Adiposity Elevates Plasma MCP-1 Levels Leading to the Increased CD11b-positive Monocytes in Mice

Obesity is currently considered as an epidemic in the western world, and it represents a major risk factor for life-threatening diseases such as heart attack, stroke, diabetes, and cancer. Taking advantage of DNA microarray technology, we tried to identify the molecules explaining the relationship between obesity and vascular disorders, comparing mRNA expression of about 12,000 genes in white adipose tissue between normal, high fat diet-induced obesity (DIO) and d-Trp34 neuropeptide Y-induced obesity in mice. Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA displayed a 7.2-fold increase in obese mice as compared with normal mice, leading to substantially elevated MCP-1 protein levels in adipocytes. MCP-1 levels in plasma were also increased in DIO mice, and a strong correlation between plasma MCP-1 levels and body weight was identified. We also showed that elevated MCP-1 protein levels in plasma increased the CD11b-positive monocyte/macrophage population in DIO mice. Furthermore, infusion of MCP-1 into lean mice increased the CD11b-positive monocyte population without inducing changes in body weight. Given the importance of MCP-1 in activation of monocytes and subsequent atherosclerotic development, these results suggest a novel role of adiposity in the development of vascular disorders. Obesity is currently considered as an epidemic in the western world, and it represents a major risk factor for life-threatening diseases such as heart attack, stroke, diabetes, and cancer. Taking advantage of DNA microarray technology, we tried to identify the molecules explaining the relationship between obesity and vascular disorders, comparing mRNA expression of about 12,000 genes in white adipose tissue between normal, high fat diet-induced obesity (DIO) and d-Trp34 neuropeptide Y-induced obesity in mice. Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA displayed a 7.2-fold increase in obese mice as compared with normal mice, leading to substantially elevated MCP-1 protein levels in adipocytes. MCP-1 levels in plasma were also increased in DIO mice, and a strong correlation between plasma MCP-1 levels and body weight was identified. We also showed that elevated MCP-1 protein levels in plasma increased the CD11b-positive monocyte/macrophage population in DIO mice. Furthermore, infusion of MCP-1 into lean mice increased the CD11b-positive monocyte population without inducing changes in body weight. Given the importance of MCP-1 in activation of monocytes and subsequent atherosclerotic development, these results suggest a novel role of adiposity in the development of vascular disorders. Obesity is now considered epidemic throughout the western world and represents a major risk factor for a variety of life-threatening diseases, such as heart attack, stroke, diabetes, and cancer (1Zimmet P. Alberti K.G. Shaw J. Nature. 2001; 414: 782-787Crossref PubMed Scopus (4594) Google Scholar, 2Ginsberg H.N. J. Clin. Invest. 2000; 106: 453-458Crossref PubMed Scopus (941) Google Scholar, 3Kopelman P.G. Nature. 2000; 404: 635-643Crossref PubMed Scopus (3698) Google Scholar, 4Calle E.E. Rodriguez C. Walker-Thurmond K. Thun M.J. N. Engl. J. Med. 2003; 348: 1625-1638Crossref PubMed Scopus (6033) Google Scholar). According to current estimates, about 30% of adults in the United States are classified as obese, roughly double the number from 20 years ago. Obesity increases the risk of developing type 2 diabetes 10-fold, cardiovascular diseases 2-fold, and colon cancer 1.6-fold. Although recent progress in understanding the molecular basis of obesity may well open new opportunities to combat this epidemic, the molecular mechanisms underlying the relationship between obesity and obesity-related comorbidities remain unclear and are currently the focus of intense investigation. Hypercholesterolemia has been considered to represent the critical factor in the development of atherosclerosis. However, a growing body of evidence suggests the importance of inflammatory processes in the pathogenesis of vascular diseases. Inflammatory processes are orchestrated by the recruitment of mononuclear leukocytes and the migration, growth, and activation of cells within atherosclerotic lesions (5Gerrity R.G. Am. J. Pathol. 1981; 103: 181-190PubMed Google Scholar, 6Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar). Attraction of circulating leukocytes to target sites is controlled by various chemokines, the presence of which is well documented in atherosclerotic lesions (7Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 8Baggiolini M. Nature. 