Non-diabetic Islets Research Articles

Real-time detection of somatostatin release from single islets reveals hypersecretion in type 2 diabetes.

Somatostatin from pancreatic δ-cells is a paracrine regulator of insulin and glucagon secretion, but the release kinetics and whether secretion is altered in diabetes is unclear. This study aimed to improve understanding of somatostatin secretion by developing a tool for real-time detection of somatostatin release from individual pancreatic islets. Reporter cells responding to somatostatin with cytoplasmic Ca2+ concentration ([Ca2+]i) changes were generated by co-expressing somatostatin receptor SSTR2, the G-protein Gα15 and a fluorescent Ca2+ sensor in HeLa cells. Somatostatin induced dose-dependent [Ca2+]i increases in reporter cells with half-maximal and maximal effects at 1.6 ± 0.4 and ~30 nM, respectively. Mouse and human islets induced reporter cell [Ca2+]i elevations that were inhibited by the SSTR2 antagonist CYN154806. Depolarization of islets by high K+, KATP channel blockade or increasing the glucose concentration from 3 to 11 mM evoked concomitant elevations of [Ca2+]i in islets and reporter cells. Exposure of islets to glucagon, GLP-1 and ghrelin also triggered reporter cell [Ca2+]i responses, whereas little effect was obtained by islet exposure to insulin, glutamate, GABA and urocortin-3. Islets from type 2 diabetic human donors induced higher reporter cell [Ca2+]i responses at 11 mM and after K+ depolarization compared with non-diabetic islets, although fewer δ-cells were identified by immunostaining. Type 2 diabetes is associated with hypersecretion of somatostatin, which has implications for paracrine regulation of insulin and glucagon secretion. The new reporter cell assay for real-time detection of single-islet somatostatin release holds promise for further studies of somatostatin secretion in islet physiology and pathophysiology.

DOC2b enrichment mitigates proinflammatory cytokine-induced CXCL10 expression by attenuating IKKβ and STAT-1 signaling in human islets.

Type 1 diabetic human islet β-cells are deficient in double C 2 like domain beta (DOC2b) protein. Further, DOC2b protects against cytokine-induced pancreatic islet β-cell stress and apoptosis. However, the mechanisms underpinning the protective effects of DOC2b remain unknown. Biochemical studies, qPCR, proteomics, and immuno-confocal microscopy were conducted to determine the underlying protective mechanisms of DOC2b in β-cells. DOC2b-enriched or -depleted primary islets (human and mouse) and β-cell lines challenged with or without proinflammatory cytokines, global DOC2b heterozygous knockout mice subjected to multiple-low-dose-streptozotocin (MLD-STZ), were used for these studies. A significant elevation of stress-induced CXCL10 mRNA was observed in DOC2b-depleted β-cells and primary mouse islets. Further, DOC2b enrichment markedly attenuated cytokine-induced CXCL10 levels in primary non-diabetic human islets and β-cells. DOC2b enrichment also reduced total-NF-κB p65 protein levels in human islets challenged with T1D mimicking proinflammatory cytokines. IKKβ, NF-κB p65, and STAT-1 are capable of associating with DOC2b in cytokine-challenged β-cells. DOC2b enrichment in cytokine-stressed human islets and β-cells corresponded with a significant reduction in activated and total IKKβ protein levels. Total IκBβ protein was increased in DOC2b-enriched human islets subjected to acute cytokine challenge. Cytokine-induced activated and total STAT-1 protein and mRNA levels were markedly reduced in DOC2b-enriched human islets. Intriguingly, DOC2b also prevents ER-stress-IKKβ and STAT-1 crosstalk in the rat INS1-832/13 β-cell line. The mechanisms underpinning the protective effects of DOC2b involve attenuation of IKKβ-NF-κB p65 and STAT-1 signaling, and reduced CXCL10 expression.

DOC2b enrichment mitigates proinflammatory cytokine-induced CXCL10 expression by attenuating IKKβ and STAT-1 signaling in human islets.

