Role of MAPK Phosphatase-1 in the Induction of Monocyte Chemoattractant Protein-1 during the Course of Adipocyte Hypertrophy
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m701549200
ISSN1083-351X
AutoresAyaka Ito, Takayoshi Suganami, Yoshihiro Miyamoto, Yasunao Yoshimasa, Motohiro Takeya, Yasutomi Kamei, Yoshihiro Ogawa,
Tópico(s)Immune cells in cancer
ResumoMonocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy. Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy. Evidence has accumulated suggesting that obesity is a state of chronic, low grade inflammation; it may represent a potential mechanism whereby obesity leads to the metabolic derangements (1Dandona P. Aljada A. Bandyopadhyay A. Trends Immunol. 2004; 25: 4-7Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar, 2Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3141) Google Scholar, 3Berg A.H. Scherer P.E. Circ. Res. 2005; 96: 939-949Crossref PubMed Scopus (1641) Google Scholar). Previous studies demonstrated that the adipose tissue is markedly infiltrated by macrophages in several models of rodent obesities and human obese subjects (4Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7308) Google Scholar, 5Xu 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. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5110) Google Scholar). Using an in vitro co-culture system composed of adipocytes and macrophages, we have provided evidence that a paracrine loop involving saturated free fatty acids (FFAs) and tumor necrosis factor-α (TNFα) derived from adipocytes and macrophages, respectively, establishes a vicious cycle that aggravates the inflammatory changes; i.e. marked up-regulation of pro-inflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) 3The abbreviations used are:ILinterleukinMCP-1monocyte chemoattractant protein-1MAPKmitogen-activated protein kinaseMKP-1mitogen-activated protein kinase phosphatase-1ERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMEKMAPK/ERK kinaseCARcoxsackie-adenovirus receptorWATwhite adipose tissueGFPgreen fluorescent proteinERendoplasmic reticulumROSreactive oxygen speciesSDstandard dietHFDhigh fat diet 3The abbreviations used are:ILinterleukinMCP-1monocyte chemoattractant protein-1MAPKmitogen-activated protein kinaseMKP-1mitogen-activated protein kinase phosphatase-1ERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMEKMAPK/ERK kinaseCARcoxsackie-adenovirus receptorWATwhite adipose tissueGFPgreen fluorescent proteinERendoplasmic reticulumROSreactive oxygen speciesSDstandard dietHFDhigh fat diet and TNFα and down-regulation of anti-inflammatory adiponectin (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar, 7Suganami T. Tanimoto-Koyama K. Nishida J. Itoh M. Yuan X. Mizuarai S. Kotani H. Yamaoka S. Miyake K. Aoe S. Kamei Y. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 84-91Crossref PubMed Scopus (614) Google Scholar). These findings have led us to speculate that macrophages, when infiltrated, may participate in the inflammatory pathways that are activated in obese adipose tissue.A previous study with bone marrow transplantation demonstrated that most macrophages in the adipose tissue are derived from the bone marrow (4Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7308) Google Scholar). In this regard, adipose tissue expression of MCP-1, a major chemokine implicated in the control of monocyte recruitment to the site of inflammation, is increased during the progression of obesity (8Chen A. Mumick S. Zhang C. Lamb J. Dai H. Weingarth D. Mudgett J. Chen H. MacNeil D.J. Reitman M.L. Qian S. Obes. Res. 2005; 13: 1311-1320Crossref PubMed Scopus (178) Google Scholar, 9Takahashi K. Mizuarai S. Araki H. Mashiko S. Ishihara A. Kanatani A. Itadani H. Kotani H. J. Biol. Chem. 2003; 278: 46654-46660Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and is roughly correlated with macrophage markers in the adipose tissue (5Xu 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. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5110) Google Scholar, 10Cancello R. Henegar C. Viguerie N. Taleb S. Poitou C. Rouault C. Coupaye M. Pelloux V. Hugol D. Bouillot J.L. Bouloumie A. Barbatelli G. Cinti S. Svensson P.A. Barsh G.S. Zucker J.D. Basdevant A. Langin D. Clement K. Diabetes. 