beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Ontogeny of insulin receptors in the rat hemochorial placenta

Binding of 125I-insulin to rat placental membranes was time and protein concentration dependent, reversible, and specific. Unlabeled porcine insulin competed for 125I-insulin binding with an IC50 of 65 nM, while IGF-I was much less potent with an IC50 of 2.12 mM. Specific binding of 125I-insulin decreased during the second half of gestation from Days 11 to 19. Scatchard analysis of the binding data for membranes prepared from Gestation Days 11 and 19 yielded typical curvilinear plots which showed a marked decrease in the number of binding sites in late gestation placenta. Beginning on Day 14, insulin binding was characterized with isolated labyrinth and basal zone portions of the hemochorial placenta. There was no evidence for differences in Kd values or the number of binding sites in these two functionally distinct portions of the rat placenta. Crosslinking of 125I-insulin followed by SDS-PAGE showed a single protein with a molecular weight of 130,000 from placental tissues on Gestation Days 11 and 19 and confirmed a gestational decrease in the number of insulin receptors. In solubilized, lectin-purified preparations from placenta and liver membranes, insulin stimulated the phosphorylation of a Mr 95,000 protein. 32P-incorporation into this 95,000 protein was stimulated fivefold by insulin in Day 11 placenta receptor, whereas no detectable 32P-incorporation was found in Day 19 placenta. Thus, while the alpha- and beta-subunits of insulin receptors in mid and late gestation placenta have molecular weights which are similar to receptors in maternal liver, data indicate the presence of a functional difference in insulin-stimulated kinase activities.     

Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorbol ester

Phorbol 12-myristate 13-acetate (PMA) was used to examine the role of insulin receptor phosphorylation in the regulation of insulin receptor internalization in vascular endothelial cells. Association of 125I-insulin in rat capillary and bovine aortic endothelial cells preincubated with PMA was increased by 80 and 64% over control, respectively. The increase was due to enhanced 125I-insulin internalization as opposed to an effect on surface-bound hormone. PMA had no significant effect on 125I-insulin degradation or on release of internalized insulin from the cells. Internalization of 125I-labeled insulin receptor was determined by the resistance of labeled receptor to trypsinization. At 10 degrees C, nearly all of the labeled receptor was sensitive to removal by trypsin, indicating that it was exposed on the cell surface. Exposure of labeled cells to insulin (100 nM) at 37 degrees C resulted in the rapid appearance of trypsin-resistant insulin receptor, indicating receptor internalization. Steady state for receptor internalization was attained at 10-15 min. When surfaced-labeled cells were preincubated with PMA at 37 degrees C, the rate of insulin receptor internalization was increased by 3.6 +/- 0.2-fold and 2.1 +/- 0.5-fold at 1 and 5 min of insulin exposure, respectively (ED50 at 16 nM PMA). This effect of PMA was associated with an increase in serine phosphorylation of the insulin receptor. Thus, PMA increased insulin internalization in the endothelial cells by modulating the insulin-induced internalization of the receptor. The additive effects of PMA and insulin on insulin receptor phosphorylation suggest that the phorbol ester and insulin act via independent signaling mechanisms.     

Kinetics of insulin binding and kinase activity of the partially purified insulin receptor from human skeletal muscle

The kinetics of insulin binding and kinase activity of soluble, partially purified insulin receptors from human skeletal muscle are considered. An equilibrium for insulin binding was obtained within 2 h at 37 degrees C. At lower temperatures the equilibrium for insulin binding was less clearly defined. Dissociation of 125I-labelled insulin was incomplete unless an excess amount of unlabelled insulin was added. Insulin-stimulatable autophosphorylation of the 95 kDa subunit was verified by gel electrophoresis. The kinase activity was measured with the synthetic polypeptide poly(Glu-Tyr(4:1] as a phosphoacceptor. The insulin receptor kinase activity correlated significantly (r = 0.92, P less than 0.0001) to the concentration of high-affinity insulin binding sites in the eluate. Autophosphorylation of the insulin receptor was necessary for the activation of the receptor kinase. When activated the receptor kinase activity was stable for at least 60 min at 21 degrees C with a pH optimum of approx. 7.8, similar to the pH optimum for insulin binding. The non-ionic detergent Triton X-100 inhibited the sensitivity of the receptor kinase to insulin. Insulin stimulated the Vmax of the kinase reaction about 3-fold, decreased the Km for ATP from 35 +/- 5 microM (mean +/- S.E.) to 8 +/- 1 microM (P less than 0.02) and induced a positive cooperativity to ATP with an increase in the Hill coefficient from 1.00 +/- 0.02 to 1.37 +/- 0.07 (P less than 0.05). According to the Hill plots, insulin itself showed no cooperativity with respect to receptor binding or kinase activation.     