1998; 392: 565-568Crossref PubMed Scopus (2407) Google Scholar). After attachment to the vessel wall, monocytes migrate into the subendothelial space, differentiating into macrophages and lipid-laden foam cells (5Gerrity R.G. Am. J. Pathol. 1981; 103: 181-190PubMed Google Scholar). These steps are likewise controlled by chemotactic cytokines; in particular, expression of monocyte chemoattractant protein-1 (MCP-1) 1The abbreviations used are: MCP-1monocyte chemoattractant protein-1DIOdiet-induced obesityWATwhite adipose tissueEWATepididymal WATPBSphosphate-buffered salineNPYneuropeptide YELISAenzyme-linked immunosorbent assayCCRC-C chemokine receptor. is enhanced in macrophages, endothelial cells, and vascular smooth muscles cells in the atheromatous plaque (9Yla-Herttuala S. Lipton B.A. Rosenfeld M.E. Sarkioja T. Yoshimura T. Leonard E.J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5252-5256Crossref PubMed Scopus (808) Google Scholar). MCP-1 is the member of the C-C chemokine β subfamily and is predominantly expressed in endothelial cells (10Strieter R.M. Wiggins R. Phan S.H. Wharram B.L. Showell H.J. Remick D.G. Chensue S.W. Kunkel S.L. Biochem. Biophys. Res. Commun. 1989; 162: 694-700Crossref PubMed Scopus (197) Google Scholar, 11Rollins B.J. Yoshimura T. Leonard E.J. Pober J.S. Am. J. Pathol. 1990; 136: 1229-1233PubMed Google Scholar). In hypercholesterolemic mice, genetic disruption of MCP-1 or its receptor, CCR2, results in markedly decreased occurrence of atheroma and the presence of fewer monocytes in vascular lesions (12Boring L. Gosling J. Cleary M. Charo I.F. Nature. 1998; 394: 894-897Crossref PubMed Scopus (1686) Google Scholar, 13Gu L. Okada Y. Clinton S.K. Gerard C. Sukhova G.K. Libby P. Rollins B.J. Mol. Cell. 1998; 2: 275-281Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar, 14Gosling J. Slaymaker S. Gu L. Tseng S. Zlot C.H. Young S.G. Rollins B.J. Charo I.F. J. Clin. Invest. 1999; 103: 773-778Crossref PubMed Scopus (598) Google Scholar). In apolipoprotein E-deficient mice, local infusion of MCP-1 induces CD11b expression on peripheral monocytes and increased formation of collateral arteries (15van Royen N. Hoefer I. Bottinger M. Hua J. Grundmann S. Voskuil M. Bode C. Schaper W. Buschmann I. Piek J.J. Circ. Res. 2003; 92: 218-225Crossref PubMed Scopus (115) Google Scholar). MCP-1 is thus considered as a chemokine regulating inflammation in atherosclerotic lesions, and manipulation of the MCP-1/CCR2 interaction may modify the pathogenetic course of such lesions. monocyte chemoattractant protein-1 diet-induced obesity white adipose tissue epididymal WAT phosphate-buffered saline neuropeptide Y enzyme-linked immunosorbent assay C-C chemokine receptor. Adipose tissue is reportedly second only to lymphatic tissue in the secretion of signaling molecules (16Loskutoff D.J. Samad F. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1-6Crossref PubMed Scopus (255) Google Scholar). The biological significance of these molecules is largely unknown, but the inflammation compartment of vascular injury or regulation of insulin resistance may be affected. Proteins that are reportedly secreted and functional in atherosclerosis include IL-6 (17Yudkin J.S. Kumari M. Humphries S.E. Mohamed-Ali V. Atherosclerosis. 2000; 148: 209-214Abstract Full Text Full Text PDF PubMed Scopus (1562) Google Scholar), tumor necrosis factor-α (18Hotamisligil G.S. Arner P. Caro J.F. Atkinson R.L. Spiegelman B.M. J. Clin. Invest. 1995; 95: 2409-2415Crossref PubMed Scopus (2991) Google Scholar), resistin (19Steppan C.M. Bailey S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (4004) Google Scholar), adiponectin (20Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (4099) Google Scholar), PAI-1 (21Juan-Vague I. Alessi M.C. Thromb. Haemostasis. 1999; 82: 832-836Crossref PubMed Scopus (51) Google Scholar), and leptin (22Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (632) Google Scholar, 23Kahn B.B. Flier J.S. J. Clin. Invest. 