Type 1 diabetic human islet β-cells are deficient in double C 2 like domain beta (DOC2b) protein. Further, DOC2b protects against cytokine-induced pancreatic islet β-cell stress and apoptosis. However, the mechanisms underpinning the protective effects of DOC2b remain unknown. Biochemical studies, qPCR, proteomics, and immuno-confocal microscopy were conducted to determine the underlying protective mechanisms of DOC2b in β-cells. DOC2b- enriched or-depleted primary islets (human and mouse) and β-cell lines challenged with or without proinflammatory cytokines, global DOC2b heterozygous knockout mice subjected to multiple-low-dose-streptozotocin (MLD-STZ), were used for these studies. A significant elevation of stress-induced CXCL10 mRNA was observed in DOC2b- depleted β-cells and primary mouse islets. Further, DOC2b enrichment markedly attenuated cytokine-induced CXCL10 levels in primary non-diabetic human islets and β-cells. DOC2b enrichment also reduced total-NF-κB p65 protein levels in human islets challenged with T1D mimicking proinflammatory cytokines. IKKβ, NF-κB p65, and STAT-1 are capable of associating with DOC2b in cytokine-challenged β-cells. DOC2b enrichment in cytokine-stressed human islets and β-cells corresponded with a significant reduction in activated and total IKKβ protein levels. Total IκBβ protein was increased in DOC2b-enriched human islets subjected to acute cytokine challenge. Cytokine-induced activated and total STAT-1 protein and mRNA levels were markedly reduced in DOC2b-enriched human islets. Intriguingly, DOC2b also prevents ER-stress-IKKβ and STAT-1 crosstalk in the rat INS1-832/13 β-cell line. The mechanisms underpinning the protective effects of DOC2b involve attenuation of IKKβ-NF-κB p65 and STAT-1 signaling, and reduced CXCL10 expression.

Beta cell-specific PAK1 enrichment ameliorates diet-induced glucose intolerance in mice by promoting insulin biogenesis and minimising beta cell apoptosis

Aims/hypothesisp21 (CDC42/RAC1) activated kinase 1 (PAK1) is depleted in type 2 diabetic human islets compared with non-diabetic human islets, and acute PAK1 restoration in the islets can restore insulin secretory function ex vivo. We hypothesised that beta cell-specific PAK1 enrichment in vivo can mitigate high-fat-diet (HFD)-induced glucose intolerance by increasing the functional beta cell mass.MethodsHuman islets expressing exogenous PAK1 specifically in beta cells were used for bulk RNA-seq. Human EndoC-βH1 cells overexpressing myc-tagged PAK1 were used for chromatin immunoprecipitation (ChIP) and ChIP-sequencing (ChIP-seq). Novel doxycycline-inducible beta cell-specific PAK1-expressing (iβPAK1-Tg) mice were fed a 45% HFD pre-induction for 3 weeks and for a further 3 weeks with or without doxycycline induction. These HFD-fed mice were evaluated for GTT, ITT, 6 h fasting plasma insulin and blood glucose, body composition, islet insulin content and apoptosis.ResultsBeta cell-specific PAK1 enrichment in type 2 diabetes human islets resulted in decreased beta cell apoptosis and increased insulin content. RNA-seq showed an upregulation of INS gene transcription by PAK1. Using clonal human beta cells, we found that PAK1 protein was localised in the cytoplasm and the nucleus. ChIP studies revealed that nuclear PAK1 enhanced pancreatic and duodenal homeobox1 (PDX1) and neuronal differentiation 1 (NEUROD1) binding to the INS promoter in a glucose-responsive manner. Importantly, the iβPAK1-Tg mice, when challenged with HFD and doxycycline induction displayed enhanced glucose tolerance, increased islet insulin content and reduced beta cell apoptosis when compared with iβPAK1-Tg mice without doxycycline induction.Conclusions/interpretationPAK1 plays an unforeseen and beneficial role in beta cells by promoting insulin biogenesis via enhancing the expression of PDX1, NEUROD1 and INS, along with anti-apoptotic effects, that culminate in increased insulin content and beta cell mass in vivo and ameliorate diet-induced glucose intolerance.Data availabilityThe raw and processed RNA-seq data and ChIP-seq data, which has been made publicly available at Gene Expression Omnibus (GEO) at https://www.ncbi.nlm.nih.gov/geo/, can be accessed in GSE239382.Graphical

