Evaluation effects of Quercetin on streptozotocin-treated RINm5F pancreatic β-cells in vitro

Background and objectives: Quercetin is a naturally occurring phenolic compound abundantly present in plants as a secondary metabolite. The purpose of this study was to investigate the effect of quercetin on improving RINm5F β-insulinemia cell viability, glucose-stimulated insulin secretion (GSIS), and cell insulin content in the presence or absence of streptozotocin (STZ). Methods: This experimental study was conducted on RINm5F β-insulinemia cell line. The cell viability was evaluated by MTT assay. The necrosis was confirmed by flowcytometry and insulin ELISA kit was used to measure the GSIS level and cell insulin content. It should be noted that for testing of cells by 50μM of quercetin, simultaneous treatment and pre-treatment of quercetin were performed in the presence of STZ (20mM). Results: The quercetin was able to improve the viability of RINm5F cells in the presence of STZ and to increase the GSIS level and cell insulin content under STZ and glucotoxic conditions Conclusion: The quercetin seems to have beneficial effects on β-cells, especially the synthesis and secretion of insulin. In addition to the therapeutic effect, given the low toxicity of this flavonoid and the results of this study, the quercetin as a preventive agent may play an important role in maintaining the health of β-cells in people at risk of diabetes.

RyR2/IRBIT regulates insulin gene transcription, insulin content, and secretion in the insulinoma cell line INS-1

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced the Ca2+ integral (AUC) stimulated by 7.5 mM glucose, and rendered it sensitive to block by the IP3 receptor inhibitor xestospongin C, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT; aka AHCYL1). Deletion of IRBIT from INS-1 cells (IRBITKO) increased the Ca2+ AUC in response to 7.5 mM glucose and induced xestospongin sensitivity. Insulin content and basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO cells and more modestly reduced in IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls. In contrast, exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells than in controls. Proteomics analysis using LC-MS/MS revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

microRNA-206 Regulates Pancreatic-β-Cell Function via Targeting Insulin-Like Growth Factor 1

Background: Diabetes mellitus (DM), a common metabolic disease, is featured with chronic hyperglycemia and dysfunction of pancreatic β-cells. Various evidences suggested that microRNAs (miR-NAs) were tightly related to the occurrence of DM and can be regulated in pancreatic-β-cells. However, whether or not microRNA-206 (miR-206) affects the development of DM and the specific mechanisms remains elusive. We designed this study to clarify the function and associated potential mechanism of miR-206 in pancreatic β-cells via the insulin-like growth factor 1 reporter (IGF1R)/phosphatidylinositide 3-kinase (PI3 K)/protein kinase B (AKT) signaling pathway, which may provide novel diagnostic or therapies for DM. Methods: In this research, the levels of miR-206 and insulin-like growth factor 1 (IGF1) were assessed using quantitative reverse transcription PCR (qRT-PCR) and/or Western bolt assay. Bioinformatics and dual luciferase reporter assays were adopted to illustrate the relationship between miR-206 and IGF1. INS-1 cells were stimulated with stimulatory glucose (16.7 mM) or basal glucose (3.3 mM) for 1 h, and the insulin content was examined by enzyme linked immunosorbent assay (ELISA). Besides, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and flow cytometry (FCM) analysis were conducted to determine the INS1 cell viability and apoptosis. Furthermore, the related proteins in IGF1R/PI3 K/AKT pathway (IGF1R, p-AKT and AKT) were tested using Western blot assay, respectively. Results: Our data suggested that miR-206 level in the blood samples of DM patients was higher than that in healthy donors. IGF1 was identified as a direct target of miR-206. Moreover, we found that miR-206 negatively regulated IGF1 expression in INS-1 cells. It was also confirmed that miR-206 inhibitor led to an increase of total insulin content, an increase of INS-1 cell viability and a decrease of cell apoptosis. Accordingly, miR-206 inhibitor significantly enhanced IGF1R and p-AKT protein expression in INS-1 cells. All these findings were reversed by IGF1-siRNA co-transfection. Conclusion: Our observations clearly demonstrated that miR-206 could regulate the IGF1R/PI3K/AKT pathway by targeting IGF1, thereby regulating the cell viability of pancreatic β cells and the ability of pancreatic β cells to secrete insulin.

