased as glucose tolerance declined during diabetes progression (89), and was absent in individuals with T2D (107). Interestingly, among individuals newly diagnosed with T2D, glucose metabolism was not different between young and older women. In contrast, 12 men exhibited a clear decline in glucose metabolism with age (107). Therefore, in T2D, an adverse metabolic profile may already exist in women at an early age. In summary, healthy females have higher insulin sensitivity, lower fasting glucose levels, higher 2-hour glucose levels following an OGGT, prolonged gut glucose absorption and a higher incidence of IGT than males (133). While it is clear that many aspects of glucose homeostasis are regulated differently in males and females, studies into the underlying mechanisms that mediate the effects of sex chromosomes and sex hormones are lacking. 1.4 Pancreatic β cell Human studies on young, healthy men and women without diabetes have also found equivalent insulin sensitivity between the sexes, yet differences in glucose-stimulated insulin secretion persisted (134). This suggests that functional differences exist in β cells of men and women which are caused by factors other than differences in insulin sensitivity. β cells are specialized cells located in the pancreatic islets of Langerhans. Islets are composed of a variety of cell types. The four main endocrine cell types are insulin-producing β cells, glucagon-producing α-cells, somatostatin-producing δ-cells, and pancreatic polypeptide-producing cells (3). β cells make up the majority of the islet cells, 50-70% and 60-80% in humans and mice, respectively. α cells are the second most prevalent cell type contributing 20-40% and 10-20% of the total cells in humans and mice, respectively (135). Together, α and β cells play a major role in the maintenance of glucose homeostasis by regulating the secretion of insulin and glucagon, respectively. 13 1.4.1 Sex Hormones Effect on β Cell Function Much of our knowledge of sex differences in β cell function comes from studies on the action of gonadal hormones. While the primary function of β cells is to produce and secrete insulin in response to glucose, insulin release from β cells can also be augmented by sex hormones. 1.4.1.1 Androgens Effect on β Cell Function Testosterone actions are mediated via the androgen receptor (AR). In mice and human islets, the addition of testosterone or dihydrotestosterone, an AR agonist, to cultured islets increased glucose-stimulated insulin secretion (GSIS) greater than in untreated islets (136, 137). Interestingly, testosterone induced GSIS in both male and female islets (137). Both male and female islets express the AR, however, its expression is more important for males than females. Indeed, β cell-specific AR knockout (βARKO) in male mice exhibited a mild worsening of glucose tolerance and a significant decrease in fasting insulin levels when fed a chow diet. Cultured male βARKO islets were less responsive to both low and high glucose stimulation than wild-type islets and were unresponsive to dihydrotestosterone (136). This phenotype was exacerbated when mice were fed a western diet and further impaired glucose tolerance (136). In contrast, in βARKO females, both chow and western diets had mild effects on glucose tolerance and GSIS. The effect of AR knockout in male β cells was reproduced in cultured human islets. The stimulatory effect of dihydrotestosterone was abolished when islets were treated with flutamide, an AR antagonist (136). The importance of the AR for proper β cell function has been demonstrated by transcriptomic analysis of βARKO male mice. Androgen receptor-deficient β cells exhibited dysregulated gene expression of β cell genes involved in the inflammatory 14 response, stress response and insulin secretion, demonstrating the AR is important for male β cell function (138). 1.4.1.2 Estrogens Effect on β Cell Function Estrogens actions are mediated via β cells two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor β (Erβ), which are expressed in both mice and human β cells (139). While ERα and Erβ have similar roles in glucose metabolism, ERα is primarily important for insulin biosynthesis, lipid homeostasis, and β cell survival, whereas Erβ enhances GSIS (64, 140, 141). In mice and human cultured islets, E2 treatment increases GSIS (64, 140–143). In contrast, mice that have reduced estradiol production or reduced estradiol action due to aromatase deficiency or ERα deficiency respectively are more susceptible to developing diabetes (61, 65, 144). This can be tested with streptozotocin which rapidly damages β cells inducing β cell death and hyperglycemia enabling the study of diabetes onset within days of injection (145–147). Indeed, aromatase knockout mice (ArKO) of both sexes are more susceptible to developing streptozotocin-induced diabetes than wild-type mice (61, 64). Estradiol treatment was shown to reduce diabetes incidence in ArKO mice and improve β cell insulin release (61). In addition, ERα knockout mice (αERKO) maintained normal insulin release, however; unlike wild-type mice, αERKO mice did not respond to E2 to increase insulin content (61, 64, 141). Together, these experiments demonstrate estrogen and ERα are important for β cell function. 1.4.2 β Cell Transcriptome RNAseq has provided significant insight into islet function through the analysis of gene expression data. Advances in single-cell RNA sequencing (scRNA-seq) made it possible to 15 examine healthy islet cell transcriptomes and transcriptomic changes associated with T2D at the level of the β cell. This advanced our understanding of how β cells are different under normal circumstances and provided insight into diabetes-induced β cell dysfunction. This made it possible to identify dysregulated transcripts and signaling pathways in diabetes (148). While many RNAseq studies have been performed on human islets (149–154), few analyzed the data separately by sex. A meta-analysis of scRNA-seq on donors with and without diabetes analyzed data from both men and women (155). Interestingly β cells from donors without diabetes revealed sexually dimorphic gene expression which was differentially impacted by age. Indeed, impaired β cell function was associated with aging in males but not females. Specifically, genes associated with β cell identity had higher expression in young adult than old adult males (155). In support of sex-specific gene expression differences in humans, RNAseq studies in mice report differential gene expression between male and female islets and β cells (156, 157). Bulk RNAseq on fluorescently sorted β cells revealed female β cells expressed higher levels of transcription factors associated with β cell function and β cell identity (157). Similar findings were uncovered with scRNA-seq on chow-fed ND mice and high-fat diet-induced T2D mice. A comparison of ND to high-fat diet-induced T2D mice revealed sex-biased genes associated with endoplasmic reticulum (ER) stress that may be altered in the progression of T2D (156). Together, these studies suggest the mechanisms underlying β cell dysfunction may not be shared between the sexes. However, a thorough analysis with more ND and T2D donors of each sex and a higher-powered mouse islet RNAseq analysis is needed to confirm and extend these initial data. 16 1.4.3 Animal Models of β cell Dysfunction β cell stress and β cell failure have been strongly linked with many forms of diabetes in humans (158–163). Interestingly, many of the observed phenotypes in mouse models of β cell dysfunction are more severe and occur with a rapid onset in males compared to female mice. A well-established mouse model of β cell failure is the Akita mouse. Abnormally high levels of misfolded insulin accumulate in β cells of Akita mice due to a missense mutation in the Ins2 gene (51, 164). Akita mice rapidly develop β cell dysfunction and hyperglycemia with a faster progression and more severe phenotype in males than females (51, 165). Another mouse model with a mutation in the Ins2 gene is the Munich mouse. Munich mice were shown to develop β cell function with a worse diabetic phenotype in males than females. Male Munich mice exhibited greater hyperglycemia, insulin resistance and reduction of β cell mass than wild-type mice. In contrast, females exhibited no changes in insulin resistance or β cell mass (166). Recently, another mouse model with an Ins2 mutation was discovered, the KINGS mouse. Like Akita and Munich mice, KINGS mice exhibited β cell dysfunction and impaired glucose tolerance with a male-biased phenotype. Male mice were hyperglycemic shortly after weaning, whereas females developed mildly elevated glycemia (50). Defects in other aspects of β cell function have also been shown to exhibit worse outcomes in male mice. Islet amyloid deposits are commonly found in patients with T2D (167), therefore transgenic mice expressing human islet amyloid polypeptide (hIAPP) have been employed to study the role of islet amyloid in the pathogenesis of T2D. hIAPP is co-secreted with insulin and plays a role in glucose homeostasis; however, in T2D hIAPP aggregates to form islet amyloid deposits (168). Islet amyloid deposits and hyperglycemia have been observed in the majority of male transgenic mice but rarely in females (169). In addition, male but not female transgenic mice experience β cell failure 17 demonstrated by insulin deficiency and loss of β cell mass (170). The worse diabetic phenotypes in males in these mice models of β cell failure suggest males and females differ in how their β cells respond to stress and/or in the progression to β cell failure. 1.4.4 β Cell Stress Responses While much remains to be learned about the mechanisms underlying β cell dysfunction, there is a growing appreciation that ER stress and the unfolded protein response (UPR) can contribute to diabetes pathogenesis (171). UPR markers are increased in β cells of T2D donors as well as in mouse models of T2D (172–175). β cells are naturally exposed to ER stress due to the large demand for insulin synthesis and processing by the ER (173, 176–178). ER stress results when unfolded and misfolded proteins including insulin accumulate and exceed the folding capacity of the ER. A process that aids in restoring ER protein homeostasis is the UPR. The UPR is an adaptive response and works to restore ER homeostasis when unfolded and misfolded proteins accumulate inducing ER stress. Activation of the UPR attenuates protein translation, induces ER-associated degradation (ERAD), and increases chaperone synthesis. When ER stress is alleviated, UPR feedback control mechanisms switch off UPR master regulators to prevent harmful UPR hyperactivation. However, when the adaptive mechanisms of the UPR are unable to alleviate ER stress and prolonged ER stress signaling occurs, the UPR switches to a maladaptive or terminal UPR that results in cell death (172, 179–181). The UPR senses ER stress through three ER transmembrane proteins. PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol requiring enzyme 1 (IRE1) (177). Under unstressed conditions, immunoglobulin binding protein (BiP) is bound to the 18 luminal domains of PERK, ATF6 and IRE1. However, upon the accumulation of misfolded proteins during ER stress, BiP is released from the ER stress sensors leading to their subsequent activation (182). 