OBESITY AND THE metabolic syndrome have reached

0021-972X/04/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 89(6):2697 2703 Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: 10.1210/jc.2003-031826 Adiponectin, Inflammation, and the Expression of the Metabolic Syndrome in Obese Individuals: The Impact of Rapid Weight Loss through Caloric Restriction ANTONIOS M. XYDAKIS, CHRISTOPHER C. CASE, PETER H. JONES, RON C. HOOGEVEEN, MINE-YINE LIU, E. O BRIAN SMITH, KATHLEEN W. NELSON, AND CHRISTIE M. BALLANTYNE Division of Endocrinology, Diabetes and Metabolism (A.M.X., C.C.C.), Center for Cardiovascular Disease Prevention, Methodist DeBakey Heart Center, and Section of Atherosclerosis, Department of Medicine (P.H.J., R.C.H., M.-Y.L., C.M.B.), and Section of Nutrition, Department of Pediatrics (E.O.S.), Baylor College of Medicine, Houston, Texas 77030; and Methodist Wellness Services (K.W.N.), The Methodist Hospital, Houston, Texas 77030 OBESITY AND THE metabolic syndrome have reached epidemic proportions in the United States, with deleterious public health consequences (1, 2). Since the publication of the National Cholesterol Education Program Adult Treatment Panel III (ATP III) diagnostic criteria for the metabolic syndrome (3), two independent studies indicated that more than one in five adults in the United States population qualifies for this diagnosis (4, 5). Insulin resistance is the hallmark of the metabolic syndrome, and it is strongly associated with excess adiposity (6, 7). Prevalence of the metabolic syndrome increases progressively from 5 and 6% of normal-weight [body mass index (BMI), 25 kg/m 2 ] men and women, respectively, to 22 and 28% of overweight (BMI, 25 29.9 kg/m 2 ) men and women to 60 and 50% of obese (BMI, 30 kg/m 2 ) men and women (5). Furthermore, caloric restriction and weight reduction are beneficial in enhancing insulin action on peripheral tissues (8, 9) and preventing the development of type 2 diabetes (10). The relationship between insulin resistance and obesity is Abbreviations: ATP III, Adult Treatment Panel III; BMI, body mass index; CI, confidence interval; GGT, -glutamyltransferase; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, insulin resistance of the homeostasis model assessment; hs-crp, high-sensitivity C-reactive protein; MS, metabolic syndrome positive; MS, metabolic syndrome negative; NEFA, nonesterified fatty acid; OR, odds ratio; PPAR-, peroxisome proliferator-activated receptor- ; TG, triglyceride; VLCD, very low-calorie diet. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. Severe obesity increases the prevalence of the metabolic syndrome, and moderate acute weight loss with a very low-calorie diet in obese subjects with the metabolic syndrome leads to significant metabolic benefits. Adiponectin has been implicated in both the pathogenesis of obesity-related insulin resistance and increased inflammation. We analyzed the relationship of the adipocyte-derived hormone adiponectin with indices of inflammation, adiposity, and insulin resistance in obese subjects with (MS, n 40) and without (MS, n 40) the metabolic syndrome and examined the acute effects of rapid weight loss. MS subjects had significantly lower adiponectin (7.6 0.6 vs. 10.4 0.6 g/ml; P 0.003) and significantly higher TNF- (3.3 0.2 vs. 2.8 0.3 pg/ml; P 0.004) levels compared with MS subjects matched for age and body mass index. Plasma adiponectin and TNF- levels were inversely related to the number of metabolic syndrome factors in a stepwise manner. After 4 6 wk of weight loss, there was marked improvement in glucose, insulin, leptin, and triglycerides, whereas adiponectin and TNF- concentrations did not change. Thus, increases in plasma levels of adiponectin or reductions in TNF- are not required for marked improvements in glucose/insulin and lipid metabolism with acute weight loss. (J Clin Endocrinol Metab 89: 2697 2703, 2004) complex, and accumulated evidence indicates that adipose tissue is a hormonally active system involved in insulin action as well as glucose and lipid metabolism (11). A variety of adipocyte-derived biologically active molecules termed adipocytokines have been identified, including leptin, resistin, TNF-, and IL-6, that may contribute to obesity-linked metabolic abnormalities (12 15). More recently, adiponectin, which is exclusively expressed in adipose tissue and is abundant in human plasma, has been found to be decreased in individuals with obesity, type 2 diabetes, and coronary heart disease (16 20). Although the physiological role of adiponectin remains unclear, it appears that it may possess insulinsensitizing and potentially antiinflammatory and antiatherogenic properties (21 26). Furthermore, different modalities of weight loss as well as peroxisome proliferator-activated receptor- (PPAR- ) agonist therapy have been shown to increase adiponectin levels longitudinally (17, 27, 28). Although the prevalence of the metabolic syndrome increases markedly with severe obesity, many individuals with severe obesity have no clinical manifestations of the metabolic syndrome. Although the diagnostic criteria for ATP III require that an individual have at least three of the five clinical criteria, risk for cardiovascular events increases with the number of clinical criteria present (29). We have previously shown that moderate acute weight loss of 5 7% in 4 6 wk with a very low-calorie diet (VLCD) in obese subjects with the metabolic syndrome is associated with dramatic improvement in all aspects of the metabolic syndrome, although the individuals remained markedly obese (30). In this study, we examined the association between adiponectin and 2697

