Overexpression of MCP-1 in adipose tissues causes ... · MCP-1 Overexpression in Adipos e Causes Insulin Resistance 1 Overexpression of MCP-1 in adipose tissues causes macrophage

MCP-1 Overexpression in Adipose Causes Insulin Resistance

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Overexpression of MCP-1 in adipose tissues causes macrophage recruitment and insulin resistance

Nozomu Kamei1,2, Kazuyuki Tobe1,3, Ryo Suzuki1,3, Mitsuru Ohsugi1, Taku Watanabe1, Naoto Kubota1,3,4, Norie Ohtsuka-Kowatari1,3, Katsuyoshi Kumagai1,3, Kentaro Sakamoto1,3, Masatoshi Kobayashi1, Toshimasa Yamauchi1,3, Kohjiro Ueki1, Yumiko Oishi4,5, Satoshi Nishimura5, Ichiro Manabe4,5, Haruo Hashimoto6, Yasuyuki Ohnishi6, Hitomi Ogata7, Kumpei Tokuyama7, Masaki Tsunoda8, Tomohiro Ide8, Koji Murakami8, Ryozo Nagai5, and Takashi Kadowaki1,3,4,9 From the 1Department of Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan, 2Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan, 3CREST of Japan Science and Technology Agency, Kawaguchi 332-0012, Japan, 4Division of Applied Nutrition, National Institute of Health and Nutrition, Tokyo 162-8636, Japan, 5Department of Cardiovascular Diseases, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan, 6Central Institute for Experimental Animals, Kawasaki 216-0001, Japan, 7Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan, 8Bioscience Division I Metabolic Discovery Research Laboratories KYORIN Pharmaceutical Co., Ltd., Shimotsuga 329-0114, Japan

Running Title: MCP-1 Overexpression in Adipose Causes Insulin Resistance 9Address correspondence to: Takashi Kadowaki, Department of Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Tel. 81-3-5800-8818; Fax. 81-3-5689-7209; E-mail: [email protected]

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity, but the role of elevated MCP-1, which may work locally or systemically, in insulin resistance, has not been directly addressed. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the aP2 promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric and gene expression analyses. TNF-α and IL-6 mRNA levels in white adipose tissue and plasma NEFA levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rate of

glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of insulin receptors, insulin receptor substrates and Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. In vitro MCP-1 treatment of myotube cells attenuated insulin-induced phosphorylation of Akt. In addition, MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 contribute to the development of insulin resistance in aP2-MCP-1 mice.

Obesity correlates closely with insulin

resistance (1, 2). We have demonstrated that the size of adipocytes is inversely related to insulin sensitivity (3-5): namely, larger adipocytes are

http://www.jbc.org/cgi/doi/10.1074/jbc.M601284200The latest version is at JBC Papers in Press. Published on June 29, 2006 as Manuscript M601284200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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associated with insulin resistance, smaller adipocytes with insulin sensitivity. Energy excess results in adipocyte hypertrophy, which in turn exerts deleterious effects on insulin sensitivity. Larger adipocytes are less insulin sensitive as shown by impaired insulin stimulated glucose uptake. Moreover, diet induced hypertrophy of adipocytes leads to changes in the profile of adipokines toward a deterioration of insulin sensitivity, particularly with decreased adiponectin levels (6, 7).

Recent studies have shown that obesity is associated not only with larger adipocytes but also with increasing numbers of infiltrating macrophages in adipose tissues (8-10). These adipose tissue macrophages are currently considered to be a major cause of obesity-associated chronic low-grade inflammation (2, 11), via secretion of a wide variety of inflammatory molecules (12), including 1tumor necrosis factor-α (TNF-α) (13), interleukin-6 (IL-6) (14), monocyte chemoattractant protein-1 (MCP-1) (15, 16) and plasminogen activator inhibitor-1 (PAI-1) (17). These inflammatory molecules may have local effects on white adipose tissue (WAT) physiology as well as potential systemic effects on other organs, which culminate in insulin resistance (12). The molecular signals that trigger the macrophage accumulation in obese WAT are, however, not yet known. How macrophage accumulation in adipose tissues causes systemic insulin resistance is currently unknown.

Among inflammatory molecules upregulated in adipose tissues of obese animals and humans, MCP-1 has been viewed as one of the likely candidate adipokines initiating macrophage infiltration of the adipose tissue and inducing systemic insulin resistance. MCP-1 is a member of the CC chemokine family and promotes migration of inflammatory cells by chemotaxis and integrin activation (18), and it has been reported to recruit monocytes from the blood into atherosclerotic lesions, thereby promoting foam cell formation (19-21). MCP-1 expression in adipose tissue and plasma MCP-1 levels have been found to correlate positively with the degree of obesity (9, 10, 16, 22). In

addition, increased expression of this chemokine in adipose tissue precedes the expression of other macrophage markers during the development of obesity (10). A recent report on mice lacking C-C motif chemokine receptor-2 (CCR2), a receptor for MCP-1 and other several chemokines, suggested the MCP-1/CCR2 pathway to influence the development of obesity and insulin resistance via adipose macrophage accumulation and inflammation (23). Thus, we hypothesized that MCP-1 may serve as a signal that triggers inflammation by attracting macrophages into adipose tissues as well as an adipokine that causes insulin resistance by directly affecting insulin signaling in other organs.

In this study, we assessed the effect of adipose overexpression of MCP-1 on the development of insulin resistance by generating transgenic (TG) mice under the adipocyte P2 (aP2) promoter. The TG mice showed increased macrophage accumulation in adipose tissues with higher plasma MCP-1 concentrations than littermate wild-type (WT) mice. The TG mice were insulin resistant as shown by insulin tolerance test (ITT), hyperinsulinemic euglycemic studies and insulin signal studies. As the TG mice displayed increased gene expression of TNF-α and IL-6 as well as higher plasma concentrations of non-esterified fatty acids (NEFA), adipocyte dysfunction caused by macrophage accumulation in adipose tissue may contribute to the development of systemic insulin resistance. In addition, we demonstrated that MCP-1 directly attenuated insulin signaling in myotube cells and insulin-stimulated glucose uptake in isolated skeletal muscle, suggesting that higher circulating MCP-1 may have a direct negative impact on insulin-stimulated glucose uptake in aP2-MCP-1 mice. Thus, we conclude that both macrophage accumulation leading to adipocyte dysfunction (local effects on adipose tissues) and direct effects of circulating MCP-1 on insulin target organs (endocrine effects) contribute to the development of insulin resistance in aP2-MCP-1 mice.

EXPERIMENTAL PROCEDURES


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Reagents - Recombinant mouse CCL2/JE/MCP-1 protein was purchased from R&D Systems Inc. (Minneapolis, MN). U0126 was purchased from Calbiochem (La Jolla, CA). 2-deoxy-D-[1-14C]-glucose and L-[1-3H]-glucose were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Mouse monoclonal anti-phosphotyrosine antibody 4G10 (αPY), rabbit polyclonal antibodies to insulin receptor substrate (IRS) -1, IRS-2 and the phosphatidylinositol 3-kinase p85 regulatory subunit were purchased from Upstate Biotechnology Inc. Rabbit polyclonal antibody to insulin receptor β (IRβ) was purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against p44/42 Map Kinase, phosphor-p44/42 Map Kinase, Akt and phospho-Akt (Ser-473) were purchased from Cell Signaling Technology. Cell Culture, Differentiation and In Vitro Assay - C2C12 mouse skeletal myoblast cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin in humidified 5% CO2, 95% air at 37 °C, and cultured to confluence. In order to induce differentiation, cells were switched to media containing DMEM, 2.5% horse serum, 100 units/ml penicillin and 100 µg/ml streptomycin for the indicated time periods. For the Western blotting analyses, cells were serum-deprived for 10 hours in media and treated with 10 nM MCP-1 for 5 minutes to detect the extracellular signal-regulated kinase (ERK) activation. C2C12 cells were treated with 1 to 10 nM MCP-1 for 30 minutes prior to 10 nM insulin stimulation to test activation of insulin signaling. In some experiments, cells were pretreated with U0126, an inhibitor of mitogen activated extracellular signal protein kinase (MEK), for 30 minutes prior to MCP-1 addition. Generation of TG Mice Expressing MCP-1 in Adipose Tissues - A murine MCP-1 coding sequence cDNA for insertion was prepared by cloning reverse transcriptase-polymerase chain reaction (RT-PCR) products from mouse macrophage mRNA into a 2.1-TOPO cloning vector (Invitrogen). For overexpression in

