Insulin Secretion In Islets Research Articles

Lysophosphatidylinositols Are Upregulated After Human β-Cell Loss and Potentiate Insulin Release.

Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with β-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E β-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of β-cells and supporting the function of the remaining β-cells.

GIP_HUMAN [22-51] Peptide Encoded by the Glucose-Dependent Insulinotropic Polypeptide (GIP) Gene Suppresses Insulin Expression and Secretion in INS-1E Cells and Rat Pancreatic Islets.

GIP_HUMAN [22-51] is a recently discovered peptide that shares the same precursor molecule with glucose-dependent insulinotropic polypeptide (GIP). In vivo, chronic infusion of GIP_HUMAN [22-51] in ApoE-/- mice enhanced the development of aortic atherosclerotic lesions and upregulated inflammatory and proatherogenic proteins. In the present study, we evaluate the effects of GIP_HUMAN [22-51] on insulin mRNA expression and secretion in insulin-producing INS-1E cells and isolated rat pancreatic islets. Furthermore, we characterize the influence of GIP_HUMAN [22-51] on cell proliferation and death and on Nf-kB nuclear translocation. Rat insulin-producing INS-1E cells and pancreatic islets, isolated from male Wistar rats, were used in this study. Gene expression was evaluated using real-time PCR. Cell proliferation was studied using a BrdU incorporation assay. Cell death was quantified by evaluating histone-complexed DNA fragments. Insulin secretion was determined using an ELISA test. Nf-kB nuclear translocation was detected using immunofluorescence. GIP_HUMAN [22-51] suppressed insulin (Ins1 and Ins2) in INS-1E cells and pancreatic islets. Moreover, GIP_HUMAN [22-51] promoted the translocation of NF-κB from cytoplasm to the nucleus. In the presence of a pharmacological inhibitor of NF-κB, GIP_HUMAN [22-51] was unable to suppress Ins2 mRNA expression. Moreover, GIP_HUMAN [22-51] downregulated insulin secretion at low (2.8 mmol/L) but not high (16.7 mmol/L) glucose concentration. By contrast, GIP_HUMAN [22-51] failed to affect cell proliferation and apoptosis. We conclude that GIP_HUMAN [22-51] suppresses insulin expression and secretion in pancreatic β cells without affecting β cell proliferation or apoptosis. Notably, the effects of GIP_HUMAN [22-51] on insulin secretion are glucose-dependent.

310.7: Single nucleotide polymorphisms associated with human islet insulin secretion

310.7: Single nucleotide polymorphisms associated with human islet insulin secretion

Central Obesity Diminishes Circulating Betatrophin Level in Middle-aged Male Subjects

Central adiposity presents an important risk factor for advancing insulin insensitivity and type 2 diabetes mellitus. Betatrophin, a liver or adipocyte-derived hormone, was assumed to improve islet insulin secretion and compensate insulin resistance but its level during obesity is still conflicted. This study aimed to explore serum betatrophin level in centrally-obese middle-aged men with diabetic potentials compared with age-matched non-obese ones. Sixty-eight male subjects of 40-60 years of age, residing in North Okkalapa Township, Yangon, Myanmar, were recruited and classified into centrally-obese group (n=34) and non-obese group (n=34). Fasting blood samples were obtained to quantify plasma glucose by glucose oxidase method, and serum insulin and betatrophin levels by ELISA. Plasma glucose levels were comparable between the two groups, while insulin concentration of obese group was significantly greater than that of non-obese group. Therefore, HOMA-IR was markedly increased in obese subjects when compared to non-obese ones (4.87±0.28 vs 1.90±0.14, p<0.001) and so did HOMA-β (310.88±26.58 vs 149.00±11.83, p<0.001). Interestingly, betatrophin hormone level was significantly reduced in obese group than non-obese group (1.72±0.21 vs 2.72±0.26 ng/ml, p<0.01). Moreover, betatrophin had a strong negative correlation with glucose and insulin levels (p<0.05) as well as with the indicator of central adiposity, waist circumference (p<0.05), among the subjects. However, significant correlation between betatrophin and HOMA-IR and HOMA-β was not observed in both groups (p=0.14 and 0.20 respectively). Taken together, betatrophin hormone has been found to decrease in adult central obesity, which is noticeably associated with insulin resistance and compensatory beta-cell hyperfunction. Betatrophin, previously regarded as beta-cell mitogen, has been denied in this study, owing to lack of correlation with HOMA indexes of diabetes.

