125I-insulin Binding Research Articles

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of 125I-insulin and of insulin-related factor-1 (125I-IGF-1) by plasma membranes of the rat liver and brain. The IC50 value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (104 ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC50 value in the case of 125I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 104 ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.

Receptor of Insulin-Like Growth Factor 1 in Tissues of the Mollusc Anodonta cygnea

To identify insulin-like receptors in the mollusc Anodonta cygnea, specific binding of 125I-insulin and 125I-IGF-1 by WGA-purified glycoprotein fractions of foot muscles and neural ganglia is studied. The binding sites for IGF-1 are detected for the first time in invertebrates, both in the muscles, and in the neural tissue of the mollusc. The level of 125I-IGF-1 binding in the muscle tissue was equal to 2.8 ± 0.1, in the neural tissue, to 4.0 ± 0.2% per 5 µg of protein. The equilibrium dissociation constant (Kd) was equal to 4.8 ± 0.3 and 4.3 ± 0.2 nM, respectively. The relative affinity of the binding sites to insulin did not exceed 1% of their affinity to IGF-1. Binding of 125I-insulin in the muscle tissue was not detected; the level of labeled insulin binding in the neural tissue was equal to 0.5% per 5 µg of protein. In the sarcolemmal fraction of the mollusc foot, IGF-1 and, to a lesser degree, insulin at a dose of 100 nM initiated phosphorylation of tyrosine in a protein with mol. mass of 70 kDa. The minor band of the phosphorylation was also detected in the zone of protein of 80 kDa. The conclusion is made about the existence in molluscan tissues of high-conserved receptors-tyrosine kinases identical by functional parameters to the mammalian receptor of IGF-1. From this, it is suggested that the peptides close by structure to vertebrate IGF-1 may be involved in physiological processes in A. cygnea. The problem of the nature of the insulin-binding sites in the molluscan neural tissue is discussed.

Functional insulin receptors on human epithelial ovarian carcinoma cells: implications for IGF-II mitogenic signaling.

The insulin receptor mediates a proliferative response in certain transformed cells, but little is known about its function in ovarian cancer. We used human epithelial ovarian carcinoma cell lines and lifespan-extended normal ovarian surface epithelial (OSE) cells to examine (125)I-insulin binding and mitogenic responses to insulin. All cancer cell and OSE cultures specifically bound (125)I-insulin. Except for OV202, the carcinoma lines had elevated insulin binding compared with OSE cells. All carcinoma lines except OV202 expressed insulin receptor as detected by flow cytometry and increased (3)H-thymidine incorporation or cell number in response to 0.1-10 nM insulin. Interestingly, similar concentrations of IGF-II also induced proliferation of the insulin-responsive cancer cell lines and displaced (125)I-insulin binding. Direct binding of (125)I-IGF-II to the insulin receptor was visualized by cross-linking and immunoprecipitation. Binding of IGF-II to the insulin receptor and a proliferative effect of insulin suggest the presence of insulin receptor isoform A. Real-time PCR analyses confirm that insulin receptor isoform A expression predominates over isoform B expression in the ovarian carcinoma cell lines. This report suggests that the insulin receptor may play a role in the regulation of ovarian cancer cell growth.

Effect of vanadate on glycogen synthesis in dexamethasone-treated 3T3 adipocytes: evidence for a novel insulin sensitizing action.

Type 2 diabetes is characterized by peripheral tissue insulin resistance. The present study was carried out to determine the insulin sensitizing action of vanadium using dexamethasone-treated 3T3 adipocytes as an in-vitro model of insulin resistance. Fully differentiated 3T3 adipocytes were incubated with or without 100 nM dexamethasone in the presence or absence of 200 nM insulin for 6 days. Sodium orthovanadate (0-1000 microM) was added on day 2 and was present during the last 4 days. At the end, insulin (100 nM) stimulated glycogen synthesis was determined. Vanadate treatment for 4 days, caused 2-3-fold increase in glycogen synthesis in dexamethasone treated adipocytes. At 100 microM, vanadate completely reversed dexamethasone-induced insulin resistance (by increasing the levels from 9.65 +/- 0.80 to 28.4 +/- 4.9 nmol/h). In cells treated with dexamethasone and insulin, vanadium was partially active and it caused only 30% increase in glycogen synthesis. Exposure of dexamethasone treated cells for 24 h with vanadium did not affect glycogen synthesis. Under identical condition, vanadium had no significant effect in the normal insulin sensitive adipocytes. Vanadium at 100 microM had no effect on 125I-insulin binding to insulin-resistant adipocytes. Glycogen synthesis in the normal and insulin-resistant adipocytes was stimulated by lithium, an inhibitor of glycogen synthase kinase 3 beta, suggesting the involvement of phosphorylation events in dexamethasone-induced insulin resistance. Since vanadium was active only in the insulin-resistant adipocytes it is likely that vanadium acts by relieving dexamethasone actions rather than having independent effects. These results provide evidence for the novel insulin sensitizing action of vanadium which might be of future clinical relevance.

