Glutathione-insulin Transhydrogenase Research Articles

Glutathione-insulin transhydrogenase activity in pregnant mouse mammary gland: hormonal influences studied in tissue culture.

Glutathione-insulin trandhydrogenase (GIT) activity has been shown to be stimulated in culture of explants of pregnant mouse mammary gland by a mixture of insulin, cortisol, and prolactin. Since this hormone mixture stimulates lactogenesis in vitro it is possible that the increase in GIT activity is functionally related to one of the processes of milk secretion or ejection. Oxytocin is degraded by GIT and the interaction of this hormone with its mammary gland receptors may be influenced by the change in enzyme activity. The increase in GIT activity caused by insulin, cortisol, and prolactin in vitro can be prevented by the addition of progesterone or oxytocin to the culture medium.

Serum Insulin Concentration, Insulin Release and Degradation, Glucose Tolerance and in Vivo Insulin Sensitivity in Cholesterol-Fed Rats

A series of experiments was conducted to elucidate the mechanism by which cholesterol feeding decreases serum insulin levels in rats and to determine the effect of this reduced insulin level on glucose metabolism. Rats were fed a casein-sucrose-soybean oil basal diet or this basal diet supplemented with 1% cholesterol (dissolved in hot oil) for periods longer than 30 days. Cholesterol feeding resulted in a decrease in serum insulin concentrations, although the decrease was not always significant. Cholesterol feeding did not affect fasting blood glucose levels, glucose tolerance, glucose-induced insulin release, pancreatic insulin content, in vivo insulin sensitivity, or in vitro glucose utilization in diaphragm and adipose tissue, but it significantly elevated the activity of liver glutathione-insulin transhydrogenase. On a per liver basis, the activity of this enzyme was approximately doubled. Results of this study suggest that cholesterol feeding has no significant effect on glucose utilization, but it can lead to a decrease in serum insulin concentration, probably by increasing the rate of insulin degradation in the liver.

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.

Insulin degradation XVIII. On the regulation of glutathione-insulin transhydrogenase in the hyperglycemic obese ( ob/ob) mouse

The occureence of insulin-degrading activity in the liver of the obese hyperglycemic mouse ( ob/ob) and its litter mate has been studied. The trichloroacetic acid-soluble product formed from insulin upon incubation with liver homogenate was identified as the A chain of insulin. In Ouchterlony double-diffusion experiments with antibody to purified rat liver glutathione-insulin transhydrogenase, mouse liver homogenate and the microsomal fraction each gave a single precipitation band of identity with the purified rat liver enzyme. These results indicate that the insulin-degrading activity preseny in the mouse liver is, in fact, glutathione-insulin transhydrogenase. Subcellular distribution studies of glutathione-insulin transhydrogenase and marker enzymes indicate that the transhydrogenase is located primarily in the microsomal fraction of mouse liver homogenate. The ob/ob mouse, which is a genetic mutant characterized by obesity, hyper-insulinism and resistance to the hypoglycemic action of insulin, contains hepatic glutathione-insulin transhydrogenase activity (per mg microsomal protein) markedly higher (40–60%) than its lean litter mates. However, a major portion of the increased hepatic enzyme in the ob/ob mouse occurs in a latent state; the increased amount of enzyme either is unavailable or is nonfunctional, although the ob/ob mouse still contains more of the functional form than the lean mouse. Thus, the results are consistent with the suggestion that the hepatic glutathione-insulin transhydrogenase is probably under a feedback control by circulating insulin.

Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.

Environmental Effects on Glutathione-Insulin Transhydrogenase in Rat Liver

The dietary and hormonal regulation of the level of glutathione-insulin tranhydrogenase in rat liver was investigated in these studies. In order to make valid comparisons, the assay of glutathione-insulin transhydrogenase was performed at near zero-order kinetics wherein enzyme rate was proportional to enzyme amount. Changing the protein content of the diet or administration of glucagon or cortisone did not significantly affect the specific activity of glutathione-insulin transhydrogenase in microsomes from rat liver. However, the Vmax and Km of this enzyme in the livers of adrenalectomized rats were increased three-and fourfold over these values in microsomes from normal liver. Administration of cortisone resulted in a return to the normal kinetic constants of microsomal GIT within 4 hr.

