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 ± T3 was used to evaluate the role of thyroid hormone on serum efflux capacity. 2D-DIGE analysis of serum proteins showed that T3 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 T3. 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 T3-treated rats stimulated efflux via ABCA1 but not by ABCG1 or SR-BI. Gel filtration chromatography revealed that T3 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 T3.
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  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␣ (peroxisome proliferator-activated receptor ␥ coactivator) enhances the T 3 induction of rPDK4. Following T 3 administration, there is an increase in the association of PGC-1␣ 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␣ mRNA and protein in the liver. In addition, we observed greater association of PGC-1␣ not only with the rPDK4 gene but also with phosphoenolpyruvate carboxykinase and CPT-1a (carnitine palmitoyltransferase 1a) genes. Knockdown of PGC-1␣ in rat hepatocytes reduced the T 3 induction of PDK4, PEPCK, and CPT-1a genes. Our results indicate that T 3 regulates PGC-1␣ abundance and association with hepatic genes, and in turn PGC-1␣ is an important participant in the T 3 induction of selected genes. Thyroid hormone (T 3 )2 plays an important role in various aspects of metabolism, development, and differentiation of cells (1). T 3 mediates its effect on gene expression through binding to the thyroid hormone receptors (TR) (2). TRs belong to the superfamily of nuclear hormone receptors, which are a class of ligand-activated transcriptional regulators (3). There are two major TR isoforms encoded on separate genes, designated as TR␣ and TR (2). TR is the most abundant isoform in liver and mediates the hepatic actions of T 3 (4, 5). The TR binds to specific DNA sequences known as T 3 -response elements (TRE), which most commonly contain a direct repeat of the AGGTCA sequence separated by four nucleotides (DR4). TR can bind to these elements in the presence or absence of ligand to mediate positive or negative regulation of T 3 target genes (2, 6). Generally, TR binds to the TRE as a heterodimer with the retinoid X receptor (RXR) (7).Lipid and glucose metabolism are among the many physiological processes that are regulated by thyroid hormone (8, 9). In hepatocytes, T 3 increases the expression of a number of genes involved in hepatic lipogenesis, including spot 14, fatty acid transporter protein, and fatty-acid synthase (10, 11). Paradoxically, T 3 simultaneously induces genes involved in fatty acid oxidation especially CPT-1a (carnitine palmitoyltransferase-1a) (12). With respect to glucose metabolism, ...
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 T3 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 NFY 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.
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.
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