Male Islets Research Articles

6524 Sex-Specific Single Cell Genetic Architecture Of Human Islets With And Without Type 2 Diabetes

Abstract Disclosure: M. Qadir: None. R.M. Elgamal: None. P. Kudtarkar: None. K.J. Gaulton: None. F. Mauvais-Jarvis: None. Type 2 diabetes (T2D) is a heterogeneous disease and biological sex affects its pathogenesis including the development of adiposity, insulin resistance, and β cell failure. For example, ketosis-prone diabetes is a phenotypically-defined form of T2D with acute β cell failure and severe insulin deficiency, predominantly observed in men. However, what mechanisms drive male predominance in β cell failure is unknown. To fully understand the sex-based pathogenesis of islet failure in diabetes, the study of sex differences in human islet biology and dysfunction is essential. To decipher sex differences in human islets genetic architecture and function, we performed an orthogonal series of experiments from 15 islets of non-diabetic (ND) donors, using single cell RNAseq (scRNAseq) and single nucleus assay for transposase-accessible chromatin sequencing (snATACseq). We also leveraged 37 ND and T2D donors for scRNAseq data from the human pancreas analysis program (HPAP) dataset. We studied over 200,000 single cells spanning the accessible genome and transcriptome. It is the largest study of sex-based single-cell genomics and transcriptomics of human islets to date. We observe that in cultured human ND islets, in the absence of the in vivo hormonal environment, islet cell transcriptome, and genome accessibility across coding regions are predominantly limited to sex chromosome genes. Of particular interest are sex differences in gene accessibility and expression of the X-linked KDM6A and Y-linked KDM5D demethylase genes in female (F) and male (M) islet cells respectively. Furthermore, analysis of T2D islets revealed a greater number of autosomal differentially expressed genes (DEGs) in F (721 upregulated and 1164 downregulated, FDR < 0.01) compared to M β cells (111 upregulated and 99 downregulated, FDR < 0.01). M β cell DEGs over-representation analysis revealed suppressed insulin secretion and processing pathways, while F β cell DEGs exhibited suppression of oxidative phosphorylation and electron transport chain pathways. Thus, although, sex differences in gene expression and accessibility of cultured ND human islets are restricted to sex chromosome genes, during the transition from ND to T2D, major sex differences in autosomal DEGs appear, with a greater number of autosomal DEGs in F compared to M T2D islets. Presentation: 6/3/2024

The GHSR1a antagonist LEAP2 regulates islet hormone release in a sex-specific manner.

LEAP2, a liver-derived antagonist for the ghrelin receptor, GHSR1a, counteracts effects of ghrelin on appetite and energy balance. Less is known about its impact on blood glucose-regulating hormones from pancreatic islets. Here we investigate whether acyl-ghrelin (AG) and LEAP2 regulate islet hormone release in a cell type- and sex-specific manner. Hormone content from secretion experiments with isolated islets from male and female mice was measured by radioimmunoassay and mRNA expression by qPCR. LEAP2 enhanced insulin secretion in islets from males (p<0.01) but not females (p<0.2), whilst AG-stimulated somatostatin release was significantly reversed by LEAP2 in males (p<0.001) but not females (p<0.2). Glucagon release was not significantly affected by AG and LEAP2. Ghsr1a, Ghrelin, Leap2, Mrap2, Mboat4 and Sstr3 islet mRNA expression did not differ between sexes. In control male islets maintained without 17-beta oestradiol (E2), AG exerted an insulinostatic effect (p<0.05), with a trend towards reversal by LEAP2 (p=0.06). Both were abolished by 72h E2 pre-treatment (10 nmol/l, p<0.2). AG-stimulated somatostatin release was inhibited by LEAP2 from control (p<0.001) but not E2-treated islets (p<0.2). LEAP2 and AG did not modulate insulin secretion from MIN6 beta cells and Mrap2 was downregulated (P<0.05) and Ghsr1a upregulated (P<0.0001) in islets from Sst-/- mice. Our findings show that AG and LEAP2 regulate insulin and somatostatin release in an opposing and sex-dependent manner, which in males can be modulated by E2. We suggest that regulation of SST release is a key starting point for understanding the role of GHSR1a in islet function and glucose metabolism.

