Decreased plasma adiponectin concentrations in nondiabetic women with elevated homeostasis model assessment ratios

Transcription

1 European Journal of Endocrinology (2003) ISSN CLINICAL STUDY Decreased plasma adiponectin concentrations in nondiabetic women with elevated homeostasis model assessment ratios Miyao Matsubara, Shinji Katayose 1 and Shoji Maruoka 1 Division of Endocrinology and Metabolism, Otaru City General Hospital, Otaru, Japan and 1 Otsuka Assay Institute, Sapporo, Japan (Correspondence should be addressed to M Matsubara, Division of Endocrinology and Metabolism, Internal Medicine, Otaru City General Hospital, Wakamatsu 1-2-1, Otaru , Japan; miyamatsuba@proof.ocn.ne.jp) Abstract Objective: Whether the adipocyte-derived protein adiponectin is associated with insulin resistance independently of the effects of adiposity and the diabetic state is an important question. We explored, in a cross-sectional study of 486 Japanese nondiabetic women, the relationship between the calculated insulin resistance (homeostasis model assessment ratio (HOMA-R)) and adiponectin levels determined using a validated sandwich ELISA. Design and methods: All participants were stratified into tertiles for HOMA-R (,, 1.5, 1.5 #,, 3.0, 3.0 #, ) and the differences across tertiles of continuous variables were tested with ANOVA. Two-way ANOVA was used to determine possible relationships for plasma adiponectin between tertiles of HOMA-R and several stratified parameters. Multiple regression analyses were performed with HOMA-R or fasting serum insulin as dependent variable, and diastolic blood pressure (BP), body mass index (BMI), serum triglyceride (TG), leptin and adiponectin as independent determinants. Results: Mean plasma adiponectin in the high HOMA-R group decreased compared with that in the low HOMA-R group both before (mean^s.e.m. 6:2^0:6 vs 9:2^0:3 mg=ml; P, 0:001) and after adjustment for body fat mass (BFM) as kg or percent ð0:31^0:04 vs 0:69^0:03; 0:18^0:02 vs 0:34^ 0:01; both P, 0:001). HOMA-R was inversely associated with adiponectin levels both before ðr ¼ 20:37; P, 0:001Þ and after adjustment for BFM ðr ¼ 20:49; 20.46, both P, 0:001Þ: After covariate adjustment for age, diastolic BP, BMI and serum TG, HOMA-R retained a significant correlation with adiponectin/bfm (kg). Both adiponectin and leptin were the significant determinants of HOMA-R or fasting insulin in multiple regression models. Conclusions: Adiponectin was inversely associated with insulin resistance in nondiabetic subjects, independently from age, BP, adiposity and serum lipids. Because adiponectin is thought to have an antiatherogenic action, the presence of hypoadiponectinemia may predispose subjects to atherosclerosis, and may progress the atherogenesis in insulin resistance. European Journal of Endocrinology Introduction Insulin resistance expressing diffuse arterial endothelial dysfunction contributing to atherosclerosis, may lead directly to arterial damage through toxic effects of hyperinsulinemia, or may act indirectly through atherogenic effects of the constellation of risk factors associated with the metabolic syndrome X (1, 2). Under normal conditions, insulin resistance is followed by peripheral hyperinsulinemia to maintain euglycemia. Obesity is often accompanied by disturbances in blood pressure (BP) or lipid metabolism related to insulin resistance. Insulin resistance or hyperinsulinemia has been associated with coronary artery disease, suggesting that previously unmeasured factors may be involved in the link between insulin resistance or hyperinsulinemia, and atherosclerosis (3, 4). The recent discovery of adiponectin, a protein produced by adipocytes (5), is potentially important for research into adiposity and related diseases (6 9). Adiponectin has been shown to reduce tumor necrosis factor (TNF)-a-induced monocyte attachment to cultured human aortic endothelial cells by inhibiting the expression of vascular cell adhesion molecule, intercellular adhesion molecule and E-selectin (7). Adiponectin was shown to suppress phagocytic activity and lipopolysacharide-induced TNF-a production in cultured macrophages (8, 9), and was detected only in injured but not intact vessel walls (6). These suggest that adiponectin may have anti-inflammatory and anti-atherogenic effects (6 10). Plasma adiponectin concentrations were demonstrated to be lower in subjects with obesity (10, 11), dyslipidemia (12) and coronary artery disease (7). q 2003 Society of the European Journal of Endocrinology Online version via

