Insulin-secreting Beta Cells Research Articles

Obesity, heart failure with preserved ejection fraction, and the role of glucagon-like peptide-1 receptor agonists.

Heart failure with preserved ejection fraction (HFpEF) has a high prevalence, affecting more than 50% of patients with heart failure. HFpEF is associated with multiple comorbidities, and obesity is one of the most common. A distinct phenotype has been proposed for obese patients with HFpEF. Recent data show the beneficial role of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) for weight loss in diabetic and non-diabetic patients with obesity or overweight when given as adjunctive therapy to diet and exercise. The mechanisms of action are related to paracrine and endocrine signalling pathways within the gastrointestinal tract, pancreas, and central nervous system that delay gastric emptying, decrease appetite, augment pancreatic beta-cell insulin secretion, and suppress pancreatic glucagon release. These drugs are therefore potentially indicated for treatment of patients with HFpEF and obesity or overweight. Efficacy and safety need to be shown by clinical trials with a first one, Semaglutide Treatment Effect in People with obesity and heart failure with preserved ejection fraction (STEP HFpEF), recently concluded. The aim of the present review is to provide the pathophysiological and pharmacological rationale for GLP-1 RA administration to obese patients with HFpEF.

Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans.

The cell-to-cell signaling role of d-amino acids (d-AAs) in the mammalian endocrine system, particularly in the islets of Langerhans, has drawn growing interest for their potential involvement in modulating glucose metabolism. Previous studies found colocalization of serine racemase [produces d-serine (d-Ser)] and d-alanine (d-Ala) within insulin-secreting beta cells and d-aspartate (d-Asp) within glucagon-secreting alpha cells. Expressed in the islets, functional N-methyl-d-aspartate receptors are involved in the modulation of glucose-stimulated insulin secretion and have binding sites for several d-AAs. However, knowledge of the regulation of d-AA levels in the islets during glucose stimulation as well as the response of islets to different levels of extracellular d-AAs is limited. In this study, we determined the intracellular and extracellular levels of d-Ser, d-Ala, and d-Asp in cultures of isolated rodent islets exposed to different levels of extracellular glucose. We found that the intracellular levels of the enantiomers demonstrated large variability and, in general, were not affected by extracellular glucose levels. However, significantly lower levels of extracellular d-Ser and d-Ala were observed in the islet media supplemented with 20 mM concentration of glucose compared to the control condition utilizing 3 mM glucose. Glucose-induced oscillations of intracellular free calcium concentration ([Ca2+]i), a proxy for insulin secretion, were modulated by the exogenous application of d-Ser and d-Ala but not by their l-stereoisomers. Our results provide new insights into the roles of d-AAs in the biochemistry and function of pancreatic islets.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

The liver-derived exosomes stimulate insulin gene expression in pancreatic beta cells under condition of insulin resistance.

An insufficient functional beta cell mass is a core pathological hallmark of type 2 diabetes (T2D). Despite the availability of several effective pharmaceuticals for diabetes management, there is an urgent need for novel medications to protect pancreatic beta cells under diabetic conditions. Integrative organ cross-communication controls the energy balance and glucose homeostasis. The liver and pancreatic islets have dynamic cross-communications where the liver can trigger a compensatory beta cell mass expansion and enhanced hormonal secretion in insulin-resistant conditions. However, the indispensable element(s) that foster beta cell proliferation and insulin secretion have yet to be completely identified. Exosomes are important extracellular vehicles (EVs) released by most cell types that transfer biological signal(s), including metabolic messengers such as miRNA and peptides, between cells and organs. We investigated whether beta cells can take up liver-derived exosomes and examined their impact on beta cell functional genes and insulin expression. Exosomes isolated from human liver HepG2 cells were characterized using various methods, including Transmission Electron Microscopy (TEM), dynamic light scattering (DLS), and Western blot analysis of exosomal markers. Exosome labeling and cell uptake were assessed using CM-Dil dye. The effect of liver cell-derived exosomes on Min6 beta cells was determined through gene expression analyses of beta cell markers and insulin using qPCR, as well as Akt signaling using Western blotting. Treatment of Min6 beta cells with exosomes isolated from human liver HepG2 cells treated with insulin receptor antagonist S961 significantly increased the expression of beta cell markers Pdx1, NeuroD1, and Ins1 compared to the exosomes isolated from untreated cells. In line with this, the activity of AKT kinase, an integral component of the insulin receptor pathway, is elevated in pancreatic beta cells, as represented by an increase in AKT's downstream substrate, FoxO1 phosphorylation. This study suggests that liver-derived exosomes may carry a specific molecular cargo that can affect insulin expression in pancreatic beta cells, ultimately affecting glucose homeostasis.

