Physiological feedback regulation of cholesterol biosynthesis: Role of translational control of hepatic HMG-CoA reductase and possible involvement of oxylanosterols.

Feedback regulation of cholesterol biosynthesis provides a mechanism to adapt to varying cholesterol input while maintaining rather constant serum and tissue cholesterol levels. The molecular mechanisms by which this occurs have been the subject of extensive investigation. This review focuses on the physiological mechanisms by which this regulation occurs. In animals that are sensitive to dietary cholesterol such as Golden Syrian hamsters, feedback regulation occurs mainly at the level of transcription of hepatic HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase). In animals like the Sprague Dawley rat that are resistant to the serum cholesterol raising action of dietary cholesterol, regulation occurs mainly at the level of translation efficiency of hepatic HMG-CoA reductase. Oxylanosterols were shown to effectively decrease translation of HMG-CoA reductase mRNA. Dietary cholesterol acts to significantly lower transcription of squalene epoxidase and lanosterol 14α demethylase favoring accumulation of the putative regulatory oxylanosterol-3ß-hydroxylanosterol-8-en-32-al. Thus, decreased transcription of enzymes occurring late in the cholesterol biosynthetic pathway appears to result in decreased translation of hepatic HMG-CoA reductase mRNA. These findings indicate that pronounced physiological feedback regulation of cholesterol biosynthesis in cholesterol resistant animals occurs at the level of translational efficiency without substantial reduction in hepatic HMG-CoA reductase transcription.

Distribution of the LDL receptor within clathrin-coated pits and caveolae in rat and human liver.

Several findings suggest that the low-density lipoprotein (LDL) receptor may internalize different lipoprotein particles via diverse pathways. Using a combination of discontinuous sucrose gradients and Triton solubilization studies, we demonstrated that the LDL receptor could be located simultaneously in clathrin-coated pits and caveolae in rat and human liver and in human hepatocyte-like C3A cells. Treatment with the cholesterol biosynthesis inhibitor, zaragozic acid A, shifted the distribution of the LDL receptor to clathrin containing fractions, whereas treatment with cholesterol or LDL shifted the receptor distribution towards caveolin-1 containing fractions. The LDL-dependent shift of the LDL receptor to caveolae coincided with a reduction in internalization of Bodipy-LDL. Redistribution within plasma membrane microdomains in response to specific treatments resulting in changes in LDL receptor function represents a novel paradigm that could be exploited in the development of a new class of therapeutic drugs.

Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin.

Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

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.

Technological advances in the study of HLA-DRA promoter regulation: extending the functions of CIITA, Oct-1, Rb, and RFX.

Several advances were established in examining the interaction of transcriptional factors with the HLA-DRA promoter. First, hydrodynamic injection was used to demonstrate the activation of the promoter by class II transactivator in a live mouse. Second, the Oct-1 DNA-binding site in the HLA-DRA promoter is a negative element in many cells, but here we show that Oct-1 activates the promoter independently of the Oct-1-binding site. Third, the retinoblastoma (Rb) protein is required for the induction of the endogenous HLA-DRA gene, due to a poorly understood, pleiotropic effect on the Oct-1 and YY1 repressive functions at the HLA-DRA promoter. There has never been an indication that direct promoter activation, by Rb, is possible. Here, we report that the first HLA-DRA intron has an Rb-responsive element, as indicated by a transient transfection/promoter reporter assay. Finally, RFX activates a methylated version of an HLA-DRA promoter reporter construct, consistent with the role of RFX in rescuing the expression of the methylated, endogenous HLA-DRA gene. Here, we report that this RFX function is not limited to a specific RFX-binding sequence or to the HLA-DRA promoter. These advances provide bases for novel investigations into the function of the major histocompatibility class II promoter.

Activation of the hepatic LDL receptor promoter by thyroid hormone.

