Effect of metformin on insulin binding to receptors in cultured human lymphocytes and cancer cells

Summary The effect of the biguanide metformin (dimethylbiguanide) on insulin binding in vitro to IM-9 lymphocytes and MCF-7 human breast cancer cells was studied. Metformin significantly increased insulin binding to both cell types: maximum increment was 47.1±7.0% > control in IM-9 and 38.0±6.1% in MCF-7 cells. The dose-response curves indicated that the latter cell line was more sensitive to metformin, with a significant effect apparent at a metformin concentration of 7.7×10^-6 mol/l, similar to the levels reached in patients treated with this drug. When compared with phenformin, metformin was less active in increasing insulin binding to cultured cells, the ratio between the two drug responses being similar to that of their therapeutic dosage in patients. Insulin binding increment due to metformin was reversible, was not dependent on new protein synthesis and was evident also in IM-9 lymphocytes that had been down-regulated by pre-incubation with insulin (10^-7 mol/l). This effect of metformin on insulin binding to receptors may contribute to the hypoglycaemic effect of this agent in patients.     

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increases insulin-like growth factor I (IGF-I) receptors in clonal osteoblastic cells. Study on interaction of IGF-I and 1,25-(OH)2D3

We previously reported a cooperative effect between insulin-like growth factor I (IGF-I) and 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] in murine clonal osteoblastic cells, MCT3T3-E1. In the present study, the possible mechanism of interaction between these hormones was investigated. The effect of IGF-I on 1,25-(OH)2D3 receptors in MC3T3-E1 cells was examined. The affinity and hormone binding capacity of 1,25-(OH)2D3 receptors were not altered by IGF-I. Immunoblot analysis showed about 54 kilodaltons (kDa) 1,25-(OH)2D3 receptors, similar to that observed for mouse fibroblasts. The synthesis of IGF-I by the cells under a serum-free condition was determined by RIA. The assay revealed immunoreactive IGF-I secreted by MC3T3-E1 cells (1.79 +/- 0.04 x 10(-9) M, mean +/- SE, n = 5). Rat GH significantly increased the concentration of IGF-I, but 1,25-(OH)2D3 did not. IGF-I radioligand-receptor assay revealed specific binding of IGF-I to MC3T3-E1 cells. The relative potency of IGF-I-related peptides to bind with the cells was in the order of IGF-I much greater than multiplication-stimulating activity (the rat homologue of IGF-II) greater than insulin, and the receptor protein migrated as a 130-kDa band in autoradiography. Scatchard analysis showed a significant increase in IGF-I binding sites by 50% after 3-day treatment with 5 x 10(-11) M 1,25-(OH)2D3, without any change in affinity. These results indicate that the interaction of IGF-I and 1,25-(OH)2D3 in the culture of MC3T3-E1 cells may be mediated by the effect of 1,25-(OH)2D3 on IGF-I receptors.     

Corticotropin positively regulates its own receptors and cAMP response in cultured bovine adrenal cells.

Bovine fasciculata adrenal cells contain specific high-affinity (KD approximately 2.3 +/- 0.4 x 10(-10) M) and low-capacity (1910 +/- 300 sites per cell) corticotropin (ACTH) receptors. Pretreatment of cells with ACTH, caused in a time-(maximum effect at 48 hr) and dose-(ED50 approximately 10(-11) M, Vmax = 10(-10) to 10(-9) M) dependent manner an increase in ACTH binding. This was due to a 4-fold increase in the number of binding sites without modification of the binding affinity. The same pretreatment also enhanced the cAMP response to further ACTH stimulation in a dose-dependent manner (ED50 approximately 10(-11) M) and to a lesser extent the response to forskolin. However, pretreatment with higher concentrations of ACTH (10(-8) M) reduced the binding and the cAMP response when compared to the effect of 10(-9) M. These ACTH effects, which were mimicked by 8-bromoadenosine 3',5'-cyclic monophosphate, required de novo protein synthesis. Pretreatment with 10(-13) to 10(-11) M ACTH also enhanced the steroidogenic responsiveness to further hormonal stimulation. However, at higher concentrations the hormone induced an apparent steroidogenic desensitization that was probably related to a depletion of endogenous cholesterol, since cortisol production in the presence of 22-(R)-hydroxycholesterol was increased. Neither angiotensin-II nor atrial natriuretic factor alone modified ACTH receptors, but angiotensin-II partially blocked the stimulatory effect of ACTH. Thus, ACTH is one of the few polypeptide hormones having a positive trophic effect on its own receptors and target-cell responsiveness.     