The effect of hypophysectomy on the glutathione-insulin transhydrogenase activity of rat liver.

The effect of hypophysectomy on the glutathione-insulin transhydrogenase activity of rat liver.

Interaction of glutathione-insulin transhydrogenase (disulfide interchange enzyme) with phospholipids

The effect of the addition of several phospholipids (lysophosphatidylcholine, α-lecithin, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, sphingomyelin, and disphosphatidylglycerol and phosphatidic acid) and related compounds (glycerophosphocholine, α- and β-glycerophosphate, choline, serine, glycerol, dipalmitoylglycerol, and stearic acid) on the ability of purified (from beef pancreas) and microsomal (rat liver) glutathioneinsulin transhydrogenase (Glutathione:protein-disulphide oxidoreductase, EC 1.8.4.2) to degrade insulin has been examined. With purified enzyme, except for phosphatidic acid and phosphatidylserine, all other phospholipids tested caused a slight activation at low concentration with phosphatidylethanolamine causing the highest activation. Lysophosphatidylcholine and phosphatidic acid are the only agents which cause inhibition of activity. The reaction rate as a function of concentration of inhibitor is hyperbolic for phosphatidic acid ( [ I] 0.5 = 25 μ M ) and biphasic for lysophosphatidylcholine ( [ I] 0.5 = 270 μ M ). Kinetic studies show that the two phospholipids are noncompetitive versus both substrates (insulin and GSH). Further, the structures of the phospholipids are quite different from the substrates and products of the reaction catalyzed by the enzyme. These data, together with the data obtained with microsomes (see below), support the possibility that phospholipids, in particular lysolecithin and phosphatidic acid, might function by an interaction at an allosteric site or sites to bring about a conformational change in the enzyme. With a microsomal fraction, four phospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, and phosphatidic acid) caused an increase in GSH-insulin transhydrogenase activity. At low concentration the addition of each of these phospholipids led to a 2.5-fold increase in GSH-insulin transhydrogenase activity. At higher concentration, lysophosphatidylcholine almost totally inhibited the microsomal GSH-insulin transhydrogenase activity, as it did with purified enzyme, while phosphatidic acid showed only a slight inhibition, in contrast to its effect on purified enzyme. With the microsomal fraction in which GSH-insulin transhydrogenase activity had been previously unmasked by Triton X-100 treatment, the addition of small amounts of lysophosphatidylcholine and phosphatidic acid produced, as expected, only slight increase in the transhydrogenase activity for both phospholipids; again, only lysophosphatidylcholine but not phosphatidic acid caused inhibition when higher levels were used. It is concluded that the four phospholipids and Triton X-100 increase the GSH-insulin transhydrogenase activity in the microsomes by unmasking the catalytic site without fully unmasking the allosteric site, the point of reaction with the phosphatidic acid.

Insulin and glucagon degradation by the kidney II. Characterization of the mechanisms at neutral pH

Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 × g pellet and 100 000 × g supernatant fractions. Further characterization of the degrading activities of the 100 000 × g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 × g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 × g pellet and the 100 000 × g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradation of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.

Insulin degradation by lysosomal extracts from rat liver; model for a role of lysosomes in hormone degradation

The presence of both glutathione-insulin transhydrogenase and “insulin specific protease” in highly purified lysosomal extracts from rat liver is presented. Although both of these enzyme activities were known to exist in the cytosol, it was difficult to understand their participation in insulin degradation, for it is known that insulin binds to plasma membrane receptors. Thus, the presence of both enzymes in lysosomes is of much interest for it suggests an explanation and/or model for the degradation via pinocytosis of insulin and possibly of other hormones which bind to receptors in the plasma membrane.

Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity.

The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the Böyum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies.

Insulin degradation. XVI. Evidence for the sequential degradative pathway in isolated liver cells.

