RESUMO
Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Músculo Esquelético/enzimologia , Estresse Oxidativo , Superóxido Dismutase/metabolismo , Regulação para Cima , Animais , Eletroporação , Técnicas de Transferência de Genes , Glutationa Peroxidase/metabolismo , Humanos , Extremidade Inferior , Masculino , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Carbonilação Proteica , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase/genéticaRESUMO
We know a great deal about the cellular response to starvation via AMPK, but less is known about the reaction to nutrient excess. Insulin resistance may be an appropriate response to nutrient excess, but the cellular sensors that link these parameters remain poorly defined. In the present study we provide evidence that mitochondrial superoxide production is a common feature of many different models of insulin resistance in adipocytes, myotubes, and mice. In particular, insulin resistance was rapidly reversible upon exposure to agents that act as mitochondrial uncouplers, ETC inhibitors, or mitochondrial superoxide dismutase (MnSOD) mimetics. Similar effects were observed with overexpression of mitochondrial MnSOD. Furthermore, acute induction of mitochondrial superoxide production using the complex III antagonist antimycin A caused rapid attenuation of insulin action independently of changes in the canonical PI3K/Akt pathway. These results were validated in vivo in that MnSOD transgenic mice were partially protected against HFD induced insulin resistance and MnSOD+/- mice were glucose intolerant on a standard chow diet. These data place mitochondrial superoxide at the nexus between intracellular metabolism and the control of insulin action potentially defining this as a metabolic sensor of energy excess.
Assuntos
Antioxidantes/metabolismo , Resistência à Insulina/fisiologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Antimicina A/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , Resistência à Insulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos/metabolismoRESUMO
Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser(473)) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3ß (Ser(9)) and AS160 (Thr(642)) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.
Assuntos
Glucose/metabolismo , Resistência à Insulina , Músculos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas Ativadoras de GTPase/metabolismo , Glucose/administração & dosagem , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Masculino , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos WistarRESUMO
Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (approximately 300 mU/l) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, 1 h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interestingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling.
Assuntos
Retroalimentação Fisiológica/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Insulina/farmacologia , Lipídeos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Animais , Glicemia/análise , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Glicogênio/metabolismo , Infusões Intravenosas , Insulina/administração & dosagem , Insulina/sangue , Resistência à Insulina/fisiologia , Lipídeos/administração & dosagem , Lipídeos/sangue , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.
Assuntos
Tecido Adiposo/enzimologia , Insulina/farmacologia , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Lipase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
Alpha-calcitonin gene-related peptide (alphaCGRP) is released mainly from sensory and motor nerves in response to physiological stimuli. Despite well-documented pharmacological effects, its primary physiological role has thus far remained obscure. Increased lipid content, particularly in skeletal muscle and liver, is strongly implicated in the pathogenesis of insulin resistance, but the physiological regulation of organ lipid is imperfectly understood. Here we report our systematic investigations of the effects of alphaCGRP on in vitro and in vivo indices of lipid metabolism. In rodents, levels of alphaCGRP similar to those in the blood markedly stimulated fatty acid beta-oxidation and evoked concomitant mobilization of muscle lipid via receptor-mediated activation of muscle lipolysis. alphaCGRP exerted potent in vivo effects on lipid metabolism in muscle, liver, and the blood via receptor-mediated pathways. Studies with receptor antagonists were consistent with tonic regulation of lipid metabolism by an endogenous CGRP agonist. These data reveal that alphaCGRP is a newly recognized regulator of lipid availability and utilization in key tissues and that it may elevate the availability of intramyocellular free fatty acids to meet muscle energy requirements generated by contraction by evoking their release from endogenous triglyceride.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP , Animais , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , AMP Cíclico/metabolismo , Ácidos Graxos/análise , Masculino , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Neurotransmissores/farmacologia , Neurotransmissores/fisiologia , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologiaRESUMO
The phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3beta phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.