2005; 54: 2277-2286Crossref PubMed Scopus (863) Google Scholar). These findings suggest that increased production of MCP-1 may be an initial event at the early stage of obesity so as to accumulate macrophages in the adipose tissue. Recently, Kanda et al. and Kamei et al. (11Kamei N. Tobe K. Suzuki R. Ohsugi M. Watanabe T. Kubota N. Ohtsuka-Kowatari N. Kumagai K. Sakamoto K. Kobayashi M. Yamauchi T. Ueki K. Oishi Y. Nishimura S. Manabe I. Hashimoto H. Ohnishi Y. Ogata H. Tokuyama K. Tsunoda M. Ide T. Murakami K. Nagai R. Kadowaki T. J. Biol. Chem. 2006; 281: 26602-26614Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 12Kanda H. Tateya S. Tamori Y. Kotani K. Hiasa K. Kitazawa R. Kitazawa S. Miyachi H. Maeda S. Egashira K. Kasuga M. J. Clin. Investig. 2006; 116: 1494-1505Crossref PubMed Scopus (1920) Google Scholar) have independently reported that MCP-1 plays a role in the recruitment of macrophages into obese adipose tissue. It is, therefore, important to know the molecular mechanism for increased production of MCP-1 at the early stage of obesity. Recent studies have demonstrated that multiple intracellular signaling pathways are activated in adipocytes during the course of adipocyte hypertrophy in vitro and in obese adipose tissue in vivo. However, how the inflammatory pathways are activated in adipocytes at the early stage of obesity is still poorly understood.Mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK) are activated in a variety of cellular processes (13Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3439) Google Scholar). Once activated by the upstream kinases, e.g. MAPK/ERK kinase (MEK), MAPKs are rapidly inactivated by a family of protein phosphatases such as MAPK phosphatase-1 (MKP-1), an inducible dual specificity phosphatase (14Farooq A. Zhou M.M. Cell. Signal. 2004; 16: 769-779Crossref PubMed Scopus (376) Google Scholar, 15Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Crossref PubMed Scopus (702) Google Scholar). Sakaue et al. showed previously that MKP-1 plays an essential role in 3T3-L1 adipocyte differentiation through ERK down-regulation (16Sakaue H. Ogawa W. Nakamura T. Mori T. Nakamura K. Kasuga M. J. Biol. Chem. 2004; 279: 39951-39957Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). On the other hand, Bost et al. (17Bost F. Aouadi M. Caron L. Even P. Belmonte N. Prot M. Dani C. Hofman P. Pages G. Pouyssegur J. Le Marchand-Brustel Y. Binetruy B. Diabetes. 2005; 54: 402-411Crossref PubMed Scopus (256) Google Scholar) reported that mice lacking ERK1 (ERK1-/- mice) are protected from high fat diet-induced obesity and insulin resistance. These findings, taken together, suggest that the MAPK pathways play an important role in the adipocyte proliferation and differentiation in vitro and in vivo (18Rosen E.D. MacDougald O.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 885-896Crossref PubMed Scopus (1903) Google Scholar).Here we show that MCP-1 mRNA expression is increased, which is followed by ERK activation and MKP-1 down-regulation in the adipose tissue from mice rendered mildly obese by a short term high fat diet, when macrophages are not infiltrated. We also demonstrate that ERK activation through MKP-1 down-regulation is involved in increased production of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. This study provides evidence that MKP-1 down-regulation is critical for the inflammatory changes in hypertrophied adipocytes at the early stage of obesity, thereby suggesting that MKP-1 activation may offer a novel therapeutic strategy to treat or reduce the inflammatory changes in adipocytes during the progression of obesity.EXPERIMENTAL PROCEDURESMaterials—Rabbit polyclonal antibodies against ERK, phospho-ERK, p38 MAPK, phospho-p38 MAPK, MEK1/2, phospho-MEK1/2, MEK inhibitors PD98059 and U0126, and a p38MAPK inhibitor SB203580 were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against JNK, phospho-JNK, and MKP-1 and a mouse monoclonal antibody against Lamin A/C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were purchased from Sigma or Nacalai Tesque (Kyoto, Japan).Animal Studies—Four-week-old male C57BL/6J mice were purchased from Charles River Laboratories Japan (Tokyo, Japan). The animals were housed in a temperature-, humidity-, and