Correlations of serum high-molecular-weight (HMW) adiponectin level with various metabolic parameters in special health checkup programs

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1 Oiginal Aticle Online edition : ISSN Pint edition : ISSN Received: Setembe 3, 2015 Acceted : Octobe 29, 2015 Published online : Decembe 31, 2015 Coelations of seum high-molecula-weight (HMW) adionectin level with vaious metabolic aametes in secial health checku ogams Hioshi Hiose 1-3), Koichio Azuma 3, 4), Ryoko Shimizu-Hiota 2), Michiyo Takayama 2), Yasushi Iwao 2, 3) 1, 3) and Hioshi Kawabe 1) Health Cente, Keio Univesity, Tokyo, Jaan 2) Cente fo Peventive Medicine, Keio Univesity Hosital, Tokyo, Jaan 3) Deatment of Intenal Medicine, Keio Univesity School of Medicine, Tokyo, Jaan 4) Institute fo Integated Sots Medicine, Keio Univesity School of Medicine, Tokyo, Jaan Abstact Objective: Adionectin (ADPN) is seceted fom adiocytes and is shown to educe blood glucose level, atheoscleosis and tumo gowth in in vito and animal exeiments. Also, succination of ADPN eotedly contibute to the decease in highmolecula-weight (HMW) ADPN level in diabetes. In the esent study, we investigated the elationshis of seum HMW-ADPN level with vaious metabolic aametes in 403 Jaanese subjects, who aticiated in secial health checku ogams. Methods: Seum HMW-ADPN levels wee analyzed in 232 men and 171 women, aged 29 to 92 yeas, who aticiated in ou secial health checku ogam fo enal and metabolic syndome, locomotive ogans o anti-aging duing a 3-yea eiod and met the inclusion citeia. Visceal fat aea (VFA) and subcutaneous fat aea (SFA) wee measued by comuted tomogahy at the umbilical level. Seum HMW-ADPN concentation was measued by chemiluminescent enzyme immunoassay (CLEIA). Results: Medians of seum HMW-ADPN level wee 2.44 μg/ml in men and 5.20 μg/ml in women. HMW-ADPN in both sex wee ositively coelated with HDL-cholesteol and age, and wee negatively coelated with BMI, waist cicumfeence, VFA, SFA, ed blood cell count (RBC), HOMA-IR, seum tiglyceides, C-eactive otein, etc. Stewise multile egession analyses evealed that log[hmw-adpn] was indeendently coelated with HDL-cholesteol, RBC, VFA, age, gende and log[homa- IR] in this ode (F 7.0,, R 2 = 0.491). Conclusion: It is suggested that seum HMW-ADPN level by CLEIA to be ositively coelated with HDL-C, age and female gende, and negatively coelated with VFA, HOMA-IR and RBC in aticiants in health checku ogams. KEY WORDS: Adionectin, high-molecula-weight fom, visceal fat aea, insulin esistance. Intoduction Adionectin (ADPN) was discoveed by eseaches at Osaka Univesity (Osaka, Jaan) in 1996 as the most abundant tanscit in adiose tissue 1). It was found to be the same substance as a 28-kD gelatin-binding otein (GBP28) extacted fom human seum by the Faculty of Phamaceutical Sciences, Showa Univesity (Tokyo, Jaan) in the same yea 2). In in vito exeiments, ADPN was shown to inhibit signals tiggeing inflammation in vascula endothelial cells and the gowth of vascula smooth muscle cells 3). Animal studies also evealed that ADPN attaches to injued blood vessel walls. Yamauchi et al. 4) and Beg et al. 5) eoted seaately, using vaious tyes of obese mice and diabetic mice, that the administation of ADPN imoves both insulin esistance and blood glucose level. Anothe study eoted that ciculating ADPN levels deceased in hesus