RESUMO
The aim of this study was to investigate the acute effects of troglitazone on several pathways of glucose and fatty acid (FA) partitioning and the molecular mechanisms involved in these processes in skeletal muscle. Exposure of L6 myotubes to troglitazone for 1 h significantly increased phosphorylation of AMPK and ACC, which was followed by approximately 30% and approximately 60% increases in palmitate oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity, respectively. Troglitazone inhibited basal ( approximately 25%) and insulin-stimulated ( approximately 35%) palmitate uptake but significantly increased basal and insulin-stimulated glucose uptake by approximately 2.2- and 2.7-fold, respectively. Pharmacological inhibition of AMPK completely prevented the effects of troglitazone on palmitate oxidation and glucose uptake. Interestingly, even though troglitazone exerted an insulin sensitizing effect, it reduced basal and insulin-stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to approximately 30%, approximately 60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin-stimulated phosphorylation of Akt(Thr308), Akt(Ser473), and GSK3alpha/beta. Troglitazone also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal ( approximately 3.5-fold) and insulin-stimulated ( approximately 5.5-fold) rates of lactate production by muscle cells. In summary, we provide novel evidence that troglitazone exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism toward lactate production in muscle cells. These effects seem to be at least partially dependent on AMPK activation and may account for potential acute PPAR-gamma-independent anti-diabetic effects of thiazolidinediones in skeletal muscle.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Cromanos/farmacologia , Insulina/farmacologia , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Complexo Piruvato Desidrogenase/antagonistas & inibidores , Tiazolidinedionas/farmacologia , Proteínas Quinases Ativadas por AMP , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacocinética , Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Ácido Láctico/biossíntese , Músculo Esquelético/citologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacocinética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Distribuição Tecidual/efeitos dos fármacos , TroglitazonaRESUMO
The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 microM of palmitate did not affect cell viability but significantly reduced FA oxidation by approximately 26.5%, approximately 43.5%, approximately 50%, and approximately 47%, respectively. Interestingly, this occurred despite significant increases in AMPK ( approximately 2.5-fold) and ACC ( approximately 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity ( approximately 38-60%). Low concentrations of palmitate (50-100 microM) caused an increase ( approximately 30%) in CPT-1 activity. However, as the concentration of palmitate increased, CPT-1 activity decreased by approximately 32% after exposure for 8 h to 800 microM of palmitate. Although FA uptake was reduced ( approximately 35%) in cells exposed to increasing palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values approximately 2.3-, approximately 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 microM palmitate, respectively. Interestingly, myotubes exposed to 400 microM of palmitate for 1 h increased basal glucose uptake and glycogen synthesis by approximately 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8 h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake ( approximately 65%) and glycogen synthesis ( approximately 30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs, such as in obesity and Type 2 Diabetes.
Assuntos
Ácidos Graxos/metabolismo , Complexos Multienzimáticos/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/enzimologia , Ácido Palmítico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Acetil-CoA Carboxilase/metabolismo , Animais , Carboxiliases/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Glucose/metabolismo , Glicogênio/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Oxirredução , Ácido Palmítico/farmacologia , Fosforilação , Ratos , Fatores de TempoRESUMO
The aim of this study was to investigate the effects of 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-induced AMP-activated protein kinase activation on glycogen metabolism in soleus (slow twitch, oxidative) and epitrochlearis (fast twitch, glycolytic) skeletal muscles. Isolated soleus and epitrochlearis muscles were incubated in the absence or presence of insulin (100 nM), AICAR (2 mM), and AICAR plus insulin. In soleus muscles exposed to insulin, glycogen synthesis and glycogen content increased 6.4- and 1.3-fold, respectively. AICAR treatment significantly suppressed ( approximately 60%) insulin-stimulated glycogen synthesis and completely prevented the increase in glycogen content induced by insulin. AICAR did not affect either basal or insulin-stimulated glucose uptake but significantly increased insulin-stimulated ( approximately 20%) lactate production in soleus muscles. Interestingly, basal glucose uptake was significantly increased ( approximately 1.4-fold) in the epitrochlearis muscle, even though neither basal nor insulin-stimulated rates of glycogen synthesis, glycogen content, and lactate production were affected by AICAR. We also report the novel evidence that AICAR markedly reduced insulin-induced Akt-Thr308 phosphorylation after 15 and 30 min exposure to insulin, which coincided with a marked reduction in glycogen synthase kinase 3 (GSK)-3alpha/beta phosphorylation. Importantly, phosphorylation of glycogen synthase was increased by AICAR treatment 45 min after insulin stimulation. Our results indicate that AICAR-induced AMP-activated protein kinase activation caused a time-dependent reduction in Akt308 phosphorylation, activation of glycogen synthase kinase-3alpha/beta, and the inactivation of glycogen synthase, which are compatible with the acute reduction in insulin-stimulated glycogen synthesis in oxidative but not glycolytic skeletal muscles.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio/biossíntese , Insulina/farmacologia , Complexos Multienzimáticos/fisiologia , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/farmacologia , Animais , Glicogênio Sintase Quinase 3 beta , Ácido Láctico/biossíntese , Masculino , Fosforilação , Ratos , Ratos WistarRESUMO
Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic ß-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.