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

12614 Induction Of Gpt2 During Metabolic Stress Is Associated With Reduced Incretin Responsiveness And Decreased β-cell Resilience To Metabolic Stress

Abstract Disclosure: S. Sen: None. A.V. Rozo: None. M. Haemmerle: None. J. Roman: None. A.V. Scota: None. J.A. Christine: None. E.M. Morrow: None. S.A. Tersey: None. C.B. Newgard: None. N.M. Doliba: None. D.A. Stoffers: None. Elevated blood glucose and lipids compromise pancreatic β-cell survival and function, but the underlying molecular mechanisms are ill-understood. We previously found that mitochondrial glutamate-pyruvate transaminase 2 (GPT2) is induced by ER and glucolipotoxic (GLT) stress. Here, we find that islets from type 2 diabetic donors exhibited markedly elevated levels of GPT2 compared to those of non-diabetic donors. GLT stress induced GPT2 in MIN6 cells, primary murine, and human non-diabetic islets. We generated β-cell specific Gpt2-deficient (Gpt2βKO) and Gpt2 floxed control (Gpt2f/f) mice to study the in vivo role of Gpt2 in β-cells. Intraperitoneal (IP) glucose tolerance (GTT) of Gpt2βKO mice was minimally affected on chow diet; therefore, we placed Gpt2f/f and Gpt2βKO mice on 45 kcal% high-fat and matched control diets. At 1 week, HFD-fed Gpt2βKO mice exhibited reduced β-cell death, measured by droplet digital PCR of circulating unmethylated preproinsulin DNA and by TUNEL analysis. Over 37 weeks, HFD-fed Gpt2βKO mice showed lower random blood glucose similar to Gpt2f/f mice under a control diet. While IPGTT was modestly improved in HFD-fed Gpt2βKO compared to HFD-fed Gpt2f/f mice, insulin secretion was not enhanced. Remarkably, oral glucose administration markedly improved glucose tolerance and enhanced insulin secretion of control- and HFD-fed Gpt2βKO mice compared to Gpt2f/f mice, while GLP-1 and GIP levels were unaltered, suggesting enhanced incretin action on the β-cell. Indeed, Gpt2βKO mice (under both control and high-fat diets) were more responsive to administration of GLP1R agonist Exendin-4 (Ex4) than Gpt2f/f mice. Isolated Gpt2βKO islets secreted more insulin when stimulated by Ex4 and GIP but not acetylcholine. Perifusion assay revealed markedly enhanced insulin secretion of Gpt2βKO islets in response to a stepwise exposure to increasing Ex4 doses, indicating enhanced incretin sensitivity. Metabolic flux analysis of Gpt2βKO and Gpt2f/f islets exposed to uniformly labeled glucose (U-13C glucose) under low (2.8mM), high (12mM) glucose, and high glucose with Ex4 conditions revealed elevated levels of key TCA cycle intermediates in Gpt2βKO islets under high glucose and high glucose+Ex4 conditions, indicating increased TCA cycle flux that likely enhances insulin secretion. Gpt2βKO mice had higher β cell mass than Gpt2f/f littermates under control diet and HFD feeding. Bulk islet RNA-seq from Gpt2f/f and Gpt2βKO mice exposed to GLT revealed upregulation of genes responsible for cell cycle and division pathways and downregulation of apoptosis and ER stress genes, consistent with enhanced survival of Gpt2βKO islets under stress conditions. In summary, β-cell GPT2 is increased during metabolic stress and T2D. Its deficiency induces a metabolic reprogramming that improves incretin sensitivity and protects pancreatic β-cells from metabolic stress. Presentation: 6/2/2024

The role of mu-opioid receptors in pancreatic islet α-cells.

