A Cysteine-rich Adipose Tissue-specific Secretory Factor Inhibits Adipocyte Differentiation

Abstract

A 12.5-kDa cysteine-rich adipose tissue-specific secretory factor (ADSF/resistin) is a novel secreted protein rich in serine and cysteine residues with a unique cysteine repeat motif of CX12CX8CXCX3CX10CXCXCX9CC. A single 0.8-kilobase mRNA coding for this protein was found in various murine white adipose tissues including inguinal and epididymal fats and also in brown adipose tissue but not in any other tissues examined. Two species of mRNAs with sizes of 1.4 and 0.8 kilobases were found in rat adipose tissue. Sequence analysis indicates that this is because of two polyadenylation signals, the proximal one with the sequence AATACA with a single base mismatch from murine AATAAA and the distal consensus sequence AATAAA. The mRNA level was markedly increased during 3T3-L1 and primary preadipocyte differentiation into adipocytes. Its expression in adipose tissue is under tight nutritional and hormonal regulation; the mRNA level was very low during fasting and increased 25-fold when fasted mice were refed a high carbohydrate diet. It was also very low in adipose tissue of streptozotocin-diabetes and increased 23-fold upon insulin administration. Upon treatment with the conditioned medium from COS cells transfected with the expression vector, conversion of 3T3-L1 cells to adipocytes was inhibited by 80%. The regulated expression pattern suggesting this factor as an adipose sensor for the nutritional state of the animals and the inhibitory effect on adipocyte differentiation implicate its function as a feedback regulator of adipogenesis. A 12.5-kDa cysteine-rich adipose tissue-specific secretory factor (ADSF/resistin) is a novel secreted protein rich in serine and cysteine residues with a unique cysteine repeat motif of CX12CX8CXCX3CX10CXCXCX9CC. A single 0.8-kilobase mRNA coding for this protein was found in various murine white adipose tissues including inguinal and epididymal fats and also in brown adipose tissue but not in any other tissues examined. Two species of mRNAs with sizes of 1.4 and 0.8 kilobases were found in rat adipose tissue. Sequence analysis indicates that this is because of two polyadenylation signals, the proximal one with the sequence AATACA with a single base mismatch from murine AATAAA and the distal consensus sequence AATAAA. The mRNA level was markedly increased during 3T3-L1 and primary preadipocyte differentiation into adipocytes. Its expression in adipose tissue is under tight nutritional and hormonal regulation; the mRNA level was very low during fasting and increased 25-fold when fasted mice were refed a high carbohydrate diet. It was also very low in adipose tissue of streptozotocin-diabetes and increased 23-fold upon insulin administration. Upon treatment with the conditioned medium from COS cells transfected with the expression vector, conversion of 3T3-L1 cells to adipocytes was inhibited by 80%. The regulated expression pattern suggesting this factor as an adipose sensor for the nutritional state of the animals and the inhibitory effect on adipocyte differentiation implicate its function as a feedback regulator of adipogenesis. CCAAT enhancer-binding protein α adipose tissue-specific secretory factor preadipocyte factor-1 fetal bovine serum dexamethasone methylisobutylxanthine fatty acid synthase peroxisome proliferator-activated receptor γ adipocyte fatty acid-binding protein stearoyl CoA desaturase Dulbecco's modified Eagle's medium thiazolidinediones base pairs nucleotide hemagglutinin polymerase chain reaction reverse transcriptase PCR expressed sequence tag Adipose tissue is the major energy reservoir in higher eukaryotes; storing triacylglycerol in periods of energy excess and its mobilization during energy shortage are its primary purposes. During adipose tissue development, genes that code for the lipid transport and lipogenic and lipolytic enzymes are induced to carry out the adipocyte function of triacylglycerol synthesis, storage, and mobilization. For the past decades, in vitro systems including preadipocyte cell lines such as 3T3-L1 cells as well as primary preadipocytes in culture have been extensively used (1Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1873) Google Scholar, 2Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (616) Google Scholar, 3Wienderer L. Loffler G. J. Lipid Res. 1987; 28: 649-658Abstract Full Text PDF PubMed Google Scholar). Transcriptional activation of adipocyte genes has been the focus of much research. CCAAT enhancer-binding protein (C/EBPα)1 and PPARγ have been shown to be critical in directing adipocyte-specific gene expression and adipogenesis (4Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar, 5Umek R.M. F Friedman A.D. McKnight S.L. Science. 