monkeys with diabetes mellitus (DM) as insulin esistance wosened 6). Studies using ADPN-knockout mice 7, 8) evealed that a high-fat diet induced insulin esistance and atheoscleosis in such mice. Futhemoe, ADPN was found to inhibit heatic fibosis 9) and gowth of cance cells 10), and ovide suvival benefits 11) in animal studies. Aai et al. eoted that the centenaians ae chaacteized to be moe sensitive to insulin, to have high blood ADPN levels, and to be less likely to have DM and/o metabolic syndome (MetS) 12). Gel filtation analyses have evealed that ADPN does not exist as a monome in seum, and that high-molecula-weight (HMW) foms of ADPN, such as a dodecame (4 3me) and octadecame (6 3me), ae moe closely associated with the onset of coonay atey disease (CAD) and weight eduction 13), and lay the ole of insulin sensitizes 14). Seveal in vito Coesonding to: Hioshi Hiose, MD, PhD Health Cente, Keio Univesity and the Cente fo Peventive Medicine, Keio Univesity Hosital, 35 Shinanomachi, Shinjuku-ku, Tokyo , Jaan Phone: (ext ), Fax: hhiose373@yahoo.co.j _ 197 _ 2015; 2 (4): (c) Society fo Glycation Stess Reseach

2 HMW-adionectin level in health checku ogams studies have eoted that HMW-ADPN mainly activates AMP kinase 15), events aotosis of vascula endothelial cells 13) and has cytostatic effects 16) ; and theefoe HMW-fom of ADPN is consideed to be an active fom. Recently, it is eoted that succination of ADPN blocks its incooation into timetic and HMW-, seceted fom of ADPN 17). They oosed that succination of oteins is a biomake of mitochondoial stess, which is inceased in diabetes, and that succination of ADPN may contibute to the decease in lasma HMW-ADPN level in diabetes 17). In the esent study tageting a total of 403 Jaanese men and women (aged 29 to 92 yeas), who aticiated in the secial health checku ogams at ou univesity hosital, elationshis of HMW-ADPN level with vaious metabolic aametes, including visceal fat aea (VFA) and subcutaneous fat aea (SFA) measued by comuted tomogahy (CT) and homeostasis model assessments of insulin esistance (HOMA-IR) and β- cell function (HOMA-β) 18) wee assessed. Subjects and Methods Subjects in this study This study tageted 502 Jaanese subjects who had fist aticiated in the secial health checku ogam fo enal and metabolic syndome, locomotive ogans o anti-aging at the Cente fo Peventive Medicine, Keio Univesity Hosital (Tokyo) duing a 3-yea eiod fom August 2012 to July 2015, whose seum HMW-ADPN level was measued. Out of these candidates, subjects who had suffeed fom malignant disease, endocine disease, inflammatoy disease, sevee heatic, enal o hematological disease, o who wee on coticosteoids wee excluded. To evaluate HOMA-IR values aoiately, we also excluded aticiants with modeate to sevee DM o on medication fo DM fom the analyses, because in subjects with DM, HOMA-IR is not eliable: FPG is abnomally high and/ o insulin level may be elevated by medication. The exclusion citeia by laboatoy data wee as follows: white blood cell count (WBC) 10 x 10 3 /μl, hemoglobin < 10 g/dl, latelet count 50 x 10 4 /μl, seum alanine amino tansfease (ALT) 100 IU/L, ceatinine 1.6 mg/dl, C-eactive otein (CRP) 2.2 mg/dl, o fasting lasma glucose (FPG) 140 mg/dl. Finally, the analyses included 232 men (age, yeas; mean, 59.0 yeas) and 171 women (age, yeas; mean, 62.5 yeas) in this study. The diagnosis of MetS