Nicolas Dif

Vanessa Euthine

Estelle Gonnet

Martine Laville

Hubert Vidal

Etienne Lefai
email: lefai@univ-lyon1.fr

Insulin activates human Sterol Regulatory Element-Binding Protein-1c (SREBP-1c) promoter through SRE motifs

Keywords: transcription, insulin, promoter, SREBP-1c

In the present work, we aimed to decipher the mechanisms involved in the transcriptional effect of insulin on the SREBP-1c specific promoter of the human srebf-1 gene. Using luciferase reporter gene constructs in HEK 293 cells, we demonstrated that the full effect of insulin requires the presence of sterol response elements (SRE) in the proximal region of the promoter. Furthermore, insulin increases the binding of SREBP-1 to this promoter region in ChIP assay. We also found that the nuclear receptor LXRs strongly activate SREBP-1c gene expression and identified the LXR response element involved in this effect. However, our data suggested that these LXREs do not play a major role in the response to insulin. Finally, using expression vectors and adenoviruses allowing ectopic overexpressions of the human mature forms of SREBP-1a or SREBP-1c we demonstrated the direct role of SREBP-1 in the control of SREBP-1c gene expression in human skeletal muscle cells. Altogether, these data strongly suggest that the transcription factors SREBP-1 are the main mediators of insulin action on SREBP-1c expression in human tissues.

Introduction

Regulation of gene expression is a major action of insulin in all its target tissues [START_REF] O'brien | Regulation of gene expression by insulin[END_REF] as it has been unlighted in a recent report showing the changes in the mRNA levels of about 800 genes during a 3 hours insulin infusion in the skeletal muscle of healthy individuals [START_REF] Rome | Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp[END_REF].

Several studies have evidenced important defects in the capability of insulin to regulate gene expression in peripheral tissues in type 2 diabetes mellitus [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF][START_REF] Patti | Gene expression in humans with diabetes and prediabetes: what have we learned about diabetes pathophysiology?[END_REF]. Understanding how insulin exerts its transcriptional actions appears thus as a key step in the quest of the molecular causes of insulin resistance. Up to date, the transcriptional mechanisms involved in the effects of insulin on gene expression are still poorly known. Studies of the promoters regions of well characterized insulin-regulated genes, such as fatty acid synthase (FAS) [START_REF] Latasa | Occupancy and function of the -150 sterol regulatory element and -65 E-box in nutritional regulation of the fatty acid synthase gene in living animals[END_REF], acetyl-CoA carboxylase-2 (ACC2) [START_REF] Oh | Acetyl-CoA carboxylase beta gene is regulated by sterol regulatory element-binding protein-1 in liver[END_REF] or hexokinase II (HKII) [START_REF] Gosmain | Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells[END_REF], led to the conclusion that sterol regulatory element binding protein-1c (SREBP-1c) is one of the key transcription factors mediating the effects of insulin [START_REF] Osborne | Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action[END_REF][START_REF] Foufelle | New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c[END_REF].

The sterol regulatory element binding proteins (SREBPs) are membrane bound transcription factors of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) family that have been shown to regulate gene expression of several enzymes implicate in cholesterol, lipid and glucose metabolism [START_REF] Foufelle | New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c[END_REF][START_REF] Brown | A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood[END_REF]. To date, three members of the SREBP family, SREBP-1a, SREBP-1c and SREBP-2, have been characterized. SREBP-1a and SREBP-1c differ in their N-terminal regions due to alternative usage of exon 1a (coding for 30 amino acids) or exon 1c (coding for 6 amino-acids) of the srebf-1 gene under the control of specific promoters. SREBP-2 is produced from a distinct gene [START_REF] Brown | The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor[END_REF]. All SREBPs are synthesized as transcriptionally inactive precursors that are bound to the endoplasmic reticulum (ER) and nuclear envelope [START_REF] Brown | The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor[END_REF]. They are activated by proteolytic cleavage in the Golgi apparatus to produce the N-terminal mature transcription factors that migrate into the nucleus where they can bind to sterol response elements (SRE) in the promoter region of target genes to modulate their transcription [START_REF] Brown | The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor[END_REF][START_REF] Amemiya-Kudo | Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes[END_REF].

A large number of studies have demonstrated that SREBP-1c is tightly regulated by nutritional and hormonal status, especially at the transcriptional level, in various tissues [START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF][START_REF] Shimomura | Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes[END_REF]. Fasting decreases SREBP-1c mRNA and protein levels, whereas they are markedly increases upon feeding a high carbohydrate diet [START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF][START_REF] Shimomura | Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes[END_REF]. Insulin itself was shown to be a potent inducer of SREBP-1c transcription in various cell models and in rodent tissues, including liver, adipose tissue and skeletal muscle [15,[START_REF] Azzout-Marniche | Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes[END_REF]. To date the mechanism by which insulin triggers the transcription of SREBP-1c is not fully defined. Studies of mouse and rat SREBP-1c promoter have shown that several motifs are likely to be involved for the full activation by insulin, corresponding to response elements for Liver-X-Receptors (LXREs), Sp1, Nuclear Factor-Y (NF-Y) as well as for SREBPs [START_REF] Amemiya-Kudo | Promoter analysis of the mouse sterol regulatory element-binding protein-1c gene[END_REF][START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF][START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF]. The presence of sterol response elements (SREs) suggests thus that SREBP-1c gene promoter can be directly activated by nuclear SREBPs in an auto-regulatory loop [START_REF] Amemiya-Kudo | Promoter analysis of the mouse sterol regulatory element-binding protein-1c gene[END_REF]. In addition, the transcription of SREBP-1c can also be induced by the activation of the nuclear receptors LXRs that have been implicated in the control of lipid and cholesterol metabolism [START_REF] Schultz | Role of LXRs in control of lipogenesis[END_REF]. LXRs can directly promote SREBP-1c transcription through two LXRE binding sites in the mice SREBP-1c promoter [START_REF] Yoshikawa | Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter[END_REF], and synthetic LXR agonists upregulate SREBP-1c gene expression both in vivo in rodents [START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF] and in vitro in cell models, including human muscle cells [START_REF] Cozzone | Activation of Liver X Receptors Promotes Lipid Accumulation but does not Alter Insulin Action in Human Skeletal Muscle Cells[END_REF][START_REF] Debose-Boyd | Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands[END_REF]. Furthermore, investigating mouse SREBP-1c promoter in rat primary hepatocytes, Chen et al [START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF] reported that LXRs might play a central role in insulin-mediated activation of SREBP-1c transcription.