Characterization of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these peptides on adrenal cell function

We have characterized insulin-like growth factor I (IGF-I) and insulin receptors in cultured bovine adrenal cells by binding and cross-linking affinity experiments. At equilibrium the dissociation constant and the number of binding sites per cell for IGF-I were 1.4 +/- (SE) 0.3 x 10(-9) M and 19,200 +/- 2,100, respectively. Under reduction conditions, disuccinimidyl suberate cross-linked [125I]iodo-IGF-I to one receptor complex with an Mr of 125,000. Adrenal cells also contain specific insulin receptors with an apparent dissociation constant (Kd) of 10(-9) M. Under reduction conditions [125I]iodo-insulin binds to one band with an approximate Mr of 125,000. IGF-I and insulin at micromolar concentrations, but not at nanomolar concentrations, slightly stimulated DNA synthesis, but markedly potentiated the mitogenic action of fibroblast growth factor. Adrenal cells cultured in a serum-free medium containing transferrin, ascorbic acid, and insulin (5 micrograms/ml) maintained fairly constant angiotensin-II (A-II) receptor concentration per cell and increased cAMP release on response to ACTH and their steroidogenic response to both ACTH and A-II. When the cells were cultured in the same medium without insulin, the number of A-II receptors significantly decreased to 65% and the increased responsiveness was blunted. Treatment of such cells for 3 days with increasing concentrations of IGF-I (1-100 ng/ml) produced a 2- to 3-fold increase in A-II receptors and enhanced the cAMP response (3- to 4-fold) to ACTH and the steroidogenic response (4- to 6-fold) to ACTH and A-II. These effects were time and dose dependent (ED50 approximately equal to 10(-9) M). Insulin at micromolar concentrations produced an effect similar to that of IGF-I, but at nanomolar concentrations the effect was far less. The enhanced steroidogenic responsiveness of IGF-I and insulin-treated cells were related to an enhanced capacity to produce pregnenolone and an increased activity of several steroid hydroxylases. These results indicate that both IGF-I and insulin, acting through their own receptor, play an important role in the maintenance of specific adrenal cell functions. However, at physiological concentrations IGF-I is more potent than insulin.     

Binding characteristics and apparent molecular size of detergent solubilized nerve growth factor receptor of sympathetic ganglia.

Nerve growth factor (NGF), a hormone-like regulator of sympathetic neuron ontogeny and metabolism affects its target cells initially by associating with specific plasma membrane receptors. We have solubilized the NGF receptor of adult rabbit superior cervical ganglia (SCG) with the nonionic detergent Triton X-100. The high-affinity equilibrium binding constant of the detergent-extracted receptor is 2-8 x 10(-10) M. Gel chromatography of the receptor or the 125I-labeled NGF receptor complex on a column of Sepharose 6B indicated, in both cases, a single component of an apparent hydrodynamic radius of 71 +/- 5 A. In parallel investigations, we have confirmed the similarity between the hydrodynamic size of the NGF receptor of rabbit SCG and that of the insulin receptor of IM-9 lymphocytes evaluated by similar methods.     

Human growth hormone stimulates somatomedin C/insulin-like growth factor I production by the human lymphoid cell line, IM-9

The human lymphoid cell line, IM-9, is known to possess receptors for human growth hormone (hGH), but the only biological response that has been shown to follow binding of this hormone to the cells is receptor down-regulation. We have studied the actions of hGH on production of insulin-like growth factor I (IGF-I) by IM-9 cells. In order to demonstrate effects cells had to be transferred to a serum-free medium in which cell multiplication almost ceased, and cell viability fell to 50-60%. hGH stimulated IGF-I production by up to 400%. The effect was dose-related, but the dose-response curve was bimodal, with peaks of activity at approximately 15 ng/ml and 1000 ng/ml hGH. The effect of hGH was of slow onset, becoming significant only after about 24 h, and approaching a maximum after 2-5 days of treatment. hGH had a much greater stimulatory effect than non-primate growth hormones. The physiological significance of the effect observed is not yet clear, but it is apparent that the IM-9 line is a potentially useful model for study of the actions of growth hormone.     

