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Peroxisome Proliferator-activated Receptor γ Agonist Rosiglitazone Increases Expression of Very Low Density Lipoprotein Receptor Gene in Adipocytes

2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês

10.1074/jbc.m109.047993

ISSN

1083-351X

Autores

Takeshi Takazawa, Toshimasa Yamauchi, Atsushi Tsuchida, Makoto Takata, Yusuke Hada, Masato Iwabu, Miki Okada‐Iwabu, Kohjiro Ueki, Takashi Kadowaki,

Tópico(s)

Adipokines, Inflammation, and Metabolic Diseases

Resumo

Apolipoprotein E (apoE) and its receptor, very low density lipoprotein receptor (VLDLR), are involved in fat accumulation in adipocytes. Here, we investigated the effect of a peroxisome proliferator-activated receptor (PPAR) γ agonist, rosiglitazone, on regulation of VLDLR expression both in white adipose tissue (WAT) of obese mice and in cultured adipocytes. Furthermore, to determine whether rosiglitazone directly regulates transcription of the VLDLR gene, we carried out luciferase assay with a reporter gene containing mouse VLDLR promoter region, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Four-day treatment with rosiglitazone increased the expression of VLDLR in WAT of ob/ob mice. Moreover, rosiglitazone increased the expression of VLDLR in cultured adipocytes. The PPAR-responsive element (PPRE)-directed mutagenesis analyses revealed that the PPRE motif in the VLDLR promoter region plays a significant role in transcriptional activation of the VLDLR gene in adipocytes. In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that endogenous PPARγ directly binds to this functional PPRE motif in the VLDLR promoter region. We also investigated the effects of rosiglitazone on insulin sensitivity and lipid accumulation in both ob/ob mice and apoE-deficient ob/ob mice. Rosiglitazone ameliorated insulin sensitivity in both ob/ob mice and apoE-deficient ob/ob mice, possibly through decreasing the expression of monocyte chemoattractant protein-1 (MCP-1), increasing the expression of superoxide dismutase 1 (SOD1) in WAT, and increasing plasma adiponectin concentration. In ob/ob mice, body weight and WAT weight were significantly higher in the mice treated with rosiglitazone than those treated with vehicle. However, in apoE-deficient ob/ob mice, no significant difference in body weight or WAT weight was observed between the vehicle-treated group and the rosiglitazone-treated group. Moreover, rosiglitazone did not increase body weight and WAT weight in VLDLR-deficient mice. These findings indicate that rosiglitazone directly increases VLDLR expression, thereby enhancing apoE-VLDLR-dependent lipid accumulation in adipocytes. Apolipoprotein E (apoE) and its receptor, very low density lipoprotein receptor (VLDLR), are involved in fat accumulation in adipocytes. Here, we investigated the effect of a peroxisome proliferator-activated receptor (PPAR) γ agonist, rosiglitazone, on regulation of VLDLR expression both in white adipose tissue (WAT) of obese mice and in cultured adipocytes. Furthermore, to determine whether rosiglitazone directly regulates transcription of the VLDLR gene, we carried out luciferase assay with a reporter gene containing mouse VLDLR promoter region, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. Four-day treatment with rosiglitazone increased the expression of VLDLR in WAT of ob/ob mice. Moreover, rosiglitazone increased the expression of VLDLR in cultured adipocytes. The PPAR-responsive element (PPRE)-directed mutagenesis analyses revealed that the PPRE motif in the VLDLR promoter region plays a significant role in transcriptional activation of the VLDLR gene in adipocytes. In addition, electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that endogenous PPARγ directly binds to this functional PPRE motif in the VLDLR promoter region. We also investigated the effects of rosiglitazone on insulin sensitivity and lipid accumulation in both ob/ob mice and apoE-deficient ob/ob mice. Rosiglitazone ameliorated insulin sensitivity in both ob/ob mice and apoE-deficient ob/ob mice, possibly through decreasing the expression of monocyte chemoattractant protein-1 (MCP-1), increasing the expression of superoxide dismutase 1 (SOD1) in WAT, and increasing plasma adiponectin concentration. In ob/ob mice, body weight and WAT weight were significantly higher in the mice treated with rosiglitazone than those treated with vehicle. However, in apoE-deficient ob/ob mice, no significant difference in body weight or WAT weight was observed between the vehicle-treated group and the rosiglitazone-treated group. Moreover, rosiglitazone did not increase body weight and WAT weight in VLDLR-deficient mice. These findings indicate that rosiglitazone directly increases VLDLR expression, thereby enhancing apoE-VLDLR-dependent lipid accumulation in adipocytes. Adipocytes are major sites of lipid storage in the body and play a critical role in maintaining lipid homeostasis. However, excess fat accumulation in adipocytes leads to obesity. Recent studies have revealed that apoE 4The abbreviations used are: apoEapolipoprotein ELDLlow density lipoproteinVLDLvery low density lipoproteinVLDLRVLDL receptorPPARperoxisome proliferator-activated receptorPPREPPAR-responsive elementWATwhite adipose tissueTZDthiazolidinedioneDMSOdimethyl sulfoxideEMSAelectrophoretic mobility shift assayChIPchromatin immunoprecipitationWTwild type. is involved in excess fat accumulation and adipogenesis in adipocytes (1Chiba T. Nakazawa T. Yui K. Kaneko E. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1423-1429Crossref PubMed Scopus (60) Google Scholar, 2Gao J. Katagiri H. Ishigaki Y. Yamada T. Ogihara T. Imai J. Uno K. Hasegawa Y. Kanzaki M. Yamamoto T.T. Ishibashi S. Oka Y. Diabetes. 2007; 56: 24-33Crossref PubMed Scopus (96) Google Scholar). ApoE is a multifunctional protein synthesized in the liver and other several peripheral tissues (3Kraft H.G. Menzel H.J. Hoppichler F. Vogel W. Utermann G. J. Clin. Invest. 1989; 83: 137-142Crossref PubMed Scopus (216) Google Scholar, 4Zechner R. Moser R. Newman T.C. Fried S.K. Breslow J.L. J. Biol. Chem. 1991; 266: 10583-10588Abstract Full Text PDF PubMed Google Scholar). ApoE is found on all lipoprotein particles except LDL and plays a key role in the receptor-mediated uptake of lipoproteins as well as in hepatic secretion of VLDL (5Breslow J.L. Annu. Rev. Biochem. 1985; 54: 699-727Crossref PubMed Scopus (108) Google Scholar, 6Hussain M.M. Maxfield F.R. Más-Oliva J. Tabas I. Ji Z.S. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1991; 266: 13936-13940Abstract Full Text PDF PubMed Google Scholar). A previous study has shown that apoE-deficient VLDL does not induce adipogenesis, whereas normal VLDL induces adipogenesis in cultured adipocytes (1Chiba T. Nakazawa T. Yui K. Kaneko E. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1423-1429Crossref PubMed Scopus (60) Google Scholar). Furthermore, it has been reported that apoE deficiency prevents diet-induced obesity in ob/ob mice and KK-Ay mice (1Chiba T. Nakazawa T. Yui K. Kaneko E. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1423-1429Crossref PubMed Scopus (60) Google Scholar, 2Gao J. Katagiri H. Ishigaki Y. Yamada T. Ogihara T. Imai J. Uno K. Hasegawa Y. Kanzaki M. Yamamoto T.T. Ishibashi S. Oka Y. Diabetes. 2007; 56: 24-33Crossref PubMed Scopus (96) Google Scholar). Thus, it appears that apoE is one of the key molecules for the development of obesity. One mechanism by which apoE modulates diet-induced obesity is through its role in lipid uptake into adipocytes. apolipoprotein E low density lipoprotein very low density lipoprotein VLDL receptor peroxisome proliferator-activated receptor PPAR-responsive element white adipose tissue thiazolidinedione dimethyl sulfoxide electrophoretic mobility shift assay chromatin immunoprecipitation wild type. There are several apoE receptors, including VLDLR and LDL receptor-related protein, which recognize VLDL in an apoE-dependent manner. The VLDLR is highly expressed in skeletal muscle, heart, and adipose tissue but only in trace amounts in the liver (7Gåfvels M.E. Paavola L.G. Boyd C.O. Nolan P.M. Wittmaack F. Chawla A. Lazar M.A. Bucan M. Angelin B.O. Strauss 3rd, J.F. Endocrinology. 1994; 135: 387-394Crossref PubMed Scopus (0) Google Scholar, 8Goudriaan J.R. Espirito Santo S.M. Voshol P.J. Teusink B. van Dijk K.W. van Vlijmen B.J. Romijn J.A. Havekes L.M. Rensen P.C. J. Lipid Res. 2004; 45: 1475-1481Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 9Sakai J. Hoshino A. Takahashi S. Miura Y. Ishii H. Suzuki H. Kawarabayasi Y. Yamamoto T. J. Biol. Chem. 1994; 269: 2173-2182Abstract Full Text PDF PubMed Google Scholar). It has previously been reported that VLDLR recognizes apoE-containing lipoprotein and mediates lipid uptake in adipose tissue as well as in skeletal muscle (10Takahashi S. Kawarabayasi Y. Nakai T. Sakai J. Yamamoto T. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 9252-9256Crossref PubMed Scopus (478) Google Scholar, 11Takahashi S. Suzuki J. Kohno M. Oida K. Tamai T. Miyabo S. Yamamoto T. Nakai T. J. Biol. Chem. 1995; 270: 15747-15754Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 12Zenimaru Y. Takahashi S. Takahashi M. Yamada K. Iwasaki T. Hattori H. Imagawa M. Ueno M. Suzuki J. Miyamori I. Biochem. Biophys. Res. Commun. 2008; 368: 716-722Crossref PubMed Scopus (18) Google Scholar). Furthermore, it has been shown that VLDLR-deficient mice are protected from diet-induced obesity and insulin resistance (13Yagyu H. Lutz E.P. Kako Y. Marks S. Hu Y. Choi S.Y. Bensadoun A. Goldberg I.J. J. Biol. Chem. 2002; 277: 10037-10043Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Goudriaan J.R. Tacken P.J. Dahlmans V.E. Gijbels M.J. van Dijk K.W. Havekes L.M. Jong M.C. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1488-1493Crossref PubMed Scopus (110) Google Scholar). This protection is most likely due to reduced lipid uptake by adipose tissue because whole-body lipid uptake is markedly decreased in VLDLR-deficient mice. These results suggest that apoE-VLDLR interaction plays an important role in the development of obesity. PPARγ is the key transcriptional regulator of adipogenesis and directly activates many genes involved in lipid storage (15Auwerx J. Diabetologia. 1999; 42: 1033-1049Crossref PubMed Scopus (580) Google Scholar, 16Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1665) Google Scholar, 17Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar). PPARγ is highly expressed in WAT and is required for adipocyte differentiation (18Chawla A. Schwarz E.J. Dimaculangan D.D. Lazar M.A. Endocrinology. 