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• W2016126865 abstract "We evaluated the hypothesis of sterol-regulatory element-binding protein (SREBP)-1c being a general mediator of the transcriptional effects of insulin, with a focus on adipocytes, in which insulin profoundly influences specific gene expression. Using real time quantitative reverse transcriptase-PCR to monitor changes in the expression of about 50 genes that cover a wide range of adipocyte functions, we have compared the impact of insulin treatment with that of adenoviral overexpression of either dominant positive or dominant negative SREBP-1c mutants in 3T3-L1 adipocytes. As expected, insulin up-regulated, dominant positive stimulated, and dominant negative decreased previously characterized direct SREBP targets (FAS, SCD-1, and low density lipoprotein receptor). We also identified three novel SREBP-1c transcriptional targets in adipocytes, which were confirmed by run-on assays: plasminogen activator inhibitor 1, CCAAT/enhancer-binding protein δ (C/EBPδ), and C/EBPβ. Because most insulin-regulated genes were also modulated by SREBP-1c mutants, our data establish that 1) SREBP-1c is an important mediator of insulin transcriptional effects in adipocytes, and 2) C/EBPβ is under the direct control of SREBP-1c, as demonstrated by the ability of SREBP-1c to activate the transcription from C/EBPβ promoter through canonical SREBP binding sites. Thus, some of the effects of insulin and/or SREBP-1c in mature fat cells might require C/EBPβ or C/EBPδ as transcriptional relays. We evaluated the hypothesis of sterol-regulatory element-binding protein (SREBP)-1c being a general mediator of the transcriptional effects of insulin, with a focus on adipocytes, in which insulin profoundly influences specific gene expression. Using real time quantitative reverse transcriptase-PCR to monitor changes in the expression of about 50 genes that cover a wide range of adipocyte functions, we have compared the impact of insulin treatment with that of adenoviral overexpression of either dominant positive or dominant negative SREBP-1c mutants in 3T3-L1 adipocytes. As expected, insulin up-regulated, dominant positive stimulated, and dominant negative decreased previously characterized direct SREBP targets (FAS, SCD-1, and low density lipoprotein receptor). We also identified three novel SREBP-1c transcriptional targets in adipocytes, which were confirmed by run-on assays: plasminogen activator inhibitor 1, CCAAT/enhancer-binding protein δ (C/EBPδ), and C/EBPβ. Because most insulin-regulated genes were also modulated by SREBP-1c mutants, our data establish that 1) SREBP-1c is an important mediator of insulin transcriptional effects in adipocytes, and 2) C/EBPβ is under the direct control of SREBP-1c, as demonstrated by the ability of SREBP-1c to activate the transcription from C/EBPβ promoter through canonical SREBP binding sites. Thus, some of the effects of insulin and/or SREBP-1c in mature fat cells might require C/EBPβ or C/EBPδ as transcriptional relays. Insulin is the main anabolic hormone in mammals and exerts its effects in liver, adipose tissue, and skeletal and cardiac muscle via the insulin receptor (for a review, see Ref. 1Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (715) Google Scholar). The cellular mechanism underlying its action on carbohydrate, lipid, and protein metabolism has been the center of major interest for many years. Active research has led to the identification of the major steps of the insulin signal transduction pathway. These include a family of soluble scaffolding molecules, known as insulin receptor substrates, which initiate downstream signaling cascades involving the phosphatidylinositol 3-kinase/Akt pathway and the mitogen-activated protein kinase pathway (for reviews, see Refs. 1Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (715) Google Scholar and 2Whitehead J.P. Clark S.F. Urso B. James D.E. Curr. Opin. Cell Biol. 2000; 12: 222-228Crossref PubMed Scopus (100) Google Scholar). In this cascade, rapid changes in the state of protein phosphorylation ultimately mediate many important actions of insulin (e.g. glucose