Metformin restores prohormone processing enzymes and normalizes aberrations in secretion of proinsulin and insulin in palmitate-exposed human islets.

To elucidate how proinsulin synthesis and insulin was affected by metformin under conditions of nutrient overstimulation. Isolated human pancreatic islets from seven donors were cultured at 5.5 mmol/L glucose and 0.5 mmol/L palmitate for 12, 24 or 72 h. Metformin (25 μmol/L) was introduced after initial 12 h with palmitate. Proinsulin and insulin were measured. Expression of prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE), was determined by western blot. Adolescents with obesity, treated with metformin and with normal glucose tolerance (n = 5), prediabetes (n = 14), or type 2 diabetes (T2DM; n = 7) were included. Fasting proinsulin, insulin, glucose, 2-h glucose and glycated haemoglobin were measured. Proinsulin/insulin ratio (PI/I) was calculated. In human islets, palmitate treatment for 12 and 24 h increased proinsulin and insulin proportionally. After 72 h, proinsulin but not insulin continued to increase which was coupled with reduced expression of PC1/3 and CPE. Metformin normalized expression of PC1/3 and CPE, and proinsulin and insulin secretion. In adolescents with obesity, before treatment, fasting proinsulin and insulin concentrations were higher in subjects with T2DM than with normal glucose tolerance. PI/I was reduced after metformin treatment in subjects with T2DM as well as in subjects with prediabetes, coupled with reduced 2-h glucose and glycated haemoglobin. Metformin normalized proinsulin and insulin secretion after prolonged nutrient-overstimulation, coupled with normalization of the converting enzymes, in isolated islets. In adolescents with obesity, metformin treatment was associated with improved PI/I, which was coupled with improved glycaemic control.

Pancreatic GLUT2 protein expression and isolated islets insulin secretion decreased in high-fat fed rat dams.

Chronic consumption of high-fat foods during the reproductive period may endanger the dams' metabolic homeostasis and might adversely affect pregnancy outcome. In this regard the present study aimed to investigate the effect of long-term high-fat feeding on pancreatic glucose transporter-2 (GLUT2) protein expression and isolated islets glucose-stimulated insulin secretion in Wistar rat dams. Female rats were randomly divided into normal (N) and high-fat (HF; containing cow butter) diet groups and consumed their respective diets for 10 weeks (from prepregnancy to the end of lactation). After lactation, fasting plasma concentrations of glucose and insulin were measured to calculate HOMA-IR index, then intraperitoneal glucose tolerance test (IPGTT) was performed. Moreover, the pancreatic GLUT2 protein expression and insulin secretion from isolated islets at basal (5.6 mM) and stimulated (16.7 mM) glucose concentrations were assessed. In HF group compared to N group, the plasma insulin level increased, whereas the plasma glucose level did not change in fasting state. Accordingly, the HOMA-IR index increased in HF fed animals. Furthermore, the IPGTT revealed glucose intolerance based on the plasma glucose and insulin results. Also, the pancreatic GLUT2 expression and isolated islets insulin secretion, in response to high glucose concentration, were decreased. The chronic consumption of high-fat foods during prepregnancy, pregnancy, and lactation periods can lead to glucose intolerance, insulin resistance, and inhibition of pancreatic GLUT2 expression, which impairs glucose homeostasis. Therefore, it is crucial to carefully monitor the diet composition of dams during this critical period. The online version contains supplementary material available at 10.1007/s40200-023-01274-6.

Glucolipotoxicity Suppressed Autophagy and Insulin Contents in Human Islets, and Attenuation of PERK Activity Enhanced Them in an ATG7-Dependent Manner.