1.4.4.1 PERK PERK is a transmembrane kinase and is activated by the release of BiP from its ER luminal domain. PERK dimerizes, autophosphorylates, then activates eukaryotic initiation factor α (eIF2α) via phosphorylation leading to global translation attenuation thereby decreasing the ER protein load. Phosphorylation of eIF2α is also required for the selective upregulation of several mRNAs, including activating transcription factor 4 (ATF4). ATF4 induces the expression of genes involved in amino acid import and resistance to oxidative stress (183). ATF4 expression also upregulates C/EBP homologous protein (CHOP). During adaptive UPR, CHOP may act in a negative feedback loop to dephosphorylate eIF2α via growth arrest and DNA damage-inducible protein (GADD34) (184). During terminal UPR, CHOP and ATF4 induce pro-apoptotic genes which leads to apoptotic cell death (181). Impaired PERK pathway signaling leads to β cell failure as evidenced by loss-of-function mutations in PERK and eIF2α (171). Mutations in PERK or eIF2α prevent the attenuation of protein synthesis, resulting in elevated protein accumulation and increased levels of ER stress (185, 186). Thus PERK/eIF2α signaling is important for the cells' ability to adapt to ER stress. PERK signaling is also important for proper proinsulin folding. Acute inhibition of PERK signaling with GSK2606414, a specific inhibitor of PERK kinase activity, resulted in a rapid accumulation of misfolded proinsulin in rat islets (187, 188). Accumulation of misfolded proinsulin following PERK inhibition has also been observed in human islets and pancreatic β 19 cell line, INS-1 (189, 190). Additionally, a PERK knockout mouse model which is a model of the human genetic disease, Wolcott-Rallison syndrome, exhibits an accumulation of misfolded proinsulin within the ER of their β cells (191, 192). Interestingly, while both PERK activity and eIF2α phosphorylation are required to prevent an abnormal increase in insulin translation in response to a high glucose load, eIF2α phosphorylation but not PERK activity is required to maintain normal levels of insulin translation in low glucose (193, 194). Lastly, PERK/eIF2α signaling has been shown to be a positive regulator of ER chaperone abundance and ERAD (177, 189, 191, 192). 1.4.4.2 ATF6α ATF6 is a transcription factor that is cleaved from the ER membrane upon the dissociation of BiP and translocated to the Golgi apparatus where it is cleaved by two proteases. The processed form of ATF6 is translocated to the nucleus where it binds to promoters of UPR-responsive genes to stimulate transcription (195–197). Specifically, ATF6 induces the expression of chaperones and UPR mediators such as BiP and X-box binding protein 1 (Xbp1) (196). While ATF6 has two isoforms, ATF6α and ATF6β, only ATF6α is responsible for the transcriptional induction of ER chaperones. Additionally, unlike ATF6β, ATF6α is a strong transcriptional activator (198, 199). Since ATF6α is a positive feedback regulator of the UPR, ATF6α is rapidly degraded to prevent UPR hyperactivation (200). Further insight into ATF6 regulation comes from mutations in the Wfs1 (wolframin ER transmembrane glycoprotein) gene, which causes Wolfram syndrome (201). WFS1 is involved in the negative regulation of UPR signaling via ATF6α degradation. WFS1 deficiency increases ATF6α protein levels, results in hyperactivation 20 of the UPR and increases β cells susceptibility to dysfunction and ER stress induced apoptosis (202–204). While excessive ATF6 signaling is detrimental to β cells, ATF6 is important in preserving β cell function when exposed to ER stress. Atf6α knockout mice show no defects in glucose homeostasis when fed a normal diet, however, when fed a high fat diet Atf6α knockout mice have impaired glucose homeostasis and reduced plasma insulin levels (205). Furthermore, examination of the impact of Atf6α knockout on β cell function revealed insulin content was significantly lower when fed a high fat diet compared to control mice, but no difference was observed on a chow diet (205). Lastly, when Atf6α was knocked out in Ins2WT/C96Y mice, a mouse model of ER stress, the Atf6α deficiency worsened the hyperglycemia observed in Ins2WT/C96Y mice, further decreased insulin content and resulted in a dilated ER, a typical phenotype of ER stress (205). Together this indicates ATF6α is required during adaptive UPR signaling when challenged by a high fat diet and under ER stress. 1.4.4.3 IRE1α IRE1 has two isoforms, IRE1α and IRE1β. IRE1α is ubiquitously expressed, while IRE1β is only expressed in gastrointestinal epithelial cells (179). Upon activation, IRE1α oligomerizes and autophosphorylates activating its cytosolic kinase domains and its endoribonuclease activity. Activated IRE1α splices XBP1 in the cytoplasm, converting it into an active transcription factor, spliced XBP1 (XBP1s). XBP1s induces gene expression of chaperone genes to assist with protein folding as well as genes involved with ERAD (177, 197). The endonuclease activity of IRE1 is also involved in IRE1-dependent decay (RIDD) that degrades ER localized mRNAs, and 21 even insulin mRNA, under IRE1α hyperactivation (206, 207). RIDD activity may aid in the recovery of ER protein homeostasis by further reducing the ER mRNA load (208). Islets treated with high glucose induced IRE1α phosphorylation and increased total Xpb1 mRNA, however, only ER stress inducers increased Xbp1 splicing (179). Specifically, under high glucose stimulation, IRE1 activation has a beneficial effect on β cells by increasing insulin biosynthesis. While high glucose increased mRNA levels of ER stress genes, BiP remained bound to IRE1α, suggesting IRE1 ER stress signaling was not activated. In contrast, sustained activation of IRE1α by chronic high glucose or unresolved ER stress led to cell death by downregulation of mRNAs involved in β cell homeostasis and survival (179). Similar to PERK/eIF2α signaling, the IRE1/XBP1 pathway is required for proper proinsulin processing. β cell specific deletion of Ire1α in mice decreases proinsulin biosynthesis, insulin content and insulin secretion, but not insulin mRNA levels (209). A similar phenotype is observed in mice with β cell specific deletion of Xbp1. Xbp1 deletion impaired proinsulin processing and insulin secretion (210). Interestingly, Xbp1 deletion also results in IRE1α hyperactivation and a reduction in mRNA of insulin and insulin processing genes (206, 210). In addition, both deletion of Ire1α and Xbp1 result in hyperglycemia and glucose intolerance in the mice (209, 210). Together the results demonstrate IRE1α has both protective and terminal roles in the UPR and both IRE1α and XBP1 are required for maintaining proinsulin processing, insulin secretion and normoglycemia. 1.4.4.4 ER stress-induced apoptosis While the primary role of the UPR is to restore ER protein homeostasis, under chronic ER stress there a several pathways in which terminal UPR results in cell death (197). Sustained 22 PERK/eIF2α signaling induces CHOP which mediates ER stress-induced apoptosis (211, 212). Indeed, CHOP deletion protects cells from ER stress induced cell death indicating CHOP signaling is important in ER stress-induced apoptosis (211, 212). An additional mode of death is signaling through IRE1α. IRE1α signaling can induce cell death through phosphorylation and activation of c-Jun N-terminal kinase (JNK) and sustained overexpression of XBP1 (171). An important family of proteins involved in IRE1α induced β cell death are proapoptotic proteins in the Bcl-2 family, Bax and Bak. CHOP signaling downregulates Bcl-2, an antiapoptotic protein, and upregulates BIM, a pro-apoptotic protein ultimately leading to apoptotic signaling (213). While the canonical ER stress signaling pathways are well described across multiple species, the majority of our knowledge of UPR pathway function is derived from studies using either cell lines, mixed-sex populations, or male mice; therefore, studies including both sexes are needed to determine if the ER stress and UPR response are different or the same in males and females. 1.4.5 Sex Hormones Effects on β Cell Stress Response An elegant body of work has outlined the effects of sex hormones on β cell dysfunction. Studies using gonadectomies and sex hormone supplementation have proposed that sex hormones may be responsible for sex differences observed in β cells stress response. 1.4.5.1 Androgens Effect on β Cell Stress Response The effect of supplemental testosterone on ER stress and apoptosis has been examined in INS-1 cells (214). Testosterone treatment on cultured INS-1 cells significantly increased proinsulin and insulin secretion, but also ER stress markers and apoptosis. Interestingly, co-treatment of 23 testosterone with an AR antagonist or an ER stress inhibitor, decreased apoptosis and apoptotic ER stress markers to similar levels. This suggests excess AR signaling may induce ER stress, potentially as a consequence of excess insulin and proinsulin production. Indeed, inhibition of protein synthesis with cycloheximide reduced apoptotic ER stress markers (214). Therefore, excess androgen signaling is detrimental to β cell function. 1.4.5.2 Estrogens Effect on β Cell Stress Response hIAPP accumulation contributes to the pathogenesis of T2D as a consequence of ER stress-induced apoptosis (215–217). To determine if endogenous estrogen in female mice is protective against the accumulation of islet amyloid deposits and the onset of hyperglycemia, ovariectomies have been performed on hIAPP transgenic mice. Interestingly, ovariectomies of hIAPP transgenic mice resulted in amyloid formation in female islets but did not result in β cell failure. Additionally, E2 treatment in male hIAPP transgenic mice protected against the progression to hyperglycemia (139). Ovariectomy in female Akita mice resulted in similar levels of hyperglycemia as seen in male Akita mice. E2 treatment in ovariectomized Akita mice protected against hyperglycemia (63). Together these results indicate a protective role of E2 in β cell function. Therefore, sex differences in β cell failure in hIAPP and Akita mice may be partially explained by the effect of sex hormones and/or differences in ER function and the ER stress response. However, it has not been clarified how the levels of testosterone, estrogen and their receptors are associated with sex differences in stress responses. Insights into the contribution of hormonal environment to β cell protection in the context of diabetes come from studies performing sex-mismatched islet transplantations. Mice with β cell specific expression of the human H-ras oncogene develop diabetes and exhibit β cell 24 degeneration indicated by islet lesions and dilated ER, indicative of ER stress, in males but not females (218, 219). Gonadectomized Insulin-ras transgenic male mice developed diabetes similarly to non-gonadectomized transgenic males, suggesting that testosterone did not confer the diabetic phenotype in males. In contrast, ovariectomy in female transgenic mice induced β cell lesions similar to those observed in male mice, suggesting estrogen may protect female β cells. Indeed, estrogen treatment in both males and ovariectomized females prevented the development of β cell degeneration. To determine the effect of the hormonal environment, transgenic islets were transplanted under the kidney capsules of non-transgenic mice of the opposite sex. Approximately 50% of the male donor islets transplanted in females exhibited β cell lesions. This was significantly lower than the near 100% of endogenous islets from transgenic male mice. In contrast, of the female transgenic donor islets transplanted into males, about 25% developed β cell lesions, a much lower proportion than the endogenous pancreatic islets from male transgenic mice. These results indicate the female hormone environment was partially protective against β cell degeneration in male transgenic islets. However, conflicting results also suggest that the female hormone environment was not required to protect female transgenic islets since a smaller fraction of transplanted female islets (25%) developed cavities than the endogenous pancreatic islets from male transgenic mice (218). Therefore, the sexual dimorphism may result from both hormonal differences and intrinsic β cell differences in male and female mice. Overall, these studies suggest substantial male-female differences in β cell function; however, it is not clear how sex hormones are associated with sex differences in disease progression. Furthermore, differences in insulin resistance can greatly impact β cell function and these studies did not account for insulin sensitivity. 