was made based on the eots in 2005 by the Jaanese Society of Intenal Medicine 19). In addition to waist cicumfeence (WC) at the umbilical level 85 cm in men o 90 cm in women, the esence of at least 2 of the following 3 abnomalities indicated MetS: Seum tiglyceides (TG) 150 mg/dl and/o high-density liootein cholesteol (HDL-C) < 40 mg/dl; Systolic blood essue (SBP) 130 mmhg and/o diastolic blood essue (DBP) 85 mmhg; FPG 110 mg/dl. We also divided male and female aticiants, esectively, into 4 age gous: age 45, 46-60, and >75 and comaed the HMW-ADPN levels, WC, and coelations of HMW- ADPN with VFA and HOMA-IR. This study was conducted accoding to the inciles exessed in the Declaation of Helsinki. Infomed consent was obtained fom each aticiant afte full exlanation of the uose, natue and isk of all ocedues used. The study otocol was aoved by the Ethical Committee, Keio Univesity School of Medicine, Tokyo (aoval numbe ). Measuements Height, weight, blood essues and laboatoy data wee measued aound 9:00 am afte an ovenight fast. Body mass index (BMI) was calculated as weight in kilogams divided by height in metes squaed. SBP, DBP and ulse ate wee measued twice using an automatic electonic shygmomanomete (BP- 900; Tanita, Inc., Tokyo, Jaan) with the subject seated afte esting fo at least 5 min, as descibed eviously 20-22). Peiheal blood cell count, lasma glucose, HbA1c, seum TG, HDL-C, heatic enzymes and ceatinine levels wee assayed by outine automated laboatoy methods, as descibed eviously 20-22). Seum C-eactive otein (CRP) level was measued by nehelomety, a latex aticle-enhanced immunoassay (N Latex CRP II, Dade Behing, Tokyo, Jaan) with both inta- and inte-assay coefficients of vaiance (CVs) < 5.0%. Seum insulin concentation was measued by an enzyme immunoassay (IRI), using a commecially available kit (Tosoh, Tokyo, Jaan), with inta- and inte-assay CVs of % and %, esectively 20-22). Seum HMW-ADPN concentation was measued by chemiluminescent enzyme immunoassay (CLEIA) 22, 23) using the catidge tye CLEIA eagent secific fo HMW-ADPN measuement in combination with Lumiulse f (Fujiebio Inc., Tokyo, Jaan). The antibody used was the same IH7, as used in enzyme-linked immunosobent assay (ELISA), which is shown to eact secifically fo HMW-ADPN 24, 25). VFA, SFA and WC wee measued in a single slice taken at the umbilical level by a CT device Aquilion CXL (Toshiba Medical Systems Cooation, Tochigi, Jaan). HOMA-IR and HOMA-β wee calculated fom FPG (mg/dl) and fasting IRI (μu/ml) levels by using the following fomulae of Matthews, et al. 18) : HOMA-IR FPG x IRI / 405, and HOMA-β 360 x IRI / (FPG - 63) (%). Statistical analyses We used the IBM SPSS Statistics vesion 22 (IBM Jaan, Ltd., Tokyo, Jaan) and the StatView ogam fo Windows vesion 5.0-J (SAS Institute, Inc., Cay, Califonia, USA) fo statistical analyses. All nomally distibuted data ae exessed as mean ± SD, while non-nomal data ae exessed as median (intequatile ange, IQR). P values of less than 0.05 wee consideed as statistically significant, though Bonfeoni coection was used to evaluate the significance of multile aametes in univaiate and multivaiate egession analyses. To assess the significance of diffeence between the nonnomal HMW-ADPN data, we used the non-aametic Mann-hitney U-test fo two gous. Because the WC, VFA/SFA atio, HOMA-IR, HOMA-β, seum TG, ALT, IRI and HMW-ADPN levels aoximated a nomal distibution afte logaithmic tansfomation, thei logtansfomed values wee used fo univaiate and multivaiate egession analyses in this study. Results 1. Medians of HMW-ADPN level and Comaison of the levels between MetS and non-mets Median levels of seum HMW-ADPN wee 2.44 μg/ml in men and 5.20 μg/ml in women. The seum HMW-ADPN level in male subjects with MetS (median, 1.89; IQR, μg/ml; n = 51) was significantly lowe than those without _ 198 _

