The effect of insulin on glucose transport in rabbit erythrocytes and reticulocytes

Insulin binding and 3-0-Methylglucose transport have been studied in erythrocyte- and reticulocyte-enriched fractions of blood cells in order to determine if the increased number of insulin binding sites in reticulocytes is associated with a glucose transport response to insulin. In these experiments rabbit reticulocytes demonstrate an eightfold increase in total insulin receptors when compared to erythrocytes. Glucose transport activity in the erythrocyte has a K_m of 3.2 mM. Reticulocytes demonstrate a saturable glucose transport activity of lower affinity, K_m 18.9 mM. Neither the erythrocyte, nor the reticulocyte glucose transport activity, was capable of an increased response to insulin. The low affinity glucose transport activity in reticulocytes could allow a fourfold increase in facilitated glucose transport at supraphysiological glucose concentrations that might occur in poorly controlled diabetes mellitus.     

Absence of covalent binding by insulin to erythrocyte and reticulocyte insulin receptors

We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.     

Evidence for a pool of non-recycling transferrin receptors in peripheral sheep reticulocytes

Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

Dissociation of beta-adrenergic receptors from hormone responsiveness during maturation of the rat reticulocyte.

Intact rat erythrocytes and reticulocytes have been studied in relation to their concentration of beta-adrenergic receptors and their responsiveness to beta-adrenergic catecholamines. Characteristics of the beta-receptor, as determined by binding of 125I-labelled hydroxybenzylpindolol, were compared among control erythrocytes and reticulocytes. The dissociation constant (Kd = 0.1--0.2 nM), association and dissociation kinetics, and stereospecificity for (--)-isomers of agonists and antagonists were similar in both cell types. The reticulocyte population contained four times more receptors per cell than the control erythrocytes. However, reticulocytes were 25 times more responsive than control cells to isoproterenol, as measured by the formation of cyclic AMP. After peak reticulocytosis, cells rapidly lost 95% of their maximum hormone responsiveness, but beta-receptors declined much more slowly. The 4-fold decrease in beta-receptors was associated with a 4-fold decrease in cell volume as the reticulocytes matured. The density of beta-receptors was unchanged. However, responsiveness to isoproterenol in the reticulocytes when expressed on the basis of cell volume was still nine times greater than the control cells. Thus, maturation of reticulocytes is associated with an uncoupling of persistent beta-receptors from catecholamine responsiveness.     

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells

Cultured human urinary bladder carcinoma cells ( JTC -32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37 degrees C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (less than 1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC -32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.     

Dissociation of beta-adrenergic receptors from hormone responsiveness during maturation of the rat reticulocyte

Intact rat erythrocytes and reticulocytes have been studied in relation to their concentration of β-adrenergic receptors and their responsiveness to β-adrenergic catecholamines. Characteristics of the β-receptor, as determined by binding of ¹²⁵I-labelled hydroxybenzylpindolol, were compared among control erythrocytes and reticulocytes. The dissociation constant (K_d=0.1?0.2 nM), association and dissociation kinetics, and stereospecificity for (?)-isomers of agonists and antagonists were similar in both cell types. The reticulocyte population contained four times more receptors per cell than the control erythrocytes. However, reticulocytes were 25 times more responsive than control cells to isoproterenol, as measured by the formation of cyclic AMP. After peak reticulocytosis, cells rapidly lost 95% of their maximum hormone responsiveness, but β-receptors declined much more slowly. The 4-fold decrease in β-receptors was associated with a 4-fold decrease in cell volume as the reticulocytes matured. The density of β-receptors was unchanged. However, responsiveness to isoproterenol in the reticulocytes when expressed on the basis of cell volume was still nine times greater than the control cells. Thus, maturation of reticulocytes is associated with an uncoupling of persistent β-receptors from catecholamine responsiveness.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Decreased binding of insulin to liver plasma membrane receptors in hereditary diabetic mice.

The interaction of insulin with its receptors was studied in liver plasma membranes of the young non-obese hereditary diabetic mouse (KK strain). Under identical conditions of preparation and incubation, the membranes of the KK mouse bind only 55-70% as much insulin per mg of protein as those of the control mouse (Swiss albino). Scatchard analysis suggests that this decrease in binding is due to a decrease in the number of receptor sites in the membrane of the diabetic mouse. However, the membranes of diabetic and control mice do not exhibit significant differences in hexosamine and sialic acid contents, enzyme activities, and protein and glycoprotein analysis. The decrease in insulin receptors in the KK mouse seems to correlate with the insulin resistance which they exhibit.     

The effect of high-glucose condition on insulin binding to IM-9 lymphocytes

The long-term effect of high-glucose condition on insulin binding to IM-9 cells was studied by incubating the cells in RPMI-1640 medium containing 450 mg/dl of glucose. Insulin binding began to decrease after incubation for 6 days in high-glucose-treated cells, and significantly decreased after 14 days due to the reduction of receptor affinity. The number of binding sites for insulin did not change by the treatment. Thus, high-glucose condition per se does not appear to induce the decrease of the number of insulin receptors on target cells, as observed in diabetic patients.     