Interventional Radiology Approaches for Managing Postpancreatic Transplant Complications and Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disorder characterized by the destruction of insulin-secreting beta cells in the pancreas, resulting in metabolic disturbances and long-term complications. While subcutaneous insulin remains the primary approach for achieving normoglycemia, pancreatic transplantation has emerged as an effective intervention for long-standing T1DM, providing insulin independence and normalized glycosylated hemoglobin levels. However, complications associated with pancreatic transplantation are frequent, necessitating thorough evaluation using diverse imaging modalities. This manuscript presents an overview of vascular complications encountered with pancreatic transplantation, including vascular complications such as arterial and venous graft thrombosis, vessel stenosis, pseudoaneurysm, arterio-enteric fistula, and arteriovenous malformations (AVM). Additionally, the manuscript discusses other associated complications such as pancreatitis, pseudocyst formation, fistulas, pseudo-thrombosis of the iliac vein, post-transplantation lymphoproliferative disorder, and fluid collections. The integration of various imaging modalities plays a crucial role in diagnosing and managing these complications, with interventional radiologists assuming a vital role in employing image-guided procedures. Moreover, the manuscript explores pancreatic islet cell transplantation as a promising cellular-based therapy for T1DM, offering stable long-term glycemic control and decreased reliance on exogenous insulin in a significant proportion of recipients. This minimally invasive procedure involves the image-guided transcatheter infusion of islet cells obtained from deceased donors into the recipient's liver. The importance of interventional radiologists in managing complications related to pancreatic transplantation is underscored, with endovascular or image-guided approaches being utilized to address the diverse spectrum of encountered complications. Furthermore, the potential of islet cell transplantation as a minimally invasive alternative to traditional pancreatic transplantation is emphasized, as it offers the prospect of alleviating many associated complications.

Effects of advanced glycation end products via oxidative stress on beta cells: insights from in vitro and in vivo studies and update on emerging therapies.

Protein, lipid, and nucleic acid glycation reactions begin and continue as a result of persistent hyperglycemia in patients with diabetes mellitus. Advanced glycated end products (AGEs) are a complex group of chemical moieties that are formed as a result of the glycation process and play an important role in the pathogenesis of diabetes mellitus. When AGEs interact with their cellular receptor (RAGE), numerous signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), are activated, increasing oxidative stress. The aim of this review was to summarize in vitro and in vivo studies underlining the involvement of AGEs on beta cell dysfunction and death via oxidative stress. A literature search of publications published between 1912 and December 2022 was conducted using MEDLINE, EMBASE, and the Cochrane Library, with restrictions on articles written in English. Recent insights have revealed that oxidative stress has a crucial role in the development of beta cell dysfunction and insulin resistance, the major hallmarks of type 2 diabetes mellitus. Studies also revealed that AGEs decrease insulin synthesis and secretion in the pancreatic beta cells and induce cell apoptosis. Experimental data have shown that both AGEs and oxidative stress contribute to beta cell dysfunction and development as well as to the progression of diabetic complications. Many anti-AGE therapies are being developed; however, it remains to be seen whether these therapies can help maintain beta cell function and prevent diabetes complications.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis

Aims/hypothesisRecovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids.MethodsIn zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids.ResultsAdjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment.Conclusions/interpretationAdjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states.Data availabilityRaw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398).Graphical

THU291 Successful Reversal Of Glucose Toxicity After DKA Precipitated By SGLT2 Inhibitor

Abstract Disclosure: H. Abdelrahman: None. B. DeMoranville: None. Case: A 40 year old man with history of NAFLD and type 2 diabetes mellitus with HbA1c 12.2% on metformin 750 mg daily, presented with diabetic ketoacidosis (DKA) with blood glucose 275 mg/dL and anion gap 24 (AG 10-12). He had been started on empagliflozin 25 mg daily four days prior as outpatient. He was treated with IV insulin and hydration per DKA protocol. Labs showed C-peptide 3.0 ng/mL (1.1-4.4 ng/mL), Anti-GAD Ab 0.01 nmol/L (<0.02 mol/L), negative anti-insulin cell Ab. He was discharged on glargine 10u qHS, lispro 2u AC plus corrective scale, and metformin 750 mg BID. On outpatient follow-up, pre-meal glucose remained under 130 mg/dL, not requiring corrective insulin. He was started on semaglutide 0.5mg weekly and insulin was discontinued. At 3 months, his HbA1c improved to 5.8%. Self-monitoring glucose levels were at goal. Discussion: This case illustrates glucotoxicity due to significant hyperglycemia causing beta-cell apoptosis and resulting in insulin deficiency. The initiation of SGLT2 inhibitors (SGLT2i) when insulin deficiency and peripheral resistance are at their peak increases risk of DKA. This patient with type 2 diabetes presented with blood glucose 275 mg/dL in DKA four days after starting empagliflozin, a SGLT2i. SGLT2i increase risk of DKA due to dehydration from glucosuria, inhibiting ketone body excretion by the kidneys, causing a state of carbohydrate deficit and hypovolemia that promotes glucagon release from alpha-cells, an increased glucagon-to-insulin ratio, and thus triggers ketogenesis.1 In glucose toxicity, intra-cellular hyperglycemia-induced molecular dysfunction results in beta-cell apoptosis. Islet cells cultured in a glucotoxic environment (20 mmol/l) had suppressed insulin content, but fully reversed the intra-cellular calcium response after one week of culturing in a euglycemic environment, demonstrating recovery of the molecular steps promoting insulin release. 2 Treatment with insulin close to euglycemic goals promotes the reversal of glucose toxicity. Our patient recovered beta-cell function several weeks after the glucotoxic episode and was able to discontinue insulin therapy. With the addition of semaglutide, a GLP-1 receptor agonist, which potentiates glucose-dependent insulin release to control post-prandial hyperglycemia, he was able to maintain euglycemia and an HbA1c 5.8%. Conclusion Patients with type 2 diabetes mellitus may present in diabetic ketoacidosis due to glucose toxicity which can be reversed with short-term insulin therapy. References 1) Nasa P, et al. Euglycemic diabetic ketoacidosis: A missed diagnosis. World J Diabetes. 2021 May 15;12(5):514-523. 2) Tariq M, et al. Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped. Diabetologia. 2022 Dec 2. Epub ahead of print. PMID: 36459178. Presentation: Thursday, June 15, 2023

SAT157 Management Of Spontaneous Hypoglycemia In Dialysis-dependent Chronic Kidney Disease Using Diazoxide