3 MetS (median, 2.77; IQR, μg/ml; n = 181, < 0.001). Although the numbe of female subjects with MetS was only 12, HMW-ADPN level in female subjects with MetS (median, 2.59; IQR, μg/ml) was also significantly lowe than those without MetS (median, 5.70; IQR, μg/ ml; n = 159, < 0.001). 2. Simle and multile egessions with HMW- ADPN as a deendent vaiable As shown in Tables 1 and 2, seum HMW-ADPN levels wee ositively coelated with HDL-C and age, and wee negatively coelated with BMI, WC, VFA, SFA, WBC, ed blood cell count (RBC), hemoglobin, HbA1c, HOMA-IR, HOMA-β, seum IRI, TG, ALT, CRP, etc. in both men and women. RBC was negatively coelated with age in both men ( = ) and women ( = ), and afte adjustment fo RBC coelations of age with log[hmw-adpn] wee still significant in males but not in females. Even afte adjustment fo age and BMI, the coelations of log[hmw-adpn] with VFA, log[iri], log[homa-ir], log[tg] and HDL-C wee significant in both men and women. 3. Stewise multile egessions with HMW-ADPN as a deendent vaiable Stewise multile egession analyses evealed that log [HMW-ADPN] was indeendently coelated with HDL-C, RBC, VFA, age, gende and log[homa-ir] in this ode (Table 3, F 7.0,, R 2 = 0.491). Even when log[wc] was enteed instead of BMI, the same esults wee obtained excet fo each F value. 4. HMW-ADPN levels when divided by 4 age gous We futhe investigated the HMW-ADPN levels when divided into 4 age gous. HMW-ADPN level linealy inceased with age in male aticiants (ue anel in Table 4). Although HMW-ADPN level in female aticiants also tended to incease with age, this elationshi was not significant (lowe anel in Table 4). Futhemoe, negative coelation of HMW- ADPN with VFA was stongest ( = , = ) in the oldest gou of age moe than 75 in the male aticiants. Discussion In the evious studies, we have investigated the 21, 26) significance of HMW-ADPN levels measued by ELISA and CLEIA 22) in health checkus of teaches and othe wokes, aged 40 to 65, in ou univesity. Although HOMA- IR was included in these studies, VFA and SFA wee not. In the esent study of comehensive health checku, VFA and SFA wee outinely measued in all the aticiants. Thee have been seveal eots of negative coelation of VFA with total ADPN level 27, 28). Also, anemia 29), 30) 29), 30), kidney disease Table 1. Relationshis between HMW-ADPN level and vaious metabolic aametes in 232 Jaanese male aticiants in the secial health checku ogams. Paametes median (IQR) o mean ± SD vs. HMW- ADPN (log) Adjusted fo RBC ' Adjusted fo age & BMI ' BMI Waist cicumfeence (log) Visceal fat aea Subcutaneous fat aea VFA/SFA (log) Systolic blood essue Diastolic blood essue White blood cell Red blood cell (RBC) Hemoglobin Fasting lasma glucose Hemoglobin A1c Insulin (log) HOMA-IR (log) HOMA-β (log) Tiglyceides (log) HDL-cholesteol ALT (log) Ceatinine CRP (log) ( yea ) ( kg/m 2 ) ( 10 3 /μl ) ( 10 6 /μl ) (μu/ml) ( IU/L ) ± 15.0 ± 3.5 ( 10.9 ) ± 50.0 ± 74.4 ( 0.46 ) ± 17.5 ± 11.1 ± 1.31 ± 0.45 ± 1.2 ± 9.8 ± 0.4 ( 4.0 ) ( 1.2 ) ( 39.1 ) ( 78.5 ) ± 13.0 ( 15 ) ± 0.17 ( 0.06 ) Values ae median (intequatile ange: IQR) fo data with non-nomal distibution, o mean ± standad deviation (SD) fo data with nomal distibution., coelation coefficient; ', standadized coelation coefficient; HMW-ADPN, high-molecula-weight adionectin; BMI, body mass index; VFA, visceal fat aea; SFA, subcutaneous fat aea; HOMA-IR, homeostasis model assessment-insulin esistance; HDL, high-density-liootein; ALT, alanine amino-tansfease; CRP, C-eactive otein. _ 199 _

4 HMW-adionectin level in health checku ogams Table 2. Relationshis between HMW-ADPN level and vaious metabolic aametes in 171 Jaanese female aticiants in the secial health checku ogams. Paametes median (IQR) o mean ± SD vs. HMW- ADPN (log) Adjusted fo RBC ' Adjusted fo age & BMI ' BMI Waist cicumfeence (log) Visceal fat aea Subcutaneous fat aea VFA/SFA (log) Systolic blood essue Diastolic blood essue White blood cell Red blood cell (RBC) Hemoglobin Fasting lasma glucose Hemoglobin A1c Insulin (log) HOMA-IR (log) HOMA-β (log) Tiglyceides (log) HDL-cholesteol ALT (log) Ceatinine CRP (log) ( yea ) ( kg/m 2 ) ( 10 3 /μl ) ( 10 6 /μl ) (μu/ml) ( IU/L ) ± 12.3 ± 3.7 ( 14.2 ) ± 40.0 ± 84.1 ( 0.21 ) ± 23.0 ± 12.2 ± 1.18 ± 0.36 ± 1.0 ± 9.9 ± 0.4 ( 4.0 ) ( 1.0 ) ( 28.2 ) ( 48.0 ) ± 14.4 ( 10 ) ± 0.12 ( 0.05 ) < Values ae median (intequatile ange: IQR) fo data with non-nomal distibution, o mean ± standad deviation (SD) fo data with nomal distibution., coelation coefficient; ', standadized coelation coefficient; HMW-ADPN, high-molecula-weight adionectin; BMI, body mass index; VFA, visceal fat aea; SFA, subcutaneous fat aea; HOMA-IR, homeostasis model assessment-insulin esistance; HDL, high-density-liootein; ALT, alanine amino-tansfease; CRP, C-eactive otein. Table 3. Stewise multile egession with log[hmw-adpn] as a deendent vaiable in 403 Jaanese aticiants in the secial health checku ogams Vaiable Standadized egession coefficient value F value Change in R 2 HDL-cholesteol % Red blood cell count % Visceal fat aea % % Gende % HOMA-IR (log) % Gende: male = 0, female = 1. R 2 = (0.701) 2 = Body mass index, log [ALT], ceatinine and log [C-eactive otein] wee also emitted to ente the egession mode. HMW-ADPN, high-molecula-weight adionectin; ALT, alanine amino-tansfease; HDL, high-density-liootein; HOMA-IR, homeostasis model assessment-insulin esistance. _ 20 0 _

5 Table 4. Seum HMW-ADPN levels, waist cicumfeence (WC), and coelations of HMW-ADPN with visceal fat aea (VFA) and HOMA-IR, when divided into 4 age gous Gous in Males ( yeas ) 1 29 ~ ~ ~ ~ 92 F n HMW-ADPN ( μg/ml ) 1.95 ( 1.76 ) 2.35 ( 1.91 ) 2.92 ( 3.19 )** 3.38 ( 3.10 )** WC 85.9 ( 15.4 ) 85.5 ( 12.3 ) 82.4 ( 11.1 ) 82.9 ( 8.0 ) Coelation with VFA Coelation with HOMA-IR Gous in Females ( yeas ) 1 32 ~ ~ ~ ~ 85 F n HMW-ADPN ( μg/ml ) 5.82 ( 5.08 ) 5.11 ( 4.45 ) 5.12 ( 5.04 )** 5.73 ( 5.64 ) WC 65.7 ( 9.1 ) 80.5 ( 17.7 ) 79.3 ( 16.1 ) 79.5 ( 9.6 )* Coelation with VFA < Coelation with HOMA-IR < Values ae median (intequatile ange) fo data with non-nomal distibution, and coelation coefficients () and values. F and values by analysis of vaiance (ANOVA) using logaithmic values. *P < 0.05 and ** < vs. Gou 1 by ost-hoc Tukey test. HMW-ADPN, high-molecula-weight adionectin; HOMA-IR, homeostasis