Characterization of thyroid hormone receptors in human IM-9 lymphocytes

Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37 degrees C, nuclear binding of [125I]T3 was saturable (Kd 34 +/- 6 pmol/l) and of finite capacity (approximately equal to 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 +/- 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 +/- 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3'-isopropylthyronine, 89; T4, 32; 3,5-dimethyl-3'isopropylthyronine 2; 3,5-T2, 0.7, rT3, 0.4; 3'5'-T2, less than 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormone receptors from IM-9 cells but not HeLa cells bind to promoters of triiodothyronine-responsive genes

We have used thyroid hormone receptors from two different human cell lines to investigate receptor binding to the promoters of thyroid hormone-responsive genes. Receptors extracted from IM-9 cells or HeLa cells displayed virtually identical affinity and specificity for [125I]triiodothyronine binding. The cells expressed a c-erbA alpha gene in the same relative proportions as the receptor concentrations. Both receptors were bound to DNA-cellulose and could be displaced with increasing concentrations of calf thymus DNA or pBR322 DNA. Relative to pBR322 DNA (designated as 1), binding to the hGH gene promoter was 8.1 +/- 1.1 using the IM-9 cell receptor. With the HeLa cell receptor relative binding was only 1.1 +/- 0.2. Similar relative differences were obtained with the mouse glandular kallikrein gene, mGK-6. In heat stability studies the IM-9 cell receptor was more resistant to heat inactivation than the HeLa receptor. Triiodothyronine receptors with identical hormone binding patterns may require the presence of an unidentified factor(s) which allows correct recognition of regulation sequences within responsive genes.     

Neurohypophysial hormone regulation of Cl- secretion: physiological evidence for V1-type receptors in sea bass gill respiratory cells in culture

Neurohypophysial hormone receptors were studied in primary cultures of sea bass (Dicentrarchus labrax) gill respiratory-like cells grown on permeable supports. This preparation was previously shown to provide a functional model for investigating the hormonal regulation of Cl- secretion. Under control conditions, the cultured monolayered epithelium had a short-circuit current (ISC) of 3.5+/-1.1 micro A x cm(-2). This current had previously been identified as an active Cl- secretion. The addition of increasing concentrations of the fish neurohypophysial hormones, arginine vasotocin (AVT) or isotocin (IT), elicited a concentration-dependent stimulation of the ISC. Maximal increases of 60.9+/-12.1% and 117.7+/-28.0% above the basal ISC value were obtained for 10(-7) M AVT and IT respectively. Half-maximal effects were obtained for 3.1 x 10(-9) M AVT and for 1.4 x 10(-9) M IT. Mucosal application of 1.0 mM diphenylalamine-2-carboxylic acid (a specific blocker of Cl- channels) after serosal addition of 5 x 10(-8) M AVT or IT inhibited not only the basal but also the stimulated current, revealing a correlation with a hormone-dependent Cl- transport. Specific V1 or V2 receptor analogues of vasopressin (mammalian hormone) were used to characterize the type of neurohypophysial hormone receptors pharmacologically. While the V1 agonist [Phe2,Orn8]-oxytocin stimulated the basal Cl- secretion with a similar profile to that of AVT or IT, the V2 agonist [Deamino1,Val4,d -Arg8]-vasopressin had no effect. The V1 antagonist [d(CH2)5 1,O-Me-Tyr2,Arg8]-vasopressin used at a concentration of 5 x 10(-7) M totally reversed the 10-8 M AVT-stimulated Cl- secretion, whereas the V2 antagonist [d(CH2)5 1,d -Ile2,Ile4,Arg8,Ala9]-vasopressin used at the same concentration had no significant effect. In contrast, similar experiments carried out in the presence of 10(-8) M IT showed that both antagonists significantly reduced the IT-stimulated Cl- secretion, with the efficiency of the V1 receptor antagonist being significantly greater than that of the V2. This study provides evidence for neurohypophysial hormone control of Cl- secretion in fish cultured gill respiratory cells. It suggests that on a physiological basis the hormonal effect is shared by the two peptides present in fish neurohypophysis (AVT and IT), acting by means of two distinct, although pharmacologically similar, V1-type receptors (according to the mammalian classification). These specific receptors are expected to play an important role in controlling ion homeostasis in seawater fish.     