Abstract Disclosure: A. Schledwitz: None. S.S. Habbsa: None. F. Sotomayor Villanueva: None. Y. Kim: None. Background: Patients with end-stage renal disease (ESRD) are at increased risk of spontaneous hypoglycemia due to decreased renal insulin clearance. Due to its inhibitory effect on pancreatic beta cell insulin secretion, oral diazoxide has been used to treat hypoglycemia due to insulinoma or congenital hyperinsulinemic hypoglycemia, but few cases detail the use of diazoxide in the ESRD population. Here, we present 4 cases of spontaneous hyperinsulinemic hypoglycemia in ESRD successfully treated with diazoxide. Clinical Cases: A 66-year-old man with ESRD presented with altered mental status secondary to severe hypoglycemia despite continuous IV 20% dextrose. Labs revealed glucose 50 mg/dL, insulin 7 (0-3 mcIU/mL), C-peptide 7.8 (0-0.6 ng/mL), proinsulin 35.7 (0-5 pmol/L), beta-hydroxybutyrate [bOH] 0.4 (0-2.7 mg/dL), negative sulfonylurea panel, normal cosyntropin stimulation test and undetectable insulin antibodies, without radiographic evidence of insulinoma. He was started on diazoxide 50 mg TID and remained euglycemic for the remainder of his course. On similar readmission 3 months later, he was restarted on diazoxide with resolution of hypoglycemia within 1 day. A 66-year-old male with ESRD and T1DM treated with pancreas transplant presented with toe gangrene and persistent hypoglycemia. Fasting labs revealed insulin 4, C-peptide 4.5, proinsulin 4.5, and bOH 4.5, without radiographic evidence of insulinoma. He maintained euglycemia 4 days after diazoxide 70 mg BID was started. A 67-year-old male with T2DM and ESRD was admitted for osteomyelitis and 2 years of episodic symptomatic hypoglycemia despite dietary adjustments. Labs showed glucose 59, insulin 7, C-peptide 5.2, proinsulin 7.5, bOH 0.04, and unremarkable imaging. Within 1 day of starting diazoxide 100 mg daily, his blood glucose stabilized, and he was discharged. Finally, a 42-year-old male with ESRD presented with 4 years of persistent episodic hypoglycemia, now refractory to continuous IV dextrose. He was found to have glucose 64, insulin 15, C-peptide 11.5, proinsulin 29, and bOH 0.1, with imaging revealing bulky pancreas without a discrete mass. His hypoglycemic episodes resolved within 1 day of starting diazoxide 100 mg daily. He was readmitted 3 months later for catheter site infection and had no episodes of hypoglycemia with continuation of diazoxide. Conclusion: Diazoxide may be a viable option for the treatment of refractory spontaneous hypoglycemia in patients with dialysis dependent ESRD. Presentation: Saturday, June 17, 2023

SAT070 The G Protein Coupled Receptor GPR31 Promotes Islet Inflammation And Diabetes

Abstract Disclosure: C. Checkcinco: None. S.A. Tersey: None. R.G. Mirmira: None. In type 1 diabetes a characteristic immune response targets insulin secreting beta cells in pancreatic islets, resulting in their destruction. This pathogenesis features inflammatory signaling cascades in both beta cells and infiltrating immune cell macrophages. 12-Lipoxygenase (12-LOX) contributes to inflammation in β cells and macrophages by producing the eicosanoid 12-HETE, which induces oxidative and ER stress resulting in cellular dysfunction. G-protein coupled receptor 31 (GPR31) was identified as a high affinity 12-HETE receptor and is increased in human islets after cytokine induced inflammation, but it remains unknown if GPR31 might mediate the downstream effects of 12-LOX. We found increased expression of GPR31 in C57Bl/6J islets, over 7 times higher relative to macrophages. To investigate the role of GPR31 during diabetic inflammation, whole body GPR31 knockout mice were generated on the C57Bl/6J background and studied for glucose homeostasis in the setting of diabetic inflammation. Islets and macrophages from these mice were studied following manipulations in vitro. Compared to wildtype (WT) littermates, Gpr31b-/- mice maintained similar body weight, beta-cell mass, and glucose homeostasis as mice aged. To mimic the setting of islet inflammation as seen in type 1 diabetes, WT and Gpr31b-/- mice were treated with the pro-diabetic toxin streptozotocin (which induces beta cell death and macrophage influx). Compared to WT, Gpr31b-/- mice showed protection from development of hyperglycemia and glucose tolerance tests showed >30% protection reduction in area-under-the-curve, suggesting that GPR31 promotes islet dysfunction and hyperglycemia. To interrogate the role of GPR31 in the beta cell, we isolated islets from WT and Gpr31b-/- mice and treated them with proinflammatory cytokines followed by RNA sequencing to interrogate gene expression pathways. Differentially expressed genes in Gpr31b-/- islets included those pertaining to oxidative stress, MAPK signaling, and cellular homeostasis by Wnt/β-catenin, a finding reminiscent of pathways that are also altered following 12-LOX inhibition. To assess how GPR31 might impact macrophage function, we induced proinflammatory M1 polarization by lipopolysaccharide and interferon gamma in bone marrow-derived macrophages. We saw no significant difference in the extent of M1 polarization between GPR31b-/- and WT macrophages by flow cytometry, as assessed by the production of iNOS. We then asked if GPR31 was involved in the mobility of macrophages using a transwell assay with chemoattractant. We saw a ∼50% reduction in migrating bone marrow-derived macrophages in GPR31b-/- compared to WT. Taken together, these data are consistent with our previously published data on the 12-LOX knockout mice, suggesting that GPR31 may be involved in mediating the proinflammatory responses of 12-HETE in both β cells and macrophages. Presentation: Saturday, June 17, 2023