2 344 M Matsubara and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 Hotta et al. (13) reported decreased plasma adiponectin levels in type 2 diabetes in humans. Experimental studies in rhesus monkeys show a parallel interrelationship between adiponectin and insulin sensitivity (14). Weyer et al. (15) reported that the degree of hypoadiponectinemia is more closely related to the degree of insulin resistance and hyperinsulinemia than to the degree of adiposity and glucose intolerance in obesity and type 2 diabetes. If there is an independent association between adiponectin and insulin resistance in nondiabetic subjects, this would support the hypothesis that adiponectin may regulate insulin resistance in humans, and thus play an important pathophysiological role in type 2 diabetes, separate from the effects of adiposity and glucose tolerance status. The purpose of this study was to examine the association between insulin resistance determined indirectly with the homeostasis model assessment ratio (HOMA-R), and plasma adiponectin concentrations using a largely overlapping Japanese nondiabetic population who have been reported previously (11, 12). Because sex differences have been reported in plasma adiponectin (10), leptin (16, 17), triglyceride (TG), high density lipoproteincholesterol (HDLC), uric acid and body fat mass (BFM) percent (11, 12), we chose to study women only. Subjects and methods Subjects Four hundred and eighty-six Japanese women residing in Hokkaido, Japan, aged (mean^s.e.m. 53:8^0:6) years excluding those with birth control pill use, diabetes mellitus (fasting plasma glucose (FPG) $7.0 mmol/l at any two previous examinations, use of hypoglycemic drug therapy, or a current FPG level $ 7.0 mmol/l or 2 h glucose level after 75 g oral glucose tolerance $11.1 mmol/l), renal failure (serum creatinine $159 mmol/l or blood urea nitrogen (BUN) $10.7 mmol/l) or untreated endocrine diseases, were included in this cross-sectional study (11, 12). Participants were recruited and examined between March 1999 and October 2000, and were in follicular phase among menstruating women. Systolic and diastolic BP were measured in the right arm of seated participants by using a mercury-column sphygmomanometer positioned near eye level. Hypertension was defined as current treatment with antihypertensive drugs or an elevated BP (diastolic $ 90 mmhg or systolic $160 mmhg) on both of two measurements. Approximately 29 and 19% of subjects had systolic and diastolic hypertension, while 64 were receiving calcium channel blockers and/or angiotensin-converting enzyme inhibitors. Approximately 36 and 12% of subjects had high serum total cholesterolemia (total cholesterol (TC) $ 5.69 mmol/l) and high triglyceridemia ($1.69 mmol/l) respectively. All anthropometric measurements were made with the participant wearing light clothes and no shoes. Body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters. Mean BMI was 23:0^0:1 (range ) kg/m 2, and overweight ðbmi $ 25:0Þ was observed in 26% of subjects. BFM was determined by bioelectrical impedance analysis; this value was the average determined using both a Tanita Body Fat Analyzer (TBF-541; Tanita, Tokyo, Japan) and an Omron Body Fat Analyzer (HBF-301; Omron, Tokyo, Japan) (16, 18). All subjects reported that their body weight had been stable (^2 kg) for at least 3 months before the study, and they provided informed consent. Blood samples after overnight fasting were collected for determination of FPG, insulin, lipids (TC, TG and HDLC) total protein, albumin, uric acid, BUN, creatinine and leptin; the serum was frozen at 280 8C until analysis. Biochemical analyses Blood glucose was measured by the glucose oxidase method, and serum lipids, total protein, albumin, uric acid, BUN and creatinine were measured using commercially available kits. Immunoreactive insulin was determined by a specific EIA with reagents from Dainabot Co. Ltd, Tokyo, Japan (18). Low density lipoproteincholesterol (LDLC) was estimated using Friedewald s formula, and an atherogenic index was calculated by the formula: (TC HDLC)/HDLC. Insulin resistance was calculated by the HOMA method, using FPG and insulin concentrations (19). Assuming that normalweight normal subjects,35 years of age have an insulin resistance of 1, the value for insulin resistance can be assessed by the formula: (FPG (mmol/l) fasting insulin (mu/ml))/22.5. The results of the HOMA-R correlated well with the measurements obtained by means of the euglycemic clamp technique, as was shown in a HOMA method validation study (18 20). Serum leptin concentration was measured with an RIA (Linco Research Inc., St Charles, MO, USA), which uses a polyclonal antibody raised in rabbits against recombinant human leptin (16 18). The assay had a sensitivity of 0.5 ng/ml. Adiponectin measurements Blood samples for measurement of fasting plasma adiponectin concentrations were drawn in 1/10 volume EDTA-aprotinin tubes, and immediately placed on ice. All tubes were centrifuged at 4 8C for collection of plasma and this was stored at 280 8C until analyses at Otsuka Assay Institute, Tokushima, Japan. Adiponectin was determined using a validated sandwich ELISA employing an adiponectin-specific monoclonal and polyclonal antibody, which has been demonstrated by Arita et al. (10). Cross-reaction with leptin, insulin and several cytokines, such as TNF-a and interleukin- 1b and -8, was not observed in this ELISA system.