2000; 106: 473-481Crossref PubMed Scopus (2488) Google Scholar). The present study identified the molecular factors explaining the relationship between obesity and atherosclerosis, with a focus on adipose tissue. The mRNA expression of epididymal white adipose tissue (EWAT) was compared among obese mice, revealing that expression of MCP-1 mRNA is increased in obese mice, leading to elevated levels of plasma MCP-1 protein. Furthermore, the higher levels of MCP-1 protein in plasma were found to increase the CD11b-positive monoycte/macrophage population among peripheral blood cells, suggesting a role for elevated MCP-1 in the vascular inflammatory process during atherosclerosis. Animals—C57BL/6N mice (6 weeks old, CLEA Japan, Tokyo, Japan) were housed in individual cages. Mice were maintained under conditions of controlled temperature (23 ± 2 °C) and light (07:00–19:00). Water and food (CA-1, CLEA Japan) were available ad libitum unless otherwise noted. For microarray analysis, 18-week-old mice were changed to an MHF diet (Oriental Bioservice, Kyoto, Japan) for 6 months to establish diet-induced obesity (DIO). The MHF diet provides 52.4% energy as carbohydrate, 15% as protein, and 32.6% as fat (4.41 kcal/g). For measurements of MCP-1 levels in plasma, normal diet was changed to the MHF diet when the mice were 9 weeks old. In food restriction experiments, body-weight changes in DIO mice were monitored after caloric intake was limited for 21 h and after food intake was restricted to a short, 3-h period for 7 days. All animal procedures complied with National Institutes of Health guidelines and were approved by the Banyu Animal Care and Usage Committee. Surgical Procedure—d-Trp34 neuropeptide Y (d-Trp34NPY, a Y5 agonist) was synthesized at Banyu Tsukuba Research Institute. Mice were anesthetized using sodium pentobarbital (80 mg/kg, intraperitoneal, Dainabot, Tokyo, Japan), and a sterile brain infusion cannula (28 gauge, Alzet, Palo Alto, CA) was stereotaxically implanted into the right lateral ventricle. The stereotaxic coordinates used were 0.4 mm posterior to the bregma, 0.8 mm lateral to the midline, and 2.0 mm from the surface of the skull, using a flat skull position. Cannulae were fixed to the skull using dental cement. The infusion cannula was connected to an osmotic minipump (model no. 2002, Alzet) filled with 10 mm phosphate-buffered saline (PBS) containing 0.05% bovine serum albumin via polyvinylchloride tubing. Pumps were implanted subdermally on the backs of mice, and antibiotic (Cefamedine A, 50 mg/kg, Fujisawa, Tokyo, Japan) was injected subcutaneously. Mice were divided into three groups, matched for average body weight: vehicle (PBS)-infused; d-Trp34NPY-infused and fed ad libitum (ad libitum-fed group); and d-Trp34NPY infused and pair-fed (pair-fed group). After 7–14 days of recovery following surgery, pumps were replaced with d-Trp34NPY-(5 μg/day) or vehicle-containing pumps. The d-Trp34NPY-pair-fed group was provided with the same amount of food as the vehicle group. Pair feeding was performed as described previously (24Mashiko S. Ishihara A. Iwaasa H. Sano H. Oda Z. Ito J. Yumoto M. Okawa M. Suzuki J. Fukuroda T. Jitsuoka M. Morin N.R. Macneil D.J. Van der Ploeg L.H.T. Ihara M. Fukami T. Kanatani A. Endocrinology. 2003; 144: 1793-1801Crossref PubMed Scopus (55) Google Scholar). Microarray Analysis—In respective model mice, total RNA was extracted from EWAT using Trizol reagents (Invitrogen) and repurified with an RNeasy purification kit (Qiagen, Hilden, Germany). To determine expression changes between models, 10 μg of RNA was utilized for microarray analysis (MG-U74U74A chip, Affymetrix, Santa Clara, CA) of ∼12,000 genes. For analysis of microarray data, GeneChip software (Affymetrix) was utilized. Appropriate control mice were used to provide baseline values for the experimental groups, and genes displaying values of p < 0.05 were identified using the Mann-Whitney test. Measurement of MCP-1 Expression by Quantitative PCR—Total RNA was extracted from EWAT as described in the previous section. Reverse transcription was performed for 500 ng of total RNA, and obtained cDNA was applied to for of MCP-1 mRNA were and using an MCP-1 expression were to expression levels in and used were as for for for for for for white adipose were in in and at were at and on Germany). were fixed in for using 0.05% Japan) for 50 and for was performed using an to or RNA was extracted from and was performed as described Measurement of of MCP-1 in plasma and EWAT were using an kit EWAT was and in 2 of with The was at for 10 at The was used for MCP-1 of CD11b Expression was infused for 2 weeks using an osmotic at a of 10 analysis was performed to determine the monocyte/macrophage population among blood blood from and mice were from the using cells were at for and using CD11b at temperature for To control were using After cells with