Divergent Effects of Peroxisome Proliferator-activated Receptor γ Agonists and Tumor Necrosis Factor α on Adipocyte ApoE Expression
2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês
10.1074/jbc.m408461200
ISSN1083-351X
AutoresLili Yue, Neda Rasouli, Gouri Ranganathan, Philip A. Kern, Theodore Mazzone,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoApoE is expressed in multiple mammalian cell types in which it supports cellular differentiated function. In this report we demonstrate that apoE expression in adipocytes is regulated by factors involved in modulating systemic insulin sensitivity. Systemic treatment with pioglitazone increased systemic insulin sensitivity and increased apoE mRNA levels in adipose tissue by 2–3-fold. Treatment of cultured 3T3-L1 adipocytes with ciglitazone increased apoE mRNA levels by 2–4-fold in a dose-dependent manner and increased apoE secretion from cells. Conversely, treatment of adipocytes with tumor necrosis factor (TNF) α reduced apoE mRNA levels and apoE secretion by 60%. Neither insulin nor a peroxisome proliferator-activated receptor (PPAR) α agonist regulated adipocyte apoE gene expression. In addition, treatment of human monocyte-derived macrophages with ciglitazone did not regulate expression of apoE. Additional analyses using reporter genes indicated that the effect of TNFα and PPARγ agonists on the apoE gene was mediated via distinct gene control elements. The TNFα effect was mediated by elements within the proximal promoter, whereas the PPARγ effect was mediated by elements within a downstream enhancer. However, the addition of TNFα substantially reduced the absolute levels of apoE reporter gene response even in the presence of ciglitazone. These results indicate for the first time that adipose tissue expression of apoE is modulated by physiologic regulators of insulin sensitivity. ApoE is expressed in multiple mammalian cell types in which it supports cellular differentiated function. In this report we demonstrate that apoE expression in adipocytes is regulated by factors involved in modulating systemic insulin sensitivity. Systemic treatment with pioglitazone increased systemic insulin sensitivity and increased apoE mRNA levels in adipose tissue by 2–3-fold. Treatment of cultured 3T3-L1 adipocytes with ciglitazone increased apoE mRNA levels by 2–4-fold in a dose-dependent manner and increased apoE secretion from cells. Conversely, treatment of adipocytes with tumor necrosis factor (TNF) α reduced apoE mRNA levels and apoE secretion by 60%. Neither insulin nor a peroxisome proliferator-activated receptor (PPAR) α agonist regulated adipocyte apoE gene expression. In addition, treatment of human monocyte-derived macrophages with ciglitazone did not regulate expression of apoE. Additional analyses using reporter genes indicated that the effect of TNFα and PPARγ agonists on the apoE gene was mediated via distinct gene control elements. The TNFα effect was mediated by elements within the proximal promoter, whereas the PPARγ effect was mediated by elements within a downstream enhancer. However, the addition of TNFα substantially reduced the absolute levels of apoE reporter gene response even in the presence of ciglitazone. These results indicate for the first time that adipose tissue expression of apoE is modulated by physiologic regulators of insulin sensitivity. Apolipoprotein E is a 35-kDa glycoprotein that circulates as a surface component of plasma lipoproteins and is expressed in multiple mammalian cell types (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3386) Google Scholar, 2Curtiss L.K. Boisvert W.A. Curr. Opin. Lipidol. 2000; 11: 243-251Crossref PubMed Scopus (196) Google Scholar, 3Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 4Driscoll D.M. Getz G.S. J. Lipid Res. 1984; 25: 1368-1379Abstract Full Text PDF PubMed Google Scholar). Reports over many years have demonstrated that apoE is involved in maintaining important aspects of organismal and cellular homeostasis. For example, disordered apoE function/expression has a well established role in the pathophysiology of important human diseases such as atherosclerosis and dementia (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3386) Google Scholar, 2Curtiss L.K. Boisvert W.A. Curr. Opin. Lipidol. 