The question of whether mature sterol regulatory element binding protein-2 (SREBP-2) mediates transcriptional activation of the hepatic low density lipoprotein (LDL) receptor by thyroid hormone was investigated. Western blotting analysis and electrophoretic mobility shift assays demonstrated that mature nuclear SREBP-2 protein could be detected in liver nuclear extracts prepared from normal animals but not in extracts prepared from rats rendered hypothyroid either by hypophysectomy (Hx) or thyroidectomy (Tx). Treatment of Hx rats with T3 restored LDL receptor mRNA levels in about 1 h and caused a 6-fold increase 2.5 h after T3 administration. However, no detectable mature SREBP-2 was seen in this time period despite a substantial reduction in serum cholesterol levels caused by the T3 treatment. Deletion of the SRE region from the LDL receptor promoter did not decrease the T3 response. Thus, the possibility that T3 may be mediating LDL receptor induction directly via a thyroid response element (TRE) was investigated. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that the rat LDL receptor promoter contains two functional TREs (US-TRE and 2H-TRE). Either one of these elements could support T3 induction. However, the stronger of these elements is US-TRE at-612 which binds TRbeta1 more tightly and when mutated results in a diminished T3 response. These results indicate that the rapid induction of the hepatic LDL receptor by thyroid hormone is likely due to direct interaction with TREs rather than indirectly by a mechanism involving SREBP-2.

Mammalian sirtuin 1 is involved in the protective action of dietary saturated fat against alcoholic fatty liver in mice.

This study was undertaken to elucidate the mechanism underlying the protective effect of a high saturated fat (HSF) diet against the development of alcoholic fatty liver in mice. We tested the effects of a HSF diet on the ethanol-mediated increase in hepatic sterol regulatory element binding protein 1 (SREBP-1) activity. Thirty-two male mice were divided into 4 groups and fed liquid diets consisting of either a high polyunsaturated fat (40% of energy from corn oil) or a HSF (40% of energy from cocoa butter) diet with or without ethanol for 4 wk. In the ethanol-containing diets, ethanol was substituted for an equivalent amount of carbohydrate to provide 27.5% of the total energy. Control mice were pair-fed the same volume of liquid diets as the ethanol-fed mice. The HSF diet suppressed the increase in mature SREBP-1 protein and prevented increased mRNA of the SREBP-1-regulated lipogenic enzymes in the ethanol-fed mice (P < 0.05). Sirtuins 1 (SIRT1), a NAD+-dependent class III histone deacetylase, was upregulated by ethanol administration in mice fed the HSF diet (P < 0.05). The HSF diet blocked histone H3 at lysine 9 (lys9) hyperacetylation and attenuated association of acetylated histone H3-Lys9 with the promoters of mitochondrial glycerol-3-phosphate acyltransferase and stearoyl-CoA desaturase 1 in the livers of the ethanol-fed mice. These results suggest that the protective effects of HSF diet against the development of alcoholic liver steatosis may occur via regulation of the hepatic SIRT1-SREBP-1-histone H3 axis, suppressing the expression of genes encoding lipogenic enzymes and slowing the synthesis of hepatic fatty acids.

Characterization of the rat LDL receptor 5'-flanking region.

A 1.5-kb genomic DNA fragment corresponding to the 5'-flanking region of the rat LDL receptor gene was cloned and putative regulatory regions were identified. A major transcription start site was identified at -154 bp relative to the ATG translation initiation codon, within a region containing two thyroid hormone response element half-site motifs (2H-TRE). Binding of thyroid hormone receptors alpha and beta1 to this element was demonstrated. Mutations within this 2H-TRE region abolished basal transcription levels of the rat LDL receptor gene. Reporter gene studies indicated that the promoter region between -300 and -200 bp, which contains one sterol response element (SRE) and two specificity protein-1 sites (Sp1) sites, is crucial for basal transcription of the rat LDL receptor gene. The functionality of the SRE motif was confirmed using electrophoretic mobility shift assays and reporter gene studies.

Functional analysis of the hepatic HMG-CoA reductase promoter by in vivo electroporation.