transport, glycogen synthesis, lipogenesis, and antilipolysis). It is also well known that, alongside these rapid nongenomic effects, important changes in gene expression play critical roles in insulin action in insulin-sensitive tissues (3O'Brien R.M. Granner D.K. Physiol. Rev. 1996; 76: 1109-1161Crossref PubMed Scopus (434) Google Scholar). The transcriptional effects of insulin and the mechanisms by which insulin can relay signal to the nucleus have remained largely unknown until recently. As described (4Flier J.S. Hollenberg A.N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14191-14192Crossref PubMed Scopus (34) Google Scholar), new light was shed by the identification of SREBP-1c 1The abbreviations used are: SREBP, sterol-regulatory element-binding protein; Ad, adenovirus; MOI, multiplicity of infection (i.e. plaque-forming units per cell); DN, dominant negative; DP, dominant positive; SRE, sterol-regulatory element; HMG, hydroxymethylglutaryl; PPARγ, peroxisome proliferator-activated receptor γ; C/EBP, CCAAT/enhancer-binding protein; RT-PCR, reverse transcriptase-PCR; LDL, low density lipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKB, protein kinase B; GFP, green fluorescent protein; PAI, plasminogen activator inhibitor. 1The abbreviations used are: SREBP, sterol-regulatory element-binding protein; Ad, adenovirus; MOI, multiplicity of infection (i.e. plaque-forming units per cell); DN, dominant negative; DP, dominant positive; SRE, sterol-regulatory element; HMG, hydroxymethylglutaryl; PPARγ, peroxisome proliferator-activated receptor γ; C/EBP, CCAAT/enhancer-binding protein; RT-PCR, reverse transcriptase-PCR; LDL, low density lipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKB, protein kinase B; GFP, green fluorescent protein; PAI, plasminogen activator inhibitor. as a transcription factor capable of mediating some of the effects of the hormone on previously identified insulin target genes. Indeed, SREBP-1c was shown not only to regulate the expression of key genes of glucose, fatty acid, and triglyceride metabolism in fibroblasts, adipocytes, hepatocytes, and the livers of transgenic mice (5Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar, 6Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar, 7Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (614) Google Scholar) but also to be able to substitute to insulin in inducing transcription of known insulin target genes like glucokinase or FAS in hepatocytes (8Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (590) Google Scholar). SREBP-1c is particularly abundant in the adipose tissue and the liver, both of which are insulin-sensitive and display quite a restricted expression pattern compared with the ubiquitously expressed SREBP-2, the other SREBP isoform that is encoded by a separate gene (9Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar). In agreement with their distinct expression pattern and regulation, SREBP-1c and SREBP-2 can also be distinguished in vivo by their ability to target different genes. Indeed, SREBP-1c and SREBP-2 assume different functions, SREBP-2 being more selective for activating genes involved in cholesterol homeostasis (reviewed in Ref. 10Osborne T.F. J. Biol. Chem. 2000; 275: 32379-32382Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), whereas SREBP-1c actions are focused on lipid synthesis and glucose metabolism. From these studies, SREBP-1c thus appears as a strong candidate to be a general mediator of the metabolic actions of insulin via the regulation of gene expression. The aim of the present study was to document further this hypothesis with a focus on adipocytes, in which specific gene expression is profoundly influenced by insulin. In the context of the adipose cell, several transcription factors that play interconnected roles ultimately determine the fully differentiated adipocyte gene expression profile. Among these factors is SREBP-1c, known also as ADD-1 (foradipocyte determination anddifferentiation factor-1 (11Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (533) Google Scholar)), a member of the basic helix-loop-helix-leucine zipper family of