Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells.

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.     

Characterization of specific insulin binding sites on chromaffin cells from bovine adrenal medulla

1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [125I]insulin in HEPES buffer, pH 7.8 at 15 degrees C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 x 10(6) cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46 nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM-1, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions.     

Insulin receptor recycling in vascular endothelial cells. Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t1/2 = 18 +/- 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.     

Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells

Micro- and macroangiopathy are major causes of morbidity and mortality in patients with diabetes. Our aim was to characterize IGF-I receptor (IGF-IR) and insulin receptor (IR) in human micro- and macrovascular endothelial cells. Cultured human dermal microvascular endothelial cells (HMVEC) and human aortic endothelial cells (HAEC) were used. Gene expression was measured by quantitative real-time RT-PCR and receptor protein by ligand-binding assay. Phosphorylation of IGF-IR beta-subunit was analyzed by immunoprecipitation and Western blot. Glucose metabolism and DNA synthesis was assessed using [(3)H]glucose and [(3)H]thymidine incorporation, respectively. We detected gene expression of IGF-IR and IR in HAEC and HMVEC. IGF-IR gene expression was severalfold higher than that of IR. The specific binding of (125)I-IGF-I was higher than that of (125)I-insulin in HAEC and HMVEC. Insulin and the new, long-acting insulin analog glargine interacted with the IGF-IR with thousand- and hundred-fold less potency than IGF-I itself. Phosphorylation of the IGF-IR beta-subunit was shown in HAEC for IGF-I (10(-8) M) and insulin (10(-6) M) and in HMVEC for IGF-I and glargine (10(-8) M, 10(-6) M). IGF-I 10(-7) M stimulated incorporation of [(3)H]thymidine into DNA, and 10(-9)-10(-7) M also the incorporation of [(3)H]glucose in HMVEC, whereas glargine and insulin had no significant effects at 10(-9)-10(-7) M. Human micro- and macrovascular endothelial cells express more IGF-IR than IR. IGF-I and high concentrations of glargine and insulin activates the IGF-IR. Glargine has a higher affinity than insulin for the IGF-IR but probably has no effect on DNA synthesis at concentrations reached in vivo.     

Evidence of the lack of receptor-mediated insulin degradation in human cultured lymphocytes (RPMI-1788 line)

We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope. Insulin degradation determined by the TCA solubility method was 64.6 +/- 1.2% (mean +/- SEM) at a trace concentration after the incubation with 2.0 x 10(7) cells (4.0 x 10(7) cells/ml) for 60 min at 37 degrees C. Because insulin degradation was 54.6 +/- 7.0% in the cell-free buffer in which 2.0 x 10(7) cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin. Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500 micrograms/ml) significantly decreased the insulin degradation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method. These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.     

Distribution of insulin receptors between plasmalemma and intracellular compartments: effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.     

Kinetics of insulin binding to rat white fat cells at 15 degrees C

The kinetics of insulin binding to isolated rat epididymal fat cells was investigated at 15 degrees C, at which temperature the system was simplified by the absence of lysosomal insulin degradation. The data were fit by maximum likelihood criteria with differential equations describing a number of models for the interaction of insulin and cells. Among those models that yielded a fit, the selection criteria were minimization of the Akaike information criterion and compatibility of the overall equilibrium constant for the system calculated from rate constants with the previously obtained experimental value. The results of the analysis indicated that insulin, I, first reversibly bound to cell surface receptors, R, whereupon this initial insulin-receptor complex, RI, reversibly altered its state or cellular location to R'I, according to the following equation. (Formula: see text) No evidence was found that insulin could either associate or dissociate from R'I directly. The association rate constant was kappa 12 = 1.6 x/divided by 1.4 X 10(5) liter mol-1 s-1, a value shown to be incompatible with diffusion control. The other rate constants were: kappa 21 = 3.4 x/divided by 1.6 X 10(-3) s-1, kappa 23 = 3.2 x/divided by 1.5 X 10(-4) s-1, and kappa 32 = 2.0 x/divided by 1.5 X 10(-4) s-1. From these rate constants, an equilibrium constant of 8.4 x/divided by 1.5 nM was calculated, in excellent agreement with the previously measured value of 8.8 x/divided by 1.3 nM (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1694-1701). The kinetic analysis also yielded receptor numbers similar to those obtained by equilibrium binding studies. The nature of the R'I state is discussed in terms of an internalized state, in terms of insulin receptor complex in caveolae, in terms of receptor aggregates, and in terms of being a Michaelis complex between insulin bound to the receptor and cell surface-bound insulin protease.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

