Rat Beta-cells Research Articles

TRAIL induces proliferation in rodent pancreatic beta cells via AKT activation.

Strategies to increase functional pancreatic beta cell mass is of great interest in diabetes-related research. TNF-related apoptosis-inducing ligand (TRAIL) is well known to promote proliferation and survival in various cell types, including vascular smooth muscle and endothelial cells. Correlation between the protective nature of TRAIL on these cells and its proliferative effect is noteworthy. TRAIL's seemingly protective/therapeutic effect in diabetes prompted us to question whether it may act as an inducer of proliferation in pancreatic beta cells. We used rat primary islet cells and MIN6 mouse beta cell line to investigate TRAIL-induced proliferation. Cell viability and/or death was analyzed by MTT, WST-1, and Annexin-V/PI assays, while proliferation rates and pathways were assessed via immunocytochemical and Western blot analyses. Receptor neutralization antibodies identified the mediator receptors. Recombinant soluble TRAIL (sTRAIL) treatment led to 1.6-fold increased proliferation in insulin-positive cells in dispersed rat islets compared to the untreated group, while adenovirus-mediated overexpression of TRAIL increased the number of proliferating beta cells up to more than six-fold. sTRAIL or adenoviral vector-mediated TRAIL overexpression induced proliferation in MIN6 cells also. TRAIL's proliferative effect was mediated via AKT activation, which was suppressed upon specific inhibition. Neutralization of each TRAIL receptor reversed the proliferative effect to some degree, with the highest level of inhibition in death receptor 5 (DR5) blockage in MIN6 cells and in decoy receptor 1 (DcR1) blockage in primary rat beta cells. Thus, TRAIL induces proliferation in rodent pancreatic beta cells through activation of the AKT pathway.

Endothelial Cell-Derived Triosephosphate Isomerase Attenuates Insulin Secretion From Pancreatic Beta Cells of Male Rats.

Insulin secretion from pancreatic beta cells is tightly regulated by glucose and paracrine signals within the microenvironment of islets of Langerhans. Extracellular matrix from islet microcapillary endothelial cells (IMEC) affect beta-cell spreading and amplify insulin secretion. This study was aimed at investigating the hypothesis that contact-independent paracrine signals generated from IMEC may also modulate beta-cell insulin secretory functions. For this purpose, conditioned medium (CMp) preparations were prepared from primary cultures of rat IMEC and were used to simulate contact-independent beta cell-endothelial cell communication. Glucose-stimulated insulin secretion (GSIS) assays were then performed on freshly isolated rat islets and the INS-1E insulinoma cell line, followed by fractionation of the CMp, mass spectroscopic identification of the factor, and characterization of the mechanism of action. The IMEC-derived CMp markedly attenuated first- and second-phase GSIS in a time- and dose-dependent manner without altering cellular insulin content and cell viability. Size exclusion fractionation, chromatographic and mass-spectroscopic analyses of the CMp identified the attenuating factor as the enzyme triosephosphate isomerase (TPI). An antibody against TPI abrogated the attenuating activity of the CMp while recombinant human TPI (hTPI) attenuated GSIS from beta cells. This effect was reversed in the presence of tolbutamide in the GSIS assay. In silico docking simulation identified regions on the TPI dimer that were important for potential interactions with the extracellular epitopes of the sulfonylurea receptor in the complex. This study supports the hypothesis that an effective paracrine interaction exists between IMEC and beta cells and modulates glucose-induced insulin secretion via TPI-sulfonylurea receptor-KATP channel (SUR1-Kir6.2) complex attenuating interactions.

Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm2 at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm2 was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.