adipose tissues, transgene expression was targeted to adipose tissue using the mouse aP2 promoter (24) kindly provided by Dr. Bruce Spiegelman (Dana Farber Institute, Boston, MA). The transgene consisted of 5.4 kb of the aP2 gene promoter linked to rabbit β globin, the 447 bp MCP-1 cDNA, and a polyadenylation sequence (Fig 2A). The construct was inserted into a pUC19 vector (Nippon Gene Co., Ltd.) and cloned. The purified AscI-AscI fragment was microinjected into the pronuclei of fertilized DBF2 eggs. The recipient eggs were [C57BL/6 × DBA2] F2 hybrids. TG founder or F2 mice were identified by Southern blot analysis of tail DNAs using the cDNA probe to the BamHI/BamHI site in MCP-1 and polymerase chain reaction (PCR). The primers used for genotyping PCR were as follows: 5' primer, 5'-CATCCTGCCTTTCTCTTTATGGTTAC-3’; and 3' primer, 5'-CTAGTTCACTGTCACACTGGTC-3'. From the 13 lines of TG mice obtained, we selected three lines showing graded expression of MCP-1 and designated them Low (L), Middle (M) and High (H). The founder and TG descendants were bred onto a C57BL/6 background for two generations. The F2 TG mice and their littermates were used in experiments. TG mice served as heterozygotes. Animal Care - ob/ob mice with a C57BL/6 background were purchased from the Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). Mice were housed under a 12 h light-dark cycle and given ad libitum access to normal chow MF consisting of 25% (w/w) protein, 53% carbohydrates, 6% fat and 8% water (Oriental Yeast Co., Ltd., Osaka, Japan) or a high fat diet 32 consisting of 25.5% (w/w) protein, 2.9% fiber, 4.0% ash, 29.4% carbohydrates, 32% fat and 6.2% water (CLEA Japan Inc., Tokyo, Japan). All experiments in this study were performed on male mice. The animal care and procedures for the experiments were approved by the Animal Care Committee of the University of Tokyo. RNA Preparation and Northern Blot Analysis - Mice were sacrificed after a 6 hour fast and the epididymal fat pad (for epididymal WAT),


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subcutaneous fat (for subcutaneous WAT), brown adipose tissue (BAT), liver, spleen, kidney, heart and muscle were excised. Total RNA was prepared from tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Northern blot analysis was performed with 15 µg of total RNA according to the standard protocol. Total RNA was loaded onto a 1.3% agarose gel then transferred to a nylon membrane (Hybond N+: Amersham Biosciences). MCP-1 coding sequence cDNA was used as the probe template. The corresponding bands were quantified by exposure of BAS 2000 to the filters and measurement with BAStation software (Fuji Film, Tokyo, Japan). Quantitative RT-PCR - Total RNA was extracted from various tissues or C2C12 cells with TRIzol reagent according to the manufacturer’s instructions. After treatment with RQ1 RNase-Free DNase (Promega, Madison, WI) to remove genomic DNA, cDNA was synthesized with MultiScribe Reverse-Transcriptase (Applied Biosystems, Foster City, CA), and Taq-Man quantitative PCR (50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min) was then performed with the ABI Prism 7900 PCR instrument (Applied Biosystems) to amplify samples for MCP-1, F4/80, CD68, matrix metallopeptidase 12 (MMP12), glucose-6-phosphatase (G6Pase), TNF-α, IL-6, resistin, adiponectin, leptin, peroxisome proliferator-activated receptor γ (PPARγ), CCR2 and cyclophilin cDNA. The primers used for cyclophilin were as described previously (25), and those for the other reactions were purchased from Applied Biosystems. The relative abundance of transcripts was normalized to constitutive expression of cyclophilin mRNA. Isolation of Adipocytes and Stromal-Vascular Cells (26) - Mice were anesthetized and epididymal white fat pads were removed. The fat pads were rinsed in saline and cut into small pieces, then digested with collagenase (Sigma-Aldrich Co., St. Louis, MO) with Krebs-Henseleit-HEPES buffer, pH 7.4, supplemented with 20 mg/ml of bovine serum albumin and 2 mmol/l of glucose at 37 °C in a

shaking water bath for 45 minutes. Then, digested tissues were filtered through mesh and fractionated by brief centrifugation (1000 rpm). Floating cells were adipocytes, the pellet nonadipocytes (stromal-vascular cells (SVCs)). Both cell types were rinsed three times with Krebs-Henseleit-HEPES buffer and used in RNA extraction or flow cytometry analysis. Flow Cytometry Analysis (9, 23) – In the SVCs, red blood cells were lysed and removed by a 15 minute incubation in Pharm Lyse (BD Biosciences) at 4 °C. The SVCs were rinsed twice and resuspended in Pharmingen Stain Buffer (BD Biosciences). The cell number was calculated by hemocytometing, and the cells were incubated with FcBlock (BD Biosciences) for 10 minutes at 4 °C before the incubation with either anti-mouse CD11b antibodies conjugated with Alexa Fluor488 (Caltag Laboratories) or anti-mouse F4/80 antibodies conjugated with phycoerythrin (Caltag Laboratories), or each of the matching isotopes as controls, for 30 minutes at 4 °C. After incubation with the antibodies, the cells were rinsed twice and resuspended in Pharmingen Stain Buffer. After labeling with TO-PRO-3 (Invitrogen), the cells were analyzed by FACSCalibur (BD Biosciences). Data acquisition and analysis were performed using CellQuest Pro software (BD Biosciences). Dead cells were gated out by a combination of FSC/SSC and TO-PRO-3 dot plots. The numbers of macrophages in epididymal white adipose tissues were calculated by multiplying the number of SVCs by the percentage of CD11d and F4/80 double positive cells. Immunoprecipitation and Western Blot Analysis - Tissues and cells were homogenized and lysed with ice-cold buffer A (25 mM Tris-HCl (pH 7.4), 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride). After centrifugation, immunoprecipitation (IP) of liver and muscle proteins was performed as described previously (27) with some modifications. Samples were separated on polyacrylamide gels and transferred to Hybond-P PVDF transfer membrane (Amersham Biosciences). After


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incubating the membrane with antibodies, bands were detected by ECL detection reagents (Amersham Biosciences). Histological and Immunohistochemical Analysis of WAT - An epididymal fat pad was removed from each animal, fixed in 10 % formaldehyde/phosphate-buffered saline, and maintained at 4 °C for 2 days. Fixed specimens were dehydrated and embedded in paraffin. The fat pad was then cut into 5 µm sections at 50 µm intervals, and then mounted on silanized slides. After deparaffinization, the sections were stained with rat monoclonal F4/80 antibody (Serotec Ltd.) at a 1:1000 concentration, followed by counter-staining with hematoxylin. The adipocyte area was manually traced and analyzed with Win ROOF software (Mitani Co. Ltd., Chiba, Japan). The area was measured in four high-power fields (275000 µm2 per field) from different sections, and the histogram was drawn by analyzing six mice per group according to methods described previously (4) with modifications. The adipocyte area was measured in 400 or more cells per mouse on normal chow or in 180 or more cells per mouse on the high fat diet. The total number of nuclei and the number of F4/80 positive nuclei were counted in four different high-power fields from each of four different sections. The nuclei of more than 2000 cells per mouse on normal chow or more than 1000 cells per mouse on the high fat diet were counted. The ratio of F4/80 positive nuclei was calculated as the number of nuclei of F4/80 expressing cells divided by the total number of nuclei in sections of a sample. Mesurement of 2-deoxyglucose (2-DG) uptake – This assay was performed as described previously (28), with some modifications. The soleus muscles of 9 week old C57BL/6 mice were removed from the hindlimbs, ligated around each tendon using silk surgical thread, and attached across a plastic holder. The muscles were incubated for 10 minutes at 37 °C in Krebs-Ringer phosphate buffer (pH7.4) containing 5 mM HEPES, 3% bovine serum albumin, and 2 mM sodium pyruvate (buffer A). For MCP-1 pretreatment, the muscles were incubated in buffer A containing 0, 0.1, 1 or 10 nM MCP-1 for 30 minutes at 37 °C prior to

insulin treatment. The muscles were incubated with or without 10 nM insulin in buffer A containing 0, 0.1, 1 or 10 nM MCP-1 at 30 °C for 10 minutes. To determine 2-DG uptake, the muscles were transferred to buffer A containing 1 mM 2-DG (0.5 µCi/mL 2-deoxy-D-[1-14C]-glucose) and 1 mM L-glucose (0.5 µCi/mL L-[1-3H]-glucose) and incubated at 30 °C for 10 minutes. For U0126 rescue experiments, 20 µM of U0126 were added to all of the buffers. The buffers were continuously gassed with 95% O2–5% CO2 in a shaking incubator. To terminate the reaction, the muscles were washed three times with chilled buffer A, and then dissolved in 5 N NaOH. The samples were neutralized with 5 N HCl and dissolved in ACSII (Amersham, UK). 14C and 3H specific activities were counted by a liquid scintillation counter (Packard Instrument Co., Meriden, CT). The specific uptake of 2-DG was calculated by subtracting the nonspecific uptake of L-glucose from total uptake 2-DG uptake. Plasma MCP-1, Adiponectin, Leptin and NEFA measures - Mice were fasted for 6 hours before plasma was obtained. Plasma MCP-1, adiponectin and leptin levels were determined with a mouse JE/MCP-1 ELISA kit (R&D Systems Inc.), mouse adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan), and mouse leptin ELISA kit (R&D Systems Inc.), respectively. Plasma NEFA (Wako Pure Chemical Industries Ltd., Osaka, Japan) were assayed by enzymatic methods. Mesurement of Tissue Triglyceride Contents - Liver and muscle tissues were homogenized, and their triglyceride contents were determined as described previously (6). Insulin Tolerance Test (ITT) - Insulin tolerance was tested in mice fasted for 4 hours. The animals were intraperitoneally injected with 0.75 mU/g (body weight) human insulin (Humulin R, Lilly). Blood samples were drawn from the tail vein at the times indicated, and glucose was measured with an automatic blood glucose meter (Glutest Pro, Sanwa Chemical, Nagoya, Japan). Glucose Tolerance Test (GTT) - Before the study, the mice were fasted for 16 hours starting at 19:00, and at the end of the fast they were orally loaded


