RESUMO
Obesity represents a major challenge to the pharmaceutical community due to the minimal availability of anti-obesity drugs and the drawbacks of current weight-loss agents. The study described herein presents lupine oil, in two pharmaceutical formulations, as a potential anti-obesity agent via its effect on different physiological, biochemical, and hormonal parameters. Rats were divided into two groups; one group was continued on a standard commercial rodent diet and served as the non-obese control. The other group was fed a high-fat diet for 7 weeks to prepare an obese rat model. Then, the obese rats were divided into groups to receive 100 mg/kg of the crude lupine oil or nanoemulsion for 10 or 20 days. Lupine oil showed a potent body weight-reducing effect and improved insulin resistance. The oil altered obesity-induced hyperlipidemia and it enhanced the leptin/adiponectin/AMPK hormonal system in epididymal fat, serum, and liver, to which all the above physiological activities could be attributed. The nanoemulsion formulation of lupine oil significantly amplified the activity for all the above physiological and hormonal parameters when compared to the crude oil formulation. Lupine oil nanoemulsion could be used as a potential drug against diet-induced obesity.
Assuntos
Animais , Masculino , Ratos , Fármacos Antiobesidade/efeitos adversos , Lupinus/efeitos adversos , Dieta/classificação , Obesidade/classificação , Fosfotransferases/administração & dosagem , Preparações Farmacêuticas , Monofosfato de Adenosina/agonistas , Adiponectina/farmacologiaRESUMO
Two muscle-specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor-1 (IGF-1) pathway inhibits muscle UL expression through Akt-mediated inhibition of FoxO transcription factors, while AMP-activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF-1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) in the presence or absence of 50 ng/ml IGF-1. IGF-1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0 mM AICAR synergized IGF-1-induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF-1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Acetil-CoA Carboxilase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Monofosfato de Adenosina/agonistas , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Fatores de Iniciação em Eucariotos , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genéticaRESUMO
Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.
Assuntos
Monofosfato de Adenosina/química , Escherichia coli/enzimologia , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/química , Frutosedifosfatos/química , Modelos Químicos , Suínos/metabolismo , Monofosfato de Adenosina/agonistas , Sítio Alostérico/fisiologia , Animais , Frutosedifosfatos/agonistas , Gluconeogênese/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
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.