Assuntos
Regulação Enzimológica da Expressão Gênica , Insulina/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas/biossíntese , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Desoxiglucose/química , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas Substratos do Receptor de Insulina , Camundongos , Modelos Biológicos , Fosforilação , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/química , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
Berberine has been shown to have antidiabetic properties, although its mode of action is not known. Here, we have investigated the metabolic effects of berberine in two animal models of insulin resistance and in insulin-responsive cell lines. Berberine reduced body weight and caused a significant improvement in glucose tolerance without altering food intake in db/db mice. Similarly, berberine reduced body weight and plasma triglycerides and improved insulin action in high-fat-fed Wistar rats. Berberine downregulated the expression of genes involved in lipogenesis and upregulated those involved in energy expenditure in adipose tissue and muscle. Berberine treatment resulted in increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 adipocytes and L6 myotubes, increased GLUT4 translocation in L6 cells in a phosphatidylinositol 3' kinase-independent manner, and reduced lipid accumulation in 3T3-L1 adipocytes. These findings suggest that berberine displays beneficial effects in the treatment of diabetes and obesity at least in part via stimulation of AMPK activity.
Assuntos
Berberina/uso terapêutico , Diabetes Mellitus/tratamento farmacológico , Resistência à Insulina , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Berberina/administração & dosagem , Linhagem Celular , Dieta , Gorduras na Dieta/administração & dosagem , Metabolismo Energético/genética , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Técnica Clamp de Glucose , Transportador de Glucose Tipo 4/metabolismo , Lipídeos/biossíntese , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Obesidade/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ratos , Ratos Wistar , Redução de Peso/efeitos dos fármacosRESUMO
Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 +/- 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism.
Assuntos
Glucose/metabolismo , Proteínas Musculares/metabolismo , Animais , Transporte Biológico Ativo , Linhagem Celular , Eletroporação , Regulação da Expressão Gênica , Masculino , Camundongos , Ratos , Ratos WistarRESUMO
Lipid accumulation in non-adipose tissues is strongly associated with the metabolic syndrome, possibly due to aberrant partitioning of intracellular fatty acids between storage and oxidation. In the present study, we administered the non-metabolizable fatty acid analog [9,10-(3)H]-(R)-2-bromopalmitate, and authentic (14)C-palmitate to conscious rats, in order to directly examine the initial intracellular fate of fatty acids in a range of insulin-sensitive tissues, including white and red muscles, liver, white adipose tissue, and heart. Rats were studied after administration of an oral glucose load to examine the effect of physiological elevation of glucose and insulin. The tracer results showed that glucose administration partitioned fatty acid toward storage in white muscle (storage:uptake ratios, vehicle vs glucose; 0.64 +/- 0.02 vs 0.92 +/- 0.09, P < 0.05), and in liver (0.66 +/- 0.07 vs 0.98 +/- 0.04, P < 0.05), but not in red muscle (1.18 +/- 0.07 vs 1.36 +/- 0.11, P = not significant). These results demonstrate the physiological relevance of the so-called 'reverse' Randle cycle, but surprisingly show that it may be more important in white rather than oxidative red muscle.
Assuntos
Ácidos Graxos/metabolismo , Glucose/administração & dosagem , Resistência à Insulina , Fibras Musculares de Contração Rápida/metabolismo , Tecido Adiposo/metabolismo , Administração Oral , Animais , Glicemia/análise , Isótopos de Carbono , Ácidos Graxos não Esterificados/sangue , Teste de Tolerância a Glucose , Glicerol/sangue , Insulina/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Miocárdio/metabolismo , Oxirredução , Palmitatos/administração & dosagem , Palmitatos/metabolismo , Ratos , Ratos Sprague-DawleyAssuntos
Dieta/efeitos adversos , Gorduras na Dieta/administração & dosagem , Resistência à Insulina , Insulina/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Humanos , Insulina/farmacologia , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestruturaRESUMO
An important regulator of fatty acid oxidation (FAO) is the allosteric inhibition of CPT-1 by malonyl-CoA produced by the enzyme acetyl-CoA carboxylase 2 (ACC2). Initial studies suggested that deletion of Acc2 (Acacb) increased fat oxidation and reduced adipose tissue mass but in an independently generated strain of Acc2 knockout mice we observed increased whole-body and skeletal muscle FAO and a compensatory increase in muscle glycogen stores without changes in glucose tolerance, energy expenditure or fat mass in young mice (12-16 weeks). The aim of the present study was to determine whether there was any effect of age or housing at thermoneutrality (29â°C; which reduces total energy expenditure) on the phenotype of Acc2 knockout mice. At 42-54 weeks of age, male WT and Acc2(-/-) mice had similar body weight, fat mass, muscle triglyceride content and glucose tolerance. Consistent with younger Acc2(-/-) mice, aged Acc2(-/-) mice showed increased whole-body FAO (24âh average respiratory exchange ratio=0.95±0.02 and 0.92±0.02 for WT and Acc2(-/-) mice respectively, P<0.05) and skeletal muscle glycogen content (+60%, P<0.05) without any detectable change in whole-body energy expenditure. Hyperinsulinaemic-euglycaemic clamp studies revealed no difference in insulin action between groups with similar glucose infusion rates and tissue glucose uptake. Housing Acc2(-/-) mice at 29â°C did not alter body composition, glucose tolerance or the effects of fat feeding compared with WT mice. These results confirm that manipulation of Acc2 may alter FAO in mice, but this has little impact on body composition or insulin action.
