Sterol Regulatory Element-binding Protein-1c Promoter Research Articles

FoxO3 regulates hepatic triglyceride metabolism via modulation of the expression of sterol regulatory-element binding protein 1c

BackgroundExcessive intrahepatic lipid accumulation is the major characteristic of nonalcoholic fatty liver disease (NAFLD). We sought to identify the mechanisms involved in hepatic triglyceride (TG) homeostasis. Forkhead box class O (FoxO) transcription factors have been shown to play an important role in hepatic metabolism. However, little is known about the effect of FoxO3 on hepatic TG metabolism.MethodsLiver biopsy samples from patients with NALFD and liver tissues from high glucose and high sucrose (HFHS) fed mice, ob/ob mice and db/db mice were collected for protein and mRNA analysis. HepG2 cells were transfected with small interfering RNA to mediate FoxO3 knockdown, or adenovirus and plasmid to mediate FoxO3 overexpression. FoxO3-cDNA was delivered by adenovirus to the liver of C57BL/6 J male mice on a chow diet or on a high-fat diet, followed by determination of hepatic lipid metabolism. Sterol regulatory element-binding protein 1c (SREBP1c) luciferase reporter gene plasmid was co-transfected into HepG2 cells with FoxO3 overexpression plasmid.ResultsFoxO3 expression was increased in the livers of HFHS mice, ob/ob mice, db/db mice and patients with NAFLD. Knockdown of FoxO3 reduced whereas overexpression of FoxO3 increased cellular TG concentrations in HepG2 cells. FoxO3 gain-of-function caused hepatic TG deposition in C57BL/6 J mice on a chow diet and aggravated hepatic steatosis when fed a high-fat diet. Analysis of the transcripts established the increased expression of genes related to TG synthesis, including SREBP1c, SCD1, FAS, ACC1, GPAM and DGAT2 in mouse liver. Mechanistically, overexpression of FoxO3 stimulated the expression of SREBP1c, whereas knockdown of FoxO3 inhibited the expression of SREBP1c. Luciferase reporter assays showed that SREBP1c regulated the transcriptional activity of the SREBP1c promoter.ConclusionsFoxO3 promotes the transcriptional activity of the SREBP1c promoter, thus leading to increased TG synthesis and hepatic TG accumulation.

The metabolic regulator small heterodimer partner contributes to the glucose and lipid homeostasis abnormalities induced by hepatitis C virus infection

BackgroundChronic hepatitis C virus (HCV) infection can predispose the host to metabolic abnormalities. The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) has been identified as a key transcriptional regulatory factor of genes involved in diverse metabolic pathways. The protective effects of SHP against HCV-induced hepatic fibrosis have been reported. However, the exact mechanisms of its role on metabolism are largely unknown. We investigated the role of hepatic SHP in regulating glucose and lipid homeostasis, particularly in the metabolic stress response caused by HCV infection. Materials and methodsGluconeogenesis and lipogenesis levels and SHP expression were measured in HCV-infected cells, as well as in liver samples from HCV-infected patients and persistently HCV-infected mice. ResultsWe demonstrated that SHP is involved in gluconeogenesis via the acetylation of the Forkhead box O (FoxO) family transcription factor FoxO1, which is mediated by histone deacetylase 9 (HDAC9). Meanwhile, SHP regulates lipogenesis in the liver via suppressing the induction of sterol regulatory element-binding protein-1c (SREBP-1c) expression by the SUMOylation of Liver X receptor α (LXRα) at the SREBP-1c promoter. In particular, SHP can be strongly reduced upon stimulation, such as by HCV infection. The SHP expression levels were decreased in the livers from the CHC patients and persistently HCV-infected mice, and a negative correlation was observed between the SHP expression levels and gluconeogenic or lipogenic activities, emphasizing the clinical relevance of these results. ConclusionsOur results suggest that SHP is involved in HCV-induced abnormal glucose and lipid homeostasis and that SHP could be a major target for therapeutic interventions targeting HCV-associated metabolic diseases.

Ursolic Acid, a Novel Liver X Receptor α (LXRα) Antagonist Inhibiting Ligand-Induced Nonalcoholic Fatty Liver and Drug-Induced Lipogenesis.