Their study led to the conclusion that insulin may induce SREBP-1c expression through the production of an unknown ligand that activates LXRs and consequently SREBP-1c promoter transcription, while SREBPs and NF-Y play only permissive roles [START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF].

Only few studies have been performed in tissues or cells from human origin. We have shown that hyperinsulinemia increases SREBP-1c gene expression in vivo in skeletal muscle and in adipose tissue [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF]. Moreover, insulin induces SREBP-1 protein in primary culture of human skeletal muscle [START_REF] Gosmain | Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells[END_REF]. However, although the organization of SREBP-1c proximal promoter is grossly similar in rodents and humans, differences regarding the presence of additional SRE motif in the human gene might suggest distinct regulatory mechanisms.

Therefore, the aim of the present study was to investigate the regulation of the human SREBP-1c promoter in order to decipher the transcriptional mechanisms trigger by insulin in human cells. Our results indicate that insulin controls SREBP-1c transcription through SRE elements and that LXRs do not seems to play a major role in this effect. This suggests that insulin activation of SREBP-1c gene expression in human skeletal muscle is mainly the result of SREBP action itself.

Materials and methods

Materials.

Human insulin was purchased from Sigma (L'isle d'Abeau, France). The synthetic LXR agonist T0901317 was a generous gift from Dr. D. Pruneau (Fournier-Pharma, Daix, France). Restriction enzymes and molecular biology products were from Promega (Charbonnières, France) and Roche-Diagnostics (Meylan, France). Plasmids for cell transfection were prepared using Plasmid Midi Kit (Qiagen, Courtaboeuf, France).

Subjects.

For the study of SREBP-1 isoforms mRNA expression in skeletal muscle and adipose tissue, we used total RNA preparations of tissues samples obtained from 8 healthy lean volunteers who participated in a global study on insulin action on gene expression [START_REF] Rome | Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp[END_REF][START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF].

All participants gave their written consent after being informed of the nature, purpose, and possible risks of the study. The experimental protocol ("Clamp-Gene Study", agreement number 2003-039/125A) was approved by the Ethical Committees of the Hospices Civils de Lyon and performed according to the French legislation (Huriet law). The description of the subjects enrolled in the study, including their anthropometric and metabolic parameters, have been presented in details elsewhere [START_REF] Cozzone | Activation of Liver X Receptors Promotes Lipid Accumulation but does not Alter Insulin Action in Human Skeletal Muscle Cells[END_REF].

To investigate insulin action on SREBP-1 mRNA and protein expression, the subjects were submitted to a 3-h euglycemic, hyperinsulinaemic clamp with an insulin infusion rate of 2 mU. kg -1 .min -1 as described previously in detail [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF] and tissue biopsies taken under local anaesthesia before and after the hyperinsulinaemic clamp, in a fasted condition [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF].

Primary cultures of human skeletal muscle cells.

For the culture of skeletal muscle cells (myotubes), muscle biopsies (about 200 mg wet weight) were taken under local anaesthesia from the vastus lateralis muscle of healthy subjects with the approval by the Ethical Committees. Differentiated myotubes were prepared according to the procedure previously described in detail [START_REF] Cozzone | Activation of Liver X Receptors Promotes Lipid Accumulation but does not Alter Insulin Action in Human Skeletal Muscle Cells[END_REF].

Quantification of messenger RNAs. Total RNA from skeletal muscle biopsies were prepared according to a procedure based on the method of Chomczynski and Sacchi, as described [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF]. Total RNA from adipose tissue were prepared using the RNeasy kit (Qiagene).

The mRNA concentrations of the target genes were determined by RT-quantitative PCR (RT-qPCR) using a Light-Cycler (Roche Diagnostics, Meylan, France), as previously described in detail [START_REF] Rome | Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp[END_REF][START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF][START_REF] Cozzone | Activation of Liver X Receptors Promotes Lipid Accumulation but does not Alter Insulin Action in Human Skeletal Muscle Cells[END_REF]. The list of the primers and real-time PCR assay conditions are available upon request (lefai@univ-lyon1.fr). The results were normalized using Cyclophilin mRNA concentration, measured as a reference gene in each samples using RT-qPCR.

Reporter plasmids and site-directed mutagenesis. A human genomic clone (NR1-B022)

which contains NotI flanking regions corresponding to the SREBP-1c promoter was obtained from Pr E.R Zabarovsky [START_REF] Zabarovsky | NotI clones in the analysis of the human genome[END_REF] and further subcloned into the luciferase reporter gene vector pGL3-Enhancer (Promega). The longest fragment of the human SREBP-1c promoter (-1470/+90, named p1c-E) was used to generate p1c-E1 (-571/+90), p1c-E2 (-257/+90), p1c-E3 (-167/+90) and p1c-E4 (-73/+90) constructs by restriction enzyme digestion (PvuII, AatII, ApaI and PstI, respectively). Mutations were created by site-directed mutagenesis using the QuickChange kit (Stratagene, La Jolla, CA). The sequences of the primers used to generate site-directed mutations are as follows (mutated bases are indicated in bold): mLXRE1, 5'-GAGGGCCAGAGTCCGCCAGATTCCCCGGCA-3' ; mLXRE2, 5'-GGCGGAAGTCCGCTAGATTCCCCAACCCC-3' ; mSRE1, 5'-CCATTCAGCGCCGCGAGATAAAACTCGAGCCCCC-3' and mSRE2, 5'-GGCCGCGCGCGCTTATCTCATGCCCGGCCCGC-3'. The sequence of each construct was verified by digestion and sequencing before use.