Administration of pancreatic endoplasmic reticulum kinase inhibitor (PERKi) improved insulin secretion and hyperglycemia in obese diabetic mice. In this study, autophagic balance was studied whether to mediate it. Human islets were isolated from living patients without diabetes. PERKi GSK2606414 effects were evaluated in the islets under glucolipotoxicity by palmitate. Islet insulin contents and secretion were measured. Autophagic flux was assessed by microtubule associated protein 1 light chain 3 (LC3) conversion, a red fluorescent protein (RFP)-green fluorescent protein (GFP)- LC3 tandem assay, and P62 levels. For mechanical analyses, autophagy was suppressed using 3-methyladenine in mouse islets. Small interfering RNA for an autophagy-related gene autophagy related 7 (Atg7) was transfected to interfere autophagy. PERKi administration to mice decreased diabetes-induced P62 levels in the islets. Glucolipotoxicity significantly increased PERK phosphorylation by 70% and decreased insulin contents by 50% in human islets, and addition of PERKi (40 to 80 nM) recovered both. PERKi also enhanced glucose-stimulated insulin secretion (6-fold). PERKi up-regulated LC3 conversion suppressed by glucolipotoxicity, and down-regulated P62 contents without changes in P62 transcription, indicating enhanced autophagic flux. Increased autophagosome-lysosome fusion by PERKi was visualized in mouse islets, where PERKi enhanced ATG7 bound to LC3. Suppression of Atg7 eliminated PERKi-induced insulin contents and secretion. This study provided functional changes of human islets with regard to autophagy under glucolipotoxicity, and suggested modulation of autophagy as an anti-diabetic mechanism of PERKi.

Vascular endothelial growth factor B regulates insulin secretion in β cells of type 2 diabetes mellitus mice via PLCγ and the IP3R‑evoked Ca2+/CaMK2 signaling pathway.

Vascular endothelial growth factor B (VEGFB) plays a crucial role in glucolipid metabolism and is highly associated with type 2 diabetes mellitus (T2DM). The role of VEGFB in the insulin secretion of β cells remains unverified. Thus, the present study aimed to discuss the effect of VEGFB on regulating insulin secretion in T2DM development, and its underlying mechanism. A high‑fat diet and streptozocin (STZ) were used for inducing T2DM in mice model, and VEGFB gene in islet cells of T2DM mice was knocked out by CRISPR Cas9 and overexpressed by adeno‑Associated Virus (AAV) injection. The effect of VEGFB and its underlying mechanism was assessed by light microscopy, electron microscopy and fluorescence confocal microscopy, enzyme‑linked immunosorbent assay, mass spectrometer and western blot analysis. The decrement of insulin secretion in islet β cell of T2DM mice were aggravated and blood glucose remained at a high level after VEGFB knockout (KO). However, glucose tolerance and insulin sensitivity of T2DM mice were improved after the AAV‑VEGFB186 injection. VEGFB KO or overexpression can inhibit or activate PLCγ/IP3R in a VEGFR1‑dependent manner. Then, the change of PLCγ/IP3R caused by VEGFB/VEGFR1 will alter the expression of key factors on the Ca2+/CaMK2 signaling pathway such as PPP3CA. Moreover, VEGFB can cause altered insulin secretion by changing the calcium concentration in β cells of T2DM mice. These findings indicated that VEGFB activated the Ca2+/CaMK2 pathway via VEGFR1‑PLCγ and IP3R pathway to regulate insulin secretion, which provides new insight into the regulatory mechanism of abnormal insulin secretion in T2DM.

Beta cell primary cilia mediate somatostatin responsiveness via SSTR3

ABSTRACT Somatostatin is a paracrine modulator of insulin secretion and beta cell function with pleotropic effects on glucose homeostasis. The mechanism of somatostatin-mediated communication between delta and beta cells is not well-understood, which we address in this study via the ciliary somatostatin receptor 3 (SSTR3). Primary cilia are membrane organelles that act as signaling hubs in islets by virtue of their subcellular location and enrichment in signaling proteins such as G-protein coupled receptors (GPCRs). We show that SSTR3, a ciliary GPCR, mediates somatostatin suppression of insulin secretion in mouse islets. Quantitative analysis of calcium flux using a mouse model of genetically encoded beta cell-specific GCaMP6f calcium reporter shows that somatostatin signaling alters beta cell calcium flux after physiologic glucose stimulation, an effect that depends on endogenous SSTR3 expression and the presence of intact primary cilia on beta cells. Comparative in vitro studies using SSTR isoform antagonists demonstrate a role for SSTR3 in mediating somatostatin regulation of insulin secretion in mouse islets. Our findings support a model in which ciliary SSTR3 mediates a distinct pathway of delta-to-beta cell regulatory crosstalk and may serve as a target for paracrine modulation.

