Ryozo Nagai

Agonist-induced activation of peroxisome proliferator-activated receptor γ (PPARγ) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPARγ was investigated by gene targeting. Homozygous PPARγ-deficient embryos died at 10.5–11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPARγ-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPARγ agonist treatment. Heterozygous PPARγ-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPARγ in high-fat diet–induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPARγ.

Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states. Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states. Adiponectin (also known as ACRP30, 1The abbreviations used are: ACRP30, adipocyte complement-related protein of 30kDa; HMW, high molecular weight; GBP28, gelatin binding protein of 28kDa; MMW, middle molecular weight; LMW, low molecular weight; AMPK, AMP-activated protein kinase; DMEM, Dulbecco's modified Eagle's Medium; ACC, acetyl-CoA carboxylase; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; WT, wild-type; BS3, Bis (sulfosuccimidyl) suberate; SP-A, Surfactant protein-A; SP-D, Surfactant protein-D.1The abbreviations used are: ACRP30, adipocyte complement-related protein of 30kDa; HMW, high molecular weight; GBP28, gelatin binding protein of 28kDa; MMW, middle molecular weight; LMW, low molecular weight; AMPK, AMP-activated protein kinase; DMEM, Dulbecco's modified Eagle's Medium; ACC, acetyl-CoA carboxylase; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; WT, wild-type; BS3, Bis (sulfosuccimidyl) suberate; SP-A, Surfactant protein-A; SP-D, Surfactant protein-D. GBP28, and AdipoQ) (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2724) Google Scholar, 2Hu E. Liang P. Spiegelman B.M. J. Biol. Chem. 1996; 271: 10697-10703Abstract Full Text Full Text PDF PubMed Scopus (1879) Google Scholar, 3Maeda K. Okubo K. Shimomura I. Funahashi T. Matsuzawa Y. Matsubara K. Biochem. Biophys. Res. Commun. 1996; 221: 286-289Crossref PubMed Scopus (1838) Google Scholar, 4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. J. Biochem. 1996; 120: 803-812Crossref PubMed Scopus (783) Google Scholar) is a hormone secreted exclusively from adipocytes and has been shown to play important roles in the regulation of glucose and lipid metabolism. Adiponectin concentrations are reduced in obese and insulin-resistant human subjects and animal models (2Hu E. Liang P. Spiegelman B.M. J. Biol. Chem. 1996; 271: 10697-10703Abstract Full Text Full Text PDF PubMed Scopus (1879) Google Scholar, 5Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. Kuriyama H. Nishida M. Yamashita S. Okubo K. Matsubara K. Muraguchi M. Ohmoto Y. Funahashi T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4039) Google Scholar, 6Yamauchi 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 (4043) Google Scholar). Genetic deletion of adiponectin in mice (7Kubota N. Terauchi Y. Yamauchi T. Kubota T. Moroi M. Matsui J. Eto K. Yamashita T. Kamon J. Satoh H. Yano W. Froguel P. Nagai R. Kimura S. Kadowaki T. Noda T. J. Biol. Chem. 2002; 277: 25863-25866Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar, 8Maeda N. Shimomura I. Kishida K. Nishizawa H. Matsuda M. Nagaretani H. Furuyama N. Kondo H. Takahashi M. Arita Y. Komuro R. Ouchi N. Kihara S. Tochino Y. Okutomi K. Horie M. Takeda S. Aoyama T. Funahashi T. Matsuzawa Y. Nat. Med. 2002; 8: 731-737Crossref PubMed Scopus (1804) Google Scholar) or adiponectin supplementation of an insulin-resistant obese murine model (6Yamauchi 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 (4043) Google Scholar, 9Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. Nat. Med. 2001; 7: 947-953Crossref PubMed Scopus (2198) Google Scholar), has demonstrated that reduced plasma adiponectin levels caused by genetic or nutritional factors to be one of the important causes of type 2 diabetes development. On the other hand, adiponectin is mapped to chromosome locus 3q27, which is reportedly the locus closely associated with type 2 diabetes based on genome-wide scans in several ethnic groups (10Mori Y. Otabe S. Dina C. Yasuda K. Populaire C. Lecoeur C. Vatin V. Durand E. Hara K. Okada T. Tobe K. Boutin P. Kadowaki T. Froguel P. Diabetes. 2002; 51: 1247-1255Crossref PubMed Scopus (229) Google Scholar, 11Vionnet N. Hani E.H. Dupont S. Gallina S. Francke S. Dotte S. De Matos F. Durand E. Lepretre F. Lecoeur C. Gallina P. Zekiri L. Dina C. Froguel P. Am. J. Hum. Genet. 2000; 67: 1470-1480Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 12Kissebah A.H. Sonnenberg G.E. Myklebust J. Goldstein M. Broman K. James R.G. Marks J.A. Krakower G.R. Jacob H.J. Weber J. Martin L. Blangero J. Comuzzie A.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14478-14483Crossref PubMed Scopus (569) Google Scholar). The G allele of single nucleotide polymorphism (SNP) 276 in adiponectin is associated with hypoadiponectinemia and type 2 diabetes (13Hara K. Boutin P. Mori1 Y. Tobe K. Dina C. Yasuda K. Yamauchi T. Otabe S. Okada T. Eto K. Kadowaki H. Hagura R. Akanuma Y. Yazaki Y. Nagai R. Taniyama M. Matsubara K. Yoda M. Nakano Y. Kimura S. Tomita M. Kimura S. Ito C. Froguel P. Kadowaki T. Diabetes. 2002; 51: 536-540Crossref PubMed Scopus (639) Google Scholar). Eight mutations in human adiponectin have been reported (13Hara K. Boutin P. Mori1 Y. Tobe K. Dina C. Yasuda K. Yamauchi T. Otabe S. Okada T. Eto K. Kadowaki H. Hagura R. Akanuma Y. Yazaki Y. Nagai R. Taniyama M. Matsubara K. Yoda M. Nakano Y. Kimura S. Tomita M. Kimura S. Ito C. Froguel P. Kadowaki T. Diabetes. 2002; 51: 536-540Crossref PubMed Scopus (639) Google Scholar, 14Takahashi M. Arita Y. Yamagata K. Matsukawa Y. Okutomi K. Horie M. Shimomura I. Hotta K. Kuriyama H. Kihara S. Nakamura T. Yamashita S. Funahashi T. Matsuzawa Y. Int. J. Obes. Relat. Metab. Disord. 2000; 24: 861-868Crossref PubMed Scopus (325) Google Scholar, 15Kondo H. Shimomura I. Matsukawa Y. Kumada M. Takahashi M. Matsuda M. Ouchi N. Kihara S. Kawamoto T. Sumitsuji S. Funahashi T. Matsuzawa Y. Diabetes. 2002; 51: 2325-2328Crossref PubMed Scopus (342) Google Scholar, 16Vasseur F. Helbecque N. Dina C. Lobbens S. Delannoy V. Gaget S. Boutin P Vaxillaire M. Lepretre F. Dupont S. Hara K. Clement K. Bihain B. Kadowaki T. Froguel P. Hum. Mol. Genet. 2002; 11: 2607-2614Crossref PubMed Scopus (443) Google Scholar). Several mutations were significantly related to diabetes and hypoadiponectinemia (15Kondo H. Shimomura I. Matsukawa Y. Kumada M. Takahashi M. Matsuda M. Ouchi N. Kihara S. Kawamoto T. Sumitsuji S. Funahashi T. Matsuzawa Y. Diabetes. 2002; 51: 2325-2328Crossref PubMed Scopus (342) Google Scholar, 16Vasseur F. Helbecque N. Dina C. Lobbens S. Delannoy V. Gaget S. Boutin P Vaxillaire M. Lepretre F. Dupont S. Hara K. Clement K. Bihain B. Kadowaki T. Froguel P. Hum. Mol. Genet. 2002; 11: 2607-2614Crossref PubMed Scopus (443) Google Scholar). Molecular mechanisms underlying the development of diabetes and hypoadiponectinemia have yet to be clarified. Adiponectin structurally belongs to the complement 1q family and consists of a carboxyl-terminal globular domain and an amino-terminal collagenous domain (17Shapiro L. Scherer P.E. Curr. Biol. 1998; 8: 335-338Abstract Full Text Full Text PDF PubMed Google Scholar, 18Yokota T. Oritani K. Takahashi I. Ishikawa J. Matsuyama A. Ouchi N. Kihara S. Funahashi T. Tenner A.J. Tomiyama Y. Matsuzawa Y. Blood. 2000; 96: 1723-1732Crossref PubMed Google Scholar). This family is also known to form characteristic multimers (19Crouch E. Persson A. Chang D. Heuser J. J. Biol. Chem. 1994; 269: 17311-17319Abstract Full Text PDF PubMed Google Scholar, 20McCormack F.X. Pattanajitvilai S. Stewart J. Possmayer F. Inchley K. Voelker D.R. J. Biol. Chem. 1997; 272: 27971-27979Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Gel filtration and velocity gradient studies revealed adiponectin circulating in serum to form several different molecular weight species; the largest species was more than several hundred kilodaltons in size (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2724) Google Scholar, 4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. J. Biochem. 1996; 120: 803-812Crossref PubMed Scopus (783) Google Scholar, 5Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. Kuriyama H. Nishida M. Yamashita S. Okubo K. Matsubara K. Muraguchi M. Ohmoto Y. Funahashi T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4039) Google Scholar, 21Tsao T-S. Murrey H.E. Hug C. Lee D.H. Lodish H.F. J. Biol. Chem. 2002; 277: 29359-29362Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In this study, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Applying this method, we investigated the molecular structure and mode of multimerization of adiponectin from human or mouse serum and cultured cells. Since we speculated that adiponectin mutations might alter multimer formations of adiponectin, we analyzed these mutants with SDS-PAGE under non-reducing and non-heat-denaturing conditions. We demonstrated impaired multimerization and secretion in these mutants to possibly contribute to the development of diabetes and hypoadiponectinemia. Adiponectin has been shown to be one of the major regulators of energy homeostasis and insulin sensitivity. This study sheds new light on the molecular structure-function relationship. Materials—Trypsin V-S, Dulbecco's Modified Eagles Medium (DMEM), anti-FLAG M2 antibody cross-linked to agarose, and FLAG peptide were purchased from Sigma. 2-Mercaptoethanol was purchased from Wako Pure Chemicals. Mammalian expression vector pcDNA3.1(+) was purchased from Invitrogen. Prokaryotic expression vector PQE30 and Ni-NTA agarose were purchased from Qiagen. Superdex S300HR 10/30 was purchased from Amersham Biosciences. Anti-phosphorylated AMP-activated protein kinase (AMPK) antibody and anti-phosphorylated acetyl-CoA carboxylase (ACC) antibody were purchased from Cell Signaling. Horseradish peroxidase-conjugated anti-rabbit antibody was purchased from Zymed Laboratories Inc.. Cell Culture—NIH-3T3 fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum in an incubator with 5% CO2 at 37 °C. 3T3-L1 adipocytes were maintained as subconfluent cultures in DMEM supplemented with 10% fetal bovine serum. 3T3-L1 preadipocytes were differentiated to mature adipocytes by a conventional method (22Miki H. Yamauchi T. Suzuki R. Komeda K. Tsuchida A. Kubota N. Terauchi Y. Kamon J. Kaburagi Y. Matsui J. Akanuma Y. Nagai R. Kimura S. Tobe K. Kadowaki T. Mol. Cell. Biol. 2001; 21: 2521-2532Crossref PubMed Scopus (170) Google Scholar). Myocyte cell line C2C12 and primary hepatocytes were cultured as described in Ref. 23Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3419) Google Scholar. Cloning, Recombinant Expression, and Purification of Adiponectin—A murine adiponectin coding sequence (NCBI accession no. U37222) flanked by a Kozak sequence was cloned from mouse white adipose tissue and inserted between EcoRI and NotI in the multiple cloning site of pcDNA3.1(+). Human adiponectin (NCBI accession no. D45371) was also cloned into pcDNA3.1(+) in the same manner. For mammalian expression, NIH-3T3 fibroblasts were transfected with expression vectors using LipofectAMINE (Invitrogen) according to the manufacturer's instruction. For the purification of recombinant adiponectin from NIN-3T3 fibroblast medium, we prepared an expression vector in which the FLAG tag sequence, 5′-GATTACAAGGATGACGACGATAAG-3′ (DYKDDDDK in amino acids) was inserted into the carboxyl terminus of adiponectin. NIH-3T3 fibroblasts were transfected with the FLAG-tagged adiponectin expression vector and recombinant protein was allowed to secrete into the medium for 48 h. Collected medium was applied to an anti-FLAG affinity column. The column was washed with Tris-buffered saline (10 mm Tris-HCl, 150 mm NaCl, pH7.5, TBS) and bound recombinant adiponectin was eluted with FLAG peptide. Adiponectin-rich fractions were collected and dialyzed against TBS. For prokaryotic expression, the coding sequence deprived of the signal sequence (corresponding to residues 18-247) of mouse adiponectin was inserted between BamHI and HindIII of the PQE30 expression vector, which expressed the His6 tag attached to the amino terminus of adiponectin. For the globular domain, the sequence corresponding the residues 104-247 was inserted. Expression and purification of His-tagged adiponectin from the Escherichia coli lysate was performed as described in Ref. 6Yamauchi 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 (4043) Google Scholar. Briefly, the soluble fraction of the E. coli lysate was applied to Ni-NTA agarose, washed thoroughly and bound adiponectin was eluted stepwise with imidazole. Fractions containing adiponectin was collected and extensively dialyzed against phosphate-buffered saline (PBS). Site-directed Mutagenesis of Adiponectin—Mouse C39S mutant adiponectin was generated using a site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The sense primer was 5′-CACCCAAGGGAACTAGTGCAGGTTGGATGG-3′ and the antisense primer was complementary to it. For human adiponectin mutagenesis, the sense primers were 5′-CATCGGTGAAACCAGAGTACCCGGGGC-3′ for G84R, 5′-CGGGGCTGAAAGTCCCCGAGGCTTTC-3′ for G90S, 5′-GCTGAAGGTCCCTGAGGCTTTCCG GG-3′ for R92X, 5′-GGTGCCTATGTACACCGCTCAGCATTCAGT G-3′ for Y111H, 5′-GGTGCCTATGTATACTGCTCAGCATTCAGTGTGG-3′ for R112C, 5′-CCTGG GCTGTACGACTTTGCCTACCACATC-3′ for Y159D, 5′-CTACTTTGCCTACCACACCACAGTCTATATGAAGG-3′ for I164T, 5′-GTATGGGGAAGGAGAGAGTAATGGACTCTATGCTG-3′ for R221S, and 5′-GGCTTTCTTCTCTACCCTGACACCAACTGATCAC-3′ for H241P. The antisense primers were complementary to these sense primers. The coding sequence corresponding to amino acids 19-71 (from the variable region to the middle of the collagenous region) was deleted for the expression of amino-terminally truncated human adiponectin (ΔH). The sense primer was 5′-GGTCTTATTGGTCCTAAGGGAGACATCGGTG-3′ with 5′-phosphorylation, and the antisense primer was 5′-CTGGTCATGCCCGGGCAGAGCTAATAGCAG-3′ with 5′-phosphorylation. The deleted construct was amplified from human adiponectin pcDNA3.1 by pfu Taq polymerase (Stratagene), and the gained blunted DNA fragment was self-ligated to form a circular plasmid. Generation of Anti-adiponectin Antibodies—Anti-mouse adiponectin globular domain antiserum was obtained by immunizing rabbits with mouse recombinant adiponectin globular domain produced in E. coli. Anti-mouse amino-terminal peptide antibody, and anti-human carboxyl-terminal peptide antibody were raised against the synthesized mouse amino-terminal peptide EDDVTTTEELAPALV and the human carboxyl-terminal peptide CYADNDSTFTGFLLYHDTN. Anti-human carboxyl-terminal peptide antibody showed good cross-reactivity against mouse adiponectin (data not shown). Preparation of Adiponectin Globular Domain by Trypsin Digestion of Full-length Adiponectin—Full-length adiponectin expressed in NIH-3T3 fibroblasts and secreted in DMEM without serum was cleaved by trypsin V-S according to Ref. 24Fruebis J. Tsao T-S. Javorschi S. Ebbets-Reed D. Erickson M.R.S. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1742) Google Scholar. Trypsin reportedly digests full-length adiponectin at the peptide bond between Arg-103 and Lys-104, generating the globular adiponectin (24Fruebis J. Tsao T-S. Javorschi S. Ebbets-Reed D. Erickson M.R.S. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1742) Google Scholar). SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting—SDS-PAGE was performed according to the standard Laemmli's method (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar). Sample buffer for reducing conditions was 3% SDS, 50 mm Tris-HCl pH 6.8, 5% 2-mercaptoethanol and 10% glycerol. For complete reduction of serum sample, 10 mm dithiothreitol was also added to the buffer. For non-reducing conditions, 2-mercaptoethanol was excluded from the sample buffer described above. The sample was mixed with 5× sample buffer and incubated for 1 hour at room temperature. For heat-denaturation, samples were heated at 95 °C for 10 min unless indicated. For immunoblotting, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes. The membranes were blocked with TBS-T (TBS, 0.1% Triton X-100) containing 3% skim milk and then incubated with 1:1000 diluted antiserum in TBS-T containing 3% skim milk for 1 h at room temperature. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:4000) for 30 min at room temperature and then washed thoroughly. The membranes were exposed to x-ray film (Fuji film) using ECL Western blotting detection reagent (Amersham Biosciences). Gel Filtration Chromatography Analysis of Adiponectin—Samples were filtered through 0.44-μm pore membranes and 200 μl were injected into a Superdex S300HR 10/30 (Amersham Biosciences) pre-equilibrated with PBS using the FPLC system (Amersham Biosciences) at 4 °C. Samples were eluted with PBS at a rate of 0.5 ml/min and monitored by absorbance at 280 nm. Fractions (0.5 ml) were collected. Analysis of Adiponectin from Human or Mouse Serum and Medium of 3T3-L1 Adipocytes—Freshly drawn human or mouse blood was coagulated for 30 min at room temperature. After centrifugation, 0.7 μl of the supernatant was diluted into non-reducing sample buffer and subjected to SDS-PAGE under non-reducing and non-heat-denaturing conditions. 3T3-L1 preadipocytes were differentiated into mature adipocytes for 10 days and washed twice in DMEM without serum. Adiponectin was allowed to secrete into DMEM without serum for 48 h and 10-μl aliquots were diluted into non-reducing sample buffer and subjected to SDS-PAGE under non-reducing and non-heat-denaturing conditions. For an analysis of serum of human subjects heterozygous for G90S mutation, subjects are identified through screening of 1373 French Caucasians (16Vasseur F. Helbecque N. Dina C. Lobbens S. Delannoy V. Gaget S. Boutin P Vaxillaire M. Lepretre F. Dupont S. Hara K. Clement K. Bihain B. Kadowaki T. Froguel P. Hum. Mol. Genet. 2002; 11: 2607-2614Crossref PubMed Scopus (443) Google Scholar). Subjects ranging from 40 to 69 of age were selected and examined. Average age ± S.E. of these groups were 56.4 ± 7.9 (wild-type female), 54.7 ± 1.7 (G90S female), 59.4 ± 4.2 (wild-type male), 52.3 ± 2.9 (G90S male). Phosphorylation of AMPK and ACC by Adiponectin—The AMPK pathway was analyzed according to Ref. 23Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3419) Google Scholar. Briefly, after cells had been incubated in serum-free RPMI 1640 medium (Sigma) for 6 h, RPMI 1640 containing E. coli recombinant adiponectin was added to the well and incubated for 5 min at 37 °C. The reaction was stopped with liquid nitrogen and cells were lysed and homogenized by a sonicator in lysis buffer (25 mm Tris-HCl, pH 7.4, 10 mm Na3VO4, 10 mm sodium pyrophosphate, 100 mm NaF, 10 mm EDTA, 10 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40). The lysate was centrifuged and the protein concentration was assayed using BCA protein assay reagent (Pierce). The same amount of lysate protein was applied to SDS-PAGE under reducing and heat-denaturing conditions, blotted onto PVDF membranes and immunostained with anti-phosphorylated AMPK antibodies or anti-phosphorylated ACC antibodies. NIH-Image was used for band quantification. SDS-PAGE under Non-reducing and Non-heat-denaturing Conditions Separates Multimer Species of Adiponectin—Adiponectin was reported to form several different molecular weight multimers by gel filtration and velocity gradient studies (1Scherer P.E. Williams S. Fogliano M. Baldini G. Lodish H.F. J. Biol. Chem. 1995; 270: 26746-26749Abstract Full Text Full Text PDF PubMed Scopus (2724) Google Scholar, 4Nakano Y. Tobe T. Choi-Miura N.H. Mazda T. Tomita M. J. Biochem. 1996; 120: 803-812Crossref PubMed Scopus (783) Google Scholar, 5Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. Kuriyama H. Nishida M. Yamashita S. Okubo K. Matsubara K. Muraguchi M. Ohmoto Y. Funahashi T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4039) Google Scholar, 21Tsao T-S. Murrey H.E. Hug C. Lee D.H. Lodish H.F. J. Biol. Chem. 2002; 277: 29359-29362Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). When human and mouse adiponectin from serum or adipocytes, and recombinant adiponectin expressed in mammalian cells, were separated by SDS-PAGE under non-reducing and non-heat-denaturing conditions, three different molecular mass species (∼67, 136, and >300 kDa) of adiponectin were detected in all preparations (Fig. 1A). We designated these species as LMW (low molecular weight), MMW (middle molecular weight), and HMW (high molecular weight) multimers, respectively. In order to demonstrate these three species seen in Fig. 1A represent different multimer species, we subjected adiponectin to gel filtration analysis in parallel. Purified mouse adiponectin expressed in NIH-3T3 resolved into three peaks in gel filtration as reported before (Fig. 1B) (21Tsao T-S. Murrey H.E. Hug C. Lee D.H. Lodish H.F. J. Biol. Chem. 2002; 277: 29359-29362Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). SDS-PAGE analysis of each fraction revealed that these three peaks represented HMW, MMW, and LMW multimers (Fig. 1C). Adiponectin in human serum and adiponectin expressed in E. coli also had three peaks, which corresponded to HMW, MMW and LMW multimers (Fig. 1D). Fig. 1, E and F are examples showing adiponectin multimer distribution in serum of representative female and male human subjects. Both non-reducing and non-heat-denaturing SDS-PAGE (Fig. 1E) and gel filtration analysis (Fig. 1F) clearly demonstrated that the levels of HMW multimers were high in a female subject and low in a male subject. These data suggested that non-reducing and non-heat-denaturing SDS-PAGE was able to represent different adiponectin multimers as accurately as the conventional gel filtration analysis. Our SDS-PAGE analysis was superior to the gel filtration analysis in terms of resolving power. SDS-PAGE was able to clearly separate each of multimer species (Fig. 1E), whereas gel filtration did not completely separate each multimers (Fig. 1F). We also noted that there were several HMW species, whereas murine adiponectin had only one (Fig. 1A). These species were not recognized by conventional gel filtration analysis (Fig. 1F). Total adiponectin concentrations are known to be higher in females than in males (5Arita Y. Kihara S. Ouchi N. Takahashi M. Maeda K. Miyagawa J. Hotta K. Shimomura I. Nakamura T. Miyaoka K. Kuriyama H. Nishida M. Yamashita S. Okubo K. Matsubara K. Muraguchi M. Ohmoto Y. Funahashi T. Matsuzawa Y. Biochem. Biophys. Res. Commun. 1999; 257: 79-83Crossref PubMed Scopus (4039) Google Scholar). HMW multimers, but not MMW and LMW multimers, were significantly less abundant in male subjects than in female subjects (Fig. 1, E and F and Table I). This suggested that not only total adiponectin concentration but also multimer distribution are different in two genders.Table IGender difference in the multimer formation of adiponectinFemaleMaleHMW100 ± 1929.0 ± 14ap < 0.05 (Student's t-test) values of the same multimer species were compared between genders.MMW100 ± 6.981.5 ± 5.2LMW100 ± 1284.0 ± 6.6a p < 0.05 (Student's t-test) values of the same multimer species were compared between genders. Open table in a new tab Influence of Reduction and Heat Denaturation on Adiponectin Multimer Formation—When adiponectin expressed in NIH-3T3 or serum adiponectin were electrophoresed after reduction and heat denaturation, all molecular mass species were converted to a single 28-kDa band, indicating all of these species to be composed of identical monomers, namely, homomultimers (Fig. 2, A and B, lane 4). 56- and 28-kDa bands were seen, when the sample was heat-denatured under non-reducing conditions, suggesting the 56-kDa band to be a dimer (Fig. 2, A and B, lane 2). On the other hand, reduction without heat denaturation converted all of the molecular mass species to a 67-kDa band, which was the smallest molecular weight component of adiponectin, that is, a LMW multimer (Fig. 2, A and B, lane 3). This 67-kDa band was deduced to represent a trimer because the collagen-like structure and the globular domain of adiponectin are known to form a trimer (17Shapiro L. Scherer P.E. Curr. Biol. 1998; 8: 335-338Abstract Full Text Full Text PDF PubMed Google Scholar). To prove the 67-kDa band to be a trimer, we co-expressed amino-terminally truncated adiponectin (ΔH) and full-length wild-type adiponectin (WT). Two heterogeneous bands were observed when samples were separated by reducing and non-heat-denaturing SDS-PAGE (Fig. 3A, lane 4). These data suggested that the 67-kDa band (Fig. 3A, lane 2, Fig. 2, A and B, lane 3) was a trimer, and the two heterogeneous bands (Fig. 3A, lane 4) were heterotrimers (i.e. WT x2/ΔH x1, WT x1/ΔH x2). The size of 67 kDa, w

