Involvement of the parasympathetic nervous system on glycemic control and pancreatic islets function in malnourished mice submitted to the high-fat diet

Orientador: Everardo Magalhães CarneiroTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A restrição proteica em camundongos, durante a infância, promove a reprogramação do metabolismo, causando prejuízo na tolerância à glicose e na ação da insulina. O sistema nervoso parassimpático (SNP) parece estar envolvido nessas alterações, visto que em modelos de programação metabólica ocorre aumento do tônus parassimpático e redução do simpático. Considerando isso, nosso objetivo foi avaliar o papel da vagotomia sobre o controle glicêmico através da secreção e ação dos principais hormônios pancreáticos: insulina e glucagon, em camundongos desnutridos precocemente e posteriormente submetidos à dieta hiperlipídica (HF). Após o desmame, camundongos C57Bl/6 foram submetidos à restrição proteica, sendo alimentados com dieta hipoproteica (6% de proteína). Após 4 semanas, estes camundongos foram divididos em três grupos: grupo LP, o qual continuou a receber a dieta hipoproteica; grupo LP+HF, o qual passou a receber dieta HF (35% de lipídios); e grupo LP+HFvag, o qual foi submetido ao procedimento de vagotomia no mesmo momento em que foi oferecido a dieta HF. Ambas as dietas foram ofertadas durante 8 semanas. Após este período, foram avaliados os seguintes parâmetros: secreção de insulina e glucagon estimulada por glicose; tolerância à glicose; sensibilidade à insulina e ao glucagon; concentração plasmática de insulina, peptídeo-c e glucagon; expressão proteica da enzima que degrada a insulina (IDE); e conteúdo de glicogênio hepático. A vagotomia melhorou a tolerância à glicose e reduziu a secreção de insulina, sem alterar a adiposidade e a sensibilidade à insulina nos animais submetidos à reprogramação metabólica. Estes efeitos foram acompanhados pelo aumento da insulinemia, provavelmente devido a redução do clearence de insulina observado no grupo LP+HFvag. Além disso, os animais LP+HF e LP+HFvag foram mais sensíveis ao glucagon e ao piruvato, contribuindo para a redução dos estoques de glicogênio hepático. Os animais LP+HF apresentaram aumento da secreção de glucagon em ilhotas isoladas em resposta à glicose, tanto em condições estimulatória (0,5mM) quanto inibitória (11,1mM). A vagotomia melhorou a supressão de glucagon, em resposta a glicose, em ilhotas isoladas e in vivo, contribuindo para a tolerância à glicose. Em conclusão, animais reprogramados metabolicamente e submetidos à vagotomia apresentam melhora da tolerância à glicose com aumento da insulinemia provavelmente devido a redução do clearance deste hormônio e também pela redução da glucagonemiaAbstract: Protein restriction in mice during early life, promotes metabolic programming in adulthood, impairing their glucose tolerance and insulin action. Parasympathetic nervous system disruption seems to be involved in this process, since increased in parasympathetic tonus and decreased in sympathetic activity was observed in metabolic programmed mice. Thus, the aim of this study was to investigate the effect of subdiaphragmatic vagotomy on glucose homeostasis through insulin and glucagon secretion and action, in metabolic programmed mice, induced by a low-protein diet early in life, followed by high fat diet (HF) in adulthood. After weaning, C57BL/6 mice were received low protein diet with 6 % of protein, composing LP group. After 4 weeks, LP group was divided in LP+HF, which started to receive high-fat diet (HF) (35% of lipids), for 8 weeks, and LP+HFvag, was submitted to the vagotomy procedure at the same time as the diet was offered. Glucose-stimulated insulin and glucagon secretion; glucose tolerance; insulin and glucagon sensitivity; plasma insulin, c-peptide and glucagonconcentration; liver insulin-degrading enzyme (IDE) protein expression and hepatic glycogen content, were evaluated. Vagotomy improved glucose tolerance and reduced insulin secretion, but did not alter adiposity and insulin sensitivity in metabolic programmed mice, compared to metabolic programmed mice. These effects were accompanied by increased in insulinemia, probably due to a diminished insulin clearance, observed in LP+HFvag group. Furthermore, LP+HF and LP+HFvag mice were more sensitive to glucagon and pyruvate, contributing to lower hepatic glycogen content. The LP+HF mice presented increased glucose-stimulated glucagon secretion in isolated islets, both in stimulatory (0.5 mM) or inhibitory (11.1 mM) conditions. Vagotomy was able to improve glucagon suppression by glucose in isolated islets and in vivo, improving glucose tolerance. In conclusion, the metabolic programmed mice, when submitted to vagotomy, showed improved glucose tolerance, associated with an increase of plasma insulin concentration as result of insulin clearance reduction and reduction on glucagonemiaDoutoradoFisiologiaDoutora em Biologia Funcional e Molecular2013/25847-6FAPES

