Insulin Binding Characteristics Research Articles

The binding and degradation of insulin by rat liver membranes: demonstration of three distinct insulin-binding components in detergent-solubilized material from liver plasma membrane.

The components responsible for insulin-binding and insulin degradation were solubilized from purified liver plasma membranes by treatment with the nonionic detergent Triton X-100. The characteristics of specific insulin binding and insulin degradation were similar to those found with the intact membranes. Polyacrylamide gel electrophoresis of the detergent extract separated the insulin-binding activity into three peaks, designated peaks I, II, and D. The majority of the insulindegrading activity was located in peak D; peak I failed to degrade insulin, while only a small amount of degradation was associated with peak II. Scatchard analysis of the insulin-binding data for peak I showed a curvilinear plot, with the initial high affinity portion of the curve having a Kd of 2 × 10−10 m. The Scatchard plot for peak II was linear, with a Kd of 8 × 10−9 m. Both peaks bound 125I-labeled insulin with high specificity, as determined by competition studies using desoctapeptide insulin, proinsulin, and glucagon. Insul...

Characteristics of insulin binding to human fetal and rat myocardial membranes

(P = 0.003). The mean number of 1,280 APC/spleen for T-treated was 45% greater than that of the U. By contrast, at 500 pg/ml and above, APC were significantly reduced; at 500 pg/ml 27 of 36 suspensions had fewer APC than U (P = 0.002). T-treated suspensions averaged 42% fewer. Comparable studies show that at C of estradiol (E0 of 500 to 5,000 pg/ml and of progesterone (P) of 500 pg to 50 ng/ml, increases in APC occur, but at C of 50 ng/ml and 500 ng/ml of these hormones respectively, APC are reduced. Thus, T, E~, and P enhance at lower C and suppress numbers of APC at higher C. On a molar basis, T is the most potent hormone, with its effects at the lowest C. Findings suggest that serum levels o f T in male infants may suppress, whereas those in female infants may enhance, the proliferation and/or differentiation of antibacterial APC.

Effects of insulin on insulin-binding components extracted from rat fat cell membranes.

WE have previously shown the presence of two distinct insulin-binding species (designated species I and II) in a Triton X-100 extract of fat cell membranes1. They were separable electrophoretically and displayed different insulin-binding characteristics. Scatchard analysis of the insulin-binding data for species I indicated a complex, non-classical association, illustrated by a curvilinear plot. The characteristics of this curve are quite similar to those of the binding curves obtained with unfractionated detergent extract or with intact plasma membranes1. In contrast to these findings, insulin-binding to species II was characterised by a relatively low affinity and a linear Scatchard plot. Although these findings indicate different insulin-binding and electrophoretic properties for the two species, the possibility remains that they are related structurally. For example, the Kd values for the low-affinity portion of the peak I Scatchard plot and for insulin binding to peak II are similar. Furthermore, following the incubation of isolated peak I with 125I-labelled insulin and subsequent electrophoresis, a small amount of radioactivity migrated in the position of peak II (ref. 1). We report here that interconversion occurs between the two binding species and that the conversion is mediated by insulin.

Hormone receptors 7. Characteristics of insulin receptors in a new line of cloned neonatal rat hepatocytes

1. 1. A new line of cloned, differentiated rat hepatocytes (RL-PR-C) was evaluated for its usefulness as an in vitro system for studying the regulation of the insulin receptor. 2. 2. Insulin rapidly reversibly and specifically bound to RL-PR-C hepatocytes. Binding of tracer 125I-labeled insulin, which was competitively inhibited by native insulin as well as by proinsulin and analogs of insulin and proinsulin in proportion to their biological activity, was not influenced by glucagon, corticotropin, or human growth hormone. Anti-insulin receptor serum from a patient with Acanthosis Nigricans Type B competed with 125I-labeled insulin for binding to cell surface sites. 3. 3. Trypsinization destroyed insulin binding sites, but these were restored by incubation under growth conditions; a 75% restoration of binding sites was achieved by one cell population doubling. 4. 4. RL-PR-C hepatocytes responded to insulin binding by an increase in glycogen synthesis from glucose. The insulin effect was maximal at 85 nM, but was detectable at lower, more physiological, concentrations. 5. 5. Chronic exposure (for at least 3 h) of hepatocytes to insulin (10 −10− 10 −8 M) reduced by up to 60% the number of binding sites for insulin (down-regulation). Down-regulation was prevented by cycloheximide at concentrations (10 μM) sufficient to inhibit markedly protein synthesis from tracer isoleucine. Recovery from down-regulation induced by native insulin at 10 −7 M or lower concentrations was complete by 18 h under growth conditions. 6. 6. Although RL-PR-C hepatocytes spontaneously transform after about 90 population doublings, no significant differences between normal and transformed cells were observed in insulin binding characteristics and in interaction or cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. 7. RL-PR-C rat hepatocytes appear, for these reasons, to be a useful model system for studying the regulation of the insulin receptor.

