SREBP-1 integrates the actions of thyroid hormone, insulin, cAMP, and medium-chain fatty acids on ACCα transcription in hepatocytes
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200283-jlr200
ISSN1539-7262
AutoresYanqiao Zhang, Liya Yin, F. Bradley Hillgartner,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoIn chick embryo hepatocytes, activation of acetyl-CoA carboxylase-α (ACCα) transcription by 3,5,3′-triiodothyronine (T3) is mediated by a cis-acting regulatory unit (−101 to −71 bp) that binds the nuclear T3 receptor (TR) and sterol regulatory element-binding protein-1 (SREBP-1). SREBP-1 directly interacts with TR on the ACCα gene to enhance T3-induced transcription. Here, we show that treating hepatocytes with T3 or insulin stimulates a 4-fold increase in the concentration of the mature, active form of SREBP-1. When T3 and insulin are added together, a 7-fold increase in the mature SREBP-1 concentration is observed. Time course studies indicate that the T3-induced increase in mature SREBP-1 abundance is closely associated with changes in ACCα transcription and that the mechanism mediating the effect of T3 on mature SREBP-1 is distinct from that mediating the effect of insulin. Transfection analyses indicate that inhibition of ACCα transcription by cAMP or hexanoate is mediated by ACCα sequences between −101 and −71 bp. Treatment with cAMP or hexanoate suppresses the increase in mature SREBP-1 abundance caused by T3 and insulin.These results establish a new interaction between the SREBP-1 and TR signaling pathways and provide evidence that SREBP-1 plays an active role in mediating the effects of T3, insulin, cAMP, and hexanoate on ACCα transcription. In chick embryo hepatocytes, activation of acetyl-CoA carboxylase-α (ACCα) transcription by 3,5,3′-triiodothyronine (T3) is mediated by a cis-acting regulatory unit (−101 to −71 bp) that binds the nuclear T3 receptor (TR) and sterol regulatory element-binding protein-1 (SREBP-1). SREBP-1 directly interacts with TR on the ACCα gene to enhance T3-induced transcription. Here, we show that treating hepatocytes with T3 or insulin stimulates a 4-fold increase in the concentration of the mature, active form of SREBP-1. When T3 and insulin are added together, a 7-fold increase in the mature SREBP-1 concentration is observed. Time course studies indicate that the T3-induced increase in mature SREBP-1 abundance is closely associated with changes in ACCα transcription and that the mechanism mediating the effect of T3 on mature SREBP-1 is distinct from that mediating the effect of insulin. Transfection analyses indicate that inhibition of ACCα transcription by cAMP or hexanoate is mediated by ACCα sequences between −101 and −71 bp. Treatment with cAMP or hexanoate suppresses the increase in mature SREBP-1 abundance caused by T3 and insulin. These results establish a new interaction between the SREBP-1 and TR signaling pathways and provide evidence that SREBP-1 plays an active role in mediating the effects of T3, insulin, cAMP, and hexanoate on ACCα transcription. In livers of avians and mammals, consumption of a high-carbohydrate, low-fat diet coordinately stimulates the transcription of genes involved in the conversion of carbohydrate to triacylglycerols (1Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar). These genes include glucokinase, l-pyruvate kinase, ATP-citrate lyase, acetyl-CoA carboxylase-α (ACCα), fatty acid synthase, malic enzyme, and glycerol-3-phosphate acyltransferase. To date, three signaling pathways have been identified that mediate the effects of dietary carbohydrate on lipogenic gene transcription. One pathway is activated by increased glucose metabolism, and its end target is carbohydrate response factor or carbohydrate response element binding protein (ChREBP) (2Kawaguchi T. Takenoshita M. Kabashima T. Uyeda K. Glucose and cAMP regulate the l-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein.Proc. Natl. Acad. Sci. USA. 