The Role of IGF-system in Vascular Insulin Resistance

Insulin and IGF-I are closely related peptides, which interact by several mechanisms. In high supraphysiological concentrations (>/=10 (-8) M), they cross-react with each other's receptors with 100- to 1000-fold lower affinity than with their cognate receptors. This can cause confusion, since in many in vitro studies, insulin has been used in high unphysiological concentrations, which activate IGF-I receptors. Due to the differences in affinity, insulin and IGF-I probably do not activate each other's receptors in vivo. IGF-I receptors are several-fold more abundant than insulin receptors in human micro- and macrovascular endothelial cells and in human vascular smooth muscle cells. Both insulin and IGF-I receptor protein can be demonstrated and they are activated by their cognate ligand at physiological concentrations of 10 (-9)-10 (-10) M. In vascular smooth muscle cells, IGF-I but not insulin stimulates metabolism and growth. IGF-I stimulates DNA-synthesis and growth in microvascular endothelial cells, but neither insulin nor IGF-I have any effect on macrovascular endothelial cells. Both insulin and IGF-I have been shown to stimulate nitric oxide production in endothelial cells, but only the effect of IGF-I was obtained at a physiological concentration. In both endothelial and vascular smooth muscle cells, insulin and IGF-I receptors occur as insulin/IGF-I hybrid receptors with high affinity to IGF-I and low for insulin. Due to the low number of insulin receptors and the presence of hybrid receptors the insulin receptor signal is probably too attenuated to elicit biological effects, explaining the insulin resistance of vascular cells in vitro. In vivo both insulin and IGF-I have been reported to increase muscle blood flow in physiological concentrations. Whether this is due to direct effects on endothelial cells or indirectly induced is not clear. The effect of insulin is attenuated by insulin resistance. In conclusion, the in vitro data suggest that endothelial cells and vascular smooth muscle cells are sensitive to IGF-I, but insensitive to insulin, and this is due to a preponderance of IGF-I receptors and the presence of insulin/IGF-I hybrid receptors.     

Glycosylation defects alter insulin but not insulin-like growth factor I binding to Chinese hamster ovary cells

Insulin binding to two Chinese hamster ovary cell lines with well-defined defects in their glycosylation pathway has been characterized and compared to insulin-like growth factor I (IGF-I) binding in the same cell lines. Insulin competition curves indicate that B4-2-1 cells, which transfer co-translationally to proteins an endoglycosidase H insensitive, truncated lipid-linked oligosaccharide, bind insulin with higher than normal affinity. Lec 1 cells, which fail to process oligosaccharide side chains to complex types, bind with a reduced affinity. The potencies of chicken and guinea pig insulins are appropriate for an insulin receptor in the control (WTB) and both mutant cell lines, whereas rat IGF-II is 3 times more potent than expected in the Lec 1 cells and human IGF-I is less potent than anticipated. Insulin bound to Lec 1 cells dissociates more quickly upon dilution than does insulin bound to either WTB or B4-2-1 cells. The Lec 1 insulin receptor is insensitive to pH change, whereas the other lines show the usual optimum of 8. 125I-IGF-I binds well to all three cell lines and is equally pH-sensitive in all three. Serum from a patient with circulating autoantibodies to the insulin receptor competes for insulin but not IGF-I binding, whereas alpha IR3, a monoclonal antibody directed toward the human IGF-I receptor inhibits IGF-I but not insulin binding. Cross-linking of either 125I-insulin or 125I-IGF-I reveals a typical alpha-subunit in the WTB and B4-2-1 cells but a band with faster mobility in the Lec 1 cells. Insulin (10(-8) M) stimulates autophosphorylation of a beta-subunit in all three lines, but again the Lec 1 subunit demonstrates an anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data demonstrate the differential effect of glycosylation on two closely related receptor molecules.     

