Number Of Insulin Binding Sites Research Articles

Glucose tolerance in amyotrophic lateral sclerosis.

Blood glucose and plasma insulin during an oral glucose tolerance test were determined in 21 patients with amyotrophic lateral sclerosis and in 10 control patients matched for age, obesity and physical activity. In addition, 125I-insulin binding to circulating erythrocytes were studied in a subgroup of 4 ALS patients and 8 controls. Both impaired glucose tolerance and diabetes mellitus were evenly distributed between the study groups, and no difference in mean blood glucose levels during the OGTT was found between ALS and control patients. Fasting plasma immunoreactive insulin concentration was significantly higher in ALS patients as compared to controls, but plasma IRI increments to the glycemic stimulus were similar in the 2 groups. The number of insulin binding sites per cell appeared lower in patients with ALS, but the difference in receptor concentration was not statistically significant. In addition, the specific bound fraction of 125I-insulin showed no difference between ALS and control patients. In conclusion, we were unable to demonstrate any marked deterioration of glucose tolerance or increase in insulin resistance in patients with ALS.

Discordance between glucokinase activity and insulin and glucagon receptor changes occurring during liver regeneration in the rat.

During regeneration of rat livers following 70% hepatectomy, insulin binding sites on hepatocyte plasma membranes are increased after 24-48 hours, glucagon binding sites are reduced on days 2-8, and the resultant insulin/glucagon binding ratio is markedly increased. An apparent paradox was the finding of a depression of the activity of an insulin associated enzyme, glucokinase, at a time when the number of insulin binding sites was increased.

Functional defects in adenylyl cyclase signaling mechanisms of insulin and relaxin in skeletal muscles of rat with streptozotocin type 1 diabetes

Abstract Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5 degrees C, cell surface [125I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37 degrees C, due toa decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [35S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37 degrees C after 2 h at 42.5 degrees C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [14C]glucose incorporation into glycogen measured at 37 degrees C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4-fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress.

Mechanisms of membranoreceptor function disturbances in pregnant patients with insulin-dependent diabetes mellitus

Studies of membranoreceptor system in patients with insulin-dependent diabetes mellitus revealed that insulin resistance in pregnant patients with type I diabetes is caused by disordered cellular sensitivity to threshold physiological and submaximal insulin doses, whereas the maximal doses of the hormone normalize glucose consumption by the cells. High insulin doses intensify lipid peroxidation, normalize the status of membranous proteins, reduce the number of thiol groups, reduce AOA level in membranes, and, hence, reduce membranous capacity to bind active peroxide radicals. Structural and functional changes in red cell membranes are associated with reduced affinity of insulin receptors, reduction in the number of insulin-binding sites in membranes, this disordering intracellular effects of insulin.

Development of hyperglycaemia and insulin resistance in conscious genetically diabetic (C57BL/KsJ-db/db) mice

A bolus injection of insulin dose-dependently reduced plasma glucose levels in genetically diabetic (db/db) mice and their normoglycaemic littermates (+/+ mice) aged 5, 8 and 12 weeks. Compared between the groups, the dose-response curves showed that insulin resistance was already present in the 5-week-old db/db mice when they were still normoglycaemic. The minimum effective dose of insulin was lower in the +/+ (32 Μg/kg) than in the db/db (100 Μg/kg) mice and the maximum response which was obtained at 320–1000 Μg/kg of the hormone was higher in the former (about 80%) than in the latter (about 55%). Although the basal plasma glucose levels in the db/db mice were significantly increased with age as compared with those in the +/+ mice, the insulin response curves were identical in the db/db mice from 5 to 12 weeks of age. The number of insulin binding sites were significantly decreased by 22–50% (5–12-week-old) in the liver plasma membrane from the db/db mice compared with that from the +/+ mice, while its affinity was not significantly changed between the groups. Streptozotocin (100 mg/kg, i.p.) treatment increased the number of insulin receptors in the db/db mice to a number comparable with those in the +/+ mice. Coinciding with the change, the hypoglycaemic action of insulin was slightly enhanced in the streptozotocin-treated db/db mice compared with that in nontreated db/db mice, but was still considerably depressed when compared with that in +/+ mice. It is concluded that a simple dose-response study using a bolus injection of insulin can detect insulin resistance in db/db mice which occurred before the manifestation of hyperglycaemia and remained constant during the course of developing hyperglycaemia. Down-regulation of insulin receptors due to the hyperinsulinaemia may play only a part in the insulin resistance.

Correlation of insulin pretreatment and insulin binding of Amoeba proteus—a new technique for evaluation

1. 1. Insulin binding sites of Amoeba proteus were analysed. The developed technique helped to compare equivalent pseudopodia membranes, according to their fluorescein-isothiocyanate (FITC)-insulin binding capacity. 2. 2. The number of insulin binding sites decrease in the control conditions as a fraction of time. 3. 3. The down regulation of binding sites (or similar phenomenon) lasts ~48 hr. 4. 4. Only low concentrations of insulin can induce a binding site elevation in the long term. 5. 5. Hormonal imprinting could be presented in Amoeba. 6. 6. The developed technique is suitable for detection of membrane dynamics in Amoeba proteus.