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca 2+-ATPase inhibitor in adrenal chromaffin cells

Long-term (≥12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA), caused a time ( t 1/2=16.3 h)- and concentration (IC 50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the K d value. TG caused a sustained increase of cytoplasmic concentration of Ca 2+ ([Ca 2+] c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) β-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR β-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca 2+] c down-regulates cell surface IR by retarding externalization of IR from the TGN.

Post-translational reduction of cell surface expression of insulin receptors by cyclosporin A, FK506 and rapamycin in bovine adrenal chromaffin cells

Long-term (≥3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface 125I-insulin binding by 62% in a concentration (IC 50=18 μM)- and time ( t 1/2=16 h)-dependent manner, but did not change the K d value. FK506 (1 μM) or rapamycin (3 μM) treatment reduced 125I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) β-subunit level ( t 1/2=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR β-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis- trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

The HIV-1 protease inhibitor indinavir impairs insulin signalling in HepG2 hepatoma cells.

Patients treated with human immunodeficiency virus-1 protease inhibitors often develop impaired glucose tolerance or diabetes, most likely due to an induction of insulin resistance. We therefore investigated whether the protease inhibitor indinavir alters insulin signalling. We incubated HepG2 cells for 48 h without or with indinavir (100 micromol/l). Subsequently 125I-insulin binding to the cells and the effects of insulin stimulation on insulin-receptor substrate-1-phosphorylation, association of phosphatidylinositol 3-kinase with insulin-receptor substrate-1 and Akt-Thr308-phosphorylation were measured. In cells not exposed to indinavir, insulin (100 nmol/l) led to rapid increases of insulin-receptor substrate-1-phosphorylation, association of phosphatidylinositol 3-kinase with insulin-receptor substrate-1 and Akt-phosphorylation during the first 75 s, followed by subsequent decreases. In indinavir-treated cells, these insulin-stimulated increases during the first 75 s were reduced by 30-60% and this was not associated with alterations in cell number or viability, insulin binding to the cells or cellular insulin-receptor substrate-1-content. Effects of indinavir on initial insulin signalling could cause, or contribute to, the metabolic effects of human immunodeficiency virus-1 protease inhibitors.

Insulin-Like Growth Factor I (IGF-I) Binding in Skeletal Muscle of Lamprey Lampetra fluviatilis Predominates over Insulin Binding and Increase in the Course of Pre-Spawning Migration

Binding of 125I-insulin and 125I-IGF-I to partially purified receptors of lamprey skeletal muscles was studied during pre-pawning migration. It has been shown that throughout this whole period the IGF-I binding to skeletal muscle predominates over the insulin binding. Besides, a certain time dynamics was observed: the insulin binding rose since October to reach maximum in February–March, then it decreased to a minimum level in May; the IGF-I binding also increased: it rose statistically significantly in March compared to October, became maximal in April, and then decreased to a minimum. The dynamics of the receptor IGF-I binding has been shown to depend on changes of receptor affinity, whereas the change of the insulin binding was determined by binding capacity (the number of binding sites). Highly specific IGF-I receptors of the lamprey skeletal muscle bound insulin with an affinity about 1% from that of IGF-I, while insulin receptors had identical affinity for the insulin and IGF-I binding. Both peptides, insulin and IGF-I, activated autophosphorylation of beta-subunits in their receptors. The increase of the IGF-I binding from October to April could be a factor that maintains a high functional activity of lamprey skeletal muscles in the course of the pre-pawning migration. It is suggested that IGF-I promotes maintaining this activity due to its property of inhibiting apoptosis.