transcription factors. Other important adipocyte transcriptional regulators include the fatty acid derivative-activated nuclear receptor zinc finger peroxisome proliferator-activated receptor γ (PPARγ) and several members of the basic leucine zipper family of CAAT/enhancer binding proteins (C/EBPs) (reviewed in Ref. 12Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307PubMed Google Scholar). In particular, C/EBPβ and C/EBPδ, when induced by appropriate stimuli, can initiate a transcriptional cascade that culminates in the induction of PPARγ and C/EBPα and the activation of the adipogenic program. In this study, we have used mature 3T3-L1 adipocytes to investigate the impact of the overexpression of mutant SREBP-1c isoforms, either dominant positive or dominant negative, on a panel of 50 adipocyte-specific genes. The latter were selected to cover a number of aspects of key fat cell functions such as lipid storage, lipolysis, glucose metabolism, energy expenditure, adipocyte-gene transcription factors, and adipocyte-derived secreted products. Our results show that genes that were up-regulated by the dominant positive and down-regulated by dominant negative SREBP-1c forms were also up-regulated by insulin, confirming that SREBP-1c is a major factor underlying the transcriptional effect of insulin. Moreover, we found out that SREBP-1c specifically mediated insulin action on some adipocyte genes, such as PAI-1, and the β and δ C/EBP isoforms, which were known as insulin-sensitive but previously unrecognized as SREBP-1c targets. Finally, we provide evidence that SREBP-1c can directly transactivate the C/EBPβ promoter through canonical SREBP binding sites. Moreover, those sites map the insulin response region of the C/EBPβ promoter. Thus, this study demonstrates the existence of an insulin-SREBP-1c-C/EBPβ axis in adipocytes and suggests that a transcriptional cascade might be initiated by SREBP-1c to mediate insulin effects on fully differentiated adipocyte gene regulation. The adenovirus vector containing the transcriptionally active dominant positive (DP) amino-terminal fragment (amino acids 1–403) of rat SREBP-1c, Ad.SREBP-1c DP, was constructed as previously described (8Foretz M. Guichard C. Ferre P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (590) Google Scholar) with homologous recombination in BJ5183 bacteria using the shuttle vector pAdTrack-CMV containing the green fluorescent protein (GFP) (13He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3221) Google Scholar). The recombinant adenovirus containing the dominant negative form of rat SREBP-1c, Ad.SREBP-1c DN, was described elsewhere (14Boizard M., Le Liepvre X. Lemarchand P. Foufelle F. Ferre P. Dugail I. J. Biol. Chem. 1998; 273: 29164-29171Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Both Ad.SREBP-1c DP and Ad.SREBP-1c DN were under control of a cytomegalovirus promoter. The adenovirus vector containing the major late promoter with no exogenous gene (Ad.null) was used as control. The adenoviral vectors were propagated in the HEK 293 cell line, purified by cesium chloride density centrifugation, and stored at −80 °C until use. The efficiency of infection in 3T3-L1 adipocytes was assessed by visualizing GFP expression using a fluorescence microscope (Eclipse E800, Nikon). Measurements of SREBP target gene expression were also performed using various MOI (from 10 to 500) and various postinfection times from 24 to 72 h. Experiments were performed 4–6 times. Various tested conditions were Ad.SREBP-1c DP with no insulin; Ad.SREBP-1c DN with insulin (100 nm); Ad.null with insulin (100 nm); and Ad.null with no insulin. 