RESUMO
The nerve growth factor (NGF) participates in cell survival and glucose-stimulated insulin secretion (GSIS) processes in rat adult beta cells. GSIS is a complex process in which metabolic events and ionic channel activity are finely coupled. GLUT2 and glucokinase (GK) play central roles in GSIS by regulating the rate of the glycolytic pathway. The biphasic release of insulin upon glucose stimulation characterizes mature adult beta cells. On the other hand, beta cells obtained from neonatal, suckling, and weaning rats are considered immature because they secrete low levels of insulin and do not increase insulin secretion in response to high glucose. The weaning of rats (at postnatal day 20 in laboratory conditions) involves a dietary transition from maternal milk to standard chow. It is characterized by increased basal plasma glucose levels and insulin levels, which we consider physiological insulin resistance. On the other hand, we have observed that incubating rat beta cells with NGF increases GSIS by increasing calcium currents in neonatal cells. In this work, we studied the effects of NGF on the regulation of cellular distribution and activity of GLUT2 and GK to explore its potential role in the maturation of GSIS in beta cells from P20 rats. Pancreatic islet cells from both adult and P20 rats were isolated and incubated with 5.6 mM or 15.6 mM glucose with and without NGF for 4 hours. Specific immunofluorescence assays were conducted following the incubation period to detect insulin and GLUT2. Additionally, we measured glucose uptake, glucokinase activity, and insulin secretion assays at 5.6 mM or 15.6 mM glucose concentrations. We observed an age-dependent variation in the distribution of GLUT2 in pancreatic beta cells and found that glucose plays a regulatory role in GLUT2 distribution independently of age. Moreover, NGF increases GLUT2 abundance, glucose uptake, and GSIS in P20 beta cells and GK activity in adult beta cells. Our results suggest that besides increasing calcium currents, NGF regulates metabolic components of the GSIS, thereby contributing to the maturation process of pancreatic beta cells.
Assuntos
Glucoquinase , Transportador de Glucose Tipo 2 , Glucose , Células Secretoras de Insulina , Fator de Crescimento Neural , Animais , Masculino , Ratos , Células Cultivadas , Glucoquinase/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Ratos WistarRESUMO
Feeding behavior is a complex process that depends on the ability of the brain to integrate hormonal and nutritional signals, such as glucose. One glucosensing mechanism relies on the glucose transporter 2 (GLUT2) in the hypothalamus, especially in radial glia-like cells called tanycytes. Here, we analyzed whether a GLUT2-dependent glucosensing mechanism is required for the normal regulation of feeding behavior in GFAP-positive tanycytes. Genetic inactivation of Glut2 in GFAP-expressing tanycytes was performed using Cre/Lox technology. The efficiency of GFAP-tanycyte targeting was analyzed in the anteroposterior and dorsoventral axes by evaluating GFP fluorescence. Feeding behavior, hormonal levels, neuronal activity using c-Fos, and neuropeptide expression were also analyzed in the fasting-to-refeeding transition. In basal conditions, Glut2-inactivated mice had normal food intake and meal patterns. Implementation of a preceeding fasting period led to decreased total food intake and a delay in meal initiation during refeeding. Additionally, Glut2 inactivation increased the number of c-Fos-positive cells in the ventromedial nucleus in response to fasting and a deregulation of Pomc expression in the fasting-to-refeeding transition. Thus, a GLUT2-dependent glucose-sensing mechanism in GFAP-tanycytes is required to control food consumption and promote meal initiation after a fasting period.