Primary Structure of Insulin of the Black Sea Rockfish Scorpaena porcus

Insulin of the Black Sea rockfish Scorpaena porcus was isolated, purified, and the primary sequence has been determined. The hormone amino acid sequence has been established: the A chain—GIVEQCCNRPCNIFDLQNYCN, and the B chain—AAGPQHLCGSHLVDALYLVCGDRGFFYNPK. The rockfish insulin, in comparison with the human one, has 14 amino acid substitutions; an additional alanine is present at the N-terminal of the B-chain, whereas the 30th amino acid at the C-terminal is absent. In in vitro experiment, the 50% inhibition of the pork 125I-insulin binding to the rat liver plasma membrane was 4 nM, i.e., 50% of the standard pork insulin affinity (2 nM) to the insulin receptors. The pork rockfish insulin biological activity as determined in the mouse convulsion test in vivo was 18 ± 2.2 ME/mg or 75% of the pork hormone activity. It is suggested that the relatively low rockfish insulin biological activity is due to the presence of A8 asparagine position in the hormone structure

Functional insulin receptors are overexpressed in thyroid tumors: is this an early event in thyroid tumorigenesis?

Insulin receptor (IR), a member of the receptor tyrosine kinase family, is expressed in normal thyroid cells and affects thyroid cell proliferation and differentiation. The authors measured IR content in benign and malignant thyroid tumors by three independent methods: a specific radioimmunoassay, 125I-insulin binding studies, and immunohistochemistry. The results obtained were compared with the IR content in paired, adjacent, normal thyroid tissue. To assess IR function in thyroid carcinoma cells, glucose uptake responsiveness to insulin was also studied in a human transformed thyroid cell line (B-CPAP) and in follicular carcinoma cells in primary culture. In 9 toxic adenomas, the average IR content was similar to that observed in the 9 paired normal thyroid tissue specimens from the same patients (2.2+/-0.3 vs. 2.1+/-0.3). In 13 benign nonfunctioning, or "cold," adenomas, the average IR content was significantly higher (P < 0.001) than in paired normal tissue specimens (4.3+/-0.5 vs. 1.8+/-0.1). In 12 papillary and 10 follicular carcinomas, IR content was significantly higher (P < 0.001) than in the adjacent normal thyroid tissue (4.0+/-0.4 vs. 1.6+/-0.2 and 5.6+/-1.0 vs. 1.8+/-0.2, respectively). The finding of a higher IR content in benign "cold" adenomas and in thyroid carcinomas was confirmed by both binding and immunostaining studies. The current studies indicate that 1) IR content is elevated in most follicular and papillary differentiated thyroid carcinomas, and 2) IR content is also elevated in most benign follicular adenomas ("cold" nodules) but not in highly differentiated, hyperfunctioning follicular adenomas ("hot" nodules), which very rarely become malignant. This observation suggests that increased IR expression is not restricted to the thyroid malignant phenotype but is already present in the premalignant "cold" adenomas. It may contribute, therefore, to thyroid tumorigenesis and/or represent an early event that gives a selective growth advantage to transformed thyroid cells.

ATP-dependent Desensitization of Insulin Binding and Tyrosine Kinase Activity of the Insulin Receptor Kinase: THE ROLE OF ENDOSOMAL ACIDIFICATION

Incubating endosomes with ATP decreased binding of 125I-insulin but not 125I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased beta-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations beta-subunit tyrosine phosphorylation and IRK activity were markedly decreased. This was not observed with nonhydrolyzable analogs of ATP, nor with plasma membrane IRK, nor with endosomal epidermal growth factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by bafilomycin A1, indicating a role for endosomal proton pump(s). The inhibition of IRK was not due to serine/threonine phosphorylation nor was it influenced by the inhibition of phosphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the beta-subunit with 1 mM ATP did not prevent the inhibition of IRK activity on incubating with 5 mM ATP. To evaluate conformational change we incubated endosomes with dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Without DTT the predominant species of IRK observed was alpha2 beta2. With DTT the alpha beta dimer predominated but on co-incubation with 5 mM ATP the alpha2 beta2 form predominated. Thus, ATP-dependent endosomal acidification contributes to the termination of transmembrane signaling by, among other processes, effecting a deactivating conformational change of the IRK.