2001; 98: 13710-13715Google Scholar, 3Koo S-H. Dutcher A.K. Towle H.C. Glucose and insulin function through two distinct transcription factors to stimulate expression of lipogenic enzyme genes in liver.J. Biol. Chem. 2001; 276: 9437-9445Google Scholar). Increased glucose metabolism enhances the ability of ChREBP to bind the l-pyruvate kinase gene and activate l-pyruvate kinase transcription (2Kawaguchi T. Takenoshita M. Kabashima T. Uyeda K. Glucose and cAMP regulate the l-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein.Proc. Natl. Acad. Sci. USA. 2001; 98: 13710-13715Google Scholar). A second pathway that signals changes in carbohydrate consumption to lipogenic genes is activated by 3,5,3′-triiodothyronine (T3), the active form of thyroid hormone (1Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar). T3 activates transcription through its interactions with nuclear T3 receptors (TRs) bound to T3 response elements (T3REs) of target genes (4Yen P.M. Physiological and molecular basis of thyroid hormone action.Physiol. Rev. 2001; 81: 1097-1142Google Scholar). Functional T3REs have been identified in the genes for ACCα (5Zhang Y. Yin L. Hillgartner F.B. Thyroid hormone stimulates acetyl-CoA carboxylase-α transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element.J. Biol. Chem. 2001; 276: 974-983Google Scholar), fatty acid synthase (6Xiong S. Chirala S.S. Hsu M.H. Wakil S.J. Identification of thyroid hormone response elements in the human fatty acid synthase promoter.Proc. Natl. Acad. Sci. USA. 1998; 95: 12260-12265Google Scholar), and malic enzyme (7Hodnett D.W. Fantozzi D.A. Thurmond D.C. Klautky S.A. MacPhee K.G. Estrem S.T. Xu G. Goodridge A.G. The chicken malic enzyme gene: structural organization and identification of triiodothyronine response elements in the 5'-flanking DNA.Arch. Biochem. Biophys. 1996; 334: 309-324Google Scholar). A third pathway that signals alterations in dietary carbohydrate status to lipogenic genes is activated by insulin, and its end target is sterol regulatory element binding protein (SREBP)-1 (8Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Google Scholar, 9Osborne T.F. Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action.J. Biol. Chem. 2000; 275: 32379-32382Google Scholar). SREBP-1 is synthesized as a 125 kDa precursor protein that is anchored to the endoplasmic reticulum. To become transcriptionally active, precursor SREBP-1 is translocated to Golgi, where it is cleaved by two proteases, resulting in the release of the N-terminal segment of SREBP-1, referred to as mature SREBP-1. Mature SREBP-1 is transported into the nucleus, where it binds the promoter/regulatory regions of several lipogenic genes, including ACCα (10Yin L. Zhang Y. Hillgartner F.B. Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase-α transcription in hepatocytes.J. Biol. Chem. 2002; 277: 19554-19565Google Scholar) and fatty acid synthase (11Magana M.M. Koo S.H. Towle H.C. Osborne T.F. Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase.J. Biol. Chem. 2000; 275: 4726-4733Google Scholar, 12Latasa M.J. Moon Y.S. Kim K.H. Sul H.S. Nutritional regulation of the fatty acid synthase promoter in vivo: sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element.Proc. Natl. Acad. Sci. USA. 2000; 97: 10619-10624Google Scholar). In rat hepatocytes, insulin increases the concentration of mature SREBP-1, resulting in an activation lipogenic gene transcription (13Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.Mol. Cell. Biol. 1999; 19: 3760-3768Google Scholar, 14Azzout-Marniche D. Becard D. Guichard C. Foretz M. Ferre P. Foufelle F. Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c) transcriptional activity in rat hepatocytes.Biochem. J. 2000; 350: 389-393Google Scholar, 15Shimomura I. Matsuda M. Hammer R.E. Bashmakov Y. Brown M.S. Goldstein J.L. Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice.Mol. Cell. 2000; 6: 77-86Google Scholar). The ChREBP, TR, and SREBP-1 signaling pathways also mediate the effects of nutrients and hormones that inhibit lipogenic gene transcription. For example, long-chain fatty acids suppress the activation of ChREBP, TR, and SREBP-1 by glucose, T3, and insulin, respectively (16Kawaguchi T. Osatomi K. Yamashita H. Kabashima T. Uyeda K. Mechanism for fatty acid "sparing" effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.J. Biol. Chem. 2002; 277: 3829-3835Google Scholar, 17Inoue A. Yamamoto N. Morisawa Y. Uchimoto T. Yukioka M. Morisawa S. Unesterified long-chain fatty acids inhibit thyroid hormone binding to the nuclear receptor. Solubilized receptor and the receptor in cultured cells.Eur. J. Biochem. 1989; 183: 565-572Google Scholar, 18Mater M.K. Thelen A.P. Pan D.A. Jump D.B. Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription.J. Biol. Chem. 1999; 274: 32725-32732Google Scholar, 19Hannah V.C. Ou J. Luong A. Goldstein J.L. Brown M.S. Unsaturated fatty acids down-regulate SREBP isoforms 1a and 1c by two mechanisms in HEK-293 cells.J. Biol. Chem. 2001; 276: 4365-4372Google Scholar, 20Xu J. Teran-Garcia M. Park J.H.Y. Nakamura M. Clarke S.D. Polyunsaturated fatty acids suppress hepatic sterol regulatory element-binding protein-1 expression by accelerating transcript decay.J. Biol. Chem. 2001; 276: 9800-9807Google Scholar). Glucagon, a hormone that signals the starved state in animals, also inhibits the activation of ChREBP by glucose (2Kawaguchi T. Takenoshita M. Kabashima T. Uyeda K. Glucose and cAMP regulate the l-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein.Proc. Natl. Acad. Sci. USA. 2001; 98: 13710-13715Google Scholar). Whether glucagon inhibits lipogenic gene transcription by suppressing positive signaling through the TR or SREBP-1 pathways is presently not known. ACC catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, which is the donor of all but two of the carbon atoms for the synthesis of long-chain fatty acids. This reaction is the pace-setting step of the fatty acid synthesis pathway (1Hillgartner F.B. Salati L.M. Goodridge A.G. Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.Physiol. Rev. 1995; 75: 47-76Google Scholar). There are two ACC isoforms that are encoded by distinct genes. ACCβ is the principal isoform expressed in heart and skeletal muscle, where it is thought to function primarily in the regulation of β-oxidation of fatty acids (21Abu-Elheiga L. Matzuk M.M. Abo-Hashema K.A. Wakil S.J. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2.Science. 2001; 291: 2613-2616Google Scholar). ACCα is the major isoform expressed in tissues such as liver and adipose tissue that exhibit high rates of fatty acid synthesis. Transcription of the ACCα gene is subject to nutritional regulation. For example, in livers of starved chickens, the rate of ACCα transcription is low; consumption of a high-carbohydrate, low-fat diet stimulates an 11-fold increase in ACCα transcription (22Hillgartner F.B. Charron T. Chesnut K.A. Alterations in nutritional status regulate acetyl-CoA carboxylase expression in avian liver by a transcriptional mechanism.Biochem. J. 1996; 319: 263-268Google Scholar). The induction of ACCα transcription caused by dietary carbohydrate is preceded or paralleled by increases in the molar ratio of insulin/glucagon and the level of T3 in the blood (23Goodridge A.G. Back D.W. Wilson S.B. Goldman M.J. Regulation of genes for enzymes involved in fatty acid synthesis.Ann. N. Y. Acad. Sci. 1986; 478: 46-62Google Scholar). In primary cultures of chick embryo hepatocytes (CEHs), addition of T3 to the culture medium stimulates a 7-fold increase in ACCα transcription (24Hillgartner F.B. Charron T. Chesnut K.A. Triiodothyronine stimulates and glucagon inhibits transcription of the acetyl-CoA carboxylase gene in chick embryo hepatocytes: glucose and insulin amplify the effect of triiodothyronine.Arch. Biochem. Biophys. 