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (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. Tumor necrosis factor-α and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid 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 rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. 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 may contribute to the development of insulin resistance in aP2-MCP-1 mice. Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (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. Tumor necrosis factor-α and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid 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 rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. 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 may contribute to the development of insulin resistance in aP2-MCP-1 mice. Obesity correlates closely with insulin resistance (1Flier J.S. Cell. 2004; 116: 337-350Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar, 2Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3225) Google Scholar). We have demonstrated that the size of adipocytes is inversely related to insulin sensitivity (3Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Investig. 1998; 101: 1354-1361Crossref PubMed Scopus (927) Google Scholar, 4Kubota 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. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar, 5Kadowaki T. J. Clin. Investig. 2000; 106: 459-465Crossref PubMed Scopus (220) Google Scholar); namely, larger adipocytes are 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 (6Yamauchi 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, 7Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. Nat. Med. 2001; 7: 947-953Crossref PubMed Scopus (2218) Google Scholar). Recent studies have shown that obesity is associated not only with larger adipocytes but also with increasing numbers of infiltrating macrophages in adipose tissues (8Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar, 9Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7562) Google Scholar, 10Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5240) Google Scholar). These adipose tissue macrophages are currently considered to be a major cause of obesity-associated chronic low grade inflammation (2Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3225) Google Scholar, 11Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2003; 112: 1785-1788Crossref PubMed Scopus (1452) Google Scholar) via secretion of a wide variety of inflammatory molecules (12Kershaw E.E. Flier J.S. J. Clin. Endocrinol. Metab. 2004; 89: 2548-2556Crossref PubMed Scopus (3754) Google Scholar), including tumor necrosis factor-α (TNF-α) 2The abbreviations used are: TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-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 acid; IR, insulin receptor; IRS, IR substrate; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated extracellular signal protein kinase; BAT, brown adipose tissue; MMP12, matrix metallopeptidase 12; PPARγ, peroxisome proliferator-activated receptor γ; SVC, stromal-vascular cell; 2-DG, 2-deoxyglucose; Rd, rate of glucose disappearance; EGP, endogenous glucose production; ANOVA, analysis of variance; eWAT, epididymal WAT; HF, high fat; NC, normal chow. (13Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Scopus (6193) Google Scholar), interleukin-6 (IL-6) (14Fernandez-Real J.M. Ricart W. Endocr. Rev. 2003; 24: 278-301Crossref PubMed Scopus (727) Google Scholar), monocyte chemoattractant protein-1 (MCP-1) (15Takahashi K. Mizuarai S. Araki H. Mashiko S. Ishihara A. Kanatani A. Itadani H. Kotani H. J. Biol. Chem. 2003; 278: 46654-46660Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 16Christiansen T. Richelsen B. Bruun J.M. Int. J. Obes. Relat. Metab. Disord. 2005; 29: 146-150Crossref PubMed Scopus (333) Google Scholar), and plasminogen activator inhibitor-1 (17Shimomura I. Funahashi T. Takahashi M. Maeda K. Kotani K. Nakamura T. Yamashita S. Miura M. Fukuda Y. Takemura K. Tokunaga K. Matsuzawa Y. Nat. Med. 1996; 2: 800-803Crossref PubMed Scopus (823) Google Scholar). 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 (12Kershaw E.E. Flier J.S. J. Clin. Endocrinol. Metab. 2004; 89: 2548-2556Crossref PubMed Scopus (3754) Google Scholar). 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 up-regulated 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 (18Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and it has been reported to recruit monocytes from the blood into atherosclerotic lesions, thereby promoting foam cell formation (19Boring L. Gosling J. Cleary M. Charo I.F. Nature. 1998; 394: 894-897Crossref PubMed Scopus (1686) Google Scholar, 20Gu 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, 21Linton M.F. Fazio S. Int. J. Obes. Relat. Metab. Disord. 2003; 27: 35-40Crossref PubMed Scopus (233) Google Scholar). MCP-1 expression in adipose tissue and plasma MCP-1 levels have been found to correlate positively with the degree of obesity (9Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7562) Google Scholar, 10Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5240) Google Scholar, 16Christiansen T. Richelsen B. Bruun J.M. Int. J. Obes. Relat. Metab. Disord. 2005; 29: 146-150Crossref PubMed Scopus (333) Google Scholar, 22Sartipy P. Loskutoff D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7265-7270Crossref PubMed Scopus (919) Google Scholar). In addition, increased expression of this chemokine in adipose tissue precedes the expression of other macrophage markers during the development of obesity (10Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5240) Google Scholar). 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 (23Weisberg S.P. Hunter D. Huber R. Lemieux J. Slaymaker S. Vaddi K. Charo I. Leibel R.L. Ferrante A.W. J. Clin. Investig. 2006; 116: 115-124Crossref PubMed Scopus (1271) Google Scholar). 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. Because the TG mice displayed increased gene expression of TNF-α and IL-6 as well as higher plasma concentrations of non-esterified fatty acids (NEFAs), 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. Reagents—Recombinant mouse CCL2/JE/MCP-1 protein was purchased from R&D Systems Inc. (Minneapolis, MN). U0126 was purchased from Calbiochem. 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 MAPK, phosphor-p44/42 MAPK, 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 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. To induce differentiation, cells were switched to media containing Dulbecco's modified Eagle's medium, 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 h in media and treated with 10 nm MCP-1 for 5 min to detect the extracellular signal-regulated kinase (ERK) activation. C2C12 cells were treated with 1-10 nm MCP-1 for 30 min before 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 min before 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-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 (24Graves R.A. Tontonoz P. Platt K.A. Ross S.R. Spiegelman B.M. J. Cell. Biochem. 1992; 49: 219-224Crossref PubMed Scopus (61) Google Scholar) kindly provided by Dr. Bruce Spiegelman (Dana Farber Institute, Boston, MA). The transgene consisted of 5.4 kilobases 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 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-h fast and the epididymal fat pad (for epididymal WAT), 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) 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 Reverse Transcriptase-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 TaqMan 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, 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 (25Suzuki 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. Kadowaki T. J. Biol. Chem. 2003; 278: 43691-43698Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), 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 (26Takahashi M. Kamei Y. Ezaki O. Am. J. Physiol. Endocrinol. Metab. 2005; 288: 117-124Crossref PubMed Scopus (138) Google Scholar)—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) with Krebs-Henseleit-HEPES buffer, pH 7.4, supplemented with 20 mg/ml of bovine serum albumin and 2 mmol/liter glucose at 37 °C in a shaking water bath for 45 min. Then digested tissues were filtered through mesh and fractionated by brief centrifugation (1000 rpm). Floating cells were adipocytes, and the pellet was 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 (9Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7562) Google Scholar, 23Weisberg S.P. Hunter D. Huber R. Lemieux J. Slaymaker S. Vaddi K. Charo I. Leibel R.L. Ferrante A.W. J. Clin. Investig. 2006; 116: 115-124Crossref PubMed Scopus (1271) Google Scholar)—In the SVCs red blood cells were lysed and removed by a 15-min 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 min 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 min 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 forward scatter side scatter (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 of liver and muscle proteins was performed as described previously (27Yamauchi T. Tobe K. Tamemoto H. Ueki K. Kaburagi Y. Yamamoto-Honda R. Takahashi Y. Yoshizawa F. Aizawa S. Akanuma Y. Sonenberg N. Yazaki Y. Kadowaki T. Mol. Cell. Biol. 1996; 16: 3074-3084Crossref PubMed Scopus (250) Google Scholar) with some modifications. Samples were separated on polyacrylamide gels and transferred to Hybond-P PVDF transfer membrane (Amersham Biosciences). After 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 (275,000 μm2/field) from different sections, and the histogram was drawn by analyzing 6 mice per group according to methods described previously (4Kubota 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. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1225) Google Scholar) 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. Measurement of 2-Deoxyglucose (2-DG) Uptake—This assay was performed as described previously (28Murakami K. Tsunoda M. Ide T. Ohashi M. Mochizuki T. Metabolism. 1999; 48: 1450-1454Abstract Full Text PDF PubMed Scopus (15) Google Scholar) 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 min at 37 °C in Krebs-Ringer phosphate buffer, pH 7.4, 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 min at 37 °C before 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 min. 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 min. For U0126 rescue experiments, 20 μm 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 3 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 Biosciences). 14C and 3H specific activities were counted by a liquid scintillation counter (Packard Instrument Co.). 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 h before plasma was obtained. Plasma MCP-1, adiponectin, and leptin levels were determined with a mouse JE/MCP-1 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems), mouse adiponectin ELISA kit (Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan), and mouse leptin ELISA kit (R&D Systems), respectively. Plasma NEFAs (Wako Pure Chemical Industries Ltd., Osaka, Japan) were assayed by enzymatic methods. Measurement of Tissue Triglyceride Contents—Liver and muscle tissues were homogenized, and their triglyceride contents were determined as described previously (6Yamauchi 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). ITT—Insulin tolerance was tested in mice fasted for 4 h. The animals were intraperitoneally injected with 0.75 milliunits/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—Before the study the mice were fasted for 16 h starting at 19:00, and at the end of the fast they were orally loaded 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 h of starvation, and 5 units of human insulin (Humulin R, Lilly) were injected into the inferior vena cava. After 5 min, 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 (29Suzuki 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. Kadowaki T. J. Biol. Chem. 2004; 279: 25039-25049Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 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-h fast. A primed-continuous infusion of insulin (Humulin R; Lilly) was given (3.0 milliunits/kg/min for normal chow (NC) fed mice and 10.0 milliunits/kg/min for high fat (HF) diet-fed mice), and the blood glucose concentration, monitored every 5 min, was maintained at ∼120 mg/dl by administration of glucose (5 g of glucose/10 ml enriched to ∼20% with [6,6-2H2]glucose (Sigma)) for 120 min. 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 non-steady-state equations (29Suzuki 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. Kadowaki T. J. Biol. Chem. 2004; 279: 25039-25049Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and endogenous glucose production (EGP) was calculated as the difference between Rd and exogenous glucose infusion rates (29Suzuki 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. Kadowaki T. J. Biol. Chem. 2004; 279: 25039-25049Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Statistical Analysis—Results were expressed as the means ± S.E. 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

Obesity is more linked to vascular disease, including atherosclerosis and restenotic change, after balloon angioplasty. The precise mechanism linking obesity and vascular disease is still unclear. Previously we have demonstrated that the plasma levels of adiponectin, an adipose-derived hormone, decreases in obese subjects, and that hypoadiponectinemia is associated to ischemic heart disease. In current the study, we investigated the in vivorole of adiponectin on the neointimal thickening after artery injury using adiponectin-deficient mice and adiponectin-producing adenovirus. Adiponectin-deficient mice showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries. Adenovirus-mediated supplement of adiponectin attenuated neointimal proliferation. In cultured smooth muscle cells, adiponectin attenuated DNA synthesis induced by growth factors including platelet-derived growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), basic fibroblast growth factor, and EGF and cell proliferation and migration induced by HB-EGF. In cultured endothelial cells, adiponectin attenuated HB-EGF expression stimulated by tumor necrosis factor α. The current study suggests an adipo-vascular axis, a direct link between fat and artery. A therapeutic strategy to increase plasma adiponectin should be useful in preventing vascular restenosis after angioplasty.

Adiponectin has been shown to stimulate fatty acid oxidation and enhance insulin sensitivity through the activation of AMP-activated protein kinase (AMPK) in the peripheral tissues. The effects of adiponectin in the central nervous system, however, are still poorly understood. Here, we show that adiponectin enhances AMPK activity in the arcuate hypothalamus (ARH) via its receptor AdipoR1 to stimulate food intake; this stimulation of food intake by adiponectin was attenuated by dominant-negative AMPK expression in the ARH. Moreover, adiponectin also decreased energy expenditure. Adiponectin-deficient mice showed decreased AMPK phosphorylation in the ARH, decreased food intake, and increased energy expenditure, exhibiting resistance to high-fat-diet-induced obesity. Serum and cerebrospinal fluid levels of adiponectin and expression of AdipoR1 in the ARH were increased during fasting and decreased after refeeding. We conclude that adiponectin stimulates food intake and decreases energy expenditure during fasting through its effects in the central nervous system.

An adipocyte-derived peptide, adiponectin (also known as GBP28), is decreased in subjects with type 2 diabetes. Recent genome-wide scans have mapped a diabetes susceptibility locus to chromosome 3q27, where the adiponectin gene (APM1) is located. Herein, we present evidence of an association between frequent single nucleotide polymorphisms at positions 45 and 276 in the adiponectin gene and type 2 diabetes (P = 0.003 and P = 0.002, respectively). Subjects with the G/G genotype at position 45 or the G/G genotype at position 276 had a significantly increased risk of type 2 diabetes (odds ratio 1.70 [95% CI 1.09-2.65] and 2.16 [1.22-3.95], respectively) compared with those having the T/T genotype at positions 45 and 276, respectively. In addition, the subjects with the G/G genotype at position 276 had a higher insulin resistance index than those with the T/T genotype (1.61 +/- 0.05 vs. 1.19 +/- 0.12, P = 0.001). The G allele at position 276 was linearly associated with lower plasma adiponectin levels (G/G: 10.4 +/- 0.85 microg/ml, G/T: 13.7 +/- 0.87 microg/ml, T/T: 16.6 +/- 2.24 microg/ml, P = 0.01) in subjects with higher BMIs. Based on these findings together with the observation that adiponectin improves insulin sensitivity in animal models, we conclude that the adiponectin gene may be a susceptibility gene for type 2 diabetes.

Peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARγ and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARγ by PPARγ agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARγ activity by heterozygous PPARγ deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARγ deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARγ deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor α, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARγ deficiency and PPARγ agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy. Peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARγ and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARγ by PPARγ agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARγ activity by heterozygous PPARγ deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARγ deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARγ deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor α, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARγ deficiency and PPARγ agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy. peroxisome proliferator-activated receptor white adipose tissue triglyceride thiazolidinediones retinoid X receptor free fatty acids tumor necrosis factor high carbohydrate sterol regulatory element-binding protein insulin receptor substrate high fat phosphatidylinositol brown adipose tissue Peroxisome proliferator-activated receptor (PPAR)1 γ is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that functions as a heterodimer with a retinoid X receptor (RXR) (1Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1701) Google Scholar, 2Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, 3Gonzalez F.J. Biochimie (Paris). 1997; 79: 139-144Crossref PubMed Scopus (97) Google Scholar, 4Auwerx J. Diabetologia. 1999; 42: 1033-1049Crossref PubMed Scopus (587) Google Scholar, 5Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar). Agonist-induced activation of PPARγ/RXR is known to increase insulin sensitivity (6Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3492) Google Scholar, 7Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti Jr., J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (578) Google Scholar). Thiazolidinediones (TZD), which have the ability to directly bind and activate PPARγ (6Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3492) Google Scholar) and to stimulate adipocyte differentiation (2Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, 8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar), are used clinically to reduce insulin resistance and hyperglycemia in type 2 diabetes (1Kersten S. Desvergne B. Wahli W. Nature. 2000; 405: 421-424Crossref PubMed Scopus (1701) Google Scholar, 2Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, 4Auwerx J. Diabetologia. 1999; 42: 1033-1049Crossref PubMed Scopus (587) Google Scholar, 5Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar). We and others (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar, 10Miles P.D. Barak Y. He W. Evans R.M. Olefsky J.M. J. Clin. Invest. 2000; 105: 287-292Crossref PubMed Scopus (377) Google Scholar) have reported that heterozygous PPARγ-deficient mice are protected from high fat (HF) diet- or aging-induced adipocyte hypertrophy, obesity, and insulin resistance. Consistent with this, the Pro-12 → Ala polymorphism in human PPARγ2, which moderately reduces the transcriptional activity of PPARγ, has been shown to confer resistance to type 2 diabetes (11Deeb S.S. Fajas L. Nemoto M. Pihlajamaki J. Mykkanen L. Kuusisto J. Laakso M. Fujimoto W. Auwerx J. Nat. Genet. 1998; 3: 284-287Crossref Scopus (1225) Google Scholar, 12Hara K. Okada T. Tobe K. Yasuda K. Mori Y. Kadowaki H. Hagura R. Akanuma Y. Kimura S. Ito C. Kadowaki T. Biochem. Biophys. Res. Commun. 2000; 271: 212-216Crossref PubMed Scopus (281) Google Scholar, 13Altshuler D. Hirschhorn J.N. Klannemark M. Lindgren C.M. Vohl M.C. Nemesh J. Lane C.R. Schaffner S.F. Bolk S. Brewer C. Tuomi T. Gaudet D. Hudson T.J. Daly M. Groop L. Lander E.S. Nat. Genet. 2000; 26: 76-80Crossref PubMed Scopus (124) Google Scholar). This apparent paradox raises the following important unresolved issue (14Kahn R.C. Chen L. Cohen S.E. J. Clin. Invest. 2000; 106: 1305-1307Crossref PubMed Scopus (151) Google Scholar) which we addressed experimentally in this study. We attempted to explain how insulin resistance could be improved by two opposite PPARγ activity states, supraphysiological activation of PPARγ and moderate reduction. We did so by using heterozygous PPARγ-deficient mice and a pharmacological activator of PPARγ in wild-type mice. We show here that supraphysiological activation of PPARγ by TZD markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARγ activity by heterozygous PPARγ deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating HF diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARγ deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARγ deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy. This results in a decrease in molecules causing insulin resistance such as free fatty acids (FFA) (15Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2220) Google Scholar) and tumor necrosis factor (TNF) α (16Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Crossref PubMed Scopus (707) Google Scholar) and up-regulation of insulin-sensitizing hormone adiponectin (17Yamauchi 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 (4165) Google Scholar), thereby leading to alleviation of insulin resistance. We conclude that, although by different mechanisms, both heterozygous PPARγ deficiency and PPARγ agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy. Rosiglitazone was synthesized as described elsewhere (6Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3492) Google Scholar). Wy-14,643 was purchased from Biomol (Plymouth Meeting, PA). All other materials were from the sources given in Refs. 8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar and9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar. Heterozygous PPARγ-deficient mice have been described (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). All other animals were purchased from Nippon CREA Co., Ltd. Six-week-old mice were fed powdered chow according to methods described previously (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). Drugs were given as food admixtures (8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar, 9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar), and there was no toxicity observed including liver damage. The area of glucose and insulin curves was calculated by multiplying the cumulative mean height of the glucose values (1 mg ml−1 = 1 cm) and insulin values (1 ng ml−1 = 1 cm), respectively, by time (60 min = 1 cm) as described in Ref. 7Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti Jr., J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (578) Google Scholar. The results are expressed as the percentage of the value of each controls. The insulin resistance index (7Mukherjee R. Davies P.J. Crombie D.L. Bischoff E.D. Cesario R.M. Jow L. Hamann L.G. Boehm M.F. Mondon C.E. Nadzan A.M. Paterniti Jr., J.R. Heyman R.A. Nature. 1997; 386: 407-410Crossref PubMed Scopus (578) Google Scholar) was calculated from the product of the areas of glucose and insulin × 10−2 in glucose tolerance test (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). The results are expressed as the ratio of the value of each wild-type controls on the high carbohydrate (HC) diet (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). Leptin was assayed with the enzyme-linked immunosorbent assay-based Quantikine M mouse leptin immunoassay kit (R & D Systems) according to the manufacturer's instructions. For leptin sensitivity (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar), leptin (PeproTech) was administered to mice as a daily intraperitoneal injection of 10 µg/g body weight/day. Isotonic sodium chloride solution was administered to the controls. Food intake and body weight were measured to assess the effects of leptin administration. Adipose tissue was removed from each animal, fixed in 10% formaldehyde/phosphate-buffered saline, and maintained at 4 °C until used. Fixed specimens were dehydrated, embedded in tissue-freezing medium (Tissue-Tek OCT compound; Miles), and frozen in dry ice and acetone. WAT was cut into 10-µm sections, and the sections were mounted on silanized slides. The adipose tissue was stained with hematoxylin and eosin. Mature white adipocytes were identified by their characteristic multilocular appearance. Total adipocyte areas were traced manually and analyzed with Win ROOF software (Mitani Co., Ltd., Chiba, Japan). White adipocyte areas were measured in 400 or more cells per mouse in each group according to the methods described previously (8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar, 9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). Sections of adipose tissues from mice treated for 14 days were stained by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique with a kit (In Situ Cell Death Detection Kit, AP; Roche Molecular Biochemicals) to detect apoptotic nuclei as described (8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar), with slight modifications. The numbers of all nuclei and apoptosis positive-stained nuclei were counted to calculate the ratio to the number of apoptotic nuclei to total number of nuclei. Total RNA was prepared from tissues with TRIzol (Life Technologies, Inc.) according to the manufacturer's instructions. Total RNA from 5 to 10 mice in each group was pooled, and aliquots were subjected to Northern blot analysis with the probes for rat acyl-CoA oxidase (Dr. T. Hashimoto), mouse CD36, UCP2, and adiponectin cDNA or RNase protection assay to measure mRNAs of TNFα performed using a standard protocol (8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar, 9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar, 18Murakami K. Tobe K. Ide T. Mochizuki T. Ohashi M. Akanuma Y. Yazaki Y. Kadowaki T. Diabetes. 1998; 47: 1841-1847Crossref PubMed Scopus (244) Google Scholar, 19Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). The radioactivity in each band was quantified, and the fold change in each mRNA was calculated after correction for loading differences by measuring the amount of 28 S rRNA. Very low levels (<10%) of adipocyte P2 mRNA were detected in muscle as compared with those in WAT. By contrast, CD36, SCD1, acyl-CoA oxidase, and UCP2 mRNAs were detected in muscle at levels comparable to those in WAT. These findings suggest that the results for muscle tissue essentially represent the results for the muscle cells, although the muscle was contaminated by a small amount (<10%) of inter-myocyte fat (20Vidal-Puig A. Jimenez-Linan M. Lowell B.B. Hamann A. Hu E. Spiegelman B. Flier J.S. Moller D.E. J. Clin. Invest. 1996; 97: 2553-2561Crossref PubMed Scopus (598) Google Scholar). The procedures used for PI3-kinase assay, immunoprecipitation, and immunoblotting have been described previously (21Yamauchi T. Tobe K. Tamemoto H. Ueki K. Kaburagi Y. Yamamoto-Honda R. Takahashi Y. Yoshizawa F. Aizawa S. Akanuma Y. Sonenberg N. Yazaki Y. Kadowaki T. Mol. Cell. Biol. 1996; 16: 3074-3084Crossref PubMed Scopus (251) Google Scholar). Representative data from one of three independent experiments are shown. The measurements of [14C]CO2 production from [1-14C]palmitic acid and lipogenesis from [1-14C]acetate were performed using liver, muscle, and WAT slices, as described (18Murakami K. Tobe K. Ide T. Mochizuki T. Ohashi M. Akanuma Y. Yazaki Y. Kadowaki T. Diabetes. 1998; 47: 1841-1847Crossref PubMed Scopus (244) Google Scholar, 22Mannaerts G.P. Debeer L.J. Thomas J. De Schepper P.J. J. Biol. Chem. 1979; 254: 4585-4595Abstract Full Text PDF PubMed Google Scholar). Liver and muscle homogenates were extracted, and their TG content was determined as described previously (18Murakami K. Tobe K. Ide T. Mochizuki T. Ohashi M. Akanuma Y. Yazaki Y. Kadowaki T. Diabetes. 1998; 47: 1841-1847Crossref PubMed Scopus (244) Google Scholar), with some modifications. To explain how insulin resistance could be improved by two opposite PPARγ activity states, supraphysiological activation of PPARγ and moderate reduction, we studied the phenotypes of untreated or PPARγ agonist-treated wild-type mice and untreated heterozygous PPARγ-deficient mice. We assessed PPARγ activity in vivoby measuring expression levels of lipoprotein lipase (23Schoonjans K. Peinado-Onsurbe J. Lefebvre A.M. Heyman R.A. Briggs M. Deeb S. Staels B. Auwerx J. EMBO J. 1996; 15: 5336-5348Crossref PubMed Scopus (1037) Google Scholar), fatty-acid translocase (FAT)/CD36 (24Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1633) Google Scholar), and adipocyte fatty acid-binding protein/adipocyte P2 (25Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3175) Google Scholar) (Fig.1A), whose promoters contain peroxisome proliferator response element, in WAT, where PPARγ is expressed most predominantly in vivo. As expected, rosiglitazone-treated wild-type mice exhibited a significant increase in PPARγ activity as compared with untreated wild-type mice (Fig.1A, lanes 1 and 2), whereas untreated heterozygous PPARγ-deficient mice showed a moderate decrease in PPARγ activity (Fig. 1A, lanes 1 and3). Untreated wild-type mice on the HF diet gained significantly more body weight than the mice on the HC diet (data not shown). Administration of rosiglitazone to wild-type mice increased significantly more body weight than vehicle on the HF diet (Fig. 1B, lanes 1 and 2). In contrast, heterozygous PPARγ deficiency reduced the increase in body weight on the HF diet (Fig. 1B, lanes 2 and 3). Treatment of wild-type mice with rosiglitazone significantly increased WAT mass (Fig. 1C,lanes 1 and 2), whereas untreated heterozygous PPARγ-deficient mice were protected from HF diet-induced increase in WAT mass (Fig. 1C, lanes 2 and 3). These data suggested that PPARγ determines the adiposity in proportion to its activity. Treatment of wild-type mice with rosiglitazone improved hyperglycemia (Fig. 1D, lanes 1) and hyperinsulinemia (Fig.1E, lane 1) on the HF diet as compared with untreated wild-type mice (Fig. 1, D and E, lane 2). Untreated heterozygous PPARγ-deficient mice were also protected from HF diet-induced hyperglycemia (Fig. 1D, lanes 2 and 3) and hyperinsulinemia (Fig. 1E, lanes 2 and 3). These findings indicate that TZD improve insulin sensitivity at the expense of obesity, whereas moderate reduction of PPARγ activity has potential as anti-obesity and anti-diabetic drugs. The rectal temperature was lower in rosiglitazone-treated wild-type mice than that in untreated wild-type mice (Fig. 2A, lanes 1 and 2); on the contrary, it was significantly higher in untreated heterozygous PPARγ-deficient mice (Fig. 2A, lanes 2 and 3). The serum leptin (26Friedman J.M. Nature. 2000; 404: 632-634Crossref PubMed Scopus (634) Google Scholar) levels were slightly but not significantly lower in rosiglitazone-treated wild-type mice than those of untreated wild-type mice (Fig. 2B,lanes 1 and 2), whereas they were significantly higher in untreated heterozygous PPARγ-deficient mice (Fig.2B, lanes 2 and 3). Thus the serum leptin levels were parallel to the rectal temperature. It was also noted that serum leptin levels were negatively correlated with PPARγ activity, suggesting that the serum leptin levels were parallel to the repression of leptin gene transcription by PPARγ/RXR (27Hollenberg A.N. Susulic V.S. Madura J.P. Zhang B. Moller D.E. Tontonoz P. Sarraf P. Spiegelman B.M. Lowell B.B. J. Biol. Chem. 1997; 272: 5283-5290Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Moreover, leptin sensitivity as assessed by reductions in food intake and body weight change in response to exogenously administered leptin was significantly increased in heterozygous PPARγ-deficient mice as compared with wild-type mice on the HF diet (Fig. 2C andD, lanes 3–6). The degree of change in body weight induced by leptin treatment differed significantly (p < 0.01) between wild-type (−0.67 ± 0.09%) and heterozygous PPARγ-deficient mice (−1.38 ± 0.11%). These data raised the possibility that increased leptin effects may contribute to the effects of the heterozygous PPARγ deficiency. In WAT from untreated heterozygous PPARγ-deficient mice, expressions of lipoprotein lipase and CD36 were reduced, which may contribute to decreased TG content. In addition, expressions of lipogenic enzymes such as sterol regulatory element-binding protein (SREBP) 1c and SCD (stearoyl-CoA desaturase) 1 were reduced, and lipid synthesis was indeed significantly decreased in WAT from heterozygous PPARγ-deficient mice as compared with that in wild-type mice on the HF diet (Fig. 2F). Expression of β3-adrenergic receptor (Fig. 2E, lanes 2 and 3) was increased presumably due to their increased leptin effects (Fig. 2, B–D) (28Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar) and decreased PPARγ/RXR effects (29Bakopanos E. Silva J.E. Diabetes. 2000; 49: 2108-2115Crossref PubMed Scopus (35) Google Scholar), and fatty acid oxidation was increased (data not shown). Decreased lipid synthesis and increased fatty acid oxidation as well as presumably decreased fatty acid influx in heterozygous PPARγ-deficient mice may in concert prevent adipocyte hypertrophy (Fig. 3A), and therefore obesity (Fig. 1, B and C, lanes 2 and 3), on the HF diet. Interestingly, supraphysiological activation of PPARγ significantly reduced the average size of adipocytes under the HF diet (Fig.3A, lane 1 and 2) as a result of a marked increase in the number of newly differentiated small adipocytes and significant decrease in the number of large adipocytes with a concomitant induction of apoptosis of adipocyte (Fig. 3B, lanes 1 and 2, and Fig. 3C) (8Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (937) Google Scholar). On the other hand, heterozygous PPARγ deficiency appeared to prevent HF diet-induced adipocyte hypertrophy without a significant change in the total number of adipocytes (Fig. 3B, lanes 2 and3 and Fig. 3C). These data suggest that the heterozygous PPARγ deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy. We next attempted to experimentally clarify the relationships between adipocyte hypertrophy and insulin resistance. To this end, we induced adipocyte hypertrophy by high fat feeding, leptin receptor deficiency, or agouti overexpression. The size of the adipose cells and the insulin resistance were increased in mice on a HF diet compared with those in mice on a HC diet (Fig. 3D). The size of the adipose cells and the insulin resistance of db/db mice were also increased compared with their wild-type controls on both the HC and HF diet (Fig. 3D). We obtained essentially similar results by using KKAy mice and their wild-type controls (KK) (Fig. 3E). These findings support a close correlation between adipocyte hypertrophy and insulin resistance, even though a cause and effect relationship is again unproven. In this context, protection from adipocyte hypertrophy due to decreased lipid synthesis in WAT from heterozygous PPARγ-deficient mice (Fig. 2F) may cause an increase in insulin sensitivity (Fig. 3F). We tried to clarify the molecular link between adipocyte hypertrophy and insulin resistance. We examined the levels of expression of molecules secreted by WAT that regulate insulin sensitivity under the following four different conditions: HC feeding, HF feeding, HF feeding with heterozygous PPARγ deficiency, and HF feeding with PPARγ agonist. The HF diet significantly increased adipocyte size and at the same time increased the molecules causing insulin resistance, such as FFA and TNFα (Fig. 3,G and H), and decreased the molecules causing insulin sensitivity, such as adiponectin (Fig. 3I), in mice that exhibited insulin resistance as compared with mice on the HC diet (Fig. 1, D and E, lanes 1 and2). (Replenishment of adiponectin in KKAy mice on the HF diet partially reverses insulin resistance even at the doses that do not significantly change adipocyte size (17Yamauchi 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 (4165) Google Scholar).) In addition, treatment of wild-type mice with the PPARγ agonist rosiglitazone or heterozygous PPARγ deficiency, both of which resulted in protection against HF diet-induced adipocyte hypertrophy, significantly decreased FFA and TNFα (Fig. 3, G and H) and increased adiponectin (Fig. 3I) and at the same time ameliorated insulin resistance (Fig. 1, D and E) on the HF diet. However, treatment of wild-type mice with a PPARγ agonist increased adipose tissue mass (Fig. 1C, lane 1) and body weight (Fig. 1B, lane 1), whereas heterozygous PPARγ deficiency significantly decreased them (Fig. 1,B and C, lane 3). These findings raised the possibility that levels of expression of molecules regulating insulin sensitivity may be more closely related to adipocyte size than PPARγ activity, adipose tissue mass, or body weightin vivo. Large adipocytes are known to be resistant to anti-lipolytic action of insulin, thereby releasing a large amount of FFA (30Askew E.W. Huston R.L. Plopper C.G. Hecker A.L. J. Clin. Invest. 1975; 56: 521-529Crossref PubMed Scopus (70) Google Scholar); however, the mechanisms underlying the correlation between larger adipocytes and up-regulation of TNFα and/or down-regulation of adiponectin remain to be elucidated. Interestingly, both supraphysiological activation of PPARγ and moderate reduction of PPARγ activity significantly reduced tissue TG content in muscle and liver (Fig.4A and Fig.5A), suggesting that insulin resistance has an excellent correlation with tissue TG content in muscle and liver (Fig. 1, D and E, Fig.4A, and Fig. 5A) (15Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2220) Google Scholar).Figure 5TZDindirectly decreases molecules involved inFFAinflux into the liver, whereas heterozygous PPARγ deficiency combusts fatty acid and decreases lipogenesis, thereby both decreasing tissueTGcontent in liver. Tissue triglyceride (A), amounts of the mRNAs of fatty-acid translocase (FAT)/CD36, SREBP 1, stearoyl Co-A desaturase (SCD) 1, acyl-CoA oxidase (ACO), and uncoupling protein (UCP) 2 (B), lipid synthesis from [14C]acetate in the liver (C), fatty acid (FA) oxidation (D), glucokinase, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (E), in liver of wild-type (WT) and heterozygous PPARγ-deficient mice (+/−) untreated (−) or treated with rosiglitazone (Rosi) for 4 weeks while on the HF diet. Rosiglitazone was given as a 0.01% food admixture. Fatty acid oxidation was assessed by the measurements of [14C]CO2 production from [14C]palmitic acid (D). Each bar represents the mean ± S.E. (n = 5–10). *, p< 0.05; **, p < 0.01; compared with untreated wild-type mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In wild-type mice treated with rosiglitazone, the decreased tissue TG content in muscle/liver, where PPARγ was less abundantly expressed as compared with what was in WAT, was presumably via reduced expression of molecules involved in FFA influx into muscle/liver (Fig. 4Eand Fig. 5B, lanes 1 and 2). On the other hand, heterozygous PPARγ deficiency reduced expression of lipogenic enzymes such as SCD1 (Fig. 4E and Fig. 5B, lanes 2 and 3) and SREBP 1 (Fig.5B, lanes 2 and 3), and indeed significantly reduced lipogenesis (Fig. 5C), presumably due to increased leptin effects (Fig. 2, B–D) (28Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar), may reduce tissue TG content in muscle/liver (Fig. 4A and Fig.5A, lanes 2 and 3). Moreover, in muscle/liver from heterozygous PPARγ-deficient mice, increased expression of enzymes involved in β-oxidation such as acyl-CoA oxidase and that of molecules involved in energy dissipation such as UCP2 (Fig. 4E and Fig.5B, lanes 2 and 3) were observed. Fatty acid oxidation was indeed significantly increased in muscle/liver from heterozygous PPARγ-deficient mice as compared with that in wild-type mice on the HF diet (Fig. 4F and Fig.5D). These alterations may be an additional mechanism for reduced TG content in muscle/liver of heterozygous PPARγ-deficient mice. Since these effects were recapitulated by treatment of wild-type mice with Wy-14,643, a PPARα agonist as reported (18Murakami K. Tobe K. Ide T. Mochizuki T. Ohashi M. Akanuma Y. Yazaki Y. Kadowaki T. Diabetes. 1998; 47: 1841-1847Crossref PubMed Scopus (244) Google Scholar, 19Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 31Kelly L.J. Vicario P.P. Thompson G.M. Candelore M.R. Doebber T.W. Ventre J. Wu M.S. Meurer R. Forrest M.J. Conner M.W. Cascier I.M.A. Moller D.E. Endocrinology. 1998; 139: 4920-4927Crossref PubMed Scopus (258) Google Scholar) (data not shown), PPARα pathways appeared to be activated in the liver of heterozygous PPARγ-deficient mice. In the BAT, where PPARα was relatively abundantly expressed compared to that in WAT, significant increases in the expression of molecules involved in fatty acid combustion presumably via activation of PPARα pathways (18Murakami K. Tobe K. Ide T. Mochizuki T. Ohashi M. Akanuma Y. Yazaki Y. Kadowaki T. Diabetes. 1998; 47: 1841-1847Crossref PubMed Scopus (244) Google Scholar, 19Motojima K. Passilly P. Peters J.M. Gonzalez F.J. Latruffe N. J. Biol. Chem. 1998; 273: 16710-16714Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 31Kelly L.J. Vicario P.P. Thompson G.M. Candelore M.R. Doebber T.W. Ventre J. Wu M.S. Meurer R. Forrest M.J. Conner M.W. Cascier I.M.A. Moller D.E. Endocrinology. 1998; 139: 4920-4927Crossref PubMed Scopus (258) Google Scholar) and β3-adrenergic receptor (Fig. 6), due to increased leptin effects (28Soukas A. Cohen P. Socci N.D. Friedman J.M. Genes Dev. 2000; 14: 963-980PubMed Google Scholar) and decreased PPARγ effects (29Bakopanos E. Silva J.E. Diabetes. 2000; 49: 2108-2115Crossref PubMed Scopus (35) Google Scholar), were observed. These alterations in concert may provide the mechanism that increased energy expenditure by heterozygous PPARγ deficiency (Fig.2A). Increased tissue TG content has been reported to interfere with insulin-stimulated phosphatidylinositol (PI) 3-kinase activation and subsequent GLUT4 translocation and glucose uptake (15Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2220) Google Scholar). Next, we tried to experimentally clarify the relationships between tissue TG content and insulin resistance. To do so, we increased tissue TG content by high fat feeding, leptin receptor deficiency, or agouti overexpression. The tissue TG content of skeletal muscle and insulin resistance were increased in mice on the HF diet compared with those in mice on the HC diet (Fig. 4B). The tissue TG content of skeletal muscle and insulin resistance of db/db mice were also increased compared with their wild-type controls on both the HC and HF diets (Fig.4B). We obtained essentially similar results by using KKAy mice and their wild-type controls (KK) (Fig. 4C). These findings raise the possibility that increases in tissue TG content are associated with insulin resistance. Conversely, decreased tissue TG content due to decreased lipid synthesis and increased fatty acid oxidation in muscle/liver from heterozygous PPARγ-deficient mice (Fig. 4F and Fig. 5, C and D) may cause an increase in insulin sensitivity (Fig. 4D). Shulman and co-workers (32Abel E.D. Peroni O. Kim J.K. Kim Y.B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (983) Google Scholar) proposed a cause and effect relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance. However, there are instances in which tissue TG content actually does not change in another scenario that also causes insulin resistance, i.e. adipose-selective targeting of the GLUT4 gene (32Abel E.D. Peroni O. Kim J.K. Kim Y.B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (983) Google Scholar). Thus, interpretation should be done with caution, and decreased tissue TG content in muscle/liver is one possible mechanism for the results of increased insulin sensitivity in heterozygous PPARγ-deficient mice. Consistent with this possibility, decreased TG content in muscle of heterozygous PPARγ-deficient mice indeed improved insulin signal transduction in muscle, as demonstrated by increases in insulin-induced tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2, and insulin-stimulated PI3-kinase activity in phosphotyrosine, IRS-1 and IRS-2 immunoprecipitates, and insulin-stimulated Akt activity in skeletal muscle (Fig.4G). The reduction of TG content in liver of heterozygous PPARγ-deficient mice was associated with increased expression of glucokinase and decreased expression of enzymes involved in gluconeogenesis such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (Fig. 5E), indicating increased insulin actions also in liver. We attempted to explain how insulin resistance could be improved by the following two opposite PPARγ activity states: a potent activation of PPARγ and its moderate reduction. We did so by using heterozygous PPARγ-deficient mice and a pharmacological activator of PPARγ in wild-type mice. On the basis of experimental results obtained in this study, we propose the following hypothesis on the mechanisms for the regulation of insulin sensitivity by PPARγ (Fig.7). As shown in the Fig. 7, panel 2, on the HF diet, “normal” amounts of PPARγ activity seen in wild-type mice increase TG content in WAT, skeletal muscle, and liver due to a combination of increased fatty acid influx into WAT, skeletal muscle, and liver and HF diet-induced leptin resistance, leading to insulin resistance and obesity. Moreover, hypertrophic adipocytes may increase the secretion of molecules causing insulin resistance, such as FFA (15Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2220) Google Scholar) and TNFα (16Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Crossref PubMed Scopus (707) Google Scholar), and decrease that of an insulin-sensitizing hormone, such as adiponectin (17Yamauchi 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 (4165) Google Scholar). As shown in Fig. 7, panel 1, supraphysiological activation of PPARγ way beyond that by TZD stimulates adipogenesis, which promotes a flux of FFA from liver and muscle into WAT, leading to a decrease in TG content in liver and muscle and improvement of insulin sensitivity at the expense of increased WAT mass, i.e.obesity. Moreover, TZD induce adipocyte differentiation and apoptosis, thereby increasing the number of small adipocytes, which finally lead to alleviation of insulin resistance presumably via a decrease in molecules causing insulin resistance, such as FFA and TNFα, and up-regulation of insulin-sensitizing hormone adiponectin, at least in part. By contrast, as shown in the Fig. 7, panel 3, moderate reduction of PPARγ activity observed in untreated heterozygous PPARγ-deficient mice decreases TG content in WAT, skeletal muscle, and liver. This effect is due to a combination of increased leptin expression by antagonism of PPARγ-mediated suppression of the gene, thereby reducing expression of lipogenic enzymes, and consequent activation of PPARα pathway in liver, BAT, and skeletal muscle, leading to an increase in expression of UCP2 and enzymes involved in β-oxidation. These observations fit well with the recently demonstrated effects of PPARα agonists on insulin resistance (33Guerre-Millo M. Gervois P. Raspe E. Madsen L. Poulain P. Derudas B. Herbert J.M. Winegar D.A. Willson T.M. Fruchart J.C. J. Biol. Chem. 2000; 275: 16638-16642Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar) and decreased fatty acid combustion in PPARα-deficient mice (34Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1390) Google Scholar). Moreover, direct antagonism of PPARγ to reduce lipogenesis in WAT prevents adipocyte hypertrophy under the HF diet, thereby reducing the molecules causing insulin resistance, such as FFA and TNFα, and up-regulating the insulin-sensitizing hormone adiponectin, at least in part. These alterations lead to prevention against HF diet-induced obesity and insulin resistance. The data showing that moderate reduction of PPARγ activity resulted in increased insulin sensitivity were further confirmed by the observation that treatment of heterozygous PPARγ-deficient mice with a low dose of TZD caused the re-emergence of insulin resistance (9Kubota 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. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar). This study has thus revealed the mechanisms whereby both PPARγ agonist and heterozygous PPARγ deficiency have a similar effect on insulin sensitivity. However, it should also be noted that PPARγ agonist and heterozygous PPARγ deficiency have an opposite effect on adiposity and energy expenditure which appear to be more directly regulated by PPARγ activity. Although both heterozygous PPARγ deficiency and PPARγ agonist finally improve insulin resistance via decreased TG content in muscle/liver and prevention of adipocyte hypertrophy, there are some important differences between them. First, although both reduced TG content in muscle/liver, heterozygous PPARγ deficiency did so via activation of fatty acid combustion and energy dissipation, whereas TZD did so via potent stimulation of adipogenesis, thereby increasing fatty acid flux from muscle/liver into WAT. Second, both prevented HF diet-induced adipocyte hypertrophy, and TZD markedly increased the number of newly differentiated small adipocytes, whereas heterozygous PPARγ deficiency appeared not to change the total number of adipocytes. Taken together, all of these differences are consistent with the notion that activation of PPARγ plays a role in energy storage and adiposity, and reduction of PPARγ causes energy dissipation and prevention of adiposity. In conclusion, although by different mechanisms, both heterozygous PPARγ deficiency and PPARγ agonist improve insulin resistance via decreased TG content in muscle/liver and prevention of adipocyte hypertrophy (Fig. 7). We thank S. Uchida, K. Kirii, S. Sakata, and T. Nagano for excellent technical assistance.

Inactivation of the klotho (kl) gene in mice results in multiple disorders that resemble human aging. The mouse kl gene encodes a novel single-pass membrane protein with homology to beta-glucosidases. In this study, we have isolated a human homologue of the kl gene and determined its gene structure. The human kl gene is composed of 5 exons and ranges over 50 kb on chromosome 13q12. We have further identified two transcripts that encode a membrane or secreted protein. These transcripts arise from a single kl gene through alternative RNA splicing. Expression of the putative secreted form predominates over that of the membrane form. The present study provides fundamental information necessary for further analyses of the human kl gene and its functions.

The high-molecular weight (HMW) form of adiponectin, an adipocyte-derived insulin-sensitizing hormone, has been reported to be the most active form of this hormone. We investigated whether measurement of plasma HMW adiponectin levels, using our newly developed enzyme-linked immunosorbent assay system for selective measurement of human HMW adiponectin level, may be useful for the prediction of insulin resistance and metabolic syndrome.A total of 298 patients admitted for diabetes treatment or coronary angiography served as study subjects. Receiver operator characteristic (ROC) curves for the HMW ratio (HMWR; ratio of plasma level of HMW adiponectin to that of total adiponectin) and plasma total adiponectin levels were plotted to predict the presence of insulin resistance and metabolic syndrome.The area under the ROC curve (AUC) of the HMWR values to predict the presence of insulin resistance was significantly larger than that of plasma total adiponectin level in total subjects (0.713 [95% CI 0.620-0.805] vs. 0.615 [0.522-0.708], P = 0.0160). The AUC for the HMWR values to predict the presence of metabolic syndrome was significantly larger than that for plasma total adiponectin levels in men (0.806 [0.747-0.865] vs. 0.730 [0.660-0.800], P = 0.0025) and in women (0.743 [0.659-0.828] vs. 0.637 [0.532-0.742], P = 0.0458).The HMWR value has better predictive power for the prediction of insulin resistance and metabolic syndrome than plasma total adiponectin level.

Adiponectin/Acrp30 is a hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic adipokine. We reported previously that AdipoR1 and -R2 serve as receptors for adiponectin and mediate increased fatty acid oxidation and glucose uptake by adiponectin. In the present study, we examined the expression levels and roles of AdipoR1/R2 in several physiological and pathophysiological states such as fasting/refeeding, obesity, and insulin resistance. Here we show that the expression of AdipoR1/R2 in insulin target organs, such as skeletal muscle and liver, is significantly increased in fasted mice and decreased in refed mice. Insulin deficiency induced by streptozotocin increased and insulin replenishment reduced the expression of AdipoR1/R2 <i>in vivo</i>. Thus, the expression of AdipoR1/R2 appears to be inversely correlated with plasma insulin levels <i>in vivo</i>. Interestingly, the incubation of hepatocytes or myocytes with insulin reduced the expression of AdipoR1/R2 via the phosphoinositide 3-kinase/Foxo1-dependent pathway <i>in vitro</i>. Moreover, the expressions of AdipoR1/R2 in ob/ob mice were significantly decreased in skeletal muscle and adipose tissue, which was correlated with decreased adiponectin binding to membrane fractions of skeletal muscle and decreased AMP kinase activation by adiponectin. This adiponectin resistance in turn may play a role in worsening insulin resistance in ob/ob mice. In conclusion, the expression of AdipoR1/R2 appears to be inversely regulated by insulin in physiological and pathophysiological states such as fasting/refeeding, insulin deficiency, and hyper-insulinemia models via the insulin/phosphoinositide 3-kinase/Foxo1 pathway and is correlated with adiponectin sensitivity.