Insulin-receptor binding characteristics in perfused SHR and WKY rat hearts

This work uses a new heart-perfusion technique to measure 125I-insulin binding on capillary endothelium and myofiber cell membranes in Wistar-Kyoto and spontaneously hypertensive rats. Ringer–Lock buffer was infused at a rate of 1 ml min −1 in the presence of 20 meq l −1 K + and 125I-insulin through an aortic cannula. The effluent was collected through a catheter introduced into the right atrium. The capillary endothelial lining was removed by detergent treatment to expose the cardiac myocyte surfaces. A physical model describing a 1:1 binding stoichiometry of 125I-insulin with its receptors is proposed and the derived mathematical equations allow for the calculation of binding constants ( k n), unbinding constants ( k −n), dissociation constants ( k d), and residency time constants ( τ). The results showed that in the spontaneously hypertensive rats’ hearts significant alterations were not noticed in the kinetics of insulin binding with its receptor at the capillary endothelial site compared to hearts of the normotensive control Wistar-Kyoto rats. However, at the myocyte site and in the spontaneously hypertensive rats, steric, configurational, and/or structural modifications for insulin binding with the receptor were observed as indicated by changes in insulin affinity for its receptor. Hence, alterations in insulin binding rather than reduction in insulin receptor number due to hyperinsulinemia, can be considered among the peculiarities of insulin resistance in the spontaneously hypertensive rats. Hyperinsulinemia, therefore, may be considered an upregulatory process as a consequence of insulin-resistance. The results support the hypothesis that insulin-resistance on the myocytes could be a pathophysiologic defect in insulin-receptor structure, function and affinity, and therefore myocardial function.

Effects of ethanol ingestion on insulin binding to rat Leydig cells.

Effects of ethanol treatment and its withdrawal on insulin binding to isolated rat Leydig cells were studied. Mature rats were given ethanol by gastric intubation for 30 days at a dose of 3.0 g/kg body weight, twice daily, as a 25% (v/v) aqueous solution and treatment was withdrawn for the subsequent 30 days in an another group. Ethanol treatment markedly increased serum insulin and reduced the 125I-insulin binding to Leydig cells and the activities of Leydig cellular steroidogenic enzymes such as 3 beta-HSD and 17 beta-HSD. Withdrawal of ethanol treatment restored these changed values to their normal levels. The results suggest the possible involvement of subnormal insulin actions, as that of LH, in the ethanol-induced impairment of Leydig cellular steroidogenesis and the resulting hypoandrogenization associated with alcohol abuse.

Insulin-Induced Protein Tyrosine Phosphorylation Cascade and Signalling Molecules Are Localized in a Caveolin-Enriched Cell Membrane Domain

The cellular localisation of time- and temperature-dependent 125I-insulin binding, insulin-sensitive signalling proteins and the insulin-induced protein tyrosine phosphorylation cascade were assessed in subcellular fractions isolated on Iodixanol gradients from control and insulin-treated H35 hepatoma cells. Western blot analysis demonstrated that the concentrations of IRS-1, Shc, GRB-2, SOS, Syp, PI 3-kinase, MAP kinase and G iα were at least 10-fold higher in cell surface–derived, caveolin-enriched fraction than in a cell surface–derived, caveolin-poor fraction (i.e., the plasma membranes). Insulin treatment caused a 15-fold increase in tyrosine phosphorylation of IRS-1 in the caveolin-enriched fraction in 5 min at 37°C compared with a 3-fold increase in plasma membranes and a 6-fold increases in the cytosol and endosomes. Insulin also increased tyrosine phosphorylation of both a 72-kDa protein and the 46-kDa Shc isoform only in the caveolin-enriched fraction. Insulin treatment did not change the concentrations of insulin receptors or Shc but increased IRS-1 in the caveolin-enriched fraction, possibly recruited from the cytosolic pool. Insulin also increased the concentrations of insulin receptors, IRS-1 and Shc in endosomes, suggesting insulin-induced internalization of the insulin receptors and proteins activated with them. Electron microscopic analysis, with the use of a combination of colloidal gold-labelled insulin to label the insulin receptor and immunolabelling to detect caveolin or IRS-1, demonstrated the co-localisation of insulin receptors in caveolin- and IRS-1–containing vesicular structures. Differences in the insulin-induced protein tyrosine phosphorylation and concentrations of these proximal signalling proteins in the caveolin-enriched fraction, plasma membranes, and cytosol suggest that insulin receptors in the caveolae play a major role in initiating insulin’s signal transduction processes.