model assessment-insulin esistance. and high BNP 30) wee eotedly associated with elevation of seum HMW-ADPN levels in atients with DM 29) and CAD 30). In this study, elationshis among HOMA-IR, VFA, comlete blood cell counts and HMW-ADPN measued by CLEIA wee investigated simultaneously in subjects with wide ange of age fom 29 to 92 yeas. HMW-fom of adionectin HMW-fom of ADPN is consideed as an active fom of ADPN in vito 15, 16). Clinical studies 13, 14) also suggest that HMW-ADPN is moe useful than total ADPN aticulaly in tye 2 diabetic atients. Fo examle, Pajvani et al. 14) eoted that the HMW/total ADPN atio was significantly moe useful fo monitoing imovements in insulin sensitivity in esonse to thiazolidinedione in tye 2 DM. Haa et al. 31) eoted that the HMW/total ADPN atio had bette owe fo edicting insulin esistance and MetS than the lasma total ADPN level. Aso et al. 32) also eoted that the HMW/total ADPN atio was moe useful fo evaluating CADs in tye 2 diabetic atients than simly measuing the seum total ADPN level. We conducted a coss-sectional study in healthy Jaanese male subjects without any medication, and eoted that HMW- ADPN measued by ELISA was as effective as the HMW/total ADPN atio fo edicting insulin esistance and/o MetS 21). Actually, negative coelations of HMW-ADPN with both HOMA-IR and MetS scoe wee stonge than those of HMW/ Total atio (Table 2 in Ref. 21) at least in subjects without medication fo DM. Also, dividing into 4 gous by HMW- ADPN level esulted in bette discimination of both HOMA- IR and MetS scoe than by HMW/Total atio (B vs. C in Figs. 2 and 3 in Ref. 21). Theefoe, we decided to measue only HMW-ADPN level instead of measuing both HMW- and total ADPN levels theeafte. HMW-ADPN levels measued by CLEIA Fom the yea 2010, CLEIA 23) has been used also in the SRL laboatoies, which method was shown to be faste and moe accuate than the conventional ELISA. Due to changes in the calibato concentation, the HMW-ADPN levels measued by CLEIA wee aoximately a half of those measued by the conventional ELISA 23) : HMW-ADPN (CLEIA) = HMW-ADPN (ELISA) x Howeve, the coelation with the levels measued by ELISA was vey high ( = 0.984, n = 297). The inta-assay CV was 1.0% to 2.2%, and the inte-assay CV was 1.7% to 3.3% by CLEIA 23), and they wee 2.4% to 3.0%, and 4.2% to 5.1% by ELISA 25). Theefoe, the CLEIA is shown to be moe accuate and faste than the conventional ELISA. _ 201 _

6 HMW-adionectin level in health checku ogams Limitations of this study The limitations of this study include selection biases fo alying the esults to geneal findings: (1) Because aticiants in this study wee selected fom secial health checku ogams in ou Univesity hosital, they may have a elatively high consciousness on health, and the eldely aticiants may be egaded as those who had lived without develoing DM, cadiovascula and/o malignant diseases. (2) The esent study excluded aticiants with modeate to sevee DM, because in subjects with DM, HOMA-IR is not eliable. Pevious studies, including ous 33), suggested that administation of PPAR gamma agonists, both ioglitazone 33) and osiglitazone 34), incease total ADPN concentation aoximately 2-fold and seum HMW-ADPN concentation 3-fold. In this study, log[hmw- ADPN] in male subjects with medication on DM (n = 29) tended to be highe (0.433±0.300 μg/ml vs ± μg/ ml) desite