Characteristics of human erythrocyte insulin receptors.

Highly specific insulin receptors have been identified on human erythrocytes. A modification of the monocyte insulin radioreceptor technique permitted distinct separation of human erythrocytes with their bound insulin from the free insulin. When incubated with 80 pg. per milliliter of 125I-insulin (pH 8.0, 3.5 hours, 15 degrees C.), erythrocytes from 17 normal volunteers specifically bound 10 per cent (+/- 1.450 S.D.) of the total 125I-insulin. Less than 15 per cent of the total 125I-insulin bound was nonspecific. Binding of 125I-insulin to human erythrocytes was dependent on pH and temperature. Less than 5 per cent of the insulin available to the plasma membrane was degraded. Both calcium and magnesium enhanced 125I-insulin binding by 100 per cent but had no synergistic effect when mixed in a 1:1 molar ratio. Scatchard analysis of the binding data resulted in a curvilinear plot with characteristics typical of negative cooperative interactions between receptor sites and with an unoccupied site affinity constant of 0.1 X 10(8) M-1. Human erythrocytes have 2,000 insulin binding sites per erythrocyte with 14 sites per square micrometer of surface area. The readily available human erythrocyte, thus, has both specific insulin binding sites and binding characteristics similar to other human cell types. These studies have provided the basis for further clinical investigation of polypeptide hormone receptors on human erythrocytes.

Insulin binding to solubilized material from fat cell membranes: Evidence for two binding species

The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the soluble preparation was shown to have insulin-binding characteristics similar to those of intact fat cells. Further studies of this preparation by polyacrylamide gel electrophoresis in the presence of (125)I-labeled insulin demonstrated two distinct insulin binding activities, designated species I and II. The two species were separated by electrophoresis in the absence of iodo-labeled hormone and eluted from the gel. Scatchard analysis of the insulin binding data for species I showed a curvilinear plot with the initial portion having a K(d) of 1.3 x 10(-10) M. The Scatchard plot for species II was linear with a K(d) of 6.0 x 10(-9) M. Desoctapeptide insulin and glucagon failed to compete for the insulin-binding sites in both species whereas desalanine insulin was an effective competitor. High concentrations of proinsulin competed with the iodo-labeled hormone for binding to species I but not to species II. In the presence of a low concentration of (125)I-labeled insulin (0.3 nM) some species I activity appeared to be converted to species II activity; there was no evidence of interconversion between the two species in the absence of insulin. Neither species degraded insulin as measured by trichloroacetic acid precipitation or rebinding to intact fat cells. These findings indicate the existence in the adipocyte plasma membrane of two insulin-binding species that have distinct physicochemical properties.

Insulin receptors in murine mammary cells: Comparison in pregnant and nonpregnant animals

1. 1. 125I-labelled insulin binds in vitro to isolated mammary cells prepared by treating freshly excised murine mammary gland with bacterial collagenase. The binding is specific for insulin, and it is saturable with respect to insulin in the range of physiologic concentrations of the hormone. Small amounts (50 pg/ml) of native insulin effectively compete with labeled insulin for binding. The specific binding of insulin to mammary cells is destroyed by digesting the cells with trypsin. 2. 2. Physiologic concentrations (about 10 −10 M of insulin stimulate the incorporation of [ 3H] uridine into trichloroacetic acid-precipitable material by isolated mammary cells from pregnant mice, but not from adult virgin mice. Although a metabolic response to insulin is observed only in cells from pregnant animals, the characteristics of 125I-labelled insulin binding to cells from pregnant and virgin animals are very similar. Lactating cells bind 3–4 times more insulin than do cells from virgin or pregnant animals. 3. 3. Although proliferation of mammary cells during pregnancy may be stimulated or regulated by insulin following prolactin priming of these cells, the normal quiescence of the virgin tissue probably cannot be explained by the absence of insulin receptors. The absence of metabolic responses to insulin in the virgin tissue is probably explained by the improper function or incomplete nature of the hormone-receptor complex in the membrane.

Heterogeneity of insulin receptors on fat cell membranes

A highly purified and well characterized fat cell membrane preparation has been shown to retain the insulin binding characteristics of intact fat cells. Two major binding sites were identified, one a high affinity, low capacity site (K D = 5 × 10 −10M) and the other a lower affinity, high capacity site (K D = 3 × 10 −9M). Insulin binding to the membranes was prevented by insulin analogues (desoctapeptide and desalanine insulin) and proinsulin in direct relationship to their biological activities.