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. 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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. 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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