Single cell regulatory architecture of human pancreatic islets suggests sex differences in β cell function and the pathogenesis of type 2 diabetes.

Biological sex affects the pathogenesis of type 2 and type 1 diabetes (T2D, T1D) including the development of β cell failure observed more often in males. The mechanisms that drive sex differences in β cell failure is unknown. Studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in β cell failure in diabetes. Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D (including 37 donors from the Human Pancreas Analysis Program [HPAP] dataset) using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. In cultured islets from nondiabetic (ND) donors, in the absence of the in vivo hormonal environment, sex differences in islet cell type gene accessibility and expression predominantly involved sex chromosomes. Of particular interest were sex differences in the X-linked KDM6A and Y-linked KDM5D chromatin remodelers in female and male islet cells respectively. Islets from T2D donors exhibited similar sex differences in differentially expressed genes (DEGs) from sex chromosomes. However, in contrast to islets from ND donors, islets from T2D donors exhibited major sex differences in DEGs from autosomes. Comparing β cells from T2D and ND donors revealed that females had more DEGs from autosomes compared to male β cells. Gene set enrichment analysis of female β cell DEGs showed a suppression of oxidative phosphorylation and electron transport chain pathways, while male β cell had suppressed insulin secretion pathways. Thus, although sex-specific differences in gene accessibility and expression of cultured ND human islets predominantly affect sex chromosome genes, major differences in autosomal gene expression between sexes appear during the transition to T2D and which highlight mitochondrial failure in female β cells.

FRI613 Sex Differences In Human Islet Transcriptional Responses To Inflammation

Abstract Disclosure: K.L. Webster: None. W. Wu: None. B. Webb-Robertson: None. E. Nakayasu: None. S. Sarkar: None. C. Evans-Molina: None. S.A. Tersey: None. J. Enriquez: None. S.R. Hammes: None. R.G. Mirmira: None. Higher female incidence of autoimmune disease is often attributed to sex hormone-immune cell interactions or X chromosome immune genes escaping inactivation. Type 1 diabetes (T1D), however, occurs slightly more often in males, and studies show that female sex protects beta cell health under conditions of stress. It is now thought that beta cells themselves trigger loss of immune tolerance in T1D and that the state of beta cell differentiation and health dictates their susceptibility to autoimmunity. Here, we hypothesized that sex influences beta cell response to inflammation during T1D development. We utilized prior bulk RNA sequencing data on human islets from 10 donors (6 males, 4 females) treated for 18h with or without proinflammatory cytokines (PIC: IL-1b, IFNy) to mimic T1D inflammation. Hierarchical clustering grouped samples largely by PIC treatment but also by sex. To identify differentially expressed genes (DEGs) for each sex in response to PIC, we filtered for genes with fold change&amp;gt;1.5 and false discovery rate&amp;lt;0.05. A total of 3754 DEGs were detected in male islets with PIC treatment, while just 1117 were detected in female islets. Of these, 1016 genes were shared between sexes, and many related to cytokine signaling and immune responses. Only 101 DEGs were unique to females, and these were enriched in terms related to leukocyte aggregation, glutamate signaling, and responses to steroid hormone stimuli. 2738 DEGs unique to males were enriched in terms related to inflammation, NOTCH signaling, differentiation, and estrogen biosynthesis. Notably, the gene encoding double-stranded RNA sensor MDA5 (Ifih1), whose activity induces antiviral type I IFN responses and has been linked to T1D, showed a greater average increase with PIC in males (9.99-fold vs. 6.64-fold in females, p&amp;lt;0.05). By gene set enrichment analysis, control and PIC-treated female islets were enriched in differentiation and development pathways, while male islets were enriched in protein translation and steroid metabolism. To probe the role of sex steroid signaling in islet inflammatory response, we measured expression of Esr1 (encoding ERa) via qRT-PCR in mouse islets in response to PIC (IFNy, IL-1b, TNFa). In non-diabetic C57BL6/J mice, islets from both males and females increased Esr1 with PIC (3.63- and 4.39-fold, p&amp;lt;0.05). However, in pre-diabetic NOD mice, in which female mice are highly susceptible to autoimmune diabetes, islets from neither sex increased Esr1. Interestingly, islets from C57BL6/J mice of both sexes decreased expression of Ar (encoding androgen receptor) upon PIC treatment, a response also absent in NOD mice. These data suggested that Esr1 might be protective- and Ar detrimental- for beta cell function, but not in a sex-dependent manner. Collectively, these data demonstrate a sexual dimorphism in islet response to inflammation, with evidence of a more aggressive inflammatory response in males. Presentation: Friday, June 16, 2023