2000; 11: 243-251Crossref PubMed Scopus (196) Google Scholar, 3Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 4Driscoll D.M. Getz G.S. J. Lipid Res. 1984; 25: 1368-1379Abstract Full Text PDF PubMed Google Scholar, 5Mayeux R. Saunders A.M. Shea S. Mirra S. Evans D. Roses A.D. Hyman B.T. Crain B. Tang M-X. Phelps C.H. N. Engl. J. Med. 1998; 338: 506-511Crossref PubMed Scopus (451) Google Scholar, 6Harris F.M. Tesseur I. Brecht W.J. Xu Q. Mullendorff K. Chang S. Wyse-Coray T. Mahley R.W. Huang Y. J. Biol. Chem. 2004; 279: 3862-3868Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). ApoE has also been implicated in regulating the expansion of the renal mesangial matrix, in regulating the differentiated function of steroidogenic tissues, and protecting cells from oxidant stress (7Chen G. Paka L. Kako Y. Singhal P. Duan W. Pillarisetti S. J. Biol. Chem. 2001; 276: 49142-49147Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Dyer C.A. Curtiss L.K. J. Biol. Chem. 1988; 263: 10965-10973Abstract Full Text PDF PubMed Google Scholar, 9Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (801) Google Scholar). In line with its diverse roles in normal mammalian physiology, apoE is expressed in multiple cell types, including vessel wall macrophages and cells of the central nervous system, in steroidogenic tissue, and in the kidney (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3386) Google Scholar, 2Curtiss L.K. Boisvert W.A. Curr. Opin. Lipidol. 2000; 11: 243-251Crossref PubMed Scopus (196) Google Scholar, 3Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 4Driscoll D.M. Getz G.S. J. Lipid Res. 1984; 25: 1368-1379Abstract Full Text PDF PubMed Google Scholar, 5Mayeux R. Saunders A.M. Shea S. Mirra S. Evans D. Roses A.D. Hyman B.T. Crain B. Tang M-X. Phelps C.H. N. Engl. J. Med. 1998; 338: 506-511Crossref PubMed Scopus (451) Google Scholar, 6Harris F.M. Tesseur I. Brecht W.J. Xu Q. Mullendorff K. Chang S. Wyse-Coray T. Mahley R.W. Huang Y. J. Biol. Chem. 2004; 279: 3862-3868Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Chen G. Paka L. Kako Y. Singhal P. Duan W. Pillarisetti S. J. Biol. Chem. 2001; 276: 49142-49147Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 8Dyer C.A. Curtiss L.K. J. Biol. Chem. 1988; 263: 10965-10973Abstract Full Text PDF PubMed Google Scholar, 9Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (801) Google Scholar). As a surface component of lipoproteins, apoE is involved in regulating organismal lipoprotein metabolism (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3386) Google Scholar, 2Curtiss L.K. Boisvert W.A. Curr. Opin. Lipidol. 2000; 11: 243-251Crossref PubMed Scopus (196) Google Scholar, 3Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 4Driscoll D.M. Getz G.S. J. Lipid Res. 1984; 25: 1368-1379Abstract Full Text PDF PubMed Google Scholar). Hepatocytes are the predominant source of circulating apoE; however, peripheral tissues can also contribute importantly to plasma apoE levels (10Hasty A.H. Linton M.F. Swift L.L. Fazio S. J. Lipid Res. 1999; 40: 1529-1538Abstract Full Text Full Text PDF PubMed Google Scholar, 11Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Crossref PubMed Scopus (109) Google Scholar). Furthermore, it has been demonstrated that circulating apoE derived from peripheral cells can play an important role in modulating systemic lipoprotein metabolism and cholesterol levels (10Hasty A.H. Linton M.F. Swift L.L. Fazio S. J. Lipid Res. 1999; 40: 1529-1538Abstract Full Text Full Text PDF PubMed Google Scholar, 11Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Crossref PubMed Scopus (109) Google Scholar, 12Zuu Y. Bellosta S. Langer C. Bernini F. Pitas R.E. Mahley R.W. Assmann G. von Eckardstein A. Proc. Natl. Acad. Sci. U. S. A. 1998; 93: 7585-7590Google Scholar). It can also modulate vessel wall atherosclerosis independent of its effects on cholesterol level. For example, in apoE knockout mice, the synthesis and