Vagotomy diminishes obesity in cafeteria rats by decreasing cholinergic potentiation of insulin release

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORHerein, we investigated whether subdiaphragmatic vagotomy has benefits on obesity, body glucose homeostasis, and insulin secretion in cafeteria (CAF)-obese rats. Wistar rats were fed a standard or CAF diet for 12 weeks. Subsequently, CAF rats were randomly submitted to truncal vagotomy (CAF Vag) or sham operation (CAF Sham). CAF Sham rats were hyperphagic, obese, and presented metabolic disturbances, including hyperinsulinemia, glucose intolerance, insulin resistance, hyperglycemia, and hypertriglyceridemia. Twelve weeks after vagotomy, CAF Vag rats presented reductions in body weight and perigonadal fat stores. Vagotomy did not modify glucose tolerance but normalized fed glycemia, insulinemia, and insulin sensitivity. Isolated islets from CAF Sham rats secreted more insulin in response to the cholinergic agent, carbachol, and when intracellular cyclic adenine monophosphate (cAMP) is enhanced by forskolin or 3-isobutyl-1-methylxanthine. Vagotomy decreased glucose-induced insulin release due to a reduction in the cholinergic action on beta-cells. This effect also normalized islet secretion in response to cAMP. Therefore, vagotomy in rats fed on a CAF-style diet effectively decreases adiposity and restores insulin sensitivity. These effects were mainly associated with the lack of cholinergic action on the endocrine pancreas, which decreases insulinemia and may gradually reduce fat storage and improve insulin sensitivity.Herein, we investigated whether subdiaphragmatic vagotomy has benefits on obesity, body glucose homeostasis, and insulin secretion in cafeteria (CAF)-obese rats. Wistar rats were fed a standard or CAF diet for 12 weeks. Subsequently, CAF rats were randomly submitted to truncal vagotomy (CAF Vag) or sham operation (CAF Sham). CAF Sham rats were hyperphagic, obese, and presented metabolic disturbances, including hyperinsulinemia, glucose intolerance, insulin resistance, hyperglycemia, and hypertriglyceridemia. Twelve weeks after vagotomy, CAF Vag rats presented reductions in body weight and perigonadal fat stores. Vagotomy did not modify glucose tolerance but normalized fed glycemia, insulinemia, and insulin sensitivity. Isolated islets from CAF Sham rats secreted more insulin in response to the cholinergic agent, carbachol, and when intracellular cyclic adenine monophosphate (cAMP) is enhanced by forskolin or 3-isobutyl-1-methylxanthine. Vagotomy decreased glucose-induced insulin release due to a reduction in the cholinergic action on beta-cells. This effect also normalized islet secretion in response to cAMP. Therefore, vagotomy in rats fed on a CAF-style diet effectively decreases adiposity and restores insulin sensitivity. These effects were mainly associated with the lack of cholinergic action on the endocrine pancreas, which decreases insulinemia and may gradually reduce fat storage and improve insulin sensitivity724625633CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORsem informaçã