1994; 135: 798-800Crossref PubMed Scopus (617) Google Scholar, 19Lowell B.B. Cell. 1999; 99: 239-242Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). PPARγ forms a heterodimer with retinoid X receptor α, and PPARγ/retinoid X receptor α heterodimer binds to a PPRE, containing direct repeats of the hexanucleotide sequence AGGTCA separated by one nucleotide (20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3121) Google Scholar). This motif, known as a direct repeat 1 (DR-1) element, is found in the promoter regions of many genes involved in lipid storage, such as the fatty acid-binding protein aP2 and the cholesterol and fatty acid transporter FATP/CD36 (21Sato O. Kuriki C. Fukui Y. Motojima K. J. Biol. Chem. 2002; 277: 15703-15711Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Thiazolidinediones (TZDs) are insulin-sensitizing antidiabetic drugs that activate the PPARγ (22Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3459) Google Scholar). TZDs have also been reported to stimulate adipogenesis by up-regulating many of the PPARγ target genes involved in fatty acid metabolism and storage (23Way J.M. Harrington W.W. Brown K.K. Gottschalk W.K. Sundseth S.S. Mansfield T.A. Ramachandran R.K. Willson T.M. Kliewer S.A. Endocrinology. 2001; 142: 1269-1277Crossref PubMed Scopus (285) Google Scholar). In fact, numerous studies in rodent models and in humans have shown that treatment with TZDs causes weight gain (24Chao L. Marcus-Samuels B. Mason M.M. Moitra J. Vinson C. Arioglu E. Gavrilova O. Reitman M.L. J. Clin. Invest. 2000; 106: 1221-1228Crossref PubMed Scopus (337) Google Scholar, 25de Souza C.J. Eckhardt M. Gagen K. Dong M. Chen W. Laurent D. Burkey B.F. Diabetes. 2001; 50: 1863-1871Crossref PubMed Scopus (297) Google Scholar). Although it has been reported that VLDL increases PPARγ expression in cultured adipocytes (1Chiba T. Nakazawa T. Yui K. Kaneko E. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1423-1429Crossref PubMed Scopus (60) Google Scholar), the possible role of PPARγ in regulation of VLDLR expression and VLDLR-mediated lipid accumulation in adipocytes remains unclear. In this study, we investigated the effect of rosiglitazone, a PPARγ agonist, on regulation of VLDLR expression both in WAT of obese mice and in cultured adipocytes. Furthermore, to determine whether rosiglitazone directly regulates transcription of the VLDLR gene, we carried out luciferase assay with a reporter gene containing mouse VLDLR promoter region. Our results showed that rosiglitazone increased the expression of VLDLR both in WAT of obese mice and in cultured adipocytes, that functional PPRE existed in the VLDLR promoter region, and that VLDLR was a direct PPARγ target gene. Further in vivo experiments showed that rosiglitazone did not increase body weight and WAT weight in apoE-deficient ob/ob mice, but it significantly increased these two parameters in ob/ob mice. In addition, rosiglitazone did not increase body weight and WAT weight in VLDLR-deficient mice. These findings indicate that VLDLR-mediated apoE-containing VLDL uptake plays an important role in rosiglitazone-induced lipid accumulation in adipocytes. Rosiglitazone was synthesized as described elsewhere (22Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3459) Google Scholar). All other materials were obtained from sources as described previously (26Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4072) Google Scholar, 27Yamauchi T. Kamon J. Waki H. Imai Y. Shimozawa N. Hioki K. Uchida S. Ito Y. Takakuwa K. Matsui J. Takata M. Eto K. Terauchi Y. Komeda K. Tsunoda M. Murakami K. Ohnishi Y. Naitoh T. Yamamura K. Ueyama Y. Froguel P. Kimura S. Nagai R. Kadowaki T. J. Biol. Chem. 2003; 278: 2461-2468Abstract Full Text Full Text PDF PubMed Scopus (803) Google Scholar). The male ob/ob mice were purchased from Charles River Japan (Yokohama, Japan). The male wild-type mice, apoE-deficient mice, ob/ob mice, and apoE-deficient ob/ob mice used in this study, which were prepared by intercross of apoE+/−ob/+ mice, were all of a C57B6/j background. The male nontransgenic wild-type mice and VLDLR-deficient mice, which were B6;129 background, were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed under a 12-h light/dark cycle in an animal room maintained at 25 °C. Mice were fed a normal chow diet until 6–8 weeks of age, and they were given a high fat diet (32% safflower oil, 33.1% casein, 17.6% sucrose, and 5.6% cellulose (28Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar)) starting 1 week before rosiglitazone administration. Rosiglitazone was given as a 0.01% food admixture. The mean dose of rosiglitazone was estimated to be 10 mg/kg/day. This dose was chosen because it has been shown to be the effective therapeutic dose in diabetic mice (24Chao L. Marcus-Samuels B. Mason M.M. Moitra J. Vinson C. Arioglu E. Gavrilova O. Reitman M.L. J. Clin. Invest. 2000; 106: 1221-1228Crossref PubMed Scopus (337) Google Scholar, 29Moore G.B. Chapman H. Holder J.C. Lister C.A. Piercy V. Smith S.A. Clapham J.C. Biochem. Biophys. Res. Commun. 