25 1.5 Thesis Investigation Although several studies have suggested that the sex difference in peripheral insulin sensitivity contributes to this differential T2D risk, recent studies have demonstrated that sex differences in insulin production and secretion persist in the context of equivalent insulin sensitivity between males and females. Given the progressive β cell dysfunction in T2D, sex differences in β cells may also contribute to the male-biased T2D risk. However, the male-bias in islet biology and diabetes studies has resulted in a large knowledge gap regarding how the function and dysfunction of β cells differs in males and females. Therefore, in this study we wanted to investigate sex differences in islet and β cell gene expression and function. The first aim was to perform an unbiased bioinformatic analysis of islet and β cell gene expression. We conducted a comprehensive multi-omics and phenotypic analysis of male and female human β cells and mouse islets. RNAseq analysis revealed male and female human islets have distinct transcriptional profiles under normal and T2D states. Our unbiased pathway enrichment analysis on mouse islet transcriptomes revealed sex differences in genes associated with ER stress and the UPR response, where females had a higher number and higher expression of genes involved in protein folding and protein synthesis. The second aim was to examine sex differences in islet and β cell function upon the induction of ER stress. Our detailed assessment of β cell function suggests female islets are more resilient to acute ER stress than male islets. Importantly, these differences exist prior to sex differences in peripheral insulin resistance. 26 Chapter 2: Materials and Methods 2.1 Animals Mice were bred in-house or purchased from Jackson laboratories. Unless otherwise stated mouse islets were isolated from C57BL/6J mice aged 20-23 weeks. Animals were housed and studied in the UBC Modified Barrier Facility using protocols approved by the UBC Animal Care Committee in accordance with international guidelines. Mice were housed in the temperature controlled UBC Modified Barrier Facility on a 12-hour light/dark cycle with food and drinking water ad libitum. Regular chow diet (LabDiet #5053); 24.5 % energy from protein, 13.1% energy from fat, and 62.4% energy from carbohydrates. 2.2 Islet Isolation, Culture, Dispersion and Treatment. Mouse islet isolations were performed by ductal collagenase injection with a 30-gauge needle. Dissected pancreata were digested in 50 mL conical centrifuge tubes containing 2 mL of collagenase solution (1000 U/ml Collagenase XI (Sigma: C7657) in 1X Hanks' Balanced Salt Solution (HBSS, GIBCO: 14175-103)) for 7-8 minutes in a water bath at 38°C, then shaken until the solution was homogenized. Two washes were performed with 30 mL of ice-cold HBSS [+1 mM CaCl2] and centrifuged at 1100 rpm for 1 minute at 4°C. The solution was filtered through a 70 μM cell strainer (BD Falcon: 352350) and rinsed with HBSS [+1 mM CaCl2]. Islets were resuspended in islet culture media (RPMI 1640 (Gibco Cat # 11875) containing 1% antibiotics (GIBCO: 15140-148) and 10% FBS (Thermo: 12483020)) then hand-picked to purity. Islets recovered overnight, in islet culture media at 37°C with 5% CO2. Islets were dispersed with 0.01% trypsin after four washes with Ca2+/Mg2+-Free Minimal Essential Medium (Gibco) then resuspended in islet culture media. Cell seedings were done as per the experimental procedure 27 (protein synthesis: 20,000 cells per well, live cell imaging: 5,000 cells per well). ER stress was induced by treating islets with thapsigargin (Tg). We found that Tg was less effective in FBS treatment therefore we used FBS free islet culture media when possible. For longer term assays >24 hour, FBS+ islet culture media was used to ensure cell viability was not impacted. 2.3 Analysis of protein synthesis Dispersed islets were seeded into an optical 96-well plate (Perkin Elmer) at a density of approximately 20,000 cells per well. 24 hours after seeding, treatments were applied in fresh islet culture medium. After incubation, fresh culture media was applied, supplemented with 20 μM O-propargyl-puromycin (OPP) (Invitrogen) and respective drug treatments. Cells were incubated at 37°C with 5% CO2, for 30m then washed with phosphate-buffered saline (PBS) and fixed using 3.7% paraformaldehyde for 15 minutes. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 minutes, washed in PBS then incubated with a Click-iT Plus OPP reaction cocktail (Invitrogen) for 30 minutes at room temperature. Cells were washed with Click-iT Reaction Rinse Buffer (Invitrogen), HCS NuclearMask Blue Stain (Invitrogen) was added at 1:2000 in PBS, then incubated for 30 minutes at room temperature, protected from light. After 2 more washes in PBS, cells were imaged at 10x with an ImageXpressMICRO high-content imager and analyzed with MetaXpress (Molecular Devices) to quantify the integrated staining intensity of OPP-Alexa Fluor 594 in cells identified by NuclearMask Blue Stain. 2.4 Live Cell Imaging Dispersed islets were seeded into 384-well plates (Perkin Elmer) at a density of approximately 5,000 cells per well and allowed to adhere for 48 hours. Cells were stained with Hoechst 33342 28 (Sigma-Aldrich) (0.05 μg/mL) and propidium iodide (Sigma-Aldrich) (0.5 μg/mL) for 1 hour prior to the addition of treatments and imaging. 384-well plates were placed into environmentally controlled (37°C, 5% CO2) ImageXpressMICRO high content imaging system. To measure cell death, islet cells were imaged every 2 hours for 84 hours; and analyzed by MetaXpress software to quantify cell death as the number of Propidium Iodide-positive/Hoechst 33342-positive cells. To measure Ins2 transcription, dispersed islets from Ins2GFP/WT mice aged 21-23 weeks were used. Islet cells were imaged every 30 minutes for 60 hours. MetaXpress analysis software and custom R scripts were used to perform single cell tracking of Ins2GFP/WT β cells as previously described (220). The following process was used to identify and study populations of high and low GFP expressing cells. Briefly, the density plot of GFP expressing cells highlighted a clear bimodal distribution (Fig. 5C, D). To identify the two populations, Mclust R package (221) was used to perform model-based clustering with finite Gaussian mixture models at time = 0. The number of clusters was set to 2, producing a mean and variance for each distribution. The equation for each normal distribution curves was determined, then the intercept of the two distributions was calculated using the curve_intersect() function from reconPlots R package (222). The intercept was used to define the threshold between low and high GFP expressing cells for each sex, per live cell imaging experiment. 2.5 Western Blot Mouse islets were sonicated in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% DOC, 0.1% SDS, 50mM Tris (pH 7.4), 2 mM EGTA, 2 mM Na3VO4, and 2 mM NaF) supplemented with complete mini protease inhibitor cocktail (Roche, Laval, QC). Protein lysates were incubated in Laemmli loading buffer (Thermo Fisher Scientific, J61337AC) at 95°C for 5 29 minutes and resolved by SDS-PAGE. Proteins were then transferred to PVDF membranes (BioRad, CA) and probed with antibodies against, HSPA5 (1:1000, Cat. #3183, Cell Signaling), eif2α (1:1000, Cat. #2103, Cell Signaling), phospho-eIF2α (1:1000, Cat. #3398, Cell Signaling), IRE1α (1:1000, Cat. #3294, Cell Signaling), phospho-IRE1α (1:1000, Cat. #PA1-16927, Thermo Fisher Scientific), DDIT3 (1:1000, #ab11419, Abcam), β-actin (1:1000, NB600-501, Novus Biologicals). The signals were detected by secondary HRP-conjugated antibodies (Anti-mouse, Cat. #7076; Anti-rabbit, Cat. #7074; CST) and either Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Forte (Immobilon). Protein band intensities were quantified using Image Studio (LI-COR). 2.6 Islet Secretion and Content Glucose stimulated insulin/proinsulin production and secretion was assessed using size-matched islets (5 islets per well in triplicates) seeded into 96-well V-bottom Tissue Culture Treated Microplates (#CLS3894, Corning). Islets were allowed to adhere for 48 hours. Islets were washed with Krebs-Ringer HEPES-Buffered Solution (KRB; 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 10 mM HEPES, 0.5% bovine serum albumin) containing 3 mM glucose then pre-incubated for 4 hours in 3 mM glucose KRB. Tg was added to the 3 mM low glucose pre-incubation buffer 4 hours prior, 2 hours prior, or at the start of the low glucose incubation period. Islets were incubated in KRB with 3 mM glucose then 20 mM glucose for 45 minutes each. Supernatant was collected after each stimulation. Islet insulin and proinsulin content was extracted by freeze-thawing in 100 μL of acid ethanol, then the plates were shaken at 1200 rpm for 10 minutes at 4°C to lyse the islets. Insulin was measured by Rodent Insulin Chemiluminescent ELISA (ALPCO: 80-INSMR) and proinsulin by 30 Rat/Mouse Proinsulin ELISA (Mercodia: 10-1232-01). Measurements were performed on a Spark plate reader (TECAN). 2.7 In vivo Analysis of Glucose Homeostasis and Insulin Secretion Mice were fasted for 6 hours prior to glucose and insulin tolerance tests. During glucose and insulin tolerance tests, tail blood was collected for blood glucose measurements using a glucometer (One Touch Ultra 2 Glucometer, Lifescan, Canada). For intraperitoneal (i.p.) glucose tolerance tests, the glucose dose was 2 g/kg. For insulin tolerance tests, the insulin dose was 0.75U insulin/kg. For measurements of in vivo glucose-stimulated insulin secretion, femoral blood was collected after intraperitoneal injection of 2 g/kg glucose. Blood samples were kept on ice during collection, centrifuged at 2000rpm for 10 minutes at 4°C and stored as plasma at -20°C. Plasma samples were analyzed for insulin using Rodent Insulin Chemiluminescent ELISA (ALPCO: 80-INSMR). 