For this study, data were collected over two separate clinical visits in the first annual visit. Children were admitted to the USC General Clinical Research Center (GCRC) at approximately 07.30 h after an overnight fast, no food or caloric beverages after 20.00 h. A licensed paediatric healthcare provider conducted a detailed medical history, including parental interview with detailed assessment of family history of diabetes; Tanner staging based on breast stage in girls and pubic hair stage in boys was assessed. Children then completed a 2-h oral glucose tolerance test on the same day. At the second clinical visit, after an overnight fast, a frequently sampled intravenous glucose tolerance test (FSIVGTT) was conducted and body composition was assessed. The evening before, the children were served dinner and an evening snack, and only water and non-caloric and non-caffeinated beverages were permitted after 20.00 h.
The methods of the study have been previously reported in detail elsewhere . Briefly, total body fat was assessed by a whole body scan using dual energy X-ray absorptiometry (Hologic QDR 4500W; Hologic, Bedford, MA, USA). Central fat distribution was measured directly by magnetic resonance imaging at the Los Angeles County/USC Imaging Science Center. A single-slice axial TR 400/16 view of the abdomen at the level of the umbilicus was analysed for cross-sectional area of adipose tissue using a General Electric 1.5 Sigma LX-Echospeed device with a General Electric 1.5-T magnet (Waukesha, WI, USA).
After an overnight fast, the FSIVGTT was performed to determine insulin dynamics. A topical anaesthetic (EMLA cream; AstraZeneca, Wilmington, DE, USA) was applied to the antecubital area of both arms and an hour later a flexible intravenous catheter was inserted into both arms. Two fasting blood samples, at −15 and −5 min, were pooled for determination of basal glucose and insulin values. At time zero, glucose (25% dextrose; 0.3 g/kg of body weight) was administered intravenously. Blood samples were then collected at the following time points: 2, 4, 8, 19, 22, 30, 40, 50, 70, 100, 120 and 180 min [21,22]. Insulin (0.02 U/kg of body weight; Humulin R—regular unmodified insulin; Eli Lilly and Co., Indianapolis, IN, USA) was injected intravenously at 20 min. Plasma was analysed for glucose and insulin, and values were entered into the Minmod Millennium 2003 computer program (version 5.16; Richard N. Bergman, University of Southern California, Los Angeles, CA, USA) for determination of insulin sensitivity (SI), the acute insulin response (AIR = insulin area under the curve above basal for the first 10 min of the FSIVGTT) and the disposition index (DI = product of AIR and SI as a measure of pancreatic β-cell function) . Blood samples from the FSIVGTT were centrifuged immediately to obtain plasma, and aliquots were frozen at −70°C until assayed. Glucose was assayed in duplicate on a Yellow Springs Instrument 2700 Analyzer (Yellow Springs Instrument, Yellow Springs, OH, USA) using the glucose oxidase method. Insulin was assayed in duplicate using a specific human insulin enzyme-linked immunosorbent assay kit from Linco Research (St Charles, MO, USA). Plasma adiponectin was measured using radioimmunoassay kits (Linco Research) with an intra-assay coefficient of variation (CV) of 3.9% and an interassay CV of 8.5%. For plasma leptin, radioimmunoassay kits were used (Linco Research) with an intra-assay CV of 3.9% and an interassay CV of 8.5%.