Modulation of the peroxiredoxin system by cytokines in insulin-producing RINm5F cells: Down-regulation of PRDX6 increases susceptibility of beta cells to oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Peroxiredoxins are a family of six antioxidant enzymes (PRDX1-6), and may be an alternative system for the pancreatic beta cells to cope with oxidative stress. This study investigated whether the main diabetogenic pro-inflammatory cytokines or the anti-inflammatory cytokine IL-4 modulate PRDXs levels and putative intracellular pathways important for this process in the insulin-producing RINm5F cells. RINm5F cells expressed significant amounts of PRDX1, PRDX3 and PRDX6 enzymes. Only PRDX6 was modulated by cytokines, showing both mRNA and protein down-regulation following incubation of RINm5F cells with TNF-alpha and IFN-gamma but not with IL-1 beta. Separately IFN-gamma or TNF-alpha decreased PRDX6 protein but not mRNA levels. The blockage of the JNK signalling and of the calpains and proteasome proteolysis systems restored PRDX6 protein levels. IL-4 alone did not modulate PRDXs levels. However, pre/co-incubation with IL-4 substantially prevented the decrease in PRDX6 induced by pro-inflammatory cytokines. Knockdown of PRDX6 increased susceptibility of RINm5F cells to the deleterious effects of pro-inflammatory cytokines and to oxidative stress. These results show that, from the PRDXs significantly expressed in RINm5F cells, only PRDX6 is modulated by the diabetogenic cytokines IFNgamma and TNF-alpha. This PRDX6 down-regulation depends on the calpain and proteasome systems and JNK signalling. PRDX6 is an important enzyme for protection against oxidative stress and the interaction between pro- and anti-inflammatory cytokines might be important to determine the antioxidant capacity of the cells. (c) 2013 Elsevier Ireland Ltd. All rights reserved.374416715664Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Requirement of NF-kappaB signalling pathway for modulation of the cholinergic muscarinic M-3 receptor expression by INGAP-PP in insulin-producing cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The pentadecapeptide comprising the 104-118 amino acid sequence of the ilotropin-derived Reg3-related islet neogenesis-associated protein (INGAP-PP) has been implicated in beta cell neogenesis and enhancement of insulin secretion in pancreatic islets. The aim of this study was to investigate intracellular pathways by which INGAP-PP signals in insulin-producing cells. Treatment with INGAP-PP increased insulin secretion and intracellular calcium levels in MIN6 cells. INGAP-PP exposure activated c-Myc, serum and particularly nuclear factor-kappaB (NF-kappa B) response elements in insulin-producing cells (1.7 +/- 0.1, 1.8 +/- 0.1, 2.4 +/- 0.3 for RINm5F, and 1.3 +/- 0.1, 1.3 +/- 0.1 and 1.6 +/- 0.1 fold for MIN6 cells compared to controls, respectively). There was an increase in the proliferation rate of viable cells (162 +/- 17% for RINm5F and 155 13% for MIN6) that was accompanied by an increase in proliferating cell nuclear antigen (PCNA) protein expression (187 +/- 19% and 170 +/- 8% for RINm5F and MIN6 cells respectively) following INGAP-PP treatment. INGAP-PP increased the expression of the muscarinic M-3 receptor subtype (169 +/- 4% for RINm5F and 222 +/- 20% for MIN6 cells). Activation of multiple serum response elements by foetal calf serum also increased muscarinic M-3 receptor expression (173 +/- 9% for RINm5F and 140 +/- 7% for MIN6 cells). The blockade of NF-kappa B signalling pathway strongly decreased muscarinic M-3 receptor expression in response to both stimuli. In summary, a network of intracellular signals that includes activation of c-Myc signalling pathway and increased PCNA expression might be related to the increased proliferation rate of insulin-producing cells following incubation with INGAP-PP. NF-kappa B signalling plays an essential role in controlling the expression of the muscarinic M-3 receptor. (C) 2010 Elsevier B.V. All rights reserved.642416993746Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Instituto Nacional de Obesidade e Diabetes (INOD, Brazil)UK Society for EndocrinologyNC3RsFONCYTCONICET (Argentina)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Insulin and glucose sensitivity, insulin secretion and beta-cell distribution in endocrine pancreas of the fruit bat Artibeus lituratus