blood cells were using a for at were with and in containing was performed using an to identify the monocyte which was using and light In white blood cells were and of and were expressed as ± S.E. were performed using a Expression of in Obesity of mice obesity were to expression changes in These the high fat DIO model and the obesity model (ad and pair-fed In the DIO mice were fed high fat energy as 52.4% as carbohydrate, and as for 6 of DIO mice by 6 at ± fed ± The model utilized d-Trp34NPY is to represent an on the Y5 receptor. of this neuropeptide is to increase food intake and body with changes in body weight by increased fat weight (24Mashiko S. Ishihara A. Iwaasa H. Sano H. Oda Z. Ito J. Yumoto M. Okawa M. Suzuki J. Fukuroda T. Jitsuoka M. Morin N.R. Macneil D.J. Van der Ploeg L.H.T. Ihara M. Fukami T. Kanatani A. Endocrinology. 2003; 144: 1793-1801Crossref PubMed Scopus (55) Google Scholar). with d-Trp34NPY only increases food intake but also energy (24Mashiko S. Ishihara A. Iwaasa H. Sano H. Oda Z. Ito J. Yumoto M. Okawa M. Suzuki J. Fukuroda T. Jitsuoka M. Morin N.R. Macneil D.J. Van der Ploeg L.H.T. Ihara M. Fukami T. Kanatani A. Endocrinology. 2003; 144: 1793-1801Crossref PubMed Scopus (55) Google Scholar), that mice pair-fed with a control group also displayed increased EWAT was and total RNA was and to a DNA GeneChip (Affymetrix) were used for DNA and were using GeneChip software of genes were increased in DIO and mice (ad and pair-fed of genes were genes are to with increased protein and protein In changes were in genes displaying in such and leptin and MCP-1 secreted with genes with EWAT in DIO and chemoattractant protein protein 2 protein subfamily in a new MCP-1 mRNA and in the expression of MCP-1 in EWAT were using in obese from DNA and well adipose various vascular endothelial cells, smooth cells, cells, and macrophages F. of D. Scholar). the of cells, reportedly MCP-1 was used to MCP-1 in high fat DIO mice. of adipose was and cells were using and was of MCP-1 mRNA as compared with control lean was in obese mice we increased levels of MCP-1 mRNA to increased levels of MCP-1 protein in Adipose tissue from DIO mice was and used for of MCP-1 protein increase in tissue levels of MCP-1 protein was in DIO mice as compared with control mice, in with the mRNA to a WAT the was at of of increases in MCP-1 levels in WAT in plasma MCP-1 protein levels be to protein levels in of plasma MCP-1 levels using this In DIO mice, plasma levels of MCP-1 were increased as compared with control mice. MCP-1 and increased plasma MCP-1 levels were in DIO mice, we plasma MCP-1 levels with body weight in DIO mice. mice were fed high fat diet for various and blood was from these mice for MCP-1 plasma MCP-1 levels were body weight in these mice between plasma MCP-1 levels and body weight were These results that MCP-1 changes circulating levels to body as a of increased To a relationship between adiposity and plasma MCP-1 the course of DIO as compared with plasma MCP-1 levels was also Mice were fed a high fat diet from the were 9 weeks old, and blood was at and 20 weeks after of plasma MCP-1 levels increased to increased body and increases were in mice, these changes were as compared with mice fed the high fat adiposity is to increase with in it is to that MCP-1 increases in mice at the are 20 weeks old. The of on plasma MCP-1 levels was also restriction represents a model for decreased fat in and we utilized DIO mice fed with of the to body weight. 7 a in body weight was in to decreased plasma MCP-1 levels These results a strong relationship between adiposity and plasma MCP-1 MCP-1 and CD11b-positive MCP-1 is of the C-C chemokine β of which to to The interaction of MCP-1 with has been well L. Gosling J. Charo I.F. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and genetic manipulation of the interaction is to the course of atherosclerosis. In this MCP-1 in the activation and recruitment of monocytes to the atherosclerotic to MCP-1 levels in the plasma of obese may functional in the development of increased and we to the significance of increases in levels of analysis was performed on mice in which plasma MCP-1 levels were by high fat diet for weeks ± for ± for The results that the CD11b a member of the of monocytes was increased among DIO mice as compared with control mice ± white blood cells for ± white blood cells for suggests that such as of plasma MCP-1 levels by a high fat diet these changes in high fat diet MCP-1 plasma which in to the changes in the CD11b-positive monocyte we to the of MCP-1 on CD11b-positive monocyte MCP-1 was infused into mice under diet analysis was performed after 2 weeks of these experimental plasma MCP-1 levels increased about as compared with ± for MCP-1 ± for with the levels with the high fat CD11b-positive monocytes increased in with study of mice on a high fat these results suggest that changes in the CD11b-positive monocyte population in DIO mice are with increases in plasma MCP-1 Obesity is well as a risk factor for of atherosclerosis (1Zimmet P. Alberti K.G. Shaw J. Nature. 