2.8 Data from the Human Pancreas Analysis Program This thesis used data acquired from the Human Pancreas Analysis Program (HPAP-RRID:SCR_016202) Database (https://hpap.pmacs.upenn.edu), a Human Islet Research Network (RRID:SCR_014393) consortium (UC4-DK-112217, U01-DK-123594, UC4-DK-112232, and U01-DK-123716). 2.9 RNAseq Islets from 20-week-old mice were snap frozen and stored at -80°C until RNA extraction. To assess Tg-induced transcriptional changes islets from each mouse were treated with DMSO or 31 Tg for 6- or 12 hours in islet culture media [-FBS] (8 groups, n=3-4 per group, each n represents pooled islet RNA from two mice). Islets were frozen at -80°C in 100 μL of RLT buffer (Qiagen) with beta mercaptoethanol (1%). RNA was isolated using RNeasy Mini Kit (Qiagen #74106) according to manufacturer's instructions. RNA Sequencing was performed at the UBC Biomedical Research Centre Sequencing Core. Sample quality control was performed using the Agilent 2100 Bioanalyzer System (RNA Pico LabChip Kit). Qualifying samples were prepped following the standard protocol for the NEBNext Ultra II Stranded mRNA (New England Biolabs). Sequencing was performed on the Illumina NextSeq 500 with Paired End 42bp × 42bp reads. De-multiplexed read sequences were then aligned to the reference sequence using STAR aligner (223). Gene differential expression was analyzed using DESeq2 R package (224). Pathway enrichment analysis were performed using Reactome (225). Over-representation analysis was performed using NetworkAnalyst3.0 (www.networkanalyst.ca) (226). 2.10 Proteomics Islets were treated with Tg for 6 hours in islet culture media [-FBS] (4 groups, n=5-7 per group, each n represents pooled islets from two mice). Islet pellets were frozen at -80°C in 100 μL of SDS lysis buffer (4% SDS, 100 mM Tris, pH 8). University of Victoria proteomics service performed non-targeted quantitative proteomic analysis using data-independent acquisition (DIA) with LC-MS/MS on an Orbitrap mass spectrometer. The proteins in the samples were cleaned up using acetone precipitation. A mouse FASTA database was downloaded from Uniprot (http://uniprot.org). This file was used with the 6 gas phase fraction files from the analysis of the chromatogram library sample to create a mouse islet specific chromatogram library using the EncyclopeDIA (v 1.2.2) software package (Searle et al, 2018). This 32 chromatogram library file was then used to perform identification and quantitation of the proteins in the 24 samples again using EncyclopeDIA with Overlapping DIA as the acquisition type, trypsin used as the enzyme, CID/HCD as the fragmentation, 10ppm mass tolerances for the precursor, fragment, and library mass tolerances. The Percolator version used was 3.10. The precursor FDR rate was set to 1%. Protein abundances were log2 transformed, imputation was performed for missing values, then proteins were normalized to median sample intensities. Gene differential expression was analyzed using limma in Perseus (227). 2.11 Statistical Analysis Data were analyzed in GraphPad Prism (GraphPad Software, San Diego, CA, USA) or R using a Student’s t-test for parametric data and a Mann-Whitney test for non-parametric data. For all statistical analyses, differences were considered significant if the p-value was less than 0.05. *: p< 0.05; ** p< 0.01; *** p< 0.001. Data are presented as means ± SEM. 33 Chapter 3: Results 3.1 Sex-Specific Transcriptional Profiles in ND and T2D Human Islets Based on newly available public scRNAseq-data, we wanted to investigate β cell gene expression changes associated with T2D in each sex. We used a recently compiled meta-analysis of all publicly available scRNA-seq datasets from male and female human islets (228). In line with prior reports (155), we found ND and T2D β cells displayed significant transcriptional differences. In β cells isolated from female T2D donors, mRNA levels of 127 genes were significantly different from ND female donors (77 down- and 50 upregulated in T2D) (Fig. 1A-C). In β cells isolated from male T2D donors, 462 genes were differentially expressed compared with ND (138 down- and 324 upregulated in T2D) (Fig. 1A-C). Of the genes that were differentially regulated by T2D in each sex, 71 were differentially expressed in both men and women (15 down- and 56 upregulated in T2D) (Fig. 1B, C); however, the magnitude of gene expression response to T2D was different between the sexes (Fig. 1D). Beyond these shared genes, our analysis showed that the genes with the largest magnitude of change in gene expression in T2D were unique to each sex (Fig. 1E, F). Together, this data suggests that the nature, and the magnitude, of the gene expression response to T2D is distinct between the sexes. To understand which biological pathways the T2D altered genes are participating in we performed pathway enrichment analysis. This revealed that upregulated T2D β cells genes are involved in similar upregulated pathways in males and females (Fig. 2A-C), including Golgi-ER transport and the unfolded protein response; however, approximately 75% of the differentially regulated genes in these pathways were unique to each sex. Downregulated T2D genes identified pathways that were mostly different between the sexes (Fig. 2A-C). Female T2D downregulated pathways were predominantly cellular responses to stress and stimuli (Fig. 2B). Male T2D 34 downregulated pathways were associated with respiratory electron transport and translation initiation (Fig 2C). Together, our analysis of scRNA-seq data suggests that the β cell gene expression response in T2D is not fully shared between the sexes, as both the magnitude of gene expression changes, and the identity of the genes with altered mRNA levels, differed in male and female donors. Since we identified a sex-specific β cell transcriptional response to T2D, we examined glucose-stimulated insulin secretion from the Human Pancreas Analysis Program of ND and T2D donor islets (229). In ND donors, islets from men and women showed similar patterns of insulin secretion in response to various stimulatory media (Fig. 3A, B). In T2D donors, we found that insulin secretion was impaired to a greater degree in islets from men than in women (Fig. 3A-E). In male islets, insulin secretion was significantly lower following stimulation with both high glucose and IBMX (Fig. 3D), which potentiates insulin secretion by increasing cAMP levels similar to the incretins (230). In contrast, islets isolated from female donors showed no significant defects in insulin secretion under identical treatment conditions (Fig. 3C). Together with our β cell gene expression data these findings suggest that β cell transcriptional and functional responses in T2D are not shared between the sexes. 3.2 Sex-Specific Transcriptional Profiles in Mouse Islets While our data suggests there are β cell differences in T2D between men and women, these differences may be affected by peripheral insulin sensitivity, disease processes, medication, and age (23). Since the impact of these factors on the sex differences β cell gene expression and functional responses in T2D were unclear (228), we used 20-week-old C57/B6J male and female mice to examine islet gene expression and function in more detail. At 20 weeks of age, mice are 35 considered to be mature adults in the mouse life stage, equivalent to a 20-30 year old human (231). Importantly, we showed that insulin sensitivity was equivalent between male and female mice at this age (Fig. 10D). We generated a well-powered islet RNAseq data set from 20-week-old C57/B6J male and female littermates (n=9, 8). Principal component analysis separated male and female islets on the basis of gene expression (Fig. 4A). Overall, 17.7% (3268/18938) of genes were differentially expressed between the sexes (1648 upregulated in females, 1620 upregulated in males), in line with estimates of sex-biased gene expression in other tissues (232, 233). Overrepresentation and pathway enrichment analysis both identified UPR-associated pathways, where the majority of genes in this category were enriched in female islets (Fig. 4B, C). Specific categories that were enriched in females include transcripts associated with the gene ontology term “Cellular response to ER stress” (GO:0034976), many of which are involved in regulating protein synthesis. Indeed, female islets had more than twice the number of transcripts associated with protein synthesis compared to male islets (Fig. 4D), and significantly higher mRNA levels of ribosomal protein genes (Fig. 4E). Additional genes enriched in females included genes associated with protein folding, protein processing, and quality control (Fig. 4D). Given that protein folding capacity is intrinsically important for β cells as insulin production constitutes a large demand on the ER (234), this suggests that female islets may have a larger protein production and folding capacity than male islets. 3.3 Female Islets are More Resilient to ER Stress Given that female islets show increased expression of genes involved in protein synthesis and protein folding, we examined global protein synthesis rates in male and female islets under normal conditions and following ER stress induction. To measure islet protein synthesis in each 36 sex, we incubated islets with OPP, which incorporates into newly translated proteins, and then ligates to an Alexa Fluor picolyl azide dye (Fig. 5A). Under normal conditions male islet cells had significantly greater protein synthesis rates compared with female islet cells (Fig. 5B). Next, we monitored protein synthesis in a context where we challenged the cellular protein folding capacity, as previous studies show that activation of PERK/eIF2α signaling attenuates protein translation (171). We used Tg, a specific inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), that results in a decrease of ER calcium levels and thereby activates ER stress and the UPR. In female islet cells, protein synthesis was repressed 2 hours post Tg treatment; however, this repression had resolved by 24 hours (Fig. 5C-E). In male islet cells, protein synthesis was similarly repressed 2 hours after Tg treatment but did not recover to control levels by 24 hours unlike female islets (Fig. 5C-E). This illustrates that female islets recover quicker from protein synthesis repression in the context of ER stress. Insulin biosynthesis accounts for approximately half the total protein production in β cells (172) and upon glucose-stimulation β cells produce up to one million molecules of insulin per minute (235); therefore, it is not surprising that even under normal physiological conditions ER stress naturally occurs (236). Furthermore, previous studies have demonstrated that high glucose induced-ER stress supresses insulin gene transcription (179, 237). Therefore, to determine if the difference in resolving protein synthesis repression was due to transcriptional changes, we assessed Ins2 gene activity. We did this using β cells from mice with GFP knocked-in to the endogenous mouse Ins2 locus (Ins2GFP/WT) (238). Quantification of the fluorescence intensity of GFP in Ins2GFP/WT β cells was used as a readout for Ins2 gene activity under normal and ER stressed conditions. In male and female β cells, ER stress induced by 1 µM Tg induced a greater repression of Ins2 gene activity than 0.1 µM Tg (Fig. 6A, B). Interestingly, beyond 30 37 hours, Tg treated cells displayed increased