The role of insulin-like growth-factor binding proteins in normal and polycystic ovaries

The insulin-like growth factor binding proteins are single chain polypeptides, that can bind insulin-like growth factors, but not insulin. They can serve as autocrine or paracrine regulators of the actions of insulin-like growth factor. The human granulosa cells produce insulin-like growth factor-II but not insulin-like growth factor-I, while the human theca cells produce insulin-like growth factor-I and II. Polycystic ovarian syndrome is a disorder which is characterised by hyperandrogenism and anovulation. In polycystic ovarian syndrome there is a disorder of follicular development, with the accumulation of antral follicles within the ovary which fail to respond appropriately to endogenous follicle-stimulating hormone. Significance is given to insulin-like growth factor binding proteins, which have an inhibitory action on follicle-stimulating hormone. No differences were found in the total level of insulin-like growth factor binding proteins follicular profiles between women with polycystic ovarian syndrome and without it. Serum insulin-like growth factor binding protein-I levels are lower in polycystic ovarian syndrome with hyperinsulinaemia, probably as a consequence of insulin-mediated suppression of insulin-like growth factor binding protein-I. Consequently, serum free insulin-like growth factor-I levels are higher. This alteration may cause an excessive thecal androgen production. The alterations in the insulin-like growth factor-insulin-like growth factor binding proteins axis may be one of several mechanisms that help to sustain the steady state of anovulation and follicular dysmaturation that are characteristic of this syndrome.     

Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.     

Pre-B acute lymphoblastic leukemia in a patient with partial lipodystrophy and acanthosis nigricans

In patients with lipodystrophies a post binding defect in insulin action has been described involving phosphorylation of the beta subunit of the insulin receptor, suggesting the presence of a genetically determined defect in insulin action; the receptor gene has been mapped to the distal short arm of chromosome 19 close to the break-point of a specific chromosome translocation frequently found in pre-B Acute Lymphoblastic Leukemia (ALL). We report on a 13 years old female patient with partial lipodystrophy, acanthosis nigricans and insulin resistance who developed a pre-B ALL. Since lipodystrophy and pre-B ALL are rare disorders, a possible causal relationship between the two diseases is suggested possibly mediated by a mutation in the insulin receptor gene.     

Comparison of the inflammatory responses of mice infected with American and Australian Trichinella pseudospiralis or Trichinella spiralis

We have examined the response of the renal insulin-like growth factor (IGF-I) axis to acute ischemic injury in the rat Key findings included a decrease in IGF-I mRNA and peptide levels, a decrease in GH receptor gene plus protein expression and a decrease in the IGF binding proteins except for IGF binding protein I. Administration of GH to compensate for the reduced GH receptor binding corrected the IGF-I mRNA levels suggesting a relative GH deficiency. Interestingly, IGF-I receptor mRNA levels were unchanged while plasma membrane IGF-I receptor number increased two fold. This appeared to be due to a redistribution of receptors to a membrane location. IGF-I receptor autophosphorylation and tyrosine kinase activity were intact despite severe uremia for up to 6 days. We propose that this increase of functional IGF-I receptors following acute tubular necrosis will sensitize the kidney to the administration of exogenous IGF-I.     

Systemic administration of kainic acid induces selective time dependent decrease in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in adult rat hippocampal formation

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1-CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [125I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [125I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [125I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [125I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [125I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1-CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment. These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.     

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.     

Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system

To determine whether insulin stimulates human ovarian testosterone production in the polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system, thecal cells from polycystic ovary syndrome women were isolated and cultured. Insulin and insulin-like growth factor I stimulated thecal testosterone biosynthesis. Antibody blockade of the insulin receptor abolished insulin's stimulatory action, whereas effective antibody blockade of the insulin-like growth factor I receptor did not alter insulin's stimulation of thecal testosterone biosynthesis. A chiro-inositol containing glycan (INS-2) increased thecal testosterone biosynthesis. Preincubation of cells with an antiinositolglycan antibody (A23939 or alpha IGP) abolished insulin's stimulatory effect, but not that of hCG. These findings suggest that inositolglycans serve as the signal transduction system for insulin's stimulation of human thecal testosterone biosynthesis.     