Early onset of obesity induces reproductive deficits in female rats

The incidence of obesity is increasing rapidly all over the world and results in numerous health detriments, including disruptions in reproduction. However, the mechanisms by which excess body fat interferes with reproductive functions are still not fully understood. After weaning, female rats were treated with a cafeteria diet or a chow diet (control group). Biometric and metabolic parameters were evaluated in adulthood. Reproductive parameters, including estradiol, progesterone, LH and prolactin during the proestrus afternoon, sexual behavior, ovulation rates and histological analysis of ovaries were also evaluated. Cafeteria diet was able to induce obesity in female rats by increasing body and fat pad weight, which resulted in increased levels of triglycerides, total cholesterol, LDL and induced insulin resistance. The cafeteria diet also negatively affected female reproduction by reducing the number of oocytes and preantral follicles, as well as the thickness of the follicular layer. Obese females did not show preovulatory progesterone and LH surges, though plasma estradiol and prolactin showed preovulatory surges similar to control rats. Nevertheless, sexual receptiveness was not altered by cafeteria diet. Taken together, our results suggest that the cafeteria diet administered from weaning age was able to induce obesity and reduce the reproductive capability in adult female rats, indicating that this obesity model can be used to better understand the mechanisms underlying reproductive dysfunction in obese subjects. (C) 2012 Elsevier Inc. All rights reserved.Fundacao Araucaria de Apoio ao Desenvolvimento Cientifico e Tecnologico do Estado do ParanaFundacao Araucaria de Apoio ao Desenvolvimento Cientifico e Tecnologico do Estado do ParanaConselho Nacional para o Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional para o Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Secretaria de Ciencia, Tecnologia e Ensino Superior do Estado do ParanaSecretaria de Ciencia, Tecnologia e Ensino Superior do Estado do Paran

1812-P: Glucagon Resistance and Decreased Susceptibility to Diabetes in a Model of Chronic Hyperglucagonemia

Elevation of glucagon levels and increase in α-cell mass are associated to states of hyperglycemia in diabetes. However, little is known about the mechanisms that control glucagon secretion and α-cell mass expansion in normal or diabetogenic conditions. The current studies investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in α-cells (αTSC2KO), we showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of α-cell proliferation, cell size and mass expansion. Hyperglucagonemia in αTSC2KO was associated to increase in glucagon content, enhanced glucagon secretion and defective adaptation to fasting. This model allowed us to identify the beneficial effects of chronic hyperglucagonemia in glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in αTSC2KO mice was characterized by reduced expression of the glucagon receptor (GCGR), phosphoenolpyruvate carboxykinase (PEPCK) and genes involved in amino acid metabolism and urea production. Surprisingly, hyperglucagonemia in αTSC2KO mice was associated to improved glucose levels in models of Streptozotocin (STZ)-induced β-cell destruction and high fat diet-induced glucose intolerance. Contrary to our current understanding of glucagon action, these studies demonstrate that endogenous chronic hyperglucagonemia improve glucose homeostasis by augmenting insulin secretion and by inducing glucagon resistance in the liver. These novel in vivo findings support the concept that enhancing glucagon action could be used as an alternative strategy to treat hyperglycemia in diabetes. Disclosure C. Lubaczeuski: None. N. Bozadjieva: None. M. Blandino-Rosano: None. E. Bernal-Mizrachi: None. Funding U.S. Department of Veterans Affairs (IBX002728A); Diabetes Research Connectio

Pancreatic and duodenal homeobox-1 (PDX1) contributes to β-cell mass expansion and proliferation induced by Akt/PKB pathway