Transient transfections and luciferase reporter assays. Human embryonic kidney 293 (HEK 293) cells were grown at 37°C in an atmosphere of 5% CO 2 in Dulbecco's modified Eagle's medium containing 25mM glucose, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate supplemented with 10% fetal calf serum. Transfection studies were carried out with cells plated on 12-well plates. HEK 293 were maintained in serum-free medium for 18-24 h before transfections that were made using Exgen 500 reagent (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions. Each culture well received 500 ng of a given SREBP-1c promoter/luciferase gene construct mixed with 1 ng of pRL-CMV vector (Promega). Six hours after transfection, the cells were incubated with the indicated agents for 24 h (insulin 10 -7 mol/l), T0901317 (1µM) or 0.1% DMSO (vehicle). Firefly and Renilla activities (Dual luciferase reporter assay system, Promega) were then measured using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) as previously reported [START_REF] Gosmain | Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells[END_REF].

Human SREBP-1 expression plasmids. Expression vectors encoding human mature nuclear forms of SREBP-1a (named pCMV-hSREBP1a) and SREBP-1c (named pCMV-hSREBP1c)

were generated by PCR amplification and ligation into the pcDNA3.1 expression vector (Invitrogen). The following primers were used : pCMV-hSREBP1a, 5'-GGCTGCGCCATGGACGAGCCAC-3' for sense and 5'-AGCGGTCCAGCATGCCCCGGCTGTGC-3' for antisense ; pCMV-hSREBP1c, 5'-GCAGATCGCGGAGCCATGGATTGC-3' for sense and 5'-AGCGGTCCAGCATGCCCCGGCTGTGC-3' for antisense. The sequence of each construct was verified before being use. For SREBP-1a or SREBP-1c overexpression, 50 ng of pCMV-hSREBP1a or pCMV-hSREBP1c was cotransfected with the promoter constructs plasmids.

The amount of DNA was kept constant by adding pcDNA3.1 plasmid when necessary. (vol/vol) glycerol at -20 C. Viral titer of stocks was higher than 10 8 plaque-forming units/ml.

Generation of Recombinant Adenoviruses and Infections

Chromatin Immunoprecipitation (ChIP) assay. ChIP assays were performed as previously reported with modifications [START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF]. For each experimental condition, 4 confluent 10-cm plates of HEK 293 were used. Cells were cross-linked for 5 min at room temperature with 1%

formaldehyde and the reaction stopped by adding glycine at a final concentration of 127 mM.

After 5 min, cells were scraped and washed with cold PBS containing protease inhibitor (1mM PMSF). Preparation and enzymatic fragmentation of chromatin was performed using the Enzymatic Shearing kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions. An aliquot was used to verify the fragmentation on agarose gels and to quantify the amount of DNA. For the ChIP assays, 50 µg of digested chromatin were first precleared for 1 h with protein A-sepharose beads (Amersham Biosciences AB, Orsay, France). After centrifugation, supernatants were incubated with 7 µg of anti-SREBP-1 antibody (H160) or anti-SREBP-2 (H164) or rabbit IgG (all three from Santa Cruz Biotechnology, Santa Cruz, CA) or no antibody (mock condition), and incubated overnight at 4°C under rotative agitation. The immunoprecipitated complexes were bound to protein Asepharose beads during 3 h at 4°C and washed successively in a low salt buffer, high salt buffer, LiCl buffer and Tris-EDTA buffer. Proteins were then eliminated using 200 µg of proteinase K (Promega) in the presence of 10% SDS by overnight incubation at 37°C.

Chromatin DNA was extracted with phenol/CHCl 3 followed by ethanol precipitation. Samples were dissolved in 80 µl of water. The sets of PCR primers used for the analysis of the human SREBP-1c proximal promoter were 5'-GCTCAGGGTGCCAGCGAACCAGTG-3' for sense and 5'-GGGTTACTAGCGGACGTCCGCC-3' for antisense. Primers sets for the analysis of the SREBP-1a proximal promoter were 5'-GGGACCCC-TATAACTTGGATCC-3' for sense and 5'-CTCGCGCACAATGGCAGCGGCCTCTG-3' for antisense. Primers sets for the analysis of the FAS proximal promoter were 5'-CGACGCTCATTGGCCTGG-3' for sense and 5'-CTGCCGTCTCTCTGGCTC-3' for antisense. Primers sets for the analysis of the LDL receptor proximal promoter were 5'-CACTTTCGAAGGACTGGAGTGG-3' for sense and 5'-CCACGTCATTTACAGCATTTC-3' for antisense. Primers sets for the analysis of a distal region of SREBP-1 gene (negative control) were 5'-CCCACTTCATCAAGGCAGACTCGC-3' for sense and 5'-CGACCATGTGGACTGTTGCCAAGATG-3' for antisense. PCR amplification products were analyzed on ethidium bromide-stained 3% agarose gels. Santa Cruz Biotechnology). The signal was detected using a horseradish peroxidaseconjugated secondary antibody and revealed with the enhanced chemiluminescence system (Pierce, Rockford, IL). After analysis, the membranes were stripped with the Re-Blot Plus

SREBP-1 protein analysis by

RESULTS

Effects of 3-h insulin perfusion on the expression of SREBP-1 isoforms in skeletal

muscle and adipose tissue. The mRNA levels of SREBP-1a and SREBP-1c, the two isoforms produced by the srebf-1 gene, were quantified using RT-quantitative PCR in tissues of healthy subjects, before and at the end of a 3 h hyperinsulinemic clamp. Figure 1 shows that insulin infusion significanlty increased SREBP-1c mRNA levels both in skeletal muscle and in subcutaneous abdominal adipose tissue, in agreement with previous report [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF]. In contrast, to SREBP-1c, the expression of SREBP-1a was only marginally affected during the clamp, with no change in skeletal muscle and only a slight increase in adipose tissue. These data indicated therefore that insulin preferentially affects the transcriptional regulation of SREBP-1c in human tissues. In order to define its mechanisms of action, we investigated the regulation of the SREBP-1c promoter region of the human srebf-1 gene.