secretion of transgenic apoE in adrenal glands to produce <2% of wild-type circulating apoE levels reduced atherosclerosis by 80–95% even though there was no effect on hypercholesterolemia (11Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Crossref PubMed Scopus (109) Google Scholar). In addition, circulating apoE has been postulated to play a role in suppressing systemic oxidant stress (13Tangirala R.K. Pratico D. FitzGerald A. Chun S. Tsukamoto K. Maugeais C. Usher D.C. Pure E. Rader D.J. J. Biol. Chem. 2001; 276: 261-266Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). ApoE expression has also been demonstrated in adipocytes. Zechner et al. (14Zechner R. Moser R. Newman T.C. Fried S.K. Breslow J. J. Biol. Chem. 1991; 266: 10583-10588Abstract Full Text PDF PubMed Google Scholar) demonstrated apoE mRNA and protein expression in 3T3-L1 adipocytes and in adipose tissue biopsies collected from the gluteal region of human subjects. In these studies, the expression of apoE was found to increase as a function of 3T3-L1 differentiation; almost no apoE was detected in preadipocytes. In addition, inhibition of adipocyte lipid accumulation by biotin deprivation reduced apoE expression. Conversely, cholesterol loading of adipocytes enhanced apoE expression. Multiple potential control elements for apoE gene expression have been described in its 5′ proximal promoter (15Smith J.D. Melian A. Leff T. Breslow J.L. J. Biol. Chem. 1998; 263: 8300-8308Google Scholar, 16Chang D.J. Paik Y-K. Leren T.P. Walker D.W. Howlett G.J. Taylor J.M. J. Biol. Chem. 1990; 265: 9496-9504Abstract Full Text PDF PubMed Google Scholar, 17Berg D.T. Calnek D.S. Grinnell B.W. J. Biol. Chem. 1998; 270: 15447-15450Abstract Full Text Full Text PDF Scopus (20) Google Scholar, 18Salero E. Perez-Sen R. Aruga J. Gimenez C. Zafra F. J. Biol. Chem. 2001; 276: 1881-1888Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19Do Carmo S. Seguin D. Milne R. Rassart E. J. Biol. Chem. 2002; 277: 5514-5523Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In addition, downstream control elements have been described that are required to direct specific expression of apoE in various tissues. Two downstream duplicated enhancers have been reported that specify expression of the apoE gene in macrophages and adipocytes of transgenic animals (20Shih S-J. Allan C. Grehan S. Tse E. Moran C. Taylor J.M. J. Biol. Chem. 2000; 275: 31567-31572Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Furthermore, an LXR 1The abbreviations used are: LXR, liver x receptor; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor; RT, reverse transcription. response element embedded within these enhancer elements has been shown to transduce the stimulatory effect of sterol/oxysterol on apoE gene expression in macrophages and adipocytes (21Mazzone T. Gump H. Diller P. Getz G.S. J. Biol. Chem. 1987; 262: 11657-11662Abstract Full Text PDF PubMed Google Scholar, 22Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (573) Google Scholar). Adipocytes are a major cell target for PPARγ agonists (23Lowell B.B. Cell. 1999; 99: 239-242Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). This class of compounds includes two drugs, pioglitazone and rosiglitazone, that are widely used to treat patients with diabetes. In view of the widespread use of these drugs in human patients, the important role for PPARγ in modulating adipocyte function, the observation that apoE is made in adipocytes, and the important and diverse roles that have been ascribed to apoE in organismal homeostasis, we evaluated a role for PPARγ agonists in regulating adipocyte apoE expression. We demonstrate that ciglitazone and pioglitazone increase adipocyte apoE expression. Furthermore, we show that TNFα markedly suppresses apoE expression in adipocytes. Materials—Cell culture media, supplements, and antibiotics were purchased from Invitrogen. Ciglitazone, insulin, dexamethasone, methylisobutylxanthine were from Sigma. Wy-14643 was purchased from Biomol Research Laboratories. TNFα was from R & D systems. All other materials were from previously identified sources (24Zhao Y. Yue L. Gu D. Mazzone T. J. Biol. Chem. 2002; 277: 29477-29483Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Zhao Y. Mazzone T. J. Biol. Chem. 2000; 275: 4759-4765Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Huang Z.H. Mazzone T. J. Lipid Res. 2002; 43: 375-382Abstract Full Text Full Text PDF PubMed Google Scholar, 27Huang Z.H. Gu D. Lange Y. Mazzone T. Biochemistry. 