In vivo antidiabetic activity of qwueous extract of Artemisia argyi (Chinese mugwort) in alloxan-induced diabetic rats

Purpose: To determine the antidiabetic, antioxidant and anti-hyperlipidemic effects of aqueous leaf extract of Artemisia argyi (Asteraceae) in alloxan (ALX)-induced diabetic rats.&#x0D; Experimental: Soxhlet apparatus was packed with grinded leaves of A. Argyi and subjected to extraction by double distillation using water as running solvent for 4 – 5 h. Male albino Wistar rats weighing 150 ± 10 g were used in this study. Diabetes was induced in overnight-fasted rats via intraperitoneal administration of freshly prepared 10 % alloxan solution at a dose of 186.9 mg/kg. Serum glucose (Glc), high-density lipoprotein cholesterol (HDL-c), triglycerides (TGs) and total cholesterol (TC) were evaluated using Randox assay kits. Serum reduced glutathione (GSH) was assayed using a slight modification of a previously reproted procedure, while histological examination was carried out microscopically after hematoxylin and eosin staining.&#x0D; Results: Oral administration of aqueous extract of Artemisia argyi significantly reduced ALX-induced increases in glycosylated hemoglobin and blood glucose, but significantly increased total protein, hemoglobin, insulin, and C-peptide levels (p &lt; 0.05). Administration of the extract also led to a significant upsurge in non-enzymic antioxidants i.e. ceruloplasmin, GSH, vitamin E and vitamin C. The extract produced a hypolipidemic effect by significantly reducing total cholesterol (TC) and serum TGs. The hypoglycemic and hypolipidemic effects of the extract were dose-dependent (p &lt; 0.05). Histological examination of the pancreas revealed that the extract protected the integrity of beta cells in ALXinduced diabetic rats.&#x0D; Conclusion: These results indicate the beneficial effects of Artemisia argyi against diabetes mellitus. Thus, Artemisia argyi may be useful in the management of diabetes mellitus.&#x0D; Keywords: Artemisia argyi, Antidiabetic, Glutathione, Histopathology, Antioxidant

ATM and P53 differentially regulate pancreatic beta cell survival in Ins1E cells.

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.

2104-P: Proinflammatory Cytokines Impair Human Islet and Beta-Cell Glucose Oxidation

Pro-inflammatory cytokines contribute to beta cell dysfunction in type 1 diabetes (T1D), but the exact underlying mechanisms remain unclear. It was previously shown that cytokines inhibit mitochondrial glucose oxidation leading to impaired glucose-stimulated insulin secretion in rat INS-1E cells, however, the effect of cytokines on mitochondrial respiration in human islets has not been explored. In this study, we investigated mitochondrial dysfunction in human islets and rat beta cells following cytokine treatment. We exposed isolated human pancreatic islets and rat INS-1E cells for up to 24 h to IL-1β and IFN-γ to mimic early beta cell destruction in T1D. Mitochondrial bioenergetics were analyzed by Seahorse XFe24 technology. Human islets from three healthy organ donors exposed to cytokines showed a reduction in glucose-induced oxygen consumption and an increase in ATP-uncoupled respiration. Higher basal mitochondrial oxygen consumption was also observed, which was not associated with an increase in ATP production. In INS-1E cells, cytokines exerted a strong inhibitory effect on mitochondrial glucose oxidation in a dose-independent manner. In acute exposure experiments, no effect on oxygen consumption was observed up to eight hours after cytokine exposure. This finding proposes that cytokine-induced inhibition of mitochondrial respiration is dependent on secondary signaling mechanism and/or changes in gene transcription. To conclude, glucose-induced oxygen consumption is reduced by cytokines in both human islets and INS-1E cells indicative of impaired mitochondrial respiration. Moreover, cytokines augment mitochondrial uncoupled respiration in human islets, indicating mitochondrial dysfunction that may contribute to islet failure in T1D. Ongoing work is investigating gene expression of genes involved in mitochondrial respiration processes. Further studies are needed to clarify the possible pathogenic role of mitochondrial dysfunction in T1D and the underlying mechanisms. Disclosure J. Melo: None. V. Hirschberg Jensen: None. J. Størling: None.