Diabetes is a complex disease that impacts more than 500 million people across the world. Many of these individuals will develop diabetic neuropathy as a comorbidity, which is historically treated with exogenous opioids, such as morphine, oxycodone, or tramadol. Although these opioids are effective analgesics, growing evidence indicates that they may directly impact the endocrine pancreas function in patients. One common feature of these exogenous opioid ligands is their preference for the mu-opioid receptor (MOPR), so we aimed to determine whether endogenous MOPRs directly regulate pancreatic islet metabolism and hormone secretion. We show that pharmacological antagonism of MOPRs enhances glucagon secretion, but not insulin secretion, from human islets under high-glucose conditions. This increased secretion is accompanied by increased cAMP signaling. mRNA expression of MOPRs is robust in nondiabetic human islets but downregulated in islets from T2D donors, suggesting a link between metabolism and MOPR expression. Conditional genetic knockout of MOPRs in murine α-cells increases glucagon secretion under high-glucose conditions without increasing glucagon content. Consistent with downregulation of MOPRs during metabolic disease, conditional MOPR knockout mice treated with a high-fat diet show impaired glucose tolerance, increased glucagon secretion, increased insulin content, and increased islet size. Together, these results demonstrate a direct mechanism of action for endogenous opioid regulation of endocrine pancreas.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Characterization of Human Pancreatic Islet Cells Using a Single-Cell Western Blot Platform.

Islet transplantation is an effective treatment for type 1 diabetes. However, transplant success depends on quick islet assessment because islets deteriorate 2-3 days after isolation. A new tool, single-cell western blot (scWestern), offers results within 1 day. In this study, we aimed to test the suitability of scWestern to detect protein markers for beta (insulin), alpha (glucagon), and delta (somatostatin) cells, the 3 major endocrine cell types in islets. We characterized the antibody specificity, signal intensity, and cell identification on the scWestern platform and then compared the islet cell composition analysis between scWestern and immunohistochemistry performed by the Integrated Islet Distribution Program. Islet cell composition is comparable for alpha and beta cells, but not delta cells. Protein expression levels of insulin, glucagon, and somatostatin in individual islet cells varied greatly, highlighting cell type heterogeneity. Surprisingly, scWestern revealed double-hormonal cells (~1%), co-expressing insulin and somatostatin or insulin and glucagon, in nondiabetic and nonobese adult human islets, which was confirmed by confocal immunofluorescence microscopy. These results demonstrate that each alpha, beta, and delta cells express varying levels of peptide hormones, and a small subpopulation co-expresses double hormones in normal human islets. The scWestern platform will enable timely assessment of beta cell mass in isolated islets before clinical transplantation.

198-OR: Pancreatic Elastase Regulates Human Beta-Cell Viability by Impairing the PAR2 Pathway

The discovery of SerpinB1 pointed to acinar cell-derived proteases such as pancreatic elastase (PE) being involved in regulating human β-cell viability. Indeed, we observed that expression of a major form of PE, namely CELA3B, was increased in acinar cells from donors with type 2 diabetes (T2D) compared to samples from nondiabetic (ND) donors. Surprisingly, CELA3B protein levels were higher in T2D vs. ND islets, although its gene expression levels were virtually undetectable. We confirmed that increasing doses of exogenous PE is able to decrease viability and function of EndoC-βH3 (βH3) cells in vitro. To evaluate the effects of PE inhibition on β-cell survival, we used telaprevir, an FDA-approved drug, that we identified as a potent PE blocker using a High-Throughput Screening assays with an IC50 of 15.6 nM to inhibit PE activity. Consistent with the effects of SerpinB1 on β-cells, telaprevir also increased human β-cell viability in in vitro and in vivo systems. By performing phospho-antibody microarrays and single-cell RNA-Seq in human islets treated with vehicle or telaprevir, we identified the Protease-Activated Receptor 2 (PAR2) pathway as a candidate pathway linking PE inhibition to β-cell viability. We validated the results by treating EndoC-βH1 (βH1) cells with a PAR2 inactivating peptide (FSLLRY-NH2) in addition to PE +/- telaprevir. We observed that PE impaired the PAR2 cascade, and these effects were reversed by telaprevir in control β-cells, but not in cells treated with the PAR2 inhibitor. Furthermore, telaprevir was able to reverse PE effects and improve viability of control human primary β-cells, but not of those in which the PAR2 pathway was pharmacologically impaired. These data point to novel pathways reflecting acinar-β-cell crosstalk that has therapeutic potential to resolve diabetes in patients. Disclosure G. Basile: None. A. Vetere: None. J. Hu: None. D.F. De Jesus: None. A. Choudhary: None. B. Wagner: Research Support; Novo Nordisk. Stock/Shareholder; Pfizer Inc., Provention Bio, Inc. Research Support; Japan Tobacco Inc. Advisory Panel; Novo Nordisk. Research Support; Ono Pharmaceutical Co., Ltd. Stock/Shareholder; Curis, Inc. Consultant; Fresh Tracks Therapeutics. R. Kulkarni: Advisory Panel; Novo Nordisk, Inversago. Research Support; Inversago. Advisory Panel; Biomea Fusion, Inc., REDD Pharma.