To investigate the role of insulin receptor substrate (IRS)-2 in vivo, we generated IRS-2-deficient mice by gene targeting. Although homozygous IRS-2-deficient mice (IRS-2-/- mice) had a body weight similar to wild-type mice, they progressively developed type 2 diabetes at 10 weeks. IRS-2-/- mice showed insulin resistance and a defect in the insulin-stimulated signaling pathway in liver but not in skeletal muscle. Despite insulin resistance, the amount of beta-cells was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% increase in the amount of beta-cells in IRS-1-deficient mice (IRS-1-/- mice) to compensate for insulin resistance. Thus, IRS-2 plays a crucial role in the regulation of beta-cell mass. On the other hand, insulin secretion by the same number of cells in response to glucose measured ex vivo was significantly increased in IRS-2-/- mice compared with wild-type mice but was decreased in IRS-1-/- mice. These results suggest that IRS-1 and IRS-2 may play different roles in the regulation of beta-cell mass and the function of individual beta-cells.

Structural remodeling of the ventricular wall is a key determinant of clinical outcome in heart disease. Such remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and apoptotic and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Recent studies have shown that the interactions between cardiac fibroblasts and cardiomyocytes are essential for the progression of cardiac remodeling. This review addresses the functional role played by cardiac fibroblasts and the molecular mechanisms that govern their activity during cardiac hypertrophy and remodeling. A particular focus is the recent progress toward our understanding of the transcriptional regulatory mechanisms involved.

Consumption of foods high in saturated fatty acids (FAs) as well as elevated levels of circulating free FAs are known to be associated with T2D. Though previous studies showed inflammation is crucially involved in the development of insulin resistance, how inflammation contributes to β cell dysfunction has remained unclear. We report here the saturated FA palmitate induces β cell dysfunction in vivo by activating inflammatory processes within islets. Through a combination of in vivo and in vitro studies, we show β cells respond to palmitate via the TLR4/MyD88 pathway and produce chemokines that recruit CD11b(+)Ly-6C(+) M1-type proinflammatory monocytes/macrophages to the islets. Depletion of M1-type cells protected mice from palmitate-induced β cell dysfunction. Islet inflammation also plays an essential role in β cell dysfunction in T2D mouse models. Collectively, these results demonstrate a clear mechanistic link between β cell dysfunction and inflammation mediated at least in part via the FFA-TLR4/MyD88 pathway.

Resistin is an adipocytokine which plays a role in the development of insulin resistance. In this study, we investigated the direct effect of resistin on vascular endothelial cells. Resistin induced the expression of adhesion molecules such as VCAM-1 and ICAM-1, and long pentraxin 3, a marker of inflammation. The induction of VCAM-1 by resistin was inhibited partially by pitavastatin. Moreover, the induction of VCAM-1 and ICAM-1 by resistin was inhibited by adiponectin, an adipocytokine that improves insulin resistance. Taken together, these results suggest that the balance in the concentrations of adipocytokines such as resistin and adiponectin determines the inflammation status of vasculature, and in turn the progress of atherosclerosis.

Clinical profiles and outcomes of patients with acute type B aortic dissection have not been evaluated in the current era.Accordingly, we analyzed 384 patients (65+/-13 years, males 71%) with acute type B aortic dissection enrolled in the International Registry of Acute Aortic Dissection (IRAD). A majority of patients had hypertension and presented with acute chest/back pain. Only one-half showed abnormal findings on chest radiograph, and almost all patients had computerized tomography (CT), transesophageal echocardiography, magnetic resonance imaging (MRI), and/or aortogram to confirm the diagnosis. In-hospital mortality was 13% with most deaths occurring within the first week. Factors associated with increased in-hospital mortality on univariate analysis were hypotension/shock, widened mediastinum, periaortic hematoma, excessively dilated aorta (>or=6 cm), in-hospital complications of coma/altered consciousness, mesenteric/limb ischemia, acute renal failure, and surgical management (all P<0.05). A risk prediction model with control for age and gender showed hypotension/shock (odds ratio [OR] 23.8, P<0.0001), absence of chest/back pain on presentation (OR 3.5, P=0.01), and branch vessel involvement (OR 2.9, P=0.02), collectively named 'the deadly triad' to be independent predictors of in-hospital death.Our study provides insight into current-day profiles and outcomes of acute type B aortic dissection. Factors associated with increased in-hospital mortality ("the deadly triad") should be identified and taken into consideration for risk stratification and decision-making.

OBJECTIVE—The expansion of adipose tissue mass seen in obesity involves both hyperplasia and hypertrophy of adipocytes. However, little is known about how adipocytes, adipocyte precursors, blood vessels, and stromal cells interact with one another to achieve adipogenesis. RESEARCH DESIGN AND METHODS—We have developed a confocal microscopy-based method of three-dimensional visualization of intact living adipose tissue that enabled us to simultaneously evaluate angiogenesis and adipogenesis in db/db mice. RESULTS—We found that adipocyte differentiation takes place within cell clusters (which we designated adipogenic/angiogenic cell clusters) that contain multiple cell types, including endothelial cells and stromal cells that express CD34 and CD68 and bind lectin. There were close spatial and temporal interrelationships between blood vessel formation and adipogenesis, and the sprouting of new blood vessels from preexisting vasculature was coupled to adipocyte differentiation. CD34+ CD68+ lectin-binding cells could clearly be distinguished from CD34− CD68+ macrophages, which were scattered in the stroma and did not bind lectin. Adipogenic/angiogenic cell clusters can morphologically and immunohistochemically be distinguished from crown-like structures frequently seen in the late stages of adipose tissue obesity. Administration of anti–vascular endothelial growth factor (VEGF) antibodies inhibited not only angiogenesis but also the formation of adipogenic/angiogenic cell clusters, indicating that the coupling of adipogenesis and angiogenesis is essential for differentiation of adipocytes in obesity and that VEGF is a key mediator of that process. CONCLUSIONS—Living tissue imaging techniques provide novel evidence of the dynamic interactions between differentiating adipocytes, stromal cells, and angiogenesis in living obese adipose tissue.

Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor known to play a pivotal role in the pathogenesis of cardiovascular disease. Here, we show that neonatal heterozygous KLF5 knockout mice exhibit a marked deficiency in white adipose tissue development, suggesting that KLF5 is also required for adipogenesis. In 3T3-L1 preadipocytes, KLF5 expression was induced at an early stage of differentiation, and this was followed by expression of PPARgamma2. Constitutive overexpression of dominant-negative KLF5 inhibited adipocyte differentiation, whereas overexpression of wild-type KLF5 induced differentiation even without hormonal stimulation. Moreover, embryonic fibroblasts obtained from KLF5+/- mice showed much attenuated adipocyte differentiation, confirming the key role played by KLF5 in adipocyte differentiation. KLF5 expression is induced by C/EBPbeta and delta. KLF5, in turn, acts in concert with C/EBPbeta/delta to activate the PPARgamma2 promoter. This study establishes KLF5 as a key component of the transcription factor network controlling adipocyte differentiation.

Recent studies have established oxidative modification of low density lipoprotein (LDL) as an important atherogenic factor. We examined the clinical relevance of circulating oxidized LDL (OxLDL) levels in atherosclerotic disease by an enzyme immunoassay with use of specific antibodies against OxLDL (FOH1a/DLH3) and apolipoprotein B. Plasma OxLDL levels were significantly higher in patients with coronary heart disease (n=65) than in control subjects (n=181; 201. 3+/-11.2 versus 112.4+/-3.3 U/dL, respectively; P<0.01). OxLDL levels were not associated with age, sex, total cholesterol, or apolipoprotein B levels in normal control subjects. Our results suggest that circulating OxLDL may be a possible biochemical risk marker for coronary heart disease.

Endothelin-1 (ET-1) is a 21-amino acid peptide with various biological activities including vasoconstriction and cell proliferation. To clarify the physiological and pathophysiological role of ET-1, we disrupted the mouse Edn1 locus encoding ET-1 by gene targeting and demonstrated that ET-1 is essential to the normal development of pharyngeal arch-derived tissues and organs. In this study, we focused on the phenotypic manifestations of Edn1-/- homozygous mice in the cardiovascular system. Edn1-/- homozygotes display cardiovascular malformations including interrupted aortic arch (2.3%), tubular hypoplasia of the aortic arch (4.6%), aberrant right subclavian artery (12.9%), and ventricular septal defect with abnormalities of the outflow tract (48.4%). The frequency and extent of these abnormalities are increased by treatment with neutralizing monoclonal antibodies or a selective ETA receptor antagonist BQ123. At an earlier embryonic stage, formation of pharyngeal arch arteries and endocardial cushion is disturbed in Edn1-/- homozygotes. In situ hybridization confirmed ET-1 expression in the endothelium of the arch arteries and cardiac outflow tract and the endocardial cushion as well as in the epithelium of the pharyngeal arches. Thus, ET-1 is involved in the normal development of the heart and great vessels, and circulating ET-1 and/or other ET isoforms may cause a functional redundancy, at least partly, through the ETA receptor.

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Obesity is a common nutritional problem often associated with diabetes, insulin resistance, and fatty liver (excess fat deposition in liver). Leptin-deficient Lep(ob)/Lep(ob) mice develop obesity and those obesity-related syndromes. Increased lipogenesis in both liver and adipose tissue of these mice has been suggested. We have previously shown that the transcription factor sterol regulatory element-binding protein-1 (SREBP-1) plays a crucial role in the regulation of lipogenesis in vivo. To explore the possible involvement of SREBP-1 in the pathogenesis of obesity and its related syndromes, we generated mice deficient in both leptin and SREBP-1. In doubly mutant Lep(ob/ob) x Srebp-1(-/-) mice, fatty livers were markedly attenuated, but obesity and insulin resistance remained persistent. The mRNA levels of lipogenic enzymes such as fatty acid synthase were proportional to triglyceride accumulation in liver. In contrast, the mRNA abundance of SREBP-1 and lipogenic enzymes in the adipose tissue of Lep(ob)/Lep(ob) mice was profoundly decreased despite sustained fat, which could explain why the SREBP-1 disruption had little effect on obesity. In conclusion, SREBP-1 regulation of lipogenesis is highly involved in the development of fatty livers but does not seem to be a determinant of obesity in Lep(ob)/Lep(ob) mice.

Fibroblasts, which are the most numerous cell type in the heart, interact with cardiomyocytes in vitro and affect their function; however, they are considered to play a secondary role in cardiac hypertrophy and failure. Here we have shown that cardiac fibroblasts are essential for the protective and hypertrophic myocardial responses to pressure overload in vivo in mice. Haploinsufficiency of the transcription factor-encoding gene Krüppel-like factor 5 (Klf5) suppressed cardiac fibrosis and hypertrophy elicited by moderate-intensity pressure overload, whereas cardiomyocyte-specific Klf5 deletion did not alter the hypertrophic responses. By contrast, cardiac fibroblast-specific Klf5 deletion ameliorated cardiac hypertrophy and fibrosis, indicating that KLF5 in fibroblasts is important for the response to pressure overload and that cardiac fibroblasts are required for cardiomyocyte hypertrophy. High-intensity pressure overload caused severe heart failure and early death in mice with Klf5-null fibroblasts. KLF5 transactivated Igf1 in cardiac fibroblasts, and IGF-1 subsequently acted in a paracrine fashion to induce hypertrophic responses in cardiomyocytes. Igf1 induction was essential for cardioprotective responses, as administration of a peptide inhibitor of IGF-1 severely exacerbated heart failure induced by high-intensity pressure overload. Thus, cardiac fibroblasts play a pivotal role in the myocardial adaptive response to pressure overload, and this role is partly controlled by KLF5. Modulation of cardiac fibroblast function may provide a novel strategy for treating heart failure, with KLF5 serving as an attractive target.

Leptin-deficient ob/ob mice show many characteristics of obesity, including excess peripheral adiposity as well as severe hepatic steatosis, at least in part, due to increased hepatic lipogenesis. Polyunsaturated fatty acids (PUFAs) are not only ligands for peroxisome proliferator-activated receptor (PPAR) alpha but are also negative regulators of hepatic lipogenesis, which is thought to be mediated by the repression of sterol regulatory element-binding protein (SREBP)-1. We have previously shown that the disruption of SREBP-1 in ob/ob mice decreased their liver triglyceride storage. To examine whether PUFAs could reduce hepatic triglyceride deposition, we challenged ob/ob mice with dietary PUFA. It is demonstrated that PUFA markedly decreased the mature form of SREBP-1 protein and thereby reduced the expression of lipogenic genes such as fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) in the livers of ob/ob mice. Consequently, the liver triglyceride content and plasma alanine aminotransferase (ALT) levels were decreased. Furthermore, both hyperglycemia and hyperinsulinemia in ob/ob mice were improved by PUFA administration, similar to the effect of PPARalpha activators. In conclusion, PUFAs ameliorate obesity-associated symptoms, such as hepatic steatosis and insulin resistance, presumably through both down-regulation of SREBP-1 and activation of PPARalpha.

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones.

*Thiazolidinediones have been shown to up-regulate adiponectin expression in white adipose tissue and plasma adiponectin levels, and these up-regulations have been proposed to be a major mechanism of the thiazolidinedione-induced amelioration of insulin resistance linked to obesity. To test this hypothesis, we generated adiponectin knock-out (<i>adipo</i>^-/-) ob/ob mice with a C57B/6 background. After 14 days of 10 mg/kg pioglitazone, the insulin resistance and diabetes of ob/ob mice were significantly improved in association with significant up-regulation of serum adiponectin levels. Amelioration of insulin resistance in ob/ob mice was attributed to decreased glucose production and increased AMP-activated protein kinase in the liver but not to increased glucose uptake in skeletal muscle. In contrast, insulin resistance and diabetes were not improved in <i>adipo</i>^-/-ob/ob mice. After 14 days of 30 mg/kg pioglitazone, insulin resistance and diabetes of ob/ob mice were again significantly ameliorated, which was attributed not only to decreased glucose production in the liver but also to increased glucose uptake in skeletal muscle. Interestingly, <i>adipo</i>^-/-ob/ob mice also displayed significant amelioration of insulin resistance and diabetes, which was attributed to increased glucose uptake in skeletal muscle but not to decreased glucose production in the liver. The serum-free fatty acid and triglyceride levels as well as adipocyte sizes in ob/ob and <i>adipo</i>^-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were significantly reduced to a similar degree after 30 mg/kg pioglitazone. Moreover, the expressions of TNFα and resistin in adipose tissues of ob/ob and <i>adipo</i>^-/-ob/ob mice were unchanged after 10 mg/kg pioglitazone but were decreased after 30 mg/kg pioglitazone. Thus, pioglitazone-induced amelioration of insulin resistance and diabetes may occur adiponectin dependently in the liver and adiponectin independently in skeletal muscle.

Abstract We previously reported the characterization of a rabbit uterus cDNA clone (SMHC29) which encoded part of the light meromyosin of smooth muscle myosin heavy chain (Nagai, R., Larson, D.M., and Periasamy, M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1047-1051). We have now characterized a second cDNA clone (SMHC40) which also encodes part of the light meromyosin but differs from SMHC29 in the following respects. Nucleotide sequence analysis demonstrates that the two myosin heavy chain mRNAs are identical over 1424 nucleotides but differ in part of the 3'-carboxyl coding region and a portion of the 3'-nontranslated sequence. Specifically, SMHC40 cDNA encodes a unique stretch of 43 amino acids at the carboxyl terminus, whereas SMHC29 cDNA contains a shorter carboxyl terminus of 9 unique amino acids which is the result of a 39-nucleotide insertion. Recent peptide mapping of smooth muscle myosin heavy chain identified two isotypes with differences in the light meromyosin fragment that were designated as SM1 (204 kDa) and SM2 (200 kDa) type myosin (Eddinger, T. J., and Murphy, R.A. (1988) Biochemistry 27, 3807-3811). In this study we present direct evidence that SMHC40 and SMHC29 mRNA encode the two smooth muscle myosin heavy chain isoforms, SM1 and SM2, respectively, by immunoblot analysis using antibodies against specific carboxyl terminus sequences deduced from SMHC40 and SMHC29 cDNA clones.

The finding of islet inflammation in type 2 diabetes (T2D) and its involvement in β cell dysfunction has further highlighted the significance of inflammation in metabolic diseases. The number of intra-islet macrophages is increased in T2D, and these cells are the main source of proinflammatory cytokines within islets. Multiple human studies of T2D have shown that targeting islet inflammation has the potential to be an effective therapeutic strategy. In this Review we provide an overview of the cellular and molecular mechanisms by which islet inflammation develops and causes β cell dysfunction. We also emphasize the regulation and roles of macrophage polarity shift within islets in the context of T2D pathology and β cell health, which may have broad translational implications for therapeutics aimed at improving islet function.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Renal tubulointerstitial damage is the final common pathway leading from chronic kidney disease to end-stage renal disease. Inflammation is clearly involved in tubulointerstitial injury, but it remains unclear how the inflammatory processes are initiated and regulated. Here, we have shown that in the mouse kidney, the transcription factor Krüppel-like factor-5 (KLF5) is mainly expressed in collecting duct epithelial cells and that Klf5 haploinsufficient mice (Klf5+/- mice) exhibit ameliorated renal injury in the unilateral ureteral obstruction (UUO) model of tubulointerstitial disease. Additionally, Klf5 haploinsufficiency reduced accumulation of CD11b+ F4/80(lo) cells, which expressed proinflammatory cytokines and induced apoptosis among renal epithelial cells, phenotypes indicative of M1-type macrophages. By contrast, it increased accumulation of CD11b+ F4/80(hi) macrophages, which expressed CD206 and CD301 and contributed to fibrosis, in part via TGF-β production--phenotypes indicative of M2-type macrophages. Interestingly, KLF5, in concert with C/EBPα, was found to induce expression of the chemotactic proteins S100A8 and S100A9, which recruited inflammatory monocytes to the kidneys and promoted their activation into M1-type macrophages. Finally, assessing the effects of bone marrow-specific Klf5 haploinsufficiency or collecting duct- or myeloid cell-specific Klf5 deletion confirmed that collecting duct expression of Klf5 is essential for inflammatory responses to UUO. Taken together, our results demonstrate that the renal collecting duct plays a pivotal role in the initiation and progression of tubulointerstitial inflammation.

Intravital visualization of thrombopoiesis revealed that formation of proplatelets, which are cytoplasmic protrusions in bone marrow megakaryocytes (MKs), is dominant in the steady state. However, it was unclear whether this is the only path to platelet biogenesis. We have identified an alternative MK rupture, which entails rapid cytoplasmic fragmentation and release of much larger numbers of platelets, primarily into blood vessels, which is morphologically and temporally different than typical FasL-induced apoptosis. Serum levels of the inflammatory cytokine IL-1α were acutely elevated after platelet loss or administration of an inflammatory stimulus to mice, whereas the MK-regulator thrombopoietin (TPO) was not elevated. Moreover, IL-1α administration rapidly induced MK rupture-dependent thrombopoiesis and increased platelet counts. IL-1α-IL-1R1 signaling activated caspase-3, which reduced plasma membrane stability and appeared to inhibit regulated tubulin expression and proplatelet formation, and ultimately led to MK rupture. Collectively, it appears the balance between TPO and IL-1α determines the MK cellular programming for thrombopoiesis in response to acute and chronic platelet needs.

<h2>Summary</h2> Distinct B cell populations, designated regulatory B (B_reg) cells, are known to restrain immune responses associated with autoimmune diseases. Additionally, obesity is known to induce local inflammation within adipose tissue that contributes to systemic metabolic abnormalities, but the underlying mechanisms that modulate adipose inflammation remain poorly understood. We identified B_reg cells that produce interleukin-10 constitutively within adipose tissue. B cell-specific <i>Il10</i> deletion enhanced adipose inflammation and insulin resistance in diet-induced obese mice, whereas adoptive transfer of adipose tissue B_reg cells ameliorated those effects. Adipose environmental factors, including CXCL12 and free fatty acids, support B_reg cell function, and B_reg cell fraction and function were reduced in adipose tissue from obese mice and humans. Our findings indicate that adipose tissue B_reg cells are a naturally occurring regulatory B cell subset that maintains homeostasis within adipose tissue and that B_reg cell dysfunction contributes pivotally to the progression of adipose tissue inflammation in obesity.

Background: Current guidelines call for high-intensity statin therapy in patients with cardiovascular disease on the basis of several previous “more versus less statins” trials. However, no clear evidence for more versus less statins has been established in an Asian population. Methods: In this prospective, multicenter, randomized, open-label, blinded end point study, 13 054 Japanese patients with stable coronary artery disease who achieved low-density lipoprotein cholesterol (LDL-C) &lt;120 mg/dL during a run-in period (pitavastatin 1 mg/d) were randomized in a 1-to-1 fashion to high-dose (pitavastatin 4 mg/d; n=6526) or low-dose (pitavastatin 1 mg/d; n=6528) statin therapy. The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal ischemic stroke, or unstable angina requiring emergency hospitalization. The secondary composite end point was a composite of the primary end point and clinically indicated coronary revascularization excluding target-lesion revascularization at sites of prior percutaneous coronary intervention. Results: The mean age of the study population was 68 years, and 83% were male. The mean LDL-C level before enrollment was 93 mg/dL with 91% of patients taking statins. The baseline LDL-C level after the run-in period on pitavastatin 1 mg/d was 87.7 and 88.1 mg/dL in the high-dose and low-dose groups, respectively. During the entire course of follow-up, LDL-C in the high-dose group was lower by 14.7 mg/dL than in the low-dose group ( P &lt;0.001). With a median follow-up of 3.9 years, high-dose as compared with low-dose pitavastatin significantly reduced the risk of the primary end point (266 patients [4.3%] and 334 patients [5.4%]; hazard ratio, 0.81; 95% confidence interval, 0.69–0.95; P =0.01) and the risk of the secondary composite end point (489 patients [7.9%] and 600 patients [9.7%]; hazard ratio, 0.83; 95% confidence interval, 0.73–0.93; P =0.002). High-dose pitavastatin also significantly reduced the risks of several other secondary end points such as all-cause death, myocardial infarction, and clinically indicated coronary revascularization. The results for the primary and the secondary composite end points were consistent across several prespecified subgroups, including the low (&lt;95 mg/dL) baseline LDL-C subgroup. Serious adverse event rates were low in both groups. Conclusions: High-dose (4 mg/d) compared with low-dose (1 mg/d) pitavastatin therapy significantly reduced cardiovascular events in Japanese patients with stable coronary artery disease. Clinical Trial Registration: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01042730.

