Daniel E. Peavy

Receptor Binding Properties of Monoiodotyrosyl Insulin Isomers Purified by High Performance Liquid Chromatography

The four monoiodinated forms of pork insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The procedure was rapid and extremely reproducible, resulting in total separation of the B16, B26, and A14 monoiodinated insulins from one another and from unreacted insulin. The A19 derivative eluted closely to the unreacted insulin, requiring an additional step on ion exchange chromatography for complete purification of this derivative. The receptor binding properties of the derivatives were examined in isolated rat adipocytes and hepatocytes and in cultured human IM-9 lymphocytes. The relative binding affinities of the derivatives varied in the different cell types. At tracer concentrations, the B26 derivative had the highest apparent affinity in IM-9 lymphocytes and in adipocytes, but the A14 had the greatest affinity in hepatocytes. At equivalent concentration of each tracer, IM-9 lymphocytes bound the B26 isomer 144 ± 9% (P < 0.05) and the B16 isomer 118 ± 3% (P < 0.05) as well as the 125I (A14) insulin. Isolated rat adipocytes also bound the B26 to a greater extent at tracer concentrations (117 ± 6%; P < 0.01) compared with A14, but bound B16 less than A14 (91 ± 2%; P < 0.01). Isolated rat hepatocytes bound more of the A14 derivative than either of the B-chain labels. The B26 bound 72 ± 2% (P < 0.01) and the B16 90 ± 1% (P < 0.01) as well as the A14 derivative to isolated hepatocytes. In contrast to the results obtained at tracer concentrations of the isomers, higher concentrations of native insulin were required for 50% competitive displacement (ED50) of the B26 derivative as compared with the other two derivatives in all three cell types examined. These results indicate a higher average affinity for the B26 isomer in the three tissues, and suggest that results obtained from tracer binding alone may not provide an accurate reflection of receptor affinity for the monoiodoinsulin isomers. Competitive displacement of the 125I-labeled derivatives by 127I (nonradioactive) labeled homologues in the IM-9 lymphocytes also showed the B26 derivative to have the highest affinity. The present study demonstrates that monoiodoinsulin selectively labeled on each of the tyrosines can be prepared and separated on HPLC and used to examine insulin receptor binding in different cell types. Furthermore, the data demonstrate different apparent affinities of the selectively-labeled monoiodoinsulin isomers in different cell types, suggesting variability in insulin binding properties in different tissues.

HPLC Analysis of Insulin Degradation Products From Isolated Hepatocytes: Effects of Inhibitors Suggest Intracellular and Extracellular Pathways

Isolated rat hepatocytes were incubated with A14-[125I]monoiodotyrosyl insulin for 30 min, and labeled material was extracted from the cells and incubation media. The medium and the cell extract were chromatographed on a Sephadex G-50 column, and radioactivity eluting in the position of intact insulin was concentrated and analyzed on HPLC. The HPLC analysis of the cell extract showed two major products eluting from the column at 19 and 23 min, whereas medium extracts showed one prominent product eluting at 14 min. Inclusion of chloroquine in the incubation blocked the formation of cellular products at 19 and 23 min and caused the accumulation of a product eluting at 41 min while not affecting the media products. After sulfitolysis all cellular products contained an intact A-chain. Dansylcadaverine increased media products and altered the cellextracted product pattern such that it had a major peak at 14 min, similar to media. These results suggest that two pathways for insulin degradation exist within hepatocytes. The extracellular process forms products that are essentially unchanged by chloroquine and dansylcadaverine. The intracellular process is altered by chloroquine and apparently inhibited by dansylcadaverine.

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.

Influence of diet on the in vivo turnover of glucose-6-phosphate dehydrogenase in rat liver

Abstract In vivo turnover of rat liver glucose-6-phosphate dehydrogenase was studied. A specific antibody was used in comparing the rates of synthesis and degradation of the enzyme between rats fed a commercial chow diet and those fed a semi-purified high-carbohydrate diet. In livers of rats fed the semi-purified diet, the rate of synthesis of the enzyme was two-fold greater than in livers of rats fed the commercial diet. The rate of degradation of glucose-6-phosphate dehydrogenase was determined by following the loss with time of [guanido-14C]-arginine from previously labelled enzyme. The rate of degradation of the enzyme was decreased in livers of rats fed the semi-purified diet, being 0.044 h−1 (half-life, 16 h) as compared to 0.120 h−1 (half-life, 6 h) in rats fed the chow diet. It was concluded that the increases in enzyme activity observed with this dietary manipulation are solely the result of changes in the rates of synthesis and degradation of enzyme. No evidence was found to support the hypothesis that an activation-inactivation process is involved as a major regulatory mechanism of glucose-6-phosphate dehydrogenase activity in rat liver.