Differential effects of OATP2B1 on statin accumulation and toxicity in a beta cell model

An increased risk of new-onset diabetes mellitus has been recently reported for statin therapy, and experimental studies have shown reduced glucose-stimulated insulin secretion (GSIS) and mitochondrial dysfunction in beta cells with effects differing among agents. Organic anion transporting polypeptide (OATP) 2B1 contributes to hepatic uptake of rosuvastatin, atorvastatin and pravastatin, three known substrates. Since OATP2B1 is present in beta cells of the human pancreas, we investigated if OATP2B1 facilitates the local accumulation of statins in a rat beta cell model INS-1 832/13 (INS-1) thereby amplifying statin-induced toxicity. OATP2B1 overexpression in INS-1 cells via adenoviral transduction showed 2.5-, 1.8- and 1.4-fold higher cellular retention of rosuvastatin, atorvastatin and pravastatin, respectively, relative to LacZ control, while absolute intracellular concentration was about twice as high for the lipophilic atorvastatin compared to the more hydrophilic rosuvastatin and pravastatin. After 24 h statin treatment at high concentrations, OATP2B1 enhanced statin toxicity involving activation of intrinsic apoptosis (caspase 3/7 activation) and mitochondrial dysfunction (NADH dehydrogenase activity) following rosuvastatin and atorvastatin, which was partly reversed by isoprenoids. OATP2B1 had no effect on statin-induced reduction in GSIS, mitochondrial electron transport chain complex expression or caspase 9 activation. We confirmed a dose-dependent reduction in insulin secretion by rosuvastatin and atorvastatin in native INS-1 with a modest change in cellular ATP. Collectively, our results indicate a role of OATP2B1, which is abundant in human beta cells, in statin accumulation and statin-induced toxicity but not insulin secretion of rosuvastatin and atorvastatin in INS-1 cells.

Genetic variants in the complement system and their potential link in the aetiology of type 1 diabetes.

Type 1 diabetes is an autoimmune disease in which one's own immune system destroys insulin-secreting beta cells in the pancreas. This process results in life-long dependence on exogenous insulin for survival. Both genetic and environmental factors play a role in disease initiation, progression, and ultimate clinical diagnosis of type 1 diabetes. This review will provide background on the natural history of type 1 diabetes and the role of genetic factors involved in the complement system, as several recent studies have identified changes in levels of these proteins as the disease evolves from pre-clinical through to clinically apparent disease.

A comparative study of the gut microbiome in Indian children with type 1 diabetes and healthy controls.

