Biomedical subjects

A C Moses

Publications and source records attributed to A C Moses.

At least 73 records · Page 4Linked to original sources

PubMed1988

Supranormal stimulation of deoxyribonucleic acid synthesis in FRTL5 cells by serum from patients with untreated acromegaly.

Insulin-like growth factor I (IGF-I) is one of a number of mitogenic factors in the serum of animals and humans. We demonstrated previously that IGF-I is a potent mitogen for FRTL5 cells, a line of rat thyroid follicular cells. In this study, we assessed the relevance of this finding with respect to the levels of IGF-I found in human serum by comparing the effects of normal serum and serum from patients with untreated acromegaly or hypopituitarism on DNA synthesis in quiescent FRTL5 cells. As expected, when added to cells maintained in Coon's modified Ham's F-12 medium containing 0.1% BSA, but devoid of insulin, transferrin, TSH, or calf serum, normal serum produced a dose-dependent stimulation of [3H]thymidine incorporation into DNA. A similar, but more marked, effect was produced by the addition of serum from patients with untreated acromegaly. In multiple experiments, a standard concentration (0.5%) of acromegaly serum was more stimulatory to DNA synthesis than was normal serum. In a single experiment designed to eliminate interassay variation and define the relationship between the ability of serum to stimulate DNA synthesis and its IGF-I concentration, studies were performed with 0.5% concentrations of serum from 9 normal subjects, 15 patients with untreated acromegaly, and 3 patients with panhypopituitarism. On the average, [3H]thymidine incorporation in the presence of serum from patients with acromegaly was approximately 3 times, and that in the presence of serum from patients with hypopituitarism only one fourth, that in the presence of serum from normal subjects; there was no overlap of individual values in the three groups. For the entire study group, we found a highly significant correlation (r = 0.86) between the serum IGF-I concentration and the ability of that serum to stimulate thymidine incorporation into the DNA of FRTL5 cells. The stimulatory effects of serum from both normal and acromegalic subjects were decreased or abolished by the addition of a monoclonal antibody against IGF-I. In hypophysectomized rats, GH increases the thyroid to body weight ratio and enhances the effect of TSH to promote thyroid growth. Further, an inordinate frequency of nontoxic goiter in patients with acromegaly has been reported. Taken together with these observations, our findings suggest that the effect of IGF-I to promote thyroid cell growth in vitro has a counterpart in the living animal or patient.

Acromegaly↗

PubMed1988

Evidence that receptor aggregation may play a role in transmembrane signaling through the insulin-like growth factor-I receptor.

alpha IR-3 is a mouse monoclonal antibody that binds to an epitope on the human insulin-like growth factor I (IGF-I) receptor and inhibits [125I]IGF-I binding to this receptor on human skin fibroblasts (HSF) and Hep G2 human hepatoblastoma cells. Unlike the natural ligand (IGF-I), neither intact alpha IR-3 nor its monovalent Fab fragment stimulate aminoisobutyric acid (AIB) uptake in HSF, and both competitively antagonize IGF-I's ability to produce this effect. However, when HSF are incubated with alpha IR-3 or its Fab' fragment, subsequent exposure to anti-mouse immunoglobulin G (IgG) produces a potent stimulation of AIB uptake. Anti-Mouse IgG by itself does not effect AIB uptake. alpha IR-3 also antagonizes IGF-I's ability to stimulate glycogen synthesis in Hep G2 cells. As with AIB uptake in HSF, the combination of alpha IR-3 followed by anti-mouse IgG stimulates glycogen synthesis in Hep G2 cells to the same extent as that produced by IGF-I. The triggering of these two biological effects depends on the concentration of both alpha IR-3 and anti-mouse IgG. These results are consistent with the possibility that local aggregation or cross-linking of IGF-I receptors plays an important role in transmembrane signaling by this receptor.

Aminoisobutyric Acids↗

PubMed1987

Effects of sodium taurodihydrofusidate on nasal absorption of insulin in sheep.