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with glucose at 1.0 mg/g (body weight). Blood samples were collected at different times, and glucose was immediately measured with an automatic blood glucose meter. Whole blood was collected and centrifuged in heparinized tubes, and the plasma was stored at -20 °C. Insulin levels were determined with an insulin radioimmunoassay kit (BIOTRAK, Amersham Biosciences) using rat insulin as the standard. Tissue Sampling for Insulin Signaling Pathway Study - Mice were anesthetized after 24 hours of starvation, and 5 units of human insulin (Humulin R, Lilly) were injected into the inferior vena cava. After 5 minutes, the liver and hindlimb muscles were removed, and the samples were then used for protein extraction as described above. Hyperinsulinemic-euglycemic clamp study - Clamp studies were carried out as described previously (29). In brief, 4-5 days before the study, an infusion catheter was inserted into the right jugular vein under general anesthesia with sodium pentobarbital. Studies were performed on mice under conscious and unstressed conditions after a 6 hour fast. A primed-continuous infusion of insulin (Humulin R: Lilly) was given (3.0 mU/kg/min for NC fed mice and 10.0 mU/kg/min for HF diet fed mice), and the blood glucose concentration, monitored every 5 minutes, was maintained at ~ 120 mg/dl by administration of glucose (5 g of glucose per 10 ml enriched to approximately 20% with [6,6-2H2]glucose (Sigma)) for 120 minutes. Blood was sampled via tail-tip bleeds at 90, 105 and 120 min for determination of the rate of glucose disappearance (Rd). Rd was calculated according to nonsteady-state equations (29), and endogenous glucose production (EGP) was calculated as the difference between Rd and exogenous glucose infusion rates (GIR) (29). Statistical Analysis - Results were expressed as means ± standard error. Differences between groups were examined for statistical significance using Student’s t test, analysis of variance (ANOVA) with Fisher’s protected least significant difference test, or ANOVA with the Games-Howell test.

RESULTS

Increased MCP-1 expression in WAT of obesity model mice

The macrophage content of adipose tissue has been reported to be increased by obesity and to correlate positively with insulin resistance (9, 10, 15, 22). To identify potential regulators of macrophage accumulation, we studied the expression profiles of epididymal adipose tissue from WT animals and PPARγ +/- mice (4) on the high fat diet using oligonucleotide microarrays (Murine Genome Array U74 ver.2, Affymetrix Inc.). The expression of MCP-1, a powerful chemokine involved in macrophage chemotaxis and recruitment, was upregulated by 2.7 fold in WT mice as compared with PPARγ +/- mice, which showed protection from obesity and insulin resistance even on the high fat diet (data not shown). MCP-1 expression was increased by 4.5 fold in ob/ob mice together with enhanced expressions of macrophage markers including F4/80, CD68 and MMP12 (Fig 1A). As reported previously, we also observed increased plasma concentrations of MCP-1 in ob/ob mice (Fig 1B). C57BL/6 mice on a high fat diet showed increased expressions of MCP-1 and CD68 (Fig 1C) in epididymal WAT as compared with control mice on a normal chow diet. These enhanced gene expressions were observed as early as 8 weeks of high fat feeding, and became even more pronounced thereafter. Increased plasma levels of MCP-1 were observed after 18 weeks of the high fat diet (Fig 1D). Thus, the accumulation of macrophages in WAT preceded the elevation of plasma MCP-1 with high fat feeding. As shown in Figure 1E, high fat diet feeding led to increased MCP-1 mRNA only in WAT and BAT. Generation of TG mice overexpressing MCP-1 in adipose tissues

Having demonstrated insulin resistance to be associated with enhanced MCP-1 expression in adipose tissue, we hypothesized that increased expression of this chemokine in these tissues may lead to systemic insulin resistance. Thus, we generated TG mice overexpressing MCP-1 in adipose tissue, utilizing the aP2 promoter (Fig 2A). We obtained three lines of TG mice with graded expressions of MCP-1, designated Low


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(L), Middle (M) and High (H), with Line L having the fewest copies of the transgene (Fig 2B). All of these lines expressed MCP-1 abundantly in WAT as well as BAT (Fig 2C), although minimal MCP-1 expressions were observed in the spleen, heart and muscle. Expression analyses revealed MCP-1 expression to be markedly increased in WAT as well as BAT in TG mice of Line M (Fig 2D). In this analysis, enhanced expression of MCP-1 mRNA was also noted in the liver, spleen and muscle. This was presumably due to aP2 activity in macrophages as well as adipocytes residing in these organs, as previously reported (30). When determined after adipose tissues had been fractioned into adipocytes and SVCs in Line M mice, MCP-1 expressions were observed mainly in adipocytes although a small but significant increase in the expression of this gene was also seen in SVCs in the Line M mice (Fig 2E).

Plasma MCP-1 concentrations were determined. All three lines had elevated MCP-1 concentrations as compared to littermate controls (Line L, 78.9±6.8 pg/ml on normal chow and 66.6±2.0 pg/ml on high fat; Line M, 161.6±6.8 pg/ml on normal chow and 213.7±14.2 pg/ml on high fat; Line H, 189.1±17.2 pg/ml on normal chow and 238.8±8.5 pg/ml on high fat) (Fig 2F). Of note, the MCP-1 elevations in aP2-MCP-1 TG mice rose to levels between those of C57BL/6 mice and ob/ob mice (Fig 1B). aP2-MCP-1 TG mice show normal adiposity and increased infiltration of macrophages into WAT

To assess the roles of MCP-1 overexpression and macrophage recruitment in adipose tissue in the development of insulin resistance, we fed either normal chow or a high fat diet to the three lines of male aP2-MCP-1 TG mice as well as their littermate controls. aP2-MCP-1 TG mice gained weight to the same extent as their WT littermates did on either normal chow or the high fat diet (Fig 3A). We further examined the weight of adipose tissue, the size of adipocytes and the number of accumulated macrophages in the epididymal WAT in Line M of the aP2-MCP-1 mice on either normal chow or after 8 weeks of high fat diet feeding. Although

neither epididymal WAT weight nor adipose cell size differed between aP2-MCP-1 mice and WT littermates (Fig 3B, 3D), immunohistochemistry showed the number and the distribution of macrophages to be altered in aP2-MCP-1 mice. As shown in Figure 3C, more macrophages were scattered throughout adipose tissue in aP2-MCP-1 mice than in WT control mice. When fed a high fat diet for 8 weeks, aP2-MCP-1 mice exhibited more areas of aggregated F4/80 positive nuclei surrounding adipocytes, which formed a typical crown. Morphometric analysis showed that aP2-MCP-1 TG mice had more F4/80 positive nuclei in adipose tissues (Fig 3E), further confirming that these mice had greater accumulation of macrophages in adipose tissues. Furthermore, flow cytometric analyses of stromal vascular fraction in Line M mice showed that the percentage of CD11b and F4/80 double positive cells to be increased in TG mice (41.0±1.0%) as compared with WT mice (34.7±1.3%; p<0.01 vs. TG mice) on normal chow diet. We also observed an increase in the total number of macrophages in the stromal vascular fraction of epididymal WAT in TG mice compared with that in WT mice (Fig 3F). Expression analyses of F4/80, CD68 and MMP12 in WAT revealed the expressions of these macrophage markers to be increased in TG mice as compared to WT mice, and the differences became significant on a high fat diet (Fig 3G). Although they were not statistically different, the expressions of macrophage markers in TG mice fed on a normal chow diet tended to be higher than those in WT mice. TG animals have high TNF-α and IL-6 expressions in WAT and high plasma NEFA

Gene expression analysis in the Line M revealed the expressions of TNF-α and IL-6 to be increased in WAT of TG mice (Fig 4A). On the contrary, however, the expressions of adipokines including resistin, adiponectin, and leptin were not altered. We also observed the plasma NEFA concentration to be increased in TG mice, while plasma adiponectin and leptin levels were not changed (Fig 4B). aP2-MCP-1 TG mice were insulin resistant and glucose intolerant