Type 1 diabetes mellitus (T1DM) occurs in genetically susceptible individuals due to certain environmental triggers causing destruction of insulin secreting beta cells. One of the environmental triggers studied recently in the pathogenesis and progression of T1DM is the role of gut microbiome. (1) To compare the gut microbiome profile of T1DM children with healthy age, gender, and body mass index (BMI) matched controls. (2) To assess the relationship of abundance of genera with glycemic control in children with T1DM. Cross-sectional, case-control study. Sixty-eight children with T1DM and 61 age-, gender-, and BMI-matched healthy controls were enrolled. QIAamp Fast DNA Stool Mini kit protocol and reagents were used for DNA isolation and Miseq sequencing platform for targeted gene sequencing. Alpha and beta diversity analysis showed no significant differences in the abundance of microbes between the groups. At phylum level, Firmicutes was the dominant phylum followed by Actinobacteria and Bacteroidota in both groups. Analysis of microbiome at the genera level showed that percentage abundance for Parasutterella was higher in children with T1DM as compared to the healthy group (p < .05). A linear regression analysis showed that increase in abundance of Haemophilus (adjusted R2 = -1.481 p < .007) was associated with a significant decrease in glycated hemoglobin (HbA1c) concentrations (p < .05). Our comparative study of gut microbiome profile showed significant differences in the taxonomial composition between Indian children with T1DM and healthy controls. Short chain fatty acid producers may play an important role in glycemic control.

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 µm diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

1773-P: Glucocorticoid Receptor Signaling Regulates Pancreatic Beta-Cell Circadian Transcriptome and Function

Glucocorticoid (GC) signaling is an important regulator of glucose homeostasis. GC secretion also serves as an entrainment signal that coordinates circadian gene expression of peripheral circadian clocks. Pancreatic beta cells are key biological controllers of glucose homeostasis through the coordinated circadian release of insulin. However, the role of GC signaling in the regulation of beta cell circadian clocks and insulin secretion remains unknown. To address this, we first performed chromatin immunoprecipitation/sequencing (ChIP-seq) in beta cell lines (MIN6 and INS1) and identified &amp;gt;1,000 glucocorticoid receptor (GR)-bound regions (FDR&amp;lt;0.01) in response to the synthetic GC dexamethasone (DEX, 100nM). Annotation of GR-bound regions showed enrichment for pathways regulating insulin secretion and circadian rhythms. To further explore the effects of GC on circadian beta cell gene expression and function in vivo, we used C57BL/6 mice housed under either 12h/12h light-dark cycles (LD) or constant light-mediated circadian disruption (CD). Mice were subsequently administered daily intraperitoneal injections of DEX (2 mg/kg) or vehicle, designed to restore circadian secretion of GC disrupted in CD group. DEX treatment normalized circadian regulation of glucose tolerance (p&amp;lt;0.05 vs. vehicle) and glucose-stimulated insulin secretion (p&amp;lt;0.05 vs vehicle) in CD group. We next utilized RNA sequencing to assess circadian transcriptional profiles in islets isolated from LD, CD, and CD + DEX mice. Consistent with previous studies, CD resulted in abrogation of circadian islet mRNA expression, whereas DEX restored circadian rhythmicity and enhanced expression of core clock genes (e.g., Bmal1, Dbp, Nr1d1) and genes regulating beta cell identity (e.g., Ins1, Nkx6.1) and insulin secretion (e.g., Gck, Irs2). These data highlight the GC/GR axis as a direct regulator of beta cell circadian gene expression, insulin secretion and glucose tolerance. Disclosure S.K.Sen: None. L.Wang: None. M.Brown: None. J.Wu: None. Z.Wei: None. A.Matveyenko: None. Funding National Institutes of Health (DK098468)

1764-P: Assessing Gene Expression Profile and Insulin Secretion in Human Beta Cells (EndoC-BH3) under Glucose Exposure