2003; 42: 3949-3955Crossref PubMed Scopus (32) Google Scholar). Cell Culture—Murine 3T3-L1 cells (ATCC, Manassas, VA; passage 3–10) were grown to confluence and differentiated to adipocytes as described (28Dalen K.T. Ulven S.M. Bamberg K. Gustafsson J-A. Nebb H.I. J. Biol. Chem. 2003; 278: 48283-48291Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 29Green A. Bumberger J.M. Stuart C.A. Ruboff M.S. Diabetes. 2004; 53: 74-81Crossref PubMed Scopus (75) Google Scholar) with minor modifications. Briefly, pre-adipocytes were cultured in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Two days after reaching confluence, cells were differentiated by treatment with 10 μg/ml insulin, 0.5 mm methylisobutylxanthine, and 1 μm dexamethasone in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 2 days. Cells were then maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5 μg/ml insulin. All experiments were performed on post-differentiation day 8 to day 10. Human monocyte-derived macrophages were isolated and cultured as previously described (24Zhao Y. Yue L. Gu D. Mazzone T. J. Biol. Chem. 2002; 277: 29477-29483Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). RNA Preparation and RT-PCR—Cytoplasmic RNA was extracted from fully differentiated adipocytes grown on six-well plates using the RNeasy mini kit (Qiagen). RT-PCR was performed using 200 ng of RNA and the one-step Calypso RT-PCR kit from DNAmp Ltd. The apoE and β-actin primer pairs were described previously (24Zhao Y. Yue L. Gu D. Mazzone T. J. Biol. Chem. 2002; 277: 29477-29483Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Reverse transcription was carried out at 50 °C for 30 min and then terminated at 94 °C for 2 min. Amplifications were pre-titrated to be within a linear range by varying template levels and number of cycles. The PCR products were resolved on agarose gels and stained with ethidium bromide. The apoE primer pair produced a 503-bp product, and the β-actin primer pair produced a 924-bp product. The images were captured using a Bio-DOC-IT system (UVP, Upland, CA) and quantitated using ImageQuant. The base-line relationship between apoE and β-actin mRNA abundance varied among experiments. However, the regulatory effects of TNFα and ciglitazone were consistent regardless of this base-line relationship. For real-time RT-PCR, one step SYBR Green RT-PCR amplification was performed using the Mx3000 quantitative PCR system (Stratagene). After optimization of each of the primer pairs, samples were assayed in a 25-μl reaction mixture containing 500 ng (for apoE) or 200 ng (for β-actin) of sample RNA and 300 nm each of the primers using Brilliant SYBR Green QRT-PCR Master Mix kit (Stratagene #600552) with 1 m betaine and 5% dimethyl sulfoxide. Relative quantitation for the apoE gene, expressed as fold increase over control, was calculated after normalization to β-actin RNA. Each sample was analyzed in triplicate. Immunoblot—3T3-L1 adipocytes were solubilized by scraping in lysis buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.05% sodium deoxycholate, 4% SDS, protease inhibitors), incubated at room temperature for 30 min, and boiled for 10 min. The lysate was clarified by centrifugation at 13,500 × g for 20 min. The protein concentration was then determined using the Bio-Rad DC protein assay kit. Twenty μg of cell lysate or 25 μl of medium were resolved by 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk, 1% bovine serum albumin, and apoE was detected by ECL kit (Amersham Biosciences) as previously described (24Zhao Y. Yue L. Gu D. Mazzone T. J. Biol. Chem. 2002; 277: 29477-29483Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 25Zhao Y. Mazzone T. J. Biol. Chem. 2000; 275: 4759-4765Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Signals on immunoblot were quantitated using Zero DScan software (Scanalytics Inc., Fairfax, VA). Plasmids—The pGL623 plasmid was constructed by cloning a –623 to +86 SmaI-XbaI fragment (30Paik Y-K. Chang D.J. Reardon C.A. Davies G.E. Mahley R.W. Taylor J.