1997; 337: 159-168Google Scholar). Insulin has no effect by itself but amplifies the increase in ACCα transcription caused by T3. Glucagon acting through cAMP suppresses the induction of ACCα transcription caused by T3 and insulin. Fatty acids containing six to eight carbons also inhibit ACCα transcription in the presence of T3 and insulin and do so within 2 h of addition of the fatty acid (25Hillgartner F.B. Charron T. Arachidonate and medium-chain fatty acids inhibit transcription of the acetyl-CoA carboxylase gene in hepatocytes in culture.J. Lipid Res. 1997; 38: 2548-2557Google Scholar, 26Yin L. Zhang Y. Charron T. Hillgartner F.B. Thyroid hormone, glucagon, and medium-chain fatty acids regulate transcription initiated from promoter 1 and promoter 2 of the acetyl-CoA carboxylase-α gene in chick embryo hepatocytes.Biochim. Biophys. Acta. 2000; 1517: 91-99Google Scholar). Hexanoate and octanoate per se are not likely to be physiological regulators of ACCα transcription, because the levels of these fatty acids in the blood are not high enough to inhibit transcription. However, the potent, rapid, and selective effects of hexanoate and octanoate on ACCα transcription suggest that the intracellular intermediates and signaling pathways mediating the effects of these fatty acids on transcription are physiologically relevant (25Hillgartner F.B. Charron T. Arachidonate and medium-chain fatty acids inhibit transcription of the acetyl-CoA carboxylase gene in hepatocytes in culture.J. Lipid Res. 1997; 38: 2548-2557Google Scholar). For example, during starvation, a condition in which ACCα transcription is inhibited, increased rates of fatty acid oxidation may cause a change in the level of a metabolic intermediate that, in turn, inhibits ACCα transcription. In hepatocytes in culture, addition of hexanoate and octanoate to the culture medium may mimic the effects of starvation on the level of this intermediate. The identity of the active intermediate(s) mediating the effects of hexanoate and octanoate on ACCα transcription is of interest because it may aid in the development of new therapies to prevent and treat obesity and cardiovascular disease. The ACCα gene is transcribed from two promoters, generating mRNAs with heterogeneity in their 5′-untranslated regions (27Kim K.H. Regulation of mammalian acetyl-coenzyme A carboxylase.Annu. Rev. Nutr. 1997; 17: 77-99Google Scholar). Alterations in the activity of the more downstream promoter (promoter 2) account for the majority of the changes in ACCα mRNA abundance caused by starvation and refeeding a high-carbohydrate diet in intact chickens and by T3, insulin, cAMP, and hexanoate in CEH (26Yin L. Zhang Y. Charron T. Hillgartner F.B. Thyroid hormone, glucagon, and medium-chain fatty acids regulate transcription initiated from promoter 1 and promoter 2 of the acetyl-CoA carboxylase-α gene in chick embryo hepatocytes.Biochim. Biophys. Acta. 2000; 1517: 91-99Google Scholar). The stimulatory effect of T3 on ACCα promoter 2 activity is mediated by a T3RE (−101 to −86 bp) that enhances ACCα transcription in both the absence and the presence of T3, with a greater stimulation observed in the presence of T3 (5Zhang Y. Yin L. Hillgartner F.B. Thyroid hormone stimulates acetyl-CoA carboxylase-α transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element.J. Biol. Chem. 2001; 276: 974-983Google Scholar). The enhancer activity in the absence of T3 is mediated by the binding of protein complexes containing liver X receptor (LXR)·retinoid X receptor (RXR) heterodimers. The increase in enhancer activity caused by T3 treatment is mediated by the binding of a different set of protein complexes. One of these complexes contains TR·RXR heterodimers, and another contains LXR·RXR heterodimers. Immediately downstream of the ACCα T3RE is a sterol regulatory element (SRE)-1 (−80 to −71 bp) that augments the ability of the ACCα T3RE to stimulate ACCα transcription in the presence of T3 (10Yin L. Zhang Y. Hillgartner F.B. Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase-α transcription in hepatocytes.J. Biol. Chem. 2002; 277: 19554-19565Google Scholar). Results from transfection, protein binding, and DNA binding assays suggest that the stimulatory effect of the SRE-1 on ACCα transcription is mediated by a direct and T3-inducible interaction between SREBP-1 and TR and that this interaction facilitates the formation of a SREBP-1·SREBP-1/TR·RXR tetrameric complex on the ACCα gene. Complex formation between TR·RXR and SREBP-1·SREBP-1 stabilizes the binding of SREBP-1 to the SRE-1. Thus, optimal induction of ACCα transcription by T3 is dependent on an interaction between TR and SREBP-1 on the ACCα gene. Both of these signaling pathways represent potential targets mediating the actions of insulin, cAMP, and medium-chain fatty acids on T3-induced ACCα transcription. The mechanisms by which insulin, cAMP, and medium-chain fatty acids control ACCα transcription remain to be determined. In the present study, we have identified a new interaction between the SREBP-1 and TR signaling pathways. T3 increases the concentration of the mature, active form of SREBP-1 in CEH. In addition, we provide evidence that insulin, cAMP, and medium-chain fatty acids regulate ACCα transcription by modulating the abundance of mature SREBP-1. Reporter plasmids are named by designating the 5′- and 3′- ends of the ACCα DNA fragment relative to the transcription start site of promoter 2. A series of 5′-deletions and 3′-deletions of ACCα promoter 2 gene in the context of p[ACC−4900/+274] chloramphenicol acetyltransferase (CAT) have been previously described (5Zhang Y. Yin L. Hillgartner F.B. Thyroid hormone stimulates acetyl-CoA carboxylase-α transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element.J. Biol. Chem. 2001; 276: 974-983Google Scholar). ACCα promoter constructs containing mutations of the SRE-1 between −79 and −72 bp have been described previously (10Yin L. Zhang Y. Hillgartner F.B. Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase-α transcription in hepatocytes.J. Biol. Chem. 2002; 277: 19554-19565Google Scholar). pBLCAT2 (pTKCAT) was obtained from B. Luckow and G. Schutz (German Cancer Research Center) (28Luckow B. Schutz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements.Nucleic Acids Res. 1987; 155490Google Scholar). p[ACC−108/−82]TKCAT, p[ACC−108/−66]TKCAT, p[ACC−84/−66]TKCAT, and pTKCAT constructs containing mutations in the −108 to −66 bp ACCα fragment are described in (10Yin L. Zhang Y. Hillgartner F.B. Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase-α transcription in hepatocytes.J. Biol. Chem. 2002; 277: 19554-19565Google Scholar). A full-length cDNA for chicken SREBP-1 was obtained by screening a chicken liver cDNA library (Stratagene) using a human SREBP-1 cDNA probe (nucleotides 721 to 1,103 relative to the start site of translation) and by 5′-rapid amplification of cDNA ends (RACE) (Y. Zhang and F. B. Hillgartner, unpublished observations). The N-terminal amino acid sequence of this chicken SREBP-1 (GenBank accession number: AY029224) more closely resembles the 1a isoform than the 1c isoform described in mammalian species (29Yokoyama 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: 187-197Google Scholar). Data from 5′-RACE and RNase protection analyses indicate that other forms of SREBP-1 containing variations in the N-terminus are not expressed in chicken cells. An expression plasmid encoding the mature form of chicken SREBP-1 was developed by subcloning an SREBP-1 cDNA fragment encoding amino acids 1 to 464 into pSV-SPORT1 (Invitrogen) to form pSV-SPORT1-SREBP-1 (). Primary cultures of CEH were prepared as previously described (30Goodridge A.G. Regulation of fatty acid synthesis in isolated hepatocytes prepared from the livers of neonatal chicks.J. Biol. Chem. 1973; 248: 1924-1931Google Scholar) and maintained in serum-free Waymouth's medium MD705/1 containing 1 μM corticosterone, 50 nM insulin (gift from Eli Lilly Corp.), and 25 mM glucose. CEHs were incubated at 40°C in a humidified atmosphere of 5% CO2 and 95% air. CEHs were transfected using a modification of the method of Baillie et al. (31Baillie R.A. Klautky S.A. Goodridge A.G. Transient transfection of chick-embryo hepatocytes.J. Nutr. Biochem. 