Dietary polyunsaturated fatty acids (PUFA) are negative regulators of hepatic lipogenesis that exert their effects primarily at the level of transcription.Sterol regulatory element-binding proteins (SREBPs) are transcription factors responsible for the regulation of cholesterol, fatty acid, and triglyceride synthesis.In particular, SREBP-1 is known to play a crucial role in the regulation of lipogenic gene expression in the liver.To explore the possible involvement of SREBP-1 in the suppression of hepatic lipogenesis by PUFA, we challenged wild-type mice and transgenic mice overexpressing a mature form of SREBP-1 in the liver with dietary PUFA.In the liver of wild-type mice, dietary PUFA drastically decreased the mature, cleaved form of SREBP-1 protein in the nucleus, whereas the precursor, uncleaved form in the membranes was not suppressed.The decreases in mature SREBP-1 paralleled those in mRNAs for lipogenic enzymes such as fatty acid synthase and acetyl-CoA carboxylase.In the transgenic mice, dietary PUFA did not reduce the amount of transgenic SREBP-1 protein, excluding the possibility that PUFA accelerated the degradation of mature SREBP-1.The resulting sustained expression of mature SREBP-1 almost completely canceled the suppression of lipogenic gene expression by PUFA in the SREBP-1 transgenic mice.These results demonstrate that the suppressive effect of PUFA on lipogenic enzyme genes in the liver is caused by a decrease in the mature form of SREBP-1 protein, which is presumably due to the reduced cleavage of SREBP-1 precursor protein.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

We and others have suggested that bone marrow-derived progenitor cells may contribute to the pathogenesis of vascular diseases. On the other hand, it was reported that bone marrow cells do not participate substantially in vascular remodeling in other experimental systems. In this study, three distinct types of mechanical vascular injuries were induced in the same mouse whose bone marrow had been reconstituted with that of GFP or LacZ mice. All injuries are known to cause smooth muscle cell (SMC) hyperplasia. At 4 weeks after wire-mediated endovascular injury, a significant number of the neointimal and medial cells derived from bone marrow. In contrast, marker-positive cells were seldom detected in the lesion induced by perivascular cuff replacement. There were only a few bone marrow-derived cells in the neointima after ligation of the common carotid artery. These results indicate that the origin of intimal cells is diverse and that contribution of bone marrow-derived cells to neointimal hyperplasia depends on the type of model.

Adult rabbit smooth muscles contain two types of myosin heavy chain (MHC) isoforms, SM1 and SM2 which are generated through alternative RNA splicing from a single gene (Nagai, R., Kuro-o, M., Babij, P. & Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). We previously reported that the expression of SM1 and SM2 during vascular development is differentially regulated at the level of RNA splicing, whereby SM1 is constitutively expressed from early development but SM2 appear after birth (Kuro-o, M., Nagai, R., Tsuchimochi, H., Katoh, H., Yazaki, Y., Ohkubo, A. & Takaku, F. (1989) J. Biol. Chem. 264, 18272-18275). We also demonstrated that embryonic vascular smooth muscles contain a third type of MHC isoform, referred to as SMemb in this report, which comigrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SM2. In the present study we have isolated and characterized a cDNA clone (FSMHC34) for SMemb. FSMHC34 encodes the light meromyosin region including the carboxyl terminus and showed 70% amino acid sequence identity with SM1 or SM2. SMemb is a nonmuscle-type MHC and identical with brain MHC, but clearly distinct from 196-kDa nonmuscle MHC in cultured smooth muscle cells. The expression of SMemb was predominant in embryonic and perinatal aortas, but down-regulated with vascular development. Interestingly SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.

We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termed klotho (kl), leads to a syndrome resembling human aging in mice. The kl gene encodes a single-pass membrane protein whose extracellular domain carries homology to beta-glucosidases. In this report, we present the entire mouse kl gene organization. The mouse kl gene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA-box and contains four potential binding sites for SP1. We further show that two kl gene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of the kl gene which may regulate aging in vivo.

There are few data available on possible independent association between uric acid and carotid atherosclerosis. Here we first sought to investigate association between uric acid levels and metabolic syndrome in Japanese; second, we assessed whether there is an independent association of uric acid with prevalence of carotid atherosclerosis in individuals subdivided according to gender and metabolic syndrome status.Cross-sectional data from 8144 individuals who underwent general health screening were analyzed. After adjusting for age, total cholesterol, and smoking status, the odds ratios (95% CI) of sex-specific quartiles of serum uric acid for metabolic syndrome were 1.0, 1.06 (0.60 to 1.87), 2.18 (1.30 to 3.64), and 4.17 (2.56 to 6.79) in women, and 1.0, 0.92 (0.74 to 1.14), 1.52 (1.25 to 1.65), and 1.97 (1.61 to 2.40) in men. After adjusting for age, serum levels, total cholesterol, and smoking status, prevalence of carotid plaque was higher in subjects in the second, third, and fourth quartiles of uric acid level with odds ratios (95% CI) of 1.24 (1.01 to 1.52), 1.37 (1.11 to 1.68), and 1.31 (1.05 to 1.63), respectively, in men without metabolic syndrome but not in men with metabolic syndrome or in women with or without metabolic syndrome.The prevalence of metabolic syndrome showed a graded increase according to serum uric acid values in both genders. In men who did not have metabolic syndrome, uric acid was found to be an independent risk factor for incidence of carotid plaque.

Atherosclerotic coronary heart disease is a common complication of the insulin resistance syndrome that can occur with or without diabetes mellitus. Thiazolidinediones (TZDs), which are insulin-sensitizing antidiabetic agents, can modulate the development of atherosclerosis not only by changing the systemic metabolic conditions associated with insulin resistance but also by exerting direct effects on vascular wall cells that express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a nuclear receptor for TZDs. Here we show that troglitazone, a TZD, significantly inhibited fatty streak lesion formation in apolipoprotein E-knockout mice fed a high-fat diet (en face aortic surface lesion areas were 6.9+/-2.5% vs 12.7+/-4.7%, P<0.05; cross-sectional lesion areas were 191 974+/-102 911 micrometer(2) vs 351 738+/-175 597 micrometer(2), P<0.05; n=10). Troglitazone attenuated hyperinsulinemic hyperglycemia and increased high density lipoprotein cholesterol levels. In the aorta, troglitazone markedly increased the mRNA levels of CD36, a scavenger receptor for oxidized low density lipoprotein, presumably by upregulating its expression, at least in part, in the macrophage foam cells. These results indicate that troglitazone potently inhibits fatty streak lesion formation by modulating both metabolic extracellular environments and arterial wall cell functions.

The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.

We investigated the relation between positivity for hepatitis C virus (HCV) and carotid-artery plaque and carotid intima-media thickening by analysing cross-sectional data of individuals undergoing a general health screening test. Of 4784 individuals enrolled, 104 (2·2%) were seropositive for HCV. After adjustment for confounding risk factors, HCV seropositivity was found to be associated with an increased risk of carotid-artery plaque (odds ratio 1·92 [95% CI 1·56–2·38], p=0·002) and carotid intima-media thickening (2·85 [2·28–3·57], p<0·0001). These findings suggest a possible role for chronic hepatitis C in the pathogenesis of carotid arterial remodelling.

The sarcoplasmic reticulum (SR) and the contractile protein myosin play an important role in myocardial performance. Both of these systems exhibit plasticity--i.e., quantitative and/or qualitative reorganization during development and in response to stress. Recent studies indicate that SR Ca2+ uptake function is altered in adaptive cardiac hypertrophy and failure. The molecular basis (genetic and phenotypic) for these changes is not understood. In an effort to determine the underlying causes of these changes, we characterized the rabbit cardiac Ca2+-ATPase phenotype by molecular cloning and ribonuclease A mapping analysis. Our results show that the heart muscle expresses only the slow-twitch SR Ca2+-ATPase isoform. Second, we quantitated the steady-state mRNA levels of two major SR Ca2+ regulatory proteins, the Ca2+-ATPase and phospholamban, to see whether changes in mRNA content might provide insight into the basis for functional modification in the SR of hypertrophied hearts. In response to pressure overload hypertrophy, the relative level of the slow-twitch/cardiac SR Ca2+-ATPase mRNA was decreased to 34% of control at 1 week. The relative Ca2+-ATPase mRNA level increased to 167% of control after 3 days of treatment with thyroid hormone. In contrast, in hypothyroid animals, the relative Ca2+-ATPase mRNA level decreased to 51% of control at 2 weeks. The relative level of phospholamban mRNA was decreased to 36% in 1-week pressure overload. Hyperthyroidism induced a decrease to 61% in the phospholamban mRNA level after 3 days of treatment, while hypothyroidism had virtually no effect on phospholamban mRNA levels. These data indicate that the expression of SR Ca2+-ATPase and phospholamban mRNA may not be coordinately regulated during myocardial adaptation to different physiological conditions.

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.

Genetically modified mice serve as a powerful tool to determine the role of specific molecules in a wide variety of biological phenomena including vascular remodeling. Several models of arterial injury have been proposed to analyze transgenic/knock-out mice, but many questions have been raised about their reproducibility and physiological significance. Here, we report a new mouse model of vascular injury that resembles balloon-angioplasty. A straight spring wire was inserted into the femoral artery via arterioctomy in a small muscular branch. The wire was left in place for one minute to denude and dilate the artery. After the wire was removed, the muscular branch was tied off and the blood flow of the femoral artery was restored. The lumen was enlarged with rapid onset of medial cell apoptosis. While the circumference of the external elastic lamina remained enlarged, the lumen was gradually narrowed by neointimal hyperplasia composed of smooth muscle cells. At 4 weeks, a concentric and homogeneous neointimal lesion was formed reproducibly in the region where the wire had been inserted. Similar exuberant hyperplasia could be induced in all strains examined (C57BL/6J, C3H/HeJ, BALB/c, and 129/SVj). This model may be widely used to study the molecular mechanism of post-angioplasty restenosis at the genetic level.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. Levels of AM are markedly increased in the fetoplacental circulation during pregnancy, although its function there remains unknown. To clarify the physiological functions of AM, we chose a gene-targeting strategy in mice.Targeted null mutation of the AM gene is lethal in utero: the mortality rate among AM(-/-) embryos was >80% at E13.5. The most apparent abnormality in surviving AM(-/-) embryos at E13.5 to E14.0 was severe hemorrhage, readily observable under the skin and in visceral organs. Hemorrhage was not detectable at E12.5 to E13.0, although the yolk sac lacked well-developed vessels. Electron microscopic examination showed endothelial cells to be partially detached from the basement structure at E12.5 in vitelline vessels and hepatic capillaries, which allowed efflux of protoerythrocytes through the disrupted barrier. The basement membrane was not clearly recognizable in the aorta and cervical artery, and the endothelial cells stood out from the wall of the lumen, only partially adhering to the basement structure. AM(+/-) mice survived to adulthood but exhibited elevated blood pressures with diminished nitric oxide production.AM is indispensable for the vascular morphogenesis during embryonic development and for postnatal regulation of blood pressure by stimulating nitric oxide production.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3x10(10) plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.

Arteriosclerosis caused by aging is recognized to be a crucial risk factor of cardiovascular disease. We recently establishedklothomouse which causes age-related disorders including arteriosclerosis. However, no information on endothelial function ofklothomouse or the physiological role of klotho protein as a circulating factor is available. In this report, we demonstrate that 50% effective dose of aortic relaxation in response to acetylcholine in heterozygousklothomice is significantly greater (4 × 10−5M) than in wild-type mice (8 × 10−6M, n = 7, p < 0.05) and that the vasodilator response of arterioles to acetylcholine is significantly attenuated in heterozygous (20% effective dose; 2 × 10−6M) and homozygousklothomice (>1 × 10−5M) as compared with wild-type mice (1 × 10−7M, n = 7, p < 0.05). Nitric oxide metabolites (NO−2and NO−3) in urine are significantly lower in heterozygousklothomice (142 ± 16 nmol/day) than wild-type mice (241 ± 28 nmol/day, n = 13, p < 0.05). Parabiosis between wild-type and heterozygousklothomice results in restoration of endothelial function in heterozygousklothomice. We conclude that the klotho protein protects the cardiovascular system through endothelium-derived NO production by humoral pathways.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

to have different carboxyl termini (Nagai, R.,

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-kappaB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL(-/-)), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.

Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent. Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-differentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in part on the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level. We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes, such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue. In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved in this activation.

We previously demonstrated that insulin receptor substrate 2 (Irs2) KO mice develop diabetes associated with hepatic insulin resistance, lack of compensatory beta cell hyperplasia, and leptin resistance. To more precisely determine the roles of Irs2 in beta cells and the hypothalamus, we generated beta cell-specific Irs2 KO and hypothalamus-specific Irs2 knockdown (betaHT-IRS2) mice. Expression of Irs2 mRNA was reduced by approximately 90% in pancreatic islets and was markedly reduced in the arcuate nucleus of the hypothalamus. By contrast, Irs2 expression in liver, muscle, and adipose tissue of betaHT-IRS2 mice was indistinguishable from that of control mice. The betaHT-IRS2 mice displayed obesity and leptin resistance. At 4 weeks of age, the betaHT-IRS2 mice showed normal insulin sensitivity, but at 8 and 12 weeks, they were insulin resistant with progressive obesity. Despite their normal insulin sensitivity at 8 weeks with caloric restriction, the betaHT-IRS2 mice exhibited glucose intolerance and impaired glucose-induced insulin secretion. beta Cell mass and beta cell proliferation in the betaHT-IRS2 mice were reduced significantly at 8 and 12 weeks but not at 10 days. Insulin secretion, normalized by cell number per islet, was significantly increased at high glucose concentrations in the betaHT-IRS2 mice. We conclude that, in beta cells and the hypothalamus, Irs2 is crucially involved in the regulation of beta cell mass and leptin sensitivity.

<h2>Summary</h2> Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.