Sex differences in islet stress responses support female β cell resilience

ObjectivePancreatic β cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in β cells, but few studies have examined male-female differences in β cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in β cell and islet gene expression and function using both human and rodent samples. MethodsIn humans, we compared β cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and β cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress. ResultsOur single-cell analysis of human β cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males. ConclusionsOur data demonstrate sex differences in β cell gene expression in both humans and mice, and that female β cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.

Sex Differences in the Molecular Programs of Pancreatic Cells Contribute to the Differential Risks of Type 2 Diabetes.

Epidemiology studies demonstrate that women are at a significantly lower risk of developing type 2 diabetes (T2D) compared to men. However, the molecular basis of this risk difference is not well understood. In this study, we examined the sex differences in the genetic programs of pancreatic endocrine cells. We combined pancreas perifusion data and single-cell genomic data from our laboratory and from publicly available data sets to investigate multiple axes of the sex differences in the human pancreas at the single-cell type and single-cell level. We systematically compared female and male islet secretion function, gene expression program, and regulatory principles of pancreatic endocrine cells. The perifusion data indicate that female endocrine cells have a higher secretion capacity than male endocrine cells. Single-cell RNA-sequencing analysis suggests that endocrine cells in male controls have molecular signatures that resemble T2D. In addition, we identified genomic elements associated with genome-wide association study T2D loci to have differential accessibility between female and male delta cells. These genomic elements may play a sex-specific causal role in the pathogenesis of T2D. We provide molecular mechanisms that explain the differential risk of T2D between women and men. Knowledge gained from our study will accelerate the development of diagnostics and therapeutics in sex-aware precision medicine for diabetes.

1456-P: Molecular Architecture of AR Action in Insulin Secretion in Male Pancreatic ß-Cells

Male mice lacking the androgen receptor (AR) in islet β cells exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of GLP-1 in mouse and human islets. To gain information on DHT-stimulated signaling networks promoting GSIS, we combined functional proteomics with highly selective pharmacological inhibitors in male human islets, and islets from genetic mouse models. We found that DHT-stimulated GSIS in human islets involved a signaling cascade including focal adhesion kinases, the nonreceptor tyrosine kinase SRC, phosphoinositide 3-kinase, and the activation of mTOR complex 2. To define the early network of AR interacting proteins, we performed DHT-stimulated AR immunoprecipitation in INS1 832/3 cells followed by mass spectrometry-based proteomics. We will discuss the AR interactome including: 1) adenylate cyclase-modulating G proteins Gαs and Gαi, cAMP-dependent PKA, and the cAMP-dependent protein LASP1 regulating actin-based cytoskeletal activities; 2) Actin cytoskeleton remodeling proteins promoting actin depolymerization, and 3) Vesicular maturation including the lysosomal vacuolar-type ATPase acidifying insulin secretory granules and the prohormone convertase 2 involved in proinsulin processing. Notably, GLP-1 addition to DHT decreased AR interaction with all these proteins and promoted AR interaction with microtubule motor proteins promoting vesicular transport, proteins involved in exocytic vesicle docking to the plasma membrane, as well as small GTPase activating proteins and guanine nucleotide exchange factors involved in actin remodeling, as well as vesicle trafficking. Together these experiments provide novel insights into the architecture of AR action in enhancing GSIS. Disclosure P.Mota de sa: None. M.Qadir: None. M.Blandino-rosano: None. J.Fan: None. W.Xu: None. E.Bernal-mizrachi: None. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

320-OR: Maternal Overnutrition Confers Increased Spare Respiratory Capacity and Glucose-Stimulated Insulin Secretion in Nonhuman Primate Offspring Islets