3 EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 The recovery rate was almost 100%, and the intra- and interassay coefficients of variation were 3.3 and 7.4% respectively (10 12). Statistical analyses Because insulin resistance was considered to be elevated at HOMA-R $3.0 (16 18), we arbitrarily divided our study subjects into tertiles (,, 1.5, 1:5 #,, 3:0; 3.0 #, ). The differences across tertiles of continuous various parameters, leptin and adiponectin before and after adjustment for BMI or BFM were tested with ANOVA. Since preliminary analyses indicated that the distributions of plasma adiponectin, leptin, TG, insulin and HOMA-R were skewed, log transformation was used, which yielded more normally distributed data. Linear regression was performed to determine the association between log-transformed HOMA-R or fasting insulin, and adiponectin before and after adjusting for BMI or BFM. Multiple regression analyses were used to identify independent determinants for HOMA-R or fasting insulin. Two-way ANOVA was used to determine possible relationships for plasma adiponectin concentration adjusting for BFM (kg) between tertiles of HOMA-R and the several stratified parameters, such as age (,, 40 years, 40 #,, 55; 55 #, ), diastolic BP (,, 80 mmhg, 80 #,, 90; 90 #, ), BMI (,, 22.0 kg/m 2, 22:0 #,, 25:0; 25.0 #, ) or serum TG (,, 1.13 mmol/l, 1:13 #,, 1:69; 1.69 #, ). Results are expressed as means^s.e.m. P, 0:05 was considered to be statistically significant. Results Adiponectin in women with elevated HOMA-R 345 As shown in Table 1, tertile 3 group subjects (HOMA-R $ 3.0) showed significantly increased BP (systolic and diastolic), BMI, BFM, serum total protein, uric acid, TG, LDLC, atherogenic index and leptin levels before and after adjustment for BMI or BFM, which decreased HDLC levels compared with tertile 1 group subjects. Plasma adiponectin concentrations by HOMA-R tertile were 9:2^0:3; 7:8^0:3; and 6:2^0:6 mg=ml: Tertile 3 subjects had lower mean plasma adiponectin before and after adjustment for BMI or BFM (kg or %) compared with tertile 1 subjects (all P, 0:001) (Table 1). The associations between log-transformed adiponectin before and after adjustment for BFM, and HOMA- R or fasting serum insulin are presented in Fig. 1. HOMA-R was inversely associated with plasma adiponectin concentration ðr ¼ 20:37; P, 0:001Þ; and Table 1 Characteristics of anthropometric and biochemical variables by tertiles of baseline HOMA-R in nondiabetic women. Data are presented as means^s.e.m. HOMA-R Variable Tertile 1 (,, 1.5) Tertile 2 (1.5 #,, 3.0) Tertile 3 (3.0 #, ) P (tertile 1 vs 3) n (%) 267 (54.9) 170 (35.0) 49 (10.1) Age (years) 54.1^ ^ ^ Systolic BP (mmhg) 141.3^ ^ ^ Diastolic BP (mmhg) 79.0^ ^ ^ Serum total protein (g/l) 73.6^ ^ ^0.9, Albumin (g/l) 46.6^ ^ ^ Uric acid (mmol/l) 268^6 280^6 297^ BUN (mmol/l) 5.1^ ^ ^ Creatinine (mmol/l) 54.8^ ^ ^ BMI (kg/m 2 ) 21.9^ ^ ^0.6, BFM (%) 28.2^ ^ ^0.8, BFM (kg) 14.9^ ^ ^0.9, TC (mmol/l) 5.27^ ^ ^ TG* (mmol/l) 1.00^ ^ ^0.11, HDLC (mmol/l) 1.72^ ^ ^0.05, LDLC (mmol/l) 3.09^ ^ ^ Atherogenic index* 2.2^ ^ ^0.3, FPG (mmol/l) 4.97^ ^ ^0.14, Fasting insulin (mu/ml)* 4.4^ ^ ^1.1, HOMA-R* 0.98^ ^ ^0.27, Serum leptin (ng/ml)* 6.0^ ^ ^1.1, Leptin/BMI* 0.27^ ^ ^0.04, Leptin/BFM (kg)* 0.40^ ^ ^0.06, Leptin/BFM (%)* 0.21^ ^ ^0.03, Plasma adiponectin (mg/ml)* 9.2^ ^ ^0.6, Adiponectin/BMI* 0.43^ ^ ^0.03, Adiponectin/BFM (kg)* 0.69^ ^ ^0.04, Adiponectin/BFM (%)* 0.34^ ^ ^0.02, * Statistical analysis was performed after log transformation.