Maintenance of pancreatic β-cell mass and function is fundamental to glucose homeostasis and to prevent diabetes. The PI3 K-Akt-mTORC1 pathway is critical for β-cells mass and function, while PDX1 has been implicated in β-cell development, maturation, and function. Here we tested whether Akt signaling requires PDX1 expression to regulate β-cell mass, proliferation, and glucose homeostasis. In order to address that, we crossed a mouse model overexpressing constitutively active Akt mutant in β-cells (β-caAkt) with mice lacking one allele of PDX1gene (β-caAkt/pdx1 +/- ). While the β-caAkt mice exhibit higher plasma insulin levels, greater β-cell mass and improved glucose tolerance compared to control mice, the β-caAkt/pdx1 +/- mice are hyperglycemic and intolerant to glucose. The changes in glucose homeostasis in β-caAkt/pdx1 +/- were associated with a 60% reduction in β-cell mass compared to β-caAkt mice. The impaired β-cell mass in the β-caAkt/pdx1 +/- mice can be explained by a lesser β-cell proliferation measured by the number of Ki67 positive β-cells. We did not observe any differences in apoptosis between β-caAkt/pdx1 +/- and β-caAkt mice. In conclusion, PDX1 contributes to β-cell mass expansion and glucose metabolism induced by activation of Akt signaling

Glucagon Resistance and Decreased Susceptibility to Diabetes in a Model of Chronic Hyperglucagonemia

Elevation of glucagon levels and increase in α-cell mass are associated with states of hyperglycemia in diabetes. Our previous studies have highlighted the role of nutrient signaling via mTOR complex 1 (mTORC1) regulation that controls glucagon secretion and α-cell mass. In the current studies we investigated the effects of activation of nutrient signaling by conditional deletion of the mTORC1 inhibitor, TSC2, in α-cells (αTSC2 ). We showed that activation of mTORC1 signaling is sufficient to induce chronic hyperglucagonemia as a result of α-cell proliferation, cell size, and mass expansion. Hyperglucagonemia in αTSC2 was associated with an increase in glucagon content and enhanced glucagon secretion. This model allowed us to identify the effects of chronic hyperglucagonemia on glucose homeostasis by inducing insulin secretion and resistance to glucagon in the liver. Liver glucagon resistance in αTSC2 mice was characterized by reduced expression of the glucagon receptor (GCGR), PEPCK, and genes involved in amino acid metabolism and urea production. Glucagon resistance in αTSC2 mice was associated with improved glucose levels in streptozotocin-induced β-cell destruction and high-fat diet-induced glucose intolerance. These studies demonstrate that chronic hyperglucagonemia can improve glucose homeostasis by inducing glucagon resistance in the liver

The regulatory G protein signaling complex, Gβ5–R7, promotes glucose- and extracellular signal–stimulated insulin secretion

G protein–coupled receptors (GPCRs) are important modulators of glucose-stimulated insulin secretion, essential for maintaining energy homeostasis. Here we investigated the role of Gβ5–R7, a protein complex consisting of the atypical G protein β subunit Gβ5 and a regulator of G protein signaling of the R7 family. Using the mouse insulinoma MIN6 cell line and pancreatic islets, we investigated the effects of G protein subunit β 5 (Gnb5) knockout on insulin secretion. Consistent with previous work, Gnb5 knockout diminished insulin secretion evoked by the muscarinic cholinergic agonist Oxo-M. We found that the Gnb5 knockout also attenuated the activity of other GPCR agonists, including ADP, arginine vasopressin, glucagon-like peptide 1, and forskolin, and, surprisingly, the response to high glucose. Experiments with MIN6 cells cultured at different densities provided evidence that Gnb5 knockout eliminated the stimulatory effect of cell adhesion on Oxo-M–stimulated glucose–stimulated insulin secretion; this effect likely involved the adhesion GPCR GPR56. Gnb5 knockout did not influence cortical actin depolymerization but affected protein kinase C activity and the 14-3-3ϵ substrate. Importantly, Gnb5−/− islets or MIN6 cells had normal total insulin content and released normal insulin amounts in response to K+-evoked membrane depolarization. These results indicate that Gβ5–R7 plays a role in the insulin secretory pathway downstream of signaling via all GPCRs and glucose. We propose that the Gβ5–R7 complex regulates a phosphorylation event participating in the vesicular trafficking pathway downstream of G protein signaling and actin depolymerization but upstream of insulin granule release