To investigate the utility of a novel adjuvant, sodium taurodihydrofusidate (STDHF), as an enhancer of mucosal permeation of drugs, experiments involving intranasal insulin:STDHF administration in sheep were performed. Rabbit erythrocyte lysis assays were employed to assess the relative membrane lytic activity of STDHF, as well as that of its glycine-conjugated analogue, compared with a nonionic detergent and a common bile salt. Equivalent weight concentrations of the fusidates were found to be 5- to 10-fold less lytic than the bile salt and at least 100-fold less lytic than the nonionic detergent laureth-9. Provided the concentration of STDHF was greater than its critical micellar concentration, formulations of insulin with STDHF greatly enhanced intranasal insulin absorption. Optimal nasal insulin absorption was attained at a molar ratio of STDHF to insulin of 5:1. In addition, intranasal absorption was linearly related to insulin dose. Compared with intravenous administration, the mean bioavailability of intranasal insulin was 16.4%. Interovine variability was low, with a coefficient of variation of 14% for 12 animals. It was found that intranasal absorption of sodium insulin was not significantly different from that of zinc insulin. However, formulations of both crystalline insulin preparations were absorbed more efficiently than a formulation prepared using commercially available solutions of U-500 insulin. The results taken together indicate that STDHF is an excellent enhancer of insulin absorption from the nasal mucosa.

Absorption↗

PubMed1987

Somatomedin-C/insulin-like growth factor-1 in infants with congenital hypothyroidism during the first week of life.

Levels of somatomedin-C/insulin-like growth factor-1 (Sm-C/IGF-1) were determined in whole blood collected on filter paper from 41 newborns with congenital hypothyroidism and 183 full-term neonates with normal thyroid function. The mean Sm-C/IGF-1 value of the untreated hypothyroid infants (0.10 U/ml) was virtually identical with that of the normal controls (0.11 U/ml). We are unable to confirm earlier findings of others that Sm-C/IGF-1 concentrations are lower in hypothyroid newborns than in normal infants.

Congenital Hypothyroidism↗

PubMed1987

Characterization and regulations of the receptor for insulin-like growth factor-I in the FRTL-5 rat thyroid follicular cell line.

In previous studies we have shown that insulin-like growth factor I (IGF-I) has a mitogenic effect in a line of rat thyroid follicular cells, the FRTL-5. In view of this effect, we undertook studies to identify and characterize some physicochemical and binding properties of the receptor for IGF-I in these cells and to determine what role it plays in the mitogenic activity of insulin and insulin-like growth factors in the FRTL-5 cell. Binding of 125I-labeled IGF-I (biosynthetic Thr59-IGF-I) to FRTL-5 was a function of time, temperature, and pH and was completely inhibited by high concentrations of unlabeled IGF-I. Scatchard plots of four saturation studies revealed a single apparent binding site with an average Ka of 4.2 +/- 0.6 X 10(9) M-1 (mean +/- SD) and an average maximum binding capacity of 20 +/- 2 pm/100 micrograms cellular protein. Rat IGF-II (rIGF-II) and insulin were far less potent that IGF-I in inhibiting the binding of [125I] IGF-I, and bovine TSH was without effect. 125I-Labeled IGF-II also bound to FRTL-5 cells. Binding was completely inhibited by unlabeled rIGF-II and, with lesser potency, by IGF-I. Even at high concentrations, insulin failed to inhibit the binding of [125I]IGF-II. Disuccinimidyl suberate cross-linked [125I]IGF-I to a moiety in FRTL-5 that had an apparent mol wt of approximately 135,000, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Cross-linking of [125I]IGF-I was inhibited in a dose-dependent manner by unlabeled IGF-I and, with far lower potency, by rIGF-II and insulin. All three peptides stimulated the incorporation of [3H]thymidine into the DNA of FRTL-5 cells, IGF-I being the most potent, followed in decreasing order of potency of rIGF-II and insulin. The mitogenic activities of these polypeptides correlated well with their abilities to inhibit the binding of [125I]IGF-I. These data indicate that the FRTL-5 cell possesses a receptor for IGF-I that resembles in its binding and physicochemical properties the receptor for IGF-I in other tissues (type I IGF receptor) and that mediates the mitogenic response to IGF-I and insulin in these cells. FRTL-5 cells also contain a receptor for IGF-II (type II IGF receptor), but its role vis-à-vis that of the type I IGF receptor in relation to the mitogenic effect of IGF-II in these cells is uncertain.

