RESUMO
The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK) inhibits its activity. The expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene is increased in fasting and other conditions associated with the switch from the utilization of glucose to fatty acids as an energy source. Transcription of the PDK4 gene is elevated by glucocorticoids and inhibited by insulin. In this study, we have investigated the factors involved in the regulation of the PDK4 gene by these hormones. Glucocorticoids stimulate PDK4 through two glucocorticoid receptor (GR) binding sites located more than 6000 base pairs upstream of the transcriptional start site. Insulin inhibits the glucocorticoid induction in part by causing dissociation of the GR from the promoter. Previously, we found that the estrogen related receptor alpha (ERRalpha) stimulates the expression of PDK4. Here, we determined that one of the ERRalpha binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRalpha binding sites. FoxO1 participates in the glucocorticoid induction of PDK4 and the regulation of this gene by insulin. Our data demonstrate that glucocorticoids and insulin each modulate PDK4 gene expression through complex hormone response units that contain multiple factors.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Insulina/farmacologia , Isoenzimas/metabolismo , Proteínas Quinases/metabolismo , Animais , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genes Reporter , Glucocorticoides/metabolismo , Humanos , Insulina/metabolismo , Isoenzimas/genética , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Peroxisomal proliferator activated receptor gamma coactivator-1 (PGC-1alpha) is a transcriptional coactivator that promotes mitochondrial biogenesis and energy metabolism in brown fat, skeletal muscle and heart. Previous studies demonstrated that PGC-1alpha is present at low levels in the liver but that the hepatic abundance of PGC-1alpha is elevated in diabetic and fasted animals. Elevated PGC-1alpha expression is associated with increased fatty acid oxidation and hepatic glucose production. Carnitine palmitoyltransferase-I (CPT-I) is a rate controlling step in the mitochondrial oxidation of long chain fatty acids. CPT-I transfers the acyl moiety from fatty acyl-CoA to carnitine for the translocation of long chain fatty acids across the mitochondrial membrane. There are two isoforms of CPT-I including a liver isoform CPT-Ialpha and a muscle isoform CPT-Ibeta. Here, we characterized the regulation of CPT-Ialpha isoform by PGC-1alpha. PGC-1alpha stimulates CPT-Ialpha primarily through multiple sites in the first intron. We found that PGC-1alpha can induce CPT-Ialpha gene expression in cardiac myocytes and primary hepatocytes. Our results indicate that PGC-1alpha elevates the expression of CPT-Ialpha via a unique mechanism that utilizes elements within the intron.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Regulação Enzimológica da Expressão Gênica , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Carnitina O-Palmitoiltransferase/biossíntese , Células Cultivadas , Indução Enzimática , Masculino , Células Musculares/metabolismo , Ratos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Thyroid hormone and cAMP stimulate transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). CCAAT enhancer-binding proteins (C/EBP(alpha) and beta) are involved in multiple aspects of the nutritional, developmental and hormonal regulation of PEPCK gene expression. Previously, we have identified a thyroid hormone response element in the PEPCK promoter and demonstrated that C/EBP proteins bound to the P3(I) site are participants in the induction of PEPCK gene expression by thyroid hormone and cAMP. Here, we identify several peptide regions within the transactivation domain of C/EBP(alpha) that enhance the ability of T(3) to stimulate gene transcription. We also demonstrate that several conserved amino acids in the transactivation domain of C/EBP(alpha) and C/EBPbeta are required for the stimulation of basal gene expression and identify amino acids within C/EBPbeta that participate in the cAMP induction of the PEPCK gene. Finally, we show that the CREB-binding protein (CBP) enhanced the induction of PEPCK gene transcription by thyroid hormone and that CBP is associated with the PEPCK gene in vivo. Our results indicate that both C/EBP proteins and CBP participate in the regulation of PEPCK gene transcription by thyroid hormone.