Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats.

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in Vmax (i.e., maximal enzyme activity) without a change in Km (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.

Nonlatent and latent hepatic glutathione-insulin transhydrogenase activity during perinatal development and liver regeneration in rats and in rat placenta.

We have studied glutathione-insulin transhydrogenase (GIT) activity in differentiating rat liver during parturition and neonatal growth and during compensatory liver growth. Parturition is characterized by a rapid but transient increase in total (i.e., nonlatent plus latent) hepatic GIT activity resulting from changes in the quantity (Vm) of the enzyme while neonatal growth is characterized by an increase in the nonlatent (active) form which persists until just prior to weaning. During liver regeneration following partial hepatectomy, GIT activity/mg protein is lowest after 12 h of regeneration and then progressively increases exceeding the control levels after 72 h of regeneration. Placenta from near-term rats contain a significant concentration of GIT which is immunologically similar to hepatic GIT.

Topology of glutathione-insulin transhydrogenase in rat liver microsomes.

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent ('inactive') state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A2 and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A2 resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A2 is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of the endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.