having DM, though it is not significant. Relations with glycative stess and succination of ADPN The esent study suggested that the eduction in seum HMW-ADPN level induce insulin esistance, which will cause ost-andial hyeglycemia. Theefoe, these cascade may influence usteam of glycative stess athways. Howeve, we could not check the effects on oduction of advanced glycation endoducts (AGEs) in this study. Futhe studies will be needed to claify the effects of ADPN level on the downsteam of glycative stess athways. As we have descibed in the Intoduction, succination of ADPN, which is inceased in DM, is eoted to block the fomation of HMW-, seceted fom of ADPN 17). It is obvious that obesity/mets educes seum ADPN level, and esulting hyeglycemia seems to educe the HMW- ADPN level futhe. Because this study was of coss-sectional study design, no cause-effect elationshi can be assessed. In the nea futue, longitudinal studies will futhe facilitate the undestanding of these imotant issues. Conclusion It is suggested that seum HMW-ADPN level measued by CLEIA to be indeendently and ositively coelated with HDL-C, age and female gende, and negatively coelated with VFA, HOMA-IR and RBC in aticiants in health checku ogams. Acknowledgments The authos thank all the staffs, medical doctos and nuses in the Cente fo Peventive Medicine, Keio Univesity Hosital, Tokyo, fo thei hels to efom the comehensive health checku ogams. The at of this study was suoted by a Gant-in-Aid fom the Ministy of Education, Cultue, Sots, Science and Technology, Jaan (No ). Conflict of Inteest Statement The authos state that efomance of this study entailed no issues eesenting a conflict of inteest. Refeences 1) Maeda K, Okubo K, Shimomua I, et al. cdna cloning and exession of a novel adiose secific collagen-like facto, am1 (AdiPose Most abundant Gene tanscit 1). Biochem Biohys Res Commun. 1996; 221: ) Nakano Y, Tobe T, Choi-Miua NH, et al. Isolation and chaacteization of GBP28, a novel gelatin-binding otein uified fom human lasma. J Biochem (Tokyo). 1996; 120: ) Matsuzawa Y, Funahashi T, Nakamua T. Molecula mechanism of metabolic syndome X: contibution of adiocytokines adiocyte-deived bioactive substances. Ann NY Acad Sci. 1999; 892: ) Yamauchi T, Kamon J, Waki H, et al. The fat-deived homone adionectin eveses insulin esistance associated with both lioatohy and obesity. Nat Med. 2001; 7: ) Beg AH, Combs TP, Du X, et al. The adiocyte-seceted otein Ac30 enhances heatic insulin action. Nat Med. 2001; 7: ) Hotta K, Funahashi T, Bodkin NL, et al. Ciculating concentations of the adiocyte otein adionectin ae deceased in aallel with educed insulin sensitivity duing the ogession to tye 2 diabetes in hesus monkeys. Diabetes. 2001; 50: ) Kubota N, Teauchi Y, Yamauchi T, et al. Disution of adionectin causes insulin esistance and neointimal fomation. J Biol Chem. 2002; 277: ) Maeda N, Shimomua I, Kishida K, et al. Diet-induced insulin esistance in mice lacking adionectin/acrp30. Nat Med. 2002; 8: ) Kamada Y, Tamua S, Kiso S, et al. Enhanced cabon tetachloide-induced live fibosis in mice lacking adionectin. Gastoenteology. 2003; 125: ) Båkenhielm E, Veitonmäki N, Cao R, et al. Adionectininduced antiangiogenesis and antitumo activity involve casase-mediated endothelial cell aotosis. Poc Natl Acad Sci USA. 2004; 101: _ 202 _

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