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3445-3449Crossref PubMed Scopus (222) Google Scholar) from the apoE gene promoter into the multiple cloning site of pGL3 basic. A 620-bp fragment containing an apoE enhancer sequence (20Shih S-J. Allan C. Grehan S. Tse E. Moran C. Taylor J.M. J. Biol. Chem. 2000; 275: 31567-31572Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 22Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (573) Google Scholar) with a conserved LXR response element (located ∼3.3 kilobases downstream of the apoE gene) was amplified by PCR from THP1 genomic DNA and cloned into the SalI/BamH1 site of pGL623 to generate pGL623enhancer. A 54-bp double-stranded oligo containing a previously defined PPARγ apoE gene response element (located ∼2 kilobases downstream of the apoE gene) in the apoE/apoC1 intergenic region (31Galetto R. Albajar M. Polanco J.I. Zakin M.M. Rodrigues-Rey J.C. Biochem. J. 2001; 357: 521-527Crossref PubMed Scopus (53) Google Scholar) was synthesized and cloned into pGL623 to generate pGL623PPAR. As a control for transfection efficacy a plasmid expressing β-galactosidase (pSVβ-galactosidase) under the control of the SV40 promoter was used. Transient Transfection and Reporter Gene Analysis—Differentiated adipocytes were electroporated using the Gene Pulser Xcell (Bio-Rad) with a setting at 180 V and 940 microfarads. Briefly, the cells were trypsinized and washed three times in Dulbecco's phosphate-buffered saline without CaCl2 and MgCl2. The cells were then suspended at 107/ml in phosphate-buffered saline, and 0.5 ml of cell suspension was transferred to a 0.4-cm gap cuvette. Twenty-five μg of apoE reporter plasmid DNA, 2.5 μgofthe β-galactosidase internal control, and 200 μg of carrier DNA (sheared herring sperm DNA, Roche Applied Science) were added. After electroporation, cells were plated on 6-well plates and allowed to recover for 24 h in 10% fetal bovine serum before use in experiments. Luciferase and β-galactosidase activity was measured in cell extracts using kits available from Promega according to the manufacturer's instructions. Firefly luciferase activities were normalized to β-galactosidase activity to calculate relative luciferase units. Human Subject Treatment Protocol—Subjects signed consents to a protocol that was approved by the Institutional Review Board of the University of Arkansas for Medical Sciences (UAMS) and was conducted on the UAMS General Clinical Research Center. Subjects were recruited who were in good health but with impaired glucose tolerance, with a fasting glucose under 100 mg/dl and a 2-h post-challenge glucose of 140 to 199 mg/dl following a standard oral 75-g glucose challenge. Subjects were weight-stable and were controlled on a 35% fat, eucaloric diet throughout the study. A subcutaneous fat biopsy was performed by incision from the lower abdominal wall, and adipose tissue was immediately placed into liquid nitrogen for subsequent RNA extraction, as described previously (32Kern P.A. Di Gregorio G.B. Lu T. Rassouli N. Ranganathan G. Diabetes. 2003; 52: 1779-1785Crossref PubMed Scopus (749) Google Scholar). Insulin sensitivity was measured using the frequently sampled intravenous glucose tolerance study using 11.4 g/m2 glucose and 0.04 units/kg insulin (33Bergman R.N. Finegood D.T. Ader M. Endocr. Rev. 1985; 6: 45-85Crossref PubMed Scopus (964) Google Scholar). Insulin was measured using an immunochemiluminescent assay (MLT Assay, Wales, UK), and glucose was measured in duplicate by a glucose oxidase assay. The insulin sensitivity index (SI) was calculated using the MinMod program. SI is an index of insulin sensitivity based on the dynamic relationship between circulating insulin and glucose levels after the intravenous injection of glucose and insulin, with low values indicating insulin resistance. The insulin sensitivity index derived from this test is highly correlated with the insulin sensitivity index derived from the euglycemic clamp (34Bergman R.N. Prager R. Volund A. Olefsky J.M. J. Clin. Investig. 