Characterization of insulin receptor from the muscle of the shrimp Penaeus japonicus (Crustacea: Decapoda)

The beta-subunit of the insulin receptor from the muscle of the shrimp Penaeus japonicus exists as multiple subtypes with M(r) of 79,000, 77,000 and 75,000. Only the subunit of M(r) 79,000 is autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the human placenta. The detergent, Triton X-100, and the metal ion, Mn2+, caused a noticeable enhancement of the autophosphorylation of shrimp insulin receptors from the muscle. Okadaic acid activated the kinase activity of the insulin-stimulated insulin receptor, but not the basal activity of the insulin receptor without the addition of insulin. Further studies comparing the insulin binding of the shrimp insulin receptor in the regulation of kinase activity of the multiple beta-subunit subtypes from the shrimp muscle are under way.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

Effects of autoantibodies to the insulin receptor on isolated adipocytes. Studies of insulin binding and insulin action

Autoantibodies to the insulin receptor have been detected in the sera of several patients with the Type B syndrome of insulin resistance and acanthosis nigricans. In this study we have used three of these sera (B-1, B-2, and B-3) as probes of the insulin receptor in isolated rat adipocytes. Preincubation of adipocytes with each of the three sera resulted in an inhibition of subsequent [(125)I]insulin binding. 50% inhibition of binding occurred with serum dilutions of 1:5 to 1:7,500. As in our previous studies with other tissues, Scatchard analysis of the insulin-binding data was curvilinear consistent with negative cooperativity. Computer analysis suggested that in each case the inhibition of binding was due to a decrease in receptor affinity rather than a change in available receptor number. In addition to the effects on insulin binding, adipocytes pretreated with antireceptor sera also showed alterations in biological responses. All three sera produced some stimulation of basal glucose oxidation. With serum B-3, maximal stimulation of glucose oxidation occurred at a serum concentration that inhibited binding by only 10-15%, whereas with serum B-2 the dilution curves for inhibition of binding and stimulation of glucose oxidation were superimposable. Serum B-1 behaved as a partial agonist; that is, it inhibited binding more effectively than it stimulated glucose oxidation. Cells pretreated with this serum in a concentration which inhibited binding by 80% also showed a five-fold shift to the right in the dose response of insulin-stimulated glucose oxidation, whereas spermine-stimulated glucose oxidation was unaffected. Serum B-2, which contained the highest titer of antireceptor antibodies, also stimulated 2-deoxy-glucose transport, as well as glucose incorporation into lipid and glycogen. Both the ability of the serum to inhibit binding and stimulate glucose utilization were enriched in purified immunoglobulin fractions and retained in the F(ab')(2) fragment of the IgG. In addition, the bioactivity was blocked by antihuman IgG but not by anti-insulin antibodies. Enzymatic digestion of adipocytes with trypsin resulted in a complete loss of insulin-stimulated bioactivity of serum B-3, but had only minor effects on the glucose oxidation produced by serum B-1 or B-2.These data suggest that the antibodies present in these three sera bind to different determinants on the insulin receptor. Thus, these antibodies may be useful probes of receptor structure and function.     

Low insulin, IGF-I and IGFBP-3 levels in children with Prader-Labhart-Willi syndrome

It is well established that insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3) and insulin are low in growth hormone deficiency, but due to their dependence on nutrition, they are elevated in healthy obese children. As the presence of growth hormone deficiency in Prader-Labhart-Willi syndrome (PWS) is still controversial, we studied insulin, IGF-I and IGFBP-3 levels in 19 children with PWS (age range 0.5-14.6 years). Serum concentrations of insulin (SDS: -0.7+/-0.9, P = 0.01) and IGF-I (SDS: -0.7+/-0.8, P = 0.002) were low, but IGFBP-3 (SDS: -0.3+/-1.2, P = 0.2) was normal compared to normal weight age-matched children. Since children with PWS are typically obese, insulin, IGF-I and IGFBP-3 levels should be compared to normal obese children who present increased levels of these hormones. In comparison to data of healthy obese children reported in the literature, not only IGF-I, but also IGFBP-3 levels are low and fasting insulin levels even very low, suggesting a growth hormone deficiency.     