Changes in granule mobility and age contribute to changes in insulin secretion after desensitization or rest

IntroductionFunctional impairment of the stimulus secretion coupling in pancreatic beta cells is an essential component of type 2 diabetes. It is known that prolonged stimulation desensitizes the secretion of insulin and thus contributes to beta cell dysfunction. Beta cell rest, in contrast, was shown to enhance the secretory response. Here, the underlying mechanisms were investigated.Research design and methodsTo characterize the consequences of desensitization or rest for the number and mobility of submembrane granules, insulin-secreting MIN6 cells were desensitized by 18-hour culture with 500 µM tolbutamide or rested by 18-hour culture with 1 µM clonidine. The granules were labeled by hIns-EGFP or hIns-DsRed E5, imaged by TIRF microscopy of the cell footprint area and analyzed with an observer-independent program. Additionally, the insulin content and secretion were measured.ResultsConcurrent with the insulin content, submembrane granules were only slightly reduced after desensitization but markedly increased after rest. Both types of pretreatment diminished arrivals and departures of granules in the submembrane space and increased the proportion of immobile long-term resident granules, but desensitization lowered and rest increased the number of exocytoses, in parallel with the effect on insulin secretion. Labeling with hIns-DsRed E5 (‘timer’) showed that desensitization did not affect the proportion of aged granules, whereas rest increased it. Aged granules showed a high mobility and made up only a minority of long-term residents. Long-term resident granules were more numerous after rest and had a lower lateral mobility, suggesting a firmer attachment to the membrane.ConclusionThe number, mobility and age of submembrane granules reflect the preceding functional states of insulin-secreting cells. Representing the pool of releasable granules, their quantity and quality may thus form part of the beta cell memory on renewed stimulation.

SGLT2 inhibitors therapy protects glucotoxicity-induced β-cell failure in a mouse model of human KATP-induced diabetes trough mitigation of oxidative and ER stress

Progressive loss of pancreatic β-cell functional mass and anti-diabetic drug responsivity are classic findings in diabetes, frequently attributed to compensatory insulin hypersecretion and β-cell exhaustion. However, loss of β-cell mass and identity still occurs in mouse models of human KATP-gain-of-function induced Neonatal Diabetes Mellitus (NDM), in the absence of insulin secretion. Here we studied the mechanisms underlying and temporal progression of glucotoxicity-induced loss of functional β-cell mass in NDM mice, and the effects of sodium-glucose transporter 2 inhibitors (SGLT2i) therapy. Upon tamoxifen induction of transgene expression, NDM mice developed severe diabetes followed by an unexpected loss of insulin content, decreased proinsulin processing and proinsulin accumulation at 2-weeks of diabetes. This was accompanied by a marked increase in β-cell oxidative and ER stress, without changes in islet cell identity. Strikingly, early treatment with the SGLT2 inhibitor dapagliflozin restored insulin content, decreased proinsulin:insulin ratio and reduced oxidative and ER stress. However, despite reduction of blood glucose, dapagliflozin therapy was ineffective in restoring β-cell function in NDM mice when tit was initiated at >40 days of diabetes, when loss of β-cell mass and identity had already occurred. These results have important clinical implications as they demonstrate that: i) hyperglycemia per se, and not insulin hypersecretion, drives β-cell failure in diabetes, ii) recovery of β-cell function by SGLT2 inhibitors is through reduction of oxidative and ER stress, iii) SGLT2 inhibitors revert/prevent β-cell failure when used in early stages of diabetes, but not when loss of β-cell mass/identity already occurred, iv) common execution pathways underlie loss and recovery of β-cell function in different forms of diabetes.

Investigation of Antidiabetogenic Effect of the Iodine-Selenium Concentrate in Animals with Chronic Alloxan Diabetes of Varying Severity