The Developmental Origins of Health and Disease hypothesis states that the intrauterine environment influences postnatal health and adult disease susceptibility. In the United States, over 30% of women of childbearing age are obese, in part due to Western Style Diet (WSD) consumption. Previous data from our group using a Japanese macaque model, revealed that maternal overnutrition increases the β:α cell ratio in the islets of offspring. WSD-exposed offspring islets also exhibit inappropriate hypersecretion of insulin in response to glucose at 1 and 3 years of age. We hypothesize that fetal exposure to WSD induces a compensatory increase in β-cell fuel metabolism resulting in an inappropriate increase in insulin secretion by offspring islets. When we assessed the effect of maternal diet on mitochondrial respiration in offspring islets, we found that spare respiratory capacity is increased in islets of one year old WSD-exposed male offspring, suggesting an increased capacity to adapt to increased metabolic demand. This could be due to increased mitochondrial abundance; however, maternal overnutrition does not appear to increase β cell mitochondrial density when analyzed via transmission electron microscopy (TEM) . Insulin granule density is increased in male offspring islets in TEM micrographs, implying that insulin production is increased in response to intrauterine WSD exposure. Transcripts for CACNA1C (voltage gated calcium channel subunit) , ABCC8 (ATP-sensitive potassium channel subunit) , and VAMP2 (vesicle fusion) are increased in response to maternal WSD exposure in offspring islets. Thus, our data suggest that increases in insulin secretion in offspring exposed to maternal overnutrition result from changes in metabolic and secretion pathways. Importantly, these mechanisms could potentiate hyperinsulinism which may predispose offspring to insulin resistance and beta cell failure in adulthood. Disclosure D.T. Carroll: None. V.F. Ricciardi: None. J.C. Dunn: None. S.R. Lindsley: None. T. Dean: None. M. Kirigiti: None. S.R. Wesolowski: None. C.E. McCurdy: None. P. Kievit: Research Support; Novo Nordisk A/S. J.E. Friedman: None. M. Gannon: None. Funding NIH-NIDDK (5T32 DK7563-30, R24DK090964-06, 1R01DK128187)

1474-P: Testosterone Amplifies Glucose-Stimulated Insulin Secretion from Male ß-Cells via Soluble Adenylate Cyclase

Male mice with elimination of the androgen receptor (AR) in islet β cells (βARKO) exhibit blunted glucose-stimulated insulin secretion (GSIS) , leading to hypoinsulinemia and hyperglycemia. We showed that dihydrotestosterone (DHT) activation of an extranuclear AR in β cells potentiates GSIS by amplifying the insulinotropic action of islet-derived and exogenous GLP-1 via GLP-1R in mouse and human islets. Using electron microscopy, we observe that DHT increases the size of granules and the frequency of multivesicular granules, suggesting that DHT promotes multivesicular exocytosis. Using live β cell models expressing organelle-specific cAMP sensors and novel allosteric inhibitors in human islets, we show that DHT enhances the ability of GLP-1 to produce cAMP at the plasma membrane and endosomes and enhances islet GSIS via the bicarbonate (HCO3 -) sensor, soluble adenylate cyclase (sAC) . The end-products of glucose metabolism in the TCA cycle are CO2 and H2O, which is followed by rapid conversion of CO2 to HCO3 - and H+ by carbonic anhydrase (CA) . Therefore, CO2 production is translated to HCO3 - and specifically sensed by sAC. Bioenergetics analysis of DHT-stimulated respiration in mouse islets using a Seahorse Analyser revealed that DHT decreases glycolytic acidification and increases mitochondrial acidification (H+ efflux rate) , suggesting that DHT increases mitochondrial production of CO2 followed by conversion to HCO3 - and H+, allowing HCO3 - to stimulate sAC. We discuss experiments of single-cell transcriptomics, proteomics and metabolomics dissecting the impact of DHT on human islets glycolysis and mitochondrial CO2 production. Consistent with DHT increasing production of CO2 followed by conversion to HCO3 - by CA and activation of sAC, in cultured human male islets, the CA inhibitor acetazolamide blunted DHT-enhancement of GSIS. Together these experiments provide novel insights into the role of DHT action via an extranuclear AR in enhancing GSIS. Disclosure M.Qadir: None. D.Hodson: Other Relationship; Celtarys Research. F.Mauvais-jarvis: Consultant; Mithra Pharmaceutical. W.Xu: None. D.Nasteska: None. M.Bhondeley: None. P.Mota de sa: None. C.R.Evans: None. J.Buck: None. L.Levin: None. C.Burant: None. Funding National Institutes of Health (DK107444 and DK074970) U.S. Department of Veterans Affairs Merit Award (BX003725)

The Effects of Photobiomodulation Therapy on Porcine Islet Insulin Secretion.