135-OR: Mapping Cis-Regulatory Programs Affecting Diabetes Risk in Pancreatic Islet Cell Types Using Single-Cell Multimodal Profiling

Genetic variants influencing type 1 diabetes (T1D) risk identified by genome-wide association studies (GWAS) are primarily non-coding and affect cis-regulatory element (cRE) activity in specific cell types. A more detailed understanding of cell type-specific cis-regulatory programs in the pancreas is therefore crucial for deciphering the functional impact of T1D risk. Recently developed assays that generate multimodal single nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) profiles from the same cell enable high-resolution mapping of cis-regulatory programs in specific cell types. We generated deeply sequenced data from 28 nondiabetic pancreatic islet samples from the Alberta Diabetes Institute IsletCore using 10x multiome assays. After extensive quality control and filtering, we clustered multimodal profiles from 174,819 cells and identified ten major cell types. For several cell types we identified evidence of heterogeneous sub-populations; for example, in beta cells there were six sub-populations corresponding to cellular states associated with insulin secretion, stress response, and others. We identified 263,223 cREs across all cell types and developed a novel method to predict target genes of cREs based on the correlation of multimodal profiles across single cells. In beta cells, there were 122,352 cRE-target gene links involving 16,907 unique genes. We mapped chromatin accessibility quantitative trait loci (caQTLs) for cREs in each cell type and identified a total of 14,535 caQTLs at FDR<.10. Finally, we annotated T1D risk variants from high-resolution fine-mapping of 136 T1D signals with cRE-target gene links and caQTLs. At 27 T1D signals including at the INS, MAPT, and DLK1 loci, variants affecting chromatin accessibility were linked to putative target genes in beta cells suggesting mechanisms of action at these loci. Experimental follow up of variant activity at these loci using genome editing in beta cell lines is ongoing. Disclosure H. Mummey: None. W. Elison: None. K. Korgaonkar: None. Y.S. Lee: None. J.M. Newsome Ashmus: None. P. Benaglio: Employee; Shoreline Biosciences. M.T. Miller: None. J.E. Manning Fox: None. A.L. Gloyn: Other Relationship; Genentech, Inc., Roche Pharmaceuticals. P. MacDonald: None. S. Preissl: None. K.J. Gaulton: Stock/Shareholder; Neurocrine Biosciences, Inc. Consultant; Genentech, Inc. Stock/Shareholder; Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (HG012059, DK105554, DK122607, DK114650)

Identification and analysis of type 2 diabetes-mellitus-associated autophagy-related genes.

Autophagy, an innate safeguard mechanism for protecting the organism against harmful agents, is implicated in the survival of pancreatic â cells and the development of type 2 diabetes mellitus (T2DM). Potential autophagy-related genes (ARGs) may serve as potential biomarkers for T2DM treatment. The GSE25724 dataset was downloaded from the Gene Expression Omnibus (GEO) database, and ARGs were obtained from the Human Autophagy Database. The differentially expressed autophagy-related genes (DEARGs) were screened at the intersection of ARGs and differentially expressed genes (DEGs) between T2DM and non-diabetic islet samples, which were subjected to functional enrichment analyses. A protein-protein interaction (PPI) network was constructed to identify hub DEARGs. Expressions of top 10 DEARGs were validated in human pancreatic â-cell line NES2Y and rat pancreatic INS-1 cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and insulin secretion were measured after cell transfection with lentiviral vector EIF2AK3 or RB1CC1 into islet cells. In total, we discovered 1,270 DEGs (266 upregulated and 1,004 downregulated genes) and 30 DEARGs enriched in autophagy- and mitophagy-related pathways. In addition, we identified GAPDH, ITPR1, EIF2AK3, FOXO3, HSPA5, RB1CC1, LAMP2, GABARAPL2, RAB7A, and WIPI1 genes as the hub ARGs. Next, qRT-PCR analysis revealed that expressions of hub DEARGs were consistent with findings from bioinformatics analysis. EIF2AK3, GABARAPL2, HSPA5, LAMP2, and RB1CC1 were both differentially expressed in the two cell types. Overexpression of EIF2AK3 or RB1CC1 promoted cell viability of islet cells and increased the insulin secretion. This study provides potential biomarkers as therapeutic targets for T2DM.