1067-P: Development of Novel Modulators of the GABAA Receptor for Diabetes Therapy

Accumulating data suggest that the GABAergic system may play a key role in beta-cell survival and regeneration in both type 1 and type 2 diabetes. Therefore, the development of new positive allosteric modulators of the GABAA receptors (GABAA-Rs) provides an interesting strategy for an antidiabetic therapy, although central nervous system side effects mediated by α1-GABAA-Rs are a potential concern. Here we synthesized and functionally analyzed a series of low molecular weight thioacrylamides (ThAcs), designed to both positively modulate GABAA-R signalling and show low blood brain barrier penetration. Beta-cell proliferation was increased by several ThAcs at nanomolar concentrations in a rat beta-cell line in vitro (i.e., ∼82% increased EdU incorporation after 24 hours treatment with ThAc HK-4 at 100 nM). The most effective candidate, HK-4, was further characterized. By using radio ligand competition assays and functional patch clamp experiments we analyzed the interaction between HK-4 and GABAA-Rs. In competition assays, we observed a dose-dependent affinity of HK-4 for GABAA-Rs in rat cortical neuron membranes. In electrophysiological patch clamp assays, we determined the modulating activity of HK-4 to individual human GABAA-Rs using HEK293 cells stably expressing different subunit combinations. HK-4 induced very strong potentiation of the current elicited by the natural ligand GABA on GABAA receptors containing combinations of α2β3γ2 (current ∼300% of GABA alone), α3β3γ2 (∼770%) and on α1β3γ2 (∼580%) subunits, respectively. Minimal blood brain barrier penetration of HK-4 was verified by mass spectrometry-based analyses of brain and plasma samples of rats. Additionally, HK-4 shows no major toxicological effects as determined in a 90-day toxicity study with daily dosing, has a favorable pharmacokinetic profile and demonstrates good oral bioavailability. Our data reveal the potential of HK-4 and other ThAcs for further development into antidiabetic drugs. Disclosure B. Hasse: Stock/Shareholder; Self; Algiax Pharmaceuticals GmbH. B. Belgardt: None. M. Hecht: None. R. Wördenweber: None. A. Piechot: None. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S. J. Eckel: None. Funding EFRE; NRW; European Union

MicroRNA-375 regulates glucose metabolism-related signaling for insulin secretion.

Enhanced beta cell glycolytic and oxidative metabolism are necessary for glucose-induced insulin secretion. While several microRNAs modulate beta cell homeostasis, miR-375 stands out as it is highly expressed in beta cells where it regulates beta cell function, proliferation and differentiation. As glucose metabolism is central in all aspects of beta cell functioning, we investigated the role of miR-375 in this process using human and rat islets; the latter being an appropriate model for in-depth investigation. We used forced expression and repression of mR-375 in rat and human primary islet cells followed by analysis of insulin secretion and metabolism. Additionally, miR-375 expression and glucose-induced insulin secretion were compared in islets from rats at different developmental ages. We found that overexpressing of miR-375 in rat and human islet cells blunted insulin secretion in response to glucose but not to α-ketoisocaproate or KCl. Further, miR-375 reduced O2 consumption related to glycolysis and pyruvate metabolism, but not in response to α-ketoisocaproate. Concomitantly, lactate production was augmented suggesting that glucose-derived pyruvate is shifted away from mitochondria. Forced miR-375 expression in rat or human islets increased mRNA levels of pyruvate dehydrogenase kinase-4, but decreased those of pyruvate carboxylase and malate dehydrogenase1. Finally, reduced miR-375 expression was associated with maturation of fetal rat beta cells and acquisition of glucose-induced insulin secretion function. Altogether our findings identify miR-375 as an efficacious regulator of beta cell glucose metabolism and of insulin secretion, and could be determinant to functional beta cell developmental maturation.

