Degradation Of 125I-labeled Insulin Research Articles

Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells.

The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or [32P]phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of 125I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.

Insulin-stimulating protein from human plasma. Chemical characteristics and biological activity.

A protein that potentiates the action of insulin in vitro was purified from human plasma. When reduced with 2-mercaptoethanol and then carboxymethylated, it yielded a single subunit, indicating that it was composed of two identical subunits connected by a single disulfide bond. This modified subunit tended to inhibit rather than stimulate insulin activity. A distinctive feature of the amino acid composition of this protein (H-ISP) was the absence of histidine, arginine, and tryptophan. The molecular mass, subunit composition, the characteristic amino acid composition and the N-terminal amino acid residue of H-ISP are very similar to those of human plasma apolipoprotein A-II (apo A-II). The isoelectric point of H-ISP was estimated to be 4.91, which is identical with that of the major apo A-II isoform. H-ISP did not itself have insulin-like activity in increasing CO2 liberation from labeled glucose and 2-deoxyglucose uptake by isolated rat adipocytes, but it potentiated the action of insulin in these parameters. It had no appreciable affect on the binding or degradation of 125I-labeled insulin by adipocytes. Like H-ISP, apo A-II isolated from human plasma also had no insulin-like activity by itself, but stimulated the effect of insulin on CO2 production from labeled glucose in isolated rat adipocytes. From these results, it is concluded that H-ISP is identical with the major apo A-II isoform. Incubation of isolated adipocytes with H-ISP resulted in marked increase in the activity of pyruvate dehydrogenase in a dose-dependent manner in the absence of added insulin. H-ISP also stimulated pyruvate dehydrogenase activity in a subcellular system consisting of plasma membranes and mitochondria from rat adipocytes. The effect of H-ISP on pyruvate dehydrogenase activity could be produced by treatment of the isolated mitochondrial fraction alone.

Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands.

Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na125I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na125I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125I-hGH or 125I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.

Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes.

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

1. 1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [ 125I]insulin. 2. 2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent. 3. 3. [ 125I]insulin was displaced by unlabelled insulin. A 1-B 29 dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity. 4. 4. Brush-border membranes showed high insulin-degrading activity with an apparent K m of 2.2 μM. 5. 5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents. 6. 6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

Insulin binding in cultured Chinese hamster kidney epithelial cells the effects of glucose concentration in the medium and tunicamycin

An epithelial cell line established from a Chinese hamster kidney, CHK-AC E, was separated into two sublines, CHK-AC E-100 and CHK-AC E-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-AC E-100 and CHK-AC E-400 cells to 125I-labeled insulin showed similar pH and time dependency; 125I-labeled insulin binding as a function of insulin concentration differed in the two sublines, however. Degradation of 125I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK_AC E-400 cells than with CHK-AC E-100 cells. When CHK-AC E-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-AC E-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in K f / K e ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 μg/ml, showed no direct effect on the assay of 125I-labeled insulin binding to CHK-AC E-100 cells; exposure of CHK-AC E-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 μg/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in K f / K e ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.

Insulin degradation by human erythrocyte lysates.

In vitro hemolysates of isolated human erythrocytes degrade 125I-labeled insulin. Ten- to 100-fold dilutions of the hemolysate give a proportionately decreased degradation of 125I-labeled insulin at 37 degrees C, while dilutions of up to eightfold do not. Like the control, diluted "Buffer G" containing 5 mmol/L Tris and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer alone, more than 500-fold dilutions of the hemolysate or boiled hemolysate (in buffer) caused negligible (less than 1%) degradation of the labeled insulin. We conclude that accurate insulin-binding data during erythrocyte insulin radioreceptor assay under optimum conditions (Clin. Chem. 23: 1590-1595, 1977) depend on avoiding or minimizing hemolysis.

Degradation of 125I-labelled insulin by isolated rat hepatocytes [proceedings

Degradation of 125I-labelled insulin by isolated rat hepatocytes [proceedings

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.

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.

Binding and degradation of 125I-labelled insulin by isolated rat fat cells

1. 1. Isolated rat fat cells were incubated at 37°C with 125I-labelled insulin and separated from the incubation medium by centrifugation through dinonyl phthalate. At low 125I-labelled insulin concentrations (7 · 10 −11−7 · 10 −10 M) the uptake of 125I-labelled insulin by fat cells reache equilibrium after 45 min of incubation. The concentration dependence of equilibrium binding of 125I-labelled insulin showed that one part of the binding could be saturated, whereas another part was proportional to the concentration in the range investigated (7 · 10 −12−7 · 10 −6 M). The saturable part of the binding of 125I-labelled insulin could be explained by a reaction with a cellular receptor resulting in the formation of a receptor-insulin complex. The dissociation constant of the receptor-insulin complex was determined as 3 · 10 −9 M and the amount of receptors as 8 · 10 −10 moles/lcell, or approximately 5 · 10 4 per cell. The rate constants of dissociation and association were 7 · 10 −2 min and 2.5 · 10 7 min −1 · M −1, respectively. Neither vasopressin, glucagon nor des-B23-B30-octapeptide insulin, in concentrations of 7 · 10 −7 M, reduced the binding of 125I-labelled insulin, whereas proinsulin inhibited the binding with an inhibition constant of 1.3 · 10 −7 M. 2. 2. 125I-labelled insulin in the medium was degraded by fat cells. The Michaelis constant of the degrading enzyme system was 3.5 · 10 −74 M and the maximal velocity was 45 nmoles/min per cell. Proinsulin was degraded less rapidly than insulin and the degradation of 125I-labelled insulin was inhibited by proinsulin (7.3 · 10 −7 M), whereas glucagon (6 · 10 −7 M) had no effect. 3. 3. Binding of 125I-labelled insulin and lipogenesis from [2- 3h]glucose were measured on the same cells. Fat cells with a dissociation constant of binding of 3 · 10 10−9 M exhibited half-maximal stimulation of lipogenesis at a 125I-labelled insulin concentration of about 7 · 10 −11 M. 4. 4. The results show that the principle binding of insulin by fat cells occurs to a group of receptors with a K d of 3 · 10 −9 M and that the degradation of insulin and the receptor binding are processes independent of each other. It is suggested that the stimulatory effect of insulin on lipogenesis from glucose in fat cells is mediated by these receptors and that the half-maximal effect of insulin is obtained when about 2% of the receptors are occupied.