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 Kikuko Hotta,1 Tohru Funahashi,1 Noni L. Bodkin,2 Heidi K. Ortmeyer,2 Yukio Arita,1 Barbara C. Hansen,2 and Yuji Matsuzawa1 Adiponectin is an adipose-specific plasma protein whose of insulin resistance, type 2 diabetes, and their complica- plasma concentrations are decreased in obese subjects tions through the secretion of a variety of biologically and type 2 diabetic patients. This protein possesses active molecules (adipocytokines) (1). Hotamisligil et al. putative antiatherogenic and anti-inflammatory proper- (2) reported that tumor necrosis factor-␣ (TNF-␣) over- ties. In the current study, we have analyzed the rela- produced in adipose tissue of obesity contributes to the tionship between adiponectin and insulin resistance in development of insulin resistance. Leptin is an adipose- rhesus monkeys (Macaca mulatta), which spontane- specific hormone contributing to the regulation of energy ously develop obesity and which subsequently fre- quently progress to overt type 2 diabetes. The plasma expenditure and food intake (3). Leptin also affects insulin levels of adiponectin were decreased in obese and dia- sensitivity and may participate in the development of betic monkeys as in humans. Prospective longitudinal hypertension (4–8). Plasminogen activator inhibitor-1 studies revealed that the plasma levels of adiponectin (PAI-1) increases in obesity and diabetes and may play a declined at an early phase of obesity and remained part in thrombosis and the development of vascular dis- decreased after the development of type 2 diabetes. ease (9–11). These adipocytokines may cause the athero- Hyperinsulinemic-euglycemic clamp studies revealed sclerotic vascular disease in type 2 diabetes directly or that the obese monkeys with lower plasma adiponectin through the development of insulin resistance. showed significantly lower insulin-stimulated periph- eral glucose uptake (M rate). The plasma levels of Adiponectin is a novel adipose-specific collagen-like .(molecule that belongs to the collectin family (12–14 ؍ adiponectin were significantly correlated to M rate (r 0.66, P < 0.001). Longitudinally, the plasma adiponectin Adiponectin bound to collagens I, III, and V (major com- decreased in parallel to the progression of insulin resis- ponents of the vascular intima) in a solid-phase binding tance. No clear association was found between the assay and accumulated in the vascular wall when the plasma levels of adiponectin and its mRNA levels in endothelial barrier was damaged (15). The concentration adipose tissue. These results suggest that reduction in of adiponectin in plasma ranged from 5 to 10 ␮g/ml in circulating adiponectin may be related to the develop- healthy humans (14). Obese patients, type 2 diabetic pa- ment of insulin resistance. Diabetes 50:1126–1133, 2001 tients, and patients with coronary artery disease showed significantly lower levels of plasma adiponectin (14,16,17). We found that administration of adiponectin decreased the attachment of monocytic cell line THP-1 cells to human nsulin resistance is one of the important risk factors aortic endothelial cells (16,18), which is an early event in associated with atherosclerosis and diabetes. Insulin atherosclerotic vascular damage. Adiponectin decreases resistance often accompanies visceral fat accumula- the expression of multiple adhesion molecules, including ␬ tion (1). Recent studies have provided evidence that in endothelial cells via the modulation of NF B signaling I (16,18). Adiponectin also dramatically suppressed the se- adipose tissue may play a crucial role in the development cretion of TNF-␣ from human monocyte-macrophages (19). These clinical and experimental observations suggest From the 1Department of Internal Medicine and Molecular Science, Graduate that adiponectin plays some protective role against the School of Medicine, Osaka University, Suita, Osaka, Japan; and the 2Obesity and Diabetes Research Center, Department of Physiology, University of atherosclerotic vascular change and that the decreased Maryland, Baltimore, Maryland. plasma adiponectin in type 2 diabetic patients may con- Address correspondence and reprint requests to Kikuko Hotta, MD, Depart- tribute to the development of atherosclerotic complica- ment of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 Japan. tions. The mechanism of decreased plasma adiponectin in E-mail: [email protected]. type 2 diabetes, however, has not yet been clarified. Received for publication 28 September 2000 and accepted in revised form 5 February 2001. Many rhesus monkeys (Macaca mulatta) spontaneously AIR, acute insulin response; ELISA, enzyme-linked immunosorbent assay; develop obesity and subsequently develop type 2 diabetes, FFM, fat-free mass; HSP83, heat shock protein 83; KG, glucose disappearance so they are excellent nonhuman primate models of human rate; M rate, insulin-stimulated glucose uptake rate; PAI-1, plasminogen activator inhibitor-1; PCR, polymerase chain reaction; RT, reverse