Interleukin-4 reduces insulin secretion in human islets from healthy but not type-2 diabetic donors

Type 2 diabetes (T2D) is associated with low-grade inflammation. Here we investigate if the anti-inflammatory cytokine interleukin-4 (IL-4) affects glucose-stimulated insulin secretion (GSIS) in human islets from non-diabetic (ND) and type-2 diabetic (T2D) donors. We first confirmed that GSIS is reduced in islets from T2D donors. Treatment with IL-4 for 48 h had no further effect on GSIS in these islets but significantly reduced secretion in ND islets. Acute treatment with IL-4 for 1 h had no effect on GSIS in ND islets which led us to suspect that IL-4 affects a slow cellular mechanism such as gene transcription. IL-4 has been reported to regulate miR-378a-3p and, indeed, we found that this microRNA was increased with IL-4 treatment. However, overexpression of miR-378a-3p in the human beta cell line EndoC-βH1 did not affect GSIS. MiR-378a-3p is transcribed from the same gene as peroxisome proliferator-activated receptor gamma co-activator 1 beta (PCG-1β) and we found that IL-4 treatment showed a clear tendency to increased gene expression of PCG-1β. PCG-1β is a co-activator of peroxisome proliferator-activated receptor gamma (PPARγ) and, the gene expression of PPARγ was also increased with IL-4 treatment. Our data suggests that the protective role of IL-4 on beta cell survival comes at the cost of lowered insulin secretion, presumably involving the PPARγ-pathway.

Palmitoylation couples insulin hypersecretion with β cell failure in diabetes

Hyperinsulinemia often precedes type 2 diabetes. Palmitoylation, implicated in exocytosis, is reversed by acyl-protein thioesterase 1 (APT1). APT1 biology was altered in pancreatic islets from humans with type 2 diabetes, and APT1 knockdown in nondiabetic islets caused insulin hypersecretion. APT1 knockout mice had islet autonomous increased glucose-stimulated insulin secretion that was associated with prolonged insulin granule fusion. Using palmitoylation proteomics, we identified Scamp1 as an APT1 substrate that localized to insulin secretory granules. Scamp1 knockdown caused insulin hypersecretion. Expression of a mutated Scamp1 incapable of being palmitoylated in APT1-deficient cells rescued insulin hypersecretion and nutrient-induced apoptosis. High-fat-fed islet-specific APT1-knockout mice and global APT1-deficient db/db mice showed increased β cell failure. These findings suggest that APT1 is regulated in human islets and that APT1 deficiency causes insulin hypersecretion leading to β cell failure, modeling the evolution of some forms of human type 2 diabetes.

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells.

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of E2f1 (E2f1β-/-) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1β-/- pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic β cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in β cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.

Detection of Antiviral Tissue Responses and Increased Cell Stress in the Pancreatic Islets of Newly Diagnosed Type 1 Diabetes Patients: Results From the DiViD Study.