4 346 M Matsubara and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 Figure 1 Relationships between log-transformed HOMA-R or fasting serum insulin and plasma adiponectin before and after adjustment for BFM (kg or %).

5 EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 this association became stronger after adjustment for BFM (kg or %) ðr ¼ 20:49 and , both P, 0:001Þ: Fasting serum insulin was also inversely associated with plasma adiponectin ðr ¼ 20:39; P, 0:001Þ; and this association became stronger after adjustment for BFM ðr ¼ 20:50 and , both P, 0:001Þ (Fig. 1). As shown in Table 2, log-transformed adiponectin was significantly and inversely related to HOMA-R ðp, 0:001Þ; and this protein was also negatively related to serum insulin ðp, 0:001Þ: The regression models used for Table 2 suggested that the relationships between HOMA-R and adiponectin, and between fasting insulin and adiponectin, are of the same magnitude. In two-way ANOVA, despite adjusting for stratified age, diastolic BP, BMI or serum TG, plasma adiponectin/bfm (kg) decreased respectively in females with the highest tertiles of HOMA-R (Fig. 2). These results were essentially unchanged when percent BFM was substituted for BFM (kg), or when BMI was substituted (data was not shown). Discussion The present cross-sectional study suggests that various determinants related to body adiposity and the metabolic syndrome X are involved in the regulation of the calculated insulin resistance, HOMA-R. Fasting plasma adiponectin concentrations were significantly decreased in proportion to the degree of calculated insulin resistance in nondiabetic women, excluding the influences of other factors such as age, BP, serum TG and body composition. Our findings suggest that insulin resistance itself was associated with decreased adiponectin levels. Insulin-resistant subjects in this study were thought to include those with considerably impaired glucose tolerance. But decreased plasma adiponectin was considered to associate with insulin resistance but not glucose intolerance, because the elevated plasma adiponectin was observed in patients with type 1 diabetes (21). As adiponectin acts to reduce atherogenic reaction (6 9), hypoadiponectinemia might accelerate Table 2 Multiple regression models showing HOMA-R or fasting serum insulin as dependent variables with diastolic BP, BMI, serum TG, leptin and adiponectin as independent variables in nondiabetic women. Independent variable Log (HOMA-R) Dependent variable Log (fasting insulin) t P t P Systolic BP BMI Log (TG) Log (leptin) 5.272, , Log (adiponectin) , , Adiponectin in women with elevated HOMA-R 347 the atherogenic reaction in insulin resistance. These results are consistent with the experimental observations of Hotta et al. (14), who showed that adiponectin was related to the insulin-stimulated peripheral glucose uptake among male rhesus monkeys. The current study extends this observation to nondiabetic humans, and uses an estimate with the adjustment for BFM. While Weyer et al. (15) reported a direct association between adiponectin and glucose clamp methods in type 2 diabetes and obesity, our results using HOMA-R as the surrogate marker of insulin sensitivity/resistance may support this in a large number of nondiabetic women; furthermore this marker has merit for discriminating easily the insulin resistance in many cases. Moreover, based on this study, together with our previous reports that decreased plasma adiponectin concentrations were observed in women with hyper-triglyceridemia, hypo-hdl cholesterolemia and obesity (11, 12), these suggest that clinically important hypoadiponectinemia is the feature of the metabolic syndrome X, potentially accounting for part of the increased risk for insulin resistance, and further studies are needed to validate these associations also in men. Interest in associations between plasma adiponectin and insulin resistance has been spurred by the search for novel metabolic factors accounting for the disturbing excess burden of atherosclerosis in the metabolic syndrome X. Genomic-wide scan studies (22, 23) have revealed linkage of the metabolic syndrome and type 2 diabetes to a region on chromosome 3q27, where the gene encoding adiponectin is located (24). Recently, associations have been reported between adiponectin gene mutation and type 2 diabetes (25, 26) and features of metabolic syndrome X, including