Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling Specificity

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-κB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-α at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state. Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-κB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-α at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state. Adipocyte complement related protein of 30 kDa (Acrp30), or adiponectin, is an adipocyte-secreted hormone found abundantly in serum (1.Scherer 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 (2686) Google Scholar). Its expression and serum concentration are decreased in obese or diabetic humans and animals (2.Berg A.H. Combs T.P. Scherer P.E. Trends Endocrinol. Metab. 2002; 13: 84-89Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, 3.Tsao T.S. Lodish H.F. Fruebis J. Eur. J. Pharmacol. 2002; 440: 213-221Crossref PubMed Scopus (252) Google Scholar). In Pima Indians, occurrence of diabetes later in life is accompanied by decreased Acrp30 levels before onset of diabetes (4.Lindsay R.S. Funahashi T. Hanson R.L. Matsuzawa Y. Tanaka S. Tataranni P.A. Knowler W.C. Krakoff J. Lancet. 2002; 360: 57-58Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). Acrp30 exerts multiple metabolic actions at multiple tissue sites. The isolated globular domain of Acrp30 (gAcrp30) simulates fatty acid oxidation in skeletal muscle, whereas fulllength Acrp30 synergizes with insulin to inhibit hepatic glucose production (5.Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. Nat. Med. 2001; 7: 947-953Crossref PubMed Scopus (2174) Google Scholar, 6.Fruebis J. Tsao T.S. Javorschi S. Ebbets-Reed D. Erickson M.R. Yen F.T. Bihain B.E. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2005-2010Crossref PubMed Scopus (1728) Google Scholar, 7.Yamauchi T. Kamon J. Waki H. Terauchi Y. Kubota N. Hara K. Mori Y. Ide T. Murakami K. Tsuboyama-Kasaoka N. Ezaki O. Akanuma Y. Gavrilova O. Vinson C. Reitman M.L. Kagechika H. Shudo K. Yoda M. Nakano Y. Tobe K. Nagai R. Kimura S. Tomita M. Froguel P. Kadowaki T. Nat. Med. 2001; 7: 941-946Crossref PubMed Scopus (3993) Google Scholar). In mice, disruption of Acrp30 locus leading to its ablation resulted in impaired fatty acid clearance, increased tumor necrosis factor α levels, and aggravated insulin resistance in animals fed a high fat diet (8.Maeda 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 (1789) Google Scholar, 9.Kubota 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 (1171) Google Scholar). How Acrp30 acts as a hormone to regulate these physiological processes remains unknown. We addressed this issue by analyzing the structure of Acrp30 secreted from cells and in serum. Acrp30 purified from transfected human embryonic kidney (HEK) 1The abbreviations used are: HEKhuman embryonic kidneyHMWhigher molecular weightPBSphosphate-buffered salineAMPKAMP-activated protein kinaseDTTdithiothreitolEDLextensor digitorum longusβMEβ-mercaptoethanol.1The abbreviations used are: HEKhuman embryonic kidneyHMWhigher molecular weightPBSphosphate-buffered salineAMPKAMP-activated protein kinaseDTTdithiothreitolEDLextensor digitorum longusβMEβ-mercaptoethanol. 293T cells or Escherichia coli exists as trimers and hexamers (10.Tsao 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 (331) Google Scholar). Transfected HEK cells also secrete an even higher molecular weight (HMW) isoform of Acrp30 (10.Tsao 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 (331) Google Scholar). All three isoforms of Acrp30 are present in mouse serum and the conditioned medium of differentiated 3T3-L1 adipocytes, albeit with different relative abundances (10.Tsao 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 (331) Google Scholar). Purified isoforms are stable in PBS and do not interconvert (10.Tsao 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 (331) Google Scholar). The HMW and hexameric Acrp30 can activate transcription factor NF-κB in undifferentiated or differentiated C2C12 cells, but trimeric Acrp30 or gAcrp30 cannot. Rather, gAcrp30, but not full-length Acrp30 hexamer, enhances muscle fatty acid oxidation by inactivating acetyl-CoA carboxylase following stimulation of AMP-activated protein kinase (AMPK) (11.Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google Scholar, 12.Yamauchi 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 (3369) Google Scholar). human embryonic kidney higher molecular weight phosphate-buffered saline AMP-activated protein kinase dithiothreitol extensor digitorum longus β-mercaptoethanol. human embryonic kidney higher molecular weight phosphate-buffered saline AMP-activated protein kinase dithiothreitol extensor digitorum longus β-mercaptoethanol. Because different isoforms of Acrp30 have different activities, is important to are Acrp30 three an signal a collagen and a globular Because the structure of the Acrp30 globular domain a trimeric to tumor necrosis factor the collagen domain of Acrp30 hexamer and HMW disulfide bonds Acrp30 because are for of Acrp30 and R. S. P. A. PubMed Scopus Google Scholar, U. PubMed Scopus Google Scholar). Acrp30 two cysteine residues are with is in the globular domain at position of mouse Acrp30 the signal The is at position in a acid with the used by Acrp30 to etch microscopy to the of each We disulfide bonds are for Acrp30 oligomerization and which cysteine residues are in their formation. Acrp30 trimer is as a on a whereas the hexamer consists of two adjacent trimers a The HMW structure is not of HMW or hexameric Acrp30 with dithiothreitol trimers. In of each of the two in Acrp30 showed cysteine at position to for formation of the HMW and hexamer In contrast, cysteine at position in the globular domain in the formation of Acrp30 with naturally Acrp30 trimers activate trimeric C22A Acrp30 trimers formed by the reduction of Acrp30 hexamers can activate showed trimeric gAcrp30 could activate and full-length C22A trimers as as trimers activated in rat extensor digitorum longus muscle, as by phosphorylation at Thr172 of In contrast, hexameric and HMW Acrp30 could not. Thus, trimeric Acrp30 and Acrp30 and the oligomerization of Acrp30 the signaling can of Acrp30 in a to the used to C22A have the and its used to are and its The by and of and Acrp30 the signal purified from E. coli as a with to the (10.Tsao 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 (331) Google Scholar). and Acrp30 of purified from conditioned medium of transfected HEK 293T cells as (5.Berg A.H. Combs T.P. Du X. Brownlee M. Scherer P.E. Nat. Med. 2001; 7: 947-953Crossref PubMed Scopus (2174) Google Scholar, 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 (331) Google Scholar). Purified protein an protein and either a or a and with of Acrp30 293T Acrp30 purified by with and in a as J. Biol. PubMed Scopus Google Scholar, Hanson J. Biol. PubMed Scopus Google Scholar). The a and for before with a of Hanson J. Biol. PubMed Scopus Google Scholar, Biol. Google Scholar). electron in with a at Hanson J. Biol. PubMed Scopus Google Scholar, Biol. Google Scholar). at for at in a before at The M.L. J. Full Text PDF PubMed Scopus Google to the following of a is the at is the at is the is the in is the is the of is the in is the molecular and is the cells with the control of U. Biol. PubMed Google and by the to the or of incubation with forms of Acrp30 or the cells and the and from and with and and to as A.K. Ruderman N.B. J. 1995; Google Scholar). The for at in containing glucose and for 30 in the medium in the or of C22A Acrp30 or hexamer, or trimer isoforms of the end of this incubation the in of muscle by and to the with serum in and for at The with or from and with to from The by and by The in which in the We etch electron microscopy to the of HEK 293T Acrp30 hexamer, and HMW isoforms following their by The trimer a structure with the globular domain the and the collagen the In the of the a structure smaller the globular domain also represents the of Acrp30 of the collagen The of the collagen domain is Because the collagen and a of the of the Acrp30 collagen domain with is to The of hexameric Acrp30 two trimers adjacent to each in and is of the because of between the globular domain with the to end of the HMW isoform in the in these HMW with the globular to the and the the the structure of HMW Acrp30 from these the disulfide bonds are responsible for formation or of hexamer or HMW of isolated hexamers and trimers purified from E. coli in following incubation with In the of the molecular of E. Acrp30 hexamer and trimer are and the molecular of hexamer and HMW isoforms secreted from transfected 293T cells are and The trimer in to as trimer in (10.Tsao 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 (331) Google Scholar). Because trimer consists of two full-length Acrp30 and the collagen domain and is not found in serum (10.Tsao 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 (331) Google is an of the production and not In the present to the trimer as in the molecular of E. hexamer and HEK hexamer and HMW Acrp30 to of a trimer of E. coli hexamer trimer could also with of or not in Acrp30 two in the between the signal and the collagen domain and the in the globular domain The Cys22 of mouse Acrp30 the or of these are responsible for oligomerization of each with of the Acrp30 secreted from transfected HEK cells a to of protein to hexamer, and trimer in to of Acrp30 of purified C22A Acrp30 this as a trimeric in the this to a trimer the molecular by this is to of the Acrp30 trimer (10.Tsao 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 (331) Google Scholar). C22A Acrp30 also by etch electron microscopy to Acrp30 trimer a large sphere the globular a collagen stalk, and a smaller sphere the could in of C22A the on oligomer formation and whereas the of Cys22 is for formation of hexamer and HMW of of and C22A Acrp30 secreted by transfected 293T cells for as a and in and disulfide of Acrp30 of C22A protein concentration in and are The a to