Electrophilic Agonists Modulate the Transient Receptor Potential Ankyrin-1 Channels Mediated by Insulin and Glucagon-like Peptide-1 Secretion for Glucose Homeostasis.

This pre-clinical study investigated the transient receptor potential ankyrin-1 (TRPA1) channels on modulating targets for glucose homeostasis using agonists: the electrophilic agonists, cinnamaldehyde (CIN) and allyl isothiocyanate (AITC), and the non-electrophilic agonist, carvacrol (CRV). A glucose tolerance test was performed on rats. CIN and AITC (5, 10 and 20 mg/kg) or CRV (25, 100, 300, and 600 mg/kg) were administered intraperitoneally (i.p.), and glycemia was measured. In the intestine, Glucagon-like peptide-1 (GLP-1) and disaccharidase activity were evaluated (in vivo and in vitro, respectively). Furthermore, in vivo and in vitro insulin secretion was determined. Islets were used to measure insulin secretion and calcium influx. CIN and AITC improved glucose tolerance and increased insulin secretion in vivo and in vitro. CRV was unable to reduce glycemia. Electrophilic agonists, CIN and AITC, inhibited disaccharidases and acted as secretagogues in the intestine by inducing GLP-1 release in vivo and in vitro and contributed to insulin secretion and glycemia. The effect of CIN on calcium influx in pancreatic islets (insulin secretion) involves voltage-dependent calcium channels and calcium from stores. TRPA1 triggers calcium influx and potentiates intracellular calcium release to induce insulin secretion, suggesting that electrophilic agonists mediate this signaling transduction for the control of glycemia.

An Enhancer Within Abcb11 Regulates G6pc2 in C57BL/6 Mouse Pancreatic Islets.

G6PC2 is predominantly expressed in pancreatic islet beta cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans, and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus: in isolated islets G6pc2 and Spc25 expression were reduced ~50%, and Gm13613 expression was abolished, whereas Cers6 and Nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.

Dietary Supplementation with Yerba Mate (Ilex paraguariensis) Infusion Increases IRS-1 and PI3K mRNA Levels and Enhances Insulin Sensitivity and Secretion in Rat Pancreatic Islets.

"Yerba mate" (YM), an aqueous extract of Ilex paraguariensis, has antioxidant, diuretic, cardio-protective and hypoglycaemic properties. Since its effect on the pancreatic islets remains unclear, we evaluated insulin sensitivity and glucose-stimulated insulin secretion (GSIS) in rats consuming YM or tap water (C) for 21 days. Glucose tolerance, glycemia, triglyceridemia, insulinemia, TBARS and FRAP serum levels were evaluated. GSIS and mRNA levels of insulin signaling pathway and inflammatory markers were measured in isolated pancreatic islets from both groups. In C rats, islets were incubated with YM extract or its phenolic components to measure GSIS. YM improved glucose tolerance, enhanced GSIS, increased FRAP plasma levels and islet mRNA levels of IRS-1 and PI3K (p110), and decreased TBARS plasma levels and islet gene expression of TNF-α and PAI-1. Islets from C rats incubated with 100 µg/mL dry YM extract, 1 µM chlorogenic acid, 0.1 and 1 µM rutin, 1 µM caffeic acid or 1 µM quercetin showed an increase in GSIS. Our results suggest that YM enhances glucose tolerance because of its positive effects on GSIS, oxidative stress rate and insulin sensitivity in rat islets, suggesting that long-term dietary supplementation with YM may improve glucose homeostasis in pre-diabetes or type 2 diabetes.