Assuntos
Células Ependimogliais , Comportamento Alimentar , Transportador de Glucose Tipo 2 , Animais , Camundongos , Células Ependimogliais/metabolismo , Jejum , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glucose/metabolismo , Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Pró-Opiomelanocortina/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transportador de Glucose Tipo 2/metabolismoRESUMO
Fructose consumption by rodents modulates both hepatic and intestinal lipid metabolism and gluconeogenesis. We have previously demonstrated that in utero exposure to dexamethasone (DEX) interacts with fructose consumption during adult life to exacerbate hepatic steatosis in rats. The aim of this study was to clarify if adult rats born to DEX-treated mothers would display differences in intestinal gluconeogenesis after excessive fructose intake. To address this issue, female Wistar rats were treated with DEX during pregnancy and control (CTL) mothers were kept untreated. Adult offspring born to CTL and DEX-treated mothers were assigned to receive either tap water (Control-Standard Chow (CTL-SC) and Dexamethasone-Standard Chow (DEX-SC)) or 10% fructose in the drinking water (CTL-fructose and DEX-fructose). Fructose consumption lasted for 80 days. All rats were subjected to a 40 h fasting before sample collection. We found that DEX-fructose rats have increased glucose and reduced lactate in the portal blood. Jejunum samples of DEX-fructose rats have enhanced phosphoenolpyruvate carboxykinase (PEPCK) expression and activity, higher facilitated glucose transporter member 2 (GLUT2) and facilitated glucose transporter member 5 (GLUT5) content, and increased villous height, crypt depth, and proliferating cell nuclear antigen (PCNA) staining. The current data reveal that rats born to DEX-treated mothers that consume fructose during adult life have increased intestinal gluconeogenesis while recapitulating metabolic and morphological features of the neonatal jejunum phenotype.
Assuntos
Dexametasona/efeitos adversos , Carboidratos da Dieta/efeitos adversos , Células Epiteliais/patologia , Frutose/efeitos adversos , Gluconeogênese , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Exposição Materna/efeitos adversos , Troca Materno-Fetal/fisiologia , Efeitos Tardios da Exposição Pré-Natal , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Animais , Feminino , Transportador de Glucose Tipo 2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Gravidez , Ratos WistarRESUMO
Obesity, metabolic syndrome, and type 2 diabetes, three strongly interrelated diseases, are associated to increased morbidity and mortality worldwide. The pathogenesis of obesity-associated disorders is still under study. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein expressed in many cell types including adipocytes, parenchymal, and non-parenchymal hepatic cells and pancreatic cells. Studies have demonstrated that SPARC inhibits adipogenesis and promotes insulin resistance; in addition, circulating SPARC levels were positively correlated with body mass index in obese individuals. Therefore, SPARC is being proposed as a key factor in the pathogenesis of obesity-associated disorders. The aim of this study is to elucidate the role of SPARC in glucose homeostasis. We show here that SPARC null (SPARC-/-) mice displayed an abnormal insulin-regulated glucose metabolism. SPARC-/- mice presented an increased adipose tissue deposition and an impaired glucose homeostasis as animals aged. In addition, the absence of SPARC worsens high-fat diet-induced diabetes in mice. Interestingly, although SPARC-/- mice on high-fat diet were sensitive to insulin they showed an impaired insulin secretion capacity. Of note, the expression of glucose transporter 2 in islets of SPARC-/- mice was dramatically reduced. The present study provides the first evidence that deleted SPARC expression causes diabetes in mice. Thus, SPARC deficient mice constitute a valuable model for studies concerning obesity and its related metabolic complications, including diabetes.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Insulina/sangue , Ilhotas Pancreáticas/metabolismo , Osteonectina/metabolismo , Envelhecimento/sangue , Animais , Biomarcadores/sangue , Diabetes Mellitus Experimental/genética , Dieta Hiperlipídica , Sacarose Alimentar , Transportador de Glucose Tipo 2/metabolismo , Homeostase , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteonectina/deficiência , Osteonectina/genética , Via SecretóriaRESUMO
Dietary phytochemicals may improve glucose metabolism while iron excess seems to be associated to impaired glucose homeostasis and insulin responses. This study investigated the effect of tucum-do-cerrado (Bactris setosa Mart.) consumption on the carbohydrate metabolism and redox response in rats supplemented or not with dietary iron. Male wistar rats were treated with one of the following diets: CT: control diet (AIN- 93G); +Fe: iron-enriched diet; Tuc: control diet +15% tucum-do-cerrado or Tucâ¯+â¯Fe: iron-enriched diet +15% tucum-do-cerrado. Iron supplementation increased muscle lipid and protein oxidation, hepatic glucokinase (GK) and phosphofrutokinase 1 (PFK1) activities and decreased hepatic glucose-6-phosphatase (G6Pase), intestinal Scl2a2 and muscle Slc2a4 and Prkaa2α mRNA levels compared to CT group. Tucum-do-cerrado consumption (Tuc) increased hepatic Slc2a2, Prkaa1α, Prkaa2α and intestinal Slc5a1 mRNA levels, also decreased hepatic G6Pase activity, muscle Slc2a4 and Prkaa2α in relation to CT group. The association of tucum-do-cerrado with iron-enriched diet increased hepatic Prkaa1 and Pck1 compared to the CT andâ¯+â¯Fe groups, intestinal Slc2a2 mRNA levels compared to the +Fe group, while decreased hepatic G6Pase activity in relation to the CT, +Fe and Tucâ¯+â¯Fe groups and muscle Slc2a4 and Prkaa2α compared to CT group. These results suggest that tucum-do-cerrado consumption might induce Prkaa1α and Prkaa2α expression, which may inhibit gluconeogenic rate limiting enzyme, G6Pase, and upregulates GLUT2 hepatic glucose uptake. In addition, moderate iron supplementation improves intracellular hepatic glucose response, stimulating the glycolytic rate limiting enzymes GK and PFK1 while inhibiting gluconeogenic enzyme G6Pase.