The fruit bat Artibeus lituratus absorbs large amounts of glucose in short periods of time and maintains normoglycemia even after a prolonged starvation period. Based on these data, we aimed to investigate various aspects related with glucose homeostasis analyzing: blood glucose and insulin levels, intraperitoneal glucose and insulin tolerance tests (ipGTT and ipITT), glucose-stimulated insulin secretion (2.8, 5.6 or 8.3 mmol/L glucose) in pancreas fragments, cellular distribution of beta cells, and the amount of pAkt/Akt in the pectoral muscle and liver. Blood glucose levels were higher in fed bats (6.88 +/- 0.5 mmol/L) than fasted bats (4.0 +/- 0.8 mmol/L), whereas insulin levels were similar in both conditions. The values of the area-under-the curve obtained from ipGTT were significantly higher when bats received 2 (5.5-fold) or 3 g/kg glucose (7.5-fold) b.w compared to control (saline). These bats also exhibited a significant decrease of blood glucose values after insulin administration during the iplTT. Insulin secretion from fragments of pancreas under physiological concentrations of glucose (5.6 or 8.3 mmol/L) was similar but higher than in 2.8 mmol/L glucose 1.8- and 2.0-fold, respectively. These bats showed a marked beta-cell distribution along the pancreas, and the pancreatic beta cells are not exclusively located at the central part of the islet. The insulin-induced Akt phosphorylation was more pronounced in the pectoral muscle, compared to liver. The high sensitivity to glucose and insulin, the proper insulin response to glucose, and the presence of an apparent large beta-cell population could represent benefits for the management of high influx of glucose from a carbohydrate-rich meal, which permits appropriate glucose utilization. (C) 2010 Elsevier Inc. All rights reserved.157214214

The adaptive compensations in endocrine pancreas from glucocorticoid-treated rats are reversible after the interruption of treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Aim: Glucocorticoid administration induces insulin resistance (IR) and enhances islet mass and insulin secretion in rodents and humans. Here, we analysed whether these effects are still present after the interruption of dexamethasone treatment. Methods: Adult Wistar rats were distributed into CTL (daily injection of saline for five consecutive days), DEX (daily injection of 1 mg kg-1 body wt of dexamethasone for five consecutive days) and DEX10 (5 days of dexamethasone treatment, followed by a period of 10 days without dexamethasone). Results: In vivo experiments indicated that the marked hyperinsulinemia found in DEX rats during fasting and fed states was normalized in the DEX10 group. Furthermore, the IR and glucose intolerance observed in DEX were restored in DEX10 rats. Islets from DEX rats secreted more insulin in response to increasing concentrations of glucose and other metabolic and non-metabolic stimuli, compared with that in the CTL group. The insulin secretion for the most compounds studied returned to CTL values in DEX10 islets. Increased insulin secretion correlated well with the augmentation in beta-cell proliferation and mass in DEX rats, and these morphological alterations were normalized in islets from DEX10 rats. In parallel, the increased levels of proteins involved in beta-cell proliferation such as Cd2 and Cdk4 observed in DEX islets were also normalized in DEX10 islets. Conclusion: These data strongly support the view that almost all the morphophysiological alterations induced by dexamethasone in the endocrine pancreas are reverted after discontinuation of the treatment. This information is important, considering the frequent use of glucocorticoids in humans.2003223235Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INOD (Instituto Nacional de Obesidade e Diabetes)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

DPP-4 is expressed in human pancreatic beta cells and its direct inhibition improves beta cell function and survival in type 2 diabetes

It has been reported that the incretin system, including regulated GLP-1 secretion and locally expressed DPP-4, is present in pancreatic islets. In this study we comprehensively evaluated the expression and role of DPP-4 in islet alpha and beta cells from non-diabetic (ND) and type 2 diabetic (T2D) individuals, including the effects of its inhibition on beta cell function and survival. Isolated islets were prepared from 25 ND and 18 T2D organ donors; studies were also performed with the human insulin-producing EndoC-βH1 cells. Morphological (including confocal microscopy), ultrastructural (electron microscopy, EM), functional (glucose-stimulated insulin secretion), survival (EM and nuclear dyes) and molecular (RNAseq, qPCR and western blot) studies were performed under several different experimental conditions. DPP-4 co-localized with glucagon and was also expressed in human islet insulin-containing cells. Furthermore, DPP-4 was expressed in EndoC-βH1 cells. The proportions of DPP-4 positive alpha and beta cells and DPP-4 gene expression were significantly lower in T2D islets. A DPP-4 inhibitor protected ND human beta cells and EndoC-βH1 cells against cytokine-induced toxicity, which was at least in part independent from GLP1 and associated with reduced NFKB1 expression. Finally, DPP-4 inhibition augmented glucose-stimulated insulin secretion, reduced apoptosis and improved ultrastructure in T2D beta cells. These results demonstrate the presence of DPP-4 in human islet alpha and beta cells, with reduced expression in T2D islets, and show that DPP-4 inhibition has beneficial effects on human ND and T2D beta cells. This suggests that DPP-4, besides playing a role in incretin effects, directly affects beta cell function and survival