The mechanism by which the liver degrades insulin has not yet been completely clarified. In intact, non-"leaky" cells the primary process seems to be mediated by initial receptor binding. We now demonstrate that isolated rat hepatocytes in primary culture are suitable for examining insulin degradation. Hepatocytes did not leak degrading activity into the medium, and thus, the degradation seen was essentially exclusively cell mediated. [125I]Iodoinsulin degradation by these cells was dependent on time and cell concentration. There was a short lag time before degradation products could be detected in the medium. After incubation with the hepatocytes, three peaks of 125I-labeled material could be separated by chromatography on Sephadex G-50. The same three peaks were seen with 125I-labeled material extracted from the cells. When [3H]insulin, labeled exclusively at the B-1 phenylalanine residue, was incubated with the cells, additional peaks of labeled material were recovered from the column. These additional peaks were intermediate in size between insulin and iodotyrosine, suggesting the production of products smaller than insulin but larger than individual amino acids. In order to begin to characterize the subcellular mechanisms for insulin metabolism, the effect of various potential inhibitors on insulin degradation were examined. The most effective inhibitors were N-ethylmaleimide, bacitracin, and Kunitz pancreatic trypsin inhibitor. Chloroquine decreased degradation only 10%, and NH4Cl had no detectable effect. The effect of the inhibitors on the purified insulin-degrading enzyme, insulin protease, was also examined. The purified enzyme responded essentially identically as the intact cells to the various inhibitors. From all these data it would seem that lysosomal degradation of insulin in the hepatocyte may be a relatively minor pathway and the neutral protease may play a major role.

Insulin-degrading activity was measured in the 100,000g supernatant fraction of muscle, liver, and kidney from rats of varying ages. Young animals (four weeks old) had the highest activity in all three tissues. By seven weeks of age the activity in both muscle and liver had decreased significantly as compared with four-week-old animals. A slight but nonsignificant decrease occurred in kidney. In animals over one year of age the insulin-degrading activity in all three tissues was significantly less than the activities at either four or seven weeks. In contrast the effect of age on degradation of albumin and parathormone was much less marked.

Six quadriplegic subjects and 6 control subjects received high-dose arginine vasopressin (AVP) infusions at rates of 500, 1,000, 2,000, and 4,000 microU.kg-1.min-1 in consecutive 10-min intervals. Six additional quadriplegic subjects received low-dose AVP infusions at rates of 50, 100, 200, 400, and 800 microU.kg-1.min-1. All subjects were studied once with and once without administration of a selective V1-receptor antagonist. During high-dose AVP infusions without V1-receptor blockade, mean arterial pressure (MAP) increased from 80 +/- 4 to 87 +/- 5 mmHg (P < 0.05) in quadriplegic subjects but was unchanged in control subjects. In the presence of V1-receptor blockade, MAP decreased from 75 +/- 4 to 58 +/- 4 mmHg (P < 0.001), and heart rate (HR) increased from 61 +/- 5 to 80 +/- 5 beats/min (P < 0.001) in quadriplegic subjects. In the studies on control subjects, MAP decreased only from 75 +/- 3 to 72 +/- 5 mmHg (P < 0.05), whereas HR increased from 64 +/- 4 to 87 +/- 4 beats/min (P < 0.001). Plasma renin activity (PRA) increased in both quadriplegic and control subjects. The effects of low-dose AVP infusions on MAP, HR, and PRA in quadriplegic subjects were similar to those observed during high-dose infusions. Thus, in the absence of baroreceptor-mediated sympathetic nervous system responses, a vasodilatory effect of AVP that is capable of producing marked reductions in MAP can be demonstrated in the presence of V1-receptor blockade.