Assuntos
Arecaceae/química , Gluconeogênese/efeitos dos fármacos , Transportador de Glucose Tipo 2/metabolismo , Glucose/metabolismo , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Animais , Glicólise/efeitos dos fármacos , Ferro/administração & dosagem , Fígado/metabolismo , Masculino , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Proteínas Quinases/metabolismo , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad-shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP-fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting-feeding transition revealed that GLUT2-knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior.
Assuntos
Comportamento Alimentar/fisiologia , Transportador de Glucose Tipo 2/metabolismo , Hipotálamo/metabolismo , Neuroglia/metabolismo , Saciação/fisiologia , Animais , Peso Corporal , Tronco Encefálico/citologia , Tronco Encefálico/metabolismo , Células Cultivadas , Jejum/metabolismo , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 2/genética , Hipotálamo/citologia , Masculino , Neuroglia/citologia , Neuropeptídeos/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Nonalcoholic fatty liver disease is one of the most common complications of obesity. The Vitis vinifera L. grape skin extract (ACH09) is an important source of polyphenols, which are related to its antioxidant and antihyperglycemic activities. We hypothesized that ACH09 could also exert beneficial effects on metabolic disorders associated with obesity and evaluated ACH09's influence on high-fat (HF) diet-induced hepatic steatosis and insulin resistance in C57BL/6 mice. The animals were fed a standard diet (10% fat, control) or an HF diet (60% fat, HF) with or without ACH09 (200mg/[kg d]) for 12weeks. Our results showed that ACH09 reduced HF diet-induced body weight gain, prevented hepatic lipid accumulation and steatosis, and improved hyperglycemia and insulin resistance. The underlying mechanisms of these beneficial effects of ACH09 may involve the activation of hepatic insulin-signaling pathway because the expression of phosphorylated insulin receptor substrate-1, phosphatidylinositol 3-kinase, phosphorylated Akt serine/threonine kinase 1, and glucose transporter 2 was increased by ACH09 and correlated with improvement of hyperglycemia, hyperinsulinemia, and insulin resistance. ACH09 reduced the expression of the lipogenic factor sterol regulatory-element binding protein-1c in the liver and upregulated the lipolytic pathway (phosphorylated liver kinase B1/phosphorylated adenosine-monophosphate-activated protein kinase), which was associated with normal hepatic levels of triglyceride and cholesterol and prevention of steatosis. ACH09 prevented the hepatic oxidative damage in HF diet-fed mice probably by restoration of antioxidant activity. In conclusion, ACH09 protected mice from HF diet-induced obesity, insulin resistance, and hepatic steatosis. The regulation of hepatic insulin signaling pathway, lipogenesis, and oxidative stress may contribute to ACH09's protective effect.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/prevenção & controle , Resistência à Insulina , Fígado/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vitis/química , Animais , Antioxidantes/farmacologia , Colesterol/sangue , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Fígado Gorduroso/tratamento farmacológico , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Lipogênese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Polifenóis/farmacologia , Triglicerídeos/sangue , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