Functional characterization of the human SREBP-1c promoter and effect of insulin. It was also noticeable in Figure 1 that SREBP-1c expression was lower in skeletal muscle than in adipose tissue. The observed acute response during hyperinsulinemic clamp (Fig. 1), together with in vivo data in rodents during nutritional transitions [START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF], indicate that SREBP-1c expression is tightly regulated in muscle, even if this tissue possesses only limited lipogenic capacities. This might suggest additional roles for SREBP-1c in skeletal muscle than only the control of lipogenesis-related genes [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF]. Up to now, the regulation of SREBP-1 has been mostly studied in cells and tissues caracterized by high rate of lipogenesis, such as hepatocytes or adipocytes. In the present study, to investigate the regulation of the human SREBP-1c promoter, we decided to utilize a human cell line with similar expression pattern of the SREBP-1 variants and comparable response to insulin than skeletal muscle (i.e similar mRNA levels of SREBP-1a and SREBP-1c, less than two-fold induction on SREBP-1c by insulin, and no induction on SREBP-1a). Among different cell lines, HEK-293 cells showed very similar SREBP-1 expression and regulation than what was observed in muscle. (Fig. 2, insert). This cell line is also easily transfectable and widely used for promoter studies and hormone action investigations. Copyright 2006 Biochemical Society sequence between -571 and -257 bp was deleted (Fig. 2), indicating that this region contains elements that are essential for the basal activity of the SREBP-1c promoter. Insulin treatment for 24 h (10 -7 M) significantly increased (1.8 ± 0.2 fold, p = 0.01) luciferase activity of the p1c-E construct (Fig. 2). This induction was similar to what was observed for the stimulation of SREBP-1c mRNA by insulin in HEK-293 cells (Fig. 2, insert). This stimulation by insulin was also observed with constructs p1c-E1 (1.6 ± 0.1 fold, p < 0.05) and p1c-E2 (1.6 ± 0.2 fold, p < 0.05), whereas further deletion of the 5'-end (p1c-E3) completely abolished insulin effect (Fig. 2). These results indicated thus that the region spanning -257 to -167 bp contains cis-acting sequences responsible for the response of the human SREBP-1c promoter to insulin.

Effects of mutations of LXR response elements (LXREs) on SREBP-1c promoter

activity. Two neighboring LXREs (located at -311/-296 bp and at -260/-245 bp) were present in the -571/-257 bp region that appeared to be essential for the basal promoter activity. Their implication in the basal activity and/or in the response to insulin was investigated by site-directed mutagenesis (Fig. 3). Mutation of the distal (p1c-E1m1), the proximal (p1c-E1m2) or both (p1c-E1m1/2) LXREs significantly reduced the basal activity (approximately 4 fold) of SREBP-1c promoter. This inhibitory effect was similar to the effect of the -257 deletion observed with the p1c-E2 construct (Fig. 2). Treatment of the cells with T0901317 (1µM, 24 h), a synthetic activator of the LXRs, produced a more than 3 fold increase in the basal activity of SREBP-1c promoter (Fig. 3A). Mutations of the two LXREs totally abolished the effect of T0901317 demonstrating the role of these motifs in the LXR response. The individual mutations of the LXREs also reduced the effect of the LXR agonist, but this was more pronounced when the proximal LXRE (-260/-245) was modified (Fig. 3A). These data indicate that the two LXR binding sites are functional and play an important role in the basal activity of the human SREBP-1c promoter.

The same mutated constructs were then used to investigate the role of the LXREs in the effect of insulin. The sequences of theses motifs (TTCACCCCGC and CTCACCCCAT for the the distal and the proximal motifs, respectively) are close to the consensus sequence ATCACCCCAC (number M00221 in Transfac) for sterol regulatory elements. Figure 4 shows the effect of mutations of these two potential SREBP binding sites. The mutations of the SREs (alone or in combination) had only minor effect on the basal activity of the p1c-E2 construct, but they completely suppressed the stimulatory effect of insulin (Fig. 4). These results strongly suggested that the SREs motifs are directly involved in the action of insulin on the human SREBP-1c gene promoter.

SREBPs proteins bind the SREs on the SREBP-1c promoter. As SREs are binding sites

for SREBPs proteins, we further aimed to demonstrate that SREBP-1 could actually bind to and activate the SREBP-1c promoter. Chromatin immunoprecipitation (ChIP) assay was used to demonstrate the interaction between SREBP-1 and the promoter region of SREBP-1c in HEK-293 cells. Chromatin was extract from untreated and insulin treated cells (10 -7 M for 6 h) and the H-160 SREBP-1 polyclonal antibody was used to immunoprecipitate the fragmented chromatin before PCR amplifications with srebf-1 gene specific primers. As identified motifs on the human SREBP-1c promoter are also potential targets for SREBP-2 proteins and because SREBP-2 is expressed in the HEK-293 (Figure 2, insert), we also made ChIP experiments using anti-SREBP2 antibody. In addition, control experiments were done using the well characterized LDL receptor (LDLr) and fatty acid synthase (FAS)

promoters. The quantitative aspect of the PCR amplifications was verified using serial dilution of the input as shown in the lower part of figure 5.