Safety and function of a new pre-vascularized bioartificial pancreas in an allogeneic rat model

Cell encapsulation could overcome limitations of free islets transplantation but is currently limited by inefficient cells immune protection and hypoxia. As a response to these challenges, we tested in vitro and in vivo the safety and efficacy of a new macroencapsulation device named MailPan®. Membranes of MailPan® device were tested in vitro in static conditions. Its bio-integration and level of oxygenation was assessed after implantation in non-diabetic rats. Immune protection properties were also assessed in rat with injection in the device of allogeneic islets with incompatible Major Histocompatibility Complex. Finally, function was assessed in diabetic rats with a Beta cell line injected in MailPan®. In vitro, membranes of the device showed high permeability to glucose, insulin, and rejected IgG. In rat, the device displayed good bio-integration, efficient vascularization, and satisfactory oxygenation (>5%), while positron emission tomography (PET)-scan and angiography also highlighted rapid exchanges between blood circulation and the MailPan®. The device showed its immune protection properties by preventing formation, by the rat recipient, of antibodies against encapsulated allogenic islets. Injection of a rat beta cell line into the device normalized fasting glycemia of diabetic rat with retrieval of viable cell clusters after 2 months. These data suggest that MailPan® constitutes a promising encapsulation device for widespread use of cell therapy for type 1 diabetes.

Upregulation of caveolin-1 and its colocalization with cytokine receptors contributes to beta cell apoptosis

Caveolin-1 (cav-1), the principal structural and signalling protein of caveolae, is implicated in various signalling events, including apoptotic cell death in type 2 diabetes. However, the precise role of beta cells in apoptosis has not been clearly defined. In this study, we investigated the involvement of cav-1 in cytokine-induced beta cell apoptosis and its underlying mechanisms in the rat beta cell line, INS-1 and isolated islets. Treatment of cytokine mixture (CM, TNFα + IL-1β) significantly increased the mRNA and protein expression of cav-1, and resulting in increased formation of caveolae. We found that IL-1 receptor 1 and TNF receptor localized to plasma membrane lipid rafts in the control cells and CM treatment recruited these receptors to the caveolae domain. After cav-1 siRNA transfection, CM-dependent NF-κB activation was reduced and consequently downregulated the mRNA expression of iNOS and IL-1β. Finally, decreased cell viability by CM treatment was ameliorated in both INS-1 cells and isolated islets treated with cav-1 siRNA. These results suggest that increased cav-1 expression and recruitment of cytokine receptors into caveolae contribute to CM-induced beta cell apoptosis.

Pengaruh Suplementasi Besi (Fe) Dosis Tinggi Terhadap Kondisi Sel Beta Pankreas pada Tikus Putih (Rattus Norvegicus) Strain Wistar Bunting

During pregnancy, pregnant women need various nutrients. One of the nutrients need is iron. Iron functions for the formation of hemoglobin. Excess iron can increase free radicals and trigger oxidative stress. The aim of this study was to determine the effect of high doses of iron supplementation on the condition of the beta cells pancreas of the pregnant wistar white rat (Rattus novergicus). The condition of pancreatic beta cells seen is the number of beta cells calculated using a microscope with 400x magnification. Iron supplementation was given 18 days with 3 different doses (0.54mg, 1.08mg, and 2.16mg). The number of pancreatic beta cells differed significantly between the control and treatment groups (p = 0,000), the mean number of the smallest pancreatic beta cells in the group (P3) 12,17. Iron supplementation can reduce the number of pancreatic beta cells in pregnant wistar white rat (Rattus novergicus).

Glucocorticoid-Induced, Morpho-Functional Alterations in Pancreatic Beta Cells of Wistar Rats