Localization of insulin degradation products to an intracellular site in isolated rat hepatocytes

In the present study, we have examined whether insulin degradation products are present on the surface of isolated rat hepatocytes and can be removed by an acid dissociation technique. Hepatocytes were incubated with [125I]insulin for 30 minutes, rapidly washed to remove unbound insulin, and then briefly exposed to acidic conditions (pH 5.0) to remove bound hormone from the cell surface. The radioactive material removed from the cell by acid dissociation and that remaining with the cells were separately analyzed by high performance liquid chromatography. The two primary degradation products of insulin present in control cell extracts were found only with the cell-associated material after acid dissociation. The insulin-sized radioactive material in the extract of acid-dissociable material consisted of only intact [125I]insulin. These results show that the two primary degradation products of insulin in rat hepatocytes are found only intracellularly and suggest that the degradation of the hormone begins after it is internalized.

In Vitro Activity of Biosynthetic Human Proinsulin: Receptor Binding and Biologic Potency of Proinsulin and Insulin in Isolated Rat Adipocytes

The receptor binding characteristics and biologic potency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15°C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 ± 0.8 × 10−7 M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 ± 0.3 × 10−9 M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37°C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 ± 0.4 × 10−8 M, whereas the same response was observed at an insulin concentration of 5.2 ± 1 × 10−11 M. Proinsulin also demonstrated an antilipolytic potency that was <1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones. It is concluded that biosynthetic human proinsulin has approximately 1% the receptor binding affinity and biologic potency of insulin in the isolated rat adipocyte system.

Immunological titration of rat liver glucose-6-phosphate dehydrogenase from animals fed high and low carbohydrate diets☆

Abstract Recent work (Hizi and Yagil [1974] Eur. J. Biochem. 45 : 211–221, and Kelly et. al. [1975] Fed. Proc. 34 : 881) suggests that the marked increase in rat liver glucose-6-phosphate dehydrogenase activity which is observed upon feeding an animal a high carbohydrate diet does not involve an increase in the total amount of enzyme present. In contrast, the data presented herein involving immunological titrations of rat liver glucose-6-phosphate dehydrogenase indicates that the increase in enzyme activity resulting from feeding a high carbohydrate diet does involve an increase in the total amount of enzyme present.

Insulin-stimulated conversion of D-[5-3H] glucose to 3HOH in the perifused isolated rat adipocyte

Characteristics of basal and insulin-stimulated glucose utilization by perifused adipocytes have been investigated by measuring the formation of 3HOH from D-(5-3H) glucose. At a glucose concentration of 0.55 mmol/L, basal glucose utilization ranged from 0.5 to 1.0 nmol/min/10(6) cells. Perifused adipocytes showed a maximal response to insulin of a threefold to fourfold increase in the conversion of (5-3H) glucose to 3HOH with a half-maximal response at an insulin concentration of 20 microU/mL. The response to insulin was blocked by phlorizin and cytochalasin B, competitive inhibitors of glucose transport, consistent with an effect of insulin on glucose transport. Insulin increased the Vmax for glucose metabolism but had no effect on the apparent affinity for glucose utilization. The characteristics of glucose utilization and the stimulation of glucose metabolism by insulin in the perifused adipocyte are therefore similar to characteristics previously observed with incubated adipocytes. Because insulin can readily be removed from the system, perifused adipocytes are especially suited for studying the termination of insulin action. The termination of insulin-stimulated glucose metabolism occurred at the same rate in the presence of tracer (1 nmol/L) (5-3H)-glucose alone as when 0.55 mmol/L glucose or 2 mmol/L pyruvate were added to the perifusion buffer. The halftime for this process in both cases was approximately 40 minutes. These data suggest that the presence of metabolizable substrate is not required for the termination of the insulin response, but the time course suggests that termination requires more than simply insulin-receptor dissociation.

Effects of Diet on the Activities of Enzymes Related to Lipogenesis in Rat Liver and Adipose Tissue

Summary The activities of glucoses-phosphate dehydrogenase, malic enzyme, and pyruvate kinase were significantly higher in the livers and adipose tissues from rats fed purified diets than in these tissues from rats fed commercial lab chow. The type of carbohydrate fed in the purified diet did not have a significant effect on the final levels of the three enzymes in epididymal adipose tissue, but did affect the levels of these enzymes in the liver: rats fed sucrose or invert sugar had higher activities than those fed glucose or starch. The activity of malic enzyme was higher in the livers of rats fed diets containing glucose than in those fed starch. No significant differences in enzyme activities were observed between rats fed diets containing sucrose or invert sugar.