Aims/hypothesisThe Diabetes Virus Detection (DiViD) study has suggested the presence of low-grade enteroviral infection in pancreatic tissue collected from six of six live adult patients newly diagnosed with type 1 diabetes. The present study aimed to compare the gene and protein expression of selected virally induced pathogen recognition receptors and interferon stimulated genes in islets from these newly diagnosed type 1 diabetes (DiViD) subjects vs age-matched non-diabetic (ND) controls.MethodsRNA was extracted from laser-captured islets and Affymetrix Human Gene 2.0 ST arrays used to obtain gene expression profiles. Lists of differentially expressed genes were subjected to a data-mining pipeline searching for enrichment of canonical pathways, KEGG pathways, Gene Ontologies, transcription factor binding sites and other upstream regulators. In addition, the presence and localisation of specific viral response proteins (PKR, MxA and MDA5) were examined by combined immunofluorescent labelling in sections of pancreatic tissue.ResultsThe data analysis and data mining process revealed a significant enrichment of gene ontologies covering viral reproduction and infectious cycles; peptide translation, elongation and initiation, as well as oxidoreductase activity. Enrichment was identified in the KEGG pathways for oxidative phosphorylation; ribosomal and metabolic activity; antigen processing and presentation and in canonical pathways for mitochondrial dysfunction, oxidative phosphorylation and EIF2 signaling. Protein Kinase R (PKR) expression did not differ between newly diagnosed type 1 diabetes and ND islets at the level of total RNA, but a small subset of β-cells displayed markedly increased PKR protein levels. These PKR+ β-cells correspond to those previously shown to contain the viral protein, VP1. RNA encoding MDA5 was increased significantly in newly diagnosed type 1 diabetes islets, and immunostaining of MDA5 protein was seen in α- and certain β-cells in both newly diagnosed type 1 diabetes and ND islets, but the expression was increased in β-cells in type 1 diabetes. In addition, an uncharacterised subset of synaptophysin positive, but islet hormone negative, cells expressed intense MDA5 staining and these were more prevalent in DiViD cases. MxA RNA was upregulated in newly diagnosed type 1 diabetes vs ND islets and MxA protein was detected exclusively in newly diagnosed type 1 diabetes β-cells.Conclusion/interpretationThe gene expression signatures reveal that pathways associated with cellular stress and increased immunological activity are enhanced in islets from newly diagnosed type 1 diabetes patients compared to controls. The increases in viral response proteins seen in β-cells in newly diagnosed type 1 diabetes provide clear evidence for the activation of IFN signalling pathways. As such, these data strengthen the hypothesis that an enteroviral infection of islet β-cells contributes to the pathogenesis of type 1 diabetes.

Research on Potential Network Markers and Signaling Pathways in Type 2 Diabetes Based on Conditional Cell-Specific Network.

Traditional methods concerning type 2 diabetes (T2D) are limited to grouped cells instead of each single cell, and thus the heterogeneity of single cells is erased. Therefore, it is still challenging to study T2D based on a single-cell and network perspective. In this study, we construct a conditional cell-specific network (CCSN) for each single cell for the GSE86469 dataset which is a single-cell transcriptional set from nondiabetic (ND) and T2D human islet samples, and obtain a conditional network degree matrix (CNDM). Since beta cells are the key cells leading to T2D, we search for hub genes in CCSN of beta cells and find that ATP6AP2 is essential for regulation and storage of insulin, and the renin-angiotensin system involving ATP6AP2 is related to most pathological processes leading to diabetic nephropathy. The communication between beta cells and other endocrine cells is performed and three gene pairs with obvious interaction are found. In addition, different expression genes (DEGs) are found based on CNDM and the gene expression matrix (GEM), respectively. Finally, ‘dark’ genes are identified, and enrichment analysis shows that NFATC2 is involved in the VEGF signaling pathway and indirectly affects the production of Prostacyclin (PGI2), which may be a potential biomarker for diabetic nephropathy.

