Experimental Design

Figure 1 provides an outline of the study design. On the morning of the study, at the onset of the light cycle, the duodenal and arterial catheters were removed from their subcutaneous pockets under local anesthesia, and a continuous 4-h (0–240 min) infusion of 20% glucose at 10 mg ⋅ kg⁻¹ ⋅ min⁻¹ was initiated via the duodenal catheter in nine dogs to mimic a morning meal (GLC group). Another nine animals received an intraduodenal infusion of normal saline at a rate identical (in mL ⋅ kg⁻¹ ⋅ min⁻¹) to the infusion received by the group receiving glucose (SAL group). Arterial glucose and insulin concentrations were monitored in the groups during the 4-h duodenal infusion. The duodenal infusions ended at 240 min. At that point, six dogs in each group were prepared for a subsequent clamp period. The flow probes and the remainder of the infusion and sampling catheters were exteriorized and peripheral venous access was established (18). A primed (38 µCi), continuous (0.38 μCi/min) infusion of [³H]glucose was initiated via a peripheral vein; after a 90-min tracer equilibration period (240–330 min), postinfusion fasting blood samples were collected from the sampling catheters from 330–360 min (“preclamp” period). Three dogs that had undergone duodenal glucose infusion and three that had received duodenal saline infusion did not receive [³H]glucose but were euthanized at 360 min to collect hepatic tissues; these are referred to as the GLC_no-clamp and SAL_no-clamp groups.

Study design. The dogs were randomly assigned to duodenal infusion of either glucose or saline. After 4 h of duodenal infusion, there was a 2-h period before the clamp, followed by a 4-h hyperinsulinemic-hyperglycemic clamp with portal glucose infusion. The dogs receiving no clamp were euthanized and tissue collected at 360 min. IV, intravenous.

From 360–600 min, all dogs in the GLC and SAL groups (n = 6 dogs/group) underwent a hyperinsulinemic-hyperglycemic clamp with portal vein glucose infusion to mimic the conditions during absorption of a later meal. During the clamp, somatostatin (0.8 µU ⋅ kg⁻¹ ⋅ min⁻¹) was infused via the peripheral vein to suppress endocrine pancreatic secretion, and regular insulin (four times basal values; 1.2 mU ⋅ kg⁻¹ ⋅ min⁻¹) and glucagon (basal; 0.57 ng ⋅ kg⁻¹ ⋅ min⁻¹) were replaced via intraportal infusion. Glucose (20%) was infused continuously at 4 mg ⋅ kg⁻¹ ⋅ min⁻¹ into the portal circulation. This provided a constant, readily quantifiable load of glucose to the liver. In conjunction with this, a primed, continuous infusion of 50% glucose was administered via a peripheral vein to create and maintain hyperglycemia; the infusion rate was adjusted as required, on the basis of 0.2-mL samples taken every 5 min, to clamp arterial blood glucose at 150 mg/dL. We previously demonstrated that this dual infusion method is effective in clamping glucose delivery to both the liver and the peripheral tissues at very constant values (see, e.g, ref. 19). Larger samples were taken every 15–30 min from the artery, portal vein, and hepatic vein catheters to allow measurement of hormones and substrates. At the end of the study, each animal was deeply anesthetized with sodium pentobarbital while all infusions continued, and tissues from three liver lobes were rapidly (within 3 min) freeze-clamped in situ and stored at −80°C for later analysis. The dogs were then euthanized.