Insulin disposition in the isolated perfused rat heart. Effect of "bound" insulin.

Abstract

Hearts from fed and fasted rats were perfused with modified Krebs-Ringer bicarbonate buffer containing 3 per cent human serum albumin, 2 mg./ml. glucose and 100 or 200 μU./ml. crystalline insulin. The concentration of immunoassayable insulin in the medium decreased, during two hours of perfusion, to approximately 50 per cent of the initial concentration. Perfusion of hearts with insulin-I-131 (100 μU./ml.) resulted in: (1) a decrease in immunoprecipitable radioactivity corresponding in rate and extent to that of crystalline insulin; (2) a significantly smaller decrease in trichloracetic acid (TCA)-precipitable radioactivity; (3) a corresponding accumulation of a fraction of radioactivity which was TCA-precipitable but not immunoprecipitable (TCA-nonimmunoprecipitable) as well as an increase in TCA-soluble radioactivity. Immunoprecipitable-I-131 and not TCA-precipitable I-131 more accurately reflects the fate of intact insulin. Analysis of the two-hour perfusate by gel nitration chromatography (Sephadex G-100) indicated that radioactivity was associated with both the insulin-I-131 peak (M.W. 12,000) and with substances exhibiting a lower molecular weight. Thus, the disappearance of insulin during heart perfusion may be largely accounted for by degradation of insulin. Both immunoassayable insulin and immunoprecipitable insulin-I-131 were demonstrated to decrease in concentration in cell-free medium which had been previously perfused through the heart for two hours, but not in unperfused medium. The extent of disappearance was less than that during a two-hour heart perfusion. Thus, a portion of the degradation of insulin occurring during heart perfusion is probably due to the action of enzymes which diffuse into the medium from the heart. “Bound” insulin inhibited the disappearance of both crystalline insulin and immunoprecipitable I-131 radioactivity from the medium during heart perfusion. “Bound” insulin was shown to inhibit the rate of disappearance of immunoprecipitable radioactivity from preperfused medium. During perfusions with “Bound” insulin alone, the level of bioassayable activity in the medium remained constant throughout the perfusion, indicating a lack of disappearance of “Bound” insulin from the medium and vascular space of the heart. Studies of glucose uptake during heart perfusion demonstrated that “Bound” insulin has minimal or no effect on basal and insulin-stimulated glucose uptake. “Bound” insulin may inhibit the disappearance of insulin by interfering with the transport of insulin across the capillary wall and/or by inhibiting the degradation of insulin in the medium.