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

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

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations.

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.

Cell-cycle regulation of insulin-stimulated tyrosine aminotranferase activity in rat hepatoma cells

Amongst the proteins that are subjected to variation during the cell division cycle few are under hormonal regulation. The variation in amount of tyrosine aminotransferase (TAT) in the hepatic tissue is under the control of glucagon, glucocorticoids and insulin. It has been reported that the inducibility of TAT activity by dexamethosone in rat hepatoma (HTC) is limited to the late G1 and the S portions of the cell cycle. Evidence is presented in this report that in the rat hepatoma Fao, insulin (which has the capability to promote both cell growth and hormonal effects via its own receptors) modulates the TAT activity during the cell cycle. The maximal insulin-stimulated induction of TAT activity was observed at the end of the G1 phase and then decreased as cells progressed through their mitotic cycle. The number of insulin binding sites per cell was decreased by only 30% during the same period of time. Furthermore, the extent of receptor autophophorylation decreased in the same proportion, suggesting that insulin receptors remained functional through the whole cell cycle. In fact, another insulin-stimulated cellular function, nertral amino-acid transport, was not modified as cells progressed into the S phase. Hydroxyurea, which is known to prevent cell progression into the S phase, stabilized the insulin-induced TAT activity at its maximal level for several hours. Reciprocally, removal of hydroxyurea resulted in a concomitant decrease in TAT activity and reinitiation of DNA synthesis.

Insulin receptor expression in Burkitt lymphoma cell lines.

The specific binding of insulin to 7 different Burkitt lymphoma cell lines containing chromosomal translocations t(8;14), t(8;2) and t(8;22) was markedly decreased when compared to binding to lymphoblastoid cells of normal karyotype derived from Burkitt lymphoma patients or the human IM-9 lymphoblastoid line. The number of insulin-binding sites on intact Burkitt cells was decreased by greater than 90% compared to lymphoblastoid cells, with no change in affinity. This decrease in binding was paralleled by reduced amounts of insulin receptor alpha (Mr 130,000) and beta (Mr 95,000) subunits detected by cell-surface-labelling and insulin receptor mRNA transcripts, indicating that transcription of receptor mRNA is decreased in Burkitt cells compared to lymphoblastoid cells and/or that receptor mRNA is less stable. Burkitt cells displayed negligible insulin-stimulated beta subunit auto-phosphorylation, which could reflect either their decreased number of receptors or a defect in signal transduction. Structural analysis also revealed that the Burkitt cells had an increase in a precursor form (Mr 210,000) of the receptor, suggesting that decreased expression of the receptor may be associated with defective processing. Four Burkitt cell lines with t(8;14) also had reductions of 45-100% in expression of class-1 major histocompatibility (MHC) antigens. The expression of insulin receptors in both Burkitt and lymphoblastoid cells correlated with the expression of class-1 MHC antigens. There was also an inverse correlation between the expression of c-myc and both insulin receptors and class-1 MHC antigens. As the insulin receptor is absent on resting B cells and is induced after cell activation, the decrease in receptor expression on Burkitt cells may reflect their less activated phenotype compared to lymphoblastoid cells.

17-β-estradiol treatment maintains differentiative potential of virgin mouse mammary gland in culture and its responsiveness to insulin

Synthesis of the milk proteins, casein and alpha-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-beta-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H]thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P less than 0.001) of that obtained in intact mice, and 17-beta-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P less than 0.001) enhanced [3H]thymidine incorporation into DNA in cultured mammary explants from both intact and 17-beta-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 +/- 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P less than 0.05) lower than those from both intact (2870 +/- 300 sites/cell) and 17-beta-estradiol-treated castrated animals (2860 +/- 190 sites/cell). The present findings suggest that 17-beta-estradiol maintains growth and differentiative responses of mammary epithelial cells to insulin, which may be through preserving the number of insulin binding sites in the cells.

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation.

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.

Concanavalin A changes not only the number of insulin binding sites but also the binding affinity in rat hepatoma cells in culture.

When rat hepatoma cells (R-Y121B) were first incubated with labeled insulin, followed by concanavalin A, at 500 micrograms/ml at 23 degrees C, the total cell-associated radioactivity increased. At 4 degrees C, however, this increase was not observed. Scatchard analysis revealed that concanavalin A increased insulin binding affinity. The bound insulin was internalized together with concanavalin A. When the cells were incubated with concanavalin A prior to insulin addition, however, the total cell-associated radioactivity decreased at both temperatures. This was caused by the masking of insulin binding sites by the lectin.