Insulin enhances the expression of the endothelial nitric oxide synthase in native endothelial cells: a dual role for Akt and AP-1

Insulin-induced vasodilatation in vivo has been attributed to the activation of the endothelial nitric oxide (NO) synthase (eNOS). The present study addressed the effects of insulin on the activity and expression of eNOS in native and cultured endothelial cells. Insulin applied to native porcine aortic endothelial cells elicited the tyrosine phosphorylation of the insulin receptor and receptor substrate, the subsequent activation of phosphatidylinositol 3-kinase (PI 3-K), Akt (protein kinase B), and ERK1/2. Insulin did not activate eNOS in cultured endothelial cells nor relax endothelium-intact arterial segments. However, 4h after application of insulin to native endothelial cells eNOS mRNA was increased 2-fold. A comparable increase in eNOS protein was detected after 18-24h and associated with an increase in intracellular cyclic GMP. In native endothelial cells, insulin enhanced the DNA-binding activity of Sp1 and AP-1, but not that of NF-kappaB. The insulin-induced increase in eNOS expression was prevented by wortmannin as well as by AP-1 decoy oligonucleotides. The MEK1 inhibitor, PD 98059, also enhanced eNOS expression in native and cultured endothelial cells, an effect which was independent of ERK1/2 and associated with an increase in the DNA-binding activity of AP-1 and Sp1. These results demonstrate that insulin activates multiple signalling pathways in endothelial cells but does not acutely activate eNOS. Insulin however enhances eNOS mRNA and protein by a mechanism involving the combined activation of a PI 3-K- and AP-1-dependent pathway.     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Insulin and analogue effects on protein degradation in different cell types. Dissociation between binding and activity

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.     

Insulin receptors and insulin effects on type II alveolar epithelial cells

Type II alveolar epithelial cells (pneumocytes) were isolated to purity from adult rabbits and analyzed for the presence of cell surface insulin receptors and for effects of insulin on cells. Assays were performed on cells cultured for 24 h in Eagle's minimum essential medium. Insulin binding to cells in culture approached a steady-state level by 180 min at 15 degrees C and remained constant for at least 1 h. Competition experiments using native insulin, proinsulin and desoctapeptide supported specificity of binding. Scatchard analysis of binding revealed a class of high-affinity receptors with Kd = 1.5 X 10(-10) M and a low-affinity component with Kd = 4 X 10(-9) M. The number of receptors was estimated at 2000-4000/cell. Insulin added to cell cultures of type II pneumocytes in concentrations from 5 X 10(-11) to 5 X 10(-8) M resulted in a dose-related increase in uptake of 2-deoxyglucose by cells. Insulin also stimulated the incorporation of choline and glucose into phosphatidylcholine and disaturated phosphatidylcholine.     

Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.     

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

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. 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. 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. 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. Chronic exposure (for at least 3h) 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 concentration (10 micron) 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. 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 of cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. RL-PR-C rat hepatocytes appear, for these reasons, to be a useful model system for studying the regulation of the insulin receptor.     

Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells

125I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R.H., LeBoeuf, R. D., Stone, G.W., and Weigel, P.H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific 125I-HA binding at 4 degrees C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cells and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (approximately 10(5)/cell). Cultured cells had 1.8 x 10(5) fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average Kd, determined by equilibrium binding studies, was 5.8 +/- 2.8 x 10(-8) M (n = 12). Dissociation of bound 125I-HA from permeable cultured cells was rapid (t1/2 = 30.9 min;kappa off = 3.7 x 10(-4) s-1). A variety of carbohydrates had essentially identical effects on 125I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for 125I-HA binding at 4 degrees C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the 125I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.     

Reconstitution of the solubilized insulin receptor in phospholipid vesicles.

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% beta-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin greater than proinsulin greater than desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15 degrees C. A high affinity association constant of 1.4 x 10(9) M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.     

High affinity insulin binding and insulin receptor-effector coupling: modulation by Ca2+.

Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to insulin action.