Ins2 gene activity compared to controls (Fig. 6A, B). Since, the Johnson lab and others have previously identified a broad range of Ins2 mRNA expression in β cells (220, 239), we examined the distribution of the β cells and identified two distinct populations of β cells (Fig. 6C, D). We next analyzed how Ins2 gene activity was altered by ER stress induction in high and low GFP β cell states. In contrast to high GFP expressing cells (Fig. 6E, F), low GFP expressing cells treated with 1 µM Tg exhibited a sustained reduction in Ins2 gene activity (Fig. 6G, H). While we found that ER stress induced a dose response reduction in Ins2 gene activity, in the total β cell population, males and females exhibited similar responses (Fig. 6A, B). This suggests that Ins2 transcriptional changes are not responsible for the male-female difference in resolving protein synthesis repression. We next investigated the impact of ER stress on apoptosis using a kinetic cell death analysis (Fig. 7A, F-I). In female islet cells, no significant increase in death was observed at either 0.1 μM or 1.0 μM Tg compared to controls after an 84-hour treatment (Fig. 7B). In contrast, in male islet cells, death was significantly increased by both doses of Tg (Fig. 7C). To test if female islets were susceptible to ER stress-induced apoptosis, we treated islet cells with a high 10 μM dose of Tg. We observed a significant increase in cell death in both male and female islet cells compared to controls (Fig. 7D, E). Overall, this suggests that female islets were more resilient to ER stress-induced apoptosis than male islets. Given that our data suggested female islets are less impacted by ER stress, we next asked if the UPR signaling response is different between the sexes. To test this, we treated mouse islets with 1 μM Tg for 24 hours, and measured protein markers that were upregulated during the UPR as readouts for its activation, including BiP, phosphorylated IRE1, phosphorylated eIF2α, and CHOP (173). We found, under unstressed conditions, protein levels of UPR markers were low 38 and there was no significant difference between male and female islets (Fig. 8A-D). Upon ER stress induction we observed a significant increase in protein levels of pIRE1α and CHOP in islets from both sexes and BiP in female islets (Fig. 8A-D). This suggests the UPR signaling response is similar in males and females. To determine whether this difference persists throughout the life course, we measured UPR protein markers in older 60-week mice under basal conditions and upon ER stress induction. In older female islets, we observed no significant induction of UPR protein markers (Fig. 8E-G). In older male islets, protein levels of BiP were significantly upregulated in response to Tg treatment (Fig. 8E-G). Overall, despite observing sex differences in ER stress associated phenotypes, the induction of ER stress was similar in male and female islets. Taken together, this suggests the relationship between ER stress activation and regulation of downstream processes differs between the sexes. 3.4 Female Islets Retain Greater β Cell Function During ER Stress To determine whether sex differences in ER stress responses impacted β cell function, we examined glucose-stimulated insulin secretion following a time course of Tg treatments (Fig. 9A). Under normal conditions, female islets had significantly lower insulin secretion than males in both basal and high glucose stimulation (Fig. 9B). In addition, male islets had significantly higher proinsulin content than females (Fig. 9C). This suggests under normal conditions, male and female islets display different behaviors. In all Tg treatment times, high glucose significantly stimulated insulin secretion in both sexes (Fig. 9D). To examine the effect of Tg treatment time on glucose-stimulated insulin secretion, we compared insulin secretion at each timepoint in low and high glucose. Female islets, in both low and high glucose, increased or maintained insulin secretion under acute and prolonged Tg treatments (Fig. 9H, I). Male islets, increased insulin 39 secretion after an acute Tg treatment in low glucose and repressed insulin secretion after a prolonged Tg treatment in high glucose (Fig. 9H, I). Insulin content measurements revealed that female, but not male islets, significantly increased in insulin content compared to controls following a prolonged Tg treatment (Fig. 9E). This suggests that male and female islets exhibit different insulin responses to Tg treatment. Measurements of proinsulin secretion revealed both male and female islets exhibited a similar response to Tg as observed in insulin secretion (Fig. 9F). In contrast to insulin content, islet proinsulin content decreased in response to increased Tg treatment time and decreased by a greater magnitude in male islets (Fig. 9G). Therefore, in response to Tg treatment, insulin and proinsulin content were altered in opposing directions. Compared to controls females showed a more prominent increase in insulin content (Fig. 9E), whereas males showed a greater reduction in proinsulin content (Fig. 9G). Together, these experiments indicate that female islets were better able to adapt and maintain insulin secretion under stress. To examine β cell function in vivo, we tested glucose-stimulated insulin secretion, glucose tolerance, and insulin tolerance in male and female mice. Male mice had significantly higher fasting plasma insulin levels than females (Fig. 10A) as in previous studies (240, 241), and female mice showed a greater glucose-stimulated increase in plasma insulin levels (Fig. 10B). Importantly, we observed these differences despite similar glucose tolerance and insulin sensitivity between the sexes (Fig. 10C, D). Collectively, these experiments showed that in vivo, female mice have greater glucose-stimulated insulin secretion than males, and ex vivo, female β cells retain greater high glucose-stimulated insulin secretion under pharmacological stress and exhibit a milder reduction in insulin release in T2D donors compared to ND donors. 40 3.5 Sex-Specific Transcriptional and Proteomic Profiles during ER Stress Next, we compared transcriptome changes in response to ER stress in male and female islets. Islets were treated with Tg or DMSO for 6 or 12 hours. Principal component analysis showed that islets cluster by sex, treatment, and treatment time (Fig. 11A). The majority of the variance is explained by treatment, followed by sex, and then time (Fig. 11B). Therefore, while male and female islets both responded strongly to treatment, unique gene expression changes also occurred due to sex and response to increasing time in Tg. As expected, pathway enrichment analysis between DMSO and Tg identified the UPR as the most upregulated pathway in Tg treated islets at both at 6- and 12 hours (Fig. 12A, B). To determine how males and females respond to Tg treatment time, we performed a two-way ANOVA treatment:time interaction to determine which genes were upregulated, downregulated or unchanged by increased time in Tg treatment. From these genes lists we again performed pathway enrichment analysis but this time we only used genes with a significant treatment:time interaction (upregulated or downregulated) or a non-significant treatment:time interaction (unchanged). In both male and female islets, pathways upregulated by increased treatment time were largely cellular responses to stimuli, stress and starvation; however, only males had a significant enrichment of translation pathways (Fig. 12C). Female, but not male islets, revealed significantly downregulated pathways in response to increased Tg treatment time of which regulation of gene expression in β cells was the most significantly downregulated (Fig. 12C). Female islets showed significant downregulation of β cell identity genes, Pklr, Rfx6, Hnf4a, Slc2a2, Pdx1, and Mafa during increased incubation time in Tg (Fig. 11C). Interestingly, the same pathway was identified in males except it was associated with genes unchanged by Tg treatment time. In male islets, β cell identity genes downregulated by Tg but unchanged by treatment time were, Pklr, Hnf4a, Pdx1, Nkx2-2, Iapp, 41 Nkx6-1, Mafa, Neurod1, Gck, Foxa2, and Foxo1 (Fig. 11D). This suggests that females, but not males, further downregulate β cell identity genes during increased Tg treatment time. β cell identity genes are well known regulators of insulin transcription and translation (242), however, they have not previously been implicated in sex differences in the ER stress response. ER stress also had sex-specific effects on islet protein. Tg-induced ER stress resulted in the majority of altered proteins being downregulated. In female islets, 47 proteins (35 down- and 12 upregulated in Tg treatment) were differentially expressed, and in male islets, 82 proteins (72 down- and 10 upregulated in Tg treatment) were differentially expressed (supplemental table). Only seven altered proteins were in common between the sexes (Fig. 11E). In females, downregulated proteins were associated with GO term endoplasmic reticulum to Golgi vesicle-mediated transport (GO:0006888) (BCAP31, COG5, COG3, GOSR1). In males, downregulated proteins were associated with GO term, insulin secretion (GO:0030073) (PTPRN2, CLTRN, PTPRN) and lysosome pathway (KEGG) (NPC2, CTSZ, LAMP2, PSAP, CLTA). Together, our multi-omics data suggests that while Tg induced ER stress in both male and female islets unique transcriptome and proteome changes occur between the sexes. This supports a model in which the relationship between ER stress activation and regulation of downstream processes differs between the sexes. 77(34%)15(7%)138(60%)MaleFemaleB T2D Downregulated Genes in Human β Cells*Indicates genes unique tofemalesTop 60 Differentially Expressed Genes in Female β Cells E50(12%)56(13%)324(75%)MaleFemaleC T2D Upregulated Genes in Human β Cells*Indicates genes unique tomalesTop 60 Differentially Expressed Genes in Male β Cells F127(19%)71(11%)462(60%)MaleFemaleA Differentially Expressed Genes in T2D Human β CellsD Non-Sex-Specific DifferentiallyExpressed Genes in β Cells4243 Figure 1: Transcriptomic analysis of ND and T2D human β cells scRNA-seq data from male and female human β cells. (A-C) Venn diagrams compare the number of significantly differentially expressed genes between non-diabetic and type 2 diabetic donors (T2D) (p adj. < 0.05). All differentially expressed genes (A), downregulated genes (B), upregulated genes (C) in human β cells. (D-F) Top 60 significantly differentially expressed genes (p adj. < 0.05). (D) Non-sex-specific. (E) Female. (F) Male. Sex-specific genes are indicated in red text. Figure 2: Pathway enrichment analysis of ND and T2D human β cells scRNA-seq data from male and female human β cells. (A-C) Top 10 significantly enriched Reactome pathways (ND vs T2D) from non-sex-specific (A), female (B), or males (C) significantly differentially expressed genes (p adj. < 0.05). Gene ratio is calculated as k/n, where k is the number of genes identified in each Reactome pathway, and n is the number of genes from the submitted gene list participating in any Reactome pathway. B CA Non-Sex-Specific β CellEnriched PathwaysFemale β Cell Enriched PathwaysMale β Cell Enriched Pathways44 Figure 3: Glucose-stimulated insulin secretion of ND and T2D human β cells (A-D) Human islet perifusion data from the Human Pancreas Analysis Program in non-diabetic and type 2 diabetic donor islets in females (A, C) and males (B, D). 