Insulin binding to differentiating muscle cell line L6

We studied insulin binding to cultured differentiating muscle cell line L6. Insulin binding to the cells reached a plateau after incubation with 125I-insulin for 4 h at 22 degrees C, and was at an optimum at pH 7.8. Preincubation with 10 microM of hydrocortisone for 36 h at 37 degrees C resulted in significantly increased insulin binding (1.73 +/- 0.12 ng/mg protein for treated cells vs. 1.13 +/- 0.025 ng/mg protein for control cells, mean +/- SD, P less than 0.001). Preincubation with 1 microM of hydrocortisone or 1 microM of dexamethasone also led to increased binding. The number of insulin-binding sites per cell increased 2.5-fold in glucocorticoid-treated cells (9.7 X 10(3) sites/cell for treated vs. 3.8 X 10(3) sites/cell for control cells). Preincubation with trifluoperazine (5 microM), a calmodulin inhibitor, did not affect insulin binding to the cells. These results indicate that glucocorticoid might have some important role in regulating the number of insulin receptors in L6 muscle cells.

Effect of training on insulin binding to rat skeletal muscle sarcolemmal vesicles.

We examined the hypothesis that the exercise training-induced increase in skeletal muscle insulin sensitivity is mediated by adaptations in insulin binding to sarcolemmal (SL) insulin receptors. Insulin binding studies were performed on rat skeletal muscle SL isolated from control and trained rats. No significant differences were noted between groups in body weight or fat. An intravenous glucose tolerance test showed an increase in whole-body insulin sensitivity with training, and specific D-glucose transport studies on isolated SL vesicles indicated that this was due in part to adaptations in skeletal muscle. Enzyme marker analyses revealed no differences in yield, purity, or contamination of SL membranes between the two groups. Scatchard analyses indicated no significant differences in the number of insulin binding sites per milligram SL protein on the high-affinity (15.0 +/- 4.1 vs. 18.1 +/- 6.4 X 10(9)) or on the low-affinity portions (925 +/- 80 vs. 884 +/- 106 X 10(9)) of the curves. The association constants of the high-affinity (0.764 +/- 0.154 vs. 0.685 +/- 0.264 X 10(9) M-1) and of the low affinity sites (0.0096 +/- 0.0012 vs. 0.0102 +/- 0.0012 X 10(9) M-1) also were similar. These results do not support the hypothesis that the increased sensitivity to insulin after exercise training is due to changes in SL insulin receptor binding.

Characteristics of insulin binding to H35 hepatoma cells

Well differentiated hepatoma cells in culture exhibit insulin binding and insulin effects. We have studied insulin binding in control and in H35 hepatoma cells down-regulated with insulin. H35 cells were grown in monolayers in alpha MEM. Insulin binding was measured with A14 mono 125I labelled insulin 72 h after seeding. Binding was time, temperature and pH-dependent. Receptor down-regulation was studied by exposing cells to increasing concentrations of unlabelled insulin. Monolayers preincubated with 10 micrograms/ml unlabelled insulin for 24 h showed a decrease of 65% in the number of insulin binding sites. There was no change in affinity.

In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix

The distribution of receptors for insulin and epidermal growth factor along the longitudinal axis of the uterine cavity was studied in 28 uteri obtained from women of reproductive age undergoing hysterectomy for benign conditions. Insulin binding to crude plasma membranes was higher (p < 0.05) in the secretory than in the proliferative phase of the menstrual cycle in all uterine segments (fundus to cervix). Epidermal growth factor binding did not change during the menstrual cycle but the number of epidermal growth factor binding sites was higher in the cervix than in the fundus (p < 0.05). Scatchard plots of binding data, obtained with crude plasma membranes from pooled uteri, were curvilinear; the high-affinity sites had dissociation constants of 1 to 4 nmol/L and receptor concentrations of 100 to 300 fmol/mg of protein, for both iodine 125-labeled insulin and 125I-labeled epidermal growth factor. In plasma membranes, obtained from another 15 uteri, mouse nerve growth factor (3.3 μg/ml) decreased the binding of insulin by an average of 17% (p < 0.005); in the decidua of a pregnant uterus at 12 weeks Scatchard analysis showed that nerve growth factor decreased the affinity but not the number of insulin-binding sites. Nerve growth factor had no effect on epidermal growth factor binding. Human prolactin (2 μg/ml) also decreased insulin binding by an average of 18% (n = 5, p < 0.025) but had no effect on epidermal growth factor binding. These “baseline” data will be useful in further studies of the possible interactions between (1) receptors for various peptide growth factors and (2) sex steroid hormones, in normal and neoplastic endometrium and cervix.

Peptide binding to intestinal epithelium: Distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [ 125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.

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 M r of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% β-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 μM unlabeled insulin, but not by 1 μM 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 M r 145 000 and an M r 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.