To assess insulin sensitivity in the


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aP2-MCP-1 TG animals, we performed ITT. In all three lines of the aP2-MCP-1 mice, whether on a normal or a high fat diet, the glucose lowering effect of insulin was impaired (Fig 5A). Oral GTT demonstrated mice of TG Line L, which had low MCP-1 expression, to not have altered glucose metabolism on either the normal diet or the high fat diet (Fig 5B). Although both the M and the H Line had normal glucose metabolism after glucose loading on a normal diet, these mice showed impaired glucose tolerance when fed a high fat diet. As shown in Figure 4C, the plasma insulin concentrations of the aP2-MCP-1 TG mice tended to be higher than those of control littermates. Hyperinsulinemic euglycemic clamp study revealed insulin resistance in skeletal muscle and liver of aP2-MCP-1 TG mice

To further examine the mechanisms of the insulin resistance in TG mice, we performed a hyperinsulinemic euglycemic clamp study on Line M mice (Fig 6A,B). The TG mice required less glucose to be infused to maintain euglycemia on both the normal chow and the high fat diet. EGP appeared to be higher and Rd tended to be lower in the TG mice than in their littermate counterparts while on normal chow. The difference in EGP and Rd increased, becoming statistically significant, when WT and TG mice were fed a high fat diet. These observations indicate insulin resistance in muscle and liver. Hepatic G6Pase expression levels were higher in TG than in WT mice on a high fat diet, which is consistent with the increased hepatic glucose production in aP2-MCP-1 mice (Fig 6C). No significant differences in triglyceride contents were observed in either muscle or hepatic tissues between WT and TG mice of Line M, whether fed normal chow or a high fat diet (data not shown). MCP-1 overexpression in WAT decreased tyrosine phosphorylation of IR and IRS proteins in both skeletal muscle and liver

To further examine the mechanisms of insulin resistance in aP2-MCP-1 mice, we studied the insulin signaling pathways in skeletal muscle and the liver in WT and TG mice of Line M on normal chow or high fat diets (Fig 7, 8). In muscle, Akt phosphorylation was decreased in TG mice

as compared with WT mice, on both normal chow and a high fat diet (Fig 7A). In addition, tyrosine phosphorylation of the IR was decreased in TG mice (Fig 7B). Likewise, insulin stimulated tyrosine phosphorylation of IRS-1 (Fig 7C) and insulin stimulated association of p85 with IRS-1 (Fig 7D) were also decreased in TG mice. TG mice showed no significant difference in insulin stimulated tyrosine phosphorylation of IRS-2 (Fig 7E) or the amount of IRS-2 associated p85 (Fig 7F) in muscle.

In the liver, we observed reductions in phosphorylated Akt and tyrosine phosphorylation of IR with insulin stimulation (Fig 8A, B), similar to the reductions seen in muscle. A significant decrease in insulin stimulated IRS-1 tyrosine phosphorylation was seen in the TG mice fed a normal diet (Fig 8C) while the amount of IRS-1 associated p85 in TG mice was not significantly decreased (Fig 8D). There was also a significant decrease in insulin stimulated tyrosine phosphorylation of IRS-2 (Fig 8E) and the amount of IRS-2 associated p85 (Fig 8F) in the liver. MCP-1 directly impaired insulin signaling in myotube cells and isolated skeletal muscle

aP2-MCP-1 mice displayed not only macrophage accumulation in adipose tissue but also higher plasma MCP-1 concentrations. We presumed that circulating MCP-1 might directly affect insulin signaling in skeletal muscle. As CCR2, the receptor for MCP-1, is expressed in skeletal muscle (Fig 9A) and in C2C12 differentiated myotube cells (Fig 9B), we assessed whether MCP-1 has a direct effect on insulin signaling via ERK phosphorylation utilizing the C2C12 cell line and isolated skeletal muscle. Incubation with 10 nM MCP-1 for 5 minutes in C2C12 myotubes stimulated ERK phosphorylation, which was reversed by a 30 minute preincubation with 100 nM U0126, a specific MEK inhibitor (Fig 9C). We found that pretreatment of the cells with 1 to 10 nM MCP-1 for 30 minutes significantly attenuated insulin-stimulated Akt phosphorylation (Fig 9D), which was restored, albeit in only partially, by preincubation with 100 nM U0126. In addition, preincubation with MCP-1 blunted insulin


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stimulated 2-deoxyglucose uptake in isolated skeletal muscle, but this uptake was also partially restored by preincubation with U0126 (Fig 9E).

DISCUSSION

In this study, we demonstrated that overexpression of MCP-1 in adipose tissue caused systemic insulin resistance, which was shown by ITT (Fig 5A), and confirmed by a hyperinsulinemic euglycemic clamp study and the assessment of insulin signaling (Fig 6-8). The aP2-MCP-1 mice had an increased number of adipose tissue macrophages with higher plasma MCP-1 levels. We assume that both the paracrine and the endocrine effects of MCP-1 contributed to the development of insulin resistance in aP2-MCP-1 mice, although it is difficult to quantify the degree to which each of these effects contributes to the development of insulin resistance.

Changes in proinflammatory adipokines may contribute to the development of insulin resistance in this murine model (Fig 10A). Although there were no changes in adiponectin or leptin levels in our aP2-MCP-1 TG mice, we observed elevations of TNF-α and IL-6 mRNA in WAT. We assume the increased expressions of TNF-α and IL-6 gene to be attributable to the increased macrophage accumulation. In our preliminary experiments, large amounts of TNF-α and IL-6 were secreted by cells in the stromal-vascular fraction while significant amounts were also from the adipocytes themselves (31, 32) (data not shown). TNF-α and IL-6 from accumulated macrophages may work locally on adipocytes (33), causing changes in metabolic and endocrine functions, as they do systemically on skeletal muscle and the liver. MCP-1 secreted by adipocytes may locally affect insulin signaling in adipocytes (22), thereby contributing to the increased release of NEFA. Elevated NEFA may also participate in the exacerbation of insulin resistance in aP2-MCP-1 mice (34-36).

Circulating MCP-1 secreted by adipocytes of aP2-MCP-1 mice may directly attenuate insulin signaling in skeletal muscle in an

endocrine fashion (Fig 10B). We have shown that MCP-1 treatment of C2C12 myotube cells blunted insulin signals, including Akt phosphorylation (Fig 9D). We further showed that MCP-1 attenuated insulin-stimulated glucose uptake in isolated skeletal muscle (Fig 9E). These negative effects of MCP-1 on insulin action were partially restored by addition of the MEK inhibitor U0126, suggesting that ERK activation is part of the mechanism by which MCP-1 blunts insulin signaling. These data indicated that circulating MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.

Results of a hyperinsulinemic euglycemic study indicating that aP2-MCP-1 mice have lower Rd and higher EGP (Fig 6). Insulin resistance in skeletal muscle and liver was confirmed by an insulin signaling study (Fig 7, 8). In fact, insulin-stimulated Akt phosphorylation was blunted in both skeletal muscle and liver. Analyses of insulin signaling revealed slight but significant attenuation of tyrosine phosphorylation of the insulin receptor in the liver as well as skeletal muscle. A difference was also seen in tyrosine phosphorylation of IRS proteins. We and others have demonstrated that IRS-1 plays a major role in insulin signaling in skeletal muscle while IRS-2 predominantly functions in the liver (27, 29, 37-41). In muscle, reduction of tyrosine phosphorylation and less IRS-1 associated p85 were apparent, while IRS-2 tyrosine phosphorylation and IRS-2 associated p85 were reduced in the liver. Decreased tyrosine phosphorylation of IRS-1 or IRS-2 in skeletal muscle or the liver, respectively, was responsible for the insulin signaling defects shown by reduced Akt phosphorylation in these two tissues.

The roles of MCP-1 in adipose tissue inflammation and systemic insulin resistance were also supported by the metabolic phenotypes of mice lacking CCR2, a receptor for MCP-1 (CCL2), MCP-2 (CCL8) and MCP-3 (CCL7) (23). The CCR2-/- mice showed improved systemic glucose homeostasis and insulin sensitivity accompanied by reduced macrophage contents and an altered inflammatory profile in adipose tissue while on a high fat diet. These mice


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showed reduced food intake with attenuated obesity. They exhibited increased adiponectin and leptin gene expressions. However, the adiposity of our aP2-MCP-1 mice was comparable to that in WT mice, and there were no significant changes in adiponectin or leptin gene expressions. Our data suggested that CCR2 ligands other than MCP-1 may be involved in CCR2 signaling, so far as metabolic phenotypes are concerned.

While this manuscript was being revised, glucose metabolism in aP2-MCP-1 transgenic mice was described by Kanda et al. (42). Their results and ours are very similar: insulin resistance in skeletal muscle as well as in the liver was found in their transgenic mice and ours. While Kanda and his colleagues focused their analysis on the effects of MCP-1 on the liver, our studies demonstrated that MCP-1 has a direct inhibitory effect on insulin signaling in skeletal muscle.

It is noteworthy that the role of MCP-1 expression in attracting macrophages in WAT and BAT seems to be different. We observed an increased expression of MCP-1 gene in BAT compared with that in WAT under both normal chow and a high fat diet (Fig 2C, D). The expression of macrophage markers in BAT, however, was not increased so much as that in WAT (supplemental Fig 1). These data suggested

that MCP-1 expression is not sufficient for macrophage accumulation, and additional factor(s), which is/are differentially expressed in between WAT and BAT, may be required for macrophage accumulation.