Assuntos
Acetil-CoA Carboxilase/fisiologia , Envelhecimento/fisiologia , Abrigo para Animais , Temperatura , Acetil-CoA Carboxilase/deficiência , Animais , Composição Corporal , Peso Corporal , Metabolismo Energético , Ácidos Graxos/metabolismo , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Glicogênio/análise , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Oxirredução , Fenótipo , Triglicerídeos/análiseRESUMO
In humans and animal models, increased lipid content of skeletal muscle is strongly associated with insulin resistance. However, it is unclear whether this accumulation is due to increased uptake or reduced utilization of fatty acids (FAs). We used (3)H-R-bromopalmitate tracer to assess the contribution of tissue-specific changes in FA uptake to the lipid accumulation observed in tissues of insulin-resistant, high fat-fed rats (HFF) compared with control rats (CON) fed a standard diet. To study FA metabolism under different metabolic states, tracer was infused under basal conditions, during hyperinsulinemic-euglycemic clamp (low FA availability) or during the infusion of intralipid and heparin (high FA availability). FA clearance was significantly increased in the red gastrocnemius muscle of HFF under conditions of low (HFF = 10.4 +/- 1.1; CON = 7.4 +/- 0.5 ml x min(-1) x 100 g(-1); P < 0.05), basal (HFF = 8.3 +/- 1.4; CON = 4.5 +/- 0.7 ml x min(-1) x 100 g(-1); P < 0.01), and high (HFF = 7.0 +/- 0.8; CON = 4.3 +/- 0.5 ml x min(-1) x 100 g(-1); P < 0.05) FA levels. This indicates an adaptation by muscle for more efficient uptake of lipid. Associated with the enhanced efficiency of FA uptake, we observed increases in CD36/FA translocase mRNA expression (P < 0.01) and acyl-CoA synthetase activity (P < 0.02) in the same muscle. FA clearance into white adipose tissue was also increased in HFF when circulating FA were elevated, but there was little effect of the high-fat diet on hepatic FA uptake. In conclusion, insulin resistance induced by feeding rats a high-fat diet is associated with tissue-specific adaptations that enhance utilization of increased dietary lipid but could also contribute to the accumulation of intramuscular lipid with a detrimental effect on insulin action.