EP3 signaling is decoupled from the regulation of glucose-stimulated insulin secretion in β-cells compensating for obesity and insulin resistance

ABSTRACT Of the β-cell signaling pathways altered by obesity and insulin resistance, some are adaptive while others contribute to β-cell failure. Two critical second messengers are Ca2+ and cAMP, which control the timing and amplitude of insulin secretion. Previous work has shown the importance of the cAMP-inhibitory Prostaglandin EP3 receptor (EP3) in mediating the β-cell dysfunction of type 2 diabetes (T2D). Here, we used three groups of C57BL/6J mice as a model of the progression from metabolic health to T2D: wildtype, normoglycemic Leptin Ob (NGOB), and hyperglycemic Leptin Ob (HGOB). Robust increases in β-cell cAMP and insulin secretion were observed in NGOB islets as compared to wildtype controls; an effect lost in HGOB islets, which exhibited reduced β-cell cAMP and insulin secretion despite increased glucose-dependent Ca2+ influx. An EP3 antagonist had no effect on β-cell cAMP or Ca2+ oscillations, demonstrating agonist-independent EP3 signaling. Finally, using sulprostone to hyperactivate EP3 signaling, we found EP3-dependent suppression of β-cell cAMP and Ca2+ duty cycle effectively reduces insulin secretion in HGOB islets, while having no impact insulin secretion on NGOB islets, despite similar and robust effects on cAMP levels and Ca2+ duty cycle. Finally, increased cAMP levels in NGOB islets are consistent with increased recruitment of the small G protein, Rap1GAP, to the plasma membrane, sequestering the EP3 effector, Gɑz, from inhibition of adenylyl cyclase. Taken together, these results suggest that rewiring of EP3 receptor-dependent cAMP signaling contributes to the progressive changes in β cell function observed in the Leptin Ob model of diabetes.

Chronic Mg2+ Deficiency Does Not Impair Insulin Secretion in Mice.

Magnesium is an essential mediator of a vast number of critical enzymatic cellular reactions in the human body. Some clinical epidemiological studies suggest that hypomagnesemia accounts for declines in insulin secretion in patients with type 2 diabetes (T2D); however, the results of various experimental studies do not support this notion. To address this discrepancy, we assessed the short- and long-term effects of hypomagnesemia on β-cell function and insulin secretion in primary mouse islets of Langerhans and in a mouse model of hypomagnesemia known as Trpm6Δ17 /fl;Villin1-Cre mice. We found that lowering the extracellular Mg2+ concentration from 1.2 mM to either 0.6 or 0.1 mM remarkably increased glucose-induced insulin secretion (GIIS) in primary islets isolated from C57BL/6 mice. Similarly, both the plasma insulin levels and GIIS rose in isolated islets of Trpm6Δ17 /fl;Villin1-Cre mice. We attribute these rises to augmented increases in intracellular Ca2+ oscillations in pancreatic β-cells. However, the glycemic metabolic profile was not impaired in Trpm6Δ17 /fl;Villin1-Cre mice, suggesting that chronic hypomagnesemia does not lead to insulin resistance. Collectively, the results of this study suggest that neither acute nor chronic Mg2+ deficiency suppresses glucose-induced rises in insulin secretion. Even though hypomagnesemia can be symptomatic of T2D, such deficiency may not account for declines in insulin release in this disease.