The findings derived from our aP2-MCP-1 mice corroborate the link between inflammation in adipose tissues and systemic insulin resistance. First, our TG mice showed greater macrophage accumulation in adipose tissue and the aggregated macrophages were more readily apparent when the transgenic mice were fed a high fat diet. Second, although adipose cell size did not differ in our aP2-MCP-1 mice, the functions of adipocytes were altered as evidenced by increased plasma concentrations of NEFA. Finally, our TG mice were insulin resistant as shown by insulin tolerance tests as well as hyperinsulinemic euglycemic clamp study.

In conclusion, transgenic overexpression of MCP-1 in adipose tissue resulted in systemic insulin resistance. We propose that both paracrine and endocrine effects of MCP-1 contribute to the development of insulin resistance in aP2-MCP-1 mice (Fig 10).

REFERENCES

1. Flier, J. S. (2004) Cell. 116, 337-350 2. Wellen, K. E., and Hotamisligil, G. S. (2005) J Clin Invest. 115, 1111-1119 3. Okuno, A., Tamemoto, H., Tobe, K., Ueki, K., Mori, Y., Iwamoto, K., Umesono, K., Akanuma, Y.,

Fujiwara, T., Horikoshi, H., Yazaki, Y., and Kadowaki, T. (1998) J Clin Invest. 101, 1354-1361 4. Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Nakano,

R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K., Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Kitamura, T., Fujita, T., Ezaki, O., Aizawa, S., Kadowaki, T., and et al. (1999) Mol Cell. 4, 597-609

5. Kadowaki, T. (2000) J Clin Invest. 106, 459-465 6. Yamauchi, 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., and Kadowaki, T. (2001) Nat Med. 7, 941-946

7. Berg, A. H., Combs, T. P., Du, X., Brownlee, M., and Scherer, P. E. (2001) Nat Med. 7, 947-953 8. Soukas, A., Cohen, P., Socci, N. D., and Friedman, J. M. (2000) Genes Dev. 14, 963-980 9. Weisberg, S. P., McCann, D., Desai, M., Rosenbaum, M., Leibel, R. L., and Ferrante, A. W., Jr.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


11

(2003) J Clin Invest 112, 1796-1808 10. Xu, H., Barnes, G. T., Yang, Q., Tan, G., Yang, D., Chou, C. J., Sole, J., Nichols, A., Ross, J. S.,

Tartaglia, L. A., and Chen, H. (2003) J Clin Invest 112, 1821-1830 11. Wellen, K. E., and Hotamisligil, G. S. (2003) J Clin Invest 112, 1785-1788 12. Kershaw, E. E., and Flier, J. S. (2004) J Clin Endocrinol Metab. 89, 2548-2556 13. Hotamisligil, G. S., Shargill, N. S., and Spiegelman, B. M. (1993) Science. 259, 87-91 14. Fernandez-Real, J. M., and Ricart, W. (2003) Endocr Rev. 24, 278-301 15. Takahashi, K., Mizuarai, S., Araki, H., Mashiko, S., Ishihara, A., Kanatani, A., Itadani, H., and

Kotani, H. (2003) J Biol Chem 278, 46654-46660 16. Christiansen, T., Richelsen, B., and Bruun, J. M. (2005) Int J Obes Relat Metab Disord 29,

146-150 17. Shimomura, I., Funahashi, T., Takahashi, M., Maeda, K., Kotani, K., Nakamura, T., Yamashita, S.,

Miura, M., Fukuda, Y., Takemura, K., Tokunaga, K., and Matsuzawa, Y. (1996) Nat Med. 2, 800-803

18. Ashida, N., Arai, H., Yamasaki, M., and Kita, T. (2001) J Biol Chem. 276, 16555-16560 19. Boring, L., Gosling, J., Cleary, M., and Charo, I. F. (1998) Nature. 394, 894-897 20. Gu, L., Okada, Y., Clinton, S. K., Gerard, C., Sukhova, G. K., Libby, P., and Rollins, B. J. (1998)

Mol Cell 2, 275-281 21. Linton, M. F., and Fazio, S. (2003) Int J Obes Relat Metab Disord. 27, S35-40 22. Sartipy, P., and Loskutoff, D. J. (2003) Proc Natl Acad Sci U S A 100, 7265-7270 23. Weisberg, S. P., Hunter, D., Huber, R., Lemieux, J., Slaymaker, S., Vaddi, K., Charo, I., Leibel, R.

L., and Ferrante, A. W. (2006) J Clin Invest. 116, 115-124 24. Graves, R. A., Tontonoz, P., Platt, K. A., Ross, S. R., and Spiegelman, B. M. (1992) J Cell Biochem.

49, 219-224 25. Suzuki, R., Tobe, K., Terauchi, Y., Komeda, K., Kubota, N., Eto, K., Yamauchi, T., Azuma, K.,

Kaneto, H., Taguchi, T., Koga, T., German, M. S., Watada, H., Kawamori, R., Wright, C. V., Kajimoto, Y., Kimura, S., Nagai, R., and Kadowaki, T. (2003) J Biol Chem. 278, 43691-43698

26. Takahashi, M., Kamei, Y., and Ezaki, O. (2005) Am J Physiol Endocrinol Metab. 288, E117-124 27. Yamauchi, T., Tobe, K., Tamemoto, H., Ueki, K., Kaburagi, Y., Yamamoto-Honda, R., Takahashi,

Y., Yoshizawa, F., Aizawa, S., Akanuma, Y., Sonenberg, N., Yazaki, Y., and Kadowaki, T. (1996) Mol Cell Biol. 16, 3074-3084

28. Murakami, K., Tsunoda, M., Ide, T., Ohashi, M., and Mochizuki, T. (1999) Metabolism. 48, 1450-1454

29. Suzuki, R., Tobe, K., Aoyama, M., Inoue, A., Sakamoto, K., Yamauchi, T., Kamon, J., Kubota, N., Terauchi, Y., Yoshimatsu, H., Matsuhisa, M., Nagasaka, S., Ogata, H., Tokuyama, K., Nagai, R., and Kadowaki, T. (2004) J Biol Chem. 279, 25039-25049

30. Barlow, C., Schroeder, M., Lekstrom-Himes, J., Kylefjord, H., Deng, C. X., Wynshaw-Boris, A., Spiegelman, B. M., and Xanthopoulos, K. G. (1997) Nucleic Acids Res. 25, 2543-2545

31. Fain, J. N., Madan, A. K., Hiler, M. L., Cheema, P., and Bahouth, S. W. (2004) Endocrinology. 145, 2273-2282

32. Lin, Y., Berg, A. H., Iyengar, P., Lam, T. K., Giacca, A., Combs, T. P., Rajala, M. W., Du, X., Rollman, B., Li, W., Hawkins, M., Barzilai, N., Rhodes, C. J., Fantus, I. G., Brownlee, M., and Scherer, P. E. (2005) J Biol Chem. 280, 4617-4626

33. Suganami, T., Nishida, J., and Ogawa, Y. (2005) Arterioscler Thromb Vasc Biol. 25, 2062-2068 34. Boden, G. (1997) Diabetes. 46, 3-10 35. McGarry, J. D. (2002) Diabetes. 51, 7-18 36. Kamei, N., Yamane, K., Nakanishi, S., Ishida, K., Ohtaki, M., Okubo, M., and Kohno, N. (2005)

Metabolism. 54, 713-718


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


12

37. Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., and et al. (1994) Nature. 372, 182-186

38. Tobe, K., Tamemoto, H., Yamauchi, T., Aizawa, S., Yazaki, Y., and Kadowaki, T. (1995) J Biol Chem. 270, 5698-5701

39. Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J., Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G. I., Bonner-Weir, S., and White, M. F. (1998) Nature. 391, 900-904

40. Kubota, N., Tobe, K., Terauchi, Y., Eto, K., Yamauchi, T., Suzuki, R., Tsubamoto, Y., Komeda, K., Nakano, R., Miki, H., Satoh, S., Sekihara, H., Sciacchitano, S., Lesniak, M., Aizawa, S., Nagai, R., Kimura, S., Akanuma, Y., Taylor, S. I., and Kadowaki, T. (2000) Diabetes. 49, 1880-1889

41. Tobe, K., Suzuki, R., Aoyama, M., Yamauchi, T., Kamon, J., Kubota, N., Terauchi, Y., Matsui, J., Akanuma, Y., Kimura, S., Tanaka, J., Abe, M., Ohsumi, J., Nagai, R., and Kadowaki, T. (2001) J Biol Chem. 276, 38337-38340

42. Kanda, H., Tateya, S., Tamori, Y., Kotani, K., Hiasa, K., Kitazawa, R., Kitazawa, S., Miyachi, H., Maeda, S., Egashira, K., and Kasuga, M. (2006) J Clin Invest. 116, 1494-1505

FOOTNOTES

We thank Dr. Shuhei Nakanishi, Dr. Kensuke Egashira, Dr. Kyoji Hioki, Dr. Kumiko Saeki-Tobe and Dr. Makoto Koyanagi for helpful discussions. We also thank Ms. C. Kohkaki, Ms. K. Maruyama, Mr. S. Furukoshi, Mr. Y. Yahagi and Mr. H. Chiyonobu for their excellent technical assistance and animal care.