Maternal diabetes is a risk factor for type 2 diabetes in the offspring, and in utero exposure to high glucose has been shown to affect pancreatic beta cell development in animal models. However, the developmental stage and specific genes which underlie these effects in humans are unknown. We aimed to understand the consequences of high glucose on the fetal pancreas by treating a human beta cell line derived from fetal pancreas, EndoC-BH3, with high, medium and normal glucose (30, 15 and 5.5mM respectively) for 3 weeks during proliferation and maturation and comparing gene expression using whole transcriptome analysis. The top differentially expressed transcripts (P&amp;lt;10−4) and the most abundant transcripts (Log2 unit&amp;gt;10.5) were validated by real-time RT-PCR. KEGG analyses of the top 500 differentially expressed transcripts identified the metabolic pathway as the top pathway. Among individual genes, high glucose exposure during the maturation upregulated EGR1 (58.6-fold), BIRC5 (37.6-fold), GPBAR1 (15.4-fold), G6PC2 (11.8-fold), CHRNA (4.8-fold), GCK (2.7-fold), INS (2.0-fold), CMIP (1.9-fold) and CHGA (1.4-fold) as compared to normal glucose exposure. EGR1 encodes early-growth response 1, a transcription factor that enhances insulin gene expression, while BIRC5 encodes an apoptotic inhibitor that plays a role in beta cell proliferation. High glucose exposure during the proliferation identified similar differentially expressed genes; however, the changes were more modest as compared with the maturation. Methylation has also been implicated in conveying risk for diabetes due to glucose exposure and several key DNA demethylation enzymes; TET1, 2, 3, were also moderately upregulated. Assessment of the effects of glucose exposure on insulin secretion and total insulin content is ongoing. Our data suggest that short term exposure to high glucose may stimulate expression of a set of genes that promote beta cell proliferation and insulin expression and secretion. Disclosure Y.L.Muller: None. K.Wiedrich: None. J.Sutherland: Stock/Shareholder; Pfizer Inc., Bristol Myers. D.Jones: None. L.Baier: None.

1480-P: Mitochondrial Haplogroup H Is Protective against Type 2 Diabetes in Arabs from Gulf Region

Background: Mitochondrial dysfunction is implicated in the pathogenesis of type 2 diabetes (T2D) via impaired glucose-stimulated insulin secretion in pancreatic beta cells and decreased oxidative phosphorylation in insulin target tissues. Several global studies have been performed to delineate the genetic associations of mitochondrial DNA (mtDNA) haplogroups and variants with T2D incidence. Maternal haplogroups F, D, M9 and N9a and a rare variant (mtDNA 3243 A&amp;gt;G) and a common variant (mtDNA 16189 T&amp;gt;C) were associated with increased risk of T2D in different populations. It is worth noting that some of these proposed associations were not consistent across populations. Since the Gulf Cooperation Council (GCC) countries are marked by high prevalence of T2D, we aim to study association of mitochondrial haplogroups and variants in Arabs from GCC region. Methods: We examined mitochondrial haplogroups and variants in 1,112 Kuwaiti and Qatari native individuals using whole exome sequencing data. The cohort was divided into T2D cases (685) and non-T2D controls (427). Results: The association tests indicated that individuals with haplogroup H have a protective effect against T2D with a suggestive significance [odds ratio (OR) = 0.65; P = 0.022]. The haplogroup H association remained significant even after adjusting for age, sex, and body mass index (BMI) (OR= 0.607; P= 0.021). We also identified mtDNA variants with suggestive significance of association (adjusting for covariates) with T2D. Conclusion: The study demonstrates that maternal H haplogroup, which we previously identified as protective towards obesity in Kuwaiti Arabs, is a protective haplogroup for T2D in Arabs from GCC. Further, the mtDNA variants delineated as associated with T2D are from genes involved in cell energy production. Disclosure M.H.Dashti: None. N.Ali: None. H.Alsaleh: None. R.Nizam: None. F.Almulla: None. A.T.Thangavel: None.