2698 J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome the number of metabolic abnormalities constituting the metabolic syndrome in obese individuals. In addition, we examined the impact of rapid weight loss on levels of adiponectin and other adipocytokines in conjunction with parameters of the metabolic syndrome and measures of insulin resistance in obese individuals with the metabolic syndrome. Subject selection Subjects and Methods Obese individuals (56 women and 24 men; age, 47.1 0.9 yr; BMI, 38.3 0.7 kg/m 2 ) enrolled in a medically supervised rapid weight loss program were recruited for this study during a 12-month period (September 2001 to September 2002). The study protocol was approved by the Baylor College of Medicine Institutional Review Board, and written informed consent was obtained from each individual. The subjects were classified as metabolic syndrome positive (MS ) if they met three or more of the National Cholesterol Education Program ATP III criteria: waist circumference greater than 40 in. (102 cm) for men or greater than 35 in. (88 cm) for women; triglycerides (TGs) of 150 mg/dl (1.69 mmol/ liter) or greater; high-density lipoprotein cholesterol (HDL-C) less than 40 mg/dl (1.03 mmol/liter) for men or less than 50 mg/dl (1.29 mmol/ liter) for women; blood pressure of 130/85 mm Hg or greater or treated hypertension; and fasting glucose of 110 mg/dl (6.1 mmol/liter) or greater (3). Sequentially enrolled MS subjects (n 40) were compared with 40 obese subjects matched for age and BMI who did not have the metabolic syndrome (MS ) (Table 1). Of the MS subjects, 26 had one criterion for the metabolic syndrome (waist), and 14 had two criteria (five with elevated blood pressure, four with low HDL-C, three with elevated TGs, and two with impaired fasting glucose). The study participants did not receive any medication to lose weight or any other medication known to affect glucose tolerance, insulin secretion, or insulin sensitivity during the active weight loss period. Weight loss protocol description The weight loss program was offered in a tertiary care medical center to individuals who were either self-referred or referred by healthcare professionals. Weight reduction was induced by a protein-sparing VLCD of approximately 600 800 kcal daily, supervised by a team of physicians, registered dietitians, and behaviorists. It consisted of meal replacement products (Nutrimed-Plus; Robard Corp., Mount Laurel, NJ; each serving contained 200 kcal, 6goffat, 26 g of protein, and 10 g of carbohydrate) alone or in combination with lean beef, fish, or poultry. TABLE 1. Anthropometric and metabolic variables of the two study groups Variables Metabolic syndrome Positive (n 40) Negative (n 40) Age (yr) 47.1 1.5 46.8 1.0 0.8 Gender (F/M) 23/17 33/7 0.02 BMI (kg/m 2 ) 38.9 1.0 37.8 0.9 0.5 Weight (lb) 257 8.3 228 7.2 0.007 Waist (in.) 45.3 1.0 41.4 0.9 0.007 SBP (mm Hg) 135 3.0 120 2.0 0.0001 DBP (mm Hg) 85 2.0 79 1.0 0.003 Glucose (mg/dl) 108 7.0 95 3.0 0.09 TG (mg/dl) 280 28 120 6.0 0.0001 HDL-C (mg/dl) 44 2.0 57 2.0 0.0001 Insulin (pmol/liter) 168 30 79 16 0.0005 HOMA-IR 7.7 1.5 3.6 1.0 0.0003 NEFA (meq/liter) 0.6 0.05 0.5 0.04 0.04 Leptin (pmol/liter) 2964 254 3111 232 0.6 Adiponectin ( g/ml) 7.6 0.6 10.4 0.6 0.003 hs-crp (mg/liter) 5.0 0.5 4.4 0.5 0.2 TNF- (pg/ml) 3.3 0.2 2.8 0.3 0.004 Values are expressed as means SE. F, Female; M, male; DBP, diastolic blood pressure; SBP, systolic blood pressure. P Daily protein intake was calculated as 1.5 g per kilogram of predetermined goal weight, and daily fluid intake was at a minimum of 2 liters. Entry criteria for the program included age greater than 18 yr and a BMI of 30 kg/m 2 or greater. Exclusion criteria were known eating disorder, cancer, use of lithium or corticosteroids, type 1 diabetes, active inflammatory bowel disease, active gout, liver disease, cardiovascular event within the past 3 months, endocrine causes of obesity, and pregnancy. Diuretic medications were discontinued before entering the program. Anthropometric and biochemical measurements All subjects were evaluated with a series of anthropometric measurements and tests for hematology, biochemistry, and hormonal functions after an overnight fast. None of the subjects was on any calorierestricting diet at the baseline evaluation. Biochemical measurements were performed at baseline for all 80 individuals and prospectively for the 40 MS subjects after 4 6 wk of active weight loss. Measurements