Determination of insulin antibodies.

Insulin antibodies were determined as percentage binding of 125I-insulin in the sera of normal persons and of diabetic subjects treated and untreated with insulin. The effect of the dilution of the serum, circulating insulin and extraction of free and total insulin was evaluated. The determination of insulin antibodies in samples at a final dilution of 1:10 clearly discriminated between insulin-treated and untreated subjects. In insulin-treated subjects, the determination of insulin antibodies in samples at a final dilution of 1:100 gave false-negative results in 28 per cent. However, the determination of insulin antibodies at a final dilution of 1:100 discriminated between insulin-resistant and non-resistant diabetic subjects. Extraction of total insulin at pH 3.0 using 0.1 N HCl increased the percentage of 125I-insulin binding significantly. Extraction of free insulin by charcoal from the samples did not increase the binding of 125I-insulin. The injection of crystalline insulin 4 hours prior to withdrawing the samples did not decrease binding of 125I-insulin.     

Characterization of a novel insulin receptor from stingray liver

The insulin receptor from the liver of stingray, a cartilaginous fish, has characteristics which are in marked contrast to those of the mammalian insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cross-linked, affinity-labeled stingray insulin receptor shows an apparent molecular mass of 210 kDa for the intact receptor. Reduction with mercaptoethanol resulted in no alteration in the apparent size of the stingray insulin receptor. Gel filtration studies of Triton X-100 solubilized stingray insulin receptor demonstrated an apparent Stokes radius of 7.6 nm. Ultracentrifugation sucrose gradient studies of cross-linked affinity labeled stingray receptor resulted in determination of a sedimentation coefficient of 13 S. Both of these parameters were greater than simultaneously obtained data for the human insulin receptor (7.2 nm and 11 S, respectively). Unlabeled insulin competed with binding of 125I-insulin and 125I-insulin growth factor (IGF) I with a half-maximal concentration of 1 nM for either. Unlabeled IGF I and II also competed, but were 4-5-fold less potent than insulin. It was found that not only did IGF I bind to the 210-kDa material, but both insulin and IGF I stimulated phosphorylation of a 210-kDa material which was immunoprecipitable by a polyclonal insulin receptor antibody. Elution of this material from the gel followed by hydrolysis and thin layer chromatography demonstrated that the 210-kDa material was specifically phosphorylated on tyrosine only. These data suggest that the insulin receptor from stingray liver is a dimer made up of 2 identical subunits of about 210 kDa size which contain both binding regions and insulin-stimulated tyrosine kinase. Specificity studies suggest that the stingray insulin receptor may represent a phylogenetic position prior to the evolutionary divergence of insulin and the insulin-like growth factors.     

Characterization of insulin binding sites in cultured smooth muscle cells of rat aorta

Insulin receptors could be demonstrated in cultured smooth muscle cells of rat aorta. The specific binding of 125I-insulin was time-, temperature- and pH-dependent. The optimal temperature for our studies was 12 degrees C. At this temperature maximal specific binding was 0.5% of total counts at 120 min incubation. The pH-optimum for the binding process was between 7.5 and 8. Degradation of 125I-insulin at 12 degrees C was 14%, no degradation of binding sites could be measured at this temperature. Dissociation of 125I-insulin was rapid. 50% of the labeled hormone remained associated with the cells. Half-maximal inhibition of 125I-insulin binding was produced by insulin at 4 X 10(-11) mol/l. Scatchard-analysis gave curvilinear plots, that may suggest negative cooperativity. Specificity of binding was studied in competition experiments between 125I-insulin, insulin, proinsulin, insulin-like growth factors and human growth hormone. Half-maximal inhibition of 125I-insulin binding was produced by proinsulin at 2 X 10(-9) mol/l and by insulin-like growth factors at 9 X 10(-9) mol/l. Human growth hormone had no significant effect on the insulin binding.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.     