2001; 286: 735-741Crossref PubMed Scopus (141) Google Scholar). The ob/ob mice were fed either a high fat diet or a high fat diet with 0.01% rosiglitazone for 4 days. The wild-type mice, apoE-deficient mice, ob/ob mice, and apoE-deficient ob/ob mice were fed either a high fat diet or a high fat diet with 0.01% rosiglitazone for 10 weeks. The wild-type mice and VLDLR-deficient mice were fed either a high fat diet or a high fat diet with 0.01% rosiglitazone for 7 weeks. The oral glucose tolerance test was performed by oral gavages of 0.75 g/kg of body weight glucose after 24 h of fasting followed by blood sampling at 0, 15, 30, 60, and 120 min. An insulin tolerance test was performed by 1.5 units/kg of body weight of intraperitoneal insulin followed by blood sampling at the 0, 30, 60, and 90 min (26Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4072) Google Scholar). Plasma glucose was determined by a glucose test (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin was measured by an insulin immunoassay (Shibayagi, Gunma, Japan), and plasma adiponectin concentration was determined by a mouse adiponectin immunoassay kit (Otsuka Pharmaceutical, Tokushima, Japan). Epididymal WAT was removed from each animal, fixed in 10% formaldehyde/phosphate-buffered saline, and maintained at 4 °C until use. Fixed specimens were dehydrated, embedded in tissue-freezing medium (Tissue-Tek OCT compound; Miles, Elkhart, IN), and frozen in dry ice and acetone. WAT was cut into 10-μm sections, and the sections were mounted on silanized slides. The adipose tissue was stained with hematoxylin and eosin (30Yamauchi T. Kamon J. Waki H. Murakami K. Motojima K. Komeda K. Ide T. Kubota N. Terauchi Y. Tobe K. Miki H. Tsuchida A. Akanuma Y. Nagai R. Kimura S. Kadowaki T. J. Biol. Chem. 2001; 276: 41245-41254Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar). Mouse 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal bovine serum. Induction of adipocyte differentiation was carried out according to a method described previously (31Yamauchi T. Waki H. Kamon J. Murakami K. Motojima K. Komeda K. Miki H. Kubota N. Terauchi Y. Tsuchida A. Tsuboyama-Kasaoka N. Yamauchi N. Ide T. Hori W. Kato S. Fukayama M. Akanuma Y. Ezaki O. Itai A. Nagai R. Kimura S. Tobe K. Kagechika H. Shudo K. Kadowaki T. J. Clin. Invest. 2001; 108: 1001-1013Crossref PubMed Scopus (285) Google Scholar). On 7 days after the induction of differentiation, 3T3-L1 adipocytes were incubated at 37 °C with rosiglitazone and pioglitazone in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal bovine serum for 24 h. The cells were harvested to isolate total RNA. Total RNA was isolated from cells or tissues with TRIzol (Invitrogen) according to the manufacturer's instructions. For quantification of mRNA, we conducted a real-time PCR using an ABI prism 7900 and sets of primer and probe for each gene (Applied Biosystems, Foster City, CA). The relative amount of each transcript was normalized to the amount of β-actin and 36B4 transcript in the same cDNA (32Yamauchi T. Kamon J. Ito Y. Tsuchida A. Yokomizo T. Kita S. Sugiyama T. Miyagishi M. Hara K. Tsunoda M. Murakami K. Ohteki T. Uchida S. Takekawa S. Waki H. Tsuno N.H. Shibata Y. Terauchi Y. Froguel P. Tobe K. Koyasu S. Taira K. Kitamura T. Shimizu T. Nagai R. Kadowaki T. Nature. 2003; 423: 762-769Crossref PubMed Scopus (2646) Google Scholar). On 7 days after the induction of differentiation, 3T3-L1 adipocytes were incubated with 1 μm rosiglitazone for 24 h. Total cellular protein (10 μg) from cells was subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. Anti-VLDLR antibody (6A6) and horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used to probe for VLDLR protein. Bands were detected with the ECL Plus Western blotting detection kit (GE Healthcare) according to the manufacturer's instructions. To generate luciferase reporter plasmids of mouse VLDLR promoter, PCR fragments (−2590 to −1 bp) from the mouse genomic DNA were inserted into the BglII and NcoI sites of the pGL3 basic luciferase expression vector (Promega, Madison, WI). Point mutation of the pGL3-VLDLR luciferase plasmid was introduced using the GeneTailor system (Invitrogen). On 3 days after the induction of differentiation, 3T3-L1 adipocytes were transfected with luciferase reporter plasmids using Lipofectamine Plus (Invitrogen) according to the instructions provided by the manufacturer. After transfection, the cells were incubated in a medium containing rosiglitazone, pioglitazone, or DMSO. At 16 h after ligand treatment, luciferase reporter assay were performed using the Luciferase Assay System (Promega). The nuclear proteins were extracted from 3T3-L1 adipocytes on 7 days after differentiation using the CelLytic NuCLEAR extraction kit (Sigma) according to the manufacturer's instructions. EMSA was performed using the gel shift assay systems (Promega). The sequences of double-stranded oligonucleotides were as follows (only one strand is shown, and the half-site of the putative PPRE and the mutated PPRE are underlined). Mouse VLDLR PPRE, 5′-TGATTTCAGTTTACAGGTCAGATGGCAGGCACAG-3′; mouse VLDLR mutant PPRE, 5′-TGATTTCGGTTTACATCGTTGATGGCTGGCACAG-3′. Double-stranded oligonucleotides were radioactively end-labeled with [γ-33P]dATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Promega) and purified from unincorporated nucleotides by gel filtration through G-25 spin columns (GE Healthcare). All of the EMSA reactions were carried out according to the manufacturer's instructions using 2 μg of nuclear extracts. DNA-protein complexes were resolved by electrophoresis through 