Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue-and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient cotransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.
The enhanced synthesis of fatty acids in the liver and adipose tissue in response to insulin is critically dependent on the transcription factor SREBP-1c (sterol-regulatory-element-binding protein 1c). Insulin increases the expression of the SREBP-1c gene in intact liver and in hepatocytes cultured in vitro. To learn the mechanism of this stimulation, we analysed the activation of the rat SREBP-1c promoter and its truncated or mutated congeners driving a luciferase reporter gene in transiently transfected rat hepatocytes. The rat SREBP-1c promoter contains binding sites for LXR (liver X receptor), Sp1, NF-Y (nuclear factor-Y) and SREBP itself. We have found that each of these sites is required for the full stimulatory response of the SREBP-1c promoter to insulin. Mutation of either the putative LXREs (LXR response elements) or the SRE (sterol response element) in the proximal SREBP-1c promoter reduced the stimulatory effect of insulin by about 50%. Insulin and the LXR agonist TO901317 increased the association of SREBP-1 with the SREBP-1c promoter. Ectopic expression of LXRalpha or SREBP-1c increased activity of the SREBP-1c promoter, and this effect is further enhanced by insulin. The Sp1 and NF-Y sites adjacent to the SRE are also required for full activation of the SREBP-1c promoter by insulin. We propose that the combined actions of the SRE, LXREs, Sp1 and NF-Y elements constitute an insulin-responsive cis-acting unit of the SREBP-1c gene in the liver.
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 (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogenrelated receptors (ERR␣ and ERR␥). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor ␥ coactivator (PGC-1␣) stimulates the expression of PDK4. Here we report that ERR␣ and ERR␥ stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1␣ to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERR␣ and ERR␥. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1␣. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1␣ from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression. The pyruvate dehydrogenase complex (PDC)3 catalyzes the irreversible oxidative decarboxylation of pyruvate to acetylCoA (1). Long term changes in PDC activity entail changes in PDC phosphorylation, whereas short term inhibition is mediated by the reaction products acetyl-CoA and NADH (1, 2). The pyruvate dehydrogenase kinases (PDK) decrease PDC activity via phosphorylation, whereas the pyruvate dehydrogenase phosphatases activate the PDC activity by dephosphorylation (3, 4). There are three serine phosphorylation sites on the ␣-subunit of pyruvate dehydrogenase (E1) that are targeted by PDKs, and phosphorylation of the ␣-subunit of the E1 element completely inhibits the activity of PDC (4). There is increased phosphorylation of PDC in the heart and skeletal muscle in starvation and diabetes, allowing pyruvate to be conserved while fatty acid oxidation is increased (5-7). In diabetes the decrease in PDC activity is due primarily to the increased PDK activity (5).Four PDK isoenzymes (PDK1, -2, -3, -4) have been identified and characterized in mammalian tissues (1). The expression patterns of the PDK isoforms are tissue-specific (8). The PDK2 and PDK4 isoforms are highly expressed in liver, heart, and skeletal muscle (9). PDK2 and PDK4 gene expression is elevated with diabetes and starvation, with PDK4 being the most highly regulated isoform (2, 4). Insulin administration and refeeding inhibit the induction of PDK4 gene expression in the skeletal muscles and heart of diabetic and fasted animals, respectively (7, 10). In Morris hepatoma cells, long chain fatty acids, glucocorticoids, and peroxisome...
Sterol regulatory element-binding proteins (SREBPs) 3 are transcription factors that regulate expression of genes controlling cholesterol homeostasis and de novo fatty acid synthesis (1-7). SREBP-1a and SREBP-1c, which differ only in their first exon, are derived from a single gene through the use of alternative promoters, whereas SREBP-2 is encoded by a separate gene (8). Although there is clearly some functional overlap among the three SREBP isoforms (5), these proteins regulate different metabolic pathways. SREBP-1c preferentially affects transcription of genes that regulate de novo lipid synthesis, whereas SREBP-2 regulates genes involved in cholesterol biosynthesis and metabolism. The SREBP-1a isoform transactivates both lipogenic and cholesterogenic genes (9). In addition, the three SREBP isoforms exhibit differential tissue-specific expression. In replicating tumor cell lines, SREBP-1a constitutes greater than 90% of the SREBP-1 pool; conversely, SREBP-1c is the predominant isoform in liver and adipose tissue (9). Increased hepatic levels of nuclear SREBP-1c are thought to mediate the development of hyperlipidemia in type II diabetes and hyperinsulinemia (10 -12). Nutritional and hormonal factors have been shown to regulate expression of SREBP-1c and its downstream regulatory targets (10,(13)(14)(15). Insulin induces the expression of SREBP-1c mRNA and nascent precursor protein (10,16,17). Glucagon opposes this effect of insulin via its second messenger cAMP (18). Newly synthesized SREBPs contain two transmembrane domains that are embedded in the endoplasmic reticulum (ER) with the NH 2 -and COOH-terminal sequences exposed to the cytoplasm. Following transport from ER to Golgi, the transcriptionally active NH 2 -terminal segments of SREBPs are liberated by two successive cleavages; the first cleavage in the loop extending into the vesicular lumen is carried out by site 1 protease (S1P), and the second cleavage is executed within the NH 2 -proximal transmembrane domain by site 2 protease (S2P).Regulation of post-translational proteolysis has been studied most extensively in the case of SREBP-2 and SREBP-1a, both of which are regulated primarily by sterols. Within the ER, the
Summary Classic cardio-metabolic risk factors such as hypertension, stroke, diabetes and hypercholesterolemia all increase the risk of Alzheimer’s disease. We found increased transcription of β-secretase/BACE1, the rate-limiting enzyme for Aβ generation, in eNOS deficient mouse brains and after feeding mice a high fat high cholesterol diet. Up- or down-regulation of PGC-1α reciprocally regulated BACE1 in vitro and in vivo. Modest fasting in mice reduced BACE1 transcription in the brains which was accompanied by elevated PGC-1 expression and activity. Moreover, the suppressive effect of PGC-1 was dependent on activated PPARγ likely via SIRT1-mediated deacetylation in a ligand-independent manner. The BACE1 promoter contains multiple PPAR/RXR sites and direct interactions among SIRT1-PPARγ-PGC-1 at these sites were enhanced with fasting. The novel interference on the BACE1 gene identified here represents a unique non-canonical mechanism of PPARγ-PGC-1 in transcriptional repression in neurons in response to metabolic signals which may involve recruitment of a corepressor NCoR.
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.
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