3 mM G – 3 mM glucose; 16.7 mM G – 16.7 mM glucose; 0.1 mM IBMX – 0.1 mM isobutylmethylxanthine; 30 mM KCl – 30 mM potassium chloride, 4 mM AAM – 4 mM amino acid mixture (mM: 0.44 alanine, 0.19 arginine, 0.038 aspartate, 0.094 citrulline, 0.12 glutamate, 0.30 glycine, 0.077 histidine, 0.094 isoleucine, 0.16 leucine, 0.37 lysine, 0.05 methionine, 0.70 ornithine, 0.08 phenylalanine, 0.35 proline, 0.57 serine, 0.27 threonine, 0.073 tryptophan, and 0.20 valine, 2 mM glutamine). (C, D) Quantification of area under the curve (AUC) is shown for the various stimulatory media in females (C) and males (D). (C) Insulin release from ND female islets was significantly higher than T2D female islets under 4 mM AAM +16.7 mM glucose (HG) + 0.1 mM IBMX stimulation (p=0.0235; unpaired student’s t test). (D) Insulin release from ND male was significantly higher than T2D male islets under 4 mM AAM + 16.7 mM glucose (HG) stimulation (p=0.0329; unpaired student’s t test) and 4 mM AAM +16.7 mM glucose (HG) + 0.1 mM IBMX stimulation (p=0.0073; unpaired student’s t test). (E) Total insulin secretion was lower in T2D male islets than ND male islets (p=0.0503; unpaired Student’s t-test). * indicates p<0.05, ** indicates p<0.01; error bars indicate SEM. Human Islet Perifusion Human Islet PerifusionBAC D EAUC – per stimulant AUC – per stimulant AUC – totalCBFemale MaleADEMouse Islet PCA Mouse Islet Network AnalysisMouse Islet Pathway Enrichment Mouse Islet ER Stress GenesMouse Islet Ribosome Gene Expression4546 Figure 4: Transcriptomic analysis of mouse islets RNAseq from 20-week-old male and female mouse islets (n=9, 8). (A) Principal component analysis (PCA) of RNAseq data (B) Over-representation analysis (ORA) of all significantly DE genes (p adj. < 0.01) from males and females. Top 30 enriched KEGG pathways (large nodes; size = proportional to connections, darker red color = greater significance) and associated genes (small nodes; green = male enriched, yellow = female enriched). (C) Top significantly enriched Reactome pathways from the top 1000 significantly differentially expressed genes. (p adj. < 0.01) for males and females. Gene ratio is calculated as k/n, where k is the number of genes identified in each Reactome pathway, and n is the number of genes from the submitted gene list participating in any Reactome pathway. (D) All transcripts of differentially expressed genes under the gene ontology term “Cellular response to ER stress” (GO:0034976) and genes labeled by their role in transcription, translation, protein processing, protein folding, secretion and protein quality control. (E) All transcripts of differentially expressed ribosomal genes. ED Protein Synthesis per Mouse Relative Islet Protein SynthesisANuclear mask OPP – Alexa Fluor 594 CompositeB CRepresentative Protein Synthesis Staining Images**** ********* ns***Protein Synthesis per Cell Protein Synthesis per Cell4748 Figure 5: ER stress-induced protein synthesis repression in mouse islets Protein synthesis measurements on dispersed islets cell from 20-week-old male and female mice (A) Representative images of dispersed islets stained with nuclear mask and OPP labeled with Alexa Fluor 594. (B, C) Integrated staining intensity of Alexa Fluor 594 in nuclear mask positive islet cells in control media or after treatment with 1 μM Tg for 2-hr or 24-hr. (B,C) Protein synthesis displayed on a per cell basis, n=4-5 mice, >1000 cells per group. Mean values are indicated under each group. (B) Protein synthesis was significantly higher in male islet cells than female islet cells in control media, 3.9% (p=0.027; unpaired student’s t test). (C) In female islet cells, protein synthesis was significantly repressed from control-2 hr treatment (p=<0.0001; unpaired student’s t test) and significantly increased from both control-24 hr treatment and 2-24 hr treatment (p=<0.0001; unpaired student’s t test). In male islet cells, protein synthesis was significantly repressed from control-2 hr treatment (p=<0.0001; unpaired student’s t test) and control-24 hr treatment (p=<0.0001; unpaired student’s t test); however, no significant recovery from 2-24 hr treatment (p=0.07; unpaired student’s t test). (D) Average protein synthesis per mouse. In female islet cells, protein synthesis was significantly lower after a 2-hr Tg treatment compared to control (p=0.0152; paired student’s t test) and significantly higher after a 24-hr Tg treatment compared to a 2-hr Tg treatment (p=0.0027; paired student’s t test). In male islet cells, protein synthesis was significantly lower after a 2-hr and 24-hr Tg treatment compared to control (p=0.0289 [0-2 hr] and p=0.0485 [0-24 hr], respectively; paired student’s t test). (E) Relative protein synthesis (within sex) showed protein synthesis was repressed after 2-hrs in both male andfemale islets cells. By 24-hr, protein synthesis repression was resolved in female, but not male islet cells. * indicatesp<0.05, ** indicates p<0.01, *** indicates p<0.001; ns indicates not significant; error bars indicate SEM.A BIns2 Gene Activity Ins2 Gene ActivityE FIns2 gene activity - High GFP β Cells Ins2 gene activity - High GFP β Cells G HIns2 gene activity - Low GFP β Cells Ins2 gene activity - Low GFP β Cells C DDistribution of β Cell GFP Intensity Distribution of β Cell GFP IntensityHigh HighLow LowFemale MaleFemale MaleFemale MaleFemale Male49Figure 6: ER stress-induced suppression of Ins2 gene activity in mouse islets Ins2 gene activity in β cells from 20-week-old Ins2GFP/WT male and female mice treated with thapsigargin (0.1 μM or 1 μM Tg) or DMSO for 60-hrs (n=6 mice per sex, > 1000 cells per treatment group) (A, B) Average change in fluorescence intensity from all GFP expressing female (A) and male (B) β cells over time. Data was normalized to the first 2-hr to examine relative change in Ins2 gene activity. (C, D) Density plot of Ins2GFP/WT β cell GFP fluorescence intensity, log transformed. Data is shown for each run for females (C) and males (D). (E-H) Average change in high (E,F) and low (G,H) GFP Ins2GFP/WT β cells fluorescence over time from females (E,G) and males (F,H). Data was normalized to the first two hours to examine relative change in Ins2 gene activity. Female Islet Cell Death Female Islet Cell DeathMale Islet Cell Death Male Islet Cell DeathB D ECA Representative Cell Death Staining ImagesHoechst 33342 Propidium Iodide CompositeF GH IFemale Islet Cell Death; 0.1, 1.0 μM Tg Female Islet Cell Death; 10 μM Tg Male Islet Cell Death; 0.1, 1.0 μM Tg Male Islet Cell Death; 10 μM Tg 5051 Figure 7: Live-cell imaging cell death assay of ER stress-induced mouse islets Kinetic cell death analysis on dispersed islet cells from 20-week-old B6 female and male mice. (A) Representative images of dispersed islet cells stained with Hoechst 33342 and Propidium Iodide (PI). (B-E) Quantification of propidium iodide (PI) cell death assay of dispersed islets treated with thapsigargin (0.1 μM, 1 μM or 10 μM Tg) or DMSO for 84-hrs (n=6-8), compared to DMSO treatment in females (B,D) and males (C,E). In female islet cells, cell death was significantly higher in 10 μM Tg compared to control (p=0.0057; paired student’s t test). In male islet cells, cell death was significantly higher in 0.1, 1.0 and 10 μM Tg compared to control (p=0.0313 [0.1 μM], p=0.0026 [1 μM] and p=0.0373 [10 μM], respectively; paired student’s t test) (D). For B-E, %PI positive cells were normalized within each run to the DMSO control run average. (F-I) Cell death analysis of dispersed islet cells from females (F,H) and males (G,I). Cells were treated with thapsigargin (0.1 μM, 1 μM or 10 μM Tg) or DMSO for 84-hrs (n=6-8). Percentage of propidium iodide positive (PI+) cells was quantified as the number of Propidium Iodide-positive cells/Hoechst 33342-positive cells. * indicates p<0.05, ** indicates p<0.01; ns indicates not significant; error bars indicate SEM. p-eIF2α Protein LevelsBiP Protein Levels p-IRE1α Protein LevelsBiP Protein Levels p-IRE1α Protein Levels CHOP Protein Levels p-eIF2α Protein LevelsA B C DE F GH IBiPt-eIF2αp-eIF2αp-IRE1ACTβCHOPMaleDMSO Tg DMSO TgFemaleRepresentative 20-week Mouse Islet Western Blot ImagesRepresentative 60-week Mouse Islet Western Blot ImagesBiPt-eIF2αp-eIF2αACTβp-IRE1FemaleDMSO Tg DMSO TgMale52Figure 8: Protein markers of ER stress in ER stress-induced mouse islets (A-D) Levels of ER stress proteins were quantified in isolated islets from 20-week-old B6 female and male mice cultured in DMSO or 1 μM Tg for 24-hrs. (A) BiP levels were significantly upregulated in females Tg vs DMSO (p=0.0011; paired Student’s t test) but not males Tg vs DMSO (p=0.1187; paired Student’s t test). (B) pIRE1α levels were significantly upregulated in females Tg vs DMSO (p=0.0001; paired Student’s t test) and in males Tg vs DMSO (p=0.0148; paired Student’s t test). (C) CHOP levels were significantly upregulated in females Tg vs DMSO (p=0.0333; paired Student’s t test) and in males Tg vs DMSO (p=0.0164; paired Student’s t test). (D) peIF2α levels were not significantly upregulated in either sex. (E-G) Levels of ER stress proteins were quantified in isolated islets from 60-week-old B6 female and male mice cultured in DMSO or 1 μM Tg for 24-hrs. (E) BiP levels were significantly upregulated in males Tg vs DMSO (p=0.0048; paired Student’s t test) but not in females (p=0.3319; paired Student’s t test). (F) pIRE1α levels were not significantly upregulated in either sex (p=0.9257 [female] and p=0.8273 [male], respectively; paired Student’s t test). (G) peIF2α levels were not significantly upregulated in either sex (p=0.8451 [female] and p=0.3076 [male], respectively; paired Student’s t test). (H) Representative western blot images of 20wk Tg treated islets. (I) Representative western blot images of 60wk Tg treated islets. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001; ns indicates not significant; error bars indicate SEM. AD EF G4 hrPre-incubation in 3 mM KRB Low Glucose 3 mM High Glucose 20 mMDiscard Media Collect Media Collect MediaExtraction BufferFreeze Plate2 hr 0 hr 0.75 hr 1.5 hr0hr Tg pre-treatment2hr Tg pre-treatment4hr Tg pre-treatmentDMSOTreatmentsMouse Islet Insulin Secretion Mouse Islet Insulin ContentMouse Islet Proinsulin ContentMouse Islet Proinsulin SecretionStatic GSIS Experimental OverviewH ILow Glucose (% baseline) High Glucose (% baseline)B CLG HG LG HGMouse Islet Insulin Secretion; controls Mouse Islet Proinsulin Secretion; controlsContent Content5354 Figure 9: In vitro mouse β-cell function (A) Experimental workflow of static GSIS on isolated islets from 20-week-old male and female mice treated withDMSO or 1 μM Tg. (B) Insulin secretion in low glucose (LG), high glucose (HG) and insulin content comparedbetween male and female controls (p=0.0085 [LG] and p=0.0012 [HG], respectively; Mann Whitney test). (C)Proinsulin secretion in LG, HG and proinsulin content compared between male and female controls (p=0.0018[Content]; Mann Whitney test). (D) Insulin secretion at basal (3 mM) and stimulatory (20 mM) glucose. Femaleislets LG secretion was significantly higher compared to control (#) after 2-hr and 4-hr Tg pre-treatments (p=0.0047[2-hr] and p=0.0003 [4-hr], respectively; Mann Whitney test). Female islets HG secretion was significantly highercompared to control (*) after 0-hr and 2-hr Tg pre-treatments (p=0.0012 [0-hr] and p=0.0061 [2-hr], respectively;Mann Whitney test). Male islets LG secretion was significantly higher compared to control (#) after a 0-hr Tg pre-treatment (p=0.0371; Mann Whitney test). Male islets HG secretion was significantly lower compared to control (*)after a 4-hr Tg pre-treatment (p=0.0012; Mann Whitney test). (E) Insulin content. Female islets insulin content wassignificantly higher compared to control after a 4-hr Tg pre-treatment (p=0.0269; Mann Whitney test). (F)Proinsulin secretion at basal (3 mM) and stimulatory (20mM) glucose. Female islets HG secretion was significantlyhigher compared to control after 0-hr and 2-hr Tg pre-treatments (p=0.0075 [0-hr] and p=0.00437 [2-hr],respectively; Mann Whitney test). Male islets HG secretion was significantly lower compared to control after a 4-hrTg pre-treatment (p=0.0025; Mann Whitney test). (G) Proinsulin content. Female islets proinsulin content wassignificantly lower compared to control after a 2-hr Tg pre-treatment (p=0.0437; Mann Whitney test). Male isletsproinsulin content was significantly lower compared to control after 2-hr and 4-hr Tg pre-treatments (p=0.0014 [2-hr] and p=0.0005 [4-hr], respectively; Mann Whitney test). (H, I) Insulin secretion data from B shown as percentincrease compared to control (DMSO) secretion in 3 mM glucose (H) and in 20 mM glucose (I). (H) In low glucose,female islets insulin secretion was significantly higher than control in all conditions (p=0.0376 [0-hr], p=0.0219 [2-hr] and p=0.0004 [4-hr], respectively; Mann Whitney test). In low glucose, male islets insulin secretion wassignificantly higher than control after a 0-hr Tg pre-treatment (p=0.0253; Mann Whitney test). (I) In high glucose,female islets insulin secretion was significantly higher than control after a 0-hr and 2-hr Tg pre-treatment (p=0.0083[0-hr] and p=0.0371 [2-hr], respectively; Mann Whitney test). In high glucose, male islets insulin secretion wassignificantly lower than control after a 4-hr Tg pre-treatment (p=0.0013; Mann Whitney test). For B-G, each greytriangle represents the concentration of insulin or proinsulin from 5 islets (triplicates per group per mouse), theaverage values per mouse are represented by black dots. * indicates p<0.05, ** indicates p<0.01, *** indicatesp<0.001; ns indicates not significant; error bars indicate SEM.A BC DGTT ITTGSIS (raw secretion) GSIS (% secretion)55Figure 10: In vivo mouse physiology(A-D) Physiology measurements after a 6-hr fast in 20-week-old male and female mice. (A, B) Insulin levels from GSIS tests (F: nM, E: % basal insulin) following a single glucose injection (2 g glucose/kg body weight, i.p). Area under the curve (AUC) calculations (n=13 females, n=18 males). (A) Insulin levels were significantly higher in male mice at 0 min and 30 min post injection (p=0.0063 [0 min] and p=0.0009 [30 min], respectively; Student’s t test). AUC was significantly higher in males (p=0.0159; Student’s t test). (B) Insulin levels (% baseline). Glucose-stimulated insulin secretion was significantly higher in female mice 15 min post injection (p=0.0279; Student’s t test). (C) Glucose levels from glucose tolerance tests (GTT) following a single glucose injection (2 g glucose/kg body weight). AUC calculations (n=11 females, n=11 males). (D) Glucose levels (% baseline) from insulin tolerance tests (ITT) following a single insulin injection (0.75U insulin/kg body weight). AUC calculations (n=11 females, n=11 males). * indicates p<0.05; ns indicates not significant; error bars indicate SEM.Log 2(Fold Change) (DMSO vs Thapsigargin)Unchanged (6-12hrs)Upregulated (6-12hrs)Downregulated (6-12hrs)FC (DMSO vs Tg) at 6 hr Significant Treatment:Time interaction FC (DMSO vs Tg) at 12 hrLog 2(Fold Change) (DMSO vs Thapsigargin)Unchanged (6-12hrs)Upregulated (6-12hrs)Downregulated (6-12hrs)FC (DMSO vs Tg) at 6 hr Significant Treatment:Time interaction FC (DMSO vs Tg) at 12 hrβ cell identity genes - Femaleβ cell identity genes - MaleBMale 6 hr DMSOMale 12 hr DMSOMale 6 hr TgMale 12 hr TgFemale 6 hr DMSOFemale 12 hr DMSOFemale 6 hr TgFemale12 hr TgACDEMouse Islet PCA PCA VarianceDifferentially ExpressedIslet Proteins 5657 Figure 11: Transcriptomic and proteomic analysis of ER stress-induced mouse islets RNAseq data from Tg treated male and female islets from 20-week-old mice. (A) Principal component analysis (PCA) of RNAseq data from female and male mouse islets treated with DMSO or 1 μM Tg for 6-hr or 12-hrs. (B) Elbow plot showing variance for the first 5 principal components. (C,D) Treatment:time interaction plots of females (C) and males (D). (E) Protein levels of proteomics data from male and female mouse islets treated with DMSO or 1 μM Tg for 6-hrs. Top 45 differentially expressed proteins are shown (p < 0.05).58 Figure 12: Pathway enrichment analysis of ER stress-induced mouse islet RNAseq data RNAseq data from thapsigargin treated male and female islets from 20-week-old mice. Top significantly enriched Reactome pathways from the top 1000 significantly differentially expressed genes (p adj. < 0.01) for females and males comparing DMSO vs Tg after a 6-hr treatment (A), 12-hr treatment (B), genes changed by treatment and time (C) or genes unchanged by treatment time (D). Entities ratio is the proportion of Reactome pathway moleculesrepresented by a given pathway.A BIslet Pathway Enrichment [6 hr, DMSO vs Tg]Islet Pathway Enrichment[12 hr, DMSO vs Tg]Islet Pathway Enrichment [up/downregulated genes 6-12 hr]Islet Pathway Enrichment [unchanged genes 6-12 hr]C D59 Figure 13: ER stress resiliency model (A) Female islets have a greater protein folding threshold to be reached prior to the onset of β cell failure. (B)Female islets can be exposed to a greater duration of ER stress conditions prior to the onset of β cell failure. (C)Female and male islets have an equivalent threshold for ER stress induction; but male and female islets exhibitdifferent downstream responses. Created with BioRender.com60 Chapter 4: Discussion The goal of this investigation was to perform a comprehensive study of sex differences in β cells in the context of equivalent insulin sensitivity, as past studies have largely identified mechanisms of β cell (dys)function in males or in mixed-sex groups. We demonstrated that female islets were more resilient to multiple ER stress-induced phenotypes. We showed that female islets have less ER stress-induced cell death, recover quicker from protein synthesis repression as well as transcriptional repression and maintain HG stimulated insulin secretion. Importantly these differences were observed in islets from mice with equivalent in vivo insulin sensitivity and UPR signaling activation in males and females under normal and ER stressed conditions. Multi-omics analysis of ER stress-induced islets supports sex-specific mRNA and protein level regulation. Together, our findings highlight that the induction of ER stress has different phenotypic outcomes in males and females. 4.1 Sex Differences in Mouse and Human Gene Expression Remarkably, our study revealed that ~20% of mouse islet genes are sex-specifically regulated. There has been a growing appreciation of the importance of analyzing gene expression data by sex. Across multiple human tissues, 37% of identified genes exhibited sex-biased gene expression in at least one of the 44 tissues analyzed (243). Early studies in mice found sex-biased gene expression across a range of tissues and recently, scRNA-seq has expanded on these findings (232, 233, 244). Similar to our finding that females have enriched UPR gene expression in islets, it has been shown in mouse kidney endothelial cells, that the UPR and associated gene ontology terms were enriched in 3-month-old females but not males (244). Furthermore, β cells from ND and T2D donors displayed sex-specific gene expression which was differentially 61 impacted by age, such that impaired β cell function was associated with aging in males but not females (155). Previous studies on sex differences in β cell gene expression found similar sex-biased gene regulation (156, 157), suggesting the sex-biased islet gene expression differences observed in our data set are conserved in β cells. It is also notable that female islets have higher levels of ribosomal genes and translation-associated pathways. Our finding of female-biased ribosomal enrichment is in line with findings in other species and tissues including zebrafish (245) as well as mouse brain and heart cells (232). Further studies are needed to determine whether female islet ribosome enrichment is observed in all islet cell types or is exclusive to β cells. In male islets, the only significantly enriched pathway was nuclear receptor transcription. Genes associated with this pathway are DNA-binding transcription factors, many of which are associated with β cell function including estrogen-related receptor gamma (Essrg) and peroxisome proliferator-activated receptor gamma (Pparg). Essrg has been shown to be required for glucose-stimulated insulin secretion and functional β cell maturation (43). Pparg has been reported to regulate key β cells genes involved in glucose sensing, insulin transcription and insulin secretion (44). Further studies are needed to determine if these genes are important only in male islets or are required in both sexes for maintaining β cell function. 4.2 Sex Differences in ER Stress-Associated Phenotypes We also found that males and females differ in ER stress-associated phenotypes. In respect to cell death, upon the induction of ER stress we observed male and female islets differed in their sensitivity to ER-stress induced apoptosis. Males had significantly increased ER stress-induced apoptosis across a dose response of Tg, whereas females where only sensitive to the highest dose 62 of Tg. Our work extends findings from other contexts from single sex and cell culture studies that demonstrate ER stress induces apoptosis in β cell lines as well as mouse and human β cells (173, 246–248). Furthermore, the diabetic Munich mouse model which experiences endogenous ER stress due to an insulin gene mutation, shows a significant reduction in β cell mass of male but not female mice (166). In line with these results, sex differences in in vivo ER stress induction have been reported in kidneys. Male mice were more susceptible to ER stress-induction and had elevated protein markers of apoptosis compared to female mice (249). This supports that there is a sex difference in a hallmark of ER stress, despite equivalent induction of ER stress signaling. Reasons for sex-specific cell death in the context of similar signaling are not entirely clear. Previous studies have proposed that estrogen plays a protective role against ER stress and oxidative stress by preserving β cell mass and reducing apoptosis in cell lines, mouse models, and human islets (61, 63, 250). In Akita mice, conjugated estrogen has been demonstrated to confer protection against diabetes incidence by preventing the onset of hyperglycemia and reducing the loss of β cell mass. Conjugated estrogens were found to exert their actions through ERα to mitigate ER stress. Specifically, conjugated estrogens stabilized the ERAD system, promoting the degradation of misfolded proinsulin. This was demonstrated using thapsigargin to increase ER stress beyond endogenous levels present in an Akita mouse cell line (63). While this study provided critical insight into a mechanism by which estrogen can mitigate ER stress, other than a blood insulin measurement during glucose tolerance tests, no measures of β cell function were assessed. Therefore, while estrogen improved glucose homeostasis in male and female mice, more remains to be understood regarding male and female β cells stress response and how stress impacts β cell function. 