197-OR: Dynorphin, a Novel Paracrine Factor That Regulates Insulin Secretion

Within the pancreatic islet, the crosstalk between different islet cells through paracrine signals orchestrates a hormonal response that controls glucose levels. The dysregulation of these signals contributes to impaired glucose homeostasis and diabetes. Therefore, understanding intra-islet communication is imperative for developing new strategies for diabetes management. Here, we show that intra-islet dynorphin (Dyn) stimulates somatostatin (SST) secretion and is a novel factor regulating islet function and insulin secretion in human and mouse islets. Dyn, an opioid peptide involved in stress response, pain, and addiction is synthesized in human and mouse β-cells and secreted in response to glucose. Dyn acts mainly through the k-opioid receptor (KOR) expressed in all islet cells with the highest expression in δ-cells. Using perifusion and static incubation studies, we show that Dyn induces SST secretion and decreases insulin secretion in mouse and human islets and that Dyn fails to induce SST secretion in islets from mice with KOR deletion in δ-cells, thereby providing a mechanism for feedback control of insulin secretion (β- to δ-cell Dyn/KOR negative feedback loop). Our data also show that Dyn is increased in islets from HFD-fed mice (8-fold) and in human β-cells from T2D donors. Remarkably, dynorphin mRNA is one of the top ten most upregulated genes in islets from HFD-fed mice, higher than several critical β-cell genes, including pdx1 or ucn3. We demonstrate that the β- to δ-cell Dyn/KOR negative feedback loop is upregulated in diabetes and pharmacological inhibition or genetic deletion of KOR in δ-cells improves glucose homeostasis in high-fat diet (HFD)-fed mice, demonstrating the therapeutic potential of this system. The novel role of the Dyn/KOR axis in islet function in healthy and diabetogenic conditions can have profound implications for glucose homeostasis and serve to develop potential targets for T2D. Disclosure M.Blandino-rosano: None. J.Panzer: None. E.Bernal-mizrachi: None.

1745-P: Voltage-Gated K+ Channel Kv7.1 Is an Important Regulator of Insulin Secretion in Normal and Hyperinsulinemic Islets

Fifty percent of infants with Beckwith-Wiedemann syndrome (BWS), an overgrowth syndrome caused by genetic / epigenetic abnormalities in imprinted genes on chromosome 11p15, have persistent hypoglycemia due to hyperinsulinism (BWS-HI). BWS-HI islets exhibit increased baseline and glucose-stimulated insulin secretion (GSIS) and are responsive to the KATP channel inhibitor glyburide, suggesting it is not caused by impaired KATP channels. KCNQ1 expression, encoding the pore-forming α subunit of voltage-gated K+ channel Kv7.1, is reduced in BWS-HI islets compared to normal islets (3-fold; p = 4.5×10−7) and KCNQ1 loss-of-function mutations in humans are associated with HI. We used genetic and pharmacologic methods to modify Kv7.1 activity and examined changes to insulin secretion. In response to glucose, Kcnq1−/− mice had lower plasma glucose and higher plasma insulin than wild-type controls. Perifusion of Kcnq1−/− pancreatic islets with increasing concentrations of glucose showed that GSIS in these islets was faster and greater than in wild-type islets. The perifusion GSIS responses in normal human, wild-type mouse and Sur1−/− mouse (KATP-HI mouse model) islets with two different Kv7.1 activators (hexachlorophene or ML277) were markedly slower and diminished. In contrast, wild-type mouse islet perifusion with the Kv7.1 inhibitor chromanol 293B strikingly fastened and increased the GSIS response compared to vehicle controls. Membrane potential (Vm) analysis of Kcnq1−/− islets demonstrated that depolarization occurs more quickly during the initiation of insulin secretion compared to wild-type controls. Treatment with Kv7.1 activators resulted in Vm changes in both wild-type mouse and normal human islets compared to controls. Our results reveal the capacity of Kv7.1 to regulate insulin secretion in pancreatic islets and that Kv7.1 is a viable target for development of therapeutics for HI. Disclosure C.Juliana: Research Support; Crinetics Pharmaceuticals, Inc., Twist Bioscience, AmideBio. D.De leon: Consultant; Zealand Pharma A/S, Crinetics Pharmaceuticals, Inc., Eiger BioPharmaceuticals, Hanmi Pharm. Co., Ltd., Research Support; Ultragenix, Crinetics Pharmaceuticals, Inc., Hanmi Pharm. Co., Ltd., Rezolute, Inc. Funding National Institutes of Health (5R01DK098517-08)

292-LB: Cholesterol Accumulation in Islets Exacerbates Insulin Secretory Defects in a Model of Mild ß-Cell Dysfunction