Nonalcoholic fatty liver disease (NAFLD) is a very common liver disease, and its incidence has significantly increased worldwide. The liver X receptor α (LXRα) is a multifunctional nuclear receptor that controls lipid homeostasis. Inhibition of LXRα transactivation may be beneficial for NAFLD and hyperlipidemia treatment. Ursolic acid (UA) is a plant triterpenoid with many beneficial effects; however, the mechanism of its action on LXRα remains elusive. We evaluated the effects of UA on T0901317 (T090)-induced LXRα activation and steatosis. UA significantly decreased the LXR response element and sterol regulatory element-binding protein-1c ( SREBP-1c) gene promoter activities, mRNA, protein expression of LXRα target genes, and hepatic cellular lipid content in a T090-induced mouse model. A molecular docking study indicated that UA bound competitively with T090 at the LXRα ligand binding domain. UA stimulated AMP-activated protein kinase (AMPK) phosphorylation in hepatic cells and increased corepressor, small heterodimer partner-interacting leucine zipper protein (SMILE) but decreased coactivator, steroid receptor coactivator-1 (SRC-1) recruitment to the SREBP-1c promoter region. In contrast, UA induced SRC-1 binding but decreased SMILE binding to reverse cholesterol transport-related gene promoters in intestinal cells, increasing lipid excretion from intestinal cells. Additionally, UA reduced valproate-induced LXRα mediated and rifampin-induced pregnane X receptor mediated lipogenesis, offering potential treatments for drug-induced hepatic steatosis. Thus, UA displays liver specificity and can be selectively repressed while RCT stimulation by LXRα is preserved and enhanced. This is a novel therapeutic option to treat NAFLD and may be helpful in developing LXR agonists to prevent atherosclerosis.

Oleanolic Acid Inhibits Liver X Receptor Alpha and Pregnane X Receptor to Attenuate Ligand-Induced Lipogenesis.

Liver X receptor α (LXRα) controls important biological and pathophysiological processes such as lipid homeostasis. Inhibiting LXRα transactivation may beneficial in the treatment of nonalcoholic fatty liver disease (NAFLD), which is one of the main causes of liver diseases and hyperlipidemia. Oleanolic acid (OA) is a naturally occurring triterpenoid found in many plants. It has several beneficial effects on biological pathways; however, the mechanisms underlying its effects on LXRα are unclear. Therefore, we evaluated the effects of OA on T0901317-induced LXRα activation and explored whether OA can attenuate hepatic lipogenesis. The results showed that OA significantly decreased the promoter activities of LXR response element and sterol regulatory element binding protein-1c (SREBP-1c). It also decreased the mRNA and protein expression of LXRα target genes. These resulted in reduced hepatocellular lipid content. Our results also revealed that the overall binding pose of OA is similar to the X-ray pose of T0901317. Furthermore, OA stimulated AMP-activated protein kinase phosphorylation in hepatic cells. Additionally, it increased small heterodimer partner-interacting leucine zipper protein (SMILE) but decreased steroid receptor coactivator-1 (SRC-1) recruitment to the SREBP-1c promoter region. OA also enhanced LXRα-mediated induction of reverse cholesterol transport (RCT)-related gene, ATP-binding cassette transporter (ABC) A1, and ABCG1 expression in intestinal cells. It was found that OA increased the binding of SRC-1 but decreased SMILE recruitment to the ABCG1 gene promoter region. Furthermore, it reduced valproate- and rifampin-induced LXRα- and pregnane X receptor-mediated lipogenesis, respectively, which indicates its potential benefit in treating drug-induced hepatic steatosis. The results also show that OA is liver-specific and can be selectively repressed of lipogenesis. Moreover, it preserves and enhances LXRα-induced RCT stimulation. The results show that OA may be a promising treatment for NAFLD. Additionally, it can be used in the development of LXRα agonists to prevent atherosclerosis.