Background: Though prolonged use of glucocorticoids has been reported to promote adverse effects, traditionally, high-dose glucocorticoids have been implicated in immune-suppression following organ transplant with Cortisone being a well-known artificial glucocorticoid.&#x0D; Objectives: This study investigated the histo-architectural and functional changes in pancreatic beta cells due to Cortisone administration.&#x0D; Materials and Methods: Forty two (42) Wistar rats (140 – 200 kg) were assigned into seven groups of six (6) rats each with group A acting as a control. While groups B and C were respectively treated with 0.1 mg/kg and 0.3 mg/kg of Cortisone, groups D and E received 0.1 mg/kg and 0.3 mg/kg of Cortisone respectively plus 33 mg/kg of Ketoconazole; whereas, groups F and G were respectively given 0.1 mg/kg and 0.3 mg/kg of Cortisone alongside 150 mg/kg of Vitamin E each for twenty-eight (28) days. After 28 days of administration, rats were euthanized and blood samples collected for insulin assay. Pancreatic tissues were also harvested and observed for histo-morphological changes.&#x0D; Results: Analysis of variance (ANOVA) found Cortisone to have significantly (p &lt; .05) increased glucose level in a dose dependent manner. This was however attenuated following co-administration of Ketoconazole and Vitamin E as Ketoconazole showed more potency in this ameliorating effect. Also, Cortisone was observed to significantly decrease (in dose dependent fashion), pancreatic β-cell functions, with attenuating effect seen following co-administration of Ketoconazole.&#x0D; Conclusion: It is recommended that caution is applied with the intake of glucocorticoids, especially in polypharmacy while treating certain ailments.

Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially.

Cell therapy for diabetes could benefit from the identification of small-molecule compounds that increase the number of functional pancreatic beta cells. Using a newly developed screening assay, we previously identified glucocorticoids as potent stimulators of human and rat beta cell proliferation. We now compare the stimulatory action of these steroid hormones to a selection of checkpoint tyrosine kinase inhibitors that were also found to activate the cell cycle-in beta cells and analyzed their respective effects on DNA-synthesis, beta cell numbers and expression of cell cycle regulators. Our data using glucocorticoids in combination with a receptor antagonist, mifepristone, show that 48h exposure is sufficient to allow beta cells to pass the cell cycle restriction point and to become committed to cell division regardless of sustained glucocorticoid-signaling. To reach the end-point of mitosis another 40h is required. Within 14 days glucocorticoids stimulate up to 75% of the cells to undergo mitosis, which indicates that these steroid hormones act as proliferation competence-inducing factors. In contrast, by correlating thymidine-analogue incorporation to changes in absolute cell numbers, we show that the checkpoint kinase inhibitors, as compared to glucocorticoids, stimulate DNA-synthesis only during a short time-window in a minority of cells, insufficient to give a measurable increase of beta cell numbers. Glucocorticoids, but not the kinase inhibitors, were also found to induce changes in the expression of checkpoint regulators. Our data, using checkpoint kinase-specific inhibitors further point to a role for Chk1 and Cdk1 in G1/S transition and progression of beta cells through the cell cycle upon stimulation with glucocorticoids.

Correction to: Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets.

As part of an institutional investigation by University of Bremen, the work carried out by Kathrin Maedler's laboratory has been reviewed.

DEXI, a candidate gene for type 1 diabetes, modulates rat and human pancreatic beta cell inflammation via regulation of the type I IFN/STAT signalling pathway.

The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved. Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Beta cell inflammation and apoptosis, driven by a synthetic viral double-stranded RNA, were evaluated by real-time PCR, western blotting and luciferase assays. DEXI-silenced beta cells exposed to a synthetic double-stranded RNA (polyinosinic:polycytidylic acid [PIC], a by-product of viral replication) showed reduced activation of signal transducer and activator of transcription (STAT) 1 and lower production of proinflammatory chemokines that was preceded by a reduction in IFNβ levels. Exposure to PIC increased chromatin-bound DEXI and IFNβ promoter activity. This effect on IFNβ promoter was inhibited in DEXI-silenced beta cells, suggesting that DEXI is implicated in the regulation of IFNβ transcription. In a mirror image of knockdown experiments, DEXI overexpression led to increased levels of STAT1 and proinflammatory chemokines. These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region, and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune responses in beta cells. Moreover, our results provide initial information on the function of DEXI.

Genome-wide profiling of histone H3K27 acetylation featured fatty acid signalling in pancreatic beta cells in diet-induced obesity in mice.