Using a set of primers that are specific of the SREBP-1c promoter region, we found that endogenous SREBP-1 proteins interact with the promoter in the control conditions and that treatment with insulin increased this association (Fig. 5). The observed increase in the PCR signal upon insulin treatment (as estimated from the input dilution test) indicated that insulin enhances the binding of SREBP-1 on the SREBP-1c promoter. The use of control PCR primers that are specific from exons 4/5 of srebf-1 gene (at about 5 kb from the SREBP-1c promoter), indicated that the amplifications obtained for the SREBP-1c promoter were not due to contaminating genomic DNA in the immunoprecipitate. Furthermore there was no amplification for the SREBP-1a promoter region, demonstrating that SREBP-1 binding to the srebf-1 gene is restricted to the SREBP-1c promoter, in the absence and in the presence of insulin. Using anti-SREBP2 antibody, a clear binding to SREBP-1c promoter was also shown. However, insulin did not increase SREBP2 binding to SREBP-1c promoter, suggesting that the insulin regulation of SREBP-1c expression involved SREBP-1 and not SREBP-2.

Ectopic expression of SREBP-1a and SREBP-1c increased SREBP-1c promoter activity.

To further support the implication of SREBP-1 transcription factors in regulating the SREBP-1c gene expression, we constructed expression plasmids for the mature active forms of the human SREBP-1a and SREBP-1c proteins. Figure 6 clearly shows that the expression of both SREBP-1a and SREBP-1c in HEK 293 cells produced a strong activation of the p1c-E2 promoter construct. Mutation of the distal SRE site (p1c-E2m1) reduced the effect of the SREBP-1 proteins, but the activations remained significant (8.3 ± 0.3 fold with SREBP-1a, p < 0.01, and 3.8 ± 0.2 fold with SREBP-1c, p < 0.01). Mutation of the proximal SRE site alone (p1c-E2m2) or in combination with the other SRE mutation (p1c-E2m1/2) completely abolished SREBP-1 proteins effects on SREBP-1c promoter (Fig. 6). Expression of the recombinant SREBP-1a and SREBP-1c active proteins also increased the promoter activity of the p1c-E1 construct (3.6 ± 0.4 fold and 3.2 ± 0.5 fold, respectively) and this effect was not affected by mutations of the LXREs (data not shown). Altogether these results confirmed the role of the SREs in the response of the human SREBP-1c promoter to SREBP-1 proteins.

Overexpression of SREBP-1 proteins in primary culture of human muscle cells.

To ascertain the role of SREBP-1 proteins in the regulation of SREBP-1c promoter in a muscle context, we produced adenoviruses expressing mature forms of SREBP-1a or SREBP-1c and overexpressed these proteins in human differentiated muscle cells (myotubes) prepared from muscle biopsies of healthy individuals. As shown in Figure 7A, increasing amounts of both adenoviruses led, as expected, to a proportional accumulation of nuclear SREBP-1, and, more importantly, to a dose-dependant increase in the SREBP-1 precursor protein (125 kDa). The induction of Insig-1 protein, another SREBP-1 target gene [START_REF] Kast-Woelbern | Rosiglitazone induction of Insig-1 in white adipose tissue reveals a novel interplay of peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding protein in the regulation of adipogenesis[END_REF], was studied as control. These experiments demonstrated therefore that the addition of mature nuclear forms of SREBP-1

DISCUSSION

In the present work, we attempted to decipher the mechanisms involved in the transcriptional effect of insulin on the SREBP-1c specific promoter of the human srebf-1 gene. We demonstrate that the full effect of insulin requires the presence of sterol response elements (SRE) in the proximal promoter and, using ChIP assay, that insulin increases the binding of SREBP-1 to this promoter region. Furthermore, we found that the nuclear receptor LXRs, that strongly activate SREBP-1c gene expression, do not play a major role in the effect of insulin, in contrast to what was recently observed with the rodent srebf-1 gene [START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF][START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF]. Our data suggest therefore that the transcription factors SREBP-1 are the main mediators of insulin action on SREBP-1c expression in human tissues.

Using primary hepatocytes, it was demonstrated that insulin activates the mouse SREBP-1c promoter primarily by increasing the activity of LXRs, the nuclear SREBP-1 playing only a permissive role [START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF]. Such finding was further confirmed with the rat SREBP-1c promoter, although this report also demonstrated the contribution of LXR-independent mechanisms involving SREBP-1 [START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF]. In agreement with these and other previous studies [START_REF] Repa | Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta[END_REF][START_REF] Yoshikawa | Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter[END_REF], we observed in the present work that activation of LXRs with a selective agonist robustly increases SREBP-1c transcription in human cell models. We also identified the LXRE motifs that mediate LXR action in the human promoter region. The mutation of these sites suppressed the effect of the LXR agonist, but did not influence the response to insulin.