Hypercholesterolemia is common in diabetes. We sought to determine whether cholesterol (Chol) can exacerbate impaired insulin secretion in islets from a mouse model of mild β-cell dysfunction. To induce β-cell dysfunction, C57BL/6J male mice were treated with low-dose (30 mg/kg, i.p.) streptozotocin (STZ) or vehicle (VEH) once daily on 3 consecutive days. Mice were then fed a 9% fat diet for 4 weeks, after which islets were isolated and cultured for 24 h ± Chol prior to endpoint measures. After 4 weeks, body weight (STZ 28.9±1.0 vs VEH 29.1±1.1 g) and fed glucose (STZ 191.2±11.0 vs VEH 177.8±8.2 mg/dl) did not differ. When cultured with 0 mM Chol, isolated islets from STZ-treated mice showed reduced glucose (20 mM)-stimulated insulin secretion (GSIS: 973.9±191.7 vs 1714.8±384.6 pM, n=5, p<0.05) and a trend for lower insulin content (180.6±18.2 vs 230.4±22.9 nM, n=5, p=0.06), compared to islets from VEH-treated mice. Having established STZ-induced β-cell dysfunction, islets from STZ-treated mice were cultured ± 0.5 mM Chol, then Chol content and GSIS measured. Culture in 0.5 mM (vs 0 mM) Chol resulted in increased islet Chol content (58.9 ± 4.3 vs 17.6 ± 3.0 µg/mg protein; n=3, p<0.01). A trend for increased basal insulin secretion (2.8 mM glucose: 185.8±64.7 vs 32.8±3.5 pM, n=5, p=0.09) and reduced GSIS (305.3±60.3 vs 973.9±191.7 pM, n=5, p<0.05) were observed in islets cultured with 0.5 mM Chol. Insulin content was also reduced (125.8±10.8 vs 180.6±18.2 nM, n=5, p<0.01) with 0.5 mM Chol culture. In sum, we have developed a STZ-based, euglycemic mouse model of subclinical β-cell dysfunction. Chol treatment of islets from these mice exacerbates β-cell dysfunction, in keeping with a deleterious effect of Chol. This model now provides the ability to determine whether hypercholesterolemia in vivo results in further impairment of β-cell dysfunction and the development of hyperglycemia, an observation that would be relevant to the progression of prediabetes to diabetes and diabetes per se. Disclosure R. Akter: None. B. Barrow: None. R. L. Hull-meichle: Employee; Veterans Administration, Research Support; Casma Therapeutics, Cystic Fibrosis Foundation, NIH - National Institutes of Health, Veterans Administration. S. Zraika: None. S. E. Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. Funding U.S. Department of Veterans Affairs (I01BX001060); National Institutes of Health (P30DK017047); Diabetes Research Connection (46)

286-OR: Using iPSC-Derived Islets to Model the Effect of an ABCC8 Arg1420His Mutation in Fetal and Adult Islets

We previously reported an Arg1420His mutation in ABCC8 in an Indigenous community that resulted in hyperinsulinemic hypoglycemia in infancy in a homozygous carrier and a doubling in type 2 diabetes (T2D) risk in heterozygous carriers (3% of the population). Previous studies of ABCC8 mutations have suggested a loss of pancreatic beta cells as a probable cause of future T2D. To understand the pathomechanism behind the increased risk of T2D due to the Arg1420His mutation, we generated two sets of isogenic iPSCs from two different Indigenous community members. These iPSCs were used to generate pancreatic islets and assess insulin secretion using a dynamic glucose stimulated insulin secretion (D-GSIS) assay when the islets were immature (resembling fetal islets) and during multiple days after maturation (resembling adult islets). In immature islets, the D-GSIS assay confirmed the clinical phenotype of significantly higher insulin secretion in islets with the His-allele under basal conditions (2mM glucose) and a lower response to diazoxide in islets homozygous for the His-allele. Both Arg1420 and His1420 mature islets had lower insulin secretion under basal conditions, however, insulin secretory response to high glucose (20mM) and Tolbutamide (sulphonylurea) was significantly lower in islets heterozygous for the His-allele whereas no response was seen in islets homozygous for the His-allele despite having similar proportion of beta cells as assessed by flow cytometry (~50% cells positive for INS and NKX6-1). This suggests a mechanism other than beta cell loss as the cause of lower insulin secretory response in these impaired islets. Single cell RNA sequencing showed higher expression of G6PC2, which inhibits insulin secretion by opposing the action of GCK, in islets with the His-allele. Our ongoing work includes knockout of G6PC2 in the isogenic iPSCs and using a GCK activator (Dorzagliatin) to correct the insulin secretory response to high glucose in the His-allele carrying islets. Disclosure A.K.Nair: None. K.Barkho: None. M.Traurig: None. R.G.Nelson: None. C.Bogardus: None. L.Baier: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

