Insulin was incubated with rat liver homogenate in the presence of glutathione. The products formed were examined by chromatography on a Sephadex G-75 column, with 50% acetic acid as eluent. The results show that insulin is degraded by rat liver homogenates in sequential order: first, a splitting of insulin into A and B chains by glutathione-insulin transhydrogenase, followed by proteolysis of the resulting polypeptides to small molecular weight components.Insulin is rapidly inactivated in vivo (1-3) and in vitro (4-6). Inactivation of insulin could take place by cleavage of insulin with glutathione(GSH)-insulin transhydrogenase (insulin transhydrogenase) (7-11), and/or by proteolysis with wellknown proteolytic enzymes (trypsin, chymotrypsin, exopeptidase) or with insulin-specific proteolytic enzymes. A and B chains of insulin are degraded by proteolytic enzymes more rapidly than is native insulin (12). It is conceivable that the inactivation of insulin takes place in a step-wise manner; the first step might be the cleavage of insulin by the transhydrogenase, followed by hydrolysis of the resulting A and B chains. This would suggest a key role for insulin transhydrogenase in the disposal (i.e., metabolism) of insulin. However, no direct experimental evidence supports these ideas.The experiments reported in this paper indicate that insulin is degraded in a step-wise manner. MATERIALS AND METHODSGSH-insulin transhydrogenase was prepared from rat liver in a highly purified state according to the method of Tomizawa and Halsey (13); the same procedure has been used for the isolation of purified transhydrogenases from beef liver (13), beef pancreas (10), human liver (9), and human kidney (11). Antiserum to the purified rat liver enzyme was produced in rabbits by a procedure used previously to produce antibodies to beef pancreatic and human liver transhydrogenases (14). In an Ouchterlony double-diffusion test with antibody, the rat enzyme showed a single precipitin band. The hr, rats were decapitated with a miniature guillotine (Harvard Apparatus Co.) and as much blood as possible was drained from the carcass. The livers were removed, rinsed with cold tap water, and placed in ice-cold beakers. A 1-g piece of liver was mixed with 4 ml of 0.25 M sucrose-5 mM EDTA (pH 7.5), and another 1-g piece was mixed with Krebs-Ringer bicarbonate buffer. The pieces were then homogenized for 40 sec with a Polytron PT-20/2 homogenizer at "position 10" of the rheostat, which was arbitrarily divided into 30 equal parts from the off position to fully on. During homogenization, the homogenization tube was kept cold. The homogenates were used without any further treatment; this procedure was chosen to minimize the role of diffusion processes (3), and at the same time retain the activity of all the liver proteins.Immunologic Procedures. These were done as described (14). In a series of tubes, different volumes of homogenate were mixed with 0.05 ml of antiserum to insulin transhydrogenase from rat liver. The final volume in each tub...

Interactions of several proteins with glutathione-insulin transhydrogenase (GIT) have been investigated by determining their ability to inhibit degradation of 125I-labeled insulin catalyzed by GIT. The inhibition by every insulin analog (des-Asn-des-Ala-pork insulin, desoctapeptide-pork insulin, des-Ala-pork insulin, pork insulin, proinsulin, and guinea pig insulin) was competitive vs. competitive vs. insulin indicating that they function as alternate substrates. The insulin analogs with the least hormonal activity showed the highest potency as inhigitors of insulin degradation. Whereas native ribonuclease and lysozyme showed little or no inhibition, their scrambled forms (i.e. reduced and randomly reoxidized) showed competitive inhibition with a potency greater than that of insulin. These results suggest that the conformation of the substrate or inhibitor is probably the major factor in determining the specificity for (or binding to) the enzyme. Studies withother peptide hormones showed competitive inhibition with vasopressin and oxytocin and noncompetitive inhibition with glycagon. The inhibition with growth hormone could be either competitive or noncompetitive. The inhibition by glucagon and growth hormone (physiologic antagonists of insulin) could serve as a control mechanism to modulate the activity of enzyme. The following showed very little or no inhibition; the native and scrambled form of pepsinogen, trypsin inhibitor of beef pancreas and of lima bean, C-peptide of pork proinsulin, and heptapeptide (B23-B29) of insulin.

The changes in the hepatic levels of glutathione-insulin transhydrogenase (GIT) in response to changes in the blood levels of insulin in rats under a variety of conditions have been determined by quantitative specific immunochemical titrations using antiserum to purified rat liver GIT. The GIT concentration was consistently lower in decreased insulin states brought about by starvation and by alloxan diabetes than in normally fed rats. Subsequent refeeding of starved rats with standard laboratory chow restored the loss in GIT content. Treatment of alloxandiabetic rats with insulin for two days increased concentration of GIT greatly above normal; administration of the A chain or B chain of insulin in the same manner was ineffective and did not augment or inhibit the effect of insulin. The insulin-mediated increase of GIT in diabetic rats was nullified by concomitant administration of either actinomycin D (an inhibitor of RNA synthesis) or cycloheximide (an inhibitor of protein synthesis). Thus, the data indicate that insulin induces synthesis of GIT protein via RNA synthesis. The biological significance of the induction of the GIT protein by insulin is interpreted as a feedback mechanism to regulate the insulin levels in the body. The results provide further evidence that GIT activity is the primary determinant of the rate of hepatic insulin metabolism. It must be assumed, however, that other factors are also involved in the regulatory process of insulin degradation, and some possibilities are suggested.

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