6% polyacrylamide gels in 0.5× Tris borate running buffer (Bio-Rad). For the competition assay, a 50-fold amount of unlabeled double-stranded DNAs was added in the binding reaction. For supershift assays, nuclear extracts were preincubated with 2 μg of mouse monoclonal anti-PPARγ antibody (E8) (Santa Cruz Biotechnology) or with 2 μg of mouse control IgG (Santa Cruz Biotechnology) for 10 min before the oligonucleotides were added. On 4 days after the induction of differentiation, 3T3-L1 adipocytes were incubated with 1 μm rosiglitazone for 24 h. The DNA and protein were cross-linked with 1% formaldehyde for 10 min. Soluble chromatin was prepared using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). After sonication, lysates were precipitated with rabbit polyclonal anti-PPARγ antibody (H100) and normal rabbit IgG (Santa Cruz Biotechnology) according to the manufacturer's instructions. Primers used for ChIP PCR were as follows: VLDLR-PPRE forward, 5′-TGAGGCCACAGATGATTTTG-3′; VLDLR-PPRE reverse, 5′-GGCTCTACACTCAACCTGGTG-3′; aP2-PPRE forward, 5′-ATGTCACAGGCATCTTATCCACC-3′; aP2-PPRE reverse, 5′-AACCCTGCCAAAGAGACAGAGG-3′; negative control primer forward, 5′-CTCCCCGATCACTGGAATAG-3′; and negative control primer reverse, 5′-ACCCTAGAGACACTGGTGGTG-3′. The negative control primers are located ∼2 kbp upstream of the PPRE for VLDLR. PCR was performed for 45 cycles. PCR products were analyzed by 2% agarose gel electrophoresis. Data are given as the means ± S.E. Student's t test was used for statistical comparison. p < 0.05 was considered as statistically significant. Previous studies have shown that rosiglitazone enhances lipid accumulation in adipose tissue by up-regulating many of the PPARγ target genes involved in fatty acid metabolism and storage (24Chao L. Marcus-Samuels B. Mason M.M. Moitra J. Vinson C. Arioglu E. Gavrilova O. Reitman M.L. J. Clin. Invest. 2000; 106: 1221-1228Crossref PubMed Scopus (337) Google Scholar, 25de Souza C.J. Eckhardt M. Gagen K. Dong M. Chen W. Laurent D. Burkey B.F. Diabetes. 2001; 50: 1863-1871Crossref PubMed Scopus (297) Google Scholar). In addition, it has been reported that VLDLR is one of the key molecules for the development of obesity (13Yagyu H. Lutz E.P. Kako Y. Marks S. Hu Y. Choi S.Y. Bensadoun A. Goldberg I.J. J. Biol. Chem. 2002; 277: 10037-10043Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Goudriaan J.R. Tacken P.J. Dahlmans V.E. Gijbels M.J. van Dijk K.W. Havekes L.M. Jong M.C. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1488-1493Crossref PubMed Scopus (110) Google Scholar). To investigate the effect of rosiglitazone on the expression of VLDLR, we treated ob/ob mice, while on the high fat diet, with rosiglitazone (0.01% food admixture) for 4 days and examined VLDLR gene expression in WAT using quantitative PCR analysis. We also examined the effect of rosiglitazone on the expression of aP2 and CD36, which are target genes of PPARγ, in WAT of ob/ob mice. Rosiglitazone significantly increased the expression of VLDLR in WAT of ob/ob mice by 2.6-fold as compared with the vehicle (Fig. 1A). In addition, treatment with rosiglitazone significantly increased the expression of aP2 and CD36 in WAT of ob/ob mice by 1.6- and 2.3-fold, respectively, as compared with the vehicle (Fig. 1, B and C). To confirm that the VLDLR gene is a target of PPARγ, we examined the effect of rosiglitazone on the expression of VLDLR in cultured 3T3-L1 adipocytes. Rosiglitazone increased the expression of VLDLR in a dose-dependent manner (Fig. 2A). The expression of aP2 and CD36, which have functional PPREs in their promoter regions, was also increased by treatment with rosiglitazone (Fig. 2, B and C). Furthermore, rosiglitazone significantly increased VLDLR protein levels as well as mRNA levels (Fig. 2D). These findings indicate that rosiglitazone directly increases VLDLR expression in 3T3-L1 adipocytes. We also examined the effect of another TZD, pioglitazone, on the expression of VLDLR in 3T3-L1 adipocytes. Pioglitazone also increased the expression of VLDLR in 3T3-L1 adipocytes (Fig. 2A), and its effect was comparable with that of rosiglitazone. To determine whether rosiglitazone directly regulates transcription of the VLDLR gene via a PPRE in its promoter region, we carried out a luciferase assay with a reporter gene including mouse VLDLR promoter region (Fig. 3A). A luciferase reporter construct containing the VLDLR promoter region was transfected into 3T3-L1 adipocytes 3 days after induction of differentiation. Incubation with rosiglitazone, as well as with pioglitazone, for 16 h significantly enhanced VLDLR promoter transcriptional activity by 2.3-fold as compared with incubation with the vehicle (Fig. 3, B and C). This result indicates that the element responsible for rosiglitazone- or pioglitazone-induced transcriptional activity is included in the VLDLR promoter region. Inspection of this region revealed a putative PPRE of the DR1 type at −2307 to −2288 bp (Fig. 3A). To determine whether this putative PPRE is involved in the rosiglitazone-mediated regulation of VLDLR gene expression, luciferase reporter constructs containing wild-type or PPRE-mutated mouse VLDLR promoter were transfected into 3T3-L1 adipocytes (Fig. 3, B–D). Transfection of the PPRE-mutated reporter construct resulted in a remarkable suppression of