RESUMO
Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser(79), an AMPK-specific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca(2+)/calmodulin-dependent protein kinase kinase-ß knockout (CaMKKß(-/-)) mice and cultured adipocytes, we further show that glucagon activates the CaMKKß/AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKKß(+/+) but not CaMKKß(-/-) mice. These results indicate that CaMKKß/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucagon/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Proteínas Quinases Ativadas por AMP , Tecido Adiposo Branco/fisiologia , Animais , Western Blotting , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Proteínas Quinases Dependentes de AMP Cíclico/genética , Indicadores e Reagentes , Lipogênese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Estimulação Química , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
Near-infrared nanoconstructs present a potentially effective platform for site-specific and deep tissue optical imaging and phototherapy. We have engineered a polymeric nanocapsule composed of polyallylamine hydrochloride (PAH) chains cross-linked with sodium phosphate and doped with indocyanine green (ICG) toward such endeavors. The ICG-doped nanocapsules were coated covalently with polyethylene glycol (5000 daltons) through reductive amination. We administrated the constructs by tail vein injection to healthy mice. To characterize the biodistribution of the constructs, we performed in vivo quantitative fluorescence imaging and subsequently analyzed the various extracted organs. Our results suggest that encapsulation of ICG in these PEGylated constructs is an effective approach to prolong the circulation time of ICG and delay its hepatic accumulation. Increased bioavailability of ICG, due to encapsulation, offers the potential of extending the clinical applications of ICG, which are currently limited due to rapid elimination of ICG from the vasculature. Our results also indicate that PAH and ICG-doped nanocapsules (ICG-NCs) are not cytotoxic at the levels used in this study.