Annual variations in the binding of insulin to hepatic membranes of the frog Rana esculenta.

Amphibia undergo regular annual cycles of metabolic activity that are influenced by both exogenous factors and hormones. Insulin binding to crude frog hepatic membranes was studied throughout the year. The general character of insulin binding was similar to that in other vertebrates; the maximum specific binding was achieved after 4 hr at 4 degrees, the optimum pH was 7.8, half-maximal displacement of bound insulin was from 9 x 10(-10) to 1 x 10(-9) M, and insulin analogs competed for the insulin receptor in line with their relative biological potencies. A biphasic Scatchard plot and negative cooperativity of the receptor were also observed in frog liver membranes. Affinity constants from Scatchard plots revealed high and low affinity binding sites which were unchanged during the year. The seasonal cycle, however, markedly affected the binding capacity for both sites. Maximum binding occurred in May-June and the minimum in November-December for both classes of receptors. Binding capacities ranged from 1.71 to 11.33 fmol/mg protein for the high affinity sites and from 432 to 3171 fmol/mg protein for the low affinity sites. It is concluded that annual cycles of insulin binding reflect modulation of receptor number rather than receptor affinity.     

Androgen and estrogen receptors in brain cytosol from male, female, and testicular feminized (tfm/y hermaphrodite) mice.

Specific binding of [3H] 5alpha-dihydrotestosterone (DHT) and [3H] estradiol by cytoplasmic extracts from whole brain of castrated male, female, and androgen-insensitive, testicular feminized (tfm/y male-female), mice has been investigated using glycerol gradient centrifugation and charcoal assay. Mouse brain cytosol contains macromolecules with the characteristics of steroid hormone receptors, binding preferentially with high-affinity androgens or estrogens. Both DHT- and estradiol-receptor complexes migrate at 8-9 S in gradients at low ionic strength and at 4-5 S in gradients containing 0.5M KCl. KD's (mean +/- SE) for DHT binding by brain cytosol from castrated males, females, and tfm/y male-female are 1.1 +/- 0.4, 0.9 +/- 0.4, and 0.8 +/- 0.1 X 10(-9)M, respectively. DHT binding activity in brain cytosol from tfm/y male-female mice is reduced to about 20-30% of that from their normal littermates, as is the case for tfm/y male-female kidney cytosol. The residual androgen receptor in tfm/y male-female brain cytosol has normal sedimentation properties. Unlike the situation for androgen binding, the number of estradiol binding sites is comparable in brain cytosol from male, female, and tfm/y male-female mice. KD's (mean +/- SE) for estradiol binding are 1.6 +/- 0.5 X 10(-10)M for castrated males, 2.4 +/- 0.4 X 10(-10)M for females, and 1.8 +/- 0.4 X 10(-10)M for tfm/y male-female. Cross-competition experiments with unlabeled estradiol, DHT, or testosterone, have shown a difference in the degree of specificity of the androgen and estrogen receptors, the estrogen receptor having considerably more specificity. For the interaction of estradiol with the androgen receptor, the Ki is 8-9 X 10(-9)M. The decrease in the number of DHT binding sites in the brain of tfm/y male-female mice without a concomitant decrease in estradiol binding sites, and the different specificities of the two sites, point to the existence of distinct androgen and estrogen receptor molecules in mouse brain cytosol.     

Reactivity of non-primate growth hormones and prolactins with human growth hormone receptors on cultured human lymphocytes.