Isolated liver cells contain insulin-degrading activity. Examination by chromatography on Sephadex G-75 of the products formed from 125I-insulin (1 nM or 1 muM) upon incubation with suspensions of hepatocytes for various time periods showed that there is at first a transient accumulation of the intermediate product, A chain, which is further hydrolyzed by protease(s) to low-molecular-weight components. These results indicate that the sequential degradative pathway is operative, both at low and high concentrations of insulin, in isolated liver cells, i.e., the insulin is first split at the disulfide bonds by glutathione-insulin transhydrogenase (GIT) into A and B chains, followed by proteolysis of the resultant polypeptides, and that this system might be used for well-defined studies of factors controlling insulin metabolism. The chelating agent ethylenediamine tetraacetic acid (EDTA) accelerated the accumulation of A chain (i.e., enhanced the activity of GIT), which is in keeping with its effect with purified GIT. In contrast to previous studies with tissue homogenates in which EDTA caused a marked inhibition of the proteolytic stage, EDTA had little or no effect with intact liver cells. Since EDTA does not appreciably penetrate the cell membrane, these data suggest that GIT activity either occurs on the cell surface or is readily available at the cell surface, whereas the proteolytic activity either occurs inside the cell or is inaccessible at the cell surface.

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 5I-labeled insulin A chain but also 1 2 5I-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.

Insulin degradation: XV. Use of different assay methods for the study of mechanism of action of glutathione-insulin transhydrogenase

Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 1 2 5I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Lineweaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent K m values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.

Interaction of insulin analogs, glucagon, growth hormone, vasopressin, oxytocin, and scrambled forms of ribonuclease and lysozyme with glytathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase): dependence upon conformation.

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.

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.

The effect of hydrocortisone phosphate, methylprednisolone and phenytoin on pancreatic insulin release and hepatic glutathione-insulin transhydrogenase activity in the rat

Following i.v. injection of glucose to rats, blood was collected from the carotid artery and the portal vein, and insulin was determined by radioimmunoassay. Pancreatic insulin release and hepatic insulin extraction were increased following the administration of glucocorticoid drugs and reduced following phenytoin. Hepatic glutathione-insulin transhydrogenase activity (GITA) was measured in each animal and found to be increased after glucocorticoid therapy but unaffected by phenytoin. In alloxan-diabetic rats, GITA was significantly increased following treatment with both methylprednisolone and phenytoin compared with control alloxan-diabetic rats. This is suggestive evidence that both these drugs can initiate an increase in GITA. It is concluded that the markedly raised GITA in steroid-treated non-diabetic rats is the combined result of a drug-induced effect plus the major inducing effect of an increased hepatic uptake of insulin degradation, whereas the main effect of phenytoin is a reduction of pancreatic insulin release.

Insulin degradation: XII. The amino acid composition, amino-terminal, and carbohydrate content of beef pancreatic glutathione-insulin transhydrogenase

The insulin-degrading enzyme, glutathione-insulin transhydrogenase, isolated from beef pancreas is a glycoprotein and has three amino-terminal residues (lysine, leucine and valine) suggesting that the enzyme might be composed of three polypeptide chains or subunits. The enzyme contained all common amino acids. The amino acid composition of this enzyme is quite similar (although not identical) to those reported for this enzyme isolated from rat liver (Ansorge, S., Bohley, P., Kirschke, H., Langner, J., Marquardt, I., Wiederanders, B. and Hanson, H. (1973) FEBS Lett. 37, 238–240) and also for the ribonuclease-reactivating enzyme of beef liver (De Lorenzo, F., Goldberger, R. F., Steers, E. Jr., Givol, D. and Anfinsen, C. B. (1966) J. Biol. Chem. 241 1562–1567).

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.

Insulin degradation. IX. On the presence of glutathione-insulin transhydrogenase in human leukocytes.

The occurrence of insulin-degrading activity in the components of human blood has been studied. Leukocytes have been found to contain such activity. On the basis of activation by reduced glutathione, complete inhibition by N-ethyl-maleimide, kinetic studies, and reaction with antibody to purified human liver glutathione-insulin transhydrogenase (GIT), it is concluded that the insulin-degrading activity present in leukocytes is, in fact, GIT. The data indicate that erythrocytes contain a very small amount of insulin-degrading activity; this activity is probably associated with the cell membrane. Preliminary studies in normal human volunteers indicate that there are apparent differences between individuals in the leukocyte GIT content. No evidence was found for any consistent changes in leukocyte GIT content during the course of a glucose tolerance test.