Background and objective: Xenotransplantation of porcine islets to human recipients has been investigated as a potential cure for type 1 diabetes. However, the porcine islets have poor insulin secretion capacity compared with human islets. The objective of this study was to evaluate the effect of photobiomodulation therapy (PBMT) in insulin secretion on isolated porcine islets. Methods: Eight pancreata were harvested from crossbred market porcine and the islets were isolated from the pancreas. The isolated islets were treated with PBMT (wavelength: 633 nm and dosages: 0.0, 15.6, and 31.3 J/cm2) followed by 30-min incubation in low (3.0 mM) or high (16.7 mM) glucose. The relative percentage differences on insulin secretion between three dosages were compared in low and high glucose, respectively. Results: Insulin secretion was higher in samples exposed to 15.6 J/cm2 PBMT in low glucose (p < 0.05), but not in high glucose. When evaluating sex differences, male islets had higher insulin secretion by 15.6 J/cm2 PBMT in low glucose compared with females (p < 0.05). No significant differences were seen in high glucose. When compared within the control groups (0.0 J/cm2 PBMT), the relative changes on insulin secretion in high glucose was significantly higher on male islets (p < 0.05), but not on female islets. Conclusions: PBMT may increase insulin secretion on isolated porcine islets in basal condition, but it may not improve islets' glucose responsiveness to secrete insulin. Male porcine islets may respond to PBMT and glucose stimuli better than female islets on insulin secretion.

226-LB: Elimination of the Progesterone Receptor in ß Cells Improves ß-Cell Function and Glucose Tolerance during Pregnancy and Diet-Induced Insulin Resistance in Female Mice

We showed that the gonadal hormones estradiol and testosterone influence islet β-cell function and survival in a sex-specific manner via estrogen and androgen receptors expressed in islet β-cells. The progesterone receptor (PR) is a ligand-activated transcription factor and is believed to be involved in the β-cell adaptation to pregnancy. Leveraging public single-cell RNA-seq datasets we observe PR mRNA expression in α- β- and γ-cells in male and female human pancreatic endocrine cells. Using immunohistochemistry, we observe that, unlike in the uterus where PR is nuclear, PR is localized in the cytoplasm of β-cells in male and female human and mouse islets. We investigated the role of progesterone and PR in β-cell physiology in male and female mice with conditional deletion of PR in β-cells (βPRKO) by crossing PR-Lox/lox and MIP-Cre transgenic mice. Male βPRKO mice exhibited no alteration in glucose homeostasis on normal chow (NC) or following western-diet (WD)-induced insulin resistance and obesity compared to littermate PR-Lox/lox controls. Similarly, NC-fed female βPRKO mice exhibited no abnormality of glucose or insulin homeostasis compared to littermate PR-Lox/lox controls. However, during pregnancy or WD feeding, female βPRKO mice developed enhanced IP-glucose-stimulated insulin secretion, associated with improved IP glucose tolerance compared to littermate PR-Lox/lox controls. These results reveal that PR is a cytosolic receptor in β-cells, and suggest that in conditions of high insulin demand, such as pregnancy and diet-induced insulin resistance, elimination of PR action in β-cells of females improves the ability of β-cells to produce insulin and lowers blood glucose. PR may be involved in the pathogenesis of gestational diabetes. Disclosure P. Mota de sa: None. M. Qadir: None. F. Mauvais-jarvis: None. Funding National Institutes of Health (DK074970, DK107444); U.S. Department of Veterans Affairs (BX003725)

123-OR: Deletion of Both Pcsk1 And Pcsk2 in Beta Cells: Lack of Mature Insulin Drives Beta-Cell Expansion and Dysfunction, but Not Severe Diabetes in Mice