of insulin resistance were obtained using the homeostasis model assessment [HOMA-IR fasting glucose (mg/dl) fasting insulin ( U/ ml)/22.5], as described previously (31). All biochemical measurements were performed on frozen plasma samples obtained by centrifugation of freshly drawn blood (3000 g for 20 min at 4 C) and subsequent storage at 70 C. Blood lipid profiles, including total cholesterol, HDL-C, calculated low-density lipoprotein cholesterol, TGs, and nonesterified fatty acids (NEFAs), as well as plasma -glutamyltransferase (GGT), concentrations were determined by enzymatic assays using a Hitachi 911 auto-analyzer (Roche Diagnostics, Indianapolis, IN). Plasma levels of adiponectin were measured by RIA according to the manufacturer s protocol (Linco Research, Inc., St. Charles, MO). High-sensitivity C-reactive protein (hs-crp) was assessed by the Denka Seiken (Tokyo, Japan) assay, which has been validated against the Dade Behring method. Plasma levels of leptin and insulin were determined on a Luminex-100 multianalyte profiling system using commercially available immunoassay panels (Linco Research, Inc.). Circulating TNF- levels were measured by high-sensitivity ELISA (R&D Systems, Inc., Minneapolis, MN). Statistical analyses Unless stated otherwise, data are expressed as the means se. Statistical analysis was made using SPSS version 11.5 (SPSS Inc., Chicago, IL). Because variables were often not normally distributed, group comparison was performed by the Mann-Whitney nonparametric test, and the Kruskal-Wallis test was used for comparison of more than two independent groups. Correlations between different variables were performed by the Spearman rank correlation test. Comparison of variables before and after weight loss intervention in the metabolic syndrome group (MS ) was assessed by the Wilcoxon signed rank test. The independent contribution of each variable was assessed by simultaneously including other variables as covariates by general linear model univariate analysis. Interactions were assessed in the general linear model, and none were detected. The TNF- data were log transformed to satisfy the assumptions of the general linear model. Multiple logistic regression was used to assess adiponectin as an independent predictor of the metabolic syndrome, while adjusting for other variables. P 0.05 was considered to be statistically significant for all analyses. Results Association of adiponectin to diagnosis and number of metabolic syndrome components Plasma adiponectin level was significantly lower in MS subjects compared with age- and BMI-matched MS individuals (7.6 0.6 vs. 10.4 0.6 g/ml; P 0.003) (Table 1; Fig. 1A). When the MS and MS groups were compared with respect to adiponectin and other variables, the difference remained significant after adjustment for age (P 0.001), gender (P 0.003), BMI (P 0.002), insulin (P 0.01), HOMA-IR (P 0.01), leptin (P 0.002), and all these together (P 0.01). Although plasma adiponectin concentra-

Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 2699 tion was higher in women compared with men in the overall group, consistent with observations by others, this difference did not reach statistical significance (9.4 0.6 vs. 8.0 0.8 g/ml; P 0.1). There was a stepwise decrease in plasma adiponectin levels in parallel to the number of metabolic syndrome components present, and subjects with four or five components of the metabolic syndrome had the lowest adiponectin concentration, 6.5 0.9 g/ml (Table 2; Fig. 1B). This stepwise decrement in adiponectin levels remained significant even after adjustment for insulin (P 0.01) and HOMA (P 0.01). Using multiple logistic regression, we calculated that the odds ratio (OR) of the metabolic syndrome as predicted by adiponectin was 0.83 [95% confidence interval (CI), 0.73 0.94; P 0.004], indicating that there was a 17% decrease in the odds of having the metabolic syndrome for each 1- g/ml increment in adiponectin levels. The OR did not change appreciably after adjustment for weight and BMI (OR, 0.85; 95% CI, 0.75 0.97; P 0.02). Furthermore, the ORs FIG. 1. The plasma adiponectin level was significantly lower in patients with the metabolic syndrome (MS ) compared with patients without the metabolic syndrome (MS ) (A) and decreased in parallel to the number of metabolic syndrome components present (B). were not substantially different when adjusted for waist (OR, 0.81; 95% CI, 0.70 0.94; P 0.005), blood pressure (OR, 0.77; 95% CI, 0.65 0.90; P 0.002), glucose (OR, 0.84; 95% CI, 0.74 0.95; P 0.009), and TG (OR, 0.81; 95% CI, 0.67 0.98; P 0.03) but became nonsignificant after adjusting for HDL-C (P 0.3). With respect to inflammatory markers, plasma TNF- levels were significantly higher in the MS group compared with the MS group (3.3 0.2 vs. 2.8 0.3 pg/ml; P 0.004) (Fig. 2A). This difference remained significant after adjustment for age (P 0.02), BMI (P 0.04), insulin (P 0.05), and leptin (P 0.02) but not when adjusted for gender (P 0.1). Levels of hs-crp were not significantly different between the two study