We therefore concluded that the LXRs do not play a major role in the transcriptional effect of insulin on the human SREBP-1c promoter. The divergence in the mechanism of action of insulin between the rodent and the human genes could eventually rely on species-related differences in promoter sequences and organizations. The two functional neighboring LXREs are rather well conserved and located at about 150 nucleotides upstream of the SRE both in rodents and in humans. In the human gene, however, there are two SRE motifs, both being involved in insulin action as assessed by mutation experiments, whereas in rodents, there is only one SRE and an E-box. Mutation of the SRE abolished insulin action whereas the E-box did not appear to participate in the effect of insulin [START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF]. Another important difference that could explain the controversial results is the experimental model used to study SREBP-1c promoter activity. LXRs, notably LXRα, are markedly expressed in liver and in hepatocytes, the cell model used for the studies of the rodents promoters [START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF][START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF]. In contrast, LXRs are expressed at rather low levels in HEK 293 and in human muscle cells [START_REF] Cozzone | Activation of Liver X Receptors Promotes Lipid Accumulation but does not Alter Insulin Action in Human Skeletal Muscle Cells[END_REF]. If the full action of insulin on the SREBP-1c promoter is mediated by the combinatorial activity of LXRE and SRE, as suggested by Cagen et al. [START_REF] Chen | Central role for liver X receptor in insulin-mediated activation of Srebp-1c transcription and stimulation of fatty acid synthesis in liver[END_REF], it is possible that the relative contribution of theses two mechanisms depends on the abundance of LXR and SREBP-1 proteins. Thus we can not exclude that the LXRs do not play a role in human hepatocytes. However, it is important to consider that insulin exerts an important effect on SREBP-1c expression not only in liver, but also in various tissues, particularly in skeletal muscle [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF][START_REF] Gosmain | Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues[END_REF]. Our data strongly suggest therefore that SREBP-1 is the major mediator of insulin action on SREBP-1c promoter in human cells with low level of LXR.

Our data confirmed, using ChIP assays, that both SREBP-1 and SREBP-2 proteins can bind the SREBP-1c promoter of the human srebf-1 gene. However, insulin only increases the binding of SREBP-1. In addition, ectopic expression of SREBP-1a or SREBP-1c nuclear proteins triggers specifically the transcription of SREBP-1c mRNA and the expression of the full-length precursor protein. These data indicate therefore that the initial event in the effect of insulin on SREBP-1c gene expression is likely an action of insulin on the nuclear form of SREBP-1 to increase either its abundance in the nucleus or its transcriptional activity. This first step is immediately followed by induction of SREBP-1c mRNA and protein production leading thus to an amplification loop. A large number of genes, including FAS, ACC-2 or HKII, are regulated by insulin through an SREBP-1 dependent mechanism [START_REF] Latasa | Occupancy and function of the -150 sterol regulatory element and -65 E-box in nutritional regulation of the fatty acid synthase gene in living animals[END_REF][START_REF] Oh | Acetyl-CoA carboxylase beta gene is regulated by sterol regulatory element-binding protein-1 in liver[END_REF][START_REF] Gosmain | Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells[END_REF]. It is not known at present whether their induction by insulin is dependent on the stimulation of SREBP-1c gene expression and protein production or whether, as we propose for SREBP-1c, an activation and/or accumulation of the nuclear form of SREBP-1c is sufficient to trigger their expression. It is likely that both mechanisms contribute to the full transcriptional effects of insulin. Previous studies have proposed a direct effect of insulin on SREBP-1 transcriptional activivity, particularly for SREBP-1a, which can be phosphorylated by various protein kinases [START_REF] Roth | MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro[END_REF]. Mutation of the phosphorylation site of SREBP-1a (Ser 117 ), which is conserved in SREBP-1c, abolished insulin action on low-density lipoprotein receptor promoter [START_REF] Roth | MAP kinases Erk1/2 phosphorylate sterol regulatory element-binding protein (SREBP)-1a at serine 117 in vitro[END_REF]. The role of SREBP-1 protein phosphorylation in the action of insulin on SREBP-1c expression is not known.

Accumulation of SREBP-1 protein in the nucleus, independently from the production of newly synthesized protein, could be due to either increased cleavage of the precursor in the endoplasmic reticulum and its migration to the nucleus or to stabilization of the nuclear form already present in the basal state. It has been demonstrated that the nuclear SREBP-1a and SREBP-2 can be degradated by the proteasome following ubiquitination, and that acetylation by co-activator p300 prevents this process thus increasing the concentration of the transcription factors in the nucleus [START_REF] Giandomenico | Coactivatordependent acetylation stabilizes members of the SREBP family of transcription factors[END_REF]. More recently, phosphorylation by glycogen synthase kinase 3 (GSK3) was reported to reduce SREBP-1c transcriptional activity [START_REF] Kim | Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c[END_REF], through a phosphorylation-dependent degradation by the ubiquitin-proteasome pathway [START_REF] Sundqvist | Control of lipid metabolism by phosphorylation-dependent Biochemical Journal Immediate Publication[END_REF]. Insulin is known to reduce GSK3 activity and thus may stabilize SREBP-1c nuclear content by inhibiting its degradation. The regulation by insulin of SREBP-1c abundance in the nucleus through the GSK3 signaling pathway appears therefore as very attractive hypothesis, but it still remains to be demonstrated in human cells and in physiological situations

An alternative mechanism to increase nuclear SREBP-1 is the regulation of the cleavage process. Indeed, the SREBPs are synthesized as transcriptionally inactive precursors bound to the endoplasmic reticulum. They undergo proteolytic cleavage in the Golgi apparatus to release the mature transcription factor that can migrate to the nucleus [START_REF] Sakai | Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment[END_REF]. The regulatory proteins essential for the cleavage process are SCAP (SREBP cleavage-activating protein) and the Insigs (insulin-induced genes). In the endoplasmic reticulum, SCAP forms a complex with newly synthesized SREBPs precursors and escorts them to the Golgi apparatus where they are processed by two proteases [START_REF] Sakai | Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembrane segment[END_REF]. Insig-1 and Insig-2 promote SREBP retention in the endoplasmic reticulum by interacting with SCAP, thus preventing SREBP translocation to the Golgi apparatus for proteolytic processing [START_REF] Yang | Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER[END_REF][START_REF] Yabe | Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins[END_REF]. Roles of Insigs proteins in the regulation of insulin action on SREBP-1c are supported by recent studies [START_REF] Hegarty | Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c[END_REF][START_REF] Engelking | Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis[END_REF]. Insulin induces Insig-1 expression, whereas it decreases Insig-2, especially the isoform Insig-2a [START_REF] Hegarty | Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c[END_REF][START_REF] Yabe | Liver-specific mRNA for Insig-2 down-regulated by insulin: implications for fatty acid synthesis[END_REF], suggesting that insulin can modulate SREBP-1 cleavage process. In agreement, it was recently reported that insulin acutely cleaves SREBP-1c precursor in vitro in rat hepatocytes and in vivo in the liver of suckling mice [START_REF] Hegarty | Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c[END_REF]. The in vivo effect was observed 20 minutes after insulin injection, before the induction of SREBP-1c mRNA, indicating that the appearance of mature SREBP-1c in the nucleus preceds the transcriptional effects of insulin [START_REF] Hegarty | Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c[END_REF].