JAZF1 ameliorates age and diet-associated hepatic steatosis through SREBP-1c -dependent mechanism

JAZF zinc finger 1 (JAZF1) is involved in glucose and lipid metabolisms. However, its role in aging- and nutrient-related hepatic steatosis is unclear. In the current study, we demonstrated that JAZF1 expression was markedly down-regulated in obesity-associated mice and nonalcoholic fatty liver disease (NAFLD) patients. During aging, JAZF1 expression was gradually down-regulated in both C57BL/6 J and JAZF1-Tg mice. In JAZF1-Tg mice, body fat content and hepatosteatosis were protected from HFD-induced steatosis, and accompanied by decreased lipogenesis gene expression. The inhibitory effects of hepatic steatosis in JAZF1-Tg mice, however, were disappeared during aging. In hepatocytes, over-expression of JAZF1 attenuated, while knockdown of JAZF1 enhanced the expression of lipogenesis genes. The over-expressing of JAZF1 in hepatocytes displayed the increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased sterol regulatory element-binding protein 1c (SREBP-1c) expression. The roles of JAZF1 were partially attenuated by Compound C. Mechanistically, JAZF1 suppressed SREBP-1c expression through the inhibition of transcriptional activity of liver X receptor response elements (LXREs) in the SREBP-1c promoter. Data illustrate that JAZF1 may have a crucial role in the regulation of age and nutrient-associated hepatosteatosis through an AMPK/SREBP-1c-dependent mechanism.

HCBP6 upregulates human SREBP1c expression by binding to C/EBPβ-binding site in the SREBP1c promoter.

Sterol regulatory element-binding protein-1c (SREBP1c) plays an important role in triglyceride (TG) homeostasis. Although our previous study showed that hepatitis C virus core-binding protein 6 (HCBP6) regulates SREBP1c expression to maintain intracellular TG homeostasis, the mechanism underlying this regulation is unclear. In the present study, we found that HCBP6 increased intracellular TG levels by upregulating SREBP1c expression. HCBP6 increased SREBP1c transcription by directly binding to the SREBP1c promoter (at the −139- to +359-bp region). Moreover, we observed that HCBP6 interacted with C/EBPβ-binding site in the SREBP1c promoter both in vitro and in vivo. These results indicate that HCBP6 upregulates human SREBP1c expression by binding to the C/EBPβ-binding site in the SREBP1c promoter.

Lithospermum erythrorhizon Root and its Naphthoquinones Repress SREBP1c and Activate PGC1α Through AMPKα.

To examine specific molecular mechanisms involved in modulating hepatic lipogenesis and mitochondria biogenesis signals by Lithospermum erythrorhizon (gromwell) root extract. Stable cell lines with luciferase reporter constructs were generated to examine sterol regulatory element binding protein 1c (SREBP1c) and peroxisome proliferator-activated receptor gamma, coactivator 1 (PGC1) α promoter activity and estrogen-related receptor (ERR) α response element activity. Gene expression of SREBP1c, stearoyl coenzyme A desaturase 1, and PGC1α was measured by using reverse transcription polymerase chain reaction. Lipogenesis was measured in human hepatoma cells with Nile red staining and flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) α was determined by using ELISA and Western blot. Gromwell root extract and its naphthoquinones dose-dependently repressed high glucose and liver X receptor α induction of SREBP1c promoter activity and gene expression. Hepatic lipogenesis was repressed, and PGC1α promoter and gene expression and ERRα response element activity were increased by gromwell root extract. Gromwell root extract, shikonin, and α-methyl-n-butyrylshikonin increased AMPKα phosphorylation, and inhibition of AMPK blunted the repression in SREBP1c promoter activity by gromwell root extract and its naphthoquinones. Data suggest that gromwell root extract and its naphthoquinones repress lipogenesis by increasing the phosphorylated state of AMPKα and stimulating mitochondrial biogenesis signals.

Nuclear receptor subfamily 1 group H member 2 (LXRB) is the predominant liver X receptor subtype regulating transcription of 2 major lipogenic genes in goat primary mammary epithelial cells.