Proinsulin (PI) is processed to mature insulin by prohormone convertases 1/3 (Pcsk1) and 2 (Pcsk2). In individuals with T1D and T2D, there is an increase in the circulating PI:insulin ratio. We hypothesized that β-cell Pcsk1 and Pcsk2 deletion would result in β-cell dysfunction, loss of insulin production, and marked hyperglycemia. Single Pcsk1flox/flox Ins1cre/+ (Pcsk1βKO), Pcsk2flox/flox Ins1cre/+ (Pcsk2βKO), and double Pcsk1flox/flox Pcsk2flox/flox Ins1cre/+ (DPCβKO) β-cell prohormone convertase knockout mice were generated (with respective Pcsk1+/+ Pcsk2+/+ Ins1cre/+ controls) and monitored until 30 weeks old. Pcsk2βKO mice displayed no glycemic phenotype, while Pcsk1βKO mice displayed mild hyperglycemia and impaired glucose tolerance by 26 weeks of age. DPCβKO mice displayed mild hyperglycemia (10.1±1.6 vs. 12.3±2.1 mM; p=0.006) and marked glucose intolerance by 10 weeks of age (AUC: 418±135 vs. 1485±360; p&amp;lt;0.0001). No animals displayed obesity relative to controls. Insulin+ pancreas area was doubled in DPCβKO male mice (p=0.0004) and DPCβKO islets showed early glucose-induced Ca2+ influx and loss of oscillatory Ca2+ flux. Pcsk2βKO islets had unaltered mature insulin, while Pcsk1βKO islets had reduced mature insulin. DPCβKO islets contained no detectable mature insulin by western blot. In contrast, Pcsk2βKO islets had markedly reduced mature islet amyloid polypeptide (IAPP), while Pcsk1βKO islets contained normal mature IAPP levels. Our results suggest that complete loss of β-cell prohormone processing, including PI processing, can drive β-cell dysfunction and expansion, but only mild diabetes and glucose intolerance. Although Pcsk2 deletion in β cells does not impact glycemia or PI processing, it is required for normal proIAPP processing. In total, our data support the idea that altered β-cell prohormone processing does not impact obesity but can contribute to the pathogenesis of diabetes via β-cell dysfunction. Disclosure A. Taylor: None. Y. Chen: None. D. Pasula: None. D. S. Luciani: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem, Stock/Shareholder; Self; Integrated Nanotherapeutics Inc. Funding Canadian Institutes of Health Research

Pronounced proliferation of non-beta cells in response to beta-cell mitogens in isolated human islets of Langerhans

The potential to treat diabetes by increasing beta-cell mass is driving a major effort to identify beta-cell mitogens. Demonstration of mitogen activity in human beta cells is frequently performed in ex vivo assays. However, reported disparities in the efficacy of beta-cell mitogens led us to investigate the sources of this variability. We studied 35 male (23) and female (12) human islet batches covering a range of donor ages and BMI. Islets were kept intact or dispersed into single cells and cultured in the presence of harmine, glucose, or heparin-binding epidermal growth factor-like growth factor (HB-EGF), and subsequently analyzed by immunohistochemistry or flow cytometry. Proliferating cells were identified by double labeling with EdU and Ki67 and glucagon, c-peptide or Nkx6.1, and cytokeratin-19 to respectively label alpha, beta, and ductal cells. Harmine and HB-EGF stimulated human beta-cell proliferation, but the effect of glucose was dependent on the assay and the donor. Harmine potently stimulated alpha-cell proliferation and both harmine and HB-EGF increased proliferation of insulin- and glucagon-negative cells, including cytokeratin 19-positive cells. Given the abundance of non-beta cells in human islet preparations, our results suggest that assessment of beta-cell mitogens requires complementary approaches and rigorous identification of cell identity using multiple markers.

Selenocysteine insertion sequence binding protein 2 (Sbp2) in the sex-specific regulation of selenoprotein gene expression in mouse pancreatic islets

Selenoproteins are a group of selenocysteine-containing proteins with major roles in cellular antioxidant defense and thyroid hormone metabolism. Selenoprotein expression is determined by hierarchical mechanisms that result in tissue-specific levels. Current data inadequately explain the abundance of various selenoproteins under normal and pathological conditions, including in pancreatic β-cells. Selenocysteine insertion sequence binding protein 2 (SBP2) is a critical protein in selenoprotein translation that also plays an essential role in stabilizing selenoprotein transcripts by antagonizing nonsense-mediated decay (NMD). Importantly, dysfunctional SBP2 is associated with endocrine disorders in humans. Here we describe the impact of induced Sbp2 deficiency in pancreatic β-cells on selenoprotein transcript profiles in the pancreatic islets of C57BL/6J mice. Sex differences were noted in control mice, in which female islets showed 5 selenoproteins decreased and one increased versus male islets. Induced Sbp2 deficiency in pancreatic β-cells altered expression of only 3 selenoprotein transcripts in male islets, whereas 14 transcripts were reduced in female islets. In all cases, decreased transcription was observed in genes known to be regulated by NMD. The differential impact of Sbp2 deletion on selenoprotein transcription between sexes suggests sex-specific hierarchical mechanisms of selenoprotein expression that may influence islet biology and consequentially metabolic disease risk.