Animals↗

PubMed1987

Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization.

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I. Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 +/- 0.30%/10(5) cells (SEM, n = 9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (alpha IR-3) inhibited 125I-IGF-I but not 125I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the alpha subunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays. IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.

Affinity Labels↗

PubMed1987

Radioimmunoassay of somatomedin-C in filter paper discs containing dried blood.

We describe a simple radioimmunoassay (RIA) for estimating concentrations of somatomedin-C (Sm-C) in dried blood on filter paper. A single 3.2-mm blood spot specimen on filter paper is eluted overnight into buffer containing antibody and 125I-labeled Sm-C. The following day, bound and free hormones are separated by addition of goat anti-rabbit gamma globulin in 60 g/L polyethylene glycol solution. The correlation between values obtained for such blood-spot discs and the corresponding wet plasma is highly significant (r = 0.90, p less than 0.001). The relative concentrations (arbitrary units) of Sm-C as determined for specimens on filter paper from mothers and infants, and for cord bloods, are similar to those reported by others using acidified serum.

Adult↗

PubMed1986

Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells.

We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c-fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c-myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP.

Animals↗

PubMed1986

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts.

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.

Antibodies, Monoclonal↗

PubMed1986

Failure to confirm the existence of short-latency, short-loop feedback regulation (autoregulation) of growth hormone secretion in the human.

That growth hormone (GH) regulates its own secretion by negative feedback both directly and indirectly via somatomedins has been well-documented in the rat and assumed, on the basis of limited studies, to be true for man as well. Prior to proceeding with studies designed to investigate the nature of this feedback in various endocrine states, we sought first to confirm the existence of direct, short-latency GH feedback in normal individuals. Primed, continuous rate infusions of human GH in normal volunteers achieved a range of steady state GH levels. After 1 h of GH infusion, arginine HCl (500 mg/kg) was infused over 30 min and the GH response assessed. Seven of 8 subjects achieved steady state GH levels in the 8-21 ng/ml range with an infusion rate of 0.0045 U/min following a 0.277 U bolus. After arginine, there was a significant increment of GH levels (range 11.6-69.5 ng/ml) in all 7 subjects. With a higher infusion rate of 0.009 U/min following a 0.54 U bolus, 1 of 2 subjects reached a steady state of 31.0 ng/ml and no response to arginine was demonstrable. Two subjects reached steady state levels of 45.0 and 58 ng/ml during a 0.018 U/min infusion after a 1.08 U bolus and had increments of 26.2 and 25.9 ng/ml following arginine. In 3 subjects achieving levels of 64.5, 107.0 and 132.0 ng/ml, there were increments of 55.0, 61.7 and 13.0 ng/ml during infusions of 0.036 U/min following boluses of 2.16 U. However, only in the first of these 3 was a true steady state achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗

PubMed1986

The development of insulin receptors and responsiveness is an early marker of differentiation in the muscle cell line L6.

After reaching confluence, mononucleated L6 myoblasts fuse into multinucleated contracting myotubes. This process is accompanied by the synthesis of characteristic skeletal muscle proteins, such as myosin heavy chain and the MM isoenzyme of creatine kinase. We have studied the development of insulin receptors and insulin responsiveness during differentiation in the L6 cells. Insulin was bound to high affinity receptors in both myoblasts and differentiated myotubes. The binding showed characteristics typical for insulin binding in other cell types, including high affinity, appropriate specificity, an upwardly concave Scatchard plot, and down-regulation. In the logarithmic growth phase, the myoblasts exhibited a low level of insulin binding, but on initiation of cell fusion, the resulting myotubes progressively developed a 2-fold increase in specific [125I]iodoinsulin binding as a result of a 2-fold increase in receptor number. The increase in insulin binding was an early differentiation event, preceding the accumulation of creatine kinase by 24 h. The development of insulin binding during differentiation correlated closely with an increased ability of the hormone to stimulate maximal 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake at physiological concentrations. The L6 cells are a useful model for studying the binding and effects of physiological insulin concentrations in skeletal muscle before and after differentiation.