In ruminants, recent research has identified a crucial role for liver X receptors (LXR) in regulating lipid metabolism in mammary cells. However, the differences between LXR subtypes in regulating ruminant lipid metabolism are unknown. We used overexpression and knockdown of LXRA and LXRB in goat primary mammary epithelial cells to distinguish subtype-specific regulation of sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FASN) mRNA expression and their promoter activity. Incubation with siRNA targeting LXR decreased expression of LXRA and LXRB. Knockdown of LXRA and LXRB in cells incubated with dimethyl sulfoxide (DMSO, control) had no effect on the expression of SREBP1c or FASN. Knockdown of LXRB in cells incubated with LXR ligand T0901317 (T09) led to decreased expression of FASN, but not SREBP1c. Overexpression of LXRB plus T09 dramatically upregulated SREBP1c and FASN to levels higher than overexpression of LXRA with T09. Luciferase reporter assays in cells with site-directed mutagenesis of LXR response elements (LXRE; LXRE1 from -286 to -251 bp or LXRE2 from -235 to -219 bp) revealed that the SREBP1c promoter with the wild type or either LXRE mutation in cells supplemented with T09 decreased markedly only when LXRB was knocked down. Knockdown of LXRA and LXRB had no effect on the SREBP1c promoter when cells had a double LXRE mutation. Overexpression of LXRA only in cells incubated with T09 increased the activity of the SREBP1c promoter with the wild type and the LXRE2 mutation. In contrast, compared with each control group, overexpression of LXRB dramatically increased SREBP1c promoter activity, regardless of LXRE mutation. Furthermore, in cells stimulated with T09, knockdown of either LXRA or LXRB did not alter wild-type FASN promoter activity. Knockdown of LXRA increased wild-type and LXRE-site-mutated (LXRE from -677 to -662 bp) FASN promoter activity. Overexpression of LXRB increased wild-type and LXRE-site-mutated FASN promoter activity regardless of treatment with DMSO or T09, but overexpression of LXRA altered LXRE-site-mutated FASN promoter activity only in cells treated with DMSO. Increased activation of SREBP1c or FASN promoters containing LXRE mutations after overexpression of LXRB suggested that LXRB activates endogenous SREBP1c, which can then bind to the promoter of SREBP1c via an auto-loop circuit regulatory mechanism. Collectively, these results highlight an important role for LXRB in the transcriptional regulation of SREBP1c and FASN in goat mammary epithelial cells. Activation of this nuclear receptor controls lipogenesis via different mechanisms.

20(S)-protopanaxatriol inhibits liver X receptor α-mediated expression of lipogenic genes in hepatocytes

20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has several interesting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT was identified as an inhibitor against the ligand-dependent transactivation of liver X receptor α (LXRα) using a Gal4-TK-luciferase reporter system. LXRα is a transcription factor of nuclear hormone receptor family and stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesterol transport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRα-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part, a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) of the SREBP-1c promoter. Furthermore, LXRα-dependent triglyceride accumulation in primary mouse hepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRα-dependent transcription of ABCA1, a crucial LXRα target gene involved in RCT. Chromatin immunoprecipitation assays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1c LXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activity against LXRα-mediated lipogenesis, but not LXRα-stimulated RCT.

Metformin attenuates palmitic acid-induced insulin resistance in L6 cells through the AMP-activated protein kinase/sterol regulatory element-binding protein-1c pathway

AMP-activated protein kinase (AMPK) is an important effector of metformin action on glucose uptake in skeletal muscle cells. We recently reported that metformin improved insulin receptor substrate-1 (IRS-1)-associated insulin signaling by downregulating sterol regulatory element-binding protein-1c (SREBP-1c) expression. In this study, we investigated whether AMPK activation and SREBP-1c inhibition contribute to the beneficial effects of metformin on IRS-1-associated insulin signaling in L6 myotubes. L6 muscle cells were incubated with palmitic acid (PA) to induce insulin resistance and then treated with metformin and/or the AMPK inhibitor, compound C. AMPK, SREBP-1c, IRS-1 and Akt protein expression levels were determined by western blot analysis. The effects of metformin on SREBP-1c gene transcription were determined by a luciferase reporter assay. Glucose uptake was evaluated using the 2-NBDG method. In the PA-treated L6 cells, metformin treatment enhanced AMPK phosphorylation, reduced SREBP-1c expression and increased IRS-1 and Akt protein expression, whereas treatment with compound C blocked the effects of metformin on SREBP-1c expression and the IRS-1 and Akt levels. Moreover, metformin suppressed SREBP-1c promoter activity and promoted glucose uptake through AMPK. The results from this study demonstrate that metformin ameliorates PA-induced insulin resistance through the activation of AMPK and the suppression of SREBP-1c in skeletal muscle cells.