rosiglitazone-induced increase in luciferase activity (Fig. 3D). This result suggests that the PPRE motif in VLDLR promoter plays a significant role in transcriptional activation of the VLDLR gene in adipocytes. To determine whether PPARγ directly binds to the PPRE motif in the VLDLR promoter region in adipocytes, we carried out EMSA. Incubation of nuclear extract from differentiated 3T3-L1 adipocytes with a radiolabeled double-stranded oligonucleotide containing the putative PPRE motif in the VLDLR promoter region resulted in a shifted complex that was effectively competed by wild-type but not mutant PPRE (Fig. 3E, lanes 1, 2, 4, and 5). In addition, incubation of the 3T3-L1 nuclear extracts with monoclonal antibody against PPARγ supershifted the complex to the higher molecular weight position (Fig. 3E, lanes 6 and 7). On the other hand, a radiolabeled double-stranded oligonucleotide containing the mutant PPRE in the VLDLR promoter region did not form a complex with nuclear extracts (Fig. 3E, lane 3). These results indicate that the shifted complex contains the endogenous PPARγ at least in part. We next used the ChIP assay to study the transcriptional regulation of the endogenous VLDLR gene in 3T3-L1 adipocytes. Primer sets were designed to span the PPRE motif. ChIP analysis demonstrated that PPARγ bound in the region of the PPRE in VLDLR promoter, whereas a region ∼2 kbp upstream of the PPRE showed no binding of PPARγ (Fig. 3F). As expected, the aP2 promoter region containing its PPRE was also bound by PPARγ (Fig. 3F). Thus, these results suggest that PPARγ directly binds in the region of functional PPRE in VLDLR promoter. It has previously been reported that VLDLR recognizes apoE-containing lipoprotein and mediates lipid uptake in adipose tissue (10Takahashi S. Kawarabayasi Y. Nakai T. Sakai J. Yamamoto T. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 9252-9256Crossref PubMed Scopus (478) Google Scholar, 11Takahashi S. Suzuki J. Kohno M. Oida K. Tamai T. Miyabo S. Yamamoto T. Nakai T. J. Biol. Chem. 1995; 270: 15747-15754Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To clarify whether the regulation of VLDLR-mediated apoE-containing VLDL uptake into adipocytes is responsible for rosiglitazone-induced weight gain, we next examined the effect of rosiglitazone on body weight and WAT weight in apoE-deficient ob/ob mice, in which VLDLR cannot recognize apoE-deficient VLDL particles. In our experiment, ob/ob mice and apoE-deficient ob/ob mice, while on the high fat diet, were treated with rosiglitazone (0.01% food admixture) for 10 weeks. Rosiglitazone significantly increased both body weight and WAT weight in ob/ob mice (Fig. 4, A–C). In contrast, no significant difference in body weight and WAT weight was observed between rosiglitazone-treated apoE-deficient ob/ob mice and the vehicle-treated apoE-deficient ob/ob mice (Fig. 4, A–C). These results indicate that apoE-dependent lipid uptake into adipocytes, at least in part, contributes to WAT weight gain by rosiglitazone. We also examined the antidiabetic effect of rosiglitazone in apoE-deficient ob/ob mice. As shown in Fig. 5, A–C, both blood glucose and plasma insulin concentrations, measured in a glucose tolerance test, in rosiglitazone-treated apoE-deficient ob/ob mice were lower than those in the vehicle-treated apoE-deficient ob/ob mice. In addition, insulin resistance index in rosiglitazone-treated apoE-deficient ob/ob mice decreased by 42% as compared with that in the vehicle-treated apoE-deficient ob/ob mice. Furthermore, blood glucose concentration, measured in the insulin tolerance test, in rosiglitazone-treated apoE-deficient ob/ob mice was significantly lower than that in the vehicle-treated apoE-deficient ob/ob mice. The extent of improvement in blood glucose and plasma insulin concentrations caused by rosiglitazone in apoE-deficient ob/ob mice was comparable with that in ob/ob mice (Fig. 5, D and E). These results indicate that treatment with rosiglitazone ameliorates glucose intolerance and insulin resistance in apoE-deficient ob/ob mice and that these effects of rosiglitazone may not depend on the apoE-VLDLR pathway. We have previously shown that PPARγ activation by a TZD prevents adipocyte hypertrophy (33Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (926) Google Scholar) and increases plasma adiponectin concentration, which results in amelioration of insulin resistance (30Yamauchi T. Kamon J. Waki H. Murakami K. Motojima K. Komeda K. Ide T. Kubota N. Terauchi Y. Tobe K. Miki H. Tsuchida A. Akanuma Y. Nagai R. Kimura S. Kadowaki T. J. Biol. Chem. 2001; 276: 41245-41254Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar, 34Tsuchida A. Yamauchi T. Takekawa S. Hada Y. Ito Y. Maki T. Kadowaki T. Diabetes. 2005; 54: 3358-3370Crossref PubMed Scopus (364) Google Scholar). Accordingly, we examined in this study the effect of rosiglitazone on adipocyte size in apoE-deficient ob/ob mice. Although treatment with rosiglitazone increased the number of small adipocytes in WAT of ob/ob mice, there was no significant difference in adipocyte size in WAT of apoE-deficient ob/ob mice between treatment with the vehicle and that with rosiglitazone (Fig. 6, A and C). On the other hand, plasma adiponectin concentration in both ob/ob mice and apoE-deficient ob/ob mice was significantly increased by treatment with rosiglitazone as compared with treatment with the vehicle (Fig. 6B). It has recently been reported that chronic inflammation and oxidative stress in WAT play a crucial role in the development of obesity-related insulin resistance (35Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7469) Google Scholar, 36Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5191) Google Scholar, 37Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Invest. 