BACKGROUND: The diabetogenic effect of alloxan is known is determined by ability to stimulate lipid peroxydation processes in B-cells of the pancreas. AIM: to investigate of the possible antidiabetic action of long time prolonged of Iodine-Selenium concentrate action in rats with alloxan diabetes. METHODS: Reproduction of experimental alloxan diabetes was carried out in rats by a single intravenous injection of alloxan 35-43 mg / kg body weight. The “iodine-selenium” concentrate was administered per os through a tube at the rate of 1.25 ml / 100 g of the concentrate. In experimental animals with mild and heavy diabetes mellitus, the level of glucose in the blood was assessed, products of LPO-AOD; the state of the histostructure of the pancreas and of insulin content in B cells were studied using of aldehyde fuchsin and diethylpseudoisocyanine methods. RESULTS: Long time prolonged administration of the “iodine-selenium” concentrate (60 days) to animals with mild experimental diabetes mellitus is accompanied by a significant decrease in blood glucose levels by 1.89 times compared to the control (p ˂ 0.05) and of LPO within normal values as by increase of the level of glutathione peroxidase (GPO) by 2.23 times compared with the initial (p˂ 0.01), by prevention of the development of histological changes in pancreatic islets and a slight decrease of insulin content in B-cells. Under similar experimental conditions in animals with severe alloxan diabetes, the level of glycemia significantly decreased from 20.23 ± 2.15 mmol / l to 12.39 ± 1.52 mmol / l as of the level of diene conjugates of erythrocytes and plasma, as decrease of ketodienes, MDA of plasma and in erythrocytes and primary lipid peroxidation products, while remaining elevated, despite an increase in GPO by 50.0% compared with control (p˂0.05) in the presence of histological changes in the pancreatic islets as in experimental diabetes. CONCLUSION: The antidiabetic effect of the “iodine-selenium” concentrate in rats with mild alloxan diabetes on the level of glycemia, LPO - AOD and state of the histostructure of the pancreas and the content of deposited insulin in pancreatic B-cells, is probably due to antioxidant effect of selenium to stimulate activity of glutathione blocking lipid peroxide and hydrogen peroxide in alloxan diabetes mellitus.

Transient antibiotic-induced changes in the neonatal swine intestinal microbiota impact islet expression profiles reducing subsequent function

Neonatal antibiotics administered to human infants initiate gut microbiota dysbiosis that may have long-term effects on body weight (BW) and metabolism. We examined antibiotic-induced adaptations in pancreatic islets of the piglet, a well-accepted model of human infant microbiota and pancreas development. Neonatal piglets randomized to amoxicillin (30mg/kg BW/day; n=7, ANTI) or placebo (vehicle control; n=7, CON) from postnatal day (PND)0-13 were euthanized at PND7, 14 and 49. The metabolic phenotype along with functional, immunohistological and transcriptional phenotypes of the pancreatic islets were studied. The gut microbiome was characterized by 16S sequencing and microbial metabolites and microbiome-sensitive host molecules were measured. Compared with CON, ANTI PND7 piglets had elevated transcripts of genes involved in GLP-1 synthesis or signaling in islets (p<0.05) coinciding with higher plasma GLP-1 (p=0.11), along with increased Tnf (p<0.05) and Npg1 (p<0.05). Antibiotic-induced relative increases in Escherichia, Coprococcus, Ruminoccocus, Dehalobacterium and Oscillospira of the ileal microbiome at PND7 normalized after antibiotic withdrawal. In ANTI islets at PND14, the expression of key regulators Pdx1, Igf2 and Tcf7l2 was down-regulated, preceding a 40% reduction of b-cell area (p<0.01) and islet insulin content at PND49 (p<0.05). At PND49, a 2-fold elevated plasma insulin concentration (p=0.07) was observed in ANTI compared with CON. We conclude that antibiotic treatment of neonatal piglets elicits gut microbial changes accompanied by phasic alterations in key regulatory genes in pancreatic islets at PND7 and 14. By PND49, reduced b-cell area and islet insulin content were accompanied by elevated non-fasted insulin despite normoglycemia, indicative of islet stress.

Lipid and endocrine status of participants of long ski crossings in the Аrctic

The paper presents the lipid and endocrine status of athletes — members of the two Arctic long ski crossings. The aim of the study was to study the main indicators of lipid metabolism, cortisol and insulin content in athletes during long skiing in the Arctic.Material and methods. A survey of sportsmen-­participants of two polar long ski crossings.Results. A significant decrease in the concentration of the total fraction of low and very low density lipoproteins in the blood serum was revealed in male athletes — participants of two long Arctic ski crossings. At the same time, a shift in the lipoprotein spectrum of blood towards an increase in the content of high density lipoproteins due to the second type of subfraction was revealed. The dynamics of the concentration of free fatty acids in the blood of participants in ski crossings in the Arctic was of a multidirectional nature, expressed in an increase in the level of free fatty acids compared to the initial level in the training trip and in a decrease in the level of free fatty acids at all stages of the trip to the North Pole of relative inaccessibility. During the training hike an increase in the level of cortisol in the blood was revealed, as well as an increase in the insulin content after the end of the hike. During the transition to the North Pole of relative inaccessibility a significant decrease in blood cortisol concentration compared with the preparatory period was revealed, as well as the absence of significant changes in insulin content at all stages of the transition.