groups. There was a stepwise increase in plasma TNF- levels in parallel to the number of metabolic syndrome components present (Fig. 2B). Univariate correlations at baseline In univariate analysis of baseline variables in all subjects (MS and MS ), plasma adiponectin was inversely correlated with surrogate measures of total and visceral adiposity (weight and waist circumference) and was reciprocally associated with insulin, HOMA-IR, and all traits of the metabolic syndrome, except blood pressure (Table 3). Furthermore, plasma adiponectin was inversely correlated with hs-crp but not with TNF-, NEFA, or leptin. Plasma adiponectin had the strongest correlation to HDL-C levels of all variables examined (Fig. 3). As shown with adiponectin, plasma TNF- was positively correlated with measures of adiposity (weight and waist circumference) and inversely correlated with insulin and HOMA-IR. There were also significant associations between TNF-, TG, and HDL-C (Table 3). Effects of rapid weight loss on anthropometric and metabolic parameters in subjects with the metabolic syndrome At 4 6 wk of intervention (mean follow-up of 36 d), the subjects lost, on average, 7% of their initial weight (17.6 1.2 lbs). Values at baseline and 4 6 wk after active weight reduction for all measured variables are displayed in Table 4. All components of the metabolic syndrome, except HDL-C, improved significantly. HDL-C decreased significantly, as seen in other studies with acute weight loss through caloric restriction (32, 33). Plasma levels of insulin, HOMA-IR, leptin, and hs-crp decreased significantly, and changes in HOMA-IR and hs-crp correlated significantly with weight loss. In contrast, adiponectin, NEFA, and TNF- levels did not change significantly after 4 6 wk of rapid weight loss. TABLE 2. Adiposity surrogate measures and biochemical parameters based on metabolic syndrome components Metabolic syndrome components BMI (kg/m 2 ) a Waist (in.) b Insulin (pmol/liter) b hs-crp (mg/liter) a Leptin (pmol/liter) a Adiponectin ( g/ml) b TNF- (pg/ml) b Obesity alone (n 26) 37.6 1.1 40.3 1.1 63.5 13.8 4.8 0.7 3138 299 11.1 0.8 2.6 0.3 Obesity 1(n 14) 38.3 1.5 43.3 1.4 108.5 38.6 3.5 0.7 3061 379 8.9 0.9 3.2 0.5 Obesity 2 (n 40) 38.9 1.0 45.3 1.0 168.0 30.5 5.0 0.5 2965 254 7.6 0.6 3.3 0.2 Values are means SE. a Nonsignificant by Kruskal-Wallis test. b P 0.006.

2700 J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome FIG. 3. In univariate analysis (Spearman rank correlation test) of baseline variables pertaining to the metabolic syndrome, the plasma adiponectin level was most strongly correlated to the HDL-C level. TABLE 4. Effect of rapid weight loss in anthropometric and metabolic variables of subjects with the metabolic syndrome FIG. 2. The plasma TNF- level was significantly higher in patients with the metabolic syndrome (MS ) compared with patients without the metabolic syndrome (MS ) (A) and increased in parallel to the number of metabolic syndrome components present (B). TABLE 3. Baseline correlations (Spearman) of several parameters with plasma adiponectin and TNF- in subjects with and without the metabolic syndrome (n 80) Variable Plasma adiponectin Discussion Plasma TNF- R P R P Weight 0.31 0.007 0.24 0.03 Waist 0.32 0.002 0.25 0.03 SBP 0.01 0.9 0.01 0.9 DBP 0.09 0.4 0.10 0.4 Glucose 0.14 0.2 0.17 0.1 TG 0.23 0.03 0.25 0.02 HDL-C 0.53 0.0001 0.23 0.03 Insulin 0.38 0.005 0.30 0.006 HOMA-IR 0.33 0.003 0.30 0.008 hs-crp 0.24 0.02 0.09 0.4 TNF- 0.12 0.2 NA NA NEFA 0.03 0.7 0.16 0.1 Leptin 0.1 0.3 0.05 0.6 DBP, Diastolic blood pressure; SBP, systolic blood pressure. Adipose tissue has been postulated to play a prominent role in both insulin resistance and the clinical expression of the metabolic syndrome, most likely mediated by increased release and peripheral tissue action of NEFA and by dysregulated production of adipocyte-secreted proteins, including leptin, adiponectin, resistin, TNF-, and IL-6 (11, 14, 15, Variables Before After P BMI (kg/m 2 ) 38.9 1.0 36.3 1.0 0.001 Weight (lb) 257 8.3 239 8.0 0.001 SBP (mm Hg) 135 3.0 129 2.0 0.01 DBP (mm Hg) 85 1.5 80 1.0 0.001 Glucose (mg/dl) 108 7.0 92 2.0 0.01 TG (mg/dl) 280 28 127 8.0 0.001 HDL-C (mg/dl) 44.0 1.7 40.5 1.4 0.001 LDL-C (mg/dl) 118 5.3 98 3.8 0.001 NEFA (meq/ml) 0.5 0.05 0.6 0.05 0.1 GGT (mg/dl) 31.1 3.7 22.4 1.5 0.001 hs-crp (mg/liter) 5.0 0.5 4.3 0.5 0.02 Insulin (pmol/liter) 168 30.5 59 10.5 0.001 HOMA-IR 7.7 1.5 2.3 0.5 0.001 Leptin (pmol/liter) 2964 254 1727 219 0.001 Adiponectin ( g/ml) 7.5 0.6 7.1 0.5 0.1 TNF- (pg/ml) 3.3 0.1 3.3 0.2 0.8 Values are expressed as means SE. P values are by the Wilcoxon signed rank test. DBP, Diastolic blood pressure; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure. 34). Of these proteins, adiponectin has recently been proposed as an important mediator of insulin action and glucose metabolism (13). Adiponectin has been reported to be decreased in obesity and type 2 diabetes and to be inversely correlated with insulin and TG levels and positively correlated with HDL-C levels (16, 17). The present study confirms and