OR09-5 Importance of Islet Intracrinology for Glucose-Stimulated Insulin Secretion (GSIS) in the Male

The importance of androgens and estrogens in preventing adiposity and insulin resistance in the male is well characterized (1, 2), but less is known about the role of androgens and estrogens in β cell function and insulin secretion. Both the androgen receptor (AR) and estrogen receptors (ERs) are present in β cells, and we showed that AR and ERs are important for pancreatic β cell health and function in the male (3, 4). In males, testosterone (T) is the main circulating source of active androgen acting on AR (dihydrotestosterone, DHT) and estrogen acting on ERs (estradiol, E2) following conversion by 5α-reductase (5α-R) and aromatase (Ar) respectively. However, whether 5α-R and Ar are expressed and active in β cells is unknown. Using qPCR and immunohistochemistry, we show that mRNA and proteins for 5α-R and Ar are expressed in male islets and β cells. In male mouse islets, T treatment enhances GSIS compared to vehicle. The effect of T is partially blocked by the 5α-R inhibitors, finasteride (FNS) and dutasteride (DTS) demonstrating that T is converted to DHT by 5α-R to enhance GSIS. Further, the insulinotropic effect of T is totally blocked by co-treatment with FNS, DTS and the Ar inhibitor, anastrozole, demonstrating that T is also converted to E2 by Ar to enhance GSIS. We detected DHT by tandem mass spectrometry in the incubations from male mouse islets with T treatment. DHT was not detectable in any of the incubations with T and 5α-R inhibitor treatment. Our study reveals the presence of functional 5α-R and Ar in male pancreatic islet β cells and identifies a novel paradigm of islet intracrinology with local conversion of T into DHT and E2, the actions of which enhance GSIS.

SAD-A Promotes Glucose-Stimulated Insulin Secretion Through Phosphorylation and Inhibition of GDIα in Male Islet β Cells.

Rho GDP-dissociation inhibitor (GDIα) inhibits glucose-stimulated insulin secretion (GSIS) in part by locking Rho GTPases in an inactive GDP-bound form. The onset of GSIS causes phosphorylation of GDIα at Ser174, a critical inhibitory site for GDIα, leading to the release of Rho GTPases and their subsequent activation. However, the kinase regulator(s) that catalyzes the phosphorylation of GDIα in islet β cells remains elusive. We propose that SAD-A, a member of AMP-activated protein kinase-related kinases that promotes GSIS as an effector kinase for incretin signaling, interacts with and inhibits GDIα through phosphorylation of Ser174 during the onset GSIS from islet β cells. Coimmunoprecipitation and phosphorylation analyses were carried out to identify the physical interaction and phosphorylation site of GDIα by SAD-A in the context of GSIS from INS-1 β cells and primary islets. We identified GDIα directly binds to SAD-A kinase domain and phosphorylated by SAD-A on Ser174, leading to dissociation of Rho GTPases from GDIα complexes. Accordingly, overexpression of SAD-A significantly stimulated GDIα phosphorylation at Ser174 in response to GSIS, which is dramatically potentiated by glucagonlike peptide-1, an incretin hormone. Conversely, SAD-A deficiency, which is mediated by short hairpin RNA transfection in INS-1 cells, significantly attenuated endogenous GDIα phosphorylation at Ser174. Consequently, coexpression of SAD-A completely prevented the inhibitory effect of GDIα on insulin secretion in islets. In summary, glucose and incretin stimulate insulin secretion through the phosphorylation of GDIα at Ser174 by SAD-A, which leads to the activation of Rho GTPases, culminating in insulin exocytosis.

Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male

Although men with testosterone deficiency are at increased risk for type 2 diabetes (T2D), previous studies have ignored the role of testosterone and the androgen receptor (AR) in pancreatic β cells. We show that male mice lacking AR in β cells (βARKO) exhibit decreased glucose-stimulated insulin secretion (GSIS), leading to glucose intolerance. The AR agonist dihydrotestosterone (DHT) enhances GSIS in cultured male islets, an effect that is abolished in βARKO(-/y) islets and human islets treated with an AR antagonist. In β cells, DHT-activated ARis predominantly extranuclear and enhances GSIS by increasing islet cAMP and activating the protein kinase A. In mouse and human islets, the insulinotropic effect of DHT depends on activation of the glucagon-like peptide-1 (GLP-1) receptor, and accordingly, DHT amplifies the incretin effect of GLP-1. This study identifies AR as a novel receptor that enhances β cell function, a finding with implications for the prevention of T2D in aging men.

Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancy-related mRNA changes.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the ‘islet pregnancy gene signature’, which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.

Sex differences in the genome-wide DNA methylation pattern and impact on gene expression, microRNA levels and insulin secretion in human pancreatic islets.

BackgroundEpigenetic factors regulate tissue-specific expression and X-chromosome inactivation. Previous studies have identified epigenetic differences between sexes in some human tissues. However, it is unclear whether epigenetic modifications contribute to sex-specific differences in insulin secretion and metabolism. Here, we investigate the impact of sex on the genome-wide DNA methylation pattern in human pancreatic islets from 53 males and 34 females, and relate the methylome to changes in expression and insulin secretion.ResultsGlucose-stimulated insulin secretion is higher in female versus male islets. Genome-wide DNA methylation data in human islets clusters based on sex. While the chromosome-wide DNA methylation level on the X-chromosome is higher in female versus male islets, the autosomes do not display a global methylation difference between sexes. Methylation of 8,140 individual X-chromosome sites and 470 autosomal sites shows sex-specific differences in human islets. These include sites in/near AR, DUSP9, HNF4A, BCL11A and CDKN2B. 61 X-chromosome genes and 18 autosomal genes display sex-specific differences in both DNA methylation and expression. These include NKAP, SPESP1 and APLN, which exhibited lower expression in females. Functional analyses demonstrate that methylation of NKAP and SPESP1 promoters in vitro suppresses their transcriptional activity. Silencing of Nkap or Apln in clonal beta-cells results in increased insulin secretion. Differential methylation between sexes is associated with altered levels of microRNAs miR-660 and miR-532 and related target genes.ConclusionsChromosome-wide and gene-specific sex differences in DNA methylation associate with altered expression and insulin secretion in human islets. Our data demonstrate that epigenetics contribute to sex-specific metabolic phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0522-z) contains supplementary material, which is available to authorized users.

Maternal high-fat diet induces insulin resistance and deterioration of pancreatic β-cell function in adult offspring with sex differences in mice

Intrauterine environment may influence the health of postnatal offspring. There have been many studies on the effects of maternal high-fat diet (HFD) on diabetes and glucose metabolism in offspring. Here, we investigated the effects in male and female offspring. C57/BL6J mice were bred and fed either control diet (CD) or HFD from conception to weaning, and offspring were fed CD or HFD from 6 to 20 wk. At 20 wk, maternal HFD induced glucose intolerance and insulin resistance in offspring. Additionally, liver triacylglycerol content, adipose tissue mass, and inflammation increased in maternal HFD. In contrast, extending previous observations, insulin secretion at glucose tolerance test, islet area, insulin content, and PDX-1 mRNA levels in isolated islets were lower in maternal HFD in males, whereas they were higher in females. Oxidative stress in islets increased in maternal HFD in males, whereas there were no differences in females. Plasma estradiol levels were lower in males than in females and decreased in offspring fed HFD and also decreased by maternal HFD, suggesting that females may be protected from insulin deficiency by inhibiting oxidative stress. In conclusion, maternal HFD induced insulin resistance and deterioration of pancreatic β-cell function, with marked sex differences in adult offspring accompanied by adipose tissue inflammation and liver steatosis. Additionally, our results demonstrate that potential mechanisms underlying sex differences in pancreatic β-cell function may be related partially to increases in oxidative stress in male islets and decreased plasma estradiol levels in males.