Obese Gene (Leptin) Receptors are Widely Distributed in Embryonic Tissues • 293

Leptin receptors (LR) mediate central (satiety) and peripheral metabolic actions of the translated and secreted product of the obese (ob) gene. While two isoforms as products of RNA splicing have been characterized, both of them bind leptin on the same epitope. Even though LR has been reported in adult tissues, limited information exists with respect to its presence and tissue-distribution in the embryo. We examined the spatial and temporal distribution of LR in the rat conceptus and placenta from d9 to d21 at 2 day intervals (term≈d21), and postnatal d15, and d30 (n=7/age/group). Employing frozen saggital embryonic and postnatal rat sections, 125I-leptin binding in the presence and absence of 0.6 × 10-6M unlabeled leptin was assessed by autoradiography and microdensitometry (Molecular Analyst Microdensitometry Program - 1.4.1, NIH). On d9, the placenta revealed LR while the known control (125I-insulin binding [IR]) was noted in amniotic membranes. At d11, LR was noted in the liver, while liver IR was present at d13. LR was present in the brain [d13], spine and long bones [d13], heart [d15], and kidney [d17]. The LR increased 2.5 fold in density from d9 to d21 (p < 0.01), with a subsequent 3-fold rise postnatally reaching a peak at d15 (p < 0.05). In contrast, IR demonstrated a 1.5-fold increase prenatally followed by a peak at postnatal d15. We conclude that 1] LR are present in embryonic tissues although the ontogenic peak is observed postnatally at d15, 2] LR are distributed in various tissues, with the earliest appearance being limited to the placenta and liver even before its appearance in the brain. We speculate that LR mediate the biological effects of leptin both centrally and peripherally in the embryo thereby regulating metabolism and growth.

Insulin binding sites in gills of the estuarine crabChasmagnathus granulata

Bovine 125I-insulin was injected into the estuarine crab Chasmagnathus granulata in order to study its distribution and specific uptake by tissues. The highest radioactivity uptake occurred in both anterior and posterior gills, which reached maximum values at 30-60 min following labeled insulin administration. Heart and hepatopancreas concentrated a very low amount of radioactivity (only 9 and 3%, respectively, of that shown by gills). A significant reduction of the uptake was observed in the gills when an excess of unlabeled insulin was injected together with the labeled hormone. In vitro studies also showed specific uptake of 125I-insulin by the gills incubated at 25 degrees C, which reached a plateau after 120-min incubation, suggesting a saturable process. The inhibition of 125I-insulin uptake was dose dependent on unlabeled insulin. Glucagon did not compete with radioactivity uptake by gills in vivo and in vitro. Further characterization of insulin-binding sites was performed in gill membrane. The amount of unlabeled insulin that prevented 50% of the 125I-insulin uptake was 7.78 micrograms/ml, and the Scatchard plot analysis established the presence of binding site with Kd of 3.11 microM and Bmax of 0.14 microM (r = 0.99). Ovine prolactin was not able to prevent. 125I-insulin binding to gill membrane. These findings seem to indicate the presence of specific binding sites for insulin or insulin-like substance in crab gills, which deserves further studies.

Binding of 125I-Insulin on Capillary Endothelial and Myofiber Cell Membranes in Normal and Streptozotocin-Induced Diabetic Perfused Rat Hearts

A heart-perfusion technique was employed to measure 125I-insulin binding on capillary endothelial and myocyte cell membranes in Sprague-Dawley rats. Animals were anesthetized, and the anterior chest wall excised to expose the mediastinal contents. The right and left superior and inferior venae cavae were dissected and tied, and another tie was passed around the aorta. A polyethylene catheter was introduced into the aortic lumen from cephalad to caudad to sit with its tip above the aortic valve. Another catheter was introduced into the cavity of the right atrium and both were anchored by sutures. Oxygenated Ringer-Lock buffer containing 20 mM/L K + and 125I-insulin was perfused at a rate of 1 mL/min via the aortic catheter. Concomitantly, the distal ascending aorta and venae cavae were ligated. The effluent was collected from the right atrial catheter at the same infusion rate. Animals were divided into two groups, the normal group and streptozotocin-induced diabetic group. Heart perfusion was done on both groups either without or after treatment with detergent (CHAPS) to remove the capillary endothelial lining. A physical model for 125I-insulin sequestration as a ligand to its receptors on endothelial and/or myocyte plasma membranes was proposed. The model described a reversible binding of ligand on cellular surface receptor concentration to fit a conservation equation and a first order Bessel function. The binding constants ( k n ), reversal constants ( k −n ), dissociation constants k d = k − n / k n , and residency time constants τ = 1/ k − n of 125I-insulin in normal untreated, normal CHAPS-treated, diabetic untreated, and diabetic CHAPS-treated hearts were estimated using a theoretically generated curve-fit to the data. Since insulin receptor binding on the capillary endothelial cell surfaces may serve to transport insulin from the intravascular to the subendothelial space, and since streptozotocin-induced diabetes was shown to diminish receptor autophosphorylation and kinase activity and hence internalization of insulin, then one can conclude the following from the data. In the normal heart, removal of the capillary endothelial lining with CHAPS did not alter k n , k −n , k d , and τ of insulin binding as compared to the normal untreated, whereas in the diabetic untreated heart these constants were altered, compared to the diabetic treated. Furthermore, the k n and k −n values in the diabetic CHAPS-treated hearts were the same as for the normals untreated and CHAPS-treated, respectively. In conclusion, the dissociation constants and residency time constants of all groups indicated the possible existence of two types of insulin receptors: the capillary endothelial cell surface insulin receptors with lower residency time (low affinity receptor or combination of insulin and IGF-1 receptors) and the myocyte plasma membrane insulin receptors with higher residency times (high affinity).