transcrip- type 2 diabetes (20–23). Sequential metabolic changes tase; TNF-␣, tumor necrosis factor-␣. during the development of obesity and diabetes can be 1126 DIABETES, VOL. 50, MAY 2001 K. HOTTA AND ASSOCIATES followed in this model. A decrease in insulin sensitivity and a progressive increase of fasting plasma insulin (fol- lowed by a decline in ␤-cell function) precedes the mani- festation of diabetes in rhesus monkeys (20–22). In the current study, we investigated the plasma concentrations and adipose mRNA expression of adiponectin during the development of obesity, insulin resistance, and type 2 diabetes in rhesus monkeys. RESEARCH DESIGN AND METHODS Male rhesus monkeys (Macaca mulatta) were individually housed and main- tained in accordance with the National Academy of Sciences guidelines for the FIG. 1. Fat weight (A) and the plasma levels of leptin (B) and care and use of laboratory animals. Either the nutritionally complete liquid adiponectin (C) in lean, hyperinsulinemic obese, and type 2 diabetic diet Ensure (Ross Laboratories, Columbus, OH) or Monkey Chow (Purina rhesus monkeys. The plasma levels of adiponectin and leptin from lean monkeys were (10 ؍ and type 2 diabetic (n ,(23 ؍ obese (n ,(14 ؍ Mills, St. Louis, MO) and fresh water were provided to monkeys ad libitum (8 (n > h/day). For measurement of adiponectin and leptin, the monkeys to be studied measured by ELISA. Values represent means ؎ SE. * P < 0.05; ** P were sorted into the following three groups: lean (body fat Ͻ22%), obese (body 0.01; *** P < 0.001. fat Ͼ22%), or type 2 diabetic. Diabetes was diagnosed according to American Ͼ Diabetes Association criteria (24) (fasting plasma glucose 7 mmol/l). mRNA levels of adiponectin and HSP83 were quantitated using a BAS5000 Procedures. Plasma samples were obtained under light anesthesia (ketamine phosphor imaging system (Fuji Photo Film, Tokyo). hydrochloride 10 mg/kg body wt) after a 16-h fast. Plasma samples were Statistical analysis. Data are expressed as means Ϯ SE. Significant group Ϫ frozen and kept at 80°C for later assays. Subcutaneous adipose tissues were differences were determined by one-way analysis of variance, and significant obtained under ketamine hydrochloride, as described above, or immediately F values were further tested by the Fisher multiple comparison method. after anesthetization by intravenous sodium pentobarbital. Tissue samples Linear relationships between key variables were tested using Pearson’s Ϫ were immediately frozen in liquid nitrogen and stored at 165°C. correlation coefficient. Multiple linear regression analysis was performed to After a 16-h fast, the monkeys were placed under light anesthesia (ket- evaluate the independent relationship of the studied variables. amine hydrochloride 10 mg/kg body wt), and an intravenous glucose tolerance test (0.5 ml/kg of 50% dextrose) was carried out with sampling at 1, 3, 5, 7, 10, RESULTS 15, and 20 min. The glucose disappearance rate (KG) was calculated using the 5- and 20-min time points (20). Acute insulin response (AIR) was determined Plasma adiponectin concentration in obese and type to be the mean change in plasma insulin per minute from basal levels for the 2 diabetic monkeys. First, we evaluated the plasma period 0–10 min during the intravenous glucose tolerance test (22). Hyperin- sulinemic-euglycemic (2,400 pmol ⅐ mϪ2 body surface area ⅐ minϪ1 insulin concentrations of adiponectin and leptin in the lean mon- infusion) clamps were carried out (21). The plasma glucose was maintained at keys (n ϭ 14, age 10 Ϯ 2 years, 10.5 Ϯ 0.7 kg body wt, 16 Ϯ ϳ4.7 mmol/l to estimate insulin-stimulated glucose uptake rate (M rate). M 1% body fat); the obese monkeys (n ϭ 23, age 16 Ϯ 1 years, rate was corrected for fat-free mass (FFM), an estimate of metabolically active 16.7 Ϯ 0.7 kg body wt, 34 Ϯ 1% body fat); and the diabetic mass, as described previously (21). Percentage body fat was determined by monkeys (n ϭ 10, age 23 Ϯ 1 years, 13.2 Ϯ 0.6 kg body wt, the tritiated water dilution method (25). Enzyme-linked immunosorbent assay of plasma adiponectin and leptin. 31 Ϯ 2% body fat). The lean group showed normal fasting We measured plasma adiponectin levels in monkeys using the enzyme-linked glucose (3.3 Ϯ 0.1 mmol/l) and insulin (351 Ϯ 31 pmol/l) immunosorbent assay (ELISA) system developed for the measurement of levels. The obese group showed normal fasting glucose human plasma adiponectin concentrations, as described previously (14). levels (4.0 Ϯ 0.2 mmol/l), but they had significantly higher Human recombinant adiponectin was used as a standard. The affinity of the Ϯ Ͻ monoclonal anti-adiponectin antibody (used as the first antibody of ELISA) to plasma insulin levels (1,009 278 pmol/l, P 0.05) the monkey adiponectin was as strong as that to human adiponectin; however, compared with normal monkeys. In the diabetic group, the affinity of the polyclonal antibody (which was used for the second fasting plasma glucose levels were elevated (11.2 Ϯ 0.9 antibody) was weaker.