1987; 79: 790-800Crossref PubMed Scopus (695) Google Scholar). Subjects were treated with pioglitazone for 10 weeks beginning at 30 mg/day for 2 weeks and then 45 mg/day for an additional 8 weeks. Compliance and laboratory tests were monitored three times during the follow-up phase, and then repeat adipose tissue biopsies and insulin sensitivity testing were performed. We measured the effect of the PPARγ agonist, ciglitazone, on apoE expression in 3T3-L1 adipocytes (Figs. 1 and 2). As shown in Fig. 1, treatment with ciglitazone increased the abundance of adipocyte apoE mRNA by 2–4-fold in a dose-dependent manner. The results in Fig. 2 demonstrate that treatment of adipocytes with ciglitazone (30 μm) increased cell-associated and secreted apoE protein as detected by immunoblot. In three independent experiments there was a 2.5-fold increase in cell-associated apoE and a 3.6-fold increase in secreted apoE after treatment with ciglitazone, similar to the magnitude of increase in the apoE mRNA level. We next performed experiments to assess the specificity of the apoE gene response to ciglitazone. Insulin is an important regulator of adipocyte gene expression (35Lay S.L. Lefrere I. Trautwein C. Dugail I. Krief S. J. Biol. Chem. 2002; 277: 35625-35634Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). PPARα is poorly expressed in adipocytes; however, PPARα agonists have been reported to modulate adipocyte gene expression (36Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1862) Google Scholar). Neither insulin nor the PPARα agonist, WY14643, influenced adipocyte apoE mRNA abundance (Fig. 3). As an additional evaluation of specificity, we examined the response of the apoE gene in human monocyte-derived macrophages to ciglitazone. When examining time points up to 48 h of treatment over a range of ciglitazone doses, there was no change in human monocyte-derived macrophage apoE expression (not shown).Fig. 2Ciglitazone increases cellular and secreted apoE in adipocytes. Adipocytes were treated with 30 μm ciglitazone in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin for 24 h. At that time 25 μl of medium or 20 μg of cellular protein was used for immunoblotting, as described under "Experimental Procedures." A representative immunoblot is shown. Results from three independent experiments were pooled to calculate -fold change. The difference between ciglitazone-treated and control cells is significant at p < 0.01.View Large Image Figure ViewerDownload (PPT)Fig. 3Insulin and PPARα activators do not influence adipocyte apoE gene expression. Differentiated adipocytes were treated with insulin or the PPARα ligand, WY14643, at the indicated concentrations in complete medium for 24 h. Insulin was withdrawn from the maintenance culture medium for 48 h before the start of the experimental insulin incubation. Agarose gels representative of results from three independent experiments with similar results are shown. Neither treatment produced a change in apoE mRNA abundance.View Large Image Figure ViewerDownload (PPT) The study of PPARγ agonist-regulated gene expression is of particular interest due to the widespread use of these agents in humans. Therefore, we next evaluated whether systemic treatment of insulin-resistant, non-diabetic subjects with the PPARγ agonist pioglitazone would influence apoE gene expression in adipose tissue. Three female subjects with impaired glucose tolerance were treated with pioglitazone for 10 weeks using the protocol described under "Experimental Procedures." For the duration of this treatment, the subjects were placed on a weight-maintaining diet containing 35% of calories from fat. Subcutaneous abdominal adipose tissue was collected before and after treatment with pioglitazone. Results obtained from these studies are given in Table I. The glycohemoglobin was in the normal range before and after treatment with pioglitazone, as