light-controlled room (12-h light and 12-h dark cycle) and allowed free access to water and chow. Five-week-old mice were fed either the standard chow (Oriental MF, 362 kcal/100 g, 5.4% energy as fat; Oriental Yeast, Tokyo, Japan) or high fat diet (D12492, 524 kcal/100 g, 60% energy as fat; Research Diets, New Brunswick, NJ) for 15 weeks. They were fasted for 1 h (12:00–13:00) and sacrificed to harvest the epididymal adipose tissue before (n = 10) and 2 weeks (n = 10), 4 weeks (n = 12), 6 weeks (n = 11), 8 weeks (n = 6), and 15 weeks (n = 4) after the experiments. All animal experiments were conducted according to the guidelines of Tokyo Medical and Dental University Committee on Animal Research (No. 0060026).Histological Analysis—The epididymal WAT was fixed with neutral-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and studied under ×200 magnification to measure the adipocyte area using Win Roof software (Mitani Corporation, Tokyo, Japan) (19Kouyama R. Suganami T. Nishida J. Tanaka M. Toyoda T. Kiso M. Chiwata T. Miyamoto Y. Yoshimasa Y. Fukamizu A. Horiuchi M. Hirata Y. Ogawa Y. Endocrinology. 2005; 146: 3481-3489Crossref PubMed Scopus (132) Google Scholar). Immunohistochemical study was carried out using 5-μm thick paraffin-embedded sections for macrophage marker F4/80 as previously described (20Suganami T. Mieda T. Itoh M. Shimoda Y. Kamei Y. Ogawa Y. Biochem. Biophys. Res. Commun. 2007; 354: 45-49Crossref PubMed Scopus (181) Google Scholar, 21Jinnouchi K. Terasaki Y. Fujiyama S. Tomita K. Kuziel W.A. Maeda N. Takahashi K. Takeya M. J. Pathol. 2003; 200: 406-416Crossref PubMed Scopus (20) Google Scholar).Cell Culture—3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were maintained as described (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar, 7Suganami T. Tanimoto-Koyama K. Nishida J. Itoh M. Yuan X. Mizuarai S. Kotani H. Yamaoka S. Miyake K. Aoe S. Kamei Y. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 84-91Crossref PubMed Scopus (614) Google Scholar). Differentiation of 3T3-L1 preadipocytes to adipocytes was described elsewhere (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar, 7Suganami T. Tanimoto-Koyama K. Nishida J. Itoh M. Yuan X. Mizuarai S. Kotani H. Yamaoka S. Miyake K. Aoe S. Kamei Y. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 84-91Crossref PubMed Scopus (614) Google Scholar). Cells at day 8 and day 21 after the induction of differentiation were used as non-hypertrophied and hypertrophied adipocytes, respectively (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar). Accumulation of triglyceride in adipocytes was detected by oil red O staining (19Kouyama R. Suganami T. Nishida J. Tanaka M. Toyoda T. Kiso M. Chiwata T. Miyamoto Y. Yoshimasa Y. Fukamizu A. Horiuchi M. Hirata Y. Ogawa Y. Endocrinology. 2005; 146: 3481-3489Crossref PubMed Scopus (132) Google Scholar).Measurement of Triglyceride Content—Triglyceride content in 3T3-L1 adipocytes was measured as previously reported (22Kamon J. Naitoh T. Kitahara M. Tsuruzoe N. Cell. Signal. 2001; 13: 105-109Crossref PubMed Scopus (10) Google Scholar). In brief, 3T3-L1 adipocytes in 35-mm dish were harvested, and cellular lipid was extracted by chloroform-methanol (2:1). After evaporation, precipitation was dissolved in isopropyl alcohol. Triglyceride content was measured using a colorimetric assay kit (triglyceride E-test Wako, Wako Pure Chemicals, Osaka, Japan) according to the manufacturer's instructions.Quantitative Real-time PCR—Quantitative real-time PCR was performed with an ABI Prism 7000 Sequence Detection System using PCR Master Mix reagent kit (Applied Biosystems, Foster City, CA) as described (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar, 19Kouyama R. Suganami T. Nishida J. Tanaka M. Toyoda T. Kiso M. Chiwata T. Miyamoto Y. Yoshimasa Y. Fukamizu A. Horiuchi M. Hirata Y. Ogawa Y. Endocrinology. 