1991; 251: 288-292Crossref PubMed Scopus (573) Google Scholar, 6Tontonoz P.E. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar, 7Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (411) Google Scholar). In animals, the activities of critical enzymes in triacylglycerol biosynthesis and lipolysis are tightly controlled by nutritional and hormonal conditions (8Joseph D. Paulauskis J.D. Sul H.S. J. Biol. Chem. 1989; 264: 574-577Abstract Full Text PDF PubMed Google Scholar). For example, feeding causes an induction whereas fasting causes suppression of lipogenic enzymes. Elevated insulin over a high carbohydrate diet feeding is thought to induce these enzymes in lipogenesis. The role of insulin can also be demonstrated by administration of insulin to diabetic animals. These enzymes have also been shown to be expressed at a high level in the adipose tissue of animal obesity models including ob/ob and db/db mice as well as Zucker rats (9Penicaud L. Ferre P. Assimacopoulos-Jeannet F. Perdereau D. Leturque A. Jeanrenaud B. Picon L. Girard J. Biochem. J. 1991; 279: 303-308Crossref PubMed Scopus (52) Google Scholar). Hyperinsulinemia may be responsible for elevated levels of the enzymes. The role of adipose tissue mainly as an organ for energy storage and mobilization has recently been expanded by the discovery of leptin (10Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11807) Google Scholar,11Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4256) Google Scholar). Leptin is primarily made and secreted by mature adipocytes. It binds to its receptor in the hypothalamus and may function in regulating body fat mass (12Maffei M. Fei H. Lee G.H. Dani C. Leroy P. Zhang Y. Proenca R. Negrel R. Ailhaud G. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6957-6960Crossref PubMed Scopus (420) Google Scholar, 13Hotamisligil G.S. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar). Other immune system-related proteins such as TNF-α, adipsin, and ACRP30/AdipoQ along with vascular function-related molecules such as angiotensinogen and plasminogen activator inhibitor type I have been shown to be secreted by adipose tissue (14Scherer 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 (2760) Google Scholar, 15He E. Liang P. Spiegelman B.M. J. Biol. Chem. 1996; 271: 10697-10703Abstract Full Text Full Text PDF PubMed Scopus (1901) Google Scholar, 16Shimomura L.T. Funahashi M. Takahashi K. Maeda K. Kotani T. Nakamura S. Yamashita M. Miura Y. Fukuda K. Takemura K. Tokunaga K. Matsuzawa Y. Nat. Med. 1996; 2: 800-803Crossref PubMed Scopus (823) Google Scholar). In addition, adipocytes also secrete factors such as Pref-1, which inhibits adipocyte differentiation (17Smas C.M. Chen L. Sul H.S. Mol. Cell. Biol. 1997; 17: 977-988Crossref PubMed Scopus (166) Google Scholar, 18Smas C.M. Sul H.S. Cell. 1993; 73: 725-734Abstract Full Text PDF PubMed Scopus (564) Google Scholar, 19Smas C.M. Kachinskas D. Liu C.-M. Xie X. Dircks L.K. Sul H.S. J. Biol. Chem. 1998; 273: 31751-31758Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Although the precise functions of these molecules are not clear, adipose tissue as a secretory organ to regulate other physiological processes as well as energy balance and homeostasis is now well established. Adipose tissue must secrete factors reflecting the nutritional status and regulating adipose tissue mass. We report here the identification and function of a serine/cysteine-rich adipocyte-specificsecretory factor (ADSF) that does not belong to known classes of cysteine-rich proteins. Its mRNA is expressed only in adipose tissue. The mRNA is induced markedly during differentiation of 3T3-L1 and primary preadipocytes. In fasted or diabetic animals, its expression is very low or non-detectable