Insulin degradation. X. Identification of insulin degrading activity of rat liver plasma membrane as glutathione-insulin transhydrogenase

The liver plasma membrane preparation devised by Neville (Biochim. Biophys. Acta, $\text{154}$, 540 (1968) contains insulin-degrading activity. Examination by chromatography on Sephadex G-75 of the products formed from ¹²⁵I-insulin upon incubation with plasma membrane showed the same products (A chain and B chain rich-A chain aggregate) as previously found with purified GSH-insulin transhydrogenase (GIT). In Ouchterlony double-diffusion experiments with antibody to purified rat liver GIT, plasma membrane gave a single precipitation band of identity with purified rat liver GIT. Thus, the insulin-degrading activity present in the plasma membrane preparation is indeed GIT.     

Stabilization of insulin receptor subunit structure by glutathione-insulin transhydrogenase

A partially purified insulin receptor preparation from rat liver was incubated at 37 degrees C with and without the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase/isomerase, EC 1.8.4.2/5.3.4.1). Insulin-binding activity was then assessed by crosslinking receptor-125I-insulin complexes and subjecting them to electrophoresis on SDS-polyacrylamide gels in the absence and presence of reductant followed by autoradiography. Prior incubation of the receptor at 37 degrees C in the absence of the enzyme markedly decreased the subsequent binding of 125I-insulin to the holoreceptor (Mr 350 000) and to its subunits (Mr 180 000 and 130 000), while addition of the enzyme to the preincubation medium served to substantially prevent this decrease. The loss in binding at 37 degrees C was not restored by subsequent addition of the enzyme, nor was the loss prevented by any of the several known inhibitors of proteolysis. The apparent stabilization of receptor by transhydrogenase, as evidenced by the increase in binding above control levels, was proportional to both the enzyme concentration and the duration of incubation. These effects seem to be specific for transhydrogenase, since several other disulfide-containing proteins were found to be ineffective. These data suggest that the stabilization of the subunit structure of the insulin receptor at physiological temperatures may take place via a disulfide interchange reaction catalyzed by glutathione-insulin transhydrogenase.     

Stimulation by insulin of cyclic AMP phosphodiesterase. Role of glutathione-insulin transhydrogenase.

In agreement with other workers, exposure of isolated rat fat cells to insulin shows a dose dependent increase in cyclic AMP phosphodiesterase (PDE) activity. However, when fat cells are pre-exposed to either guinea pig antiserum against insulin, rabbit antiserum against glutathione-insulin transhydrogenase (GIT), or immunogamma globulin against GIT, each antibody preparation totally or almost totally abolished the insulin-dependent increase in PDE activity. In control experiments, appropriate normal (non-immune) sera, normal gamma globulin, or the GIT-antiserum or the GIT-immunogamma globulin which had been previously neutralized with purified rat liver GIT were found to be completely ineffective in abolishing the insulin-dependent PDE activity of fat cells. These results suggest that the GIT-catalyzed sulfhydryl-disulfide inter-change reaction with insulin might be part of the mechanism by which insulin regulates the intracellular cyclic AMP concentration.     

Kinetic analysis of the mechanism of insulin degradation by glutathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase).

Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.     

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 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.     

Insulin degradation in insulinoma: evidence for the occurrence of an inactive form of glutathione-insulin transhydrogenase and for the absence of insulin A and B chains degrading protease(s)

Islet cell tumors (insulinomas) have been found to contain insulin-degrading activity. Apparent K_m values for insulin obtained with tumor extracts were similar to those found for other tissues and for purified glutatione-insulin transhydrogenase (GIT). In Ouchterlony double diffusion experiments with antibody to purified human liver GIT, each tumor extract gave a single precipitation band of identity with purified human liver GIT. Examination by chromatography on Sephadex G-75 of the products formed from ¹²⁵I-insulin upon incubation with tumor extracts showed the same products (A chain, and B chain rich-A chain aggregate) as previously found with purified GIT; however, there was no further degradation (i.e., proteolysis) of A chain to low molecular weight components. These results indicate that the insulin-degrading activity present in the islet tumor is, in fact, GIT and that the protease(s) that further catabolizes the insulin A and B chains is apparently missing in the insulinoma. These data could be interpreted to indicate that the function of GIT in this tissue is to promote the biosynthesis of proinsulin and insulin rather than their degradation. Data are also presented which indicate that in insulinoma GIT is present in an inactive state as a divalent metal ion complex since it could be activated with EDTA and/or GSH.     