This work was supported by a grant-in-aid for the development of innovative technology and a grant-in-aid for creative basic research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. K. and K. T.) and Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (to T. K.).

1 The abbreviations used are: TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1; WAT, white adipose tissue; CCR2, C-C motif chemokine receptor 2; TG, transgenic; aP2, adipocyte P2; WT, wild-type; ITT, insulin tolerance test; NEFA, non-esterified fatty acids; IRS, insulin receptor substrate; IR, insulin receptor; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; MEK, mitogen activated extracellular signal protein kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; BAT, brown adipose tissue; MMP12, matrix metallopeptidase 12; G6Pase, glucose-6-phosphatase; PPARγ, peroxisome proliferator-activated receptor γ; SVCs, stromal-vascular cells; IP, immunoprecipitation; GTT, glucose tolerance test; 2-DG, 2-deoxyglucose; Rd, rate of glucose disappearance; EGP, endogenous glucose production; GIR, glucose infusion rate; ANOVA, analysis of variance.

FIGURE LEGENDS Fig. 1 Increased MCP-1 expression in adipose tissues from ob/ob mice and mice on a high fat (HF) diet. (A-B), Expressions of MCP-1 (Ccl2), F4/80 (Emr1), CD68 (Cd68) and MMP12 (Mmp12) genes in epididymal WAT (eWAT) (A) and plasma levels of MCP-1 (B) from control C57BL/6 (B6) mice (open bars, n=5) and ob/ob mice (closed bars, n=4) at 13 weeks of age. For comparison, the expressions of these genes in lean mice were arbitrarily set at 1 (A). Values are means ± S.E. **, p < 0.01 by Student’s t-test. (C-D), MCP-1 and CD68 mRNA expressions in eWAT (C) and plasma concentrations of MCP-1 (D) in mice on a HF diet for 5 weeks, 8 weeks or 18 weeks (open bars, lean


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mice; closed bars, obese mice; n=5 per group). For comparison, the expressions of these genes in lean mice on a normal chow (NC) diet were arbitrarily set at 1 (C). *, p < 0.05, **, p < 0.01 compared with lean mice of the same age, by ANOVA. (E), MCP-1 mRNA expressions in various tissues in B6 mice on NC or a HF diet at 23 weeks of age (n=5 per group). For comparison, the MCP-1 gene expression level in eWAT of NC mice was arbitrarily set at 1. *, p < 0.05 compared with the same tissue of normal chow mice, by ANOVA. Fig. 2 Generation of aP2-MCP-1 TG mice. (A), Schematic representation of the aP2 promoter/MCP-1 cDNA fusion gene. Murine MCP-1 cDNA cloned from macrophage cDNA was inserted into an aP2 promoter cassette as described in experimental procedures. Hatched blocks, rabbit β globin. Stippled blocks, polyadenylation signal. (B), Southern blot analysis of Bam HI-digested mouse genomic DNA from 3 lines of aP2-MCP-1 TG and nontransgenic littermate mice hybridized with MCP-1 cDNA as a probe. Genomic DNA was prepared from each F2 mouse obtained by crossing WT and TG mice. The band at 7.5 kb corresponds to the endogenous gene and that at 3.2 kb to the transgene. The copy numbers of each line were measured by BAS2000 and estimated to be 3, 7 and 16 copies, respectively. (C), Northern blot analysis of MCP-1 mRNA in 3 lines of aP2-MCP-1 TG and WT mice. mRNA was prepared from three independent pairs of F2 mice from various tissues including epididymal WAT (eWAT), subcutaneous WAT (sWAT), BAT and the spleen. (D), Expressions of MCP-1 mRNA in various tissues in WT (open bars) and TG (closed bars) mice of Line M on normal chow (NC) or a high fat (HF) diet (n=6 per group). For comparison, the MCP-1 expression level in eWAT from WT mice on the NC diet was arbitrarily set at 1. Values are means ± S.E. *, p < 0.05 compared with littermates for each tissue and diet, by ANOVA. (E), Expression of MCP-1 mRNA in adipocytes and SVCs of eWAT in WT (open bars) and TG (closed bars) mice (n=4 per group) of Line M. For comparison, the MCP-1 expression level in the adipocyte fraction from WT mice on the NC diet was arbitrarily set at 1. Values are means ± S.E. *, p < 0.05 compared with littermates for each fraction and diet, by ANOVA. (F), Plasma levels of MCP-1 in WT (open bars) and TG (closed bars) mice of each of the 3 lines on the NC and HF diets (n=9-10 per group). Values are means ± S.E. **, p < 0.01 compared with littermates for each genetic line and diet, by ANOVA. Fig. 3 aP2-MCP-1 TG mice show normal adiposity and increased macrophages in WAT. (A), Body weights of three lines of aP2-MCP-1 transgenic mice. All lines of aP2-MCP-1 TG (F2) and control mice were fed normal chow (NC) (open circle, WT; closed circle, TG) or a high fat (HF) diet (open square, WT; closed square, TG) (n=10-12 per group). Values are means ± S.E., compared by ANOVA. (B), Weights of epididymal WAT (eWAT) from WT (open bars) and TG (closed bars) mice of Line M on NC or an 8 week HF diet (n=6 per group). Values are means ± S.E., compared by ANOVA. (C), The morphologies of eWAT of WT or TG mice of Line M on NC or a HF diet are shown (X200). Immunoreactivity for F4/80, a macrophage marker, is visualized as brown while routine hematoxylin staining appears blue. (D), Histogram of adipocyte size (n=6 per group) of Line M. (E), The ratios of F4/80 antibody positive nuclei in adipose tissues of WT (open bars) and TG (closed bars) mice of Line M on NC and a HF diet (n=6 per group). Values are means ± S.E. *, p < 0.05 compared with littermates on the same diet, by ANOVA. (F), Flow cytometric analyses of the number of CD11b and F4/80 double positive cells from the stromal vascular fractions in eWAT of WT (open bars) and TG (closed bars) mice of Line M on NC and a HF diet (n=4 per group). Values are means ± S.E. **, p < 0.01 compared with littermates on the same diet, by ANOVA. (G), The mRNA expressions of macrophage markers including F4/80, CD68, and MMP12 in TG (closed bars) mice of Line M as compared to WT (open bars) mice (n=14-15 per group). Values are means ± S.E. *, p < 0.05, **, p < 0.01, compared with littermates on the same diet, by ANOVA.


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Fig. 4 TG animals have high TNF-α and IL-6 expressions in WAT and high plasma NEFA concentrations. (A), Expressions of TNF-α, IL-6, resistin, adiponectin, leptin and PPARγ mRNA in eWAT of WT (open bars) and TG (closed bars) mice (n=14-15 per group) of Line M. Values are means ± S.E. *, p < 0.05, **, p < 0.01, compared with littermates on the same diet, by ANOVA. (B), Plasma concentrations of NEFA, adiponectin and leptin in WT and TG mice of Line M on normal chow and high fat diets (n=9-10 per group). Values are means ± S.E. **, p < 0.01 compared with littermates on the same diet, by ANOVA. Fig. 5 aP2-MCP-1 TG mice showed insulin resistance and glucose intolerance as compared with WT mice. (A-C), plasma glucose levels (A) during ITT (0.75 mU/g of body weight); plasma glucose levels (B) and plasma insulin levels (C) during GTT (1.0 mg/g of body weight). Values are means ± S.E. (n=8-11 per group). *, p < 0.05, **, p < 0.01, by ANOVA. In figure (A-C), data from Line L, M and H mice are presented in the left, middle and the right panels respectively. In each figures (A)-(C), the upper three panels are data derived from mice fed on a normal chow (NC) diet, and the lower three panels are data from mice on a high fat (HF) diet. Fig. 6 Hyperinsulinemic euglycemic clamp studies demonstrate insulin resistance in skeletal muscle and liver of aP2-MCP-1 TG mice. (A-B), Hyperinsulinemic euglycemic clamp study showed glucose infusion rates (GIR), endogenous glucose production (EGP), and rates of glucose disappearance (Rd) in aP2-MCP-1 TG (closed bars) and WT (open bars) mice of Line M on normal chow (NC) (A) or a high fat (HF) diet (B) (n=6 per group). Values are means ± S.E. *, p < 0.05, by Student’s t-test. (C), Expressions of G6Pase in the livers of WT (open bars) and TG (closed bars) mice (n=6 per group) of Line M on the HF diet after the clamp studies. Values are means ± S.E. *, p < 0.05, **, by Student’s t-test. Fig. 7 Insulin signaling in skeletal muscle of aP2-MCP-1 TG mice of Line M was attenuated as compared to that in WT mice. (A), Equal amounts of muscle protein in total lysates were immunoblotted with anti-phospho-Akt and anti-Akt antibodies (n=4 per group). Phosphorylated-Akt and the Akt signal were quantified with a scanning imager. The relative ratio of Akt phosphorylation was calculated after normalization with the Akt signal, and then normalized in WT mice without insulin. (B), Lysates were immunoprecipitated with anti-insulin receptor β subunit antibodies (αIRβ) and the immunoprecipitates were subjected to Western blotting with αPY and αIRβ. (C-D), Lysates were immunoprecipitated with αIRS-1 and immunoblotted with αPY (C), αp85 (D), and αIRS-1 (C-D). (E-F), Lysates were immunoprecipitated with αIRS-2 and immunoblotted with αPY (E), αp85 (E) and αIRS-2 (E-F). Values are means ± S.E. *, p < 0.05, **, p < 0.01, compared with WT mice under the same conditions, by ANOVA. Fig. 8 Insulin signals in the livers of aP2-MCP-1 TG mice of Line M were also attenuated as compared to those of WT mice. (A), Equal amounts of liver protein in total lysates were immunoblotted with anti-phospho-Akt and anti-Akt antibodies (n=4 per group). Phosphorylated-Akt and the Akt signal were quantified with a scanning imager. The relative ratio of Akt phosphorylation was calculated after normalization with the Akt signal, and then normalized in WT mice without insulin. (B), Lysates were immunoprecipitated with anti-insulin receptor β subunit antibodies (αIRβ) and the immunoprecipitates were subjected to Western blotting with αPY and αIRβ. (C-D), Lysates were immunoprecipitated with αIRS-1 and immunoblotted with αPY (C), αp85 (D) and αIRS-1 (C-D). (E-F), Lysates were immunoprecipitated with αIRS-2 and immunoblotted with αPY (E), αp85 (E) and αIRS-2 (E-F). Values are means ± S.E. *, p < 0.05, **, p < 0.01, compared with WT mice under the same conditions, by ANOVA.