2001; 414: 782-787Crossref PubMed Scopus (4594) Google Scholar, 2Ginsberg H.N. J. Clin. Invest. 2000; 106: 453-458Crossref PubMed Scopus (941) Google Scholar, 3Kopelman P.G. Nature. 2000; 404: 635-643Crossref PubMed Scopus (3698) Google Scholar). progress in understanding of the pathogenesis underlying obesity has to the also as cardiovascular or insulin resistance N. Y. Acad. Sci. 1999; PubMed Scopus Google Scholar). However, the molecular of has been in and the genes are under investigation. The present study to identify such by on secreted from increased adiposity represents the in obesity and a risk factor for vascular In in secreted from the that such a strong for on DNA microarray are well for analysis of and using such expression we were to identify secreted from the of obesity DIO and mice. All changes in pair-fed were in the changes in ad libitum-fed d-Trp34 pair-fed were used to focus on genes expression changed with increased MCP-1 protein changed expression levels in obese as only protein, is in with a recent that MCP-1 mRNA is elevated in the adipose tissue of obese mice P. D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). were to protein levels in EWAT and circulating of the correlation between body weight and plasma MCP-1 levels a strong levels to that displayed by the correlation between body weight and plasma leptin levels S. R.L. Am. J. 1997; Google Scholar). plasma MCP-1 levels increased when body weight and decreased when body weight decreased These suggest that changes in adipose weight with are the of in plasma levels of In an to the functional of changes to plasma MCP-1 levels in obese mice, we CD11b-positive monocytes in circulating blood using MCP-1 reportedly induces CD11b expression on monocytes and of monocytes to blood (15van Royen N. Hoefer I. Bottinger M. Hua J. Grundmann S. Voskuil M. Bode C. Schaper W. Buschmann I. Piek J.J. Circ. Res. 2003; 92: 218-225Crossref PubMed Scopus (115) Google Scholar, K. J. Google Scholar). CD11b are expressed when monocytes are to monocytes and represent the molecular of the W. J. PubMed Scopus Google Scholar). In analysis, circulating CD11b-positive monocytes increased in obese DIO mice as compared with control mice, that increased MCP-1 in obese mice the CD11b-positive monocyte population in obese mice. In a has been a study in in which the number of monocytes increased by about in obese and Am. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Although that study CD11b-positive this represent the of increased CD11b-positive increase in CD11b-positive monocyte was also following of MCP-1 in mice. MCP-1 was infused into mice to the same levels in DIO mice, the that increased CD11b-positive monocytes were by obesity-related factors MCP-1 be We that increases in MCP-1 to changes in the CD11b-positive monocyte population in circulating However, study the sites of monocyte of monocyte activation the attachment and of monocyte to the MCP-1 secreted local sites high of MCP-1 protein in obese mice and the of activation for monocyte to macrophages represent of investigation. recent on the of of MCP-1 on atherosclerotic development also (15van Royen N. Hoefer I. Bottinger M. Hua J. Grundmann S. Voskuil M. Bode C. Schaper W. Buschmann I. Piek J.J. Circ. Res. 2003; 92: 218-225Crossref PubMed Scopus (115) Google Scholar). study that MCP-1 infusion in mice of monocytes in collateral arteries and increases The increased macrophages in obese mice described in the present thus to the of inflammatory processes in atheromatous to the development of atherosclerotic lesions. the we that obesity in mice increases MCP-1 mRNA in EWAT and MCP-1 plasma protein of which are well with changes in body weight. In these changes to increases in the population of CD11b-positive monocytes in circulating We MCP-1 protein as of the molecular factors obesity and atherosclerosis. of in to manipulation of MCP-1 between obese and normal in of atherosclerotic be of