Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.     

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.     

Identification of insulin receptors on the mucosal surface of colon epithelial cells

Brush-border membranes were isolated from the mucosal surface of rabbit proximal colon epithelial cells by a procedure involving Ca2+ precipitation. Ouabain-insensitive K+-phosphatase, a marker enzyme for the colon brush-border membrane, was enriched 17-fold by this technique, while no enrichment was observed in the activity of ouabain-sensitive K+-phosphatase, a marker for the basal-lateral membrane. Insulin binding studies revealed a dose-dependent inhibition of 125I-insulin binding with porcine insulin and approximately 4 X 10(-9) M insulin was required to produce 50% inhibition of 125I-insulin binding, while desoctapeptide insulin, insulin-like growth factor I, and A chain of insulin had less effect on 125I-insulin binding. This is the first demonstration of the existence of high-affinity insulin binding sites on the brush-border membrane of mammalian colon epithelial cells. Subsequent studies with the cross-linking agent disuccinimidyl suberate confirmed the presence of insulin binding sites in these membranes and autoradiography of polyacrylamide gels revealed that the binding subunit of the colon epithelial cell brush-border insulin receptor is similar in size to that observed in hepatic tissue. Interestingly, the insulin binding capacity/mg of protein of this preparation is high, suggesting that large numbers of insulin receptors are present in vivo on the mucosal surface of colon epithelial cells. The potential physiological role of these previously unrecognized insulin receptors is discussed.     

Characterization of insulin receptors in the bovine adrenal cortex and medulla.

In order to identify insulin receptors in the bovine adrenal cortex and medulla, we have studied 125I-porcine insulin binding to the membrane preparations from the bovine adrenal cortex and medulla. 125I-porcine insulin bound not only to the bovine adrenal cortex but to the medulla in time-, temperature-, and pH-dependent manners. The maximum levels of 125I-porcine insulin binding in the two tissues were observed at 4 degrees C for 24 h of incubation, and its optimum pH ranged from 7.6 to 8.0. Under these conditions, at tracer concentration of porcine insulin (200 pg/ml), 10.4% and 6.6% of 125I-porcine insulin added to each reaction tube bound specifically to 10(5) x g-pellet fractions (microsomal membrane) from the cortical tissue (0.3 mg of protein) and from the medullary tissue (2 mg of protein), respectively. 125I-porcine insulin binding was observed predominantly in the microsomal membrane from the bovine adrenal cortex, and in a 15,000 x g- pellet fraction (synaptosomal membrane) from the bovine adrenal medulla. Scatchard analysis of binding data yielded curvilinear plots in each tissue. Analysis of curvilinear plots based on two sites model revealed similar affinity constant between the cortex and medulla. Receptor concentration of the cortex was several times higher than that of the medulla. In the two bovine adrenal tissues, human proinsulin and insulin-like growth factor I (IGF-I) had about 1/100 potency compared to porcine insulin in displacing 125I-porcine insulin binding. Porcine glucagon added with concentration up to 10(-6) M did not inhibit 125I-porcine insulin binding to both the cortex and the medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

Regulation of insulin receptor activity of human erythrocyte membrane by prostaglandin E1

Incubation of human erythrocyte membrane with low concentration of prostaglandin E1 or prostacyclin increased the binding of 125I-labeled insulin to the membrane. The binding of the radioiodinated hormone was maximally stimulated at 3 nM prostaglandin E1 and the use of higher concentrations (above 8 nM) of the autacoid tended to reverse its own effect at lower concentrations. While prostaglandins A1, A2, B1, B2, D2, F1 alpha, F2 alpha or 6-keto-prostaglandin F1 alpha had no effect on the binding of insulin to the erythrocyte membrane, prostaglandin E2 at similar concentrations decreased the binding of the hormone. The effect of prostaglandin E1 on the increased binding of the insulin was found to be reversible and depended on the occupancy of the autacoid molecules on the membrane and showed positive cooperativity. Scatchard analysis of the binding of 125I-labeled insulin to the erythrocyte ghosts indicated that in the presence of the autacoid, the binding capacity of the insulin receptor increased 2-fold (from 207 to 424 fmol/mg protein) without any change in the ghosts affinity for the ligand (Kd 2.4 X 10(-9) versus 2.49 X 10(-9) M). As a consequence of increased binding of insulin to the erythrocyte membrane in the presence of prostaglandin E1 (3.0 nM), the optimal concentration of the peptide hormone for the maximal reduction of the membrane microviscosity decreased from approx. 1.6 to approx. 0.4 nM. Addition of prostaglandin E1 alone at the above concentration to the assay mixture had no effect on the membrane microviscosity.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

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.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

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.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Inhibition of insulin receptor binding by dimethyl sulfoxide.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125-I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor was affected to a much lesser extent.