Aminoisobutyric Acids↗

PubMed1986

Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves'-IgG.

The present studies were undertaken to examine the factors that influence the growth of cells of endocrine gland origin, particularly the possible interactions between "nonspecific" growth factors and the trophic hormone for a target endocrine cell. As a model system, we explored the individual and conjoint effects of insulin-like growth factor-I (IGF-I) and TSH on the growth of FRTL5 cells, a nontransformed line of cloned rat thyroid follicular epithelium. In these cells, IGF-I and TSH each produced a dose-dependent enhancement of DNA synthesis and cell proliferation. When added together, IGF-I and TSH were markedly synergistic in stimulating DNA synthesis, producing increases in 3H-thymidine incorporation that were far greater than the sum of the effects of each alone. A similar effect of IGF-I was evident in the case of the stimulation of DNA synthesis produced by immunoglobulin G (IgG) preparations from the blood of patients with Graves' disease. Such IgG bind to the TSH receptor and mimic the actions of TSH therein. It is suggested, therefore, that there exist in the FRTL5 cell line at least two mechanisms for the regulation of growth, one activated at the level of the IGF-I receptor and the other at the level of the TSH receptor. When the two pathways are activated concurrently, a synergistic enhancement of DNA synthesis takes place. The findings indicate that the FRTL5 cell line is an excellent model in which to study these complex interactions and that IGF-I may be a determinant of thyroid cell growth, both normally and in certain thyroid diseases.

Animals↗

PubMed1986

An autocrine/paracrine role for insulin-like growth factors in the regulation of human placental growth.

Tissue derived from preterm (9-19 weeks gestation) and term (38-41 weeks gestation) human placentae were examined for their ability to synthesize and secrete insulin-like growth factors (IGFs) in organ culture. IGF-I was measured by a specific RIA, and IGF-II by a rat placental membrane radioreceptor assay. First, explants of placental tissue were maintained in organ culture. These explants secreted immunoreactive IGF-I (IR-IGF-I). There were no differences in the IR-IGF-I content of media conditioned by term and preterm placentae under these conditions. The similarity of this material to authentic human IGF-I was supported by parallel displacement in a specific RIA and coelution during Sephadex G-50 gel filtration. Second, monolayer cultures of fibroblasts from normal human preterm placentae (15-19 weeks gestation) were established. Confluent monolayers of these fibroblasts secreted IR-IGF-I (3-10 pg/10(5) cells X 40 h). IR-IGF-I secretion was reversibly inhibited by 5.3 microM cycloheximide, suggesting that the IR-IGF-I was the result of de novo protein synthesis. IR-IGF-I secretion was stimulated 5-fold by platelet-derived growth factor (0.6 U/ml). The response of monolayers of placental fibroblasts to IGF-I also was tested. IGF-I stimulated alpha-[3H]aminoisobutyric acid transport in these fibroblasts, with half-maximal stimulation occurring at 2-3 ng/ml. Stimulation of alpha-[3H]aminoisobutyric acid uptake by IGF-I correlated with specific binding of [125I]iodo-IGF-I. Half-maximal inhibition of [125I]iodo-IGF-I binding occurred at 2-3 ng/ml IGF-I. Placental tissue also secreted IGF-II-like activity, as measured by radioreceptor assay. Media conditioned by placental explants contained 15-20 ng/mg protein X 48 h, and media conditioned by placental fibroblasts contained 3-7 ng/10(5) cells X 40 h IGF-II determined by radioreceptor assay. These data support the hypothesis that the human placenta produces IGFs (IGF-II and/or IGF-I) that act locally to regulate placental growth.

Aminoisobutyric Acids↗

PubMed1986

Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors.