in adipose tissue but increases markedly upon feeding or insulin administration. Furthermore, when treated with the conditioned medium from COS cells transfected with the expression vector, adipose conversion of 3T3-L1 cells was inhibited, indicating its potential role as a feedback regulator of adipogenesis. During the submission of this manuscript, the Lazar laboratory (20Steppan C.M. Balley S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (4004) Google Scholar) reported this protein as a TZD down-regulated factor contributing to insulin resistance. Adipose tissue-specific genes were examined by microarray analysis using rat Genefilter membranes (Research Genetics). Filters were hybridized with α-33P-labeled cDNAs synthesized with 5 μg of total RNA. Spots exclusively hybridized with cDNA probes prepared from adipose tissue RNA were used for sequence analysis. Mice were fasted for 48 h, or fasted mice were refed a 58% carbohydrate fat-free diet (21Moon Y.S. Latasa M.J. Kim K.-H. Wang D. Sul H.S. J. Biol. Chem. 2000; 275: 10121-10127Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Induction of diabetes and insulin treatments were carried out as described previously (21Moon Y.S. Latasa M.J. Kim K.-H. Wang D. Sul H.S. J. Biol. Chem. 2000; 275: 10121-10127Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). A hemagglutinin (HA)-tagged full-length mouse ADSF/resistin fragment was prepared by PCR amplification. The 5′ primer, 5′-TGGGACAGGAGCTAATACCCAGA-3′ and 3′ primer, 5′-GTCAAGCATAATCTGGAACATCATATGGATAGGAAGCGACCTGCAGCTT-3′ were used. The resultant PCR product was originally ligated into pCR3.1 (Invitrogen), and a BamHI-XhoI fragment was then subcloned into pcDNA3.1 (Invitrogen). The sequence of the resultant plasmid was confirmed by restriction mapping and sequencing. 3T3-L1 preadipocytes were maintained in DMEM containing 10% FBS. For adipocyte differentiation, confluent cells were treated with 1 μm Dex and 0.5 mm MIX as described previously (22Rubin C.S. Hirsch A. Fung C. Rosen O.M. J. Biol. Chem. 1978; 253: 7570-7578Abstract Full Text PDF PubMed Google Scholar). For experiments in which conditioned medium was used, cells were cultured in medium composed of 75% conditioned medium and 25% DMEM, 10% FBS plus Dex and MIX. The adipose-derived stromal vascular fractions from rats were prepared as has been described previously (23Gregoire F.M. Johnson P.R. Greenwood M.R. Int. J. Obes. Relat. Metab. Disord. 1995; 19: 664-670PubMed Google Scholar). Briefly, the subcutaneous inguinal fat deposits from female Zuker rats were dissected and the lymph nodes were removed. The stromal vascular cells were obtained by collagenase (Sigma, 540 units/mg) digestion at 1 mg/ml at 37 °C for 45 min in Hepes-phosphate buffer (10 mm HEPES, pH 7.4, 135 mm NaCl, 2.2 mm CaCl2, 1.25 mm MgSO4, 0.45 mmKH2PO4, 2.17 mmNa2HPO4, 5 mmd-glucose, and 2% w/v bovine serum albumin). The cell suspension was filtered through a 100-μm nylon filter and centrifuged at 400 × g for 10 min. The pellets were washed, filtered through a 25-μm nylon filter, and plated at a density of 2.5 × 104 cells/cm2 in DMEM, 10% FBS. At confluence, differentiation was initiated by the addition of 0.1 μm Dex, 0.25 mm MIX, and 17 nminsulin. After 2 days, the medium was replaced by DMEM, 10% FBS plus insulin only. The pcDNA3.1 expression vector was transiently transfected into COS cells using DEAE-dextran in DMEM with 10% serum plus (JRH Biosciences) as described previously (24Gulick T. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1996: 9201-9210Google Scholar). Twenty-four hours after transfection, the medium was changed to DMEM supplemented with 10% FBS. The conditioned medium was collected 72 h after transfection, centrifuged at 500 × g for 5 min, and stored at 4 °C for less than a week before use. The total RNA from the rat tissues was prepared by guanidine isothiocyanate/cesium chloride centrifugation. The total RNA from the cells was prepared using TriZOL reagent (Life Technologies, Inc.). RNA was electrophoresed in 1% formaldehyde-agarose gel in 2.2 m formaldehyde, 20 mm MOPS, 1 mm EDTA, and transferred to Hybond N (Amersham Pharmacia Biotech). After UV cross-linking, the membranes were hybridized with the α-32P-labeled cDNA probes in ExpressHyb solution (CLONTECH). The membranes were exposed to x-ray film with an intensifying screen, and the signals were scanned using the Molecular Analyst (Bio-Rad). Cells were lysed in a buffer containing 50 mm Tris-HCl, pH 8.0, 120 mm NaCl, 0.5% Nonidet P-40, 1 mm EDTA, and 2 mmphenylmethylsulfonyl fluoride for 30 min on ice. The protein content was determined by Bradford assay (Bio-Rad). Thirty μg of protein were subjected to SDS-polyacrylamide gel electrophoresis, electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore), and immunodetected using mouse anti-HA antiserum (Covance) and goat anti-mouse IgG-horseradish peroxidase conjugate (Bio-Rad) by using an enhanced chemiluminescence detection kit (Bio-Rad). RNA was reverse transcribed at 37 °C for 60 min, and the products were used as the template for two PCR reactions in one tube containing target genes with actin as an internal control. The primers used were PPARγ (PCR product of 375 bp in length), 5′-ATGCCATTCTGGCCCACCAAC-3′ and 5′-CCTTGCATCCTTCCAAAGCAT-3′; aFABP (PCR product of 310 bp in length) 5′-CTTGTCTCCAGTGAAAACTT-3′ and 5′-ACCTTCTCGTTTTCTTTAT-3′; FAS (PCR product of 120 bp in length), 5′-AGGGGTCGACCTGGTCCTCA-3′ and 5′-GGGTGGTTGTTAGAAAGAT-3′; and actin (PCR product of 282 bp in length), 5′-TCCTATGTGGGTGACGAGGC-3′ and 5′-CATGGCTGGGGTGTTGAAGG-3′. To identify novel genes that are only expressed in adipocytes and are induced during adipocyte differentiation, we compared expression levels of rat expressed sequence tag (EST) sequences by cDNA microarray. RNA samples were prepared from white adipose tissue as well as brown adipose tissue, liver, muscle, and brain. The RNAs were used to synthesize cDNAs for hybridization, and those sequences that were expressed only in white and brown adipose tissues were identified. Candidate EST clones were sequenced, and one such sequence was identified as that coding for a novel adipose tissue-specific, serine- and cysteine-rich secreted protein. This gene was originally named as FIZZ3, which belongs to a gene family whose founding member, FIZZ1, is implicated as a possible mediator of neuronal function and airway hyperactivity B. A. C. Wright J. 2000; 19: PubMed Scopus Google Scholar). this was the protein was also identified as a TZD down-regulated adipocyte which may to insulin (20Steppan C.M. Balley S.T. Bhat S. Brown E.J. Banerjee R.R. Wright C.M. Patel H.R. Ahima R.S. Lazar M.A. Nature. 2001; 409: 307-312Crossref PubMed Scopus (4004) Google Scholar). now we to the novel serine/cysteine-rich adipocyte-specific secretory factor as The cDNA sequence of rat ADSF/resistin is shown in A. It a cDNA with two potential polyadenylation the proximal one at and the distal one at analysis two mRNAs with sizes of 1.4 and 0.8 found in rat adipose tissue with the in to the In murine fat on the other we only one which to the rat we murine we found that the rat the mouse was in the with only one polyadenylation sequence to the rat The mRNA species in analysis and sequence analysis that the in the of rat and murine mRNAs is in the mouse cDNA sequence a consensus AATAAA. In rat cDNA sequence the proximal polyadenylation with a single base which is a and causes the of the mRNA using the distal polyadenylation of AATAAA. rat and murine sequences is in nucleotide sequence and in coding and The a acid protein with a mass of and 75% the rat and murine sequence 1 is by a the 20 residues and is of a To this protein is we a expression vector and transiently transfected into COS analysis demonstrated that the COS cells synthesized a protein with a to the in vitro synthesized and the secreted protein was in the conditioned medium The is the of and of the acid residues with a Although are classes of cysteine-rich protein the of the does not the known classes of cysteine-rich proteins. the motif possible We examined the tissue of the mRNA by analysis using various tissues from mice and rats two mRNA species of 1.4 and 0.8 were only in rat adipose tissue but not in any other tissues examined including liver, and A single mRNA was only in murine adipose tissue not We also the two mRNAs at levels in of rat white adipose tissues including epididymal and inguinal fat these mRNAs were also found in brown adipose tissue in than in white adipose tissue. We adipose tissue into