2005; 146: 3481-3489Crossref PubMed Scopus (132) Google Scholar). Primers used were described in supplemental Table S1. Levels of mRNAs were normalized to those of housekeeping gene 36B4 mRNA.ELISA—The MCP-1, IL-6, and adiponectin levels in culture supernatants were determined by the commercially available ELISA kits (MCP-1 and IL-6, R&D systems, Minneapolis, MN; adiponectin, Otsuka Pharmaceutical, Tokyo, Japan).Immunoblot Assay—Nuclear and cytosolic extracts were prepared by using the Nuclear/Cytosol fractionation kit (Bio-Vision, Mountain View, CA). Separation of nuclear and cytosolic proteins was confirmed by immunoblots with α-tubulin and lamin A/C antibodies, respectively. Whole cell lysates were prepared using buffer containing 50 mmol/liter HEPES (pH7.5), 150 mmol/liter NaCl, 100 mmol/liter sodium fluoride, 1 mmol/liter EGTA, 1 mmol/liter EDTA, 1% Triton X-100, 2 mmol/liter sodium vanadate, 2 mmol/liter phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Immunoblot assay was performed as described (6Suganami T. Nishida J. Ogawa Y. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2062-2068Crossref PubMed Scopus (807) Google Scholar). Samples (10–20 μg protein/lane) were separated by 12.5% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride filter membrane (PolyScreen; PerkinElmer, Wellesley, MA). After membranes were incubated with primary antibodies for 1 h at room temperature, immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-Sciences, Piscataway, NJ) and a chemiluminescence kit (GE Healthcare Bio-Sciences). The signals were detected with LAS3000 (Fuji Photo Film, Tokyo, Japan).Generation of 3T3-L1 Adipocytes Stably Expressing Coxsackie-Adenovirus Receptor (CAR)—A mouse CAR-expressing plasmid pcDNA3-CAR (23Hosono T. Mizuguchi H. Katayama K. Koizumi N. Kawabata K. Yamaguchi T. Nakagawa S. Watanabe Y. Mayumi T. Hayakawa T. Gene (Amst.). 2005; 348: 157-165Crossref PubMed Scopus (38) Google Scholar) was kindly provided by Dr. Hiroyuki Mizuguchi (National Institute of Biomedical Innovation, Osaka, Japan). The CAR retroviral expression vector (pMRX-CAR) was constructed by ligating the full-length CAR cDNA into the EcoR1 site of pMRX vector (24Saitoh T. Nakayama M. Nakano H. Yagita H. Yamamoto N. Yamaoka S. J. Biol. Chem. 2003; 278: 36005-36012Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar) and transfected into Plat-E packaging cells (25Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1338) Google Scholar) using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Viral supernatants were harvested from 24 to 48 h after transfection and applied to 3T3-L1 adipocytes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5 μg/ml of polybrene (Nacalai Tesque) in a final volume of 5 ml. The stable CAR-expressing 3T3-L1 adipocytes (CAR-3T3-L1 adipocytes) were obtained by 2 μg/ml of puromycin (Nacalai Tesque) selection.Adenovirus-mediated Expression of MKP-1—The adenoviral vector expressing mouse MKP-1 (Ad-MKP-1) (26Bueno O.F. De Windt L.J. Lim H.W. Tymitz K.M. Witt S.A. Kimball T.R. Molkentin J.D. Circ. Res. 2001; 88: 88-96Crossref PubMed Scopus (142) Google Scholar), kindly provided by Dr. Jeffery D. Molkentin (University of Cincinnati, Cincinnati, OH), was prepared using HEK293 cells and purified by VIRAPREP adenovirus purification kit (Virapur, LLC, San Diego, CA) as previously described (27Suganami E. Takagi H. Ohashi H. Suzuma K. Suzuma I. Oh H. Watanabe D. Ojima T. Suganami T. Fujio Y. Nakao K. Ogawa Y. Yoshimura N. Diabetes. 2004; 53: 2443-2448Crossref PubMed Scopus (131) Google Scholar). The GFP adenovirus (Ad-GFP; Clontech Laboratories, Palo Alto, CA) was used as a control. The CAR-3T3-L1 adipocytes at day 5 and day 18 after the induction of differentiation were transfected with Ad-MKP-1, incubated for 3 days, and harvested to be used for quantitative real-time PCR and immunoblot assay.Statistical Analysis—Data are shown as means ± S.E. Statistical analysis was performed using the Student's t test and analysis of variance followed by Scheffe's test. p < 0.05 was considered statistically significant.RESULTSMCP-1 mRNA Expression in the Adipose Tissue from Mice with Diet-induced Obesity—Body weight was increased significantly in mice fed high fat diet for 2 weeks relative to those fed standard diet (p < 0.01) (Fig. 1A). The mice fed high fat diet weighed ∼20% more than those fed standard diet for 15 weeks (29.7 ± 0.3 g versus 34.8 ± 1.9 g, p < 0.05). The weight of epididymal white adipose tissue (WAT) was significantly increased in mice fed high-fat diet for 2 weeks relative to those fed standard diet (0.26 ± 0.01 g versus 0.49 ± 0.04 g, p < 0.01). Histological examination revealed