Assuntos
Metabolismo Energético , Glucose/farmacologia , Insulina/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Glucose/metabolismo , Teste de Tolerância a Glucose , Secreção de Insulina , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/sangue , Nucleobindinas , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
This study investigated the effects of obesity induced by high-fat (HF) diet on thyroid function and whole-body energy balance. To accomplish that, we assessed the effects of 8 wk of HF diet on several parameters of hypothalamus-pituitary-thyroid axis function. Serum total T(4) and T(3), rT(3), and TSH, the activity of type 1 and type 2 deiodinases in central and peripheral tissues were determined. Also, we measured in vivo energy balance, substrate partitioning, and markers of leptin resistance. Here we provide novel evidence that prolonged positive energy balance acquired by feeding a HF diet induced hyperactivation of the hypothalamus-pituitary-thyroid axis, which was characterized by 2.24-, 1.6-, and 3.7-fold elevations in hypothalamic TRH expression, thyroid iodide uptake, and serum TSH, respectively. Serum T(4) and T(3) were normal together with augmented deiodinase type 1 activity in liver (1.3-fold) and kidney (1.2-fold) and increased (1.5-fold) serum rT3 in HF rats. Despite no increase in circulating levels of T(3) and T(4), whole-body oxygen consumption was increased, and substrate metabolism was shifted toward fat oxidation in HF rats. These in vivo metabolic adjustments were mainly driven by the fat content of the diet. Furthermore, spontaneous dark cycle physical activity was reduced by 30% in rats fed a HF diet, which limited energy expenditure and favored the development of obesity. Our findings provide new insight into the endocrine and physiological mechanisms that underlie the alterations in thyroid hormone availability, energy balance, and metabolic partitioning in HF diet-induced obesity.
Assuntos
Gorduras na Dieta/farmacologia , Metabolismo Energético/efeitos dos fármacos , Iodeto Peroxidase/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/sangue , Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/metabolismo , Animais , Western Blotting , Composição Corporal/efeitos dos fármacos , Composição Corporal/genética , Teste de Tolerância a Glucose , Masculino , Reação em Cadeia da Polimerase , Ratos , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismoRESUMO
The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK phosphorylation and fatty acid oxidation were measured. A 2-fold increase in both AMPK and ACC phosphorylation was observed in the presence of palmitate concentrations as low as 10 microM, which was also accompanied by a significant increase in fatty acid oxidation. The effect of palmitate on AMPK and ACC phosphorylation was dose-dependent, reaching maximum increases of 3.5- and 4.5-fold, respectively. Interestingly, ACC phosphorylation was coupled with AMPK activation at palmitate concentrations ranging from 10 to 100 microM; however, at concentrations >200 microM, ACC phosphorylation and fatty acid oxidation remained high even after AMPK phosphorylation was completely prevented by the use of a selective AMPK inhibitor. This indicates that LCFAs regulate ACC activity by AMPK-dependent and -independent mechanisms, based on their abundance in skeletal muscle cells. Here, we provide novel evidence that the AMPK/ACC pathway may operate as a mechanism to sense and respond to the lipid energy charge of skeletal muscle cells.