Ontogeny of insulin binding during chick skeletal myogenesis in vitro.

Our studies show that insulin receptors exist on chicken skeletal muscle cells at all developmental stages in culture. ¹²⁵I-labeled insulin binding at physiological concentrations to mature myotubes demonstrated saturability, binding proportional to cell number, reversibility, and specificity by competition with native hormone which reduced specific binding by 40% with 1 ng/ml and was maximal with 10 μg/ml. Further evidence for specificity was shown by no competition of insulin specific binding with insulin A chain, insulin B chain, growth hormone, and thyrotropin. Two binding sites were detected, with affinity constants of 10¹⁰M^?1 and 2 × 10⁹M^?1. The hormone receptor complex showed rapid dissociation (70% in 30 min) after equilibrium binding. During myogenesis, an increase in insulin receptors occurs from 500 per proliferating myoblast to 3000 per cell equivalent in mature (6 day) myotubes. Since these studies demonstrate that insulin receptors are present and other studies have shown that insulin is present during most of chicken embryogenesis, insulin may regulate muscle development in vivo to a greater degree than previously suspected.     

Increased surface binding sites of insulin in ML236B (compactin)-resistant mutants of Chinese hamster cell line

Mutants resistant to ML236B (compactin) were isolated from the Chinese hamster lung V79 cell line (1). Three ML236B-resistant mutants, MF-1, MF-2 and MF-3, were enhanced in insulin-specific binding activity about 2 to 3 times over the parental V79 cell lines. Compared to V79, endocytosis of insulin was also increased 2 to 3-fold in ML236B-resistant mutants than V79. Scatchard analysis showed that 5,000 insulin binding sites per cell in V79 and 16,000 in a NL236B-resistant clone, MF-2. Insulin receptors in mutant and parental strains are down-regulated to a similar extent in the parental V79 treated with an excess insulin. This is the first somatic cell mutant with increased surface binding sites for insulin.     

Domain-specific monoclonal antibodies to ovotransferrin indicate conservation of determinants involved in avian transferrin receptor recognition

1. Three of five monoclonal antibodies produced to chicken ovotransferrin bound quail ovotransferrin but none of the antibodies bound human, bovine or equine serum transferrin. 2. Equilibrium binding experiments indicate that both quail and chicken ovotransferrin bind to transferrin receptors on chick reticulocytes although the quail protein binds to 40% fewer sites with an affinity which is three times lower than chicken ovotransferrin. 3. The antibodies that recognize quail ovotransferrin block binding of both radiolabelled chicken and quail ovotransferrin to chick reticulocytes. 4. Quail NH2-terminal half-molecule domain appears to be unable to form a functional hybrid holo-ovotransferrin with chicken C-terminal half-molecule domain.     

PKC site mutations reveal differential modulation by insulin of NMDA receptors containing NR2A or NR2B subunits

Insulin modulates N-methyl-d-aspartate (NMDA) receptors in the CNS and potentiates currents of recombinant NMDA receptors in a subunit-specific manner in Xenopus oocytes. Previously we identified two sites in the NR2B C-terminus as targets for direct phosphorylation by C-type protein kinases (PKCs). Mutating these sites reduced insulin potentiation of currents by one half, reflecting the PKC-mediated portion of the NR2B insulin effect. The PKC-proline rich tyrosine kinase (Pyk2)-Src family kinase pathway may also mediate insulin potentiation. A dominant negative Pyk2 mutant significantly reduced insulin potentiation when co-expressed with NR2B-containing receptors, suggesting that Pyk2 and downstream Src-family tyrosine kinases are involved, along with PKCs, in insulin potentiation of NR2B. The NR2A C-terminus contains two residues homologous to the NR2B PKC targets. Mutating both these sites eliminated insulin potentiation of NR2A-containing receptors, while co-expression of dominant negative Pyk2 had no effect. Together, these data indicate that PKCs alone mediate the NR2A insulin effect. When tested individually for importance in insulin potentiation, the two PKC sites showed an additive effect in potentiation of NR2A-containing receptors. Insulin modulation of NR2A-containing receptors is mediated solely by PKCs, whereas insulin modulation of NR2B-containing receptors is mediated by PKCs and tyrosine kinases (PTKs).     

Insulin receptors in dog lymphocytes

The circulating lymphocyte because of its easy accessibility provides a useful model for the study of insulin receptors in dog. Consequently, we have studied the binding of insulin to lymphocytes prepared from Ficoll gradient. Our results demonstrate that circulating dog lymphocytes bind 125I-Insulin and this binding is inhibited by unlabeled insulin. This binding is function of the cell concentration and a saturable function of 125I-Insulin concentration. At 4 degrees C the equilibrium conditions are reached in 30 mn. Scatchard analysis revealed a cirvilinear plot. The number of receptors per cell was calculated as 1000 to 1500. This study have also revealated a decrease in the number of sites in diabetic dogs. We believe that the study of insulin receptor in lymphocyte's dog is important to understand some aspects of pathological states in diabetes.     

Insulin Binding to the Blood-Brain Barrier in the Streptozotocin Diabetic Rat

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)