1746-P: The Role of Cell–Cell Junction Associated Mediator in Glucose-Stimulated Insulin Secretion of Pancreatic Islets

Glucose-stimulated insulin secretion from β cells is essential in maintaining glucose homeostasis. Cell-cell communication within the pancreatic islets, mediated by cell-cell junctions and their mediators, regulates insulin secretion dynamics and glucose homeostasis. It has been reported that Nephrin-mediated intercellular junction between β cells is implicated in the regulation of insulin secretion, however, the underlying mechanisms are not fully understood. Here, we investigated the effects of Girdin, a cell-cell junction associated mediator, on insulin release. We found that Girdin was expressed in mouse pancreatic β cells and mediated glucose-stimulated insulin secretion, especially for the second phase. Next, we demonstrated that glucose-stimulated tyrosine phosphorylation of Girdin mediated Nephrin phosphorylation by recruiting Src kinase, which leads to the endocytosis of Nephrin. Furthermore, we observed the glucose-induced Girdin/Nephrin/Src signaling induced downstream Akt phosphorylation and activated Rac1-mediated cytoskeleton reorganization, allowing insulin secretory granules to access the plasma membrane for the second-phase secretion. Finally, we found that Girdin is downregulated in the diabetic islets, and rescue of Girdin restored impaired glucose-stimulated insulin secretion. Taken together, these data provide evidence that the cell-cell junction associated mediator play important roles in fusion of insulin secretory vesicles within the cell and establish a role for Girdin in regulating this process. Disclosure H.Wang: None. Funding National Natural Science Foundation of China

1756-P: Extracellular Matrix Stiffness Mediates Insulin Secretion in Pancreatic Islets via Phosphofructokinase Activity

Pancreatic islets are surrounded by extracellular matrix (ECM) which provides mechanical and biochemical cues to the β-cells that regulate survival and insulin secretion. Changes to ECM stiffness have been correlated to dysfunction in insulin secretion that is directly coupled to glucose metabolism; however, the mechanisms of mechanotransduction regulating insulin secretion have not been studied in the islet. We hypothesize that increasing matrix stiffness will increase islet glucose sensitivity by increasing phosphofructokinase (PFK) activity. To determine the effect of changes in ECM stiffness on islet function we encapsulated mouse islets in a reverse thermal gel (RTG) scaffold with increasing stiffness as determined by rheological analysis. Glucose-stimulated insulin secretion, PFK activity, and PFK expression was measured after 24 hours of culture. Proliferation was analyzed by immunohistochemistry of Ki-67. Reactive oxygen species (ROS) were measured via confocal microscopy of islets transfected with an adenoviral ROS sensor. We found that increasing RTG wt% yielded increasing stiffness at 40°C. Insulin secretion increased as the matrix stiffness increased in basal (2mM) and high glucose (20mM) conditions. Insulin secretion at 20mM glucose normalized to 2mM glucose (stimulation index) decreases with matrix stiffness indicating dysfunction to insulin secretion. PFK activity increased in islets encapsulated in stiffer RTGs. No changes in proliferation were observed in any samples compared to controls. Our results support a role for ECM stiffness in regulating insulin secretion via PFK activity. This work provides insight into how changes in ECM stiffness contribute to islet dysfunction, as in pancreatitis and T2D. Our results will inform future development of mechanically tuned microenvironments for differentiation of iPSCs into functional β-cells and provide novel targets to protect against islet dysfunction in T2D. Disclosure C.Garcia: None. K.Holcomb: None. N.L.Farnsworth: None. Funding American Diabetes Association (7-21-JDF-020 to N.L.F.); National Institutes of Health (1F31DK132926-01A1); JDRF (3-APF-2019-749-A-N, 1-FAC-2020-891-A-N)