HMG-CoA reductase (HMGR) catalyzes the rate-controlling step in cholesterol production. This enzyme is highly expressed in the liver, where it is subject to extensive hormonal and dietary regulation. Although much is known about the regulation of the HMGR promoter in cultured cells, this issue has not been directly addressed in liver. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at -104 to -96 and an NF-Y site at -70 to -65 resulted in marked reduction of promoter activity. Sterol regulation of this promoter was investigated by raising tissue cholesterol levels by feeding cholesterol and by decreasing them through administration of a statin (lovastatin). Using this approach, we found that HMGR promoter constructs were sterol responsive in live animals, adding in vivo relevance to previous findings in cultured cells. We also conclude that in vivo electroporation is a convenient and powerful technique for the analysis of promoter elements in the livers of live animals.

Selective compensatory induction of hepatic HMG-CoA reductase in response to inhibition of cholesterol absorption.

The effect of the potent inhibitor of cholesterol absorption, ezetimibe, on serum cholesterol levels was tested in diabetic and thyroidectomized male Sprague-Dawley rats. Feeding diets supplemented with 1% cholesterol to the diabetic rats raised serum cholesterol levels from 132 to 514 mg/dl while decreasing hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein and mRNA levels. Addition of 10 mg/kg/day of ezetimibe to the diets of these animals lowered the serum cholesterol level to 90 mg/dl and produced a large compensatory increase in hepatic HMG-CoA reductase protein without significantly increasing mRNA levels, indicating a post-transcriptional effect. Hepatic LDL receptor protein levels in these diabetic rats were unaffected by ezetimibe treatment. In contrast, ezetimibe treatment of these young normal Sprague-Dawley rats, known to express high levels of hepatic HMG-CoA reductase, did not lower serum cholesterol levels. In thyroidectomized rats, dietary cholesterol increased serum cholesterol levels from 116 to 135 mg/dl and ezetimibe treatment lowered these elevated cholesterol levels to 85 mg/dl. Cholesterol feeding of thyroidectomized rats severely reduced hepatic HMG-CoA reductase protein, while ezetimibe treatment restored reductase protein to normal levels. Again, hepatic LDL receptor protein levels were unaffected by ezetimibe treatment of cholesterol-fed thyroidectomized rats. The data demonstrate that the cholesterol absorption inhibitor ezetimibe profoundly lowers serum cholesterol levels in animals expressing very low rates of hepatic cholesterol synthesis and produces large compensatory increases in hepatic HMG-CoA reductase expression without significantly affecting expression of hepatic LDL receptors. This indicates that ezetimibe should be most effective in lowering serum cholesterol levels in people with low rates of cholesterol synthesis/high rates of cholesterol absorption.

Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent.

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.

Target size analysis by radiation inactivation: the use of free radical scavengers.

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.

Increased sensitivity to dietary cholesterol in diabetic and hypothyroid rats associated with low levels of hepatic HMG-CoA reductase expression.

We recently postulated that hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase functions as a cholesterol buffer to protect against the serum and tissue cholesterol raising action of dietary cholesterol. This postulate predicts that diminished basal expression of hepatic HMG-CoA reductase results in increased sensitivity to dietary cholesterol. Because diabetic and hypothyroid animals are known to have markedly reduced hepatic HMG-CoA reductase, these animals were selected as models to test our postulate. When rats were rendered diabetic with streptozotocin, their hepatic HMG-CoA reductase activity decreased from 314 to 22 pmol. min(-1). mg(-1), and their serum cholesterol levels increased slightly. When the diabetic animals were challenged with a diet containing 1% cholesterol, their serum cholesterol levels doubled, and their hepatic reductase activity decreased further to 0.9 pmol. min(-1). mg(-1). Hepatic low-density lipoprotein (LDL) receptor immunoreactive protein levels were unaffected in the diabetic rats whether fed cholesterol-supplemented diets or not. In rats rendered hypothyroid by thyroparathyroidectomy, serum cholesterol levels rose from 100 to 386 mg/dl in response to the 1% cholesterol challenge, whereas HMG-CoA reductase activity dropped from 33.8 to 3.4 pmol. min(-1). mg(-1). Hepatic LDL receptor immunoreactive protein levels decreased only slightly in the hypothyroid rats fed cholesterol-supplemented diets. Taken together, these results show that rats deficient in either insulin or thyroid hormone are extremely sensitive to dietary cholesterol largely due to low basal expression of hepatic HMG-CoA reductase.

