Co-expression and regulation of connexins 36 and 43 in cultured neonatal rat pancreatic islets

Fetal and neonatal pancreatic islets present a lower insulin secretory response as compared with adult islets. Prolonged culturing leads to an improvement of the glucose-induced insulin secretion response in neonatal pancreatic islets that may involve regulation of gap junction mediated cell communication. In this study, we investigated the effect of culturing neonatal islet cells for varying periods of time and with different glucose medium concentrations on the cellular expression of the endocrine pancreatic gap junction associated connexin (Cx) 36 and Cx43. We report here that the 7-d culture induced upregulation of the expression of these junctional proteins in neonatal islets in a time-dependent manner. A correlation was observed between the increased mRNA and protein expression of Cx36 and Cx43 and the increased insulin secretion following islet culturing. In addition, increasing glucose concentration within the culture medium induced a concentration-dependent enhancement of Cx36 islet expression, but not of Cx43 expression in cultured neonatal islets. In conclusion, we suggest that the regulation of gap junctional proteins by culture medium containing factors and glucose may be an important event for the maturation process of P cells observed at in vitro conditions.83214215

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

Decreased insulin secretion in islets from rats fed a low protein diet is associated with a reduced PKA alpha expression

A low protein diet has been shown to affect the amount and activity of several enzymes and to decrease insulin secretion by islets isolated from rats fed such a diet. To understand the mechanisms involved in this phenomenon, we investigated the effects of forskolin, a stimulator of adenylyl cyclase, on insulin secretion by pancreatic islets from rats fed a normal (17%; NP) or low (6%; LP) protein diet for 8 wk. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 8.3 mmol glucose/L, with or without 10 mumol forskolin/L. The forskolin-induced insulin secretion was higher in islets from NP rats than in those from LP rats (P < 0.05). Western blotting revealed that the amount of the alpha catalytic subunit of protein kinase A (PKAalpha) was 35% lower in islets from LP rats than in islets from NP rats (P < 0.05). Moreover, PKAalpha mRNA expression was reduced by 30% in islets from LP rats (P < 0.05). Our results indicated a possible relationship between a low protein diet and a reduction in PKAalpha expression. These alterations in PKAalpha may be responsible in part for the decreased insulin secretion by islets from rats fed a low protein diet.1341636

Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets

Cell-cell coupling mediated by gap junctions formed from connexin (CX) contributes to the control of insulin secretion in the endocrine pancreas. We investigated the cellular production and localisation of CX36 and CX43, and gap junction-mediated beta cell coupling in pancreatic islets from rats of different ages, displaying different degrees of maturation of insulin secretion. The presence and distribution of islet connexins were assessed by immunoblotting and immunofluorescence. The expression of connexin genes was evaluated by RT-PCR and quantitative real-time PCR. The ultrastructure of gap junctions and the function of connexin channels were assessed by freeze-fracture electron microscopy and tracer microinjection, respectively. Young and adult beta cells, which respond to glucose, expressed significantly higher levels of Cx36 (also known as Gjd2) than fetal and newborn beta cells, which respond poorly to the sugar. Accordingly, adult beta cells also showed a significantly higher membrane density of gap junctions and greater intercellular exchange of ethidium bromide than newborn beta cells. Cx43 (also known as Gja1) was not expressed by beta cells, but was located in various cell types at the periphery of fetal and newborn islets. These findings show that the pattern of connexins, gap junction membrane density and coupling changes in islets during the functional maturation of beta cells.5371428143