The Effect of Insulin on Xylose Uptake by the Intact Hemidiaphragm of the Hypophysectomized Rat

Hjalmarson (1) has reported a doubling of xylose incorporation into the intact hemidiaphragm of the hypophysectomized rat by incubation with 100 μU/ml of insulin. These data suggested that xylose incorporation into the intact hemidiaphragm of the hypophysectomized rat might be used to develop an improved bioassay for insulin. Such an assay would differ from standard insulin bioassays (2) in the use of hypophysectomized rats, an intact hemidiaphragm, and radioactive xylose. Materials and Methods. Conditions described by Hjalmarson (1) were duplicated, except as noted. Only female Sprague—Dawley rats which gained less than 10 g in the 14—19 days after hypophysectomy were used. Radioactive 14C-D-xylose (Amersham) was used with specific activity of 0.2 μCi/μmole at a final concentration of 1.0 mM. Porcine insulin (5 times recrystallized, Lilly) was dissolved in acetic acid pH 2.3 and diluted with buffer. The rats were decapitated and intact hemidiaphragms were dissected (3), preincubated (1) in Krebs-bicarbonate buffer (pH 7.3) with glucose (2.5 mg/ml), and equilibrated with 95% O2-5% CO2. Preincubation was for 15 min. Then one intact hemidiaphragm was incubated with insulin and one was used as control. The uptake of 14C-D-xylose was measured after 15 min, because the insulin effect was largest at this time. The dissection from the rib cage, washing, blotting, weighing, homogenization with 10% TCA, and centrifugation were as described by Hjalmarson (1, 4). Scintillation fluid was prepared by dissolving 60 g of naphthalene, 4 g of PPO (2,5-diphenyloxazole), 0.2 g of POPOP {1,4-bis [2-5-(phenyloxazolyl)]benzene} in p-dioxane to make 1 liter. All reagents were scintillation grade and counting was done in a Picker Nuclear Liquimat 220 liquid scintillation counter. One hundred microliter aliquots of supernatant were counted in 10 ml of scintillation fluid.