The Hexane Fraction of cyperus rotundus Prevents Non-Alcoholic Fatty Liver Disease Through the Inhibition of Liver X Receptor α-Mediated Activation of Sterol Regulatory Element Binding Protein-1c.

The goals of this study were (1) to examine the effects of Cyperus rotundus (CR) rhizome on cellular lipogenesis and non-alcoholic/diet-induced fatty liver disease, and (2) to elucidate the molecular mechanism behind its actions. The present investigation showed that the hexane fraction of CR rhizome (CRHF) reduced the elevated transcription levels of sterol regulatory element binding protein-1c (SREBP-1c) in primary hepatocytes following exposure to the liver X receptor α (LXRα) agonist. The SREBP-1c gene is a master regulator of lipogenesis and a key target of LXRα. CRHF inhibited not only the LXRα-dependent activation of the synthetic LXR response element (LXRE) promoter, but also the activation of the natural SREBP-1c promoter. Moreover, CRHF decreased (a) the recruitment of RNA polymerase II to the LXRE of the SREBP-1c gene; (b) the LXRα-dependent up-regulation of various lipogenic genes; and (c) the LXRα-mediated accumulation of triglycerides in primary hepatocytes. Furthermore, CRHF ameliorated fatty liver disease and reduced the expression levels of hepatic lipogenic genes in high sucrose diet (HSD)-fed mice. Interestingly, CRHF did not affect the expression of ATP-binding cassette transporter A1, another important LXR target gene that is required for reverse cholesterol transport (RCT) and protects against atherosclerosis. Taken together, these results suggest that CRHF might be a novel therapeutic remedy for fatty liver disease through the selective inhibition of the lipogenic pathway.

Ezetimibe prevents hepatic steatosis induced by a high-fat but not a high-fructose diet

Nonalcoholic fatty liver disease is the most frequent liver disease. Ezetimibe, an inhibitor of intestinal cholesterol absorption, has been reported to ameliorate hepatic steatosis in human and animal models. To explore how ezetimibe reduces hepatic steatosis, we investigated the effects of ezetimibe on the expression of lipogenic enzymes and intestinal lipid metabolism in mice fed a high-fat or a high-fructose diet. CBA/JN mice were fed a high-fat diet or a high-fructose diet for 8 wk with or without ezetimibe. High-fat diet induced hepatic steatosis accompanied by hyperinsulinemia. Treatment with ezetimibe reduced hepatic steatosis, insulin levels, and glucose production from pyruvate in mice fed the high-fat diet, suggesting a reduction of insulin resistance in the liver. In the intestinal analysis, ezetimibe reduced the expression of fatty acid transfer protein-4 and apoB-48 in mice fed the high-fat diet. However, treatment with ezetimibe did not prevent hepatic steatosis, hyperinsulinemia, and intestinal apoB-48 expression in mice fed the high-fructose diet. Ezetimibe decreased liver X receptor-α binding to the sterol regulatory element-binding protein-1c promoter but not expression of carbohydrate response element-binding protein and fatty acid synthase in mice fed the high-fructose diet, suggesting that ezetimibe did not reduce hepatic lipogenesis induced by the high-fructose diet. Elevation of hepatic and intestinal lipogenesis in mice fed a high-fructose diet may partly explain the differences in the effect of ezetimibe.