extends these associations by demonstrating that plasma adiponectin levels are significantly lower in obese individuals with the metabolic syndrome when compared with obese individuals, matched for age and BMI, who do not have the metabolic syndrome. Furthermore, stepwise decreases in adiponectin were observed with increasing numbers of metabolic syndrome components. The predictive ability of adiponectin to distinguish the MS from the MS obese subjects remained significant after adjusting for both weight and BMI, suggesting that adiponectin may contribute to the expression of the metabolic syndrome phenotype independent of total adiposity. When each component of the metabolic syndrome was introduced separately as a covariate, the predictive ability of adiponectin to distinguish the MS from the MS obese subjects did not change substantially after adjusting for each variable but became nonsig-

Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 2701 nificant after adjusting for HDL-C, suggesting an important role of adiponectin in modulating HDL metabolism. Surprisingly, no change in adiponectin levels was observed after 4 6 wk of weight loss. Despite the persistence of obesity, there was a marked improvement in some aspects of the metabolic syndrome, such as blood pressure, glucose, and TG, but no significant change in TNF- and only a modest (statistically but not clinically significant) drop in hs-crp. As reported previously with acute weight loss associated with ongoing caloric restriction, HDL-C decreased. There are several reports suggesting that adiponectin directly modulates glucose tolerance and peripheral tissue insulin sensitivity, possibly through AMP kinase activation (35, 36). Weight loss studies in humans have been used to support the hypothesis that increases in adiponectin may mediate improvement in insulin action and carbohydrate metabolism observed with weight loss (17, 28, 37). Three studies have demonstrated that a 10 22% weight reduction in obese individuals with VLCD or bariatric surgery was associated with a 36 51% increase in adiponectin levels (17, 28, 37). One study of 22 patients showed a substantial increase in adiponectin levels after 2 months of VLCD with an average weight loss of more than 10% (28), whereas the other studies with bariatric surgery observed improved adiponectin levels with even greater weight loss (17, 37). In contrast, in our study of acute VLCD-induced weight loss, which specifically assessed adipocytokines in an obese population with the metabolic syndrome, we observed marked improvements in glucose, insulin levels, and HOMA-IR within 4 6 wk that were not associated with any significant change in adiponectin levels. In general, expression of adiponectin in plasma correlates well with expression in adipose tissue. Therefore, it is unlikely that the improvements in glucose, insulin, and HOMA-IR, which are all reflective of improved whole-body insulin sensitivity, were related to changes in adiponectin. Our data are consistent with the data from a 6-month weight loss study that achieved a 6% reduction in BMI with diet and exercise and had no significant change in adiponectin levels but did have modest significant reductions in insulin (15%) and glucose (6%) (38). This 6-month study differs from our study in numerous aspects: TG was not reported, patients were not selected on the basis of the metabolic syndrome, average BMI was lower (32 vs. 37), and only postmenopausal women were included. The data from our study are consistent with a proposed model in which reductions in adiponectin levels reflect adipocyte dysregulation or malfunction rather than being causative of insulin resistance. Although unlikely, we cannot exclude the possibility that specific metabolic consequences induced by the VLCD resulted in the lack of change in adiponectin. In the same context, one potential limitation of our study is the lack of an isocaloric steady state at the follow-up evaluation, because subjects were in persistent caloric deficit at that time point. In agreement with our observations, Abbasi et al. (39) have recently shown that there is a significant overlap in adiponectin values between insulin-sensitive and insulin-resistant individuals (assessed by steady state plasma glucose concentration at the end of a 180-min infusion of octreotide, insulin, and glucose) and that a low adiponectin concentration is not uncommon in insulin-sensitive subjects. Furthermore, intervention modalities known to increase peripheral glucose disposal and insulin sensitivity, such as exercise or metformin therapy, do not alter adiponectin levels (40, 41). Thiazolidinediones or PPAR- agonists have been shown to increase adiponectin levels in vitro and in vivo, although the exact mechanism for this action has not been elucidated (27, 42, 43). It has been suggested that the up-regulation of PPAR- transcriptional activity and the resulting effect on adipocyte differentiation positively influences adiponectin secretion independent of the insulinsensitizing properties of these compounds (41). An emerging paradigm proposed to explain the link between excess adiposity and insulin resistance involves the metabolic diversion and ectopic storage of energy substrates (predominantly TG) from adipose to nonadipose tissues, particularly muscle and liver (44, 45). Our results are consistent with this hypothesis, because early mobilization of intracellular lipid from muscle (46) and liver (47) in response to rapid weight loss would be expected to enhance insulin action and decrease plasma levels of insulin, glucose, TG, and GGT acutely, by augmenting substrate transport and utilization (9, 48), despite the fact that our patients remained markedly obese. It is possible that a substantially greater and sustained weight reduction is necessary to correct altered adipocyte function, which could be determined by increased levels of adiponectin and reduced levels of TNF- and NEFA (49). Our study confirmed the association between adiponectin and dyslipidemia in nondiabetic obese subjects, demonstrating a reciprocal association with TG and a strong positive correlation with HDL-C. Although the precise mechanism for this association is unknown, it has been postulated that insulin resistance and/or hyperinsulinemia might offer an explanation in the case of insulin-resistant states, such as obesity, type 2 diabetes, and the metabolic syndrome. Interestingly, in our study the strong positive correlation between adiponectin and HDL-C remained significant even after adjusting for all components of the metabolic syndrome, insulin resistance, and adiposity measures both at baseline and after weight reduction. The adiponectin receptors AdipoR1 and AdipoR2 are associated with PPAR- activity and fatty acid oxidation (50), and thus the strong positive correlation between adiponectin and HDL-C levels may be secondary to modulation of HDL metabolism by adiponectin (51). Adiponectin may also play a role in regulating inflammatory responses. We confirmed the reciprocal association between adiponectin and hs-crp. Although we did not find a clear association of adiponectin and TNF-, which differs from in vitro data (52, 53), the relationship between TNF- and adiponectin in vivo may be complex because it has been suggested that TNF- may regulate adiponectin expression through autocrine and paracrine pathways (37). TNF-, in addition to adiponectin, has been postulated to play a causal role in the development of obesity-induced insulin resistance. To our knowledge, this is the first study demonstrating the stepwise increase in TNF- levels in parallel to the number of metabolic syndrome components present. In vitro studies have shown that adiponectin can decrease endothelialleukocyte adhesion through inhibition of endothelial nuclear factor- B signaling (18, 23). Mice deficient in adiponectin

2702 J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome have increased neointimal proliferation after injury and increased atherosclerosis (24). In summary, we have shown that adiponectin is inversely associated with the expression of the metabolic syndrome and its individual traits in obese individuals, with a stepwise decrease in adiponectin levels with greater numbers of metabolic syndrome-defining criteria. Rapid weight loss through ongoing caloric restriction (7%, on average, over 4 6 wk) by a protein-sparing VLCD in obese individuals with the metabolic syndrome led to impressive reductions in insulin, glucose, TG, and blood pressure without the expected concomitant change in adiponectin or TNF- and only a modest reduction in hs-crp. These data are supportive of a proposed model that relates rapid weight loss to early mobilization of ectopic fat from liver and skeletal muscle, thereby reducing GGT, TG, glucose, insulin, and HOMA-IR, although subjects remained markedly obese after weight loss. We postulate that the lack of change in adiponectin and TNF- and the very modest decrease in hs-crp reflect persistent adipocyte dysregulation despite initiation of a rapid weight loss intervention and that a critical amount of total adiposity must be lost before the adipocyte resumes a more balanced function. The association of adiponectin with HDL and inflammation merits additional studies in humans to examine whether pharmacological manipulation of adiponectin levels would favorably influence these parameters. Acknowledgments Received October 20, 2003. Accepted February 24, 2004. Address all correspondence and requests for reprints to: Christie M. Ballantyne, M.D., Baylor College of Medicine, 6565 Fannin Street, MS A-601, Houston, Texas 77030. E-mail: cmb@bcm.tmc.edu. This work was supported by National Institutes of Health General Clinical Research Center Grant 5M01RR00350 and Texas Applied Technology Program Grant 004949-0093-2001. The atherosclerosis laboratory was supported by donations from George and Cynthia Mitchell, Nijad Fares, and Jeffrey Hines. References 1. Flegal KM, Carroll MD, Ogden CL, Johnson CL 2002 Prevalence and trends in obesity among US adults, 1999 2000. JAMA 288:1723 1727 2. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, Salonen JT 2002 The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA 288:2709 2716 3. Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults 2001 Executive summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 285:2486 2497 4. Ford ES, Giles WH, Dietz WH 2002 Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 287:356 359 5. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, Heymsfield SB 2003 The metabolic syndrome: prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey, 1988 1994. Arch Intern Med 163:427 436 6. Kahn BB, Flier JS 2000 Obesity and insulin resistance. J Clin Invest 106:473 481 7. Maison P, Byrne CD, Hales CN, Day NE, Wareham NJ 2001 Do different dimensions of the metabolic syndrome change together over time? Evidence supporting obesity as the central feature. Diabetes Care 24:1758 1763 8. Goodpaster BH, Kelley DE, Wing RR, Meier A, Thaete FL 1999 Effects of weight loss on regional fat distribution and insulin sensitivity in obesity. Diabetes 48:839 847 9. Kelley DE, Goodpaster B, Wing RR, Simoneau JA 1999 Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss. Am J Physiol Endocrinol Metab 277:E1130 E1141 10. Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, Nathan DM 2002 Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 346:393 403 11. Trayhurn P, Beattie JH 2001 Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proc Nutr Soc 60:329 339 12. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB 2002 Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339 343 13. Matsuzawa Y, Funahashi T, Nakamura T 1999 Molecular mechanism of metabolic syndrome X: contribution of adipocytokines adipocyte-derived bioactive substances. Ann NY Acad Sci 892:146 154 14. McTernan CL, McTernan PG, Harte AL, Levick PL, Barnett AH, Kumar S 2002 Resistin, central obesity, and type 2 diabetes. Lancet 359:46 47 15. Kern PA, Ranganathan S, Li C, Wood L, Ranganathan G 2001 Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance. Am J Physiol Endocrinol Metab 280:E745 E751 16. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y 1999 Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257:79 83 17. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y 2000 Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20:1595 1599 18. Ouchi N, Kihara S, Arita Y, Maeda K, Kuriyama H, Okamoto Y, Hotta K, Nishida M, Takahashi M, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 1999 Novel modulator for endothelial adhesion molecules: adipocytederived plasma protein adiponectin. Circulation 100:2473 2476 19. Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA 2001 Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86:1930 1935 20. Kumada M, Kihara S, Sumitsuji S, Kawamoto T, Matsumoto S, Ouchi N, Arita Y, Okamoto Y, Shimomura I, Hiraoka H, Nakamura T, Funahashi T, Matsuzawa Y 2003 Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler Thromb Vasc Biol 23:85 89 21. Yamauchi T, Kamon J, Waki H, Imai Y, Shimozawa N, Hioki K, Uchida S, Ito Y, Takakuwa K, Matsui J, Takata M, Eto K, Terauchi Y, Komeda K, Tsunoda M, Murakami K, Ohnishi Y, Naitoh T, Yamamura K, Ueyama Y, Froguel P, Kimura S, Nagai R, Kadowaki T 2003 Globular adiponectin protected ob/ob mice from diabetes and apoe-deficient mice from atherosclerosis. J Biol Chem 278:2461 2468 22. 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 ML, Kagechika H, Shudo K, Yoda M, Nakano Y, Tobe K, Nagai R, Kimura S, Tomita M, Froguel P, Kadowaki T 2001 The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941 946 23. Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 2000 Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF- B signaling through a camp-dependent pathway. Circulation 102:1296 1301 24. Okamoto Y, Kihara S, Ouchi N, Nishida M, Arita Y, Kumada M, Ohashi K, Sakai N, Shimomura I, Kobayashi H, Terasaka N, Inaba T, Funahashi T, Matsuzawa Y 2002 Adiponectin reduces atherosclerosis in apolipoprotein E-deficient mice. Circulation 106:2767 2770 25. Okamoto Y, Arita Y, Nishida M, Muraguchi M, Ouchi N, Takahashi M, Igura T, Inui Y, Kihara S, Nakamura T, Yamashita S, Miyagawa J, Funahashi T, Matsuzawa Y 2000 An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls. Horm Metab Res 32:47 50 26. Matsuzawa Y, Funahashi T, Nakamura T 2002 Molecular mechanism of vascular disease in metabolic syndrome X. J Diabetes Complications 16:17 18 27. Yu JG, Javorschi S, Hevener AL, Kruszynska YT, Norman RA, Sinha M, Olefsky JM 2002 The effect of thiazolidinediones on plasma adiponectin levels in normal, obese, and type 2 diabetic subjects. Diabetes 51:2968 2974 28. Yang WS, Lee WJ, Funahashi T, Tanaka S, Matsuzawa Y, Chao CL, Chen CL, Tai TY, Chuang LM 2001 Weight reduction increases plasma levels of an adipose-derived anti-inflammatory protein, adiponectin. J Clin Endocrinol Metab 86:3815 3819 29. Ridker PM, Buring JE, Cook NR, Rifai N 2003 C-reactive protein, the metabolic syndrome, and risk of incident cardiovascular events: an 8-year follow-up of 14,719 initially healthy American women. Circulation 107:391 397 30. Case CC, Jones PH, Nelson K, O Brian Smith E, Ballantyne CM 2002 Impact of weight loss on the metabolic syndrome. Diabetes Obes Metab 4:407 414 31. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC 1985 Homeostasis model assessment: insulin resistance and -cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412 419 32. Henry RR, Wiest-Kent TA, Scheaffer L, Kolterman OG, Olefsky J 1986