Demonstration of insulin degradation by thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) and proteinases of pancreatic islets

Homogenate preparations of pancreatic islets have been found to degrade insulin by cleavage of the interchain disulfide bonds, followed by proteolysis of the resulting A and B chains. A proteolytic system of the pancreatic islets splitting not only ^{1 2 5}I-labeled insulin A chain but also ^{1 2 5}I-labeled glucagon at pH 7.0, was shown to be activated by glutathione and inhibited by EDTA. The results suggest that pancreatic islets contain both the thiol-protein disulfide oxidoreductase (glutathione : protein-disulfide oxidoreductase, EC 1.8.4.2) and the A and B chain-degrading enzyme(s). The effects of EDTA argue against the implication of cathepsins in insulin breakdown under the experimental conditions employed.     

Demonstration of insulin degradation by thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) and proteinases of pancreatic islets.

Homogenate preparations of pancreatic islets have been found to degrade insulin by cleavage of the interchain disulfide bonds, followed by proteolysis of the resulting A and B chains. A proteolytic system of the pancreatic islets splitting not only 125I-labeled insulin A chain but also 125I-labeled glucagon at pH 7.0, was shown to be activated by glutathione and inhibited by EDTA. The results suggest that pancreatic islets contain both the thiol-protein disulfide oxidoreductase (glutathione : protein-disulfide oxidoreductase, EC 1.8.4.2) and the A and B chain-degrading enzyme(s). The effects of EDTA argue against the implication of cathepsins in insulin breakdown under the experimental conditions employed.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Studies on the subcellular localization and role of glutathione-insulin transhydrogenase in rat liver

₁₂₅I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subceltular processing of receptor-bound internalized insulin.     

Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase

Five monoclonal antibodies specific for glutathione-insulin transhydrogenase were characterized. None of the monoclonal antibodies cross-reacted with another insulin-degrading enzyme, neutral thiopeptidase. The isotype of four antibodies was IgG1 and of the fifth IgG2b. Affinity studies, competitive binding studies and immunoblot analysis of CNBr and trypsin cleavage products of glutathione-insulin transhydrogenase demonstrated that the four IgG1 antibodies were directed to an epitope of the enzyme which was distinct from the epitope recognized by the IgG2b antibody. Inhibition studies indicated that each monoclonal antibody, when added singly to glutathione-insulin transhydrogenase, was unable to inhibit the insulin-degrading activity of the enzyme. However, when monoclonal antibodies directed against separate epitopes of glutathione-insulin transhydrogenase were presented together (i.e., the IgG2b with any one of the four IgG1 antibodies), a loss in enzymatic activity was noted. Immunoblot analysis of rat organ extracts with the IgG1 antibodies demonstrated one immunoreactive protein band of Mr 56,000 in all tissues examined (liver, fat, pancreas and kidney) except the spleen, which demonstrated two immunoreactive protein bands of Mr 56,000 and 51,000. The same immunoblots, when probed with the IgG2b antibody, demonstrated the same immunoreactive protein banding pattern as above plus an additional immunoreactive protein band of Mr 67,000 in all tissues. Studies with spleen extracts from steptozotocin-induced diabetic rats demonstrated that there was a loss of the 51,000 immunoreactive band in diabetes. This 51,000 protein was restored upon insulin treatment of the diabetic rats and nullified upon concomitant administration of cycloheximide or actinomycin D with insulin. Immunoblots of human liver, adipose and skeletal muscle extracts indicated that each monoclonal antibody cross-reacted with the human form of the enzyme which had a molecular weight of Mr 63,000; a second minor immunoreactive band of 67,000 was detected with the IgG2b antibody. The physiological significance of additional molecular forms of the enzyme (i.e., 67,000 and 51,000) remains to be determined.     

Immunological identity between bovine preparations of thiol:protein-disulphide oxidoreductase, glutathione-insulin transhydrogenase and protein-disulphide isomerase

Five preparations of bovine thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase (EC 1.8.4.2) and one preparation of bovine liver protein-disulphide isomerase (EC 5.3.4.1) from four different laboratories showed immunological identity in double immunodiffusion and rocket-line immunoelectrophoresis. Consequently, thiol:protein-disulphide oxidoreductase/glutathione-insulin transhydrogenase and protein-disulphide isomerase, formerly classified as two separate enzymes, should be considered as alternative activities of the same enzyme.     

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.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase / oxidoreductase)

A human liver cDNA expression library in λ-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase / oxidoreductase (EC 5.3.4.1 / 1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3′-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.