Inhibition of Ca2+ signaling and glucagon secretion in mouse pancreatic alpha-cells by extracellular ATP and purinergic receptors

Glucagon secreted from pancreatic alpha-cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since alpha-cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and beta-cell secretory granules. Using immunocytochemistry, we identified in alpha-cells the P2 receptor subtype P2Y(1), as well as the P1 receptors A(1) and A(2A). In contrast, only P2Y(1) and A(1) receptors were localized in beta-cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca2+ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca2+ signals that lead to secretion. Application of ATP (1-10 mu M) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATP gamma S, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 mu M) was also found to reduce Ca2+ signals. ATP-mediated inhibition of Ca2+ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y(1) and A(2A) were likely involved in the inhibitory effect on Ca2+ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.2945E952E96

Characterization of the peroxidase system at low H2O2 concentrations in isolated neonatal rat islets

B cell destruction during the onset of diabetes mellitus is associated with oxidative stress. In this work, we attempted to further trace the fate of H2O2 inside the pancreatic islets and determine whether it is mediated by enzymatic (peroxidase) activity or by chemical reaction with thiols from any protein chain. Our results suggest that the islet cells have a very similar peroxidase activity at the hydrophilic (cytoplasm) and hydrophobic compartments (organelles and nucleus), independent of the catalase content of the samples. This activity is composed of sacrificial thiols and by proteins with Fe3+/Mn3+ ions at non-heme catalytic sites. The capacity of the hydrophobic fraction to scavenge O-2(-) was increased in the presence of high concentrations of NADP and RS and was highly dependent on RSH. On the contrary, the hydrophilic fraction exhibited a low RSH-dependent activity where the O-2(-) scavenging is related to metal Cu2+/Fe3+/Mn3+ ions attached to the protein molecules. (C) 2004 Elsevier B.V. All rights reserved.1690215916

Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for I h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. Particularly, the decrease in S6K- I expression might be an important factor affecting either glucose- or leucine-induced insulin secretion. (c) 2008 Published by Elsevier Inc.824192154254

Impaired Insulin Secretion And Decreased Expression Of The Nutritionally Responsive Ribosomal Kinase Protein S6k-1 In Pancreatic Islets From Malnourished Rats

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. 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Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2

Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. lmmunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially re- stored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.1811253

Decreased Cholinergic Stimulation Of Insulin Secretion By Islets From Rats Fed A Low Protein Diet Is Associated With Reduced Protein Kinase Cα Expression

Undernutrition has been shown to affect the autonomic nervous system, leading to permanent alterations in insulin secretion. To understand these interactions better, we investigated the effects of carbamylcholine (CCh) and phorbol 12-myristate 13-acetate (PMA) on insulin secretion in 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 PMA (400 nmol/L) and CCh. Increasing concentrations of CCh (0.1-1000 μmol/L) dose dependently increased insulin secretion by islets from both groups of rats. However, insulin secretion by islets from rats fed the NP diet was significantly higher than that of rats fed the LP diet, and the dose-response curve to CCh was shifted to the right in islets from rats fed LP with a 50% effective concentration (EC50) of 2.15 ± 0.7 and 4.64 ± 0.1 μmol CCh/L in islets of rats fed NP and LP diets, respectively (P < 0.05). PMA-induced insulin secretion was higher in islets of rats fed NP compared with those fed LP. Western blotting revealed that the protein kinase (PK)Cα and phospholipase (PL)Cβ1 contents of islets of rats fed LP were 30% lower than those of islets of rats fed NP (P < 0.05). In addition, PKCα mRNA expression was reduced by 50% in islets from rats fed LP. In conclusion, a reduced expression of PKCα and PLCβ1, may be involved in the decreased insulin secretion by islets from LP rats after stimulation with CCh and PMA.1333695699Becker, D.J., The endocrine responses to protein calorie malnutrition (1983) Annu. Rev. 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Diet., 30, pp. 107-147Okitolonda, W., Brichard, S.M., Henquin, J.C., Repercussions of chronic protein-calorie malnutrition on glucose homeostasis in the rat (1987) Diabetologia, 30, pp. 946-951Picarel-Blanchot, F., Alvarez, C., Bailbe, D., Pascual-Leone, A.M., Portha, B., Changes in insulin action and insulin secretion in the rat after dietary restriction early in life: Influence of food restriction versus low-protein food restriction (1995) Metabolism, 44, pp. 1519-1526Escriva, F., Kergoat, M., Bailbe, D., Pascual-Leone, A.M., Portha, B., Increased insulin action in the rat after protein malnutrition early in life (1991) Diabetologia, 34, pp. 559-564Levine, L.S., Wright, P.G., Marcus, F., Failure to secrete immunoreactive insulin by rats fed a low protein diet (1983) Acta Endocrinol., 102, pp. 240-245Swenne, I., Crace, C.J., Milner, R.D., Persistent impairment of insulin secretory response to glucose in adult rats after limited period of protein-calorie malnutrition early in life (1987) Diabetes, 36, pp. 454-458Swenne, I., Crace, C.J., Jansson, L., Intermittent protein-calorie malnutrition in the young rat causes long-term impairment of the insulin secretory response to glucose in vitro (1988) J. 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Decreased cholinergic stimulation of insulin secretion by islets from rats fed a low protein diet is associated with reduced protein kinase C alpha expression