3T3-L1 cells (ATCC number CL-173) were grown in 6-cm diameter dishes and differentiated at 37 °C in an atmosphere of air/CO2 (90:10, v/v) in Dulbecco's modified Eagle's medium (Invitrogen) with 4.5 g/liter glucose, 10% fetal calf serum, penicillin/streptomycin (50 units penicillin/50 μg of streptomycin per ml of medium). Two days after reaching confluence, cells were induced into differentiation with a 2-day incubation in Dulbecco's modified Eagle's medium, 10% fetal-calf serum containing insulin (1 μg/ml), dexamethasone (0.25 μm), and isobutylmethylxanthine (0.1 mm) (all from Sigma). Then preadipocytes were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum supplemented with insulin (1 μg/ml). After 10 days, when adipocytes have accumulated numerous lipid droplets as judged by Oil Red O staining, cells were placed for 16–18 h in a defined medium consisting of Dulbecco's modified Eagle's medium/F-12 (1:1, v/v), 4.5 g/liter glucose, glutamine, penicillin/streptomycin, free fatty acid bovine serum albumin (5%) (Sigma), in the absence or in the presence of insulin (100 nm), and then treated for various times (24–72 h) at an MOI of 10–500 with the different recombinant adenoviruses. Total RNA was prepared as described (15Krief S. Feve B. Baude B. Zilberfarb V. Strosberg A.D. Pairault J. Emorine L.J. J. Biol. Chem. 1994; 269: 6664-6670Abstract Full Text PDF PubMed Google Scholar). cDNA was synthesized from 5 μg of total RNA in 20 μl using random hexamers and murine Moloney leukemia virus reverse transcriptase (Invitrogen). The design of primers was done using either Primer Express (Applied Biosystems) or Oligo (MedProbe, Olso, Norway) software. Real time quantitative RT-PCR analyses for the genes described in Table I were performed starting with 50 ng of reverse transcribed total RNA (diluted in 5 μl of 1× Sybr Green buffer), with a 200 nm concentration of both sense and antisense primers (Genset) in a final volume of 25 μl using the Sybr Green PCR core reagents in an ABI PRISM 7700 Sequence Detection System instrument (Applied Biosystems). Fluorescence is generated after laser excitation by bound Sybr Green to double-stranded DNA. Because we used Sybr Green in measurements of amplification-associated fluorescence for real time quantitative RT-PCR, it was important to verify that generated fluorescence was not overestimated by contaminations resulting from residual genomic DNA amplification (using controls without reverse transcriptase) and/or from primer dimers formation (controls with no DNA template nor reverse transcriptase). RT-PCR products were also analyzed on ethidium bromide-stained agarose to ensure that a single amplicon of the expected size was indeed obtained. To measure PCR efficiency, serial dilutions of reverse transcribed RNA (0.1 pg to 200 ng) were amplified, and a line was obtained by plotting cycle threshold (C T) values as a function of starting reverse transcribed RNA, the slope of which was used for efficiency calculation using the formula E = 10‖(1/slope)‖ - 1 (16PerkinElmer Life Sciences (1999) Relative Quantification of Gene Expression: Bulletin 2, Boston, MA.Google Scholar). Ribosomal 18 S RNA amplifications were used to account for variability in the initial quantities of cDNA. The relative quantitation for any given gene, expressed as -fold variation over control (untreated cells), was calculated after determination of the difference between C T of the given gene A and that of the calibrator gene B (GAPDH) in treated cells (ΔC T1 = C T1A −C TB) and control cells (ΔC T0 = C T0A −C TB) using the 2 − ΔΔC T(1–0) formula (16PerkinElmer Life Sciences (1999) Relative Quantification of Gene Expression: Bulletin 2, Boston, MA.Google Scholar). GAPDH expression of a control cDNA was used as interplate calibrator. Variation over controls was determined using the above-mentioned formula as follows. The effect of insulin was calculated by comparing meanC T values obtained in the Ad.null with insulin condition and that obtained in the Ad.null with no insulin condition; the effect of Ad.SREBP-1c DP by comparing Ad.SREBP-1c DP with no insulin and the Ad.null with no insulin conditions; and the effect of Ad.SREBP-1c DN by comparing Ad.SREBP-1c DN with insulin and the Ad.null with insulin conditions. C T values are means of triplicate measurements. Experiments were repeated 4–6 times. All primers are presented in Table I. In a given cDNA population, relative expression level between genes could be calculated based on individual C T, provided that PCR efficiencies were close to 1. The latter were calculated according to Ref. 16PerkinElmer Life Sciences (1999) Relative Quantification of Gene Expression: Bulletin 