Insulin Interactions with Liver Plasma Membranes

Insulin interactions with purified plasma membranes of rat liver were studied with respect to insulin degradation and specific binding to receptors. W-insulin was rapidly degraded upon exposure to liver membranes. After only 5 min of incubation at 30” of 1251-insulin (0.3 nlvr) and liver membranes (1 to 2 mg of protein per ml), 40 to 60% of the labeled hormone was degraded as measured by its ability to specifically bind to a second aliquot of membranes. After 90 min of exposure, less than 10% of the lz51-insulin was intact when measured by its ability to bind specifically to membranes. Binding by anti-insulin antibody, precipitation by trichloroacetic acid, and adsorption by talc were less sensitive methods of measuring degradation. Degradation of 1251-insulin was dramatically reduced at 1”. No significant deiodination was associated with the degradation process. Gel filtration patterns suggested that lzsIinsulin degradation products are composed mainly of small peptide fragments that loosely adsorb to the gel and are eluted after the salt peak. The independence of binding to receptors and degradation is strongly suggested by the following findings. (a) ‘=Idesalanine-desaparagine insulin, which has an affinity for receptors that is only 2% that of insulin, is degraded to the same extent as 1251-insulin. (b) There is no relationship between the bioactivity of an insulin analogue and its ability to prevent the degradation of 1251-insulin. (c) The apparent K, for insulin degradation is 1.7 X 1O-7 M, which is 40-fold more than that concentration of insulin that produces halfmaximal inhibition of specific binding of 1251-insulin to receptors in the liver membrane. (d) Insulin that is recovered from membranes upon dissociation of the hormone-receptor complex is undegraded. Proinsulin is very slowly degraded by the liver membranes, but may act as a competitive inhibitor of insulin degradation. This is similar to the findings by others of the insulin-specific protease of muscle and liver. These studies provide evidence that interaction of insulin with liver plasma membranes is a complex phenomenon