a single of purified HEK 293T C22A Acrp30 by electron for and electron microscopy are and in J. Biol. PubMed Scopus Google Biol. Google is of C22A Acrp30 and the three different Acrp30 purified from transfected HEK the to which Acrp30 monomers are in each of the three the of Acrp30 and monomers in reduction of the Acrp30 as monomers on not from and Acrp30 and monomers in an disulfide bond between two monomers and a Cys22 in the and HMW and hexamer isoforms Acrp30 are linked by disulfide Because HMW and hexamer have the to as the Acrp30 the disulfide bonds monomers formed by the C22A trimer in and consists of monomers are not disulfide showed HMW and hexameric Acrp30 activated but trimer could not (10.Tsao 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 (331) Google Scholar). the between the oligomerization and NF-κB the of E. hexamer as as C22A and in transfected cells to activate as by an with at at and for to the concentration of E. Acrp30 hexamer to trimer not and its to activate NF-κB of concentration by did not Acrp30 not with the HMW and hexamer activated NF-κB the trimer the C22A trimer activated NF-κB the hexamer and higher of Acrp30 can activate We gAcrp30 activated in isolated rat skeletal muscle but full-length Acrp30 hexamer in E. coli did not (11.Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google Scholar). We of also depends on the oligomerization of of isolated rat with trimeric C22A Acrp30 or with purified trimer led to and in the of at Thr172 phosphorylation at Thr172 is a of activation. changes in phosphorylation of not to changes in HMW hexamer not isoform of Acrp30 to phosphorylation in isolated rat Thus, gAcrp30 and trimeric but not hexameric or HMW Acrp30 activate in skeletal muscle, whereas hexameric or HMW Acrp30 but not gAcrp30 or trimeric activate NF-κB the of oligomerization of Acrp30 the signal transduction of and NF-κB by Acrp30 from E. coli or 293T and HMW hexamer trimer trimer 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 (331) Google E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google and 12.Yamauchi 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 (3369) Google in a the of two Acrp30 isoforms and the of Cys22 disulfide bonds in formation of the hexamer and HMW showed hexameric and HMW but not trimeric Acrp30 can activate and gAcrp30 and trimeric but not hexameric or HMW Acrp30 activate in skeletal with the C22A are with the of Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; Scholar). Although Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; substitution of Cys22 with resulted in of did not in the C22A these individual Acrp30 have different activities, because also showed trimeric Acrp30 the isoform in glucose production Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; Scholar). of the of different Acrp30 purified from different Because trimeric full-length Acrp30 and gAcrp30 activate but not NF-κB and because Acrp30 have the opposite actions in activate NF-κB but not the signaling of Acrp30 depends upon its oligomerization state. Acrp30 represents a novel example the signaling of a hormone is by its oligomerization state. All three isoforms of Acrp30 are present in serum (10.Tsao 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 (331) Google but at in the relative of the different depends on serum from a higher of HMW isoforms from Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; Scholar). of with glucose or insulin a of the HMW isoform Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; Scholar). is to the of Acrp30 different activate different signaling pathways, the levels of Acrp30 with the of insulin resistance and (2.Berg A.H. Combs T.P. Scherer P.E. Trends Endocrinol. Metab. 2002; 13: 84-89Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, 3.Tsao T.S. Lodish H.F. Fruebis J. Eur. J. Pharmacol. 2002; 440: 213-221Crossref PubMed Scopus (252) Google In do not the or used to Acrp30 levels J. Metab. PubMed Scopus Google Scholar, Y. Kihara S. Ouchi N. Takahashi M. K. J. K. Shimomura I. T. K. H. M. Yamashita S. K. K. M. Y. Funahashi T. Matsuzawa Y. PubMed Scopus Google exhibit or of these insulin via of of oligomer between and insulin which signaling or is important for of insulin and the of Acrp30 electron the collagen of Acrp30 trimers and hexamers to extended and with or The of this collagen to a globular domain the of the in an large molecular weight in Acrp30 hexamers are composed of two trimers in a to We HMW The the trimeric globular are adjacent to each in the hexamer is because could the hexamer and HMW to with the bonds are present in three isoforms of the hexamer and the HMW isoforms are composed of also each trimer consists of and The structure of the trimeric Acrp30 globular domain of the three residues in disulfide Scherer P.E. Biol. 8: Full Text Full Text PDF PubMed Google Scholar). Because Cys22 is the cysteine in the responsible for disulfide formation. We this by the of in the trimeric C22A In contrast, Acrp30 the to as Acrp30 in The disulfide