Assuntos
Ácidos Graxos/farmacocinética , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Tecido Adiposo/metabolismo , Animais , Peso Corporal , Antígenos CD36/genética , Antígenos CD36/metabolismo , Gorduras na Dieta/sangue , Gorduras na Dieta/farmacocinética , Ingestão de Alimentos/fisiologia , Ácidos Graxos/sangue , Expressão Gênica/fisiologia , Insulina/metabolismo , Masculino , Fibras Musculares de Contração Rápida/metabolismo , Músculo Esquelético/citologia , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
Insulin-stimulated glucose uptake is increased in white but not red muscle of insulin-resistant high-fat-fed (HF) rats after administration of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). To investigate whether a lesser AICAR effect on glucose uptake in red muscle was offset by a greater effect on fatty acid (FA) uptake, we examined acute effects of AICAR on muscle glucose and FA fluxes in HF rats. HF rats received AICAR (250 mg/kg) subcutaneously. At 30 min, a mixture of either (3)H-(R)-2-bromopalmitate/(14)C-palmitate or (3)H-2-deoxyglucose/(14)C-glucose was administered intravenously to assess muscle FA and glucose uptake. AICAR decreased plasma levels of glucose (approximately 25%), insulin (approximately 60%), and FAs (approximately 30%) at various times over the next 46 min (P < 0.05 vs. controls). In white muscle, AICAR increased both FA (2.4-fold) and glucose uptake (4.9-fold), associated with increased glycogen synthesis (6-fold). These effects were not observed in red muscle. We conclude that both glucose and FA fluxes are enhanced by AICAR more in white versus red muscle, consistent with the relative degree of activation of AMPK. Therefore, a lesser effect of AICAR to alleviate muscle insulin resistance in red versus white muscle is not explained by a relatively greater effect on FA uptake in the red muscle.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP , Animais , Gorduras na Dieta/administração & dosagem , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Resistência à Insulina , Masculino , Ratos , Ratos WistarRESUMO
Increased hypothalamic neuropeptide-Y (NPY) action and disruption of the melanocortin (MC)-4 receptor both result in hyperphagia and obesity. To determine whether similar hormonal and metabolic mechanisms are involved in these two obesity syndromes, we investigated the time course of effects induced by 6-day intracerebroventricular (ICV) infusion of NPY (3.5 nmol/day) or the MC4 receptor antagonist HS014 (4.8 nmol/day) in rats pair-fed with vehicle-infused controls. The weight of white adipose tissue (WAT) deposits was increased after 6-day NPY and HS014 infusion compared with controls, and the increase was significantly greater in HS014- than in NPY-infused rats (retroperitoneal WAT: NPY 0.57 +/- 0.05; HS014 0.80 +/- 0.05; control 0.43 +/- 0.03% body wt, n = 8-13, P < 0.05). Plasma leptin was also increased in both experimental groups (NPY 10.6 +/- 1.9; HS014 4.4 +/- 0.9; control 2.0 +/- 0.1 ng/ml, n = 8-13, P < 0.05 for all comparisons). Basal plasma corticosterone and insulin levels were increased by ICV NPY infusion, whereas HS014-infused rats showed no significant increase in these parameters on any of 1-6 days of infusion. Both NPY and HS014 infusion potentiated intravenous glucose-induced (300 mg/kg) plasma insulin levels, and there was no difference in glycemia among groups. In NPY-infused rats, the plasma free fatty acid levels were decreased and triglyceridemia was increased compared with controls, but these parameters were unchanged in HS014-infused rats. Hepatic triglyceride content was significantly increased by HS014 but not by NPY infusion. Levels of uncoupling protein-1 mRNA in brown adipose tissue were significantly decreased after 6 days of HS014 infusion, similar to the effect of central NPY. Because ICV HS014 induced at least as great an increase in fat mass as ICV NPY and yet had divergent hormonal and metabolic effects, we conclude that MC4 receptor antagonism does not induce obesity solely by regulation of the endogenous NPY-ergic system.
Assuntos
Tecido Adiposo/citologia , Neuropeptídeo Y/farmacologia , Receptores de Peptídeos/antagonistas & inibidores , Tecido Adiposo/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Proteínas de Transporte/genética , Corticosterona/sangue , Regulação da Expressão Gênica , Infusões Parenterais , Insulina/sangue , Canais Iônicos , Cinética , Masculino , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Neuropeptídeo Y/administração & dosagem , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Wistar , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/antagonistas & inibidores , Receptores de Peptídeos/efeitos dos fármacos , Valores de Referência , Transcrição Gênica , Proteína Desacopladora 1RESUMO
Metformin reduces the incidence of progression to type 2 diabetes in humans with obesity or impaired glucose tolerance. We used an animal model to investigate whether metformin could prevent acute lipid-induced insulin resistance and the mechanisms involved. Metformin or vehicle was administered to rats daily for 1 week. Rats were studied basally, after 3.75 h of intralipid-heparin or glycerol infusion, or after 5 h of infusion with a hyperinsulinemic-euglycemic clamp between 3 and 5 h. Metformin had no effect on plasma triacylglycerol or nonesterified fatty acid concentrations and did not alter glucose turnover or gluconeogenic enzyme mRNA after lipid infusion. However, metformin normalized hepatic glucose output and increased liver glycogen during lipid infusion and clamp. Basal liver (but not muscle or fat) AMP-activated protein kinase activity was increased by metformin (by 310%; P < 0.01), associated with increased phosphorylation of acetyl CoA carboxylase. Postclamp liver but not muscle phosphorylated/total Akt protein was increased, whereas basal c-Jun NH2-terminal kinase-1 and -2 protein expression were reduced (by 39 and 53%, respectively; P < 0.05). Metformin also increased hepatic basal IkappaBalpha levels (by 260%; P < 0.001) but had no effect on tyrosine phosphorylation or expression of insulin receptor substrate-1 (IRS-1). In summary, metformin opposes the development of acute lipid-induced insulin resistance in the liver through alterations in multiple signaling pathways.
