Mice are surgically implanted with dual carotid and jugular catheters 5-7 days before the clamp and subjected to the clamp procedures under conscious and unrestrained conditions after fasted for 5-6 hours (15 hours for rats).
Animals are placed in metabolic chambers during fasting and experimental periods while VO2 and VCO2 are continuously measured. The protocol consists of a 90 min of tracer equilibration and blood sampling (t = 0 to 90 min). At t = 0, a bolus-continuous infusion of 1.0 μCi of tracer infusate ([1-14C]palmitate and [2-3H]glycerol ), followed by a 0.05 μCi/min infusion for 90 minutes. Blood samples (30~40 μl) are collected at the baseline and during the steady-state of the infusion at t = -10, 70, 80, and 90 min. Plasma samples are immediately separated and stored at -80°C for later analysis.
To determine the plasma [3H]-NEFA, [3H]- H2O and [3H]-TG radioactivity, lipid extraction and separation procedures are performed using a mixture of isopropanol-hexane-0.5M H2SO4 (40:10:1) followed by polarity separation steps under alkaline (0.5N KOH in methanol) and acid (acid hexane) conditions. Tissue samples are homogenized in water and [3H]-TG is extracted and counted for NEFA incorporation rates into tissue-specific storage during 120-140 min.