2, Boston, MA.Google Scholar and were 1.1 + 0.07 (mean ± S.E., n = 21), indicating an approximate doubling of DNA at each PCR cycle, as theoretically expected. The percentage of relative expression between several genes of a given family (e.g. for the three C/EBP isoforms) was calculated as follows; mean C T of C/EBPα, C/EBPβ, and C/EBPδ in the absence of insulin were 24.9, 22.24, and 30.31, respectively. Using the 2 − ΔC Tformula, these could be expressed as two equations (C/EBPβ = 6.32 × C/EBPα and C/EBPβ = 268 × C/EBPδ) plus another equation as C/EBPα + C/EBPβ + C/EBPδ = 100. It could thus be calculated that the percentage expression of the C/EBPα, C/EBPβ, and C/EBPδ isoforms was 13.6, 86.1, and 0.3%, respectively.Table IPrimer sequences of the selected genes involved in key pathways of adipose metabolismThe abbreviations of the genes, full name, accession number or locus, corresponding primer numbers, and 5′ to 3′ nucleotide sequences of the sense and antisense primers are presented. Open table in a new tab The abbreviations of the genes, full name, accession number or locus, corresponding primer numbers, and 5′ to 3′ nucleotide sequences of the sense and antisense primers are presented. Differentiated 3T3-L1 cells were treated with insulin or were infected with the adenovirus encoding the DP SREBP-1c mutant. After 24 h, nuclei were prepared as previously described (17Lacasa D. Le L., X Ferre P. Dugail I. J. Biol. Chem. 2001; 276: 11512-11516Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and were incubated with [α-32P]CTP (3000 Ci/mmol) for 45 min at 32 °C. Incubations were terminated by the addition of RNase-free DNase and proteinase K, and labeled RNA was extracted by phenol/chloroform. Labeled transcripts were hybridized for 72 h with plasmid cDNA immobilized on nylon membranes. Blots were washed to high stringency, and hybridized RNA was quantified with an optical scanner (Storm 860;Amersham Biosciences). A 1.4-kb promoter fragment of the rat C/EBPβ gene, cloned in front of the luciferase reporter in the p19-Luc vector, has been described elsewhere (18Niehof M. Manns M.P. Trautwein C. Mol. Cell. Biol. 1997; 17: 3600-3613Crossref PubMed Google Scholar). From this construct, a series of 5′ deletions was derived, encompassing regions from −441 to +16, −183 to +16, and −136 to +16 relative to the transcription start site. Point mutations on sterol-regulatory element (SRE) sites in the 1.4-kb promoter fragment were introduced using the QuikChange multisite-directed mutagenesis kit (Promega) as recommended by the manufacturer. The sequences of the mutagenic primers were 5′-GGGCGGAGGTCGTACCAGCTCAGCAfor the SRE1 site located at −1124 and 5′-AAGGTTGAGCAACGTACCACCAGCTTGCC for the SRE2 at −1064. In both cases, the disruption of the SRE motif was performed by replacing the ACC triplet by GTA as underlined in the sequences. Growing 3T3L1 preadipocytes in 60-mm dishes were transfected using the calcium phosphate precipitation method, with a mixture of plasmid DNA containing 1 μg of a promoter luciferase construct, 1 μg of Rous sarcoma virus-β-galactosidase as an internal standard, and 50 ng of pSV Sport1-ADD1 expression vector encoding an active form of the rat SREBP-1c transcription factor (19Kim J.B. Spotts G. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1998; 15: 2582-2588Crossref Scopus (294) Google Scholar). The total amount of DNA was kept constant in each experiment by adding empty pSV Sport1 when necessary. Reporter gene activities were assayed 24 h after transfection, and luciferase data were normalized to galactosidase. Differentiated 3T3-L1 cells were placed in serum-free medium containing 2% bovine serum albumin and no insulin for 24 h and transfected by electroporation as described previously for mature adipocytes (20Rolland V. Dugail I., Le Liepvre X. Lavau M. J. Biol. Chem. 1995; 270: 1102-1106Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Briefly, 1–2 × 106 cells in 200 μl were shocked electrically in the presence of 20 μg of promoter luciferase constructs and 1 μg of Rous sarcoma virus-chloramphenicol acetyltransferase internal control. Cells were then