Leptin-deficient mice (ob/ob) are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study, we show an unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein receptor (LDLR) deficiency (−/−). Doubly mutant mice (LDLR−/−;ob/ob) exhibited striking elevations in both total plasma cholesterol (TC) and triglyceride (TG) levels (1715 ± 87 and 1016 ± 172 mg/dl, respectively), at age 3–4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months. Lipoprotein analyses revealed the elevated TC and TG levels to be due to a large increase in an apoB-containing broad-β remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin treatment significantly lowered TG levels, they caused only slight changes in TC levels. Hepatic cholesterol and triglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence that the hypertriglyceridemia and hypercholesterolemia in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might have some impact on plasma cholesterol metabolism, possibly through an LDLR-independent pathway. This model will be an excellent tool for future studies on the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis. Leptin-deficient mice (ob/ob) are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study, we show an unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein receptor (LDLR) deficiency (−/−). Doubly mutant mice (LDLR−/−;ob/ob) exhibited striking elevations in both total plasma cholesterol (TC) and triglyceride (TG) levels (1715 ± 87 and 1016 ± 172 mg/dl, respectively), at age 3–4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months. Lipoprotein analyses revealed the elevated TC and TG levels to be due to a large increase in an apoB-containing broad-β remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin treatment significantly lowered TG levels, they caused only slight changes in TC levels. Hepatic cholesterol and triglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence that the hypertriglyceridemia and hypercholesterolemia in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might have some impact on plasma cholesterol metabolism, possibly through an LDLR-independent pathway. This model will be an excellent tool for future studies on the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis. leptin-deficient mice low density lipoprotein receptor total plasma cholesterol total plasma triglycerides LDLR-related protein high density lipoproteins low density lipoproteins very low density lipoprotein intermediate density lipoproteins sterol regulatory element-binding protein LDLR−/−, SREBP-1a transgenic mice crossed with LDLR(−/−) mice apolipoprotein high performance liquid chromatography phosphate-buffered saline Dyslipidemia, diabetes, and obesity are among the many risk factors associated with coronary artery disease. Although many human patients present with varying combinations of these risk factors, most animal models produced to date have focused on only one or two and their effects on atherogenesis. Leptin-deficient mice (ob/ob)1 are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis (1Coleman D.L. Diabetologia. 1978; 14: 141-148Crossref PubMed Scopus (1085) Google Scholar, 2Zhang Y. Proenca R. Maffel M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11755) Google Scholar). These mice also have fatty livers, presumably due to hepatic overproduction of triglycerides, reflecting an imbalanced energy metabolism. However, they show only a modest increase in plasma triglyceride and HDL cholesterol levels (3Nishina P.M. Lowe S. Wang J. Paigen B. Metabolism. 1994; 43: 549-553Abstract Full Text PDF PubMed Scopus (101) Google Scholar) and thus do not develop atherosclerosis on a regular chow diet as shown in the current study. It has been reported that ob/ob mice might have an impaired secretion of very low density lipoproteins (VLDL) (4Li X. Grundy S.M. Patel S.B. J. Lipid Res. 1997; 38: 1277-1288Abstract Full Text PDF PubMed Google Scholar); however, extensive studies on apoB-containing lipoprotein metabolism in these mice have not been undertaken. Remnant lipoprotein particles are the apolipoprotein B (apoB)-containing lipoproteins that remain in the circulation after hepatic VLDL and intestinal chylomicrons are lipolyzed. They include lipoproteins in the size range of VLDL, intermediate density lipoproteins (IDL), and low density lipoproteins (LDL). Remnant particles can exist in individuals in a normolipidemic state, especially postprandially, but are generally processed by lipoprotein lipase and quickly cleared from plasma. However, the triglyceride-rich lipoproteins that are increased in diabetic patients as well as cholesterol-rich remnants referred to as β-VLDL that are found in patients with type III hyperlipidemia are also remnant lipoproteins and in these cases are known to be atherogenic. Several mouse models of chronically increased remnant lipoproteins have recently been created by targeted disruption of genes that are crucial in lipoprotein metabolism. These include apoE-deficient mice (5Plump A.S. Smith J.D. Hayek T. Aalto-Setälä K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1874) Google Scholar, 6Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Science. 1992; 258: 468-471Crossref PubMed Scopus (1841) Google Scholar), LDL receptor-deficient mice on a high fat diet (7Ishibashi S. Goldstein J.L. Brown M.S. Herz J. Burns D.K. J. Clin. Invest. 1994; 93: 1885-1893Crossref PubMed Scopus (600) Google Scholar), and LDLR/LDLR-related protein (LRP) double knockout mice (8Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (400) Google Scholar), confirming the importance of apoE as a ligand and LDLR and LRP as receptors for plasma remnant clearance. Recently, it was reported that disruption of the LDLR gene unmasked severe hyperlipidemia with increased remnant particles in sterol regulatory element-binding protein (SREBP)-1a transgenic mice (TgSREBP-1a;LDLR−/−), providing another model for studying the effects of excessive remnant lipoproteins in plasma (9Horton J.D. Shimano H. Hamilton R.L. Brown M.S. Goldstein J.L. J. Clin. Invest. 1999; 103: 1067-1076Crossref PubMed Scopus (158) Google Scholar). In this model, hepatic overexpression of nuclear SREBP-1a, a potent transcription activator for both cholesterol and triglyceride synthesis, caused overproduction of lipids and large VLDL in the liver. Normally, these large particles can be taken up by the LDLR very efficiently, which prevented the development of hyperlipidemia in the TgSREBP-1a mice; however, in the absence of the LDLR, remnant lipoproteins accumulated in plasma, providing support for the importance of the LDLR in remnant clearance. The TgSREBP-1a;LDLR−/− mouse model is different from other hyperlipidemic animal models because hepatic lipid overproduction, rather than simply a lack of lipoprotein clearance, contributes to the pathological mechanism. This observation prompted us to study the metabolic consequences of hepatic triglyceride overproduction as seen in ob/ob mice, on the background of a state of impaired remnant clearance. We sought to analyze the effects of leptin deficiency on remnant lipoprotein metabolism by producing mice that were deficient in both leptin and the LDLR (LDLR−/−;ob/ob). These doubly mutant mice showed extreme elevations in both plasma cholesterol and triglyceride levels and also had extensive atherosclerotic lesions throughout the aorta. While fasting, diet restriction, and low level leptin treatment of LDLR−/−;ob/ob mice significantly lowered TG levels, they caused only slight changes in total plasma cholesterol (TC) levels. This model supports the idea that a relationship exists between energy imbalance/hepatic overproduction of triglycerides and plasma remnant accumulation during impaired lipoprotein clearance. LDLR(−/−) mice (10Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1273) Google Scholar) were back-crossed onto a C57Bl/6 background to the 10th generation. ob/+ mice on a C57Bl/6 background were purchased from Jackson Laboratories and were also maintained in our colonies. To obtain leptin deficient (ob/ob) on a background of LDLR deficiency, ob/+ mice were crossed with LDLR(−/−) mice, and the F1 progeny of these matings (LDLR+/−;ob/+) were then crossed to obtain mice that had either zero, one, or both normal LDLR alleles and were leptin-deficient (LDLR−/−;ob/ob, LDLR+/−;ob/ob, and LDLR+/+;ob/ob, respectively) as well as control LDLR(−/−), LDLR(+/−), and wild type mice. Male and female mice were caged separately, and data was combined for all experiments, since no gender difference was noted. Mice were maintained and cared for according to the regulations of the Tokyo University Animal Care Committee. Mice were kept in microisolator cages with 12-h light/dark cycles and were fed ad libitum except during food restriction procedures. All mice were fed a normal rodent chow diet containing 0.075% cholesterol (MF; Oriental Yeast Co., Ltd. (Osaka, Japan)). A cholesterol-free diet was also purchased from Oriental Yeast Co. and fed to mice ad libitum for the cholesterol-free diet studies. For caloric restriction, mice were given 1.6 g of normal chow diet per day, and control age-matched mice were fed ad libitum. Blood collections were performed via optical venous plexus puncture with EDTA-treated tubes. Collections were performed between 10 and 12 a.m. on animals fedad libitum. Samples were preserved with EDTA and NaN3. Plasma cholesterol, triglyceride, and HDL cholesterol levels were measured with colorimetric assays by Determiner TC555, TG555, and HDL-C (Kyowa Medex, Co., Ltd. Tokyo, Japan), respectively. Glucose was measured by a standard enzymatic method. Insulin levels were tested with an enzyme-linked immunosorbent assay kit by LABIS (Japan) for mouse insulin. For analysis of lipoprotein distribution, pooled plasma samples from three mice per group were subjected to high performance liquid chromatography (HPLC) (SRL, Japan). Estimation of HDL levels in some samples were obtained from HPLC plots by calculation of the area under the curve. Agarose gel electrophoresis for lipoproteins was performed per standard protocol on gels purchased from Ciba/Corning. Livers were immediately collected and snap frozen in liquid nitrogen. A 50-mg piece of liver was homogenized in PBS. Folch's reagent (CHCl3/MeOH, 2:1) (0.75 ml) was added to the homogenate. The nonaqueous phase was collected, and 30 μl of 200 mg/ml Triton X-100 in CHCl3 was added. Samples were dried and used for cholesterol and triglyceride analysis as described above for plasma samples. Total RNA was prepared from the snap frozen livers with TRIzol reagent (Life Technologies, Inc.). RNA (12 μg) was electrophoresed through formalin-denatured agarose gels and transferred to Hybond-N membranes (Amersham Pharmacia Biotech). cDNA probes were prepared as described (11Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M. Goldstein J.L. J. Clin. Invest. 1996; 7: 1575-1584Crossref Scopus (698) Google Scholar, 12Shimano H. Yahagi N. Amemiya-Kudo M. Hasty A. Osuga J. Tamura Y. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Gotoda T. Ishibashi S. Yamada N. J. Biol. Chem. 1999; 274: 35832-35839Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). Probes were labeled with [α-32P]dCTP using Megaprime DNA Labeling System kit (Amersham Pharmacia Biotech). Membranes were hybridized with the radiolabeled probes in Rapid-hyb Buffer (Amersham Pharmacia Biotech) and washed in 0.1× SSC, 0.1% SDS at 65 °C. Membranes were exposed to Eastman Kodak Co. XAR-5 film for 2–5 h at −80 °C. Lipoprotein lipase measurements in postheparin plasma (10 min after intravenous injection of 0.1 mg/Kg heparin) were performed using a modification of the standard technique by Muir (21Muir J.R. Clin. Chim. Acta. 1967; 17: 312-314Crossref PubMed Scopus (4) Google Scholar). Leptin was purchased from Calbiochem and was prepared according to the manufacturer's instructions. Leptin was diluted in PBS for individual mice according to their weight, and 0.3 μg/g body weight was injected in 100-μl doses per day. Whole aortae were collected and stained with Sudan IV as described (7Ishibashi S. Goldstein J.L. Brown M.S. Herz J. Burns D.K. J. Clin. Invest. 1994; 93: 1885-1893Crossref PubMed Scopus (600) Google Scholar, 13Yagyu H. Ishibashi S. Chen Z. Osuga J. Okazaki M. Perrey S. Kitamine T. Shimada M. Ohashi K. Harada K. Shionoiri F. Yahagi N. Gotoda T. Yazaki Y. Yamada N. J. Lipid Res. 1999; 40: 1677-1685Abstract Full Text Full Text PDF PubMed Google Scholar). Cross-sections of proximal aorta were prepared and stained with Oil-Red-O by SKK (Japan) per standard techniques as described (14Paigen B. Morrow A. Holmes P.A. Mitchell D. Williams R.A. Atherosclerosis. 1987; 68: 231-240Abstract Full Text PDF PubMed Scopus (789) Google Scholar). All values are stated as mean ± S.E., and differences between groups were evaluated with Student'st test. Blood was collected from wild type, LDLR(−/−), LDLR(+/−), LDLR+/+;ob/ob, LDLR+/−;ob/ob, and LDLR−/−;ob/ob mice that were 3–4 months of age. Plasma was analyzed for TG and TC levels (Table I). LDLR+/+;ob/ob mice had slightly elevated TC levels (119 ± 25 mg/dl), as was previously reported (3Nishina P.M. Lowe S. Wang J. Paigen B. Metabolism. 1994; 43: 549-553Abstract Full Text PDF PubMed Scopus (101) Google Scholar). LDLR+/−;ob/ob mice showed a significant elevation in TC levels to 282 ± 29 mg/dl with no increase in TG levels; however, the LDLR−/−;ob/ob mice showed a dramatic increase in TC levels with an average of 1715 ± 87 mg/dl with values ranging from 1430 to 2030 mg/dl. TG levels in these mice were also highly elevated (1016 ± 172 mg/dl).Table IBlood TC and TG levels in 12–16-week-old miceGenotypenCholesterolTriglyceridemg/dlWild type481 ± 1660 ± 12LDLR(+/−)3149 ± 11100 ± 12LDLR(−/−)6265 ± 40161 ± 25LDLR+/+;ob/ob5119 ± 25120 ± 12LDLR+/−;ob/ob6282 ± 2978 ± 8LDLR−/−;ob/ob61715 ± 871016 ± 172Mice were maintained on a chow diet (0.075% cholesterol). TC and TG (mg/dl) of 3–4-month-old mice were measured as described under “Experimental Procedures.” Values are given as the mean ± S.E. Open table in a new tab Mice were maintained on a chow diet (0.075% cholesterol). TC and TG (mg/dl) of 3–4-month-old mice were measured as described under “Experimental Procedures.” Values are given as the mean ± S.E. Since well known phenotypes of ob/ob mice such as obesity, increased plasma glucose, and insulin levels are age-dependent, a time course study of TC and TG levels from additional animals was performed (Fig. 1). At 1 month (upon weaning), the LDLR−/−;ob/ob mice already displayed modest elevations in both TC and TG levels (369 ± 20 mg/dl and 137 ± 26 mg/dl, respectively; n = 6). These values increased significantly to 716 ± 48 and 393 ± 97 after mice were placed on a normal diet for 2 weeks. The LDLR−/−;ob/ob mice showed an age-dependent increase in TC and TG levels between 1 and 4 months, with values becoming maximal at 3–4 months, followed by a gradual decrease to 585 ± 44 and 306 ± 36 mg/dl (n = 3) by 8 months. LDLR+/−;ob/ob mice also showed a similar pattern of reaching peak TC levels at 3–4 months of age and maintained stable TC levels of ∼280 mg/dl thereafter. TG levels in LDLR+/−;ob/ob mice were not different from wild type animals at any time (Fig. 1). HDL levels in all groups of mice were estimated from HPLC results and remained unchanged over the course of the study. Wild type mice had HDL levels of 54 mg/dl at 3 months and 47 mg/dl at 8 months. LDLR(−/−) HDLs were slightly elevated to 69 and 65 mg/dl at 3 and 8 months. LDLR+/+;ob/ob mice were 89 and 56 mg/dl, LDLR+/−;ob/ob were 69 and 62 mg/dl, and LDLR−/−;ob/ob had levels of 90 and 94 mg/dl at 3 and 8 months, respectively. Glucose levels in LDLR−/−;ob/ob mice were ∼200 mg/dl at 2 months and rose to between 300 and 400 mg/dl from 2 to 7 months. There were no significant differences between LDLR−/−;ob/ob and LDLR+/−;ob/ob mice during the study (data not shown). Insulin levels in LDLR−/−;ob/ob and LDLR+/−;ob/ob mice rose steadily from ∼30 to ∼300 ng/ml blood between 1 and 7 months (data not shown). Plasma samples from 12–16-week-old mice were further analyzed for the distribution of lipoproteins (Fig.2). As expected, wild type mouse plasma contained primarily HDL-sized lipoproteins. LDLR+/+;ob/ob mice showed a unique profile containing mostly HDL with a shoulder on the HDL peak with particles sized between LDL and HDL, as has been recently described (15Silver D.L. Jiang X.-C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This peak most likely contains HDL1 particles and will be referred to as such. LDLR(−/−) mice displayed elevated levels of LDL, similar to previous reports (10Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1273) Google Scholar). The distribution of lipoproteins in LDLR+/−;ob/ob mice was similar to the LDLR+/+;ob/ob mice except that the HDL1 peak was higher and broader. In contrast, plasma from LDLR−/−;ob/ob mice contained a severely elevated and broadened lipoprotein peak ranging from VLDL/IDL-sized particles to LDL-sized particles, presumably remnant lipoproteins, and retained HDL levels similar to wild type animals. Further analysis of lipoprotein profiles was performed by lipoprotein-agarose gel electrophoresis (data not shown). As expected, LDLR+/+;ob/ob mouse plasma had a pattern similar to wild type mice, although the α-band was slightly broader, presumably due to the HDL1 particles. Interestingly, LDLR+/−;ob/ob mice had both α and β migrating particles, and LDLR−/−;ob/ob plasma contained a broad β-migrating band similar to that seen from apoE(−/−) lipoproteins. Whole lipoprotein fractions (d < 1.21) were separated from pooled plasma samples of each group (n = 3) and were subjected to SDS-polyacrylamide gel electrophoresis to analyze apoproteins (Fig. 3). LDLR(−/−) mice had elevated apoB-100 levels as compared with wild type mice (lanes 1 and 2). LDLR+/+;ob/ob mouse plasma contained barely detectable apoBs (lane 3); however, LDLR+/−;ob/ob plasma did have apoB-100 and low levels of B-48 (lane 4). The amounts of apoBs in these groups were consistent with their LDL peaks in the HPLC. In striking contrast, the LDLR−/−;ob/ob lipoproteins consisted of highly elevated levels of both apoB-100 and apoB-48, with a greater ratio of B-100 to B-48 particles (lane 5), indicating that their elevated TC and TG levels may be at least partially due to an increase in particle number. In addition, the LDLR−/−;ob/ob mice had an apparent increase in apoAIV and apoE. This is probably due to the increased remnant lipoproteins. In comparison, apoE(−/−) mouse lipoproteins are enriched in apoAIV, and LDLR(−/−) mice on a high fat diet show increases in apoE (7Ishibashi S. Goldstein J.L. Brown M.S. Herz J. Burns D.K. J. Clin. Invest. 1994; 93: 1885-1893Crossref PubMed Scopus (600) Google Scholar). Lipoprotein lipase activities were measured in pooled samples from mice to determine if lipolytic activity was decreased in the LDLR−/−;ob/ob mice. Postheparin lipolytic activity for wild type mice was 0.45 μmol of FFA/ml/min. LDLR(−/−), LDLR+/+;ob/ob, and LDLR−/−;ob/ob all had slightly elevated lipolytic activity (0.61, 0.72, and 0.57 μmol of FFA/ml/min). To determine if the increased plasma TC and TG in the LDLR−/−;ob/ob mice was related to hepatic cholesterologenic and lipogenic enzymes, livers were collected from 6-month-old animals and analyzed for both cholesterol and triglyceride content as well as various enzyme RNA levels. The livers of LDLR(−/−) mice as well as all three ob/ob groups were found to contain slightly, but significantly, higher levels of cholesterol than wild type mice (Table II), which is consistent with previous reports (9Horton J.D. Shimano H. Hamilton R.L. Brown M.S. Goldstein J.L. J. Clin. Invest. 1999; 103: 1067-1076Crossref PubMed Scopus (158) Google Scholar, 16Shimomura I. Bashmakov Y. Horton J. J. Biol. Chem. 1999; 274: 30028-30032Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). LDLR−/−;ob/ob mice contained slightly higher levels of cholesterol than the other two ob/ob groups and the LDLR(−/−) mice (p < 0.05) (Table II). Triglyceride levels were ∼10 times higher in the three ob/ob groups compared with the wild type and LDLR(−/−) mice, but there was no association between LDLR genotype and liver triglyceride content in the ob/ob mice. These data indicate that leptin deficiency causes a marked increase in liver triglyceride levels but only a small increase in liver cholesterol content.Table IILiver cholesterol and triglyceride contentGenotypeCholesterol content2-ap < 0.05 wild type versus all other groups; LDLR(−/−) versus LDLR−/−;ob/ob.Triglyceride content2-bp < 0.005 wild type and LDLR(−/−) versus all ob/ob groups.mg/g of liverWild type1.77 ± 0.3227.0 ± 5.2LDLR(−/−)2.91 ± 0.2224.2 ± 3.3LDLR+/+;ob/ob4.13 ± 0.9244 ± 4.2LDLR+/−;ob/ob3.45 ± 0.1222 ± 40LDLR−/−;ob/ob5.01 ± 0.5278 ± 40Liver cholesterol and triglyceride content was measured as described under “Experimental Procedures.” For all groups, n= 3. Values are given as mean ± S.E. in mg/g of liver.2-a p < 0.05 wild type versus all other groups; LDLR(−/−) versus LDLR−/−;ob/ob.2-b p < 0.005 wild type and LDLR(−/−) versus all ob/ob groups. Open table in a new tab Liver cholesterol and triglyceride content was measured as described under “Experimental Procedures.” For all groups, n= 3. Values are given as mean ± S.E. in mg/g of liver. Northern blot analyses on liver RNAs revealed that cholesterologenic enzymes HMG-CoA synthase (Fig.4), HMG-CoA reductase and SREBP-2 (data not shown), were unchanged among all five groups. In contrast, lipogenic enzyme RNAs (SREBP-1, fatty acid synthase, ATP citrate lyase, and stearoyl-CoA desaturase-1) were increased in the ob/ob groups compared with wild type and LDLR(−/−); however, there was very little difference in the ob/ob mice between the three LDLR genotypes. ApoAI was slightly decreased in ob/ob mice; however, apoAII, and apoE RNA levels were unchanged in the ob/ob mice compared with controls. A control probe for the ribosomal enzyme 36B4 demonstrates equal loading (Fig. 4). To determine the possible mechanism by which lipoproteins become elevated in the LDLR−/−;ob/ob mice, initial studies were focused on the possibility that the increased caloric intake of ob/ob mice was overwhelming and surpassed backup lipoprotein clearance systems when the LDLR was not present. To test this hypothesis, LDLR+/−;ob/ob and LDLR−/−;ob/ob mice were fasted for 2 days, after which glucose, TC, and TG levels were analyzed (n = 3 in each group). Glucose levels were reduced by 57 and 44% in LDLR+/−;ob/ob and LDLR−/−;ob/ob mice, respectively. Furthermore, TG levels fell by 58 and 53%, respectively, in the fasted mice. In contrast, TC levels increased slightly (although not significantly; p = 0.12) in the LDLR−/−;ob/ob mice from 585 ± 44 to 712 ± 47 mg/dl, while TC levels in the LDLR+/−;ob/ob mice were unchanged (265 ± 14 to 244 ± 12 mg/dl). These data are consistent with the faster turnover rate of fatty acids and triglycerides than cholesterol. Following this short term fasting experiment, a long term caloric restriction diet, in which 4-week-old LDLR−/−;ob/ob mice were given only 50% of what a wild type mouse would consume, was initiated. Both diet-restricted mice (n = 2) and mice fed ad libitum (n = 3) showed a significant increase in TC over the 2-week study, confirming the age-dependent increase in TC; however, this increase was more drastic in the mice fedad libitum, so that after 2 weeks there was a significant difference in TC levels between the two groups (Fig.5). TG levels increased significantly in the control group but remained unchanged in the diet-restricted group (Fig. 5), a pattern that paralleled changes in glucose levels in the mice (data not shown). There was a slight reduction in HDL levels in both control and diet-restricted LDLR−/−;ob/ob mice (92 ± 35 to 89 ± 28 and 90 ± 35 to 79 ± 32, respectively); however, there were no significant differences, reflecting the smaller contribution of HDL to the hyperlipidemia in these mice. Based on these studies, while the elevated TG levels in the LDLR−/−;ob/ob mice were basically associated with the amount of short term food consumption, the increased caloric intake of these mice, due to their leptin deficiency, cannot account for the elevations in TC levels. To determine whether the increase in TC levels was derived from the cholesterol that was present in the normal chow diet, these mice were placed on a cholesterol-free diet. After 2 weeks on the cholesterol-free diet, there were no detectable changes in TC (542 ± 20 to 560 ± 102 mg/dl) or TG levels (272 ± 19 to 281 ± 34 mg/dl) of LDLR−/−;ob/ob animals. HDL levels in LDLR−/− mice were unchanged by the cholesterol-free diet (83 ± 12 to 86 ± 13.5 mg/dl). HDL levels in LDLR−/−;ob/ob mice were decreased from 75 ± 5 to 65 ± 13 mg/dl; however, it was not statistically significant. Glucose levels were reduced by 29% in LDLR+/−;ob/ob and LDLR−/−;ob/ob mice. To determine whether the high plasma TC and TG levels observed in the LDLR−/−;ob/ob were reversible with leptin treatment, 6–8-week-old LDLR(−/−), LDLR+/−;ob/ob, and LDLR−/−;ob/ob mice were injected with a low dose of leptin (0.3 μg of leptin/g body weight) daily for 10 days. This low dose of leptin has been shown to be suboptimal for weight reduction so that body weight in the treated mice was not significantly changed by this treatment (15Silver D.L. Jiang X.-C. Tall A.R. J. Biol. Chem. 1999; 274: 4140-4146Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Control mice were injected daily with a similar volume of PBS. The LDLR(−/−) mice did not show any significant changes in TC, TG, or glucose levels (data not shown). Mice in all groups gained weight within the 10-day treatment (predictable from their age), but there were no significant differences due to the leptin treatment (TableIII). Insulin levels were typical of ob/ob mice of this age (20–30 ng/ml) throughout the study. Insulin levels in the PBS-treated mice increased during the treatment, while leptin treatment tended to retard this increase in both LDLR−/−;ob/ob and LDLR+/−;ob/ob groups, although there was no significant difference due to a high variability (data not shown). Glucose levels were slightly decreased in the leptin-treated LDLR−/−;ob/ob and LDLR+/−;ob/ob mice (Table III). TG levels were substantially decreased in the leptin-treated LDLR−/−;ob/ob mice, while they tended to be increased in the PBS-treated mice during the course of the study (TableIII). In contrast, TC levels in the leptin-treated LDLR−/−;ob/ob mice were only slightly reduced, whereas those of the PBS-treated LDLR−/−;ob/ob increased significantly (p < 0.05) during the course of the study (Table III). HPLC analyses of pooled serum samples from leptin and PBS-injected LDLR−/−;ob/ob mice are shown are Fig. 6. Leptin treatment caused a substantial decrease in the VLDL/IDL fraction compared with little change seen in the PBS-injected mice (data not shown). This noticeable shift from VLDL/IDL size particles to particles in the LDL size range is consistent with the marked reduction in TG and minimal decrease in TC detected from total serum. Interestingly, HDL levels appeared to increase upon leptin injection. The leptin-injected LDLR+/−;ob/ob mice also displayed a lowering in TG levels, which was concomitant with a reduction in the HDL1 peak compared with their base-line profile and with PBS-injected animals (data not shown). These data indicate that leptin treatment in LDLR−/−;ob/ob mice can revert their hypertriglyceridemia but not their hypercholesterolemia, since the remnant cholesterol was presumably converted to LDL cholesterol.Table IIIEffect of leptin treatment in LDLR−/−;ob/ob miceGroupControlLeptin-treatedBase line10 daysBase line10 daysLDLR+/−;ob/obBody weight (g)21 ± 330 ± 232.5 ± 636 ± 5Glucose (mg/dl)194 ± 26201 ± 31303 ± 41215 ± 67TG (mg/dl)84 ± 17193 ± 273-ap < 0.05 compared with base line.128 ± 17114 ± 42TC (mg/dl)2

Background —Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Reactive oxygen species induce apoptosis in many types of cells including cardiomyocytes. Because insulin has been reported to have protective effects, we examined whether insulin prevents cardiomyocytes from oxidative stress–induced apoptotic death. Methods and Results —Cultured cardiomyocytes of neonatal rats were stimulated by hydrogen peroxide (H 2 O 2 ). Apoptosis was evaluated by means of the TUNEL method and DNA laddering. Incubation with 100 μmol/L H 2 O 2 for 24 hours increased the number of TUNEL-positive cardiac myocytes (control, ≈4% versus H 2 O 2 , ≈23%). Pretreatment with 10 − 6 mol/L insulin significantly decreased the number of H 2 O 2 -induced TUNEL-positive cardiac myocytes (≈12%) and DNA fragmentation induced by H 2 O 2 . Pretreatment with a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin, and overexpression of dominant negative mutant of PI3K abolished the cytoprotective effect of insulin. Insulin strongly activated both PI3K and the putative downstream effector Akt . Moreover, a proapoptotic protein, Bad , was significantly phosphorylated and inactivated by insulin through PI3K. Conclusions —These results suggest that insulin protects cardiomyocytes from oxidative stress–induced apoptosis through the PI3K pathway.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor whose activation has been linked to several physiologic pathways including those related to the regulation of intestinal inflammation. We sought to determine whether PPAR gamma could function as an endogenous anti-inflammatory pathway in a murine model of intestinal ischemia-reperfusion (I/R) injury.PPAR gamma-deficient and wild-type mice were examined for their response to I/R procedure. Treatment with a PPAR gamma-specific ligand was also performed.In a murine model of intestinal I/R injury, we observed more severe injury in PPAR gamma-deficient mice and protection against local and remote tissue injury in mice treated with a PPAR gamma-activating ligand, BRL-49653. Activation of PPAR gamma resulted in down-regulation of intercellular adhesion molecule 1 expression by intestinal endothelium and tissue tumor necrosis factor alpha messenger RNA levels most likely by inhibition of the NF-kappa B pathway.These data strongly suggest that an endogenous PPAR gamma pathway exists in tissues that may be amenable to therapeutic manipulation in I/R-related injuries.

Recently, several groups have carried out whole-genome association studies in European and European-origin populations and found novel type 2 diabetes-susceptibility genes, fat mass and obesity associated (FTO), solute carrier family 30 (zinc transporter), member 8 (SLC30A8), haematopoietically expressed homeobox (HHEX), exostoses (multiple) 2 (EXT2), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), which had not been in the list of functional candidates. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) in these genes and type 2 diabetes in participants from the Japanese population.Sixteen previously reported SNPs were genotyped in 864 Japanese type 2 diabetes individuals (535 men and 329 women; age 63.1 +/- 9.5 years (mean+/-SD), BMI 24.3 +/- 3.9 kg/m(2)) and 864 Japanese control individuals (386 men and 478 women; age 69.5 +/- 6.8 years, BMI 23.8 +/- 3.7 kg/m(2)).The SNPs rs5015480 [odds ratio (OR) = 1.46 (95% CI 1.20-1.77), p = 2.0 x 10(-4)], rs7923837 [OR = 1.40 (95% CI 1.17-1.68), p = 2.0 x 10(-4)] and rs1111875 [OR = 1.30 (95% CI 1.11-1.52), p = 0.0013] in HHEX were significantly associated with type 2 diabetes with the same direction as previously reported. SNP rs8050136 in FTO was nominally associated with type 2 diabetes [OR = 1.22 (95% CI 1.03-1.46), p = 0.025]. SNPs in other genes such as rs7756992 in CDKAL1, rs10811661 in CDKN2B and rs13266634 in SLC30A8 showed nominal association with type 2 diabetes. rs7756992 in CDKAL1 and rs10811661 in CDKN2B were correlated with impaired pancreatic beta cell function as estimated by the homeostasis model assessment beta index (p = 0.023, p = 0.0083, respectively).HHEX is a common type 2 diabetes-susceptibility gene across different ethnic groups.

Obesity and insulin resistance have been recognized as leading causes of major health issues. We have endeavored to depict the molecular mechanism of insulin resistance, focusing on the function of adipocyte. We have investigated a role of PPARgamma on the pathogenesis of Type II diabetes. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Moreover, a Pro12Ala polymorphism in the human PPARgamma2 gene was associated with decreased risk of Type II diabetes in Japanese. Taken together with these results, PPARgamma is proved to be a thrifty gene mediating Type II diabetes. Pharmacological inhibitors of PPARgamma/RXR ameliorate high-fat diet-induced insulin resistance in animal models of Type II diabetes. We have performed a genome-wide scan of Japanese Type 2 diabetic families using affected sib pair analysis. Our genome scan reveals at least 9 chromosomal regions potentially harbor susceptibility genes of Type II diabetes in Japanese. Among these regions, 3q26-q28 appeared to be very attractive one, because of the gene encoding adiponectin, the expression of which we had found enhanced in insulin-sensitive PPARgamma-deficient mice. Indeed, the subjects with the G/G genotype of SNP276 in the adiponectin gene were at increased risk for Type II diabetes compared with those having the T/T genotype. The plasma adiponectin levels were lower in the subjects with the G allele, suggesting that genetically inherited decrease in adiponectin levels predispose subjects to insulin resistance and Type II diabetes. Our work also confirmed that replenishment of adiponectin represents a novel treatment strategy for insulin resistance and Type II diabetes using animal models. Further investigation will be needed to clarify how adiponectin exerts its effect and to discover the molecular target of therapies.

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, we established embryonic fibroblast cells with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1(-/-)), IRS-2 deficient (IRS-2(-/-)), and IRS-1 IRS-2 double deficient (IRS-1(-/-) IRS-2(-/-)), from mouse embryos of the corresponding genotypes. The abilities of IRS-1(-/-) cells and IRS-2(-/-) cells to differentiate into adipocytes are approximately 60 and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1(-/-) IRS-2(-/-) cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) is severely decreased in IRS-1(-/-) IRS-2(-/-) cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1(-/-) IRS-2(-/-) cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1(-/-) IRS-2(-/-) cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPalpha and PPARgamma, a result which is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1(-/-) IRS-2(-/-) double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. Our results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPalpha and PPARgamma expression and adipocyte differentiation.

The tumor suppressor p53 is a transcription factor that activates or represses its target genes after various genotoxic stresses. We have previously shown that sterol regulatory element-binding protein-1 (SREBP-1), a key transcriptional regulator of triglyceride synthesis, and the lipogenic enzymes under its control are markedly suppressed in adipocytes from genetically obese <i>ob/ob</i> mice. Here we demonstrate that p53 and its target genes are highly induced in adipocytes of <i>ob/ob</i> mice in a fed state, leading to the negative regulation of SREBP-1 and thereby lipogenic genes. In fact, disruption of p53 in <i>ob/ob</i> mice completely suppressed the p53-regulated genes to wild-type levels and partially restored expression of lipogenic enzymes. Consistently, reporter gene analysis showed that p53 overexpression suppressed the promoter activity of the <i>SREBP-1c</i> gene and its downstream genes. Thus, the activation of p53 might constitute a negative feedback loop against excess fat accumulation in adipocytes. In conclusion, we discovered a novel role of p53 in the pathophysiology of obesity.

Obesity is associated with a high incidence of cardiovascular complications. However, the molecular link between obesity and vascular disease is not fully understood. Most previous studies have focused on the association between cardiovascular disease and accumulation of visceral fat. Periadventitial fat is distributed ubiquitously around arteries throughout the body.Here, we investigated the impact of obesity on inflammation in the periadventitial adipose tissue and on lesion formation after vascular injury.High-fat, high-sucrose feeding induced inflammatory changes and decreased adiponectin expression in the periadventitial adipose tissue, which was associated with enhanced neointima formation after endovascular injury. Removal of periadventitial fat markedly enhanced neointima formation after injury, which was attenuated by transplantation of subcutaneous adipose tissue from mice fed on regular chow. Adiponectin-deficient mice showed markedly enhanced lesion formation, which was reversed by local delivery, but not systemic administration, of recombinant adiponectin to the periadventitial area. The conditioned medium from subcutaneous fat attenuated increased cell number of smooth muscle cells in response to platelet derived growth factor-BB.Our findings suggest that periadventitial fat may protect against neointimal formation after angioplasty under physiological conditions and that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular disease accelerated by obesity.