Xydakis et al. Adiponectin, TNF, and Metabolic Syndrome J Clin Endocrinol Metab, June 2004, 89(6):2697 2703 2703 Metabolic consequences of very-low-calorie diet therapy in obese non-insulindependent diabetic and nondiabetic subjects. Diabetes 35:155 164 33. Dattilo AM, Kris-Etherton PM 1992 Effects of weight reduction on blood lipids and lipoproteins: a meta-analysis. Am J Clin Nutr 56:320 328 34. Hotamisligil GS, Spiegelman BM 1994 Tumor necrosis factor : a key component of the obesity-diabetes link. Diabetes 43:1271 1278 35. Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang Cc C, Itani SI, Lodish HF, Ruderman NB 2002 Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-coa carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci USA 99:16309 16313 36. 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 BB, Kadowaki T 2002 Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMPactivated protein kinase. Nat Med 8:1288 1295 37. Bruun JM, Lihn AS, Verdich C, Pedersen SB, Toubro S, Astrup A, Richelsen B 2003 Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans. Am J Physiol Endocrinol Metab 285: E527 E533 38. Ryan AS, Nicklas BJ, Berman DM, Elahi D 2003 Adiponectin levels do not change with moderate dietary induced weight loss and exercise in obese postmenopausal women. Int J Obes Relat Metab Disord 27:1066 1071 39. Abbasi F, Chu J, McLaughlin T, Lamendola C, Reaven G, Hayden J, Reaven P 2003 Obesity versus insulin resistance in modulation of plasma adioponectin concentration. Diabetes 52(Suppl 1):A81 (Abstract) 40. Hulver MW, Zheng D, Tanner CJ, Houmard JA, Kraus WE, Slentz CA, Sinha MK, Pories WJ, MacDonald KG, Dohm GL 2002 Adiponectin is not altered with exercise training despite enhanced insulin action. Am J Physiol Endocrinol Metab 283:E861 E865 41. Phillips SA, Ciaraldi TP, Kong AP, Bandukwala R, Aroda V, Carter L, Baxi S, Mudaliar SR, Henry RR 2003 Modulation of circulating and adipose tissue adiponectin levels by antidiabetic therapy. Diabetes 52:667 674 42. Maeda N, Takahashi M, Funahashi T, Kihara S, Nishizawa H, Kishida K, Nagaretani H, Matsuda M, Komuro R, Ouchi N, Kuriyama H, Hotta K, Nakamura T, Shimomura I, Matsuzawa Y 2001 PPAR ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 50:2094 2099 43. Yang WS, Jeng CY, Wu TJ, Tanaka S, Funahashi T, Matsuzawa Y, Wang JP, Chen CL, Tai TY, Chuang LM 2002 Synthetic peroxisome proliferator-activated receptor- agonist, rosiglitazone, increases plasma levels of adiponectin in type 2 diabetic patients. Diabetes Care 25:376 380 44. Ravussin E, Smith SR 2002 Increased fat intake, impaired fat oxidation, and failure of fat cell proliferation result in ectopic fat storage, insulin resistance, and type 2 diabetes mellitus. Ann NY Acad Sci 967:363 378 45. Lewis GF, Carpentier A, Adeli K, Giacca A 2002 Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocr Rev 23:201 229 46. Gray RE, Tanner CJ, Pories WJ, MacDonald KG, Houmard JA 2003 Effect of weight loss on muscle lipid content in morbidly obese subjects. Am J Physiol Endocrinol Metab 284:E726 E732 47. Tiikkainen M, Bergholm R, Vehkavaara S, Rissanen A, Hakkinen AM, Tamminen M, Teramo K, Yki-Jarvinen H 2003 Effects of identical weight loss on body composition and features of insulin resistance in obese women with high and low liver fat content. Diabetes 52:701 707 48. Williams KV, Bertoldo A, Kinahan P, Cobelli C, Kelley DE 2003 Weight loss-induced plasticity of glucose transport and phosphorylation in the insulin resistance of obesity and type 2 diabetes. Diabetes 52:1619 1626 49. Esposito K, Pontillo A, Di Palo C, Giugliano G, Masella M, Marfella R, Giugliano D 2003 Effect of weight loss and lifestyle changes on vascular inflammatory markers in obese women: a randomized trial. JAMA 289:1799 1804 50. Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, Sugiyama T, Miyagishi M, Hara K, Tsunoda M, Murakami K, Ohteki T, Uchida S, Takekawa S, Waki H, Tsuno NH, Shibata Y, Terauchi Y, Froguel P, Tobe K, Koyasu S, Taira K, Kitamura T, Shimizu T, Nagai R, Kadowaki T 2003 Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature 423:762 769 51. Matsubara M, Maruoka S, Katayose S 2002 Decreased plasma adiponectin concentrations in women with dyslipidemia. J Clin Endocrinol Metab 87:2764 2769 52. Kappes A, Loffler G 2000 Influences of ionomycin, dibutyryl-cycloamp and tumour necrosis factor- on intracellular amount and secretion of apm1 in differentiating primary human preadipocytes. Horm Metab Res 32:548 554 53. Fasshauer M, Klein J, Neumann S, Eszlinger M, Paschke R 2002 Hormonal regulation of adiponectin gene expression in 3T3 L1 adipocytes. 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