On the day of the tracer experiments animals were placed in a clean cage, without food but with continued access to water, at 07:00 h, and the final substance/vehicle gavage was given. Animals were weighed and anesthetized at ~09:00 h (thiobutabarbital sodium salt, Inactin, RBI, Natick, MA; lean 120, obese 180 mg·kg−1, i.p.). Rats were tracheotomized with PE 240 tubing and breathed spontaneously. One catheter (PE 50 tubing) was placed in a carotid artery for blood sampling and recording of arterial blood pressure and heart rate. Up to five catheters (PE 10) were placed together in one jugular vein for infusions of tracers, insulin, and glucose and for administering top-up doses of anesthetic, if needed. The catheters were filled with Na citrate solution (20.6 mM) in normal saline to prevent clotting. The arterial catheter patency was maintained by continuous infusion of Na citrate (20.6 mM in saline, 5 μL/min) from shortly after carotid catheterization until the conclusion of the experiment. Body temperature was monitored using a rectal thermocouple and maintained at 37.5°C by means of servo controlled external heating. A stabilization period of ~150 min elapsed between completion of the surgical preparation and commencement of metabolic studies at ~12:00 h.