2004; 114: 1752-1761Crossref PubMed Scopus (4017) Google Scholar). Moreover, it has been shown that rosiglitazone suppresses the expression of inflammatory genes in WAT of ob/ob mice (37Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Invest. 2004; 114: 1752-1761Crossref PubMed Scopus (4017) Google Scholar). To investigate the mechanism by which rosiglitazone ameliorates insulin resistance in apoE-deficient ob/ob mice without preventing adipocyte hypertrophy, we investigated the effects of rosiglitazone on the expression of inflammatory gene and antioxidant enzyme gene in WAT of apoE-deficient ob/ob mice. The expression of MCP-1 was significantly increased in WAT of apoE-deficient ob/ob mice as compared with that in WAT of the wild-type mice (Fig. 6D). Rosiglitazone significantly suppressed the expression of MCP-1 in WAT of apoE-deficient ob/ob mice as compared with the vehicle (Fig. 6D). The expression of SOD1 was decreased in apoE-deficient ob/ob mice as compared with that in the wild-type mice (Fig. 6E). Rosiglitazone significantly increased the expression of SOD1 in apoE-deficient ob/ob mice as compared with the vehicle (Fig. 6E). Taken together, these findings indicate that rosiglitazone may attenuate inflammation and oxidative stress in WAT of apoE-deficient ob/ob mice through the apoE-VLDLR-independent pathway. To further investigate a role for VLDLR in rosiglitazone-induced weight gain, we also examined the effect of rosiglitazone on body weight and WAT weight in VLDLR-deficient mice. Wild-type mice and VLDLR-deficient mice, while on the high fat diet, were treated with rosiglitazone (0.01% food admixture) for 7 weeks. Rosiglitazone significantly increased both body weight and WAT weight in wild-type mice (Fig. 7, A–D). In contrast, no significant difference in body weight and WAT weight was observed between rosiglitazone-treated VLDLR-deficient mice and the vehicle-treated VLDLR-deficient mice (Fig. 7, A–D). To confirm that rosiglitazone activated PPARγ in WAT of both wild-type mice and VLDLR-deficient mice, we next examined the effect of rosiglitazone on plasma adiponectin concentration and the expression of aP2 and VLDLR in WAT. Plasma adiponectin concentration in both wild-type mice and VLDLR-deficient mice was significantly increased by treatment with rosiglitazone as compared with treatment with the vehicle (Fig. 7E). In addition, the expression of aP2 in WAT was also increased by treatment with rosiglitazone in both wild-type mice and VLDLR-deficient mice (Fig. 7F). Furthermore, in wild-type mice, rosiglitazone significantly increased the expression of VLDLR in WAT by 1.6-fold as compared with the vehicle (data not shown). These results indicate that VLDLR-mediated lipid uptake into adipocytes plays an important role in rosiglitazone-induced lipid accumulation in adipocytes. TZDs activate PPARγ, which regulates the transcription of genes involved in fatty acid metabolism and storage (15Auwerx J. Diabetologia. 1999; 42: 1033-1049Crossref PubMed Scopus (580) Google Scholar, 16Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1665) Google Scholar, 17Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar). The weight gain associated with TZDs treatment is likely to be due to an increase of lipid accumulation in adipose tissue (28Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 29Moore G.B. Chapman H. Holder J.C. Lister C.A. Piercy V. Smith S.A. Clapham J.C. Biochem. Biophys. Res. Commun. 2001; 286: 735-741Crossref PubMed Scopus (141) Google Scholar). Recent studies have revealed that apoE-VLDLR interaction plays an important role in the development of obesity and excess fat accumulation (1Chiba T. Nakazawa T. Yui K. Kaneko E. Shimokado K. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1423-1429Crossref PubMed Scopus (60) Google Scholar, 2Gao J. Katagiri H. Ishigaki Y. Yamada T. Ogihara T. Imai J. Uno K. Hasegawa Y. Kanzaki M. Yamamoto T.T. Ishibashi S. Oka Y. Diabetes. 2007; 56: 24-33Crossref PubMed Scopus (96) Google Scholar). However, it remains unclear whether VLDLR-mediated lipid accumulation plays an important role in the weight gain associated with TZD treatment. In this study, we demonstrated that rosiglitazone increases the expression of VLDLR both in WAT in vivo and in cultured adipocytes (Figs. 1A and 2A). Moreover, the use of VLDLR promoter reporter constructs in 3T3-L1 adipocytes led us to identify functional PPRE in the promoter region (Fig. 3, A–D). Mutagenesis analyses, EMSA, and the ChIP assay also demonstrated that endogenous PPARγ directly binds to this functional PPRE in the VLDLR promoter region (Fig. 3, A–F). These results clearly show that rosiglitazone directly regulates transcription of the VLDLR gene in adipocytes. To clarify the association between rosiglitazone-induced weight gain and apoE-containing VLDL uptake through VLDLR, we investigated the effects of rosiglitazone on body weight and WAT weight in both apoE-deficient ob/ob mice and VLDLR-deficient mice. In our experiments, rosiglitazone did not increase body weight and WAT weight in apoE-deficient ob/ob mice and VLDLR-deficient mice, but it significantly increased these two parameters in their wild-type control mice (Figs. 4, A–C, and 7, A–D). It has been shown that apoE deficiency in genetically obese Ay mice prevents the development of obesity and that this phenotype is reversed by adenoviral apoE replenishment (2Gao J. Katagiri H. Ishigaki Y. Yamada T. Ogihara T. Imai J. Uno K. Hasegawa Y. Kanzaki M. Yamamoto T.T. Ishibashi S. Oka Y. Diabetes. 