Supplementary Material
Methods
Measurements and assays before and after the overfeeding period
Body weight was measured each morning before breakfast with the subject wearing a light exercise short. Body density was determined before and after the overfeeding protocol by underwater weighing1, and percent body fat was derived from a standard equation2. Pulmonary residual volume was estimated from a helium-dilution technique3. FM and FFM were computed from body mass and percentage of body fat.
Day 1 of testing. Resting metabolic rate (RMR) was assessed by indirect calorimetry in a 12-hour fasted state in the morning. The subject sat in a comfortable reclined seat with the head inside a Beckman ventilated hood system4. After resting for 30 min, the concentrations of oxygen and carbon dioxide were measured using paramagnetic and infrared analyzers (Beckman OM-II and LB-2), and ventilatory volume was determined with a Fleish pneumotacograph. After resting measurements had been completed, the subject ate a 4.2 MJ (1 000 kcal) meal with the following composition: 15 percent protein. 35 percent lipid, and 50 percent carbohydrate. The test meal was consumed within 15 min, after which the indirect calorimetric measurements were resumed for the next 240 min to assess the thermic effect of the meal (TEM). The energy equivalent of VO2 was calculated using the formula of Weir5. The respiratory quotient (RQ) was derived from VO2 and VCO2 following standard procedures.
Day 2 of testing. A skeletal muscle biopsy was obtained in the fasted state from the vastus lateralis muscle6. Fiber type distribution was assessed based on a standard histochemical technique7. The maximal activities of creatine kinase, phosphofructokinase (PFK), and oxoglutarate dehydrogenase (OGDH) were determined as previously described6. The ratio of PFK to OGDH activities was also calculated. The procedures and the assays have been described earlier8. Afterward, an oral glucose tolerance test (OGTT) was performed9. Briefly, the fasted subject drank a beverage containing 75 g of glucose over a 3-min period. Blood samples were collected in the fasted state and 15, 30, 45, 60, 90, 120, 150, and 180 min after glucose ingestion for the determination of plasma glucose, insulin, and glucagon.
Day 3 of testing. Adipose tissue biopsies were performed in a fasting state from the abdominal and femoral areas. Adipocytes were isolated by collagenase digestion10. Fat cell size was assessed from isolated adipocytes as previously described11. From the abdominal tissue, basal and epinephrine- and isoproterenol-stimulated (at 10-5 M for both agents) lipolysis was determined as reported elsewhere for these subjects12. Adipose tissue lipoprotein lipase (LPL) was determined using a modification13 of the method described earlier by Taskinen et al.14. Fat cell size was also calculated from mid-femoral fat cells. Subjects then ate a light meal, completed, questionnaires and rested. Later, after insertion of a venous catheter in an antecubital vein, a progressive test to exhaustion was performed on a treadmill around noon15. Heart rate was continuously monitored by an electrocardiogram derivation. Oxygen uptake and carbon dioxide output were determined using an open gas circuit system (for O2, Applied Electrochemistry S-3A, and for VCO2, Anarad R1 analyzer). Pulmonary ventilation was measured with a Fleisch pneumotacograph. Maximal oxygen update (VO2max) was defined as the highest VO2 recorded during 1 min. One hour after the treadmill test, with the subject resting comfortably in a semi-reclined position, an IV injection (400 µg) of thyrotropin-releasing hormone (TRH test) was performed, and blood samples were obtained before and 20, 30, and 45 min post-injection15.
Plasma assays. From blood obtained in the fasted state, the following hormones were assayed. Plasma epinephrine (Epi) and norepinephrine (Norepi) (from day 3 samples) were measured by a radioenzymatic procedure16. Epi and Norepi were also assayed at exhaustion at the end of the maximal treadmill exercise bout. From samples drawn at day 3, thyroid hormones were assayed by radioimmunoassay: T317, T4 and free T4 (FT4)18, and thyroid-stimulating hormone (TSH)19. From blood drawn on day 2, fasting and OGTT plasma glucose was assayed with an enzymatic technique20. Plasma insulin21 and glucagon22 were determined by radioimmunoassays. Insulin-like growth factor 1 (IGF-l) was assayed from plasma obtained early in the morning in the fasted state on day 3 using the SM-C radioimmunoassay kit of Nichols Institute Diagnostics (San Juan Capistrano, CA). Growth hormone (hGH) was measured with a double antibody radioimmunoassay23. Plasma leptin was measured with an enzyme-linked immunoabsorbent assay specific for human leptin24. Plasma adiponectin was quantified with a commercial radioimmunoassay (Linco Research, Missouri). Plasma ghrelin was measured with a commercial radioimmunoassay (Phoenix Pharmaceuticals, Belmont, CA) that uses 125I-labeled bioactive ghrelin as a tracer molecule and a polyclonal antibody raised in rabbits against full-length, octanoylated human ghrelin, as described before25.
Steroids. The assays have been described before26, 27. Plasma steroids were extracted twice with ethanol. The solvent was evaporated under nitrogen, resuspended in methanol:water (5:95), and separated on methanol/methanol:water-conditioned C-18 columns. Glucuronide and sulfate derivatives were eluted with methanol/water. Non-conjugated steroids were extracted with methanol/water. Both fractions were evaporated. The steroid glucuronide fraction was solubilized in 0.1 M phosphate buffer and hydrolyzed with β-glucuronidase. The steroids released were extracted twice with ethyl ether. The organic phase was evaporated, and the residue was used to assess glucuronide conjugate levels. The water phase was used for sulfate solvolysis with HCl and ethyl ether saturated with HCl. The organic phase was evaporated and the residue solubilized with 0.2 M phosphate buffer and further extracted twice with ethyl ether. The organic phase was evaporated, and the residue was used to assess sulfate steroid levels. Steroid fractions were solubilized in isooctane/toluene and eluted on Sephadex LH-20 columns (Pharmacia, Uppsala, Sweden). Steroids were collected using solvent mixtures with increasing polarities28 and assessed by radioimmunoassay29. Assay results are the mean of triplicate determinations in each individual sample. Reliability of the methodology was previously assessed and described in repeated samples from the HERITAGE Family Study30. Plasma sex-hormone-binding-globulin (SHBG) was measured with a commercial immunoradiometric assay (Framos, Munich, Germany).
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