appreciable increase in adipocyte cell size in mice fed a high fat diet during the initial 2 weeks, which reached up to ∼4-fold larger than that in mice fed standard diet after 15 weeks (Fig. 1B). There were no appreciable infiltration of macrophages in the adipose tissue up to 8 weeks after the experiment, after which interstitial cells stained with F4/80, a marker of activated macrophages, appeared in mice fed high fat diet (Fig. 1C). Correspondingly, F4/80 mRNA expression was also increased in the epididymal WAT in mice fed high fat diet for 15 weeks relative to those fed standard diet (Fig. 1D, left). In mice fed high fat diet, MCP-1 mRNA expression was increased as early as 4 weeks and gradually increased up to 15 weeks after the experiment (Fig. 1D, right). These observations indicate that MCP-1 mRNA expression is increased prior to macrophage infiltration at the early stage of obesity.Dysregulation of Adipocytokine Production during the Course of Adipocyte Hypertrophy—To explore the molecular mechanisms underlying adipocyte hypertrophy, we cultured 3T3-L1 adipocytes up to 21 days after the induction of differentiation; they exhibited a gradual increase in lipid accumulation from day 8 to day 21 during the course of adipocyte hypertrophy as revealed by oil-red O staining (Fig. 2A) and triglyceride content (Fig. 2B). In this study, insulin-induced glucose uptake was preserved up to day 21 (supplemental Fig. S1).FIGURE 2Changes in adipocytokine expression during the course of adipocyte hypertrophy in vitro. A, morphological changes of 3T3-L1 adipocytes during the course of adipocyte hypertrophy (day 8-day 21) as revealed by oil-red O staining. Original magnification, ×200. Scale bars, 100 μm. B, triglyceride accumulation in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. C and D, changes in adipocytokine mRNA expression (C) and secretion (D) in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. **, p < 0.01 versus day 8. n = 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative real-time PCR analysis revealed that MCP-1 mRNA expression was significantly increased up to day 21, ∼6-fold higher than that in 3T3-L1 adipocytes (day 8) (p < 0.01), in parallel with increased cell size and lipid accumulation (Fig. 2C). Expression of IL-6 mRNA was also increased during the course of adipocyte hypertrophy. The IL-6 mRNA levels in 3T3-L1 adipocytes (day 21) were ∼5-fold higher than those in 3T3-L1 adipocytes (day 8) (p < 0.01). By contrast, adiponectin mRNA expression showed significant reduction (up to 30%) during the course of adipocyte hypertrophy (p < 0.01). The MCP-1, IL-6, and adiponectin concentrations in the culture media were roughly parallel to their respective mRNA levels (Fig. 2D). The expression patterns of adipocytokines in hypertrophied 3T3-L1 adipocytes (day 21) were similar to those found in obese adipose tissue. We also confirmed that mRNA expression patterns of adipogenesis-related markers such as peroxisome proliferator-activated receptor γ2 (PPARγ2), adipocyte fatty acid-binding protein (aP2), fatty-acid transport protein 1 (FATP1), and CCAAT/enhancer-binding protein α (C/EBPα) in hypertrophied 3T3-L1 adipocytes were consistent with those in obese adipose tissue (supplemental Fig. S2). In this study, we used 3T3-L1 adipocytes cultured for 8 and 21 days after differentiation as non-hypertrophied (day 8) and hypertrophied (day 21) adipocytes, respectively.Activation of MAPK Pathways during the Course of Adipocyte Hypertrophy—To explore the role of MAPK activation in the dysregulation of MCP-1 production during the course of adipocyte hypertrophy, we examined phosphorylation of ERK, p38 MAPK, and JNK in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. Immunoblot analysis revealed that phosphorylation of ERK and p38 MAPK is increased in hypertrophied adipocytes relative to non-hypertrophied adipocytes (Fig. 3A). In this study, there was no significant induction of phosphorylation of JNK during the course of adipocyte hypertrophy (Fig. 3A). Treatment of hypertrophied adipocytes with MEK inhibitors, PD98059 and U0126, for 24 h significantly reduced MCP-1 mRNA levels (Fig. 3B left, p < 0.01) and secretion in the culture media (Fig. 3B right, p < 0.01). Moreover, the effect of the MEK inhibitors