1993; 4: 431-439Google Scholar). Briefly, CEHs were isolated as described above and incubated on 60 mm petri dishes (Fisher Scientific). At 6 h of incubation, the medium was replaced with one containing 20 μg of lipofectin (Invitrogen), 2.5 μg of p[ACC−4900/+274]CAT or an equimolar amount of another reporter plasmid and pBluescript KS(+) to bring the total amount of transfected DNA to 3.0 μg per plate. At 18 h of incubation, the transfection medium was replaced with fresh medium containing corticosterone, insulin, 25 mM glucose, and T3 (1.5 μM). On some cells, the medium was supplemented with dibutyryl cAMP (50 μM) or hexanoate (2.5 mM). At 66 h of incubation, CEHs were harvested and cell extracts were prepared (31Baillie R.A. Klautky S.A. Goodridge A.G. Transient transfection of chick-embryo hepatocytes.J. Nutr. Biochem. 1993; 4: 431-439Google Scholar). CAT activity (32Gorman C.M. Moffat L.F. Howard B.H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.Mol. Cell. Biol. 1982; 2: 1044-1051Google Scholar) and protein (33Sedmak J.J. Grossberg S.E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250.Anal. Biochem. 1977; 79: 544-552Google Scholar) were assayed by the indicated methods. All DNAs used in transfection experiments were purified using the Qiagen endotoxin-free kit. All procedures were carried out at 4°C. To prevent proteolysis, a mixture of protease inhibitors (Complete, Roche Molecular Biochemicals) was included in all the buffers. Nuclear extracts were prepared from CEHs by a modification of the method described by Dignam et al. (34Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). Briefly, CEHs from four 100 mm plates were pooled and centrifuged at 1,000 g for 5 min at 4°C. The resulting cell pellet was homogenized in Buffer 1 [10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol] using 20 strokes in a Dounce homogenizer. The homogenate was centrifuged at 1,100 g for 10 min, and the resulting nuclear pellet was washed once in buffer 1. The nuclear pellet was resuspended in Buffer 2 [20 mM Hepes (pH 7.9), 420 mM NaCl, 25% (v/v) glycerol, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol]. This suspension was rotated for 30 min and then centrifuged 15,000 g for 30 min. The resulting supernatant is designated as the nuclear extract fraction. The membrane extract fraction was prepared by centrifuging the supernatant of the original 1,100 g spin for 1 h at 100,000 g. The resulting membrane pellet was dissolved in Buffer 3 [10 mM Tris (pH 6.8), 100 mM NaCl, 1% (w/v) SDS, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol]. The protein content of the nuclear and membrane extracts was determined as described (33Sedmak J.J. Grossberg S.E. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250.Anal. Biochem. 1977; 79: 544-552Google Scholar). Double-stranded oligonucleotides were prepared by combining equal amounts of the complementary single-stranded DNA in a solution containing 10 mM Tris (pH 8.0) and 1 mM EDTA followed by heating to 90°C for 2 min, and then cooling to room temperature. The annealed oligonucleotides were labeled by filling in overhanging 5′-ends using the Klenow fragment of Escherichia coli DNA polymerase in the presence of [α-32P]dCTP and/or [α-32P]dGTP. Binding reactions were carried out in 20 μl of 20 mM Tris (pH 7.9), 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol (v/v), 0.3 mg/ml BSA, and 2 μg of poly[d(I·C)]. A typical reaction contained 20,000 cpm of labeled DNA and 20 μg of nuclear extract. The reactions were performed at 20°C for 20 min (ACCα SRE-1 probe) (Figs. 1A, B, 7B) or on ice for 60 min (ACCα T3RE probe, Fig. 6). DNA and DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gels at 4°C in 0.5 × TBE (45 mM Tris (pH 8.3), 45 mM boric acid, 1 mM EDTA). Following electrophoresis, the gels were dried and subjected to storage phosphor autoradiography. For competition experiments, unlabeled competitor DNA was mixed with radiolabeled oligomer prior to addition of nuclear extract. For antibody supershift experiments, nuclear extracts were incubated with antibodies for 30 min prior to addition of the oligonucleotide probe. Mouse monoclonal antibodies against SREBP-1 (IgG-2A4) and SREBP-2 (IgG-1D2) were obtained from American Type Tissue Collection (Manassas, VA). The sequence of ACCα SRE-1 probe and ACCα T3RE probe was 5′-TCGCATCACACCACCGCGG-3′ and 5′-AGGTGGTTGACCCGA GGTTAACCCCTCG-3′, respectively.Fig. 7cAMP and hexanoate suppress the stimulatory effects of T3 and insulin on the concentration of mature SREBP-1 in CEH. Eighteen hours after being placed in culture, CEHs were incubated in Waymouth's medium containing corticosterone, insulin, 25 mM glucose, and T3 with or without dibutyryl cAMP or hexanoate for the indicated times. Cell extracts or total RNA were prepared as described in Experimental Procedures. A: The abundance of precursor SREBP-1 in membrane fractions and mature SREBP-1 in nuclear extracts was measured by Western analyses. These data are from a representative experiment. B: Gel mobility shift assays were performed using nuclear extracts from hepatocytes and an oligonucleotide probe containing the ACCα SRE-1 (−84 to −66 bp). Positions of specific protein-DNA complexes (arrows) and nonspecific complexes (asterisk) are indicated. C: The abundance of SREBP-1 mRNA and 18S rRNA was measured using an RNase protection assay. These data are from a representative experiment. D: Signals for precursor SREBP-1 protein and mature SREBP-1 protein from Western analyses and SREBP-1 mRNA from RNase protection analyses were quantitated. Levels of precursor SREBP-1 protein, mature SREBP-1 protein, and SREBP-1 mRNA in hepatocytes treated with T3 and insulin without cAMP and hexanoate were set at 1. Values are the means ± SEM of four experiments. a: mean is significantly (P < 0.05) different from that of cells treated with T3 and insulin without cAMP and hexanoate.View Large Image Figure ViewerDownload (PPT)Fig. 6Effect of cAMP and hexanoate on the binding of hepatic nuclear proteins to the ACCα T3RE. Eighteen hours after being placed in culture, CEHs were incubated in Waymouth's medium containing corticosterone, insulin, and 25 mM glucose with or without T3, T3 plus dibutyryl cAMP, or T3 plus hexanoate for the indicated times. Cells were harvested and nuclear extracts were prepared as described in Experimental Procedures. Nuclear extracts were subjected to gel mobility shift analyses using an oligonucleotide probe containing the ACCα T3RE (−108 to −82 bp). Specific protein-DNA complexes are indicated by arrows. Previous studies have shown that complexes 1, 2, and 3 contain liver X receptor (LXR)·retinoid X receptor (RXR) heterodimers, whereas complex 4 contains nuclear T3 receptor (TR)·RXR heterodimers (5Zhang Y. Yin L. Hillgartner F.B. Thyroid hormone stimulates acetyl-CoA carboxylase-α transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element.J. Biol. Chem. 2001; 276: 974-983Google Scholar). These data are representative of five experiments employing independent preparations of nuclear extract.View Large Image Figure ViewerDownload (PPT) Proteins in nuclear extract and membrane fractions were subjected to electrophoresis in 8% SDS-polyacrylamide gels and then transferred to PDVF membranes (Amersham Pharmacia) using an electroblotting apparatus (Owl Scientific). Immunoblot analyses were performed as described in the Western blotting protocol from Santa Cruz Biotechnology. Briefly, the blots were blocked in Blotto [5% nonfat dry milk, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl] at 4°C overnight, and then incuba
Referência(s)