Undernutrition has been shown to affect the autonomic nervous system, leading to permanent alterations in insulin secretion. To understand these interactions better, we investigated the effects of carbamyl-choline (CCh) and phorbol 12-myristate 13-acetate (PMA) on insulin secretion in 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 PMA (400 nmoI/Q and CCh. Increasing concentrations of CCh (0.1 -1000 mumol/L) dose dependently increased insulin secretion by islets from both groups of rats. However, insulin secretion by islets from rats fed the NP diet was significantly higher than that of rats fed the LIP diet, and the dose-response curve to CCh was shifted to the right in islets from rats fed LIP with a 50% effective concentration (EC50) of 2.15 +/- 0.7 and 4.64 +/- 0.1 mumol CCh/L in islets of rats fed NP and LP diets, respectively (P < 0.05). PMA-induced insulin secretion was higher in islets of rats fed NP compared with those fed LIP. Western blotting revealed that the protein kinase (PK)Calpha and phospholipase (PL)Cbeta(1) contents of islets of rats fed LP were 30% lower than those of islets of rats fed NP (P < 0.05). In addition, PKCalpha mRNA expression was reduced by 50% in islets from rats fed LIP. In conclusion, a reduced expression of PKCa and PLCbeta(1), may be involved in the decreased insulin secretion by islets from LIP rats after stimulation with CCh and PMA. J. Nutr. 133: 695-699, 2003.133369569

Exercise at anaerobic threshold intensity and insulin secretion by isolated pancreatic islets of rats

To evaluate the effect of acute exercise and exercise training at the anaerobic threshold (AT) intensity on aerobic conditioning and insulin secretion by pancreatic islets, adult male Wistar rats were submitted to the lactate minimum test (LMT) for AT determination. Half of the animals were submitted to swimming exercise training (trained), 1 h/day, 5 days/week during 8 weeks, with an overload equivalent to the AT. The other half was kept sedentary. At the end of the experimental period, the rats were submitted to an oral glucose tolerance test and to another LMT. Then, the animals were sacrificed at rest or immediately after 20 minutes of swimming exercise at the AT intensity for pancreatic islets isolation. At the end of the experiment mean workload (% bw) at AT was higher and blood lactate concentration (mmol/L) was lower in the trained than in the control group. Rats trained at the AT intensity showed no alteration in the areas under blood glucose and insulin during OGTT test. Islet insulin content of trained rats was higher than in the sedentary rats while islet glucose uptake did not differ among the groups. The static insulin secretion in response to the high glucose concentration (16.7 mM) of the sedentary group at rest was lower than the sedentary group submitted to the acute exercise and the inverse was observed in relation to the trained groups. Physical training at the AT intensity improved the aerobic condition and altered insulin secretory pattern by pancreatic islets