Solid tumors require neovascularization for their growth. Recent evidence indicates that bone marrow-derived endothelial progenitor cells (EPCs) contribute to tumor angiogenesis. We show here that granulocyte colony-stimulating factor (G-CSF) markedly promotes growth of the colon cancer inoculated into the subcutaneous space of mice, whereas G-CSF had no effect on cancer cell proliferation in vitro. The accelerated tumor growth was associated with enhancement of neovascularization in the tumor. We found that bone marrow-derived cells participated in new blood vessel formation in tumor. Our findings suggest that G-CSF may have potential to promote tumor growth, at least in part, by stimulating angiogenesis in which bone marrow-derived EPCs play a role.

Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate in vascular tissues in aging and diabetes. Receptors for AGEs include scavenger receptors, which recognize acetylated low density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SR-BI), and lectin-like oxidized low density lipoprotein receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs have prompted us to examine whether AGEs are also recognized by the novel scavenger receptors, which we have recently isolated from a cDNA library prepared from human umbilical vein endothelial cells, such as the scavenger receptor expressed byendothelial cells-I (SREC-I); thefasciclin EGF-like, laminin-typeEGF-like, and link domain-containing scavenger receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At 4 °C, 125I-AGE-bovine serum albumin (BSA) exhibited high affinity specific binding to Chinese hamster ovary (CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) withKd of 2.55 and 1.68 μg/ml, respectively, but not to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At 37 °C, 125I-AGE-BSA was taken up and degraded by CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The 125I-AGE-BSA binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids, and dextran sulfate but not by native LDL, oxidized LDL, or HDL. FEEL-1, which is expressed by the liver and vascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis. Advanced glycation end products (AGEs) are nonenzymatically glycosylated proteins, which accumulate in vascular tissues in aging and diabetes. Receptors for AGEs include scavenger receptors, which recognize acetylated low density lipoproteins (Ac-LDL) such as scavenger receptor class AI/AII (SR-A), cell surface glycoprotein CD36, scavenger receptor class B type I (SR-BI), and lectin-like oxidized low density lipoprotein receptor-1. The broad ligand repertoire of these receptors as well as the diversity of the receptors for AGEs have prompted us to examine whether AGEs are also recognized by the novel scavenger receptors, which we have recently isolated from a cDNA library prepared from human umbilical vein endothelial cells, such as the scavenger receptor expressed byendothelial cells-I (SREC-I); thefasciclin EGF-like, laminin-typeEGF-like, and link domain-containing scavenger receptor-1 (FEEL-1); and its paralogous protein, FEEL-2. At 4 °C, 125I-AGE-bovine serum albumin (BSA) exhibited high affinity specific binding to Chinese hamster ovary (CHO) cells overexpressing FEEL-1 (CHO-FEEL-1) and FEEL-2 (CHO-FEEL-2) withKd of 2.55 and 1.68 μg/ml, respectively, but not to CHO cells expressing SREC (CHO-SREC) and parent CHO cells. At 37 °C, 125I-AGE-BSA was taken up and degraded by CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and parent CHO cells. Thus, the ability to bind Ac-LDL is not necessarily a prerequisite to bind AGEs. The 125I-AGE-BSA binding to CHO-FEEL-1 and CHO-FEEL-2 cells was effectively inhibited by Ac-LDL and polyanionic SR-A inhibitors such as fucoidan, polyinosinic acids, and dextran sulfate but not by native LDL, oxidized LDL, or HDL. FEEL-1, which is expressed by the liver and vascular tissues, may recognize AGEs, thereby contributing to the development of diabetic vascular complications and atherosclerosis. advanced glycation end products low density lipoprotein acetylated low density lipoprotein oxidized low density lipoprotein high density lipoprotein scavenger receptor class AI/AII scavenger receptor class B type I scavenger receptor expressed by endothelial cells epidermal growth receptor fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor-1 receptor for advanced glycation end product bovine serum albumin polyinosinic acid Chinese hamster ovary human umbilical endothelial cells phosphate-buffered saline Advanced glycation end products (AGEs)1 are generated by nonenzymatic glycosylation of proteins or lipids after prolonged exposure to glucose (1Brownlee M. Annu. Rev. Med. 1995; 46: 223-234Crossref PubMed Scopus (1141) Google Scholar). AGEs elicit a wide variety of cellular responses including induction of growth factors and cytokines (2Lu M. Kuroki M. Amano S. Tolentino M. Keough K. Kim I. Bucala R. Adamis A.P. J. Clin. Invest. 1998; 101: 1219-1224Crossref PubMed Scopus (404) Google Scholar), adhesion molecules (3Schmidt A.M. Hori O. Chen J.X. Li J.F. Crandall J. Zhang J. Cao R. Yan S.D. Brett J. Stern D. J. Clin. Invest. 1995; 96: 1395-1403Crossref PubMed Scopus (813) Google Scholar), oxidant stress (4Yan S.D. Schmidt A.M. Anderson G.M. Zhang J. Brett J. Zou Y.S. Pinsky D. Stern D. J. Biol. Chem. 1994; 269: 9889-9897Abstract Full Text PDF PubMed Google Scholar), and chemotaxis (5Higashi T. Sano H. Saishoji T. Ikeda K. Jinnouchi Y. Kanzaki T. Morisaki N. Rauvala H. Shichiri M. Horiuchi S. Diabetes. 1997; 46: 463-472Crossref PubMed Google Scholar). These proinflammatory responses are implicated to contribute to the development of pathologies associated with aging, diabetes mellitus, and Alzheimer's disease (6Vlassara H. Palace M.R. J. Intern. Med. 2002; 251: 87-101Crossref PubMed Scopus (615) Google Scholar). Indeed, AGEs were shown to be present in atherosclerotic lesions (7Nakamura Y. Horii Y. Nishino T. Shiiki H. Sakaguchi Y. Kagoshima T. Dohi K. Makita Z. Vlassara H. Bucala R. Am. J. Pathol. 1993; 143: 1649-1656PubMed Google Scholar) and diabetic kidney (8Niwa T. Katsuzaki T. Miyazaki S. Miyazaki T. Ishizaki Y. Hayase F. Tatemichi N. Takei Y. J. Clin. Invest. 1997; 99: 1272-1280Crossref PubMed Scopus (190) Google Scholar). The AGE-elicited proinflammatory reactions are mediated by its receptors or binding proteins, which include the receptor for advanced glycation end product (RAGE) (9Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar, 10Schmidt A.M. Hasu M. Popov D. Zhang J.H. Chen J. Yan S.D. Brett J. Cao R. Kuwabara K. Costache G. Simonescu N. Simonescu M. Stern D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8807-8811Crossref PubMed Scopus (279) Google Scholar), OST-48 (ARE-R1)/80K-H (AGE-R2)/galectin-3 (AGE-R3) (11Vlassara H. Li Y.M. Imani F. Wojciechowicz D. Yang Z. Liu F.T. Cerami A. Mol. Med. 1995; 1: 634-646Crossref PubMed Google Scholar), scavenger receptor class AI/AII (SR-A) (12Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar), scavenger receptor class B type I (SR-BI) (13Ohgami N. Nagai R. Miyazaki A. Ikemoto M. Arai H. Horiuchi S. Nakayama H. J. Biol. Chem. 2001; 276: 13348-13355Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), cell surface glycoprotein CD36 (14Ohgami N. Nagai R. Ikemoto M. Arai H. Kuniyasu A. Horiuchi S. Nakayama H. J. Biol. Chem. 2001; 276: 3195-3202Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), lectin-like oxidized low density lipoprotein receptor-1 (15Jono T. Miyazaki A. Nagai R. Sawamura T. Kitamura T. Horiuchi S. FEBS Lett. 2002; 511: 170-174Crossref PubMed Scopus (115) Google Scholar), lactoferin (16Li Y.M. Tan A.X. Vlassara H. Nat. Med. 1995; 1: 1057-1061Crossref PubMed Scopus (143) Google Scholar), and lysozyme (16Li Y.M. Tan A.X. Vlassara H. Nat. Med. 1995; 1: 1057-1061Crossref PubMed Scopus (143) Google Scholar). The broad ligand repertories of these AGE-binding proteins as well as the diversity of receptors for AGEs have prompted us to examine whether AGEs are recognized by novel members of scavenger receptors that Adachiet al. (17Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) have recently cloned from a cDNA library prepared from human umbilical vein endothelial cells as receptors for acetylated low density lipoproteins (Ac-LDL), such as thescavenger receptor expressed byendothelial cells-I (SREC-I) (17Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and thefasciclin EGF-like, laminin-typeEGF-like, and link domain-containing scavenger receptor-1 (FEEL-1) (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These receptors are structurally unrelated to other scavenger receptors. SREC is a protein of 830 amino acids with five epidermal growth factor-like cysteine pattern signatures. FEEL-1 is a protein of 2570 amino acids including 7 fasciclins, 16 EGF-like, 2 laminin-type EGF-like, and 1 link domain near the transmembrane region. FEEL-2 is a paralogous gene of FEEL-1 whose amino acid sequence is ∼40% identical to FEEL-1. Quantitative PCR analyses showed that both FEEL-1 and FEEL-2 are expressed in the spleen and lymph node, whereas only FEEL-1 is detectable in CD14+-mononuclear cells and vascular endothelial cell lines (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Here we show that FEEL-1 and FEEL-2, but not SREC, are endocytic receptors for AGEs. Because FEEL-1 is expressed by the liver, macrophages, and endothelial cells in an amount comparable with other receptors for AGEs, FEEL-1 may play a significant role in the elimination of AGEs from the circulation as well as in the development of diabetic vascular complications and atherosclerosis. Ham's F-12 medium, Dulbecco's modified Eagle medium (DMEM), penicillin G, streptomycin sulfate, G418, and TriZOL were purchased from Invitrogen. Bovine serum albumin (BSA), MOPC21 (mouse IgG), fucoidan, polyinosinic acid (poly I), and dextran sulfate were purchased from Sigma. Heparin sodium salt was purchased from Mitsubishi Pharma (Osaka, Japan). Glucose 6-phosphate was purchased from Oriental Yeast (Tokyo, Japan). Endothelial cell growth supplement was purchased from BD Biosciences. Oligotex-dT30TM was purchased from Roche Molecular Biochemicals. Hybond N was purchased from Amersham Biosciences. Na-125I was purchased from Daiichi Chemical (Osaka, Japan). IodogenTM and BCA protein assay reagent kit were purchased from Pierce. Human umbilical vein endothelial cells (HUVECs) were purchased from Kurabo (Tokyo, Japan). A murine monoclonal antibody against human FEEL-1 (FE-1-1) was described previously (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). cDNA probes for murine FEEL-1, FEEL-2 SREC, RAGE, galectin-3, SR-A, CD36, and SR-B1 were prepared by reverse transcriptase-PCR using primers designed based on the reported nucleotide sequences. AGE-BSA was prepared as described previously (19Ichikawa K. Yoshinari M. Iwase M. Wakisaka M. Doi Y. Iino K. Yamamoto M. Fujishima M. Atherosclerosis. 1998; 136: 281-287Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). 600 mg of BSA was incubated with 50 mm glucose 6-phosphate in 10 ml of sterile sodium phosphate buffer or phosphate-buffered saline (PBS) for 10 weeks at 37 °C and dialyzed overnight against PBS. AGE-specific fluorescence was measured at 450 nm after excitation at 360 nm at a concentration of 1 mg/ml with a fluorescence spectrometer (Biolumin, Amersham Biosciences). The AGE-BSA exhibited ∼38-fold higher fluorescence intensity than BSA. AGE-BSA was labeled with 125I by using Iodogen according to the manufacturer's instruction. Protein concentrations were determined by BCA protein assay reagent kit. LDL (d = 1.019–1.063 g/ml) and high density lipoprotein (HDL) (d = 1.063–1.21 g/ml) were prepared by stepwise ultracentrifugation from plasma obtained from healthy volunteers. LDL was acetylated with acetic anhydrate as described previously (20Ishibashi S. Inaba T. Shimano H. Harada K. Inoue I. Mokuno H. Mori N. Gotoda T. Takaku F. Yamada N. J. Biol. Chem. 1990; 265: 14109-14117Abstract Full Text PDF PubMed Google Scholar) and oxidized by incubation in a buffer containing 5 μm CuSO4 for 16 h at 37 °C (21Steinbrecher U.P. Witztum J.L. Parthasarathy S. Steinberg D. Arteriosclerosis. 1987; 7: 135-143Crossref PubMed Google Scholar). The lipoproteins were dialyzed against a buffer containing 10 mm sodium phosphate, pH 7.4, and 150 mm NaCl. CHO cells overexpressing human FEEL-1 (CHO-FEEL-1), human FEEL-2 (CHO-FEEL-2), and human SREC (CHO-SREC) were obtained as described previously (17Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). These cells were maintained at 37 °C with 5% (v/v) CO2 in medium A (Ham's F-12 supplemented with 100 units/ml penicillin and 100 units/ml streptomycin) containing 10% (v/v) fetal calf serum and 0.2 mg/ml G418 (medium B). Untransfected CHO cells designated as CHO-Wild were maintained in medium B without G418. The four lines of CHO cells were cultured for 2 days to confluence in 12-well plates and used for the following experiments. Thioglycolate-elicited mouse peritoneal macrophages were prepared as described previously (22Perrey S. Ishibashi S. Kitamine T. Osuga J. Yagyu H. Chen Z. Shionoiri F. Iizuka Y. Yahagi N. Tamura Y. Ohashi K. Harada K. Gotoda T. Yamada N. Atherosclerosis. 2001; 154: 51-60Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and maintained in DMEM containing 10% (v/v) fetal calf serum. HUVECs were cultured with DMEM containing 20% fetal calf serum, 30 μg/ml endothelial cell growth supplement, and 10 IU/ml heparin. Confluent dishes were used for the studies within the 15 passages. Total RNA was prepared by TriZOL. Poly(A)+ RNA was purified using Oligotex-dT30 and subjected to 1% (w/v) agarose gel electrophoresis in the presence of formalin. The fractionated RNA was transferred to Hybond N, hybridized to32P-labeled cDNA probes, and analyzed by BAS2000 (FUJI XEROX, Tokyo, Japan). The expression levels of each gene were compared between various organs after adjusting to the expression level of 36B4. After washing twice with PBS, the confluent cells were incubated with 0.5 ml of medium C (medium A supplemented with 3% (w/v) BSA) containing the indicated concentrations of iodinated AGE-BSA with or without 20-fold excess of unlabeled AGE-BSA for 2 h at 4 °C. After washing three times with ice-cold PBS containing 2 mg/ml BSA and three times further with PBS, the cells were dissolved with 0.1 n NaOH and the cell-bound radioactivity and cellular proteins were determined. One hour prior to the study, the media of the confluent cells were changed to medium C. The media were replaced with medium C containing the indicated concentrations of 125I-AGE-BSA with or without 20-fold excess of unlabeled AGE-BSA and incubated for 6 h at 37 °C. The amounts of 125I-AGE-BSA either degraded by or associated with the cells were measured using trichloroacetic acid and AgNO3 as described previously (22Perrey S. Ishibashi S. Kitamine T. Osuga J. Yagyu H. Chen Z. Shionoiri F. Iizuka Y. Yahagi N. Tamura Y. Ohashi K. Harada K. Gotoda T. Yamada N. Atherosclerosis. 2001; 154: 51-60Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The differences of the means were compared by Student's t test. Northern blot analyses were performed to compare the mRNA expression of FEEL-1, FEEL-2, and SREC in CHO-FEEL-1, CHO-FEEL-2, and CHO-SREC cells, respectively (Fig.1). CHO-FEEL-1 and CHO-SREC expressed comparable amounts of the respective mRNA, which was 2-fold higher than the mRNA of FEEL-2 mRNA in CHO-FEEL-2 cells. 125I-AGE-BSA bound to both CHO-FEEL-1 and CHO-FEEL-2 cells in a saturable manner at 4 °C (Fig.2, A and B). Scatchard analysis (insets) showed the presence of single binding site for AGE-BSA with an apparent Kd of 2.55 and 1.68 μg/ml for FEEL-1 and FEEL-2, respectively. CHO-FEEL-2 cells bound ∼17 times larger amounts of 125I-AGE-BSA than CHO-FEEL-1 cells (Bmax = 315.5versus 18.9 ng/mg). On the other hand, the specific125I-AGE-BSA binding to CHO-SREC cells was 30% of that to CHO-FEEL-1 cells with lower affinity (Fig. 2C). Untransfected CHO cells bound negligible amounts125I-AGE-BSA (Fig. 2D). To examine whether the binding is associated with cellular uptake and degradation of125I-AGE-BSA, we incubated the cells with125I-AGE-BSA at 37 °C and determined the amounts of125I-AGE-BSA bound (Fig. 3,A–D) and degraded by the cells (Fig. 3, E–H). Significant amounts of 125I-AGE-BSA were taken up and degraded by both CHO-FEEL-1 and CHO-FEEL-2 cells but not by CHO-SREC and untransfected CHO cells. The activity was larger in CHO-FEEL-2 cells than in CHO-FEEL-1 cells. These results indicate that FEEL-1 and FEEL-2 but not SREC are endocytic receptors for AGE-BSA. To characterize the ligand specificity for the AGE-BSA binding site of the FEEL-1 and FEEL-2, we performed a competition study. First, we examined whether the binding of 125I-AGE-BSA is inhibited by lipoproteins (Fig.4, A and B). The binding of 125I-AGE-BSA to CHO-FEEL-1 (Fig. 4A) and CHO-FEEL-2 (Fig. 4B) cells was inhibited by Ac-LDL but not by LDL and HDL. It is noteworthy that Ac-LDL inhibited the binding more potently than AGE-BSA itself in CHO-FEEL-1 cells, whereas AGE-BSA inhibited the binding more potently than Ac-LDL in CHO-FEEL-2 cells. Table I shows concentrations required to inhibit the binding by 50% (IC50). Ox-LDL partially inhibited the binding only in CHO-FEEL-1 cells (∼40% reduction at 100 μg/ml). We next examined the inhibitory effect of various materials known as SR-A inhibitors (Fig. 4, C and D). Fucoidan, poly I, and dextran sulfate inhibited the 125I-AGE-BSA binding almost completely in both CHO-FEEL-1 (Fig. 4C) and CHO-FEEL-2 cells (Fig. 4D). Heparin was also effective in suppressing the binding, but the effect was weaker than the other three compounds (Table I).Table IConcentrations of various lipoproteins and SR-A inhibitors to inhibit the binding of 125I-AGE-BSA to CHO-FEEL-1 and CHO-FEEL-2 cells (IC50)CompetitorsCHO-FEEL-1CHO-FEEL-2μg/ml (nm)AGE-BSA22.2 (337)<6.9 (104)AcLDL<5.9 (11)16.8 (31)Fucoidan<5.4 (81)<5.0 (76)Polyinosinic acid<5.3 (11∼26)<5.0 (10∼25)Dextran sulfate<5.5 (11)<5.1 (10)Heparin8.9 (443∼1,770)7.2 (358∼1,434)CHO-FEEL-1 and CHO-FEEL-2 cells were incubated with 2 μg/ml125I-AGE-BSA for 2 h at 4 °C with AGE-BSA, LDL, Ac-LDL, Ox-LDL, HDL, fucoidan, polyinosinic acid (poly I), dextran sulfate, or heparin at the concentrations of 0, 10, 50, or 100 μg/ml. The IC50 value were calculated and shown for competitors that inhibited the binding of 125I-AGE-BSA >50% at 100 μg/ml. Values in parentheses are IC50 in molar concentrations, which were calculated based on the assumption that molecular masses of AGE-BSA, AcLDL, fucoidan, poly I, dextran sulfate, and heparin are 66, 549, 66.4, 200∼500, 500, and 5∼20 kDa, respectively. Open table in a new tab CHO-FEEL-1 and CHO-FEEL-2 cells were incubated with 2 μg/ml125I-AGE-BSA for 2 h at 4 °C with AGE-BSA, LDL, Ac-LDL, Ox-LDL, HDL, fucoidan, polyinosinic acid (poly I), dextran sulfate, or heparin at the concentrations of 0, 10, 50, or 100 μg/ml. The IC50 value were calculated and shown for competitors that inhibited the binding of 125I-AGE-BSA >50% at 100 μg/ml. Values in parentheses are IC50 in molar concentrations, which were calculated based on the assumption that molecular masses of AGE-BSA, AcLDL, fucoidan, poly I, dextran sulfate, and heparin are 66, 549, 66.4, 200∼500, 500, and 5∼20 kDa, respectively. We determined the effects of anti-FEEL-1 monoclonal antibody (FE-1-1) as well as other compounds on the endocytic uptake and degradation of125I-AGE-BSA by CHO-FEEL-1 cells (Fig.5). We have previously reported that FE-1-1 effectively suppressed the cellular uptake of Ac-LDL in CHO-FEEL-1 cells (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). AGE-BSA and Ac-LDL effectively inhibited the cellular uptake and degradation of125I-AGE-BSA by CHO-FEEL-1 cells. No inhibitory effect was observed with 100 μg/ml native LDL, Ox-LDL, HDL, and 30 μg/ml control IgG. FE-1-1 inhibited both cellular uptake and degradation of125I-AGE-BSA by 72 and 48%, respectively. The extent of inhibition by the antibody was slightly smaller than that by AGE-BSA or Ac-LDL but was almost the same as observed in inhibition study for cellular uptake of Ac-LDL (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). To estimate the contribution of FEEL-1 and FEEL-2 to the endocytosis of AGEs in various tissues in comparison with other receptors for AGEs, we performed Northern blot analyses (Fig.6). FEEL-1 was expressed in a wide variety of organs in the following order: liver > lung = heart = white adipose tissue > aorta = kidney > spleen. FEEL-2 whose expression was much lower than that of FEEL-1 was detectable only in the liver and spleen. In contrast to organs like lung, spleen, and white adipose tissue in which RAGE, galectin-3, and CD36 were most highly expressed, respectively, organs like heart, liver, and aorta appeared to express significant amounts of FEEL-1 whose expression level was as comparable as that of other receptors for AGEs. We further performed Northern blot analysis to evaluate the expression of FEEL-1 in mouse peritoneal macrophages and HUVECs (Fig. 7). Mouse peritoneal macrophages and HUVECs expressed FEEL-1 to a degree that was comparable with galectin-3 and SR-A, respectively. We have recently reported the cloning of novel scavenger receptors expressed on vascular endothelial cells by expression cloning strategy using 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbo-cyanine perchlorate (DiI)-labeled Ac-LDL as the ligand, such as SREC (17Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 31217-31220Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), FEEL-1, and its paralogous gene, FEEL-2 (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Structures of these receptors are unique and unrelated to other scavenger receptors. Although the precise functions of these receptors are currently unknown, in vitro studies have suggested their involvement in cellular interaction and host defense. For example, both FEEL receptors bind to Gram-negative and Gram-positive bacteria (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, anti-FEEL-1 antibody inhibited the in vitrovascular tube formation (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). On the other hand, SREC-I and its isoform, SREC-II, showed a strong heterophilic trans-interaction through their extracellular EGF-like repeat domains (23Ishii J. Adachi H. Aoki J. Koizumi H. Tomita S. Suzuki T. Tsujimoto M. Inoue K. Arai H. J. Biol. Chem. 2002; 277: 24014-24021Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This study has first revealed that FEEL-1 and FEEL-2 are endocytic receptors for AGEs, implicating the involvement of these receptors in the pathologies of aging or diabetes. Some of other receptors for AGEs are involved in these pathologies. For example, Park et al.(24Park L. Raman K.G. Lee K.J. Lu Y. Ferran Jr., L.J. Chow W.S. Stern D. Schmidt A.M. Nat. Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1018) Google Scholar) have shown that intravenous administration of the soluble extracellular domain of RAGE, which was originally cloned from bovine pulmonary endothelial cells, efficiently suppressed diabetic atherosclerosis in apolipoprotein E-deficient mice. Second, Pugliese et al. (25Pugliese G. Pricci F. Iacobini C. Leto G. Amadio L. Barsotti P. Frigeri L. Hsu D.K. Vlassara H. Liu F.T. Di Mario U. FASEB J. 2001; 15: 2471-2479Crossref PubMed Scopus (161) Google Scholar) have shown that diabetic glomerulopathy was rather accelerated in mice lacking galectin-3, a critical component of OST-48/80-K-H/galectin-3 complex (11Vlassara H. Li Y.M. Imani F. Wojciechowicz D. Yang Z. Liu F.T. Cerami A. Mol. Med. 1995; 1: 634-646Crossref PubMed Google Scholar) that is ubiquitously expressed in mammalian cells, suggesting its protective role. Third, Suzuki et al. (26Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. et al.Nature. 1997; 386: 292-296Crossref PubMed Scopus (1002) Google Scholar) and Matsumoto et al. (27Matsumoto K. Sano H. Nagai R. Suzuki H. Kodama T. Yoshida M. Ueda S. Smedsrød B. Horiuchi S. Biochem. J. 2000; 352: 233-240Crossref PubMed Scopus (49) Google Scholar) observed that peritoneal macrophages obtained from SR-A knock-out mice had the reduced capacity for endocytic degradation of AGE-BSA, which was 30% of that in wild-type cells. However, liver sinusoidal endothelial cells obtained from SR-A knock-out mice had capacity for endocytic degradation of AGE-BSA, which was indistinguishable from that of wild-type liver sinusoidal endothelial cells (27Matsumoto K. Sano H. Nagai R. Suzuki H. Kodama T. Yoshida M. Ueda S. Smedsrød B. Horiuchi S. Biochem. J. 2000; 352: 233-240Crossref PubMed Scopus (49) Google Scholar). These results suggest that SR-A is the primary endocytic receptor for AGE-BSA in macrophages, whereas other receptors mediate the uptake of AGEs in endothelial cells. Although FEEL-1 and FEEL-2 are structurally unrelated to SR-A, their ligand specificity is similar to that of SR-A. CHO-FEEL-1 cells bind not only Ac-LDL but also Ox-LDL to a lesser degree (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The binding was competitively inhibited by SR-A inhibitors, such as maleyl-BSA and dextran sulfate (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, both FEEL-1 and FEEL-2 bind AGE-BSA (Figs. 1 and 2) as does SR-A (12Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar). Ac-LDL and negatively charged compounds such as fucoidan, dextran sulfate, and poly I competitively inhibited the binding of AGE-BSA to CHO-FEEL-1 or CHO-FEEL-2 cells (Figs. 4 and 5) as is the case for SR-A (12Araki N. Higashi T. Mori T. Shibayama R. Kawabe Y. Kodama T. Takahashi K. Shichiri M. Horiuchi S. Eur. J. Biochem. 1995; 230: 408-415Crossref PubMed Scopus (223) Google Scholar). These results suggest that AGE-BSA and Ac-LDL share a same binding site on the receptors. Importantly, the binding affinity of AGE-BSA to FEEL-1 and FEEL-2 cells appeared to exceed that to other receptors for AGEs. CalculatedKd was 25 nm for FFEL-2; 39 nm for FEEL-1; 85 nm for CD36 (14Ohgami N. Nagai R. Ikemoto M. Arai H. Kuniyasu A. Horiuchi S. Nakayama H. J. Biol. Chem. 2001; 276: 3195-3202Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar); 100 nm for RAGE (9Neeper M. Schmidt A.M. Brett J. Yan S.D. Wang F. Pan Y.C. Elliston K. Stern D. Shaw A. J. Biol. Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar); 126 nm for SR-BI (13Ohgami N. Nagai R. Miyazaki A. Ikemoto M. Arai H. Horiuchi S. Nakayama H. J. Biol. Chem. 2001; 276: 13348-13355Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar); 148 nm for lectin-like oxidized low density lipoprotein receptor-1 (15Jono T. Miyazaki A. Nagai R. Sawamura T. Kitamura T. Horiuchi S. FEBS Lett. 2002; 511: 170-174Crossref PubMed Scopus (115) Google Scholar); and 350 nm for galectin3 (11Vlassara H. Li Y.M. Imani F. Wojciechowicz D. Yang Z. Liu F.T. Cerami A. Mol. Med. 1995; 1: 634-646Crossref PubMed Google Scholar), assuming that molecular mass of AGE-BSA is 66 kDa. The binding activity of CHO-FEEL-1 cells was ∼20-fold lower than that of CHO-FEEL-2 cells. A similar although less significant difference in the binding activity between the isoforms was observed for Ac-LDL (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Together with the fact that the mRNA expression of FEEL-2 in CHO-FEEL-2 cells was lower than that of FEEL-1 in CHO-FEEL-1, these results indicate that a significant proportion of the binding sites for AGEs on FEEL-1 was inactive or masked in CHO-FEEL-1 cells. This possibility warrants further investigation. In many organs, the mRNA expression levels of FEEL-1 were higher than those of FEEL-2 (Fig. 6). Mouse peritoneal macrophages and HUVECs also expressed FEEL-1 to a degree comparable with other receptors for AGEs (Fig. 7). Furthermore, FEEL-2 mRNA was barely detectable in several vascular endothelial cell lines or in monocyte/macrophages at nonstimulated conditions (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Taken together, we propose that FEEL-1 and possibly FEEL-2 serve as functional endocytic receptors for AGEsin vivo. This hypothesis needs to be verified by further studies, such as gene targeting, that are currently in progress in our laboratory. As suggested by the presence in the aorta (Fig. 6), macrophages, and endothelial cells (Fig. 7) (18Adachi H. Tsujimoto M. J. Biol. Chem. 2002; 277: 34264-34270Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), FEEL-1 may scavenge AGEs accumulated in vascular tissues in diabetes and aging, thereby directly contributing to the development of diabetic vascular complications and atherosclerosis. Because Smedsrød et al.(28Smedsrød B. Melkko J. Araki N. Sano H. Horiuchi S. Biochem. J. 1997; 322: 567-573Crossref PubMed Scopus (170) Google Scholar) reported that >90% of intravenously injected AGE-BSA was distributed in the liver in rats, it is also possible that FEEL-1, which was expressed in the liver (Fig. 6), may be involved in the elimination of AGEs from the circulation. In conclusion, we demonstrate that FEEL-1 and FEEL-2, but not SREC-I, function as endocytic receptors for AGEs when overexpressed in CHO cells. Although further studies will be needed to determine the role of these two receptors in vivo, they may play a pivotal role in the pathogenesis of diabetic vascular complications and could be the promising target molecules for their prevention. FEEL-1 is identical to stabilin-1 (hyaluronan-scavenger receptor; HA/S-R) first identified as MS-1 antigen (29Politz O. Gratchev A. McCourt P.A. Schledzewski K. Guillot P. Johansson S. Svineng G. Franke P. Kannicht C. Kzhyshkowska J. Longati P. Velten F.W. Johansson S. Goerdt S. Biochem. J. 2002; 362: 155-164Crossref PubMed Scopus (241) Google Scholar). AGE uptake by liver sinusoidal endothelial cells is partially inhibited by an antibody against HA/S-R (30Hansen B. Svistounov D. Olsen R. Nagai R. Horiuchi S. Smedsrød B. Diabetologia. 2002; 45: 1379-1388Crossref PubMed Scopus (37) Google Scholar).

