Details of biochemical assays were described previously (31 (link)). Briefly, blood glucose concentrations were determined by the glucose oxidase method using a glucose analyzer (YSI 2300 STAT Plus; Yellow Springs Instruments, Yellow Springs, OH). Plasma immunoreactive insulin levels were measured using a double-antibody radioimmunoassay (Millipore, St. Charles, MO) (32 (link)). Plasma AG and desacylated ghrelin (DAG) levels were measured using separate sensitive and specific two-site sandwich ELISAs (16 (link)). The sensitivity of the AG assay was 6.7 pg/mL with intra- and interassay coefficients of variation of ∼14 and 18% (16 (link)). The sensitivity of the DAG assay was 4.6 pg/mL with intra- and interassay coefficients of variations of ∼13 and 20% (16 (link)). Glucagon was measured by radioimmunoassay (Millipore Life Sciences, Billerica, MA), and serum concentrations of human growth hormone were measured by a sandwich immunoassay using the automated Immulite 2000 chemiluminescent assay system (Siemens, Bad Nauheim, Germany) (31 (link)). Plasma free fatty acids were measured using a specific colorimetric assay (Wako Diagnostics, Richmond, VA). All samples were assayed in duplicate, and specimens from the four studies in each participant were run in the same assay.