Adiponectin Expression From Human Adipose Tissue: Relation to Obesity, Insulin Resistance, and Tumor Necrosis Factor-α Expression

Philip A. Kern Gina B. Di Gregorio Tong Lu Negah Rassouli Gouri Ranganathan - sion and have not determined the relation between adiponectin and other cytokines. This study examined adiponectin expression in plasma and adipose tissue in nondiabetic subjects with varying degrees of obesity and insulin resistance. We found a significant inverse association between plasma adiponectin and insulin resistance, an effect that was independent of obesity. In addition, there was an inverse association between adipose tissue adiponectin and TNF- expression, but there was no association between adiponectin and other cytokines. These data suggest that adiponectin, like TNF- and IL-6, plays an important role in obesityassociated insulin resistance. RESEARCH DESIGN AND METHODS Subjects. This study involved 62 weight-stable subjects ages 23 61 years. All subjects gave informed consent, and the research was approved by the institutional review board. Subjects initially underwent a 75-g oral glucose tolerance test, and subjects with diabetes (fasting glucose 126 mg/dl, 2-h glucose 200 mg/dl) were excluded. Of the 62 subjects, 15 had impaired glucose tolerance based on a 2-h glucose test of 140 200 mg/dl, and 4 of these subjects had impaired fasting glucose based on a fasting glucose of 110 126 mg/dl. Subjects then underwent a frequently sampled intravenous glucose tolerance test (FSIVGT) and an adipose tissue biopsy, which were performed on separate days. Characteristics of the study subjects are shown in Table 1. Blood lipids, glucose, and HbA1c were measured using standard clinical assays. Of the 62 subjects studied, 50 were women and 12 were African American. The subjects ranged from lean to very obese (BMI range 19 65 kg/m2). Some subjects demonstrated moderate dyslipidemia, but no subject demonstrated fasting triglycerides 400 mg/dl. Body composition was determined using bioelectric impedance (18). Insulin sensitivity measurements. The measurement of in vivo insulin sensitivity was performed in the fasting state using the classic tolbutamidemodified minimal model analysis of the FSIVGT (19,20), which has been validated against the euglycemic clamp (21,22). Four basal blood samples were obtained at time 0. Patients were then given an intravenous glucose bolus (11.4 g/m2) and, 20 min later, an injection of tolbutamide (125 mg/m2). Frequent blood sampling was then performed according to the standard protocol. Glucose was measured using the glucose oxidase method in a glucose analyzer and insulin was measured using radioimmunoassay (RIA; Endocrinology Laboratory of the Indiana University School of Medicine, Indianapolis, IN). The insulin sensitivity index (SI) was calculated using the MINMOD program, along with the acute insulin response to glucose (acute insulin response to glucose [AIRglu]) (20). Adipose tissue biopsy. Abdominal subcutaneous adipose tissue (5 g) was removed from each patient by incision. Some of the tissue was immediately frozen in liquid N2 for later RNA extraction, whereas the rest of the tissue was placed into cold Dulbeccos modified Eagles medium (DMEM) for other assays. To measure the secretion of adiponectin and TNF-, 500 mg of adipose tissue were minced and placed into serum-free DMEM (pH 7.4, 10 mmol/l HEPES) at 37 for varying times, as previously described (23). To compare cytokine secretion among different subjects, we measured cytokine levels in the medium after 2 h at 37. All data were normalized to either FIG. 1. Adiponectin expression in lean and obese subjects. A: Plasma adiponectin levels expressed in relation to subject BMI. B: Representative Northern blot from three lean and three obese women (see text for explanation). adipose DNA content (24) or cell number to control for differences in fat cell size. Cell number was measured using the method of DiGirolamo et al. (25). Measurement of cytokine expression. The measurement of adiponectin protein used a radioimmunoassay (Linco Research, St. Charles, MO). This assay demonstrates a 4.3% intra-assay variation and a 7.1% interassay variation. This assay method was used to measure adiponectin in fasting plasma as well as secretion by adipose tissue, as described above. Adiponectin mRNA levels were measured by Northern blotting using the cDNA to human adiponectin, and the same blots were reprobed with the cDNA to 18S RNA as a constitutive probe (Fig. 1B). To quantitate adiponectin mRNA expression, the blots were analyzed by densitometry, and the adiponectin/18S RNA ratio was given as arbitrary units. TNF- expression. TNF- mRNA levels were measured as previously described using competitive RT-PCR (26). In brief, 0.4 g of total RNA from adipose tissue were added to increasing quantities of a competing RNA construct containing an internal 49-nt deletion. After the RT and PCR reactions, the products were resolved on a 2% agarose gel and the ethidium bromidestained gel was quantitated. Data are expressed as the number of copies per microgram of total RNA, where number of copies refers to the number of copies of cRNA added at the equivalence point between TNFmRNA product and cRNA. TNF- and IL-6 protein were measured using enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN), and leptin was measured using an RIA (Linco Research). Statistics. All data are expressed as means SE. Analysis of trends was performed using linear regression. When comparing two groups, a Students t test was used, and to analyze data among groups of three or more, a one-way ANOVA was performed and secondary analysis was performed with the Students t test with Bonferroni correction. Adiponectin expression and relation to obesity. Adiponectin was measured in plasma, in the medium secreted from adipose tissue, and adiponectin mRNA levels were measured by Northern blotting. To examine the relation between adiponectin and obesity, adiponectin expression FIG. 2. Plasma adiponectin levels expressed in relation to percent body fat. The regression lines are drawn using a least-squares fit. was measured in subjects covering a wide range of BMI and percent body fat. When all subjects were considered together, there was a significant inverse association between BMI and plasma adiponectin (r 0.39, P 0.002, n 62) (Fig. 1A). In addition, adiponectin mRNA was assessed by Northern blotting, which yielded a single band for adiponectin (Fig. 1B). There was a strong correlation between adiponectin mRNA levels and plasma adiponectin levels (r 0.80, P 0.001, n 24; data not shown), and likewise a significant inverse correlation between adiponectin mRNA and BMI (r 0.55, P 0.01, n 24; data not shown). Gender differences in adiponectin expression. Among subjects with similar BMIs, there was considerable variation in plasma adiponectin level. As can be seen in Fig. 1A, some of this variation could be attributed to lower adiponectin levels in men. To more appropriately analyze this association and account for gender differences, adiponectin exp (...truncated)