Ovine placental lactogen is as reactive as human growth hormone with the human growth hormone receptor of cultured human (IM-9) lymphocytes, which confirms the findings of Carr and Friesen with receptors of human liver. We now also show that bovine and ovine growth hormones and ovine prolactin have reactivity for the human growth hormone receptor on IM-9 lymphocytes that is of the same order of magnitude (0.03%) as that previously reported for human placental lactogen. The binding studies predict that these non-primate hormones will have biological effects on skeletal growth in primates, either as agonists or antagonists. Previous studies have shown that when IM-9 lymphocytes are exposed to human growth hormone for 18 h at 37 C, there is a time and concentration dependent loss of human growth hormone receptors, and the magnitude of the loss of receptors after preincubation for 18 h at 37 C is greater than the average occupancy of receptors under steady state conditions for 90 min at 30 C. In the present study we show that human and ovine placental lactogens, ovine prolactin, and bovine and ovine growth hormones also produce this effect on the human growth hormone receptor. Since the cellular process by which a hormone induces loss of its own receptors appears to require binding of the hormone to its receptor as well as one or more subsequent steps in hormone action, it is likely that all of the preparations that induce receptor loss will be shown to have some agonist activity of human growth hormone in promoting skeletal growth in primates. Further, these studies extend the interrelationships between primate and non-primate pituitary and placental hormones from what has been suggested previously from biological and structural studies.     

Growth hormone specific binding sites : characteristics of lactogenic receptors induced by estrogens (author's transl)]

In vitro binding of growth hormone was characterized in rat liver. Microsomal preparations were found more active than membranes purified with an aqueous two-phase polymer system. Binding conducted at 4 degrees C was found optimal after 72 hours of incubation in 5 mM Tris-Maleate buffer pH 6.4 with 25 mM CaCl2. Injecting estrone (25 microgram/100 g B.W.) for one week induced the formation of lactogenic receptors, and increased the specific binding of hGH from 2.8 +/- 1.9 to 22.3 +/- 7.1%. Prolactin and hGH, but not rGH or other pituitary hormones, could displace radioactive hGH. With incubations conducted at 37 degrees C, equilibrium was reached more rapidly but at the cost of a more extensive degradation. The presence of membrane receptors in the medium partly protected the hormone against aggregation and degradation. Scatchard plots were obtained from experiments conducted under optimal conditions and analyzed on computer using a program based on an iterative method. Data indicated that lactogenic receptors possessed a single specific binding site for hGH with a constant (Ka) of 2.18 +/- 0.22 x 10(9) M-1 and a binding capacity of 304 +/- 91 fm/mg proteins.     

Intracellular potassium depletion in IM-9 lymphocytes suppresses the slowly dissociating component of human growth hormone binding and the down-regulation of its receptors but does not affect insulin receptors.

We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin-receptor binding kinetics and down-regulation.     

Detection of glucocorticoid receptors on Friend erythroleukemia cells.

The inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells by steroids led us to examine these cells for the presence of glucocorticoid receptors. Direct assessement of dexamethasone binding revealed high-affinity dexamethasone receptors on the untreated cells. The specific binding of [3H]dexamethasone was dose dependent. At a concentration of 10(-8) M, almost all binding sites were occupied. The mean number of binding sites per cell in two separate experiments was 8045 and 7191, respectively, and the Kd varied between 3.38 and 3.49 x 10(-9) M. Dimethyl sulfoxide treatment led to a decrease in the number of dexamethasone binding sites on the cells induced to differentiate. After 5 days of treatment, the mean number of sites per cell was reduced to 1216 and 896 in two experiments, with a Kd of 5 x 10(-9) M. Dexamethasone treatment resulted in a moderate decrease in the efficiency of colony formation within 72 hr after the cells were plated in methylcellulose. The mechanism of this inhibitory effect is unknown. However, it was also dose dependent and could be abrogated by appropriate concentrations of progesterone or 11-deoxycortisone. These results suggest that the steroid effects on growth and differentiation of the erythroleukemia cells may be mediated via glucocorticoid receptors.     

Hormone responsive human breast cancer in long-term tissue culture: effect of insulin.