on MCP-1 mRNA expression was observed as early as 6 h after the treatment (Fig. 3C). Furthermore, phosphorylation of ERK was increased in the nuclear fraction rather than in the cytosolic fraction from hypertrophied adipocytes (Fig. 3D, p < 0.01). We also confirmed that phosphorylation of MEK is increased in hypertrophied adipocytes (Fig. 3E, p < 0.01). On the other hand, no such inhibitory effect was observed when treated with a p38 MAPK inhibitor, SB203580 (Fig. 3, B and C). These observations suggest that increased mRNA expression and secretion of MCP-1 in hypertrophied adipocytes are due at least in part to MEK-ERK activation.FIGURE 3Role of MAP kinases in MCP-1 mRNA expression in hypertrophied adipocytes. A, phosphorylation of MAP kinases during the course of adipocyte hypertrophy. Representative immunoblots of ERK, p38 MAPK and JNK quantification of phosphorylation levels. *, p < 0.05; **, p < 0.01 versus day 8. n = 4. B, effect of 24-h-treatment with MAP kinase inhibitors on MCP-1 mRNA expression (left) and secretion (right) in hypertrophied 3T3-L1 adipocytes (day 21). PD, PD98059, 20 μmol/liter; U, U0126, 10 μmol/liter; SB, SB203580, 10 μmol/liter. **, p < 0.01 versus vehicle treated day 21. n = 6. N.S., not significant. C, effect of 6-h-treatment with MAPK inhibitors on MCP-1 mRNA expression in hypertrophied 3T3-L1 adipocytes (day 21). D, phosphorylation of ERK in the cytosolic and nuclear fractions from non-hypertrophied (day 8) and hypertrophied (day 21) 3T3-L1 adipocytes. Representative immunoblots of ERK and quantification of phosphorylation levels. E, phosphorylation of MEK during the course of adipocyte hypertrophy. Representative immunoblots of MEK and quantification of phosphorylation levels. **, p < 0.01 versus day 8. n = 4–6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MKP-1 Down-regulation during the Course of Adipocyte Hypertrophy—We next examined expression of members of the MKP family during the course of adipocyte hypertrophy. Interestingly, we detected substantial amounts of MKP-1 mRNA and protein in non-hypertrophied adipocytes, which are markedly down-regulated in hypertrophied adipocytes (Fig. 4, A and B, p < 0.05). There were no obvious changes in MKP-2 and MKP-3 mRNA levels during the course of adipocyte hypertrophy (Fig. 4A). We also observed that MKP-1 mRNA expression is significantly down-regulated in the adipose tissue from mice fed high fat diet for 2- and 4-weeks relative to those fed standard diet (Fig. 4C, p < 0.05). In addition, phosphorylation of ERK was significantly increased in the adipose tissue from mice that received 4-, 6-, 8-, and 15-week high fat diet relative to those fed standard diet (Fig. 4D, p < 0.05). These observations, taken together, suggest that MKP-1 is down-regulated in hypertrophied adipocytes, which is accompanied by ERK activation in vivo.FIGURE 4Changes in MKP-1 expression during the course of adipocyte hypertrophy in vitro and in vivo. A, changes in mRNA expression of MKP family during the course of adipocyte hypertrophy. n = 6. B, changes in MKP-1 protein levels in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. Representative immunoblots of MKP-1 and quantification of protein levels. n = 4. *, p < 0.05; **, p < 0.01 versus day 8. C and D, time course of MKP-1 mRNA expression (C) and ERK phosphorylation (D) levels in mice with diet-induced obesity. Representative immunoblots of ERK and quantification of phosphorylation levels. Data in C and D are expressed as the ratio of changes in mice fed HFD to those in mice fed SD. *, p < 0.05; **, p < 0.01 versus SD. n = 4–12 at each time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Generation of CAR-3T3-L1 Adipocytes—Because MKP-1 down-regulation may be responsible for the induction of MCP-1 during the course of adipocyte hypertrophy, we next examined the effect of MKP-1 restoration on ERK activity and MCP-1 mRNA expression in hypertrophied adipocytes. Because hypertrophied 3T3-L1 adipocytes are difficult to transfect with plasmid- or even virally encoded genes, we generated CAR-3T3-L1 adipocytes as described under "Experimental Procedures." There was no obvious difference in lipid accumulation between CAR-3T3-L1 and 3T3-L1 adipo
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