insulin resistance (27). In multiple regression analyses performed with log-transformed HOMA-R or fasting insulin as the dependent variable and systolic BP, BMI, log-transformed TG, leptin and adiponectin as independent variables, only adiponectin and leptin were significant independent determinants in women. Although Yamauchi et al. (28) reported that the concomitant replenishment of adiponectin and leptin completely improved insulin resistance in lipoatrophic and obese diabetic mice, our results may support this. In numerous models of obesity diabetes syndromes, TNF-a is overexpressed in adipose and muscle tissues compared with tissues from lean animals (29, 30). TNF-a blocks the action of insulin in cultured cells and in whole animals (29, 30). In humans, TNF-a is also overexpressed in the adipose and muscle tissues of obese subjects in proportion to their degree of insulin resistance (31, 32). At the early stages of atherosclerosis, endothelial cell activation by various inflammatory stimuli, including TNF-a, results in the synthesis of adhesion molecules and increases the adherence of monocytes. Adiponectin has been shown to inhibit both the production and action of TNF-a, a cytokine which has direct effects on the adhesion molecules

6 348 M Matsubara and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 Figure 2 Age- (A), diastolic BP- (B), BMI- (C), and serum TG- (D) adjusted plasma adiponectin/bfm (kg) by tertiles of HOMA-R. Data are presented as means^s.e.m. Statistical analyses were performed after log transformation.

7 EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) 148 (7, 8). Because hypoadiponectinemia accelerates the TNF-a reaction, the possibility has been considered that insulin resistance may be partially induced by hypoadiponectinemia. However, as hyperinsulinemia has been shown to downregulate adiponectin gene expression in 3T3-L1 adipocytes (33), decreased adiponectin secretion in the insulin-resistant state might be induced by hyperinsulinemia. Adiponectin may be considered to have implications for clinical prevention. Experimental studies in mice showed that adiponectin affects improving insulin resistance (28), and that recombinant adiponectin administration influences blood glucose, insulin resistance and TG metabolism (12, 34). Supplements of adiponectin may be beneficial for increasing plasma adiponectin levels, improving insulin sensitivity, and reducing risk for the development of type 2 diabetes and atherosclerosis. In conclusion, we have found that fasting plasma adiponectin levels are significantly decreased in insulin resistance in nondiabetic subjects, and this protein has an independent effect on insulin resistance that is not entirely explained by adiposity, age, BP and serum TG. There may be a different physiological mechanism to explain the adiponectin/insulin resistance association apart from the adiposity/insulin resistance link. Because adiponectin has anti-atherogenic properties, the presence of hypoadiponectinemia may predispose subjects to atherosclerosis, and may progress the atherogenesis in insulin resistance. References 1 Reaven GM. Role of insulin resistance in human disease. Diabetes Stout RW. Insulin and atheroma: a 20-yr perspective. Diabetes Care Pyorala M, Miettinen H, Laakso M & Pyorala K. Hyperinsulinemia predicts coronary heart disease risk in healthy middle-aged men: the 22-year follow-up results of the Helsinki Policemen Study. Circulation Bonora E, Tessari R, Micciolo R, Zenere M, Targher G, Padovani R et al. Intimal-medial thickness of the carotid artery in nondiabetic and NIDDM patients: relationship with insulin resistance. Diabetes Care Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y & Matsubara K. cdna cloning and expression of a novel adipose specific collagen-like factor, apmi (adipose most abundant gene transcript 1). Biochemical and Biophysical Research Communications Okamoto Y, Arita Y, Nishida M, Muraguchi M, Ouchi N, Takahashi M et al. An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. Hormone and Metabolic Research Ouchi N, Kihara S, Arita Y, Maeda K, Kuriyama H, Okamoto Y et al. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation Yokota T, Oritani K, Takahashi I, Ishikawa J, Matsuyama A, Ouchi N et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages. Blood Adiponectin in women with elevated HOMA-R Ouchi N, Kihara S, Arita Y, Nishida M, Matsuyama A, Okamoto Y et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochemical and Biophysical Research Communications Matsubara M, Maruoka S & Katayose S. Inverse relationship between plasma adiponectin and leptin concentrations in normal-weight and obese women. European Journal of Endocrinology Matsubara M, Maruoka S & Katayose S. Decreased plasma adiponectin concentrations in women with dyslipidemia. Journal of Clinical Endocrinology and Metabolism Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y et al. Plasma concentrations of a novel, adiposespecific protein, adiponectin, in type 2 diabetic patients. Arteriosclerosis and Thrombosis and Vascular Biology Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen BC et al. Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys. Diabetes Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Platley RE et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. Journal of Clinical Endocrinology and Metabolism Matsubara M, Yoshizawa T, Morioka T & Katayose S. Serum leptin and lipids in patients with thyroid dysfunction. Journal of Atherosclerosis and Thrombosis Mantzoros CS. The role of leptin in human obesity and disease: a review of current evidence. Annals of Internal Medicine Matsubara M, Chiba H, Maruoka S & Katayose S. Elevated serum leptin concentrations in women with components of multiple risk factor clustering syndrome. Journal of Atherosclerosis and Thrombosis Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF & Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia Bonora E, Saggiani F, Targher G, Zenere MB, Alberiche M, Monauni T et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity. Diabetes Care Imagawa A, Funahashi T, Nakamura T, Moriwaki K, Tanaka S, Nishizawa H et al. Elevated serum concentration of adiposederived factor, adiponectin, in patients with type 1 diabetes. Diabetes Care Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG et al. Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. PNAS Vionnet N, Hani El-H, Dupont S, Gallina S, Francke S, Dotte S et al. Genome wide search for type 2 diabetes-susceptibility gene in French white: evidence for a novel susceptibility locus for earlyonset diabetes on chromosome 3q27-qter and independent replication of a type 2-diabetes locus on chromosome 1q21-q24. American Journal of Human Genetics Takahashi M, Arita Y, Yamagata K, Matsukawa Y, Okutomi K, Horie M et al. Genomic structure and mutations in adiposespecific gene, adiponectin. International Journal of Obesity Hara K, Boutin P, Mori Y, Tobe K, Dina C, Tasuda K et al. Genetic variation in the gene encoding adiponectin is associated with an increased risk of type 2 diabetes in the Japanese population. Diabetes

8 350 M Matsubara and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2003) Kondo H, Shimomura I, Matsukawa Y, Kumada M, Takahashi M, Matsuda M et al. Association of adiponectin mutation with type 2 diabetes. Diabetes Menzaghi C, Ercolino T, Paola RD, Berg AH, Warram JH, Scherer PE et al. A haplotype at the adiponectin locus is associated with obesity and other features of the insulin resistance syndrome. Diabetes Yamauchi T, Kamon J, Waki H, Terauchi Y, Kubota N, Hara K et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nature Medicine Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF & Spiegelman BM. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance. Science Cheung AT, Ree D, Koll JK, Fuselier J, Coy DH & Bryer-Ash M. An in vivo model for elucidation of the mechanism of tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance: evidence for differential regulation of insulin signalling by TNF-alpha. Endocrinology Hotamisligil GS, Arner P, Caro JF, Atkinson RL & Spiegelman BM. Increased adipose tissue expression of tumor necrosis factor-a in human obesity and insulin resistance. Journal of Clinical Investigation Saghizadeh M, Ong JM, Garvey WT, Henry RR & Kern PA. The expression of TNFa by human muscle: relationship to insulin resistance. Journal of Clinical Investigation Fasshauer M, Klein J, Neumann S, Eszlinger M & Paschke R. Hormonal regulation of adiponectin gene expression in 3T3-L1 adipocytes. Biochemical and Biophysical Research Communications Berg AH, Combs TP, Du X, Brownlee M & Scherer PE. The adipocyte-secreted protein Acrp 30 enhances hepatic insulin action. Nature Medicine Received 14 June 2002 Accepted 4 December 2002