In summary, the present study demonstrates that the effect of insulin on SREBP-1c gene expression is mediated by the transcription factors SREBP-1, and more probably by SREBP-1c itself. Together with several recent data, our results also indicate that the initial event in the effect of insulin on SREBP-1c gene expression is likely an action of the hormone on the nuclear form of SREBP-1 to increase either its transcriptional activity or its abundance in the nucleus. Further works are now needed to elucidate whether SREBP-1 nuclear accumulation is due to the stabilization of the endogenous protein, the stimulation of the precursor cleavage, or a combination of both mechanisms. Because the regulation by insulin of the expression of a number of SREBP-1 target genes, including SREBP-1c itself, is altered in the muscle and the adipose tissue of type 2 diabetic patients [START_REF] Ducluzeau | Regulation by insulin of gene expression in human skeletal muscle and adipose tissue. Evidence for specific defects in type 2 diabetes[END_REF][START_REF] Gosmain | Sterol regulatory element-binding protein-1 mediates the effect of insulin on hexokinase II gene expression in human muscle cells[END_REF], understanding the mechanisms controlling the nuclear abundance of the mature SREBP-1c in human tissues is now an important issue. The luciferase reporter gene p1c-E2 and SRE mutated constructs were obtained as described in materials and methods. After transfection, HEK 293 cells were incubated for 24 h with or without insulin (10 -7 mol/l). Data are expressed by reference to the basal activity of p1c-E2

and are presented as the mean ± SEM (n=3). *P < 0.05 in the presence of insulin vs. basal conditions. Human skeletal muscle cells were infected with increasing concentrations of Ad-SREBP1a or Ad-SREBP1c as indicated in materials and methods. 24 hours after infection, total cell lysates were prepared and analysed for the presence of precursor and mature forms of SREBP-1 and for INSIG-1 using specific antibodies (A). α-tubulin is shown as a loading control. In parallel, total RNA were prepared from muscle cells overexpression the SREBP-1 isoforms (24 h after adenofection) and analysed for the expression of SREBP-1a and SREBP-1c transcripts using RT-qPCR (B). Due to sequence identities between the endogenous gene and the adenoviruses, SREBP-1c transcripts were measured when SREBP-1a adenovirus was used and conversely, SREBP-1a mRNA was determined when SREBP-1c mature protein was overexpressed. Data are presented as mean ± SEM (n=3). *P < 0.05.

SREBP-1a

SREBP-1c

  Recombinant adenoviral genomes carrying the human mature forms of SREBP-1a and SREBP-1c were generated by homologous recombination as described[START_REF] Chaussade | Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport[END_REF]. Briefly, CaCl2 competent E. coli BJ5183 were co-transformed with 200 ng SwaI-linearized VmAdcDNA3 plasmid (a generous gift from Dr. S. Rusconi, Fribourg, Switzerland) and 600 ng linearized pCMV-hSREBP1a or pCMV-hSREBP1c. Recombinants were screened by PCR with the following set of primers: A = 5'-GACGGATGTGGCAAAAGTGA-3' annealing to the leftmost part of the adenoviral genome; and B = 5'-ATGGGGTGGAGACTTGGAAATC-3' annealing to portion of the CMV promoter, which is brought in by homologous recombination. Positive clones were further analyzed by restriction analysis. Positive recombinants were large-scale amplified in E. coli XL-1 blue, digested with PacI and transfected by the calcium phosphate methods in HEK 293 cells. Cytopathic effect due to virus production was observed 8-10 days after transfection. Adenoviruses were extracted by 3 freeze/thaw cycles and stored in PBS, 10%

  Western-blotting. Cells were lysed at 4°C in 200 mM NaF, 20 mM NaH 2 PO 4 , 150 mM NaCl, 50 mM Hepes, 4 mM NaVO 4 , 10 mM EDTA, 1% Triton X, 10% Glycerol and 2mM PMSF and proteins were quantified by the Bradford assay (Biorad). Aliquots of proteins (40 µg for cell extracts) were separated by 10% SDS-PAGE and transferred to PVDF membrane. Efficiency of the transfer was confirmed by Ponceau staining. Membranes were then incubated with SREBP-1 antibody (H160; Santa Cruz Biotechnology). When indicated, Insig-1 protein was also analyzed (Insig-1 antibody N-19;

Figure 2

 2 Figure2shows the activity of various fragments of the 5'-flanking region of the human SREBP-1c promoter in HEK-293 cells. For the presentation of the results, the luciferase activity of the different constructs, measured in absence or in presence of insulin, was expressed by reference to p1c-E, the longest promoter fragment (-1470/+90). In the absence of insulin, the deletion of the 5'-end dramatically reduced luciferase activity when the

  Figure3Bclearly shows that the mutations of the LXREs did not modify the stimulatory effect of insulin on SREBP-1c promoter activity. Despite major reduction of the basal activity, insulin significantly increased luciferase activity with all the mutated constructs to a similar extend to what was obtained with the wild-type construct (1.5 ± 0.1 fold with p1c-E1; 1.5 ± 0.2 fold with p1c-E1m1; 1.5 ± 0.1 fold with p1c-E1m2, and 1.5 ± 0.1 fold with p1c-E1m1/2, respectively). These results demonstrated therefore that the functional LXREs are not involved in the stimulatory effect of insulin of the human SREBP-1c promoter.Effect of mutations in the sterol response elements (SREs) on SREBP-1c promoteractivity. Data from figure2suggested that insulin response relies on cis-acting elements that are located within the -257/-73 region of the SREBP-1c promoter. Two putative SREs are located in this region, at -228/-218 bp and at -127/-117 bp from the transcription start.