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Fig. 9 MCP-1 attenuated the insulin signaling in C2C12 myotubes and the insulin-stimulated glucose uptake in isolated skeletal muscle. (A), Expressions of CCR2 mRNA in various tissues in B6 mice on normal chow (NC) or a high fat (HF) diet at 23 weeks of age (n=5 per group). For comparison, the expression level of the CCR2 gene in eWAT of NC mice was arbitrarily set at 1. (B), CCR2 mRNA expressions in C2C12 myoblast cells before and after 5 to 12 days of differentiation (n=4 per group). CCR2 expression was seen in C2C12 myotubes after differentiation. For comparison, the CCR2 expression level in C2C12 cells which had differentiated for 5 days was arbitrarily set at 1. (C), Serum-starved C2C12 cells were incubated with or without 100 nM U0126, an inhibitor of MEK, for 30 minutes before stimulation by 10nM MCP-1. Whole cell lysates were analyzed by Western blotting with anti-phospho ERK and ERK antibodies. The relative ratio of ERK phosphorylation was calculated after normalization with the ERK signal, and then normalized in control cells without MCP-1. Values are means ± S.E. **, p < 0.01, by ANOVA. (D), Serum-starved C2C12 cells were incubated with or without 1 to 10 nM MCP-1 or 100 nM U0126 for 30 minutes before stimulation with 10 nM insulin. Whole cell lysates were analyzed by Western blotting with anti-phospho Akt and Akt antibodies. The relative ratio of Akt phosphorylation was calculated after normalization with the Akt signal, and then normalized in control cells without insulin. Values are means ± S.E. *, p < 0.05, **, p < 0.01, by ANOVA. (E), Isolated soleus muscles were incubated with or without 0.1 to 10 nM MCP-1 or 20 µM U0126 for 30 minutes before stimulation by 10 nM insulin. 2-deoxy-[3H]glucose (2-DG) uptake was measured as described in experimental procedures. Values are means ± S.E. *, p < 0.05, by ANOVA. Fig. 10 Overexpression of MCP-1 in adipose tissues causes insulin resistance in both a paracrine and an endocrine manner. (A-B), We propose that both increased accumulation of macrophages in adipose tissue (A) and higher plasma levels of MCP-1 (B) contributed to the development of insulin resistance in aP2-MCP-1 TG mice. (A), MCP-1 produced by adipocytes induced recruitment of macrophages to adipose tissue, thereby causing inflammation. In fact, increased gene expression of inflammatory secretory molecules such as TNF-α and IL-6 was observed in adipose tissue of aP2-MCP-1 mice. In addition, plasma NEFA levels were increased in TG mice. Increased NEFA levels as well as TNF-α and IL-6 gene expressions may contribute to the development of insulin resistance. (B), Circulating plasma MCP-1 may also contribute to the development of systemic insulin resistance in aP2-MCP-1 mice. We demonstrated that MCP-1 blunted insulin signaling and insulin-stimulated glucose uptake in myotube cells and isolated skeletal muscle, respectively. Alternation of these two pathways may cause insulin resistance.


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0

1

2

3

4

5

6

7CD68

0

50

100

150

200

250

300

NC HF5 NC HF8 NC HF18

Pla

sma

leve

ls (p

g/m

l)

MCP-1

0

1

2

3

4

5

6

10weeks 13weeks 23weeks

**

** **

**

*

0

10

20

30

40

50

60MCP-1

** ** **ar

bitra

ry u

nit

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

NC HF5 NC HF8 NC HF18

10weeks 13weeks 23weeks NC HF5 NC HF8 NC HF18

10weeks 13weeks 23weeks

eWAT BAT Liver Spleen Heart Muscle Aorta

NC HF

*

*

arbi

trary

uni

t

NC HF NC HF NC HF NC HF NC HF NC HF


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TG TG

Line H

*

Fig. 2A

B

C

Southern blot

Northern blot

aP2 Promoter MCP-1 cDNA

5.4 kb700 bp 600 bp

447 bp

7.5 kb (endogenous)

3.2 kb (transgene)

WT TG WT TG WT TGLine L Line M Line H

MCP-1

Line L

18S

MCP-1

WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG

Liver Spleen Kidney eWAT sWAT BAT Heart Muscle

Line M

Line H

18S

18S

MCP-1

D E

F

0

400

800

1200

1600

WT

TG WT

TG WT

TG WT

TG WT

TG WT

TG WT

TG WT

TG WT

TG WT

TG

Liver Spleen eWAT BAT MuscleNC HF NC HF NC HF NC HF NC HF

* *

*

** *

* *0

100

200

300

400

500

600

WT TG WT TG WT TG WT TG

Adipocyte SVCsNC HF NC HF

*

*

* *

MCP-1 MCP-1

WTTG WTTG WTTG WTTG WTTG WT WT WTTG


WT TG WT TG WT TG WT TG WT TG WT TG WT TG WT TG


0

100

200

300

Pla

sma

leve

ls (p

g/m

l)

****

Line L

WT TG WT TGNC HF

0

100

200

300

WT TG WT TGNC HF

Pla

sma

leve

ls (p

g/m

l)

**

**

Line M

arbi

trary

uni

t

arbi

trary

uni

t

0

100

200

300

Pla

sma

leve

ls (p

g/m

l) ****

WT TG WT TGNC HF

Line M Line M


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Fig. 3

DC

E

0

10

20

0 6000 120000

10

20WT-NC TG-NC

WT-HF TG-HF

0 6000 120000 6000 12000

0 6000 12000

WT-NC TG-NC

0

10

20

0

10

20

Freq

uenc

y (%

)

(μm2) (μm2)

(μm2) (μm2)

WT-HF TG-HF

0

20

40

60

80

Pos

itive

nuc

lei (

%)

*

WT TG WT TGNC HF

0

1.0

2.0

3.0

4.0

Freq

uenc

y (%

)

*

WT TG WT TGNC HF

MMP12

0

1.0

2.0

3.0* **

0

1

2

3

4

5

0

4

8

12

16

F4/80 CD68

arbi

trary

uni

t

WT TG WT TGNC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

arbi

trary

uni

t

arbi

trary

uni

t

G

F

8 10

A B

0

10

20

30

40

WT-NC TG-NC WT-HF TG-HF

5 6 7 8 9 10 11 12(weeks)

10

Line L Line H

0

0.5

1.0

1.5

2.0

2.5

eWA

Tw

eigh

t (g)

WT TG WTNC HF

TG0

10

20

30

40

5 6 7 9 11 12(weeks)

0

10

20

30

40

5 6 7 8 9(weeks)

Line M

Body

wei

ght (

g)

Body

wei

ght (

g)

ns

nsBody

wei

ght (

g)

**

**

cell

num

ber/e

WAT

(x10

6 )


