Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARα) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARα ligands. Here, we characterized a binding site for PPARα in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARα binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1α) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1α did not enhance CPT-1A transcription through the PPARα binding site in the second intron. Following knockdown of PGC-1α with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARα and PGC-1α stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-controlling step in the pathway of mitochondrial fatty acid oxidation. Thyroid hormone will stimulate the expression of the liver isoform of CPT-I (CPT-I␣). This induction of CPT-I␣ gene expression requires the thyroid hormone response element in the promoter and sequences within the first intron. The peroxisomal proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣) is a coactivator that promotes mitochondrial biogenesis, mitochondrial fatty acid oxidation, and hepatic gluconeogenesis. In addition, PGC-1␣ will stimulate the expression of CPT-I␣ in primary rat hepatocytes. Here we report that thyroid hormone will increase PGC-1␣ mRNA and protein levels in rat hepatocytes. In addition, overexpression of PGC-1␣ will enhance the thyroid hormone induction of CPT-I␣ indicating that PGC-1␣ is a coactivator for thyroid hormone. By using chromatin immunoprecipitation assays, we show that PGC-1␣ is associated with both the thyroid hormone response element in the CPT-I␣ gene promoter and the first intron of the CPT-I␣ gene. Our data demonstrate that PGC-1␣ participates in the stimulation of CPT-I␣ gene expression by thyroid hormone and suggest that PGC-1␣ is a coactivator for thyroid hormone.

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 6,000 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 (ERRα) stimulates the expression of PDK4. Here, we determined that one of the ERRα binding sites contributes to the insulin inhibition of PDK4. A binding site for the forkhead transcription factor (FoxO1) is adjacent to the ERRα 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.

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Carnitine palmitoyltransferase-I (CPT-I) catalyzes the rate-controlling step of fatty acid oxidation. CPT-I converts long-chain fatty acyl-CoAs to acylcarnitines for translocation across the mitochondrial membrane. The mRNA levels and enzyme activity of the liver isoform, CPT-I␣, are greatly increased in the liver of hyperthyroid animals. Thyroid hormone (T3) stimulates CPT-I␣ transcription far more robustly in the liver than in nonhepatic tissues. We have shown that the thyroid hormone receptor (TR) binds to a thyroid hormone response element (TRE) located in the CPT-I␣ promoter. In addition, elements in the first intron participate in the T3 induction of CPT-I␣ gene expression, but the CPT-I␣ intron alone cannot confer a T3 response. We found that deletion of sequences in the first intron between ؉653 and ؉744 decreased the T3 induction of CPT-I␣. Upstream stimulatory factor (USF) and CCAAT enhancer binding proteins (C/EBPs) bind to elements within this region, and these factors are required for the T3 response. The binding of TR and C/EBP to the CPT-I␣ gene in vivo was shown by the chromatin immunoprecipitation assay. We determined that TR can physically interact with USF-1, USF-2, and C/EBP␣. Transgenic mice were created that carry CPT-I␣-luciferase transgenes with or without the first intron of the CPT-I␣ gene. In these mouse lines, the first intron is required for T3 induction as well as high levels of hepatic expression. Our data indicate that the T3 stimulates CPT-I␣ gene expression in the liver through a T3 response unit consisting of the TRE in the promoter and additional factors, C/EBP and USF, bound in the first intron.