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.     

Site-site interactions among insulin receptors. Characterization of the negative cooperativity.

By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds.     

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.     

Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites

Mutations in the insulin receptor gene can cause insulin resistance. Previously, we have identified a mutation substituting glutamic acid for lysine at position 460 in the alpha-subunit of the insulin receptor in a patient with a genetic form of insulin resistance. In the present work, we have investigated the effect upon receptor function of amino acid substitutions at position 460. Decreasing the pH from 8.0 to 5.5 caused a progressive acceleration of the dissociation of 125I-insulin from the wild-type insulin receptor. Substitution of acidic amino acids (Glu or Asp) for Lys460 decreased the ability of acid pH to accelerate dissociation of 125I-insulin. In contrast, substitution of Arg or neutral amino acids (Val, Met, Thr, or Gln) had no effect upon the sensitivity to acid pH. Correlated with decreased sensitivity to acid pH, substitution of Glu or Asp at position 460 retarded the dissociation of 125I-insulin from intracellular receptors subsequent to receptor-mediated endocytosis. Furthermore, retardation of dissociation of 125I-insulin from the internalized receptor was associated with a decreased half-life of the receptor. In summary, the Glu460 mutation appears to cause insulin resistance by accelerating receptor degradation and, thereby, decreasing the number of insulin receptors on the cell surface. Additional studies suggested that Lys460 may provide the amino groups whereby disuccinimidyl suberate cross-links the two alpha-subunits to each other. Consistent with the hypothesis that Lys460 is located at the interface between adjacent alpha-subunits, substitutions at position 460 impair cooperative interactions among insulin binding sites. The Glu460 mutation decreases positively cooperative binding interactions; the Arg460 mutation impairs negative cooperativity. Mutations at position 460 in the alpha-subunit did not decrease the ability of insulin to stimulate receptor tyrosine kinase.     

Insulin receptors and insulin modulation of norepinephrine uptake in neuronal cultures from rat brain

Neuronal cells from 1-day-old rat brain in primary culture have been utilized in the present study to characterize insulin-binding sites and a possible action of insulin on these cells. Binding of 125I-insulin to neuronal cultures was 90% specific and time-dependent and reached equilibrium in 120 min. Specific binding was reversible with greater than 90% of binding dissociable within 120 min with a t1/2 of dissociation of 15 min. Various insulin analogues competed for 125I-insulin binding to neuronal cultures according to their known biological potencies. Scatchard analysis of competition data yielded a typical curvilinear plot providing a class of high affinity (Kd = 11 nM) and low affinity (Kd = 65 nM) binding sites. Light microscopic autoradiographic analysis of 125I-insulin bound to neuronal cultures revealed the presence of silver grains predominantly on the neurites with occasional occurrence on the cell soma. Insulin had no effect on neuronal 2-deoxyglucose uptake in contrast with our previous findings demonstrating a 2-fold stimulation of 2-dGlc uptake into astrocyte glial cells from rat brain (Clarke, D.W., Boyd, F.T., Jr., Kappy, M.S., and Raizada, M. K. (1984) J. Biol. Chem. 259, 11672-11675). Incubation of neuronal cultures with insulin caused a dose-dependent inhibition of [3H]norepinephrine uptake with significant inhibition occurring at 1.67 X 10(-11) M. These findings demonstrate that: 1) neuronal cells in primary culture possess specific insulin receptors which are predominantly located on neurites and 2) insulin modulates monoamine uptake in these cultures which suggests that insulin may modulate neural signaling via specific neuronal insulin receptors.     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Distinct receptors for IGF-I, IGF-II, and insulin are present on bovine capillary endothelial cells and large vessel endothelial cells

