RESUMEN
The metabolic syndrome is a collection of risk factors including obesity, insulin resistance and hepatic steatosis, which occur together and increase the risk of diseases such as diabetes, cardiovascular disease and cancer. In spite of intense research, the complex etiology of insulin resistance and its association with the accumulation of triacylglycerides in the liver and with hepatic steatosis remains not completely understood. Here, we performed quantitative measurements of 144 proteins involved in the insulin-signaling pathway and central metabolism in liver homogenates of two genetically well-defined mouse strains C57BL/6J and 129Sv that were subjected to a sustained high-fat diet. We used targeted mass spectrometry by selected reaction monitoring (SRM) to generate accurate and reproducible quantitation of the targeted proteins across 36 different samples (12 conditions and 3 biological replicates), generating one of the largest quantitative targeted proteomics data sets in mammalian tissues. Our results revealed rapid response to high-fat diet that diverged early in the feeding regimen, and evidenced a response to high-fat diet dominated by the activation of peroxisomal ß-oxidation in C57BL/6J and by lipogenesis in 129Sv mice.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/metabolismo , Insulina/metabolismo , Lipogénesis/genética , Obesidad/metabolismo , Peroxisomas/metabolismo , Proteoma/metabolismo , Transducción de Señal , Adipogénesis/genética , Animales , Hígado Graso/etiología , Hígado Graso/genética , Regulación de la Expresión Génica , Resistencia a la Insulina/genética , Espectrometría de Masas , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/genética , Oxidación-Reducción , Peroxisomas/genética , Proteoma/genética , Especificidad de la EspecieRESUMEN
Early adaptive responses to hypoxia are essential for cell survival, but their nature and underlying mechanisms are poorly known. We have studied the post-transcriptional changes in the proteome of mammalian cells elicited by acute hypoxia and found that phosphorylation of eukaryotic elongation factor 2 (eEF2), a ribosomal translocase whose phosphorylation inhibits protein synthesis, is under the precise and reversible control of O(2) tension. Upon exposure to hypoxia, phosphorylation of eEF2 at Thr(56) occurred rapidly (<15 min) and resulted in modest translational arrest, a fundamental homeostatic response to hypoxia that spares ATP and thus facilitates cell survival. Acute inhibitory eEF2 phosphorylation occurred without ATP depletion or AMP kinase activation. Furthermore, eEF2 phosphorylation was mimicked by prolyl hydroxylase (PHD) inhibition with dimethyloxalylglycine or by selective PHD2 siRNA silencing but was independent of hypoxia-inducible factor α stabilization. Moreover, overexpression of PHD2 blocked hypoxic accumulation of phosphorylated eEF2. Therefore, our findings suggest that eEF2 phosphorylation status (and, as a consequence, translation rate) is controlled by PHD2 activity. They unravel a novel pathway for cell adaptation to hypoxia that could have pathophysiologic relevance in tissue ischemia and cancer.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hipoxia/enzimología , Hipoxia/genética , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Biosíntesis de Proteínas , Adenosina Trifosfato/metabolismo , Línea Celular , Humanos , Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Procolágeno-Prolina Dioxigenasa/genéticaRESUMEN
The Ca(2+)- and voltage-dependent K+ (maxi-K) channel beta(1)-subunit mRNA is particularly abundant in cardiomyocytes but its functional role is unknown. This is intriguing because functional maxi-K channels are not found in cardiomyocyte plasmalemma, although they have been suggested to be in the inner mitochondrial membrane and participate in cardioprotection. We report here that beta(1) protein may interact with mitochondrial proteins and that the beta(1)-subunit gene (KCNMB1) is repressed by sustained hypoxia in dispersed cardiomyocytes as well as in heart intact tissue. The effect of hypoxia is time- and dose-dependent, is mimicked by addition of reactive oxygen species, and selectively requires hypoxia inducible factor-2alpha (Hif-2alpha) stabilization. We have observed that adaptation to hypoxia exerts a protective role on cardiomyocytes subjected to ischemia and that, unexpectedly, this form of preconditioning absolutely depends on Hif-2alpha. Interference of the beta(1)-subunit mRNA increases cardiomyocyte resistance to ischemia. Therefore, Hif-2alpha-mediated beta(1)-subunit gene repression is a previously unknown mechanism that could participate in the gene expression program triggered by sustained hypoxia to prevent deleterious mitochondrial depolarization and ATP deficiency in cardiac cells. Our work provides new perspectives for research on cardiac preconditioning.