Assuntos
Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Lipídeos/farmacologia , Fígado/fisiologia , Metformina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Glucose/metabolismo , Técnica Clamp de Glucose , Hiperinsulinismo , Fígado/efeitos dos fármacos , Masculino , Síndrome Metabólica/prevenção & controle , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
HIV protease inhibitor-related lipodystrophy is characterized by peripheral fat loss, hyperlipidemia, and insulin resistance. Increased availability of lipid to muscle may be one of the mechanisms that induce insulin resistance. Regional fat, intramyocellular lipid (by (1)H-magnetic resonance spectroscopy), serum lipids, and insulin-stimulated glucose disposal (by hyperinsulinemic-euglycemic clamp) were quantified in 10 men who had HIV-1 infection with moderate to severe lipodystrophy and a control group of 10 nonlipodystrophic men who had HIV-1 infection and were naïve to protease inhibitors to examine the effects of lipodystrophy on glucose and lipid metabolism. Lipodystrophic subjects showed lower insulin-stimulated glucose disposal than control subjects (P = 0.001) and had increased serum triglycerides (P = 0.03), less limb fat (P = 0.02), increased visceral fat as a proportion of total abdominal fat (P = 0.003), and increased intramyocellular lipid (1.90 +/- 0.15 vs. 1.23 +/- 0.16% of water resonance peak area; P = 0.007). In both groups combined, visceral fat related strongly to intramyocellular lipid (r = 0.83, P < 0.0001) and intramyocellular lipid related negatively to insulin-stimulated glucose disposal (r = -0.71, P = 0.0005). Fasting serum cholesterol and triglycerides related positively to intramyocellular lipid and visceral fat in lipodystrophic subjects only. The data indicate that lipodystrophy is associated with increased lipid content in muscle accompanying impaired insulin action. The results do not establish causation but emphasize the interrelationships among visceral fat, myocyte lipid, and insulin action.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Tecido Adiposo/anatomia & histologia , Inibidores da Protease de HIV/efeitos adversos , Lipodistrofia/induzido quimicamente , Absorciometria de Fóton , Síndrome da Imunodeficiência Adquirida/patologia , Tecido Adiposo/efeitos dos fármacos , Adulto , Fármacos Anti-HIV/uso terapêutico , Glicemia/metabolismo , Composição Corporal/efeitos dos fármacos , Homeostase , Humanos , Indinavir/efeitos adversos , Insulina/fisiologia , Leptina/sangue , Lipídeos/sangue , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Nelfinavir/efeitos adversos , Ritonavir/efeitos adversos , Saquinavir/efeitos adversosRESUMO
Exercise improves insulin sensitivity. As AMP-activated protein kinase (AMPK) plays an important role in muscle metabolism during exercise, we investigated the effects of the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) on insulin action in insulin-resistant high-fat-fed (HF) rats. Rats received a subcutaneous injection of 250 mg/kg AICAR (HF-AIC) or saline (HF-Con). The next day, euglycemic-hyperinsulinemic clamp studies were performed. Glucose infusion rate during the clamp was enhanced (50%) in HF-AIC compared with HF-Con rats. Insulin-stimulated glucose uptake was improved in white but not in red quadriceps, whereas glycogen synthesis was improved in both red and white quadriceps of HF-AIC rats. HF-AIC rats also showed increased insulin suppressibility of hepatic glucose output (HGO). AICAR-induced responses in both liver and muscle were accompanied by reduced malonyl-CoA content. Clamp HGO correlated closely with hepatic triglyceride content (r = 0.67, P < 0.01). Thus, a single dose of AICAR leads to an apparent enhancement in whole-body, muscle, and liver insulin action in HF rats that extends beyond the expected time of AMPK activation. Whether altered tissue lipid metabolism mediates AICAR effects on insulin action remains to be determined. Follow-up studies suggest that at least some of the post-AICAR insulin-enhancing effects also occur in normal rats. Independent of this, the results suggest that pharmacological activation of AMPK may have potential in treating insulin-resistant states and type 2 diabetes.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Ribonucleotídeos/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal , Gorduras na Dieta/farmacologia , Técnica Clamp de Glucose , Masculino , Ratos , Ratos WistarRESUMO