63 Our data suggests that another potential contributing factor is a sex difference in protein synthesis. Protein synthesis repression is a classical phenotype of ER stress (171); however, it has not previously been reported to be sexually dimorphic. Our study has identified for the first time a sex difference in protein synthesis repression, where female islets restore protein synthesis to basal rates quicker than male islets. Perhaps this ability to reverse the repression of protein synthesis is important in female islets ability to evade ER stress-induced cell death since the inability to recover from protein synthesis repression increases ER-stress induced apoptosis (52). A large body of work from single-sex studies and cell culture have expanded our knowledge of ER stress-induced cell death and UPR-mediated recovery from protein translation repression (193, 251–255). Future studies will need to determine whether these mechanisms are sex-specific. 4.3 Male-Female Differences in β Cell Function The primary function of β cells is to produce and release insulin, as insulin biosynthesis accounts for approximately half the total protein production in β cells (172). To facilitate their need to accommodate the demands of insulin production, β cells possess an extensive ER (179, 256). The ER is responsible for synthesis, folding, modifying and transporting proteins, and functions as a major site for lipid synthesis and calcium storage (257). Insulin is produced at a rate of up to one million molecules per minute in response to glucose stimulation (258). Therefore, it is critical for β cells to maintain a balance between the protein load and folding capacity of the ER to maintain insulin biosynthesis. 64 When we examined β cell function, we found that males and females exhibited differences in their response to ER stress. Female islets maintain HG stimulated insulin secretion and increase insulin content under stress, whereas males displayed a repression of HG stimulated insulin secretion. Similarly, there was a greater decrease in insulin release between T2D and ND human islets in males than females. This suggests male and female islets exhibit fundamentally different behaviors in response to stress. Other studies have attributed male-female differences in β cells function to differences in peripheral insulin sensitivity and that observed differences in β cell response are caused by insulin resistance. However, in our data we identified sex differences in mouse β cell function and responses to stress which arise prior to the onset of insulin resistance, as male and female mice have equivalent insulin sensitivity. Therefore, the β cells differences we describe are intrinsic to the β cell that pre-date insulin resistance. In line with these findings, human studies have shown that glucose responses during the oral glucose tolerance test differ between healthy men and women without diabetes (1–3). Importantly, it has been reported that these differences in glucose stimulated insulin secretion persist in the context of equivalent insulin sensitivity (1, 2). This supports findings where there are functional differences between male and female β cells which are not solely caused by differences in insulin resistance. While estrogen has been shown to influence peripheral insulin sensitivity in males and females (4), its effects on β cell function has not been studied in the context of equivalent insulin sensitivity. Therefore, our study extends findings into intrinsic β cell sex differences. 65 4.4 Male-Female Transcriptional Differences in Response to ER Stress The final main finding of our study was that under ER stress induction, male and female islets exhibit unique gene expression changes. Both males and females experience ER stress as evidenced by data in this study and by others (173), and when we performed RNAseq on islets treated with ER stress inducing Tg, the UPR was identified as the most significantly differentially expressed pathway in both sexes and treatment times. Interestingly, while the majority of altered UPR genes are shared between the sexes, 18/62 UPR genes are differentially regulated by sex and treatment time. Specifically, Arfgap1, Creb3l1 and Xbp1, are uniquely upregulated by Tg in females. In males Sec31a is uniquely upregulated by Tg at 6 hours as well as Nfyb and Exosc6 in males at 12 hours. When we expanded our analysis of unique differentially expressed genes, we found significantly enriched pathways in males, but not females. At both 6- and 12-hour treatment times males displayed enriched translation pathways, predominantly associated with ribosomal genes. Comparing ribosome gene expression between males and females we observed that the sex difference in ribosomal gene expression was abolished by ER stress induction. This was due to the greater upregulation of ribosomal genes in males than females (55 male, 12 female). Lastly, we identified that downregulation of β cell identity genes occurs in both sexes but shows differences in gene expression over time. A greater number of β cell identity genes were further downregulated by increasing time in Tg in females than males. Insight into dedifferentiation and β cell failure has come from mixed sex studies (176, 259, 260), therefore more studies are needed to determine if the phenotypes associated with reduced β cell maturity markers are sex-specific. Previous studies have examined gene expression to ER stress in human B lymphocytes cells (261), and in female mouse embryonic 66 fibroblasts (262), however, this is the first study to examine the effect of ER stress on the islet transcriptome in male and female mice. Overall, our data suggests that female islets are more resilient to ER stress. We propose potential models to explain how male-female β cells differ in their ER stress response. Firstly, female islets have a greater threshold prior to experiencing β cell failure (Fig. 13). Compared to males, female islets possess a greater complement of protein folding and UPR pathway genes that may prime female islets with the capacity to mount a greater adaptive ER stress response. The threshold may be either time based (Fig. 13A) in which a longer ER stress exposure is required to induce β cell failure in females, and/or accumulation based (Fig. 13B) in which female islets can cope with a greater misfolded and unfolded protein burden in the ER. Supporting evidence comes from the insulin secretion assays where female islets retained insulin secretory function throughout Tg-induced ER stress whereas male islets exhibited a repression of glucose stimulated insulin secretion. An alternative model is that female and males have an equivalent threshold for ER stress induction; but male and female islets exhibit a different downstream response (Fig. 13C). Supporting evidence comes from the finding of equivalent induction of ER stress markers in both sexes, female islets’ ability to recover faster from protein synthesis repression and lower cell death. A time-course assessment of ER stress-signaling markers will be required to determine if the onset of ER stress occurs within a similar timeframe and to a similar degree of activation. Furthermore, while female islets recovered quicker from protein synthesis repression, this does not conclude they had recovered from ER stress, as protein markers of ER stress were upregulated compared to controls at the same timepoint. Lastly, RNAseq data from the Tg treated islets supports this model; despite similar ER stress pathway activation, males and females exhibited unique gene expression changes in response to ER stress. 67 4.5 Overall Significance Despite known differences in β cells between males and females, the relationship between ER stress, protein synthesis, insulin production and secretion, and cell death has not been thoroughly examined in both sexes (171, 263–265). These findings further our understanding of male-female differences in islet stress responses and provide insight into potential mechanisms underlying the differential risk of developing T2D between men and women. 68 4.6 Limitations While studying islets ex vivo limits confounding factors such as differences insulin sensitivity and glucose homeostasis, there are limitations to this method. The process of islet isolations themselves can trigger stress responses in islets involving apoptosis and larger islets are susceptible to hypoxia (266, 267). This can be mitigated by gentler pancreas digestions, reducing islet culture media to increase oxygenation, and removing unhealthy islets prior to overnight culture. Our islet culture media contains fetal bovine serum (FBS) which is the universal growth supplement of cell and primary tissue culture media; however, it contains low concentrations of sex hormones and the composition of FBS varies between lots (268). Charcoal stripped FBS is an alternative to regular FBS since charcoal-stripping reduces sex hormone levels (269). Insulin resistance has been shown to vary throughout the estrus cycle (117); however, we did not control for the stage of the estrus cycle. We did use age-matched siblings to reduce litter variability and limited female mice to a single pregnancy to minimize the effect of the higher fat content in breeder diets (270). In our physiology experiments, we performed intraperitoneal glucose tolerance tests. A recent study has highlighted that oral administration of glucose promotes a greater insulin secretion response and is more physiologically relevant (271). It is unknown if Tg affects males and females differently. In our RNAseq data Serca1 and Serca2 differ by less than 0.2 log2FC between male and females (Serca2; -0.15 log2FC, upregulated in females, Serca3; 0.17 log2FC, upregulated in males), therefore it is unlikely the subtle gene expression differences impact the effect of thapsigargin inhibiting SERCA activity. 69 4.7 Future Directions To gain further insight into the reduction in β cell identity genes and potential dedifferentiation upon ER stress induction, future work would involve histological analysis and scRNA-seq. This would allow us to determine if a change in the proportion of β cell occurs and the power of scRNA-seq would permit the analysis of sub populations of β cells to determine if dedifferentiation occurs on a population or individual cell level. To extend our understanding of sex differences in β cells ER stress, further studies would include assessing male-female differences in β cell function in mouse models of endogenous ER stress. 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Thesis/Dissertation
eng
Cell and Developmental Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Rideout, Elizabeth J.
Johnson, James D.
Sex differences in islet stress responses support female beta cell resilience
Text