EARLY DETECTION AND TREATMENT OF IMPAIRED GLUCOSE AND HYPERINSULINEMIA IN EARLY PREGNANCY TO PREVENT DIABETES AND CARDIOVASCULAR DISEASE IN ADULTS

Objective: So far, no attempt has been made the Prevention of Diabetes and CVD in the community. There is a concept that GDM is the mother of Non-Communicable Diseases. Hence, the aim is to decide the glycemia level which predicts the development of GDM. The suggestion is that HbA1C of more than 5.3 (110mg/dl) during the 10th week of pregnancy predicts GDM. HBA1C 5.7 during pregnancy establishes GDM in Asian ethnicity, and HbA1c measured in the first trimester is helpful for early prediction of GDM. A blood test performed at the 10th week of pregnancy and if PPBS &gt;110 mg% may help identify a pregnant woman at risk of GDM. Hence in the 8th week itself, PPBS has to be estimated. If the PPBS is &gt;110 mg%, a grace period of two weeks is available to maintain PPBS&lt;110mg% at the 10th week. Another essential physiology is that at the 11th week, Fetal beta cell insulin secretion starts. If PPBS remains, more than 110 mg% fetal hyperinsulinemia occurs, which is undesirable. Design and method: Testing for PPBS at the 8th week with 110mg/dl so that enough time is available before fetal insulin secretion starts in the 11th week. If PPBS more than 110mg/dl in the 8th week, the PPBS should be controlled with MNT and Exercise and, if necessary, with Metformin so that PPBS should be maintained at less than 110mg/dl (99 +/- 10) till the 12th week. At the 16th week, 75gm OGCT (DIPSI Test) should be done to know whether she develops GDM and, if negative, be repeated at the 24th and 32nd weeks. Points to Remember:PPBS more than 110mg/dl around the 10th week predicts GDM Fetal Beta Cell starts secreting insulin in the 11th week. The maternal PPBS should be less than 110mg/dl till the 12th week. Results: The Pregnant woman is unlikely to develop Gestational Diabetes if 2-hour PPBS around the 10th Week is less than &lt;110 mg/dl with MNT or Metformin Conclusions: Early Pregnancy hyperglycemia at 8th Weeks detection and treatment may prevent future Diabetes and NCDs especially CVD.

Study of Lipid Profile in Type 2 Diabetes Mellitus Patients in South Chennai Population.

Diabetes Mellitus is caused by the decreased utilization of glucose by tissues and is attributed either to hypoinsulinemia or hyperinsulinemia induced by the overstimulation of insulin-secreting beta cells. Patients with diabetes mellitus, obesity and dyslipidemia are prone to Metabolic syndrome. The study aims to estimate and compare the lipid profile in type 2 diabetes mellitus patients and healthy individuals. This case-control study was undertaken in type II Diabetic patients, where 48 were male, and 45 were female. The diabetic subjects were compared with 33 male and 55 Healthy female subjects who volunteered as controls. Venous blood samples from healthy and diabetic subjects were collected and used to measure routine parameters such as fasting blood sugar (FBS), post-prandial blood sugar(PPBS), Total cholesterol, HDL, and TGL. The Fasting Plasma Glucose and post-prandial glucose were significantly increased among diabetic subjects than the healthy controls. The triglyceride levels in cases had elevated levels in comparison to controls. HDL showed a marginal decrease in the diabetic group than the control group. Mean LDL levels in people with diabetes increase compared to the healthy controls. Besides, the VLDL levels also significantly increased among people with diabetes. There was a substantial increase in FBS and PPBS levels among the diabetic subjects compared to the healthy controls. The lipid profile constituting Total cholesterol, Triglycerides, HDL, LDL and VLDL levels showed the following changes: There was a significant increase in Total cholesterol, TGL, VLDL, and LDL levels, whereas the HDL levels were decreased among the diabetic subjects. Patients with type 2 diabetes mellitus must keep their blood sugar levels under control through follow-ups because uncontrolled diabetes mellitus leads to lipid dysfunction, which ultimately increases the risk of coronary artery disease. Keywords: , , Triglyceride (TGL), Low-Density Lipoprotein (LDL).

PIMT Controls Insulin Synthesis and Secretion through PDX1.

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.