The mechanisms of steroid and peptide hormone action in human breast cancer are poorly understood. We have previously characterized a cell line of human breast cancer in long-term tissue culture that possesses various steroid hormone receptors and responses, providing a model for the study of steroid hormone action. The present studies describe a human breast cancer in vitro that responds to physiologie concentrations of insulin with an increased rate of macromolecular synthesis and growth. Thymidine and uridine incorporation in cells in serum-free medium are stimulated by 10(-11) M insulin and are maximal with 10(-8) M. Leucine incorporation is stimulated by 5 X 10(-11) M insulin and is maximal with 10(-9) M. Significant stimulation of uridine and leucine incorporation is evident by 3 hr and maximal by 10 hr. A 10-hr lag period exists for insulin stimulation of thymidine incorporation, which is maximal form 14 to 24 hr. The effect of 10(-8) M insulin on macromolecular synthesis is accompanied by a 69% increase above controls in the number of cells after 24 hr. The effect on macromolecular synthesis is observed in glucose-free medium. Insulin's effect on protein synthesis is not blocked by inhibition of RNA synthesis with actinomycin D. Glucocorticoids partially inhibit the action of insulin in these cells. This system provides a model for studying insulin action, and suggests that some human breast cancer may show growth regulation by insulin.     

Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes.

Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide shown to be cosecreted with insulin from the pancreatic beta-cells. We have investigated the existence and characteristics of IAPP binding sites in the rat. Specific binding sites for [125I]IAPP were found to be highest in the lung followed by the stomach fundus, spleen, brain stem, hypothalamus, and the liver, respectively. The interaction of [125I]IAPP with its binding site was rapid and temperature dependent, displaying optimum binding at 4 C. This may be explained by the rapid degradation of the label observed at 22 C and 37 C, as determined by fast protein liquid chromatography analysis, and also degradation of the receptor at 37 C. Binding of [125I]IAPP was rapidly dissociated by the addition of 200 nM unlabeled peptide. The presence of nonmetabolizable GTP-gamma-S (0.5 microM) reduced binding, thus suggesting the coupling of the binding site to a G protein. Rat IAPP displaced [125I]IAPP displaying an IC50 of 5.75 x 10(-9) M (mean, n = 4). Displacement was also seen with human IAPP (IC50 = 5.53 x 10(-8) M), human alpha-calcitonin gene-related peptide (CGRP) (IC50 = 3.8 x 10(-8) M), rat alpha-CGRP (IC50 = 9.0 x 10(-7) M), and rat beta-CGRP (IC50 = 5.53 x 10(-8) M); suggesting an IAPP-specific binding site. Scatchard plots for rat IAPP binding in the lung gave a dissociation constant of 10.4 +/- 2.63 nM (mean +/- SE, n = 4) and maximal binding of 3.1 +/- 0.97 pmol/mg (mean +/- SE, n = 4), displaying a single class of binding site. Chemical cross-linking analysis showed binding of IAPP to sites of Mr 67,000, 64,000, and 38,000. These findings suggest that specific IAPP binding sites exist which differ from the CGRP receptors in rat tissues. This indicates a possible novel autocrine/paracrine role for IAPP.     

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.     

Human growth hormone and cortisol response to insulin stimulation in aging.

The influence of age on plasma growth hormone (HGH) and cortisol response to i.v. insulin (0.1 U/kg of body weight) was evaluated in 32 healthy subjects whose ages ranged between 20 and 84 years. A significant reduction in HGH response to insulin was observed with aging. In the young (20-34 years), middel-aged (35-49 years), and elderly (53-84 years) groups, average HGH peaks were 46.51 +/- 7.37, 29.95 +/- 5.35, and 14.31 +/- 2.39 ng/ml while average HGH areas were 2.911 +/- 0.484, 1.654 +/- 0.316, and 0.699 +/- 0.149 mug-min, respectively. Since insulin's hypoglycemic effect became less rapid with aging, this could, in part, explain the progressive decline in the HGH response to insulin. This phenomen may also be attributed to histological changes occurring in the pituitary with aging. Moreover, cortisol response was similar to all three age groups. These findings suggest that, while HGH response to insulin is correlated with age, adrenal response does not show any important modifications with aging.     

Insulin receptor down-regulation, prevention at a post-receptor site

Nicotinamide (50mM) prevented insulin-mediated down-regulation of insulin receptors in IM-9 lymphoblastoid cells. Half-maximum effectiveness was between 10 and 33mM. Nicotinamide did not influence insulin binding to the cells, cell viability, insulin degradation or protein synthesis. A variety of inhibitors of ADP-ribosylation reactions besides nicotinamide, most of them pyridine analogues, similarly prevented insulin-induced receptor loss. Spermine decreased the number of insulin receptors in IM-9 cells, but this effect was not inhibited by nicotinamide.