bond in Acrp30 trimers to the collagen the by the disulfide bond at the is by an of the three monomers the trimeric globular domain in the structure Scherer P.E. Biol. 8: Full Text Full Text PDF PubMed Google Scholar). bonds are for assembly of hexameric and HMW Acrp30 because reduction of either isoform with trimers. Although not disulfide bonds in the structure of the trimeric gAcrp30 Scherer P.E. Biol. 8: Full Text Full Text PDF PubMed Google in hexamer or HMW forms of the can hexamer and HMW isoforms this disulfide bonds Cys22 residues are for the formation of Acrp30 hexamer and HMW because Acrp30 with the substitution forms trimers. The trimeric of C22A Acrp30 by and in the of the of Acrp30 a of acid Cys22 in this important in the formation of in hexameric and HMW reduction of high Acrp30 isoforms with or substitution of Cys22 with alanine the of Acrp30 to activate hexameric and HMW Acrp30 isoforms can activate Acrp30 and is for of NF-κB by The a hexameric but not trimeric is for extended to a of the tumor necrosis factor trimeric ligand is to a hexameric ligand by with the Acrp30 collagen domain could signal N. A. M. S. O. F. A. D. S. T. J. P. J. Biol. PubMed Scopus Google Scholar). of the and NF-κB by the of NF-κB by hexameric and HMW Acrp30 is NF-κB not to fatty acid oxidation or of hepatic glucose because gAcrp30 enhances fatty oxidation but not activate NF-κB (10.Tsao 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 (331) Google Scholar, E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google Scholar). Du X. Combs T.P. A.H. T. J. Brownlee M. Scherer P.E. J. Biol. Chem. 2002; the trimer of Acrp30 is active of glucose the higher molecular weight to gAcrp30, trimeric Acrp30 is not in NF-κB NF-κB is in glucose NF-κB in increased expression and from C2C12 I. T. A. S. A. C. J. Biol. 2002; PubMed Scopus Google Scholar, G. J. PubMed Scopus Google Scholar). is at high levels by skeletal muscle J. 2002; PubMed Scopus Google and is to increased fatty acid and glucose production from tissue and an in for by J. 2002; PubMed Scopus Google Scholar). hexameric and HMW Acrp30 a in the of from skeletal muscle NF-κB and from fat tissue and glucose production from the hexamer and HMW gAcrp30 not activate NF-κB (10.Tsao 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 (331) Google Scholar). (11.Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google Scholar, 12.Yamauchi 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 (3369) Google Scholar). full-length Acrp30 can also is controversial. We found Acrp30 hexamer could not activate (11.Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google whereas Yamauchi 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 (3369) Google full-length Acrp30 for the is used by Yamauchi of different Acrp30 In the present showed trimeric fulllength but not the higher molecular weight could activate The of trimer in the used by Yamauchi could for the between these (11.Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang Cc C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (830) Google Scholar, 12.Yamauchi 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 (3369) Google Scholar). two as albeit with different for gAcrp30 and full-length Acrp30 to of in muscle T. Kamon J. Ito Y. A. T. Kita S. T. M. Hara K. M. Murakami K. T. Uchida S. S. Waki H. Y. Terauchi Y. Froguel P. Tobe K. S. K. T. T. Nagai R. Kadowaki T. PubMed Scopus Google Scholar). is which fulllength Acrp30 hexamer, or HMW to each of these The of the in of disulfide bonds to to a in the of the globular bonds the the of to U. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). and have a at the in of these R. S. P. A. PubMed Scopus Google Scholar). Because collagen the collagen domains are for of this of We the of disulfide bonds formed by Cys22 in the assembly of hexameric and HMW Acrp30 but have not these disulfide bonds are as disulfide bonds are the trimer of the hexamer to linked in the formation. disulfide bonds to the high of to the of the is by as as with S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. PubMed Scopus Google Scholar). Because Acrp30 hexamer is composed of two collagen these not by to the hexamer these to the of the hexameric molecular formation to the of the HMW of In disulfide bonds formed by Cys22 are necessary for the oligomerization of Acrp30 the trimer and formation of the hexamer and HMW in etch electron are to the structure of the HMW Acrp30 and molecular the disulfide bonds to the of each Acrp30 in of different Acrp30 activate different signal transduction pathways, the physiological of the different oligomerization in animals In of the of Acrp30 in human serum are used in Acrp30 levels with diabetes and (2.Berg A.H. Combs T.P. Scherer P.E. Trends Endocrinol. Metab. 2002; 13: 84-89Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, 3.Tsao T.S. Lodish H.F. Fruebis J. Eur. J. Pharmacol. 2002; 440: 213-221Crossref PubMed Scopus (252) Google whereas the of or of these isoforms We J. J. S. and for this