SREBP-1-independent regulation of lipogenic gene expression in adipocytes
2007; Elsevier BV; Volume: 48; Issue: 7 Linguagem: Inglês
10.1194/jlr.m700033-jlr200
ISSN1539-7262
AutoresMotohiro Sekiya, Naoya Yahagi, Takashi Matsuzaka, Yoshinori Takeuchi, Yoshimi Nakagawa, Haruka Takahashi, Hiroaki Okazaki, Yoko Iizuka, Ken Ohashi, Takanari Gotoda, Shun Ishibashi, Ryozo Nagai, Tsutomu Yamazaki, Takashi Kadowaki, Nobuhiro Yamada, Jun-ichi Osuga, Hitoshi Shimano,
Tópico(s)Lipid metabolism and biosynthesis
ResumoSterol regulatory element-binding protein (SREBP)-1c is now well established as a key transcription factor for the regulation of lipogenic enzyme genes such as FAS in hepatocytes. Meanwhile, the mechanisms of lipogenic gene regulation in adipocytes remain unclear. Here, we demonstrate that those in adipocytes are independent of SREBP-1c. In adipocytes, unlike in hepatocytes, the stimulation of SREBP-1c expression by liver X receptor agonist does not accompany lipogenic gene upregulation, although nuclear SREBP-1c protein is concomitantly increased, indicating that the activation process of SREBP-1c by the cleavage system is intact in adipocytes. Supportively, transcriptional activity of the mature form of SREBP-1c for the FAS promoter was negligible when measured by reporter analysis. As an underlying mechanism, accessibility of SREBP-1c to the functional elements was involved, because chromatin immunoprecipitation assays revealed that SREBP-1c does not bind to the functional SRE/E-box site on the FAS promoter in adipocytes. Moreover, genetic disruption of SREBP-1 did not cause any changes in lipogenic gene expression in adipose tissue. In summary, in adipocytes, unlike in hepatocytes, increments in nuclear SREBP-1c are not accompanied by transactivation of lipogenic genes; thus, SREBP-1c is not committed to the regulation of lipogenesis. Sterol regulatory element-binding protein (SREBP)-1c is now well established as a key transcription factor for the regulation of lipogenic enzyme genes such as FAS in hepatocytes. Meanwhile, the mechanisms of lipogenic gene regulation in adipocytes remain unclear. Here, we demonstrate that those in adipocytes are independent of SREBP-1c. In adipocytes, unlike in hepatocytes, the stimulation of SREBP-1c expression by liver X receptor agonist does not accompany lipogenic gene upregulation, although nuclear SREBP-1c protein is concomitantly increased, indicating that the activation process of SREBP-1c by the cleavage system is intact in adipocytes. Supportively, transcriptional activity of the mature form of SREBP-1c for the FAS promoter was negligible when measured by reporter analysis. As an underlying mechanism, accessibility of SREBP-1c to the functional elements was involved, because chromatin immunoprecipitation assays revealed that SREBP-1c does not bind to the functional SRE/E-box site on the FAS promoter in adipocytes. Moreover, genetic disruption of SREBP-1 did not cause any changes in lipogenic gene expression in adipose tissue. In summary, in adipocytes, unlike in hepatocytes, increments in nuclear SREBP-1c are not accompanied by transactivation of lipogenic genes; thus, SREBP-1c is not committed to the regulation of lipogenesis. acetyl-coenzyme A carboxylase N-acetyl-leucyl-leucyl-norleucinal chromatin immunoprecipitation liver X receptor retinoid X receptor sterol regulatory element-binding protein The fatty acid biosynthetic pathway, composed of some 25 enzymes, has been elucidated in detail (1.Goodridge A.G. Fatty Acid Synthesis in Eucaryotes. Elsevier Science, Amsterdam, The Netherlands1991Google Scholar). Among these enzymes, FAS, the main synthetic enzyme that catalyzes the condensation of malonyl-CoA to produce the 16 carbon saturated fatty acid palmitate, and acetyl-coenzyme A carboxylase (ACC), which synthesizes malonyl-CoA from acetyl-CoA, are of particular importance. The regulation of these lipogenic enzymes has two remarkable features. First, their overall enzymatic activities largely depend on the amount of expressed protein that is primarily controlled at the transcriptional level, although regulation through phosphorylation is also important for some enzymes, such as ACC. Second, their rates of transcription are coordinately regulated (2.Goodridge A.G. Dietary regulation of gene expression: enzymes involved in carbohydrate and lipid metabolism.Annu. Rev. Nutr. 1987; 7: 157-185Crossref PubMed Scopus (160) Google Scholar). Therefore, it has been presumed that these genes share a regulatory sequence in their promoters that interacts with common trans-acting factors. In the liver, the most likely factor conducting this coordinated transcriptional regulation has been revealed to be sterol regulatory element-binding protein (SREBP)-1 (3.Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. Nuclear sterol regulatory element-binding proteins activate genes responsible for the entire program of unsaturated fatty acid biosynthesis in transgenic mouse liver.J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 4.Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. et al.Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). SREBPs are transcription factors that belong to the basic helix-loop-helix leucine zipper family and are considered to be profoundly involved in the transcriptional regulation of cholesterogenic and lipogenic enzymes (5.Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene.Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (797) Google Scholar, 6.Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar). Unlike other members of the basic helix-loop-helix leucine zipper family, SREBPs are synthesized as precursors bound to the endoplasmic reticulum and nuclear envelope. Upon activation, SREBPs are released from the membrane into the nucleus as mature protein by a sequential two-step cleavage process. To date, three SREBP isoforms, SREBP-1a, -1c, and -2, have been identified and characterized. SREBP-1a and -1c are transcribed from the same gene, each by a distinct promoter, and the predominant SREBP-1 isoform in liver and adipose tissue is 1c rather than 1a (7.Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (642) Google Scholar). The role of SREBP-1c for the regulation of lipogenesis in the liver has been well established by several lines of evidence, especially from transgenic and knockout mouse models (4.Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. et al.Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 8.Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (688) Google Scholar). In these models, mature hepatic SREBP-1c protein levels determine mRNA expression levels for a battery of lipogenic genes in the liver. Moreover, mature hepatic SREBP-1c is physiologically regulated by nutrient availability (i.e., it is downregulated when animals are starved and upregulated when they are refed), thereby adjusting lipogenic gene expression levels to the nutritional conditions (9.Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.J. Clin. Invest. 1998; 101: 2331-2339Crossref PubMed Google Scholar). Thus, regarding its role in the liver, SREBP-1c is now well established as a key transcription factor for the regulation of lipogenic gene expression and by extension triglyceride storage in liver (10.Yahagi N. Shimano H. Hasty A.H. Matsuzaka T. Ide T. Yoshikawa T. Amemiya-Kudo M. Tomita S. Okazaki H. Tamura Y. et al.Absence of sterol regulatory element-binding protein-1 (SREBP-1) ameliorates fatty livers but not obesity or insulin resistance in Lep(ob)/Lep(ob) mice.J. Biol. Chem. 2002; 277: 19353-19357Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 11.Sekiya M. Yahagi N. Matsuzaka T. Najima Y. Nakakuki M. Nagai R. Ishibashi S. Osuga J. Yamada N. Shimano H. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP-1 suppression.Hepatology. 2003; 38: 1529-1539Crossref PubMed Scopus (342) Google Scholar). Despite the extensive knowledge gained in recent years regarding the role of SREBP-1 in lipogenesis in liver, its physiological role in adipocytes remains obscure. Although the mRNA expression of SREBP-1c in adipocytes is also drastically altered by dietary conditions, we have reported that targeted disruption of the SREBP-1 gene scarcely affected the dynamic changes of lipogenic gene expression in adipose tissue (4.Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. et al.Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Conversely, the impact of SREBP-1c overexpression in adipocytes was also evaluated in transgenic mice; however, it disrupted the differentiation processes of adipocytes, resulting in lipodystrophy. Hence, the effect of SREBP-1c on lipogenic gene regulation in adipocytes was not able to be evaluated (12.Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy.Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (680) Google Scholar), although ectopic overexpression of SREBP-1a drives fatty acid synthesis in the adipose tissue of transgenic mice (13.Horton J.D. Shimomura I. Ikemoto S. Bashmakov Y. Hammer R.E. Overexpression of sterol regulatory element-binding protein-1a in mouse adipose tissue produces adipocyte hypertrophy, increased fatty acid secretion, and fatty liver.J. Biol. Chem. 2003; 278: 36652-36660Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). These situations prompted us to investigate the effects of SREBP-1c activation in adipocytes by stimulating the SREBP-1c promoter. We and others have found that liver X receptors (LXRs), nuclear receptor-type transcription factors, activate the transcription of the SREBP-1c gene through binding to LXR elements in the promoter, together with retinoid X receptors (RXRs) (14.Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta.Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1423) Google Scholar, 15.Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. et al.Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter.Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (435) Google Scholar). Here, we show that the stimulation of the SREBP-1c gene with LXR agonist has negligible effects on the expression of lipogenic enzymes in adipocytes, despite a concomitant increase in nuclear active SREBP-1c, indicating that the transcriptional activity of SREBP-1c against lipogenic genes is almost nil in adipocytes in contrast to hepatocytes. In fact, luciferase reporter gene assays demonstrated that the recombinant nuclear active form of SREBP-1c had far lower activity for the FAS promoter in 3T3-L1 adipocytes than in HepG2 hepatoma cells. Consistent with these observations, chromatin immunoprecipitation (ChIP) assays revealed that SREBP-1 is not recruited to the functional cis element on the FAS promoter in LXR-activated adipocytes. Therefore, lipogenic genes are controlled almost independently of SREBP-1c in adipocytes, and the triglyceride biosynthetic pathway is differently regulated between liver and adipose tissue. The synthetic LXR agonist T0901317 was purchased from Cayman Chemical (Ann Arbor, MI). The RXR agonist LG100268 was synthesized as described elsewhere (16.Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. et al.Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists.Nature. 1997; 386: 407-410Crossref PubMed Scopus (576) Google Scholar). Standard laboratory chow (MF; composed of 60% carbohydrate, 13% fat, and 27% protein on a caloric basis) was obtained from Oriental Yeast (Tokyo, Japan). Other materials were purchased from Sigma unless indicated otherwise. Eight week old male Wister rats and 8 week old male C57BL/6J mice were purchased from CLEA (Tokyo, Japan). All animals were maintained in a temperature-controlled environment with a 12 h light/dark cycle and were given free access to standard chow and water. The dosage of T0901317 (suspended with 0.9% carboxymethylcellulose, 9.95% polyethylene glycol 400, and 0.05% Tween 80, administered orally) was 10 mg/kg for rats and 50 mg/kg for mice. Animals were euthanized at 12 h after administration. The protocol of dietary manipulation was as follows: for the fasting group, mice were starved for 24 h and rats were starved for 48 h; for the refeeding group, they were refed at 12 h or 24 h after a 24 h (for mice) or 48 h (for rats) starvation, respectively, unless stated otherwise. All groups of animals in one experiment were euthanized at the same time. SREBP-1-null mice (16 weeks of age, female) on the C57BL/6J background have been reported previously (4.Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. et al.Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar). Total RNA from liver, epididymal fat pads, and cultured cells was isolated with Trizol Reagent (Invitrogen), and a 10 μg RNA sample equally pooled among each group was run on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. cDNA probes were cloned as described previously (4.Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A.H. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. et al.Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes.J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 17.Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar). The probes were labeled with [α-32P]dCTP using the Megaprime DNA Labeling System (Amersham Biosciences). The membranes were hybridized with the radiolabeled probe in Rapid-hyb buffer (Amersham Biosciences) at 65°C and washed in 0.1× SSC buffer with 0.1% SDS at 65°C. Blots were exposed to both Kodak XAR-5 film and the imaging plate for the BAS2000 BIO Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). The quantification results obtained from the BAS2000 system were normalized to the signal generated from 36B4 (acidic ribosomal phosphoprotein P0) mRNA. Two micrograms of total RNA was reverse-transcribed using the ThermoScript RT-PCR system (Invitrogen). Quantitative real-time PCR was performed using SYBR Green dye (Applied Biosystems, Foster City, CA) in an ABI Prism 7900 PCR instrument (Applied Biosystems). The relative abundance of each transcript was calculated by a standard curve of cycle thresholds for serial dilutions of a cDNA sample and normalized to 36B4. Primer sequences are available upon request. Nuclear extract protein from mouse or rat liver was prepared as described previously (18.Sheng Z. Otani H. Brown M.S. Goldstein J.L. Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver.Proc. Natl. Acad. Sci. USA. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar). Briefly, excised livers (0.5 g) were homogenized in a Polytron in 5 ml of buffer A, which consisted of 10 mM HEPES at pH 7.9, 25 mM KCl, 1 mM EDTA, 2 M sucrose, 10% glycerol, 0.15 mM spermine, and 2 mM spermidine, supplemented with protease inhibitors [6 μg/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN; Calbiochem), 2.5 μg/ml pepstatin A, 2 μg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 2.5 μg/ml aprotinin]. Pooled homogenate was then subjected to one stroke of a Teflon pestle in a Potter-Elvejiem homogenizer, followed by filtration through two layers of cheesecloth, and layered over 10 ml of buffer A. After centrifugation at 24,000 rpm on a Beckman SW28 rotor for 1 h at 4°C, the resulting nuclear pellet was resuspended in a buffer containing 10 mM HEPES at pH 7.9, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol, supplemented with protease inhibitors, after which 0.1 volume of 5 M NaCl was added. Each mixture was agitated gently for 30 min at 4°C and then centrifuged at 89,000 rpm on a Himac S120AT2 rotor (Hitachi, Tokyo, Japan) for 30 min at 4°C. The supernatant was used as nuclear extract. Nuclear protein from white adipose tissue was prepared as described previously (19.Soukas A. Cohen P. Socci N.D. Friedman J.M. Leptin-specific patterns of gene expression in white adipose tissue.Genes Dev. 2000; 14: 963-980Crossref PubMed Google Scholar). In brief, fresh epididymal fat pads (∼3 g) were rinsed in ice-cold PBS, minced, and homogenized with 10 strokes of a Teflon homogenizer in 15 ml of NDS buffer at 4°C (10 mM Tris-HCl at pH 7.5, 10 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 14 mM mercaptoethanol, 0.5 mM EGTA, 2 mM EDTA, 0.5% Nonidet P-40, and 1 mM dithiothreitol) supplemented with protease inhibitors (6 μg/ml ALLN, 2 μg/ml leupeptin, 2.5 μg/ml aprotinin, 2.5 μg/ml pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride). The Nonidet P-40 concentration was increased to 1%, and nuclei were pelleted at 700 g for 10 min, washed once with 25 ml of NDS buffer (1% Nonidet P-40), filtered through 70 μm mesh, pelleted at 500 g for 10 min, resuspended in 1 volume of 1% citric acid, lysed by the addition of 2.5 volumes of 0.1 M Tris-HCl, 2.5% SDS, and 0.1 M dithiothreitol, sonicated briefly, and heated to 90°C for 5 min. Aliquots of nuclear protein (20 μg) were subjected to SDS-PAGE. Fibroblastic preadipocytes were isolated from epididymal fat pads of Wister rats by collagenase digestion as described previously (20.Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (504) Google Scholar). In brief, the epididymal fat pads from male Wister rat were removed and minced in KRBH buffer (130 mM NaCl, 5.2 mM KCl, 1.3 mM KH2PO4, 2.7 mM CaCl2, 1.3 mM MgSO4, 24.8 mM NaHCO3, and 10 mM HEPES at pH 7.4) supplemented with 3% (w/v) BSA, 2 mM glucose, and 200 nM adenosine. After digestion by collagenase (type II; 1.5 mg/ml) at 37°C for 1 h in a shaking water bath, the digest was filtered through sterile 250 μm nylon mesh. The adipocytes were allowed to float to the top of the tube and the infranatant was collected, passed through a 25 μm stainless steel filter, and centrifuged at 250 g for 10 min. The pellet was resuspended in MEM-α medium (Invitrogen) with 10% FBS supplemented with penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively; Invitrogen). Red blood cells were lysed by hypotonic shock (21.Atkin S.L. Hipkin L. Radcliffe J. White M.C. Hypotonic lysis of red blood cell contamination from human anterior pituitary adenoma cell preparations.In Vitro Cell. Dev. Biol. Anim. 1995; 31: 657-658Crossref PubMed Scopus (7) Google Scholar). The cells were plated on 60 mm culture dishes at a density of 6 × 105 cells/well. Medium was changed every 2 days. After the cells reached confluence, differentiation was induced in MEM-α medium with 10% FBS by the addition of 0.5 mM isobutylmethylxanthine (Wako), 0.25 μM dexamethasone, 5 μg/ml bovine insulin, and 1 μM pioglitazone (provided by Takeda Pharmaceutical). The differentiation of cells was morphologically confirmed. Differentiated adipocytes were treated with either vehicle (ethanol) or 10 μM T0901317 for 12 h in MEM-α medium containing the indicated amounts of glucose and insulin. Hepatocytes were isolated from nonfasted 4 week old Wister rats by the collagenase perfusion method as described previously (22.Horton J.D. Bashmakov Y. Shimomura I. Shimano H. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice.Proc. Natl. Acad. Sci. USA. 1998; 95: 5987-5992Crossref PubMed Scopus (540) Google Scholar). Cells were resuspended in DMEM containing 25 mM glucose supplemented with 5% FBS and seeded on collagen-coated 100 mm dishes at a final density of 4 × 104 cells/cm2. After 4 h, attached cells were cultured with Medium 199 containing Earle's salts (Invitrogen) and 5% FBS. After incubation for 20 h, cells were treated with either vehicle (ethanol) or 10 μM T0901317 for 12 h in similar medium containing the indicated amounts of glucose and insulin. Nuclear proteins from cultured cells were extracted as described previously (23.Hasty A.H. Shimano H. Yahagi N. Amemiya-Kudo M. Perrey S. Yoshikawa T. Osuga J. Okazaki H. Tamura Y. Iizuka Y. et al.Sterol regulatory element-binding protein-1 is regulated by glucose at the transcriptional level.J. Biol. Chem. 2000; 275: 31069-31077Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In brief, 2 h before collection, ALLN (6 μg/ml) was added to the medium. After collection, cells were resuspended in buffer A (10 mM HEPES at pH 7.6, 1 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) and passed through a 26 gauge needle 20 times and then briefly centrifuged. The pellet, containing the nuclei, was resuspended in buffer B (20 mM HEPES at pH 7.6, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 0.5 mM dithiothreitol) and rotated at 4°C for 15 min and then centrifuged at 15,000 g for 20 min. The supernatant was collected as nuclear extract. Whole cell lysates were harvested with a buffer (20 mM Tris-HCl at pH 7.4) containing detergents (1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) and protease inhibitors according to a standard protocol. Membrane fractions from livers and epididymal fat pads were prepared as described previously (18.Sheng Z. Otani H. Brown M.S. Goldstein J.L. Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver.Proc. Natl. Acad. Sci. USA. 1995; 92: 935-938Crossref PubMed Scopus (279) Google Scholar). Aliquots of nuclear extract (20 μg) and membrane fraction (50 μg) proteins were subjected to SDS-PAGE. Immunoblot analysis was performed using the ECL Western Blotting Detection System (Amersham Biosciences) and exposed to Kodak XAR-5 film. The primary antibodies (rabbit polyclonal; No. 931 for mouse SREBP-1a and -1c, No. 772 for SREBP-1c, which does not bind to SREBP-1a, and No. 528 for SREBP-2) were used as described previously (11.Sekiya M. Yahagi N. Matsuzaka T. Najima Y. Nakakuki M. Nagai R. Ishibashi S. Osuga J. Yamada N. Shimano H. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP-1 suppression.Hepatology. 2003; 38: 1529-1539Crossref PubMed Scopus (342) Google Scholar, 17.Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (699) Google Scholar, 24.Shimano H. Shimomura I. Hammer R.E. Herz J. Goldstein J.L. Brown M.S. Horton J.D. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene.J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (356) Google Scholar). The precursor and mature SREBP-1 bands are ∼125 and 65 kDa, respectively. An expression plasmid for the human nuclear form of SREBP-1c constructed in pcDNA3.1(+) (Invitrogen) was described previously (25.Yamamoto T. Shimano H. Nakagawa Y. Ide T. Yahagi N. Matsuzaka T. Nakakuki M. Takahashi A. Suzuki H. Sone H. et al.SREBP-1 interacts with hepatocyte nuclear factor-4{alpha} and interferes with PGC-1 recruitment to suppress hepatic gluconeogenic genes.J. Biol. Chem. 2004; 279: 12027-12035Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Luciferase reporter plasmids for the SRE promoter (SRE-Luc) and the fatty acid synthase gene promoter (FAS-Luc) were prepared as described previously (8.Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (688) Google Scholar, 24.Shimano H. Shimomura I. Hammer R.E. Herz J. Goldstein J.L. Brown M.S. Horton J.D. Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene.J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (356) Google Scholar). Human hepatoma HepG2 cells were cultured in DMEM containing 25 mM glucose, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate supplemented with 10% FBS. On day 0, cells were plated on a 12-well plate at 4.5 × 104 cells/well. On day 2, the indicated amounts of mature SREBP-1c expression plasmids, mock plasmids [empty pcDNA3.1(+)] to adjust total DNA amount, and luciferase reporter plasmids (FAS-Luc or SRE-Luc; 0.25 μg each) mixed with an SV-β-galactosidase reference plasmid (0.1 μg; p-SV-β-gal; Promega) were cotransfected into HepG2 cells using SuperFect Transfection Reagent (Qiagen) according to the manufacturer's protocol. The luciferase activity in transfectants was measured on a luminometer and normalized to β-galactosidase activity measured by standard kits (Promega). 3T3-L1 adipocytes were transfected by electroporation as described previously (26.Min J. Okada S. Kanzaki M. Elmendorf J.S. Coker K.J. Ceresa B.P. Syu L.J. Noda Y. Saltiel A.R. Pessin J.E. Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.Mol. Cell. 1999; 3: 751-760Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 27.Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.Proc. Natl. Acad. Sci. USA. 2003; 100: 7569-7574Crossref PubMed Scopus (312) Google Scholar). In brief, after cells became confluent (day 0), adipose conversion was induced in DMEM containing 25 mM glucose, 10% FBS, 0.5 mM isobutylmethylxanthine, 5 μg/ml bovine insulin, 0.25 μM dexamethasone, 1 μM pioglitazone, 33 μM biotin (Wako), 17 μM pantothenate (Wako), and antibiotics. After 48 h, the induction medium was removed and replaced by DMEM containing 25 mM glucose, 10% FBS, insulin, pioglitazone, biotin, and pantothenate. This medium was changed every 2 days. The adipocytes at day 8 of differentiation grown on 60 mm dishes were detached from dishes with 0.25% trypsin and 0.5 mg/ml collagenase (Wako) in PBS, washed twice, and resuspended in PBS. The indicated amounts of SREBP-1c expression plasmids, mock plasmids to adjust total DNA amount, luciferase reporter plasmids (FAS-Luc or SRE-Luc; 15 μg each), and control plasmids (pSV-β-gal; 10 μg) were cotransfected to the cells by a pulse current generated from the electroporator (Cell-Porator; Invitrogen) at 160 V with 880 μF capacitance and a low ohm setting. After electroporation, cells were immediately mixed with fresh medium for 10 min before being reseeded onto 24-well collagen-coated plates and assayed at 40 h after transfection. ChIP assays were performed as described by Boyd and Farnham (28.Boyd K.E. Farnham P.J. Coexamination of site-specific transcription factor binding and promoter activity in living cells.Mol. Cell. Biol. 1999; 19: 8393-8399Crossref PubMed Scopus (147) Google Scholar) with minor modifications. The supernatant of soluble chromatin derived from 1 × 107 cells was used. Briefly, rat primary hepatocytes and adipocytes were treated with 1% formaldehyde for 10 min. The cross-linking reaction was stopped by the addition of glycine to a final concentration of 0.125 M for 5 min. After washing with cold PBS, the cells were suspended in cell lysis buffer containing 10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, and protease inhibitors. After incubation for 30 min on ice, the cells were passed through a 26 gauge needle 20 times on ice. The nuclei were collected by m
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