Hepatic HMG-CoA reductase expression and resistance to dietary cholesterol.

The premise that the intrinsic level of expression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase determines the relative sensitivity to the serum cholesterol raising action of dietary cholesterol was examined in 9 strains of rat. For further comparison purposes, hamsters were also examined. The basal expression of hepatic HMG-CoA reductase, extent of feedback regulation by cholesterol, and changes in serum cholesterol levels and the hepatic low-density lipoprotein (LDL) receptor in response to cholesterol challenge were determined in these animals. The Sprague-Dawley, Wistar-Furth, Spontaneously Hypertensive, Lewis, and Wistar-Kyoto rats were all very resistant to dietary cholesterol and exhibited hepatic HMG-CoA reductase activities above 150 pmol / min(-1) / mg(-1). The Buffalo, Brown Norway, and Copenhagen 2331 rats had hepatic HMG-CoA reductase activities below 90 pmol / min(-1) / mg(-1) and had increases in serum cholesterol levels ranging from 12 to 33 mg/dl when given a 4-day, 1% cholesterol challenge. The extent of feedback regulation was reduced to only 3-fold in the Fisher 344 and Brown Norway rats that exhibited significant increases in serum cholesterol levels when given a cholesterol challenge. The Golden Syrian hamsters exhibited the largest increase (197 mg/dl) in serum cholesterol levels in response to dietary cholesterol and the lowest basal expression of hepatic HMG-CoA reductase (3.3 pmol / min(-1) / mg(-1)). Hepatic LDL receptor levels were not significantly decreased by dietary cholesterol in any of the animals. The data from these inbred rats and the hamsters strongly support the conclusion that the animals expressing the highest levels of hepatic HMG-CoA reductase are the most resistant to the serum cholesterol raising action of dietary cholesterol.

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).

Peroxisome proliferator activated receptor alpha (PPARalpha) and PPAR gamma coactivator (PGC-1alpha) induce carnitine palmitoyltransferase IA (CPT-1A) via independent gene elements.

Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARalpha) 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 PPARalpha ligands. Here, we characterized a binding site for PPARalpha 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 PPARalpha 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-1) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1alpha did not enhance CPT-1A transcription through the PPARalpha binding site in the second intron. Following knockdown of PGC-1alpha with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARalpha and PGC-1alpha stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

Involvement of mammalian sirtuin 1 in the action of ethanol in the liver.

Chronic ethanol feeding causes liver steatosis in animal models by upregulating the sterol regulatory element-binding protein 1 (SREBP-1), which subsequently increases the synthesis of hepatic lipid. SREBP-1 activity is regulated by reversible acetylation at specific lysine residues. The present study tests the hypothesis that activation of SREBP-1 by ethanol may be mediated by mammalian sirtuin 1 (SIRT1), a NAD(+)-dependent class III protein deacetylase. The effects of ethanol on SIRT1 were determined in cultured rat hepatoma cells and in the livers of ethanol-fed mice. In rat H4IIEC3 cells, we observed that ethanol exposure induced SREBP-1c lysine acetylation and SREBP-1c transcriptional activity. The effect of ethanol was abolished by expression of wild-type SIRT1 or by treatment with resveratrol, a known potent SIRT1 agonist. Conversely, knocking down SIRT1 by the small silencing SIRT1 plasmid SIRT1shRNA or expression of a SIRT1 mutant, SIRT1(H363Y), did not negate the ethanol effect. These findings suggest that the effect of ethanol on SREBP-1 is mediated, at least in part, through SIRT1 inhibition. Consistent with the in vitro findings, chronic ethanol feeding substantially downregulated hepatic SIRT1 in mice. Inhibition of hepatic SIRT1 activity was associated with an increase in the acetylated active nuclear form of SREBP-1c in the livers of ethanol-fed mice. Our results indicate an essential role for SIRT1 in mediating the effects of ethanol on SREBP-1 and hepatic lipid metabolism, as well as the development of alcoholic fatty liver. Hence, SIRT1 may represent a novel therapeutic target for treatment of human alcoholic fatty liver disease.