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Fig. 4

A

B

TNF-α

0

1.0

2.0

3.0

WT TG WT TG

IL-6

0

1.0

2.0

Resistin

0

0.4

0.8

1.2

Adiponectin

0

0.4

0.8

1.2

1.6Leptin

0

1

2

3

4PPARγ

0

0.4

0.8

1.2

1.6

NEFA

0

0.5

1.0

1.5

2.0

2.5Adiponectin

0

10

20

30

40

50Leptin

0102030405060

arbi

trary

uni

tar

bitra

ry u

nit

NC HFWT TG WT TG

NC HFWT TG WT TG

NC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

WT TG WT TGNC HF

arbi

trary

uni

t

arbi

trary

uni

t

plas

ma

leve

ls (m

Eq/

l)

*

*

** **

****

plas

ma

leve

ls (μ

g/m

l)

plas

ma

leve

ls (n

g/m

l)

ns ns

ns

ns

arbi

trary

uni

t

arbi

trary

uni

t


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Fig. 5A

B

C

Line L Line M Line H

NC

HF

020406080

100120

020406080

100120

020406080

100120

020406080

100120

020406080

100120

020406080

100120

0

100

200

300

400

500

0

100

200

300

400

500

0

1.0

2.0

3.0

4.0

0 15 30 (min)0

1.0

2.0

3.0

4.0

0 15 30 (min) 0

1.0

2.0

3.0

4.0

0 15 30 (min)

0

1.0

2.0

3.0

4.0

0 15 30 (min)0

1.0

2.0

3.0

4.0

0 15 30 (min) 0

1.0

2.0

3.0

4.0

0 15 30 (min)



Bloo

d G

luco

se (%

)

Bloo

d G

luco

se (%

)

Bloo

d G

luco

se (%

)

Bloo

d G

luco

se (%

)

Bloo

d G

luco

se (%

)

Bloo

d G

luco

se (%

)

NC

HF

Bloo

d G

luco

se(m

g/dl

)

NC

HF

Pla

sma

Insu

lin

(ng/

ml)

Bloo

d G

luco

se

(mg/

dl)

Bloo

d G

luco

se

(mg/

dl)

Bloo

d G

luco

se(m

g/dl

)

Bloo

d G

luco

se(m

g/dl

)Bl

ood

Glu

cose

(mg/

dl)

*** **

**** ** ***

** * *

** **

** *** **

**** *

*

****

*

0 20 40 60 80 100 120(min)

0 20 40 60 80 100 120(min)

0 20 40 60 80 100 120(min)

0 20 40 60 80 100 120(min)

0 20 40 60 80 100 120(min)

0 20 40 60 80 100 120(min)

Pla

sma

Insu

lin

(ng/

ml)

Pla

sma

Insu

lin

(ng/

ml)

Pla

sma

Insu

lin

(ng/

ml)

Pla

sma

Insu

lin(n

g/m

l)

Pla

sma

Insu

lin

(ng/

ml)

0 30 6015 120

0 30 6015 120

(min)

(min)

0

100

200

300

400

500

0 30 6015 120(min)

0

100

200

300

400

500

0 30 6015 120(min)

* *

0

100

200

300

400

500

0 30 6015 120(min)

0

100

200

300

400

500

0 30 6015 120(min)

** **

WT-NC TG-NC WT-HF TG-HF


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Fig. 6

*

0

20

40

60

80

0

5

10

15

20 100

*

0

20

40

60

80

BGIR RdEGP

mg/

kg/m

in

mg/

kg/m

in

mg/

kg/m

in

A

WT TG

*

0

20

40

60

80

WT TG0

5

10

15

20 100

WT TG0

20

40

60

80

GIR RdEGP

mg/

kg/m

in

mg/

kg/m

in

mg/

kg/m

in

*

WT TG WT TG WT TG

P=0.0734

NCNCNC

HFHFHF

WT TG

HF

0

1.0

2.0

*

arbi

trary

uni

t

G6PaseC


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Fig. 7

Normal Chow

pAkt

Akt

A BpY

IR

C

E

DpY

IRS-1p85

IRS-1

FpY

IRS-2

p85

IRS-2

0

5

10

15

- + - +WT TG

Insulin

arbi

trary

uni

t

0

5

10

15

20

25

0

4

8

12

16

20

05

1015202530

02

4

6

8

10

0

1

2

3

4

0

1

2

3

4

5

0

1

2

3

0

1

2

3

0

0.5

1.0

1.5

2.0

0

0.5

1.0

1.5

2.0

Insulin

0

2

4

6

8 * ** **

High Fat Diet

- + - +WT TG

Normal Chow

- + - +WT TG

Insulin

High Fat Diet

- + - +WT TG

IP: αIRβblot: αPY

total lysateblot: αpAkt

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + +

arbi

trary

uni

t

Normal Chow

- + - +WT TG

Insulin

High Fat Diet

- + - +WT TG

Normal Chow

- + - +WT TG

Insulin

High Fat DietWT TG

IP: αIRS-1blot: αp85

IP: αIRS-1blot: αPY

arbi

trary

uni

t

arbi

trary

uni

t



Normal Chow

- + - +WT TG

High Fat Diet

- + - +WT TG

Normal Chow

- + - +WT TG

Insulin

High Fat DietWT TG

- + - +

- + - +

arbi

trary

uni

t

arbi

trary

uni

t

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + + - - + + - - + + - - + + - - + +

***

Ins

**

****

Ins

Ins

Ins

Ins Ins


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IRS-2

Ins

IRS-2

- - + + - - + +

pAktAkt

pY

IR

pY

IRS-1

p85

IRS-1

pY p85

0

10

20

30

40

0

10

20

30

40

0

4

8

12

16

0

2

4

6

8

0

1

2

3

4

0

1

2

3

4

0

0.5

1.0

1.5

2.0

0

0.5

1.0

1.5

2.0

1.0

2.0

3.0

0

0.5

1.0

1.5

2.0

**** ** **

*

**

0 0

1.0

2.0

3.0* **

0

0.5

1.0

1.5

2.0*

Fig. 8A B

C

E

D

F

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + +

- - + + - - + + - - + + - - + + - - + + - - + +

Normal Chow

- + - +WT TG

Insulin

Insulin

High Fat Diet

- + - +WT TG

Normal Chow

- + - +WT TG

Insulin

Normal Chow

- + - +WT TG

Insulin

High Fat Diet

- + - +WT TG

Normal Chow

- + - +WT TG

Insulin

High Fat DietWT TG

Normal Chow

- + - +WT TG

High Fat Diet

- + - +WT TG

Normal Chow

+ - +WT TG

Insulin

High Fat DietWT TG

- + - +

- + - +

High Fat Diet

- + - +WT TG

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

arbi

trary

uni

t

Ins

Ins

Ins

Ins

Ins

IP: αIRβblot: αPY






-


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Fig. 9A

D

B

E

0

1.0

2.0

3.0

WAT BAT Liver Spleen Heart Muscle AoNC HF NC HF NC HF NC HF NC HF NC HF NC HF

arbi

trary

uni

t

CCR2

0 5 8 10 12 Day0

1.0

2.0

3.0

arbi

trary

uni

t

not d

etec

ted

CCR2

pERK

ERK

MCP-1U0126

C

--

--

+-

+-

++

++

MCP-1U0126

MCP-1U0126

insulin

pAkt

Akt

---

---

+--

+--

+-+

+-+

+1-

+1-

+10-

+10-

+10+

+10+


---

+--

+-+

+1-

+10-

+10+

** **

2

4

0--

+-

++

arbi

trary

uni

t

0

3

4

5*

**

*

2

1

2-DG uptake

0

20

40

60

InsulinMCP-1U0126

---

+--

+0.1-

+1-

+10-

+10+

nmol

/g/m

in

**

*

1

3

5

InsulinMCP-1U0126

total lysateblot: αpERK

arbi

trary

uni

t


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Fig. 10

Systemic effects (endocrine)

Plasma MCP-1

Local MCP-1

Macrophage recruitment

Adipocyte ; Insulin signaling

TNF-αIL-6NEFA

A

B

AdipocyteMuscle ; Rd

Liver ; EGP

Local effects (paracrine)

GIR


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KadowakiTokuyama, Masaki Tsunoda, Tomohiro Ide, Koji Murakami, Ryozo Nagai and Takashi

Ichiro Manabe, Haruo Hashimoto, Yasuyuki Ohnishi, Hitomi Ogata, Kumpei Kobayashi, Toshimasa Yamauchi, Kohjiro Ueki, Yumiko Oishi, Satoshi Nishimura,

Kubota, Norie Ohtsuka-Kowatari, Katsuyoshi Kumagai, Kentaro Sakamoto, Masatoshi Nozomu Kamei, Kazuyuki Tobe, Ryo Suzuki, Mitsuru Ohsugi, Taku Watanabe, Naoto

insulin resistanceOverexpression of MCP-1 in adipose tissues causes macrophage recruitment and

published online June 29, 2006J. Biol. Chem.

10.1074/jbc.M601284200Access the most updated version of this article at doi:

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Overexpression of MCP-1 in adipose tissues causes ... · MCP-1 Overexpression in Adipos e Causes Insulin Resistance 1 Overexpression of MCP-1 in adipose tissues causes macrophage