2007; 56: 24-33Crossref PubMed Scopus (96) Google Scholar). In addition, it has been reported that VLDLR-deficient mice do not show body weight gain when fed a high fat, high calorie diet (13Yagyu H. Lutz E.P. Kako Y. Marks S. Hu Y. Choi S.Y. Bensadoun A. Goldberg I.J. J. Biol. Chem. 2002; 277: 10037-10043Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Goudriaan J.R. Tacken P.J. Dahlmans V.E. Gijbels M.J. van Dijk K.W. Havekes L.M. Jong M.C. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1488-1493Crossref PubMed Scopus (110) Google Scholar). Our results in the present study as well as those of previous reports (2Gao J. Katagiri H. Ishigaki Y. Yamada T. Ogihara T. Imai J. Uno K. Hasegawa Y. Kanzaki M. Yamamoto T.T. Ishibashi S. Oka Y. Diabetes. 2007; 56: 24-33Crossref PubMed Scopus (96) Google Scholar, 13Yagyu H. Lutz E.P. Kako Y. Marks S. Hu Y. Choi S.Y. Bensadoun A. Goldberg I.J. J. Biol. Chem. 2002; 277: 10037-10043Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Goudriaan J.R. Tacken P.J. Dahlmans V.E. Gijbels M.J. van Dijk K.W. Havekes L.M. Jong M.C. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1488-1493Crossref PubMed Scopus (110) Google Scholar) indicate that induction of lipid uptake by rosiglitazone in adipocytes might not work in VLDLR-deficient mice and apoE-deficient ob/ob mice because of lack of the apoE-VLDLR pathway. It is therefore suggested that the apoE-VLDLR pathway plays a crucial role in rosiglitazone-induced lipid accumulation in adipose tissue. Because our results do not exclude the possibility that other receptors contribute to the uptake of VLDL particles into adipocytes, further studies are needed to elucidate the extent to which the apoE-VLDLR pathway is responsible for rosiglitazone-induced lipid uptake. We also investigated in this study the mechanism by which rosiglitazone ameliorates insulin resistance in apoE-deficient ob/ob mice without affecting WAT weight. It has been reported that rosiglitazone increases the number of small adipocytes and plasma adiponectin concentration, thereby ameliorating insulin resistance (26Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4072) Google Scholar, 31Yamauchi T. Waki H. Kamon J. Murakami K. Motojima K. Komeda K. Miki H. Kubota N. Terauchi Y. Tsuchida A. Tsuboyama-Kasaoka N. Yamauchi N. Ide T. Hori W. Kato S. Fukayama M. Akanuma Y. Ezaki O. Itai A. Nagai R. Kimura S. Tobe K. Kagechika H. Shudo K. Kadowaki T. J. Clin. Invest. 2001; 108: 1001-1013Crossref PubMed Scopus (285) Google Scholar, 33Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (926) Google Scholar). Although there was no significant difference in the size of adipocytes in WAT between the vehicle- and rosiglitazone-treated apoE-deficient ob/ob mice, plasma adiponectin concentration was increased by treatment with rosiglitazone (Fig. 6B). These results suggest that, in addition to the increase in the number of small adipocytes, the increase in plasma adiponectin levels considerably contributes to the insulin-sensitizing action of TZDs. Furthermore, it has been reported that obesity-associated metabolic syndrome leads to increase in oxidative stress and macrophages accumulation in WAT, which are causes of adipocytes dysfunction (35Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Invest. 2003; 112: 1796-1808Crossref PubMed Scopus (7469) Google Scholar, 36Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Invest. 2003; 112: 1821-1830Crossref PubMed Scopus (5191) Google Scholar, 37Furukawa S. Fujita T. Shimabukuro M. Iwaki M. Yamada Y. Nakajima Y. Nakayama O. Makishima M. Matsuda M. Shimomura I. J. Clin. Invest. 2004; 114: 1752-1761Crossref PubMed Scopus (4017) Google Scholar). In the present study, rosiglitazone increased the expression of antioxidant enzyme and reduced the expression of macrophage-specific gene in WAT, suggesting the reduction of oxidative stress and inflammation in WAT (Fig. 6, D and E). Thus, normalization of adipocyte function might be another possible mechanism by which rosiglitazone improves glucose intolerance and insulin resistance in apoE-deficient ob/ob mice. In conclusion, we demonstrated in this study that PPARγ directly binds to the functional PPRE in the VLDLR promoter region and that the PPARγ agonist rosiglitazone increases VLDLR expression, which may be involved in lipid accumulation in WAT. We therefore propose that VLDLR-mediated apoE-containing VLDL uptake plays an important role in lipid accumulation induced by rosiglitazone in adipocytes. We are grateful to A. Okano, A. Itoh, and K. Miyata for excellent technical assistance.

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