The Adipose Obese Gene Product, Leptin: Evidence of a Direct Inhibitory Role in Ovarian Function

Leptin, a recently-discovered hormonal product of the obese gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety and improves reproductive performance if injected into mice lacking circulating leptin. Therefore, we tested the hypothesis that leptin signals metabolic information to the reproductive system by directly affecting granulosa cell function. Doses of 10–300 ng/ml leptin had no effect (P > 0.10) on basal or insulin-induced numbers of granulosa cells cultured from small (1–5 mm) or large (≥8 mm) bovine follicles. Similarly, 30 and 300 ng/ml leptin had no effect (P > 0.10) on basal estradiol production. However, leptin, in a dose-dependent manner, inhibited (P < 0.05) insulin-induced progesterone and estradiol production by granulosa cells from small and large follicles. Leptin did not compete for specific 125I-insulin binding to granulosa cells. Furthermore, specific binding of 125I-leptin was demonstrable in granulosa cells. In conclusion, leptin, at physiological levels, can directly attenuate insulin-induced steroidogenesis of granulosa cells without affecting proliferation of this ovarian cell type. These results provide evidence to support the hypothesis that leptin can act as a metabolic signal to the reproductive system via direct action at the ovarian level.

The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function.

Leptin, a recently-discovered hormonal product of the obese gene, circulates in the blood at levels paralleling those of fat reserves and regulates satiety and improves reproductive performance if injected into mice lacking circulating leptin. Therefore, we tested the hypothesis that leptin signals metabolic information to the reproductive system by directly affecting granulosa cell function. Doses of 10-300 ng/ml leptin had no effect (P > 0.10) on basal or insulin-induced numbers of granulosa cells cultured from small (1-5 mm) or large (> or = 8 mm) bovine follicles. Similarly, 30 and 300 ng/ml leptin had no effect (P > 0.10) on basal estradiol production. However, leptin, in a dose-dependent manner, inhibited (P < 0.05) insulin-induced progesterone and estradiol production by granulosa cells from small and large follicles. Leptin did not compete for specific 125I-insulin binding to granulosa cells. Furthermore, specific binding of 125I-leptin was demonstrable in granulosa cells. In conclusion, leptin, at physiological levels, can directly attenuate insulin-induced steroidogenesis of granulosa cells without affecting proliferation of this ovarian cell type. These results provide evidence to support the hypothesis that leptin can act as a metabolic signal to the reproductive system via direct action at the ovarian level.

Expression of insulin receptors and of 60-kDa receptor substrate in rat mature and immature enterocytes.

The mechanism(s) by which rat immature enterocytes exhibit increased responsiveness to insulin before weaning is unknown. Therefore, we have analyzed the distribution, ontogeny, and molecular properties of insulin receptors (IR) and of related substrates in immature and mature enterocytes. IR were studied by radioligand binding assays, cross-linking labeling, immunohistochemistry, and in vitro phosphorylated substrates by immunoprecipitation. Regardless of age, 125I-insulin binding to IR was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. Binding capacity to villus cells from sucklings (day 14) exceeded three times that of older animals (day 30 and day 60). Scatchard analysis of equilibrium binding data confirmed an age-related decrease in low- and high-affinity receptor classes without change in affinity constants. In concordance, both alpha- and beta-IR subunits were more abundant in immature than in mature membranes. In vitro, insulin elicited the phosphorylation of three membrane proteins (96, 60 and 42 kDa), whose signals were virtually inhibited by preincubating membranes with antireceptor monoclonal antibodies. By immunoprecipitation, the 60-kDa signal was rapidly detected as a tyrosine-phosphorylated protein, expressed in mature and immature membranes, and identified as a receptor substrate phosphorylated in vitro by the IR tyrosine kinase. In conclusion, 1) increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of IR and 2) normal rat enterocytes express a 60-kDa phosphotyrosine protein identified as a direct substrate of the IR tyrosine kinase.