High glucose-induced oxidative stress and increased NADPH oxidase-2 (NOX2) activity may contribute to the progressive decline of the functional ß-cell mass in type 2 diabetes. To test that hypothesis, we characterized, in islets from male NOX2 knockout (NOX2-KO) and wild-type (WT) C57BL/6J mice cultured for up to 3 weeks at 10 or 30 mmol/l glucose (G10 or G30), the in vitro effects of glucose on cytosolic oxidative stress using probes sensing glutathione oxidation (GRX1-roGFP2), thiol oxidation (roGFP1) or H2O2 (roGFP2-Orp1), on ß-cell stimulus-secretion coupling events and on ß-cell apoptosis. After 1-2 days of culture in G10, the glucose stimulation of insulin secretion (GSIS) was â¼1.7-fold higher in NOX2-KO vs. WT islets at 20-30 mmol/l glucose despite similar rises in NAD(P)H and intracellular calcium concentration ([Ca2+]i) and no differences in cytosolic GRX1-roGFP2 oxidation. After long-term culture at G10, roGFP1 and roGFP2-Orp1 oxidation and ß-cell apoptosis remained low, and the glucose-induced rises in NAD(P)H, [Ca2+]i and GSIS were similarly preserved in both islet types. After prolonged culture at G30, roGFP1 and roGFP2-Orp1 oxidation increased in parallel with ß-cell apoptosis, the glucose sensitivity of the NADPH, [Ca2+]i and insulin secretion responses increased, the maximal [Ca2+]i response decreased, but maximal GSIS was preserved. These responses were almost identical in both islet types. In conclusion, NOX2 is a negative regulator of maximal GSIS in C57BL/6J mouse islets, but it does not detectably contribute to the in vitro glucotoxic induction of cytosolic oxidative stress and alterations of ß-cell survival and function.
Assuntos
Glucose/toxicidade , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , NADPH Oxidase 2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2/deficiência , Oxirredução , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Compostos de Sulfidrila/metabolismo , Técnicas de Cultura de TecidosRESUMO
In a previous study, we showed that saccharin can induce weight gain when compared with sucrose in Wistar rats despite similar total caloric intake. We now question whether it could be due to the sweet taste of saccharin per se. We also aimed to address if this weight gain is associated with insulin-resistance and to increases in gut peptides such as leptin and PYY in the fasting state. In a 14 week experiment, 16 male Wistar rats received either saccharin-sweetened yogurt or non-sweetened yogurt daily in addition to chow and water ad lib. We measured daily food intake and weight gain weekly. At the end of the experiment, we evaluated fasting leptin, glucose, insulin, PYY and determined insulin resistance through HOMA-IR. Cumulative weight gain and food intake were evaluated through linear mixed models. Results showed that saccharin induced greater weight gain when compared with non-sweetened control (p = 0.027) despite a similar total caloric intake. There were no differences in HOMA-IR, fasting leptin or PYY levels between groups. We conclude that saccharin sweet taste can induce mild weight gain in Wistar rats without increasing total caloric intake. This weight gain was not related with insulin-resistance nor changes in fasting leptin or PYY in Wistar rats.
Assuntos
Ingestão de Energia , Resistência à Insulina , Sacarina/efeitos adversos , Paladar , Aumento de Peso , Animais , Glicemia/metabolismo , Água Potável , Jejum , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Insulina/sangue , Leptina/sangue , Masculino , Peptídeo YY/sangue , Ratos , Sacarina/administração & dosagem , IogurteRESUMO
Glucose transporter proteins 2 and 4 (GLUT2 and GLUT4) play important roles in glucose transport and energy metabolism. Changes in the levels of GLUT2 and GLUT4 mRNA were measured in longissimus dorsi muscle from the lean Yorkshire and fat Tibetan pig breeds at six different time points (1, 2, 3, 4, 5, and 6 months) with quantitative real-time polymerase chain reactions. The results showed that GLUT2 and GLUT4 mRNA were abundantly expressed in the longissimus dorsi muscle and that the developmental expression patterns were similar in both breeds. Tibetan pigs exhibited higher intramuscular fat and GLUT2 mRNA levels, while Yorkshire pigs exhibited a higher myofiber cross-sectional area (CSA) and GLUT4 mRNA levels. Furthermore, the changes in the GLUT4 mRNA levels were strongly and positively correlated with the CSA over a period of six months. These results exhibit time- and breed-specific expression patterns of GLUT2 and GLUT4, which highlight their potential as candidate genes for assessing adipose deposition and muscle development in pigs. These differences in the expression of GLUT family genes may also have indications for meat quality.