253-LB: Ethnic Differences in Pancreatic Hormone Secretion in Health and T2D

Purpose: We tested the hypothesis that a critical biologic determinant of T2D disparities is based on ethnicity-related differences in pancreatic islet function. Methods: Utilizing donor tissues from the NIDDK-funded Human Pancreas Analysis Program (HPAP) , we examined both insulin and glucagon secretion from nondiabetic and diabetic Caucasian and African American donor islets. Initially, a physiological amino acid mixture was used to stimulate glucagon secretion, followed by low and high glucose to stimulate insulin secretion and suppress glucagon secretion. During the high glucose step, IBMX (0.1 mM) is added to potentiate secretion of both hormones. Insulin and glucagon concentration in perifusates and islet extracts was measured by radioimmunoassay or ELISA. Statistical comparisons were drawn by repeated measures ANOVA. Results: Comparing nondiabetic islets, we observed overlapping insulin secretion profiles between both ethnicities; however, glucagon secretion was distinctly greater in both nondiabetic African American (+111%) islets compared to nondiabetic Caucasian islets, under all interventions. In T2D, the reduction in insulin secretion was significantly greater in African American T2D donors (high glucose -76%; IBMX -76%) compared to Caucasian T2D donors (high glucose -47%; IBMX -50%) . More strikingly, glucagon secretion in T2D donors was markedly different based on ethnicity. Whereas Caucasian T2D islets exhibited a similar baseline of glucagon secretion compared to Caucasian controls, there was a marked reduction of glucagon secretion overall in African American T2D donors (-70%) compared to their corresponding controls. The largest detected difference was in IBMX-potentiation of glucagon secretion, which is higher in Caucasian T2D donors (+103%) but significantly decreased in African American T2D donors (-76%) . Preliminary analysis of hormone content in a subset of the donor islet preparations revealed no differences according to ethnicity. Conclusions: We propose that ethnicity-related differences in nondiabetic islet function may contribute to the enhanced risk of T2D. Disclosure N. M. Doliba: None. M. Brissova: None. K. H. Kaestner: None. D. A. Stoffers: Other Relationship; Biomarin, Correlagen. J. Roman: None. A. V. Rozo: None. W. Qin: None. C. Liu: None. A. Naji: None. M. R. Rickels: Advisory Panel; Sernova, Corp., Vertex Pharmaceuticals Incorporated, Zealand Pharma A/S, Consultant; L-Nutra Inc. M. A. Atkinson: None. A. C. Powers: None. Funding This work is supported by 3UC4-112217-01S1 and U01-DK123594-02, UC4-DK-112217, UC4-DK-112232, and U01-DK-123716.

247-LB: Pancreatic Elastase Regulates ß-Cell Viability by Impairing the Mechanosignaling Pathway

The discovery of SerpinB1 as an inducer of human β-cell proliferation pointed to acinar cell-derived proteases such as pancreatic elastase (PE) being involved in regulating β-cell viability. Indeed, we observed that expression of a major form of PE, namely CELA3B, was increased in acinar cells from donors with type 2 diabetes (T2D) compared to samples from non-diabetic (ND) donors. Surprisingly, CELA3B protein levels were higher in T2D vs. ND islets, although its gene expression levels were virtually undetectable. We confirmed that increasing doses of exogenous PE is able to decrease viability of EndoC-βH3 (βH3) cells in vitro. To evaluate the effects of PE inhibition on β-cell survival, we used telaprevir, an FDA-approved drug, that we identified as a potent PE blocker using a High-Throughput Screening assays with an IC50 of 15.6 nM to inhibit PE activity. Consistent with the mitogenic effects of SerpinB1 on β-cells, telaprevir also increased human β-cell viability in in vitro and in vivo systems. By performing phospho-antibody microarrays and single-cell RNA-Seq in human islets treated with vehicle or telaprevir, we identified the mechano-signaling pathway, which integrates the signals generated by the extracellular matrix (ECM) , as a candidate pathway linking PE inhibition to β-cell viability. We validated the results by treating EndoC-βH1 (βH1) cells lacking focal adhesion kinase (siFAK) with PE +/- telaprevir. We observed that PE impaired the mechano-signaling cascade, and these effects were reversed by telaprevir in control cells, but not in siFAK cells. Furthermore, using the novel approach of Brillouin microscopy we observed that PE increased the intracellular stiffness in βH1 cells, as a readout of cytoskeleton arrangements, whereas telaprevir blunted these effects. These data point to novel pathways underlying the acinar-β-cell crosstalk that could be harnessed for therapeutic approaches to resolve diabetes in patients. Disclosure G. Basile: None. B. Wagner: Research Support; Japan Tobacco Inc., Novo Nordisk Foundation, Ono Pharmaceutical Co., Ltd., Stock/Shareholder; Moderna, Inc., Pfizer Inc. R. Kulkarni: Advisory Panel; Biomea, Novo Nordisk, REDD Pharma, Consultant; Relay Therapeutics, Research Support; Inversago, SerPlus. A. Vetere: None. J. Hu: None. O. Ijaduola: None. D. F. De jesus: n/a. K. Fukuda: None. A. M. Eltony: None. A. Yun: None. A. Choudhary: None. Funding JDRF: 3-PDF-2020-935-A-NNIH: U01 DK123717NIH: UC4 DK116255