Epigenetic regulation of gene expression has been implicated in the pathogenesis of obesity and type 2 diabetes. However, detailed information, such as key transcription factors in pancreatic beta cells that mediate environmental effects, is not yet available. To analyse genome-wide cis-regulatory profiles and transcriptome of pancreatic islets derived from a diet-induced obesity (DIO) mouse model, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) of histone H3 lysine 27 acetylation (histone H3K27ac) and high-throughput RNA sequencing. Transcription factor-binding motifs enriched in differential H3K27ac regions were examined by de novo motif analysis. For the predicted transcription factors, loss of function experiments were performed by transfecting specific siRNA in INS-1, a rat beta cell line, with and without palmitate treatment. Epigenomic and transcriptional changes of possible target genes were evaluated by ChIP and quantitative RT-PCR. After long-term feeding with a high-fat diet, C57BL/6J mice were obese and mildly glucose intolerant. Among 39,350 islet cis-regulatory regions, 13,369 and 4610 elements showed increase and decrease in ChIP-Seq signals, respectively, significantly associated with global change in gene expression. Remarkably, increased H3K27ac showed a distinctive genomic localisation, mainly in the proximal-promoter regions, revealing enriched elements for nuclear respiratory factor 1 (NRF1), GA repeat binding protein α (GABPA) and myocyte enhancer factor 2A (MEF2A) by de novo motif analysis, whereas decreased H3K27ac was enriched for v-maf musculoaponeurotic fibrosarcoma oncogene family protein K (MAFK), a known negative regulator of beta cells. By siRNA-mediated knockdown of NRF1, GABPA or MEF2A we found that INS-1 cells exhibited downregulation of fatty acid β-oxidation genes in parallel with decrease in the associated H3K27ac. Furthermore, in line with the epigenome in DIO mice, palmitate treatment caused increase in H3K27ac and induction of β-oxidation genes; these responses were blunted when NRF1, GABPA or MEF2A were suppressed. These results suggest novel roles for DNA-binding proteins and fatty acid signalling in obesity-induced epigenomic regulation of beta cell function. The next-generation sequencing data in the present study were deposited at ArrayExpress. RNA-Seq: Dataset name: ERR2538129 (Control), ERR2538130 (Diet-induced obesity) Repository name and number: E-MTAB-6718 - RNA-Seq of pancreatic islets derived from mice fed a long-term high-fat diet against chow-fed controls. ChIP-Seq: Dataset name: ERR2538131 (Control), ERR2538132 (Diet-induced obesity) Repository name and number: E-MTAB-6719 - H3K27ac ChIP-Seq of pancreatic islets derived from mice fed a long-term high-fat diet (HFD) against chow-fed controls.

A novel inhibitor of inducible NOS dimerization protects against cytokine-induced rat beta cell dysfunction.

Beta cell apoptosis is a major feature of type 1 diabetes, and pro-inflammatory cytokines are key drivers of the deterioration of beta cell mass through induction of apoptosis. Mitochondrial stress plays a critical role in mediating apoptosis by releasing cytochrome C into the cytoplasm, directly activating caspase-9 and its downstream signalling cascade. We aimed to identify new compounds that protect beta cells from cytokine-induced activation of the intrinsic (mitochondrial) pathway of apoptosis. Diabetogenic media, composed of IL-1β, IFN-γ and high glucose, were used to induce mitochondrial stress in rat insulin-producing INS1E cells, and a high-content image-based screen of small molecule modulators of Casp9 pathway was performed. A novel small molecule, ATV399, was identified from a high-content image-based screen for compounds that inhibit cleaved caspase-9 activation and subsequent beta cell apoptosis induced by a combination of IL-1β, IFN-γ and high glucose, which together mimic the pathogenic diabetic milieu. Through medicinal chemistry optimization, potency was markedly improved (6-30 fold), with reduced inhibitory effects on CYP3A4. Improved analogues, such as CAT639, improved beta cell viability and insulin secretion in cytokine-treated rat insulin-producing INS1E cells and primary dispersed islet cells. Mechanistically, CAT639 reduced the production of NO by allosterically inhibiting dimerization of inducible NOS (iNOS) without affecting its mRNA levels. Taken together, these studies demonstrate a successful phenotypic screening campaign resulting in identification of an inhibitor of iNOS dimerization that protects beta cell viability and function through modulation of mitochondrial stress induced by cytokines.