was the fasting blood glucose, although the fasting blood glucose modestly decreased after treatment in all three subjects. Improved insulin sensitivity, expected with pioglitazone treatment, was demonstrated by the increase in the insulin sensitivity index, SI, measured at 10 weeks in all three subjects. With respect to apoE gene expression, treatment of subjects with pioglitazone produced a 2–3-fold increase in apoE mRNA abundance, as measured using real time RT-PCR. The magnitude of the apoE gene response measured in adipose tissue exposed to a PPARγ agonist in vivo was similar to that produced in isolated 3T3-L1 adipocytes treated with ciglitazone.Table ITreatment of IGT subjects with PPARγ agonists increases apoE mRNA in adipose tissueBMIHgbA1CFBGSIApoE mRNAkg/m2%mg/dl×10-4 (min-1 × μg-1 × ml-1)-Fold change (post/pre)Subject 1Pre375.0920.372.2 ± 0.1Post37.65.0900.78Subject 2Pre375.2861.102.6 ± 0.2Post37.95.9711.94Subject 3Pre25.25.3811.211.9 ± 0.1Post25.35.1781.95 Open table in a new tab The above results demonstrated the in vivo effect of a PPARγ agonist on increasing systemic insulin sensitivity and adipose tissue apoE gene expression. TNFα has been implicated in reducing insulin sensitivity and is an important modulator of adipocyte gene expression in vivo and in vitro. We, therefore, next evaluated the effect of TNFα on apoE expression in 3T3-L1 adipocytes (Figs. 4 and 5). Treatment of adipocytes with 20 ng/ml TNFα led to an approximate 60% reduction in apoE mRNA abundance (Fig. 4). Furthermore, this reduction was reflected in an approximate 60% decrease in apoE protein secretion into the medium during TNFα treatment at 20 ng/ml (Fig. 5).Fig. 5TNFα reduces apoE secretion from adipocytes. Differentiated adipocytes were treated with TNFα for 24 h in 0.2% bovine serum albumin at the doses indicated. At that time, 25 μl of culture medium was utilized for immunoblot analysis as described under "Experimental Procedures." A representative immunoblot is shown. -Fold change in secreted apoE was calculated from two independent experiments.View Large Image Figure ViewerDownload (PPT) Although most TNFα in adipose tissue may derive from macrophages, low levels of TNFα may be expressed by adipocytes in the presence of obesity (37Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7473) Google Scholar, 38Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2003; 112: 1785-1788Crossref PubMed Scopus (1439) Google Scholar, 39Spiegelman B.M. Choy L. Hotamisligil G.S. Graves R.A. Tontonoz P. J. Biol. Chem. 1993; 268: 6823-6826Abstract Full Text PDF PubMed Google Scholar, 40van Wijk J.P.H. Rabelink T.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 798-800Crossref PubMed Scopus (12) Google Scholar). PPARγ agonists have been shown to suppress TNFα expression in adipocytes (40van Wijk J.P.H. Rabelink T.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 798-800Crossref PubMed Scopus (12) Google Scholar). It has further been shown that suppression of most adipocyte genes by TNFα is mediated via NFκB, and PPARγ agonists can interfere with transcriptional activity of NFκB (40van Wijk J.P.H. Rabelink T.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 798-800Crossref PubMed Scopus (12) Google Scholar, 41Ruan H. Pownall H.J. Lodish H.F. J. Biol. Chem. 2003; 278: 28181-28192Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Therefore, the opposing effects of TNFα and ciglitazone on apoE expression in adipocytes could result from regulatory interactions that impinge on a single gene regulatory locus. We addressed this mechanism and obtained additional insight into how PPARγ agonists influence apoE expression in adipocytes by analyzing portions of the apoE gene control elements for their responsiveness to TNFα and ciglitazone. The results of the first series of experiments are shown in Fig. 6. The proximal promoter portion of the apoE gene (pGL623) has been shown to contain numerous potential regulatory elements and binding sites for transcription factors (15Smith J.D. Melian A. Leff T. Breslow J.L. J. Biol. Chem. 1
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