stromal vascular which mainly preadipocytes and adipocyte We found that in adipocyte these mRNAs were not in stromal vascular fractions suggesting that ADSF/resistin expression may be induced during adipogenesis. To ADSF/resistin expression is regulated during adipocyte differentiation, mRNA levels were examined in 3T3-L1 preadipocytes during differentiation into adipocytes 2 The ADSF/resistin mRNA was not in the preadipocyte levels of the murine as by the murine of 3T3-L1 were markedly increased during differentiation into adipocytes. expression of aFABP was induced during adipose whereas actin expression was by We also carried out in vitro differentiation of stromal vascular cells from rat adipose tissue. of adipose cells from the epididymal fat may processes of adipogenesis. Two species and 0.8 of rat mRNAs were in plated preadipocytes of stromal vascular but expression was increased during adipose conversion initiated by Dex and MIX The in mRNA levels during adipogenesis of 3T3-L1 and primary preadipocytes was to or than that of other adipocyte such as adipocyte fatty acid-binding protein and CoA The of 3T3-L1 and primary preadipocytes in culture along with its tissue that the mRNA levels during of adipose tissue development, and ADSF/resistin is expressed only in adipose tissue in mature To the of of ADSF/resistin expression in adipose tissue, we examined mRNA levels and hormonal shown in the mRNA levels were low when mice were Upon a high carbohydrate fat-free the mRNA levels increased 25-fold after FAS mRNA levels increased by with the treatment that lipogenesis. of causes an increased of We the in mRNA levels during is by an in insulin We The mRNA levels were low in the adipose tissue of diabetic mice Upon insulin the mRNA level was increased 23-fold after 30 min. FAS mRNA levels increased after 30 min. mRNA levels not by or by administration. These that ADSF/resistin is regulated in a to the lipogenic fatty acid the lipogenic which are induced in and adipose tissue during feeding and by ADSF/resistin is expressed only in adipose tissue and induced by and that is expressed only in adipose tissue and induced when the animals are in a we that ADSF/resistin of preadipocytes. is that this protein may be a to adipocytes for the increased to excess We collected medium from COS cells transfected with expression vector containing full-length mouse The conditioned medium was to 3T3-L1 cells to effect on adipogenesis. 3T3-L1 cells maintained in conditioned medium collected from COS cells transiently transfected with vector into adipocytes as shown in those cells maintained in medium from COS cells transfected with expression vector not adipose conversion as by lipid expression of adipocyte and fatty acid synthase was by when cells were treated with conditioned medium from COS cells transfected with expression vector 4 The actin mRNA level of these cells was as These an inhibitory effect of ADSF/resistin on adipose the of adipose tissue, are to function in energy storage and balance under tight hormonal control. with the that adipocytes secrete factors known to a role in immune and vascular a much and role for adipose tissue has The known is a that is primarily made and secreted by mature adipocytes to regulate adipose fat mass. adipocyte-specific factors with functions including adipsin, and are secreted along with other well factors such as TNF-α, and plasminogen activator inhibitor of these factors to be to immune or vascular These that the adipocytes as as well as The role of the ADSF/resistin secreted from adipose tissue is not It is composed of cysteine-rich with unique cysteine and may in It is exclusively made in adipose tissue and is secreted to the Its expression in its during the of and its induction during and by insulin administration to animals that this factor may be in the nutritional status of the animals to adipogenesis. of these are to those with which is secreted only by adipocytes and is induced by and by We that this factor may be a that as a feedback to adipose tissue It is also possible that functions in an to known differentiation such as Dex, MIX, and insulin during adipocyte differentiation The report of ADSF/resistin as a TZD down-regulated adipose tissue which may insulin by obesity to is also and