Insulin Degradation by Hepatocytes in Primary Culture*

Abstract

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 Enzyme in Brain Microvessels

“…IDE is a 110-kDa zinc-and thiol-dependent peptidase present in the cytosol and peroxisomes and on the cell surface of a wide variety of cell types including neurons (25)(26)(27). Because of its K m for insulin in the nanomolar range, IDE is involved in the degradation of this hormone in vivo (28). Yet IDE is also capable of degrading many small peptides of unrelated sequence, several of which show amyloidogenic potential in vitro and in vivo such as glucagon, amylin, calcitonin, atrial natriuretic peptide, and A␤, including all the genetic variants of A␤ associated with dementia or stroke (for reviews, see Refs.…”

Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease.

“…Finally, several agents (i.e., bacitracin and suifhydryl modifying reagents), which inhibit the activity of this enzyme, inhibit insulin degradation in the intact cell (24)(25)(26). However, these agents are not specific for the IDE.…”

Possible role of cell surface insulin degrading enzyme in cultured human lymphocytes

“…Lysosomes may be implicated in the intracellular degradation of insulin. However, current studies have demonstrated that chloroquine, a lysosomal inhibitor, has little effect on 125I-insulin degradation in various tissues [4,15,20,21], suggesting that lysosomes may have only a small role in the process of insulin degradation. On the contrary, IDE accounts for most of the insulin-degrading activity of various cell extracts [9,12,15].…”

“…This loss of cell surface receptors may be linked with hormone internalization, because after insulin binds to its receptors, insulin-receptor complexes are intemalized by absorptive endocytosis [2,3]. Following this, insulin seems to be degraded in the intracellular sites [4][5][6]. Although the degradation of insulin has been extensively studied, the sites and enzymes of intracellular degradation have still not been determined.…”