Endothelial cells were cultured from bovine fat capillaries, aortae and pulmonary arteries and their interactions with 125I-IGF-I, 125I-MSA (an IGF-II), 125I-insulin and the corresponding unlabeled hormones were evaluated. Each endothelial culture showed similar binding parameters. With 125I-insulin, unlabeled insulin competed with high affinity while IGF-I and MSA were approximately 1% as potent. With 125I-MSA, MSA was greater than or equal to IGF-I in potency and insulin did not compete for binding. Using 125I-IGF-I, IGF-I was greater than or equal to MSA whereas insulin decreased 125I-IGF-I binding by up to 72%. Exposing cells to anti-insulin receptor antibodies inhibited 125I-insulin binding by greater than 90%, did not change 125I-MSA binding, while 125I-IGF-I binding was decreased by 30-44%, suggesting overlapping antigenic determinants between IGF-I and insulin receptors that were not present on MSA receptors. We conclude that cultured capillary and large vessel endothelial cells have distinct receptors for insulin, IGF-I and MSA (IGF-II).     

A radioimmunoassay for human insulin receptor correlation between insulin binding and receptor mass.

We have developed a radioimmunoassay for human insulin receptor. Serum from a patient with Type B severe insulin resistance was used as anti-insulin receptor antiserum. Pure human placental insulin receptor was used as reference preparation and 125I labeled pure insulin receptor as trace. The radioimmunoassay was sensitive (limit of detection less than 17 fmol), reproducible (inter and intra-assay coefficients of variation 12.5% and 1.6% respectively) and specific (no crossreactivity with pure placental IGF-1 receptor, insulin and glucagon). The anti-insulin receptor antibody was, however, able to differentiate between insulin receptor from human placenta and from rat liver. To determine the number of insulin binding sites per receptor, we measured insulin binding (by insulin binding assay) and insulin receptor mass (by radioimmunoassay) in solubilized aliquots from 5 human placentas. The molar ratio of insulin binding to receptor mass was 0.86 +/- 0.12 when binding was determined with monoiodinated 125I-Tyr A 14-insulin. It was 1.94 +/- 0.27 when randomly iodinated 125I-insulin was used. In conclusion, using a sensitive, reproducible and specific radioimmunoassay, we have measured insulin receptor mass independent of insulin binding. Our data are most compatible with binding of one insulin molecule per human placental insulin receptor.     

Production of antibodies that inhibit the binding of insulin to its receptor

In this study, we report a procedure for producing antisera that block the binding of ¹²⁵I-insulin to its receptor. After 2 injections with intact IM-9 cultured human lymphocytes, the antisera from 8 of 17 $\text{Balb}\text{C}$ mice inhibited the binding of ¹²⁵I-insulin to its receptor on IM-9 cells by 50% or greater. One antiserum at dilutions of 1:200 and 1:50 inhibited the binding of ¹²⁵I-insulin by 50% and 80%, respectively. Four lines of evidence indicated that the inhibition of ¹²⁵I-insulin binding by this antiserum was due to a specific immunoglobulin directed against the insulin receptor. First, removal of the immunoglobulin fraction of the antiserum resulted in a complete loss of its inhibitory activity. Second, the antiserum inhibited the binding of ¹²⁵I-insulin to its receptor on both human cultured lymphocytes and human placenta particles. Third, the antisera bound solubilized insulin-receptor complexes. Finally, the antiserum did not inhibit the binding of ¹²⁵I-human growth hormone to its receptor on IM-9 lymphocytes. These studies demonstrate therefore, a simple method for producing antibodies that block the binding of ¹²⁵I-insulin to the human insulin receptor.     

Inhibition of insulin receptor binding by phorbol esters

Phorbol esters inhibit the binding of insulin to its receptors on U-937 monocyte-like and HL-60 promyelocytic leukemia human cell lines. Within 20-30 min, exposure of these cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 37 degrees C results in a 50% reduction of the specific binding of 125I-insulin. Half-maximal inhibition occurs at 1 nM TPA. Other tumor-promoting phorbol esters also inhibit 125I-insulin binding in a dose-dependent manner which parallels their known promoting activity in vivo. TPA does not alter the degradation of the hormone nor does it induce any shedding of its receptors in the medium. The effect of phorbol esters is dependent on temperature and cell type. It is less prominent at 22 degrees C than at 37 degrees C. It is reversible within 2 h at 37 degrees C. TPA reduces the binding of insulin predominantly by increasing its dissociation rate. This effect results in an accelerated turnover of the hormone on its receptors.     

Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.