Differential Regulation of Gene Expression by LXRs in Response to Macrophage Cholesterol Loading

The ability of cells to precisely control gene expression in response to intracellular and extracellular signals plays an important role in both normal physiology and in pathological settings. For instance, the accumulation of excess cholesterol by macrophages initiates a genetic response mediated by the liver X receptors (LXRs)-α (NR1H3) and LXRβ (NR1H2), which facilitates the transport of cholesterol out of cells to high-density lipoprotein particles. Studies using synthetic LXR agonists have also demonstrated that macrophage LXR activation simultaneously induces a second network of genes that promotes fatty acid and triglyceride synthesis that may support the detoxification of excess free cholesterol by storage in the ester form. We now show that treatment of human THP-1 macrophages with endogenous or synthetic LXR ligands stimulates both transcriptional and posttranscriptional pathways that result in the selective recruitment of the LXRα subtype to LXR-regulated promoters. Interestingly, when human or mouse macrophages are loaded with cholesterol under conditions that mimic the development of atherogenic macrophage foam cells, a selective LXR response is generated that induces genes mediating cholesterol transport but does not coordinately regulate genes involved in fatty acid synthesis. The gene-selective response to cholesterol loading occurs, even in the presence of LXRα binding to the promoter of the gene encoding the sterol regulatory element-binding protein-1c, the master transcriptional regulator of fatty acid synthesis. The ability of promoter bound LXRα to recruit RNA polymerase to the sterol regulatory element-binding protein-1c promoter, however, appears to be ligand selective.

FoxO1 Inhibits Sterol Regulatory Element-binding Protein-1c (SREBP-1c) Gene Expression via Transcription Factors Sp1 and SREBP-1c

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

Early growth response-1 transcription factor promotes hepatic fibrosis and steatosis in long-term ethanol-fed Long-Evans rats.

Previous studies demonstrated that the Long-Evans (LE) rats exhibited liver injury and lipid metabolic abnormalities after 8weeks of ethanol feeding. The goal of this study was to investigate if the LE rats develop more advanced hepatic abnormalities (e.g., fibrosis) after long-term feeding with an ethanol-containing Lieber-DeCarli diet. In addition, the contribution of early growth response-1 (EGR1) transcription factor to these pathological changes was assessed. Long-Evans rats were fed an ethanol-containing or isocaloric control liquid diet for 18months. Livers were processed for histological analyses, studies of fibrosis-related gene expression, cell fractionation and triglyceride measurement. Serum alanine aminotransferase (ALT) levels were assessed. DNA binding activities of p53 and the sterol regulatory element-binding protein-1c (SREBP1c) were analysed. The abundance of EGR1 and enzymes involved in fatty acid synthesis were determined. Chromatin immunoprecipitation was employed to study EGR1 binding to the SREBP1c promoter region. Ethanol feeding generated steatosis, chicken wire fibrosis and ALT elevations in the LE rats. Fibrosis was associated with the upregulation of EGR1 and its downstream target genes. EGR1 upregulation was associated with enhanced p53 activity and an increase in the cellular p66(shc) abundance. Steatosis was linked to the activation of SREBP1c. Importantly, EGR1 upregulation paralleled the expression and transcriptional activity of SREBP1c. Finally, EGR1 was shown to bind to the SREBP1c promoter region. Long-term ethanol feeding promoted steatosis and fibrosis in LE rats via EGR1 activation. The highly abundant EGR1 bound to the SREBP1c promoter and contributed to the steatosis observed in the LE rat model.

Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

Protein kinase Cbeta mediates hepatic induction of sterol-regulatory element binding protein-1c by insulin

Sterol-regulatory element binding protein-1c (SREBP-1c) is a transcription factor that controls lipogenesis in the liver. Hepatic SREBP-1c is nutritionally regulated, and its sustained activation causes hepatic steatosis and insulin resistance. Although regulation of SREBP-1c is known to occur at the transcriptional level, the precise mechanism by which insulin signaling activates SREBP-1c promoter remains to be elucidated. Here we show that protein kinase C beta (PKCbeta) is a key mediator of insulin-mediated activation of hepatic SREBP-1c and its target lipogenic genes. Activation of SREBP-1c in the liver of refed mice was suppressed by either adenoviral RNAi-mediated knockdown or dietary administration of a specific inhibitor of protein kinase C beta. The effect of PKCbeta inhibition was cancelled in insulin depletion by streptozotocin (STZ) treatment of mice. Promoter analysis indicated that PKCbeta activates SREBP-1c promoter through replacement of Sp3 by Sp1 for binding to the GC box in the sterol regulatory element (SRE) complex, a key cis-element of SREBP-1c promoter. Knockdown of Sp proteins demonstrated that Sp3 and Sp1 play reciprocally negative and positive roles in nutritional regulation of SREBP-1c, respectively. This new understanding of PKCbeta involvement in nutritional regulation of SREBP-1c activation provides a new aspect of PKCbeta inhibition as a potential therapeutic target for diabetic complications.