Figure 1 :

 1 Figure 1 : Effects of insulin infusion on the mRNA levels of SREBP-1 isoforms in skeletal muscle and adipose tissue. The mRNA levels of SREBP-1a and SREBP-1c were determined by quantitative real-time PCR in skeletal muscle and adipose tissue biopsies of healthy subjects before (open bars) and at the end (black bars) of a 3-h hyperinsulinemic euglycemic clamp. Data are presented as mean ± SEM. *P < 0.05 after vs. before 3 h of insulin infusion and ** P < 0.01 after vs. before 3 h of insulin infusion.

Figure 2 :

 2 Figure 2 : Activation of human SREBP-1c promoter by insulin. HEK 293 cells were transfected with a luciferase reporter gene driven by different constructs of the human SREBP-1c gene promoter. Cells were incubated for 24 h with (black bars) or without insulin (10 -7 mol/l) (open bars). Relative luciferase activity was calculated using a dual luciferase assay as indicated in materials and methods. Data are expressed by reference to the basal luciferase activity of p1c-E and presented as the means ± SEM for at least three independent transfection experiments. *P < 0.05 in the presence of insulin vs. basal conditions.Insert: Effect of insulin (10 -7 mol/l) on the mRNA levels of SREBP-1a, SREBP-1c and SREBP-2 in HEK 293 cells. The mRNA levels were determined by quantitative real-time PCR. Data are presented as mean ± SEM (n=4). *P < 0.05 vs. untreated cells.

Figure 3 :

 3 Figure 3 : Mutations of the LXREs in the SREBP-1c promoter reduce its response to T0901317 but not to insulin.The luciferase reporter gene p1c-E1 and LXRE mutated constructs were obtained as described in materials and methods. After transfection, HEK 293 cells were incubated for 24 h with or without T0901317 (1µM) (A) and with or without insulin (10 -7 mol/l) (B). Data are expressed by reference to the basal activity of p1c-E1 and are presented as the mean ± SEM (n=3). *P < 0.05 in the presence of T0901317 or insulin vs. basal conditions.

Figure 4 :

 4 Figure 4 : Mutations of the SREs in the proximal SREBP-1c promoter suppress transcriptional stimulation by insulin.

Figure 5 :

 5 Figure 5 : Chromatin immunoprecipitation (ChIP) assay of SREBP-1 protein association with the SREBP-1c promoter.HEK 293 cells were incubated for 6 h in control medium or in medium supplemented with insulin (10 -7 mol/l). After cross-linking chromatin DNA to the interacting proteins, specific immunoprecipitations with anti-SREBP-1 or anti-SREBP-2 or non-specific (IgG) antibodies were performed as indicated in materials and methods. The PCR products were generated by the amplifications of the SREBP-1c promoter, the SREBP-1a promoter, the FAS promoter, the LDL receptor promoter and the SREBP-1 exons 4/5 (negative control). Amplification products were resolved in 3% agarose gels stained with ethidium bromide. The figure is representative of three independent experiments. Mock : no antibody condition ; C : untreated cells ; I : cells treated with insulin. The lower part of the figure shows the quantitative aspect of the PCR amplification using serial dilutions of the input.

Figure 6 : 1 (

 61 Figure 6 : Effects of human SREBP-1a or SREBP-1c overexpression on the SREBP-1c promoterEach reporter gene construct was cotransfected with an empty expression vector pcDNA3.1 (control condition) or an expression vector for mature form of the human SREBP-1a (pCMV-hSREBP1a) or the human SREBP-1c (pCMV-hSREBP1c) in HEK 293 cells. Luciferase activity was measured 24 h after transfection. Data are expressed by reference to the basal activity of p1c-E2 and are presented as the mean ± SEM (n=3). *P < 0.05 in the presence of SREBP-1a or SREBP-1c vs. control conditions.

Figure 7 :

 7 Figure 7 : Effects of adenoviral expression of human SREBP-1a or SREBP-1c in differentiated skeletal muscle cells

  Figure 1

ACKNOWLEDGMENTS

We thank Dr. R. Rabasa-Lhoret, Dr. F. Guebre-Egziabher and Dr. D. Jacquet for performing the clamp studies and the tissue biopsies used in the present work. This work was supported in part by research grants from INSERM and INRA. N. Dif is the recipient of a doctoral fellowship from the Ministère de l'Enseignement Superieur et de la Recherche, France.

protein (either 1a or 1c) induces the expression of srebf-1 gene in human muscle cells, as evidenced by the production of SREBP-1 precursor protein. The lack of specific SREBP-1a and SREBP-1c antibody did not allow to determine which isoform was induced. To do so, we monitored the changes in SREBP-1a and SREBP-1c mRNAs upon adenoviral infections using RT-quantitative PCR. Due to sequence identities between the endogenous gene and the adenoviruses, SREBP-1c transcripts were measured when SREBP-1a adenovirus was used and conversely, SREBP-1a mRNA was determined when SREBP-1c mature protein was overexpressed. Figure 7B shows a clear induction of SREBP-1c mRNA and no change in SREBP-1a transcript when overexpressing the mature active forms of SREBP-1. Together with the ChIP experiment (Fig. 5), these data strongly suggest that the regulation of srebf-1 gene by SREBP-1 proteins is restricted to the SREBP-1c isoform.