A monoclonal antibody to the human type I insulin-like growth factor (IGF-I) receptor (alpha IR-3) was used to distinguish actions of insulin and IGF-I that are mediated through insulin as opposed to IGF-I receptors on human skin fibroblasts. Both insulin and IGF-I stimulate uptake of the nonmetabolized alanine analog alpha-aminoisobutyric acid (AIB) in these cells. alpha IR-3 inhibited AIB uptake stimulated by both of these hormones in a dose-dependent manner. However, the pattern of hormone action in the presence of alpha IR-3 differed for the two hormones. In the case of IGF-I, alpha IR-3 potently inhibited AIB uptake at low hormone concentrations, but this inhibition was overcome by high hormone concentrations, consistent with impairment of IGF-I action through the IGF-I receptor. In the case of insulin, the action of low concentrations (i.e. 10 ng/ml) was not inhibited, but that of higher insulin concentrations was, suggesting a dual receptor mechanism of cell stimulation by insulin. alpha IR-3 will be an important tool in further studies of the biology of the IGF-I receptor in normal and abnormal human cells.

Aminoisobutyric Acids↗

PubMed1985

Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells.

The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation.

Affinity Labels↗

PubMed1985

Characterization of monoclonal antibodies to the IGF-I receptor that inhibit IGF-I binding to human cells.

A mouse monoclonal antibody directed against the human placental IGF-I receptor is shown to completely inhibit IGF-I binding to both human placental membranes and circulating human mononuclear leukocytes. Insulin binding is unaffected. Studies with 125I-labelled anti-receptor antibody indicate that the antibody reacts with an epitope distinct from the IGF-I binding site. These antibodies will be powerful probes of IGF-I binding and action in human cells.

Animals↗

PubMed1985

Nasal absorption of insulin: enhancement by hydrophobic bile salts.

We demonstrate that therapeutically useful amounts of insulin are absorbed by the nasal mucosa of human beings when administered as a nasal spray with the common bile salts. By employing a series of bile salts with subtle differences in the number, position, and orientation of their nuclear hydroxyl functions and alterations in side chain conjugation, we show that adjuvant potency for nasal insulin absorption correlates positively with increasing hydrophobicity of the bile salts' steroid nucleus. As inferred from studies employing various concentrations of unconjugated deoxycholate and a constant dose of insulin, insulin absorption begins at the aqueous critical micellar concentration of the bile salt and becomes maximal when micelle formation is well established. These and other data are consistent with the complementary hypotheses that bile salts act as absorption adjuvants by producing high juxtamembrane concentrations of insulin monomers via solubilization in mixed bile salt micelles and forming reverse micelles within nasal membranes, through which insulin monomers can diffuse through polar channels from the nares into the blood stream.

Administration, Intranasal↗

PubMed1985

Characterization of the receptors for insulin and the insulin-like growth factors on micro- and macrovascular tissues.

Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.e. pericytes) and aorta (i.e. smooth muscle cells) responded equally to insulin, IGF-I, and IGF-II. The structure and ligand specificities of the receptor for these peptides were studied by covalently cross-linking 125I-labeled peptide hormones to their respective receptors using disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. The binding subunit of the insulin receptor, alpha-subunit, for all cell types was found to have a mol wt 145,000 under reduced conditions. Labeling of this band was inhibited by 10(-9) M insulin, antiinsulin receptor antibodies, and 10(-8) M IGF-I, but not by multiplication-stimulating activity (IGF-II). The beta-subunit of the insulin receptor in endothelial cells was identified by its ability to be autophosphorylated when stimulated by insulin and was found to have a mol wt of 99,000. Covalent cross-linking of IGF-I to its receptor revealed a mol wt of 145,000, similar to that of insulin receptor, except that IGF-I was 100-fold more potent than insulin in competing with [125I]IGF-I for binding. [125I]IGF-II in all cells was cross-linked to receptor with mol wt of 260,000 and 230,000 under reduced and nonreduced conditions, respectively. IGF-I competed weakly with [125I]IGF-II, whereas insulin was ineffective. [125I]IGF-II also bound to the band with alpha mol wt of 135,000, which was inhibited by insulin, IGF-I, and IGF-II. In summary, receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels are biochemically similar to those in other cells. Interestingly, the finding of large numbers of IGF-I and IGF-II receptors on endothelial cells suggests that these growth factors play a physiological role and are involved in vascular complications associated with diabetes.

Animals↗