OBJECTIVE: To examine the effect of moderate intensity physical activity on the interactions between central abdominal adiposity, myocyte lipid content, and insulin action in overweight and obese, sedentary men. RESEARCH DESIGN AND METHODS: Myocyte lipid (biochemical triglyceride and long-chain acyl CoA [LCAC] from vastus lateralis biopsy and soleus and tibialis anterior intramyocellular lipid by (1)H-magnetic resonance spectroscopy), regional body and abdominal fat (dual-energy X-ray absorptiometry and magnetic resonance imaging), serum lipids, insulin action (hyperinsulinemic-euglycemic clamp), and substrate oxidation were measured in 18 nondiabetic, sedentary, and overweight to obese men (aged 37.4 +/- 1.3 years and BMI 30.9 +/- 0.7 kg/m(2), range 26.4-37.6) at baseline, after the first two to four bouts of aerobic exercise (55-70% of VO(2max) for 40 min/session), and at completion of 4.1 +/- 0.2 exercise sessions/week for 9.7 +/- 0.5 weeks (postexercise measurements performed 24-36 h after the last exercise bout). RESULTS: Mean whole body insulin-stimulated glucose uptake and basal fat oxidation rate increased 16 and 41%, respectively, after two to four bouts of exercise, without further increase at program end. Mean aerobic capacity increased 11%, and central abdominal fat decreased 5% at program end, but myocyte lipid levels were not significantly changed. Posttraining increases in insulin-stimulated glucose uptake were predicted by increase in aerobic capacity (r = 0.726, P = 0.001) and magnitude of reduction in visceral fat (r = -0.544, P = 0.02) and not by changes in myocyte lipid or LCAC levels. CONCLUSIONS: These results suggest that in overweight and obese sedentary men, increase in insulin sensitivity with moderate intensity exercise is predicted by improvement in aerobic capacity and reduction in visceral fat but is independent of myocyte triglyceride or LCAC levels.
Assuntos
Tecido Adiposo/anatomia & histologia , Exercício Físico/fisiologia , Metabolismo dos Lipídeos , Células Musculares/metabolismo , Músculo Esquelético/fisiopatologia , Obesidade/fisiopatologia , Consumo de Oxigênio/fisiologia , Abdome , Acil Coenzima A/metabolismo , Adulto , Glicemia/metabolismo , Composição Corporal , Técnica Clamp de Glucose , Humanos , Lipídeos/sangue , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Oxirredução , Análise de Regressão , Inquéritos e Questionários , Triglicerídeos/metabolismoRESUMO
SIRT1 is a NAD+-dependent deacetylase thought to regulate cellular metabolic pathways in response to alterations in nutrient flux. In the current study we investigated whether acute changes in SIRT1 expression affect markers of muscle mitochondrial content and also determined whether SIRT1 influenced muscle insulin resistance induced by acute glucose oversupply. In male Wistar rats either SIRT1 or a deacetylase inactive mutant form (H363Y) was electroprated into the tibialis cranialis (TC) muscle. The other leg was electroporated with an empty control vector. One week later, glucose was infused and hyperglycaemia was maintained at ~11mM. After 5 hours, 11mM glucose induced significant insulin resistance in skeletal muscle. Interestingly, overexpression of either SIRT1 or SIRT1 (H363Y) for 1 week did not change markers of mitochondrial content or function. SIRT1 or SIRT1 (H363Y) overexpression had no effect on the reduction in glucose uptake and glycogen synthesis in muscle in response to hyperglycemia. Therefore we conclude that acute increases in SIRT1 protein have little impact on mitochondrial content and that overexpressing SIRT1 does not prevent the development of insulin resistance during hyperglycaemia.