Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by thyroid hormone: role of the peroxisome proliferator-activated receptor gamma coactivator (PGC-1 alpha).

PDK4 (pyruvate dehydrogenase kinase 4) regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). PDC catalyzes the conversion of pyruvate to acetyl-CoA and is an important control point in glucose and pyruvate metabolism. PDK4 gene expression is stimulated by thyroid hormone (T(3)), glucocorticoids, and long chain fatty acids. The effects of T(3) on gene expression in the liver are mediated via the thyroid hormone receptor. Here, we have identified two binding sites for thyroid hormone receptor beta in the promoter of the rat PDK4 (rPDK4) gene. In addition, we have investigated the role of transcriptional coactivators and found that the PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator) enhances the T(3) induction of rPDK4. Following T(3) administration, there is an increase in the association of PGC-1 alpha with the rPDK4 promoter. Interestingly, this increased association is with the proximal rPDK4 promoter rather than the distal region of the gene that contains the T(3) response elements. Administration of T(3) to hypothyroid rats elevated the abundance of PGC-1 alpha mRNA and protein in the liver. In addition, we observed greater association of PGC-1 alpha not only with the rPDK4 gene but also with phosphoenolpyruvate carboxykinase and CPT-1a (carnitine palmitoyltransferase 1a) genes. Knockdown of PGC-1 alpha in rat hepatocytes reduced the T(3) induction of PDK4, PEPCK, and CPT-1a genes. Our results indicate that T(3) regulates PGC-1 alpha abundance and association with hepatic genes, and in turn PGC-1 alpha is an important participant in the T(3) induction of selected genes.

In vivo identification of promoter elements and transcription factors mediating activation of hepatic HMG-CoA reductase by T3.

The promoter elements and transcription factors necessary for triiodothyronine (T3) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T3 response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T(3) treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T3 induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T3-induction of hepatic HMGR transcription.

Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3.

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp - 612 and - 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRß1 to the - 612 and - 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRß1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

Mechanism of resistance to dietary cholesterol.

Background. Alterations in expression of hepatic genes that could contribute to resistance to dietary cholesterol were investigated in Sprague-Dawley rats, which are known to be resistant to the serum cholesterol raising action of dietary cholesterol. Methods. Microarray analysis was used to provide a comprehensive analysis of changes in hepatic gene expression in rats in response to dietary cholesterol. Changes were confirmed by RT-PCR analysis. Western blotting was employed to measure changes in hepatic cholesterol 7α hydroxylase protein. Results. Of the 28,000 genes examined using the Affymetrix rat microarray, relatively few were significantly altered. As expected, decreases were observed for several genes that encode enzymes of the cholesterol biosynthetic pathway. The largest decreases were seen for squalene epoxidase and lanosterol 14α demethylase (CYP 51A1). These changes were confirmed by quantitative RT-PCR. LDL receptor expression was not altered by dietary cholesterol. Critically, the expression of cholesterol 7α hydroxylase, which catalyzes the rate-limiting step in bile acid synthesis, was increased over 4-fold in livers of rats fed diets containing 1% cholesterol. In contrast, mice, which are not resistant to dietary cholesterol, exhibited lower hepatic cholesterol 7α hydroxylase (CYP7A1) protein levels, which were not increased in response to diets containing 2% cholesterol.

Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter.

OBJECTIVE: The goal of this study was to examine the effects of thyroid hormone status on the ability of serum to accept cellular cholesterol. METHODS AND RESULTS: Sera from hypophysectomized rats treated ± T(3) was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T(3) treated rats had increased ApoA-I, ApoA-IV and fetuin A levels with decreased Apo E levels. Microarray and real-time RT-PCR analysis of rat liver revealed large increases in ApoA-I, ApoA-IV, ABCG5, and ABCG8 in response to T(3). J774 macrophages, BHK cells, and Fu5AH rat hepatoma cells were used to measure cholesterol efflux mediated by ABCA1, ABCG1 transporters or SR-BI. Sera from T(3)-treated rats stimulated efflux via ABCA1 but not by ABCG1 or SR-BI. Gel filtration chromatography revealed that T(3) treatment caused a decrease in HDL particle size accompanied by higher levels of lipid-poor ApoA-I. CONCLUSIONS: Thyroid hormone enhances the ability of serum to accept cellular cholesterol via the ABCA1 transporter. This effect is most likely attributable to increases in small HDL and lipid poor ApoA-I in response to T(3).