Regulation of aging and oxidative stress pathways in aged pancreatic islets using alpha-lipoic acid.

Oxidative stress has been involved in the aging process and the pathogenesis of type-2 diabetes, which is a serious health problem worldwide. This study investigates the anti-aging, anti-apoptotic, and antioxidant properties of alpha-lipoic acid (ALA), aiming to improve aged rat pancreatic cells. In this regard, half maximal effective concentration (EC50) of ALA based on the survival of aged pancreatic islet cells was determined as 100µM. Following this, p38 and p53 genes expression as key factors in aging, oxidative stress biomarkers, insulin secretion, and Pdx1 protein expression were evaluated using real-time PCR, ELISA reader, and fluorescence microscope. It was revealed that ALA reduces and controls the effects of aging on beta cells mainly by suppressing p38 and p53 at the gene level (P < 0.001 and P < 0.01), respectively, reducing reactive oxygen species (P < 0.001) and enhancing levels of thiols (P < 0.05) compared with the aged islets. Furthermore, both qualitative and quantitative investigations of insulin secretion have shown that ALA can improve aged cells' function and increase insulin secretion specially in the stimulating concentration of glucose. Also, the expression of Pdx1 was considerably increased by ALA in comparison to the aged pancreatic islets (P < 0.001). As far as the authors of the present study are concerned, this is the first study, which evaluated aging associated with p38 and p53 pathways, oxidative stress parameters, and the expression of insulin in beta cells of an aged rat and reaffirmed the fact that ALA has a significant antioxidant role in reducing the aging process.

Prolactin protects against cytokine-induced beta-cell death by NFκB and JNK inhibition.

Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.

Catalase and glutathione reductase co‐localize with insulin in pancreatic beta cells of normal and diabetic rats

BackgroundLong‐term diabetes mellitus (DM) is associated with hyperglycemia. Hyperglycemia causes oxidative stress which may subsequently reduce the level of endogenous antioxidants. Hyperglycemia‐induced tissue stress has been implicated in the formation of advanced glycated end products and other factors responsible for the development of short and long‐term complications of DM.AimsOur study aims to determine whether catalase (CAT)‐ and glutathione reductase (GR) are present in pancreatic islet cells and if yes, which of the islet cells contain CAT and or GR?MethodsDM was induced in adult male Wistar rats with streptozotocin (60 mg/kg body wt., intraperitoneal). Four weeks after the induction of DM, the rats were anesthetized and part of the pancreas was removed, incubated overnight in Zamboni's fixative and processed for double‐labelled immunofluorescence using antibodies against insulin, glucagon, somatostatin, pancreatic polypeptide, CAT and GR. A portion of the fresh pancreatic was also processed for immunoelectron microscopy using different gold particle sizes as probes for either pancreatic hormone or antioxidant.ResultsThe number of insulin‐, CAT‐ and GR‐immunoreactive cells was significantly (p &lt; 0.05) reduced after the onset of DM. In contrast, the percentage distribution of glucagon‐, somatostatin‐, and pancreatic polypeptide‐positive cells increased in pancreatic islets of diabetic rats, when compared to non‐diabetic controls. CAT and GR were observed in insulin‐containing pancreatic beta cells. A decrease in the number of insulin‐secreting cells was associated with a concomitant reduction in either CAT or GR. Immunoelectron microscopy showed that CAT‐ and GR‐labelled immunogold particles are present in association with insulin in secretory granules of pancreatic beta cell. The number of insulin‐, CAT‐ and GR‐coated gold particles was markedly (p &lt; 0.05) reduced in pancreatic beta cells of diabetic rats.ConclusionCo‐localization of CAT and GR with insulin suggests that these antioxidants may play role in the maintenance of pancreatic beta cell structure and function.Support or Funding InformationSupported by research grants from the CMHS (Grant # NP‐14‐32) NP and UAEU (Grant # ZCHS‐7‐2014)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.