Mechanical stress activates various hypertrophic responses, including activation of mitogen-activated protein kinases (MAPKs) in cardiac myocytes. Stretch activated extracellular signal-regulated kinases partly through secreted humoral growth factors, including angiotensin II, whereas stretch-induced activation of c-Jun NH(2)-terminal kinases and p38 MAPK was independent of angiotensin II. In this study, we examined the role of integrin signaling in stretch-induced activation of p38 MAPK in cardiomyocytes of neonatal rats. Overexpression of the tumor suppressor PTEN, which inhibits outside-in integrin signaling, strongly suppressed stretch-induced activation of p38 MAPK. Overexpression of focal adhesion kinase (FAK) antagonized the effects of PTEN, and both tyrosine residues at 397 and 925 of FAK were necessary for its effects. Stretch induced tyrosine phosphorylation and activation of FAK and Src. Stretch-induced activation of p38 MAPK was abolished by overexpression of FAT and CSK, which are inhibitors of the FAK and Src families, respectively, and was suppressed by overexpression of a dominant-negative mutant of Ras. Mechanical stretch-induced increase in protein synthesis was suppressed by SB202190, a p38 MAPK inhibitor. These results suggest that mechanical stress activates p38 MAPK and induces cardiac hypertrophy through the integrin-FAK-Src-Ras pathway in cardiac myocytes.

To reveal genetic risk factors of nonfamilial idiopathic cardiomyopathy (IDC) in Japanese, polymorphisms in the SOD2 and HLA-DRB1 genes were investigated in 86 patients and 380 healthy controls. There was a significant excess of homozygotes for the V allele [Val versus Ala (A allele), a polymorphism in the leader peptide of manganese superoxide dismutase at position 16] of the SOD2 gene in the patients compared with the controls (87.2% versus 74.7%, odds ratio = 2.30, p = 0.013, pc < 0.03), and a significant increase in the frequency of HLA-DRB1*1401 in the patients was confirmed (14.0% vs 4.5%, odds ratio = 3.46, p = 0.001, pc < 0.03). A two-locus analysis suggested that these two genetic markers (SOD2-VV genotype and DRB1*1401) may play a synergistic role in controlling the susceptibility to nonfamilial IDC. In addition, processing efficiency of Val-type SOD2 leader peptide in the presence of mitochondria was siginificantly lower than that of the Ala-type by 11 +/- 4%, suggesting that this lower processing efficiency was in part an underlying mechanism of the association between the SOD2-VV genotype and nonfamilial IDC.

Adrenomedullin (AM) is a peptide involved both in the pathogenesis of cardiovascular diseases and in circulatory homeostasis. The high-affinity AM receptor is composed of receptor activity-modifying protein 2 or 3 (RAMP2 or -3) and the GPCR calcitonin receptor-like receptor. Testing our hypothesis that RAMP2 is a key determinant of the effects of AM on the vasculature, we generated and analyzed mice lacking RAMP2. Similar to AM-/- embryos, RAMP2-/- embryos died in utero at midgestation due to vascular fragility that led to severe edema and hemorrhage. Vascular ECs in RAMP2-/- embryos were severely deformed and detached from the basement membrane. In addition, the abnormally thin arterial walls of these mice had a severe disruption of their typically multilayer structure. Expression of tight junction, adherence junction, and basement membrane molecules by ECs was diminished in RAMP2-/- embryos, leading to paracellular leakage and likely contributing to the severe edema observed. In adult RAMP2+/- mice, reduced RAMP2 expression led to vascular hyperpermeability and impaired neovascularization. Conversely, ECs overexpressing RAMP2 had enhanced capillary formation, firmer tight junctions, and reduced vascular permeability. Our findings in human cells and in mice demonstrate that RAMP2 is a key determinant of the effects of AM on the vasculature and is essential for angiogenesis and vascular integrity.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is one of the nuclear receptors that plays a central role in adipocyte differentiation and insulin sensitivity. PPAR gamma has also recently been recognized as an endogenous regulator of intestinal inflammation. However, its levels are decreased during chronic inflammation in human and mice, thus limiting PPAR gamma ligand therapy during established disease. We sought to determine whether this decrease in PPAR gamma could be counteracted by a gene therapy approach.We characterized PPAR gamma levels in experimental colitis associated with dextran sodium sulfate administration to mice. In this model, the therapeutic benefits of PPAR gamma gene therapy using a replication-deficient adenovirus vector expressing PPAR gamma (Ad-PPAR gamma) was assessed.PPAR gamma protein levels were decreased in whole colonic tissue, lamina propria lymphocytes, and peritoneal exudate cells during the course of colitis. PPAR gamma gene delivery using Ad-PPAR gamma restored responsiveness to a PPAR gamma ligand, resulting in marked amelioration of tissue inflammation associated with the colitis, which included attenuation of intercellular adhesion molecule-1, cyclooxygenase-2 and tumor necrosis factor-alpha expression.Our results suggest that gene delivery of PPAR gamma can be used to restore and/or enhance endogenous anti-inflammatory processes that are normally operative in mammalian tissues such as in the colon.

Cardiotoxicity leading to congestive heart failure is a complication of the anthracyclines. Biochemical methods to diagnose and monitor cardiac function after anthracycline administration would be most useful. We examined the diagnostic role of B-type natriuretic peptide (BNP), a potent biochemical marker of left ventricular dysfunction, in patients administered anthracyclines.Twenty-seven consecutive patients receiving anthracyclines were investigated by serial measurements of BNP levels and other cardiac neurohormones (A-type natriuretic peptide, renin, aldosterone, angiotensin II, norepinephrine, and epinephrine) and myocardial markers (creatine kinase-MB and myosin light chain). Echocardiography was done to assess systolic (ejection fraction) and diastolic (mitral inflow A/E ratio) functions.Of the examined cardiac biochemical markers, BNP levels alone showed marked elevations to abnormal levels after anthracycline administration. Most patients showed transient increases (peak at 3 to 7 days). Patients with persistent elevations showed a poor prognosis. A/E ratio also correlated with increases in BNP levels in selected patients, which may suggest that raised BNP levels are reflective of induced diastolic dysfunction.Our studies suggest the possible use of BNP levels to assess the cardiac state after anthracycline administration. BNP levels most likely reflect cardiac tolerance to the cardiotoxic agent. Serial BNP profiles also suggest persistent elevations to be associated with potentially decompensatory states in contrast to tolerable transient increases. Diagnosis of degree of cardiac tolerance by response to drug administration may be analogous to use of stress testing (exercise) to help define underlying left ventricular dysfunction.

Adiponectin, one of the insulin-sensitizing adipokines, has been shown to activate fatty acid oxidation in liver and skeletal muscle, thus maintaining insulin sensitivity. However, the precise roles of adiponectin in fatty acid synthesis are poorly understood. Here we show that adiponectin administration acutely suppresses expression of sterol regulatory element-binding protein (SREBP) 1c, the master regulator which controls and upregulates the enzymes involved in fatty acid synthesis, in the liver of +Lepr(db)/+Lepr(db) (db/db) mouse as well as in cultured hepatocytes. We also show that adiponectin suppresses SREBP1c by AdipoR1, one of the functional receptors for adiponetin, and furthermore that suppressing either AMP-activated protein kinase (AMPK) via its upstream kinase LKB1 deletion cancels the negative effect of adiponectin on SREBP1c expression. These data show that adiponectin suppresses SREBP1c through the AdipoR1/LKB1/AMPK pathway, and suggest a possible role for adiponectin in the regulation of hepatic fatty acid synthesis.

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.

Endothelial PAS domain protein 1 (EPAS1) is a basic-helix-loop-helix/PAS domain transcription factor that is expressed preferentially in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and is reported to transactivate vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), and Tie2 promoters. In this study, we analyzed the role of EPAS1 in the process of angiogenesis. Using microarray technology, we looked for target genes regulated by EPAS1 in vascular endothelial cells. A total of 130 genes were upregulated by EPAS1, including fms-like tyrosine kinase-1 (Flt-1). Reporter analysis using human Flt-1 promoter and gel mobility shift assays showed that the heterodimer of EPAS1 and aryl hydrocarbon receptor nuclear translocator binds directly to HIF-1-binding site upstream of Flt-1 promoter and transactivates it. Small interfering RNA targeted to EPAS1 but not HIF-1alpha attenuated desferrioxamine-induced Flt-1 mRNA expression, thus EPAS1 is thought to play an essential role in hypoxic induction of Flt-1 gene. Furthermore, using mouse wound healing models, we demonstrated that adenovirus-mediated delivery of EPAS1 gene significantly induced the expression of VEGF, Flt-1, Flk-1, and Tie2 mRNA at the wound site and promoted mature angiogenesis. The proportion of the number of mural cells in newly formed vessels was significantly higher in EPAS1-treated wound area than VEGF-treated area. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2, leading to enhancement of mature angiogenesis in vivo. Thus, EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.

Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription.

Accumulating evidence suggests that adipose tissue not only stores energy but also secretes various bioactive substances called adipocytokines. Periadventitial fat is distributed ubiquitously around arteries throughout the body. It was reported that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular diseases accelerated by obesity. We investigated the effect of endovascular injury on the phenotype of perivascular fat.Endovascular injury significantly upregulated proinflammatory adipocytokines and downregulated adiponectin within periadventitial fat tissue in models of mouse femoral artery wire injury and rat iliac artery balloon injury. Genetic disruption of tumor necrosis factor (TNF)-alpha attenuated upregulation of proinflammatory adipocytokine expression, with reduced neointimal hyperplasia after vascular injury. Local delivery of TNF-alpha to the periadventitial area enhanced inflammatory adipocytokine expression, which was associated with augmented neointimal hyperplasia in TNF-alpha-deficient mice. Conditioned medium from a coculture of 3T3-L1 and RAW264 cells stimulated vascular smooth muscle cell proliferation. An anti-TNF-alpha neutralizing antibody in the coculture abrogated the stimulating effect of the conditioned medium.Our findings indicate that endovascular injury induces rapid and marked changes in perivascular adipose tissue, mainly mediated by TNF-alpha. It is suggested that the phenotypic changes in perivascular adipose tissue may have a role in the pathogenesis of neointimal hyperplasia after angioplasty.

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.

It has been suggested that transcription factor 7-like 2 protein (TCF7L2) plays an important role in glucose metabolism by regulating the production level of glucagon-like peptide-1, a hormone which modifies glucose-dependent insulin secretion. Recently, variants of TCF7L2 gene were reported to confer an increased risk of type 2 diabetes in three different samples from European and European-origin populations. We studied whether the single nucleotide polymorphisms (SNPs) in TCF7L2 were associated with type 2 diabetes in samples from a Japanese population.Five SNPs were genotyped in three different sample sets. Association with type 2 diabetes was investigated in each, as well as in combined sample sets.The SNP rs7903146 was nominally associated with type 2 diabetes in the initial (p = 0.08) and two replication sample sets (p = 0.05 and 0.06). For the combined sample set, in which we successfully genotyped 1,174 type 2 diabetes patients and 823 control subjects, rs7903146 showed a significant association with type 2 diabetes (odds ratio = 1.69 [95% CI 1.21-2.36], p = 0.002) with the same direction as the previous reports in samples from European and European-origin populations. SNPs rs7903146 and rs7901695 were in complete linkage disequilibrium. The rest of the five SNPs (rs7895340, rs11196205 and rs12255372) did not show any significant associations with type 2 diabetes.The consistent association between rs7903146 in TCF7L2 and type 2 diabetes in different ethnic groups, including the Japanese population, suggests that TCF7L2 is a common susceptibility gene for type 2 diabetes.

Alteration in the differentiated state of smooth muscle cells (SMCs) is known to be integral to vascular development and the pathogenesis of vascular disease. However, it is still largely unknown how environmental cues translate into transcriptional control of SMC genes. We found that deltaEF1 is upregulated during SMC differentiation and selectively transactivates the promoters of SMC differentiation marker genes, SM alpha-actin and SM myosin heavy chain (SM-MHC). DeltaEF1 physically interacts with SRF and Smad3, resulting in a synergistic activation of SM alpha-actin promoter. Chromatin immunoprecipitation assays and knockdown experiments showed that deltaEF1 is involved in the control of the SMC differentiation programs induced by TGF-beta signaling. Overexpression of deltaEF1 inhibited neointima formation and promoted SMC differentiation, whereas heterozygous deltaEF1 knockout mice exhibited exaggerated neointima formation. It thus appears deltaEF1 mediates SMC differentiation via interaction with SRF and Smad3 during development and in vascular disease.

Accumulating evidence demonstrates that dietary intake of n-3 polyunsaturated fatty acids (PUFAs) is associated with reduced incidence of cardiovascular events. However, the molecular mechanisms by which n-3 PUFAs prevent atherosclerosis are not fully understood. Here, we examined the effect of eicosapentaenoic acid (EPA), a major n-3 PUFA, on the pathogenesis of atherosclerosis in ApoE-deficient mice. Five-week-old ApoE-deficient male mice were fed on western-type diet supplemented with 5% (w/w) EPA (EPA group, n=7) or not (control group, n=5) for 13 weeks. An analysis of the fatty acid composition of liver homogenates revealed a marked increase of the n-3 PUFA content in the EPA group (n-3/n-6 ratio: 0.20+/-0.01 vs. 2.5+/-0.2, p<0.01). En face Sudan IV staining of the aorta and oil red O-staining of the aortic sinus revealed that EPA significantly suppressed the development of atherosclerotic lesions. We also observed anti-atherosclerotic effects of EPA in LDL-receptor-deficient mice. The lesions of the EPA group contained more collagen (19.6+/-2.4% vs. 32.9+/-3.9%, p<0.05) and smooth muscle cells (1.3+/-0.2% vs. 3.6+/-0.8%, p<0.05) and less macrophages (32.7+/-4.1% vs. 14.7+/-2.0%, p<0.05). Pretreatment with EPA attenuated the up-regulation of VCAM-1, ICAM-1 and MCP-1 in HUVECs as well as the expression of MMP-2 and MMP-9 in macrophage-like cells induced by TNF-alpha. The anti-inflammatory effects of EPA were abrogated when the expression of peroxisome proliferator-activated receptor alpha (PPARalpha) was suppressed. EPA may potentially reduce and stabilize atherosclerotic lesions through its anti-inflammatory effects.

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.

Similar to the healthcare systems in other industrialized countries, the Japanese healthcare system is facing the problem of increasing medical expenditure. In Japan, this situation may be primarily attributed to advanced technological developments, an aging population, and increasing patient demand. Japan also faces the problem of a declining youth population due to a low birth rate. Taken together, these problems present the healthcare system with a very difficult financial situation. Several reforms have been undertaken to contain medical expenditure, such as increasing employee copayment for health insurance from 10% to 20% in 1997 and from 20% to 30% in 2003 in order to curb unnecessary visits to medical institutions. Since the aging of the Japanese population is inevitable, a suitable method to contain medical expenditure may be to screen individuals who are likely to develop lifestyle-related diseases and conduct early intervention programs for them to prevent the development of diseases such as myocardial infarction or stroke that are costly to treat. If this goal is attained, it may contribute to the containment of medical expenditure as well as to improving the quality of life of the elderly. Therefore, the Japanese Ministry of Health, Labor and Welfare has decided to introduce a nationwide health screening and intervention program specifically targeting the metabolic syndrome commencing April 2008. Here, we discuss (1) the background of the Japanese healthcare system and the problems facing it, (2) the underlying objective and details of the new screening program, and (3) the expected impact of the program.

A decrease in plasma adiponectin levels has been shown to contribute to the development of diabetes. However, it remains uncertain whether adiponectin plays a role in the regulation of insulin secretion. In this study, we investigated whether adiponectin may be involved in the regulation of insulin secretion in vivo and in vitro.The effect of adiponectin on insulin secretion was measured in vitro and in vivo, along with the effects of adiponectin on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentration and state of 5'-AMP-activated protein kinase (AMPK). In addition, insulin granule transport was measured by membrane capacitance and total internal reflection fluorescence (TIRF) analysis.Adiponectin significantly stimulated insulin secretion from pancreatic islets to approximately 2.3-fold the baseline value in the presence of a glucose concentration of 5.6 mmol/l. Although adiponectin had no effect on ATP generation, membrane potentials, Ca2+ currents, cytosolic calcium concentrations or activation status of AMPK, it caused a significant increase of membrane capacitance to approximately 2.3-fold the baseline value. TIRF analysis revealed that adiponectin induced a significant increase in the number of fusion events in mouse pancreatic beta cells under 5.6 mmol/l glucose loading, without affecting the status of previously docked granules. Moreover, intravenous injection of adiponectin significantly increased insulin secretion to approximately 1.6-fold of baseline in C57BL/6 mice.The above results indicate that adiponectin induces insulin secretion in vitro and in vivo.

Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBP-binding element on the promoter ("autoloop regulatory circuit"), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukaemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to the Fli1 silencing-dependent collagen induction, the simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B-cell activation and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.