Nuclear Receptor Liver X Receptor Is O-GlcNAc-modified in Response to Glucose

Post-translational modification of nucleocytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRalpha and LXRbeta are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRalpha O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRalpha O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.

C/EBP transcription factors regulate SREBP1c gene expression during adipogenesis

The transcription factor SREBP1c (sterol-regulatory-element-binding protein 1c) is highly expressed in adipose tissue and plays a central role in several aspects of adipocyte development including the induction of PPARgamma (peroxisome-proliferator-activated receptor gamma), the generation of an endogenous PPARgamma ligand and the expression of several genes critical for lipid biosynthesis. Despite its significance, the regulation of SREBP1c expression during adipogenesis is not well characterized. We have noted that in several models of adipogenesis, SREBP1c expression closely mimics that of known C/EBPbeta (CCAAT/enhancer-binding protein beta) targets. Inhibition of C/EBP activity during adipogenesis by expressing either the dominant-negative C/EBPbeta LIP (liver-enriched inhibitory protein) isoform, the co-repressor ETO (eight-twenty one/MTG8) or using siRNAs (small interfering RNAs) targeting either C/EBPbeta or C/EBPdelta significantly impaired early SREBP1c induction. Furthermore, ChIP (chromatin immunoprecipitation) assays identified specific sequences in the SREBP1c promoter to which C/EBPbeta and C/EBPdelta bind in intact cells, demonstrating that these factors may directly regulate SREBP1c expression. Using cells in which C/EBPalpha expression is inhibited using shRNA (short hairpin RNA) and ChIP assays we show that C/EBPalpha replaces C/EBPbeta and C/EBPdelta as a regulator of SREBP1c expression in maturing adipocytes. These results provide novel insight into the induction of SREBP1c expression during adipogenesis. Moreover, the findings of the present study identify an important additional mechanism via which the C/EBP transcription factors may control a network of gene expression regulating adipogenesis, lipogenesis and insulin sensitivity.

N-3 polyunsaturated fatty acids suppress insulin-induced SREBP-1c transcription via reduced trans-activating capacity of LXRα

Insulin coordinately up-regulates lipogenic gene transcription via induction of sterol regulatory element binding protein-1c (SREBP-1c). Conversely, polyunsaturated fatty acids (PUFA) decrease lipogenic gene transcription via suppression of SREBP-1c. We therefore examined the ability of n-3 PUFA to mitigate induction of SREBP-1c and its downstream lipogenic targets by insulin in primary rat hepatocyte cultures. Insulin induced expression of SREBP-1c mRNA 5-6 fold as well as rat SREBP-1c promoter activity. These effects were prevented by the n-3 fatty acids eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3, DHA), but not by the monounsaturated fatty acid oleic acid (18:1 n-6, OLA). N-3 fatty acids also effectively prevented insulin induction of the downstream lipogenic enzyme targets fatty acid synthase (FAS) and acetyl carboxyl coenzyme acetyltransferase-1 (ACC-1), and reduced de novo lipogenesis. The SREBP-1c promoter contains an insulin response unit consisting of tandem LXRα response elements (LXREs) as well as sites for NF-Y, Sp1, and SREBP-1c itself. The LXREs were identified as a primary site mediating suppression of SREBP-1c transcription by n-3 PUFA. DHA effectively prevented LXRα-dependent activation of both the wild type SREBP-1c promoter and the synthetic LXRE-driven promoter, and significantly blunted LXRα-dependent activation of a Gal4-LXRα chimeric protein thus demonstrating that n-3 PUFA effectively mitigate induction of SREBP-1c by insulin via reduced trans-activation of LXRα.