RESUMEN
As a master regulator of metabolism, AMP-activated protein kinase (AMPK) is activated upon energy and glucose shortage but suppressed upon overnutrition. Exaggerated negative regulation of AMPK signaling by nutrient overload plays a crucial role in metabolic diseases. However, the mechanism underlying the negative regulation is poorly understood. Here, we demonstrate that high glucose represses AMPK signaling via MG53 (also called TRIM72) E3-ubiquitin-ligase-mediated AMPKα degradation and deactivation. Specifically, high-glucose-stimulated reactive oxygen species (ROS) signals AKT to phosphorylate AMPKα at S485/491, which facilitates the recruitment of MG53 and the subsequent ubiquitination and degradation of AMPKα. In addition, high glucose deactivates AMPK by ROS-dependent suppression of phosphorylation of AMPKα at T172. These findings not only delineate the mechanism underlying the impairment of AMPK signaling in overnutrition-related diseases but also highlight the significance of keeping the yin-yang balance of AMPK signaling in the maintenance of metabolic homeostasis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus/enzimología , Glucosa/farmacología , Proteínas de la Membrana/metabolismo , Músculo Esquelético/efectos de los fármacos , Obesidad/enzimología , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/genética , Animales , Glucemia/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Macaca mulatta , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Músculo Esquelético/enzimología , Obesidad/sangre , Obesidad/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
Exogenous overexpression of target genes in both general and specific cell types is important for mechanistic studies of gene function. Here, we provide a step-by-step protocol for cell culture, plasmid transfection in HEK293T, and adenoviral infection in C2C12 cells for gene overexpression in vitro, using MG53 as an example. This protocol enables sufficient and efficient gene expression for the downstream functional analysis. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2021).
Asunto(s)
Técnicas de Transferencia de Gen , Animales , Diferenciación Celular , Línea Celular , Expresión Génica , Células HEK293 , Humanos , RatonesRESUMEN
The AMP-activated protein kinase (AMPK) is a principal nutrient sensor and a master regulator of cellular energy homeostasis. Once activated, AMPK induces glucose uptake, which leads to a transient decrease in blood glucose level and can be used as an indicator of AMPK activity. Here, we present a protocol accessing AMPK activity in mice by measuring glucose uptake induced by AMPK activators, MK8722 and A769662. This protocol can be used to evaluate AMPK signaling in vivo under various pathophysiological conditions. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2021).