replated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in the presence or absence of 1 μg/ml insulin. Reporter gene activities were measured after 24 h. Nuclear extracts were obtained from differentiated 3T3-L1 adipocytes as described previously (20Rolland V. Dugail I., Le Liepvre X. Lavau M. J. Biol. Chem. 1995; 270: 1102-1106Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and used for Western blotting with a commercially available antibody against C/EBPβ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For statistical analyses of real time RT-PCR experiments, results for a given gene were expressed as differences from the mean C T value obtained in the Ad.Null with no insulin condition. Statistical significance was assessed by analysis of variance followed by Newman-Keuls comparison tests (Statistica, StatSoft Inc.). In transfection experiments, statistical differences were assessed by Student's t test. A p value of <0.05 was considered as the threshold of statistical significance. As a first step to study the impact of changes in SREBP-1c content in 3T3-L1 adipocytes and its potential correlation with insulin-induced gene expression profile, we assessed the ability of insulin to modulate endogenous SREBPs expression in these cells. In agreement with initial studies (21Kim J.B. Sarraf P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Invest. 1998; 101: 1-9Crossref PubMed Scopus (607) Google Scholar), we observed a stimulatory effect of insulin on SREBP-1c mRNA, increasing its levels by 3-fold (Fig.1 A). The effect of insulin was restricted to the SREBP-1c isoform, with no change in SREBP-1a or SREBP-2. Similar results were obtained in livers of streptozotocin-induced diabetic rats (7Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (614) Google Scholar) and in cultured hepatocytes (22Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C., Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (450) Google Scholar). Thus, insulin specifically targets SREBP-1c in adipocytes. It remains to be determined whether the isoform-specific effect of the hormone equally occurs in adipose in vivo and in other insulin-sensitive tissues (i.e. skeletal, cardiac muscle, and brown fat). Having shown that insulin stimulated SREBP-1c, we examined the conditions to achieve optimal expression of SREBP-1c mutants (DP or DN forms) following adenoviral infection of differentiated 3T3-L1 adipocytes. First, using Ad.SREBP-1c DP, which co-expresses GFP, optimal conditions for transduction of 3T3-L1 adipocytes with recombinant adenoviruses were assessed. After 24 h, nearly 100% of GFP-expressing adipocytes was achieved with an MOI of 500 (Fig. 1 B, bottom panel). Second, the steady state levels of SREBP-1 mRNA were monitored by real time RT-PCR using primers designed to differentially target endogenous SREBP-1c, endogenous SREBP-1a, or total SREBP-1 expression (including endogenous as well as adenovirus-mediated DN or DP mutant forms of SREBP-1c; see Table I for primer sequences). Ad.Null was used as control and exerted no effects on gene expression, whatever the MOI (not shown). Fig. 1 B shows that the infection of adipocytes with increasing titers of adenoviruses encoding mutant forms of SREBP-1c (either dominant negative or positive) produced, as expected, a dose-dependent increase in total SREBP-1 mRNA expression, demonstrating significant transgene expression in adipocytes. Because we observed that the endogenous expressions of SREBP-1a and SREBP-1c were not altered by Ad.SREBP-1c DP or DN (Fig. 1 B), the increase in total SREBP-1 observed following adenovirus infection was solely accounted for by an increase in the expression of the transgene. As shown in Fig.1 B, total SREBP-1 expression increased by ∼15-fold in cells infected with the Ad.SREBP-1c DP (MOI of 500) and was stimulated to a similar extent (∼10-fold) using the same titer of Ad.SREBP-1c DN. This was confirmed by Western blot analysis with an anti-SREBP-1 antibody that showed a huge increase in SREBP transgene protein content in nuclear extracts prepared from cells infected with Ad.SREBP-1c DP (data not shown). To ensure that SREBP-mediated transcriptional activity was significantly altered in adipocytes infected with the adenoviruses encoding the DP or DN SREBP-1c mutants, we measured the steady state levels of known SREBP target genes such as FAS (5Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar, 14Boizard M., Le Liepvre X. Lemarchand P. Foufelle F. Ferre P. Dugail I. J. Biol. Chem. 1998; 273: 29164-29171Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar,23Bennett M.K. Lopez J.M. Sanchez H.B. Osborne T.F. J. Biol. Chem. 1995; 270: 25578-25583Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), SCD-1 (24Tabor D.E. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1998; 273: 22052-22058Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and LDL receptor (25Yokoyama C. Wang X. Briggs M.R. Admon A., Wu, J. Hua X.X. Goldstein J. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (776) Google Scholar). Fig. 1 C shows that increasing titers of Ad.SREBP-1c DP mutant dose-dependently stimulated the expression of FAS, SCD-1, and LDL receptor genes (left panel). The induction of FAS, SCD-1, and LDL receptor gene expression started at 10–100 plaque-forming units/cell, and a plateau was reached after 250, the 500 plaque-forming units/cell condition being optimal. The mRNAs encoding FAS and SCD-1 were increased with higher efficiencies (up to 10-fold) than that of the LDL receptor, which was stimulated only 3-fold. This agreed well with the ability of SREBP-1c to stimulate lipogenesis in preference to cholesterol uptake in vivo (26Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar, 27Pai J.T. Guryev O. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 26138-26148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) (reviewed in Ref. 10Osborne T.F. J. Biol. Chem. 2000; 275: 32379-32382Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). In reciprocal experiments, cells were infected with increasing titers of Ad.SREBP-1c DN mutant (Fig. 1 C, right panel). We observed, as expected, a gradual decline in the steady state levels of FAS, SCD-1, and LDL receptor mRNAs. The observed changes in DN-expressing cells were of lesser magnitude than those in cells expressing the DP form. Since the dominant negative mutant inhibits SREBP-1c transcriptional activity by titrating endogenous SREBP-1c into inactive heterodimers (5Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (824) Google Scholar), it is possible that Ad DN expression might not reach sufficient levels to completely inhibit endogenous SREBP-1c. Alternatively, because transcriptional activation of these genes requires in addition to SREBP other transcription factors such as NFY or Sp1 (24Tabor D.E. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1998; 273: 22052-22058Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 28Bennett M.K. Osborne T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6340-6344Crossref PubMed Scopus (100) Google Scholar), it remained plausible that the presence of these untitrated factors or that of other co-activators allows sufficient residual transcriptional activity, thus obviating total inhibition of transcription. Taken together, all these results establish that SREBP-1c transcriptional activity can be efficiently manipulated in 3T3-L1 cells by means of adenovirus-mediated overexpression of dominant positive or negative SREBP-1c mutants. The 3T3-L1 differentiated adipocyte cell system was used to compare the effects of insulin with that of SREBP-1c manipulations on the expression of various adipocyte genes. Specific primers were designed for real time fluorescent RT-PCR analyses of a panel of genes that covers a wide range of fat cell functions,i.e. lipid storage or lipolysis," @default.
• W2016126865 created "2016-06-24" @default.
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• W2016126865 creator A5006242340 @default.
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• W2016126865 date "2002-09-01" @default.
• W2016126865 modified "2023-09-29" @default.
• W2016126865 title "Insulin and Sterol-regulatory Element-binding Protein-1c (SREBP-1C) Regulation of Gene Expression in 3T3-L1 Adipocytes" @default.
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