Role of the juxtamembrane tyrosine in insulin receptor-mediated tyrosine phosphorylation of p60 endogenous substrates

Prior studies have demonstrated that a juxtamembrane tyrosine (tyrosine 972) in the insulin receptor is required for the receptor to elicit various biological responses and to stimulate the tyrosine phosphorylation of two endogenous substrates, the insulin receptor substrate-1 and the adaptor protein called Shc. In the present studies the role of this tyrosine was examined in the insulin-stimulated tyrosine phosphorylation of a group of 60-kDa endogenous proteins. These include a 60-kDa protein which, when phosphorylated, becomes associated with the GTPase activating protein of Ras, a distinct 60-kDa protein that associates with either the phosphatidylinositol 3-kinase or the tyrosine phosphatase Syp, as well as a 58/53-kDa protein that is tyrosine phosphorylated in response to insulin but has no known associated protein. In each case, a mutant insulin receptor in which tyrosine 972 has been changed to phenylalanine was found to be defective in its ability to phosphorylate these three endogenous substrates, although the mutant receptor exhibited the same level of insulin-stimulated autophosphorylation as the wild type receptor. These results further demonstrate the critical role that the juxtamembrane tyrosine 972 plays in downstream signaling by the insulin receptor.     

Effect of captopril, losartan, and bradykinin on early steps of insulin action

Insulin initiates its metabolic and growth-promoting effects by binding to the alpha subunit of its receptor, thereby activating the kinase in the beta subunit. This event leads to tyrosyl phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1), which in turn associates with and activates phosphatidylinositol (PI) 3-kinase. The clinical use of ACE inhibitors has been associated with increased insulin sensitivity. However, the exact molecular mechanism is unknown. In the present study, we examined the phosphorylation status of the insulin receptor and IRS-1, as well as the association between IRS-1 and PI 3-kinase in the liver and muscle of 20-month-old rats treated acutely with captopril, using immunoprecipitation with antipeptide antibodies to the insulin receptor and IRS-1, and immunoblotting with antiphosphotyrosine and anti-PI 3-kinase antibodies. Insulin stimulation increased receptor autophosphorylation to 462 +/- 253% (P < 0.05) in the liver and 697 +/- 78% (P < 0.001) in the muscle of ACE inhibitor-treated rats. There were also increases to 250 +/- 17% (P < 0.001) and 280 +/- 50% (P < 0.05) in the insulin-stimulated IRS-1 phosphorylation levels in the liver and muscle, respectively, of animals treated with captopril. The insulin-stimulated IRS-1 association with PI 3-kinase rose to 305 +/- 20% (P < 0.001) in liver and 267 +/- 48% (P < 0.05) in muscle. Losartan, an ANG receptor blocker, had no significant effect on insulin-stimulated IRS-1 phosphorylation in both tissues. The acute administration of bradykinin increased insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 in the liver and muscle. These data demonstrate that ACE inhibitors modulate the early steps of insulin signaling, and that this effect may be simulated by the administration of bradykinin.     

Reduced tyrosine kinase activity of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha

Insulin resistance is an important metabolic abnormality often associated with infections, cancer, obesity, and especially non-insulin-dependent diabetes mellitus (NIDDM). We have previously demonstrated that tumor necrosis factor-alpha produced by adipose tissue is a key mediator of insulin resistance in animal models of obesity-diabetes. However, the mechanism by which TNF-alpha interferes with insulin action is not known. Since a defective insulin receptor (IR) tyrosine kinase activity has been observed in obesity and NIDDM, we measured the IR tyrosine kinase activity in the Zucker (fa/fa) rat model of obesity and insulin resistance after neutralizing TNF-alpha with a soluble TNF receptor (TNFR)-lgG fusion protein. This neutralization resulted in a marked increase in insulin-stimulated autophosphorylation of the IR, as well as phosphorylation of insulin receptor substrate 1 (IRS-1) in muscle and fat tissues of the fa/fa rats, restoring them to near control (lean) levels. In contrast, no significant changes were observed in insulin-stimulated tyrosine phosphorylations of IR and IRS-1 in liver. The physiological significance of the improvements in IR signaling was indicated by a concurrent reduction in plasma glucose, insulin, and free fatty acid levels. These results demonstrate that TNF-alpha participates in obesity-related systemic insulin resistance by inhibiting the IR tyrosine kinase in the two tissues mainly responsible for insulin-stimulated glucose uptake: muscle and fat.     

The internal symmetry of the primary structure of the beta-subunits of the tyrosine kinase receptors in insulin superfamily peptides and its relation to their functional activity

Using the author's original graphic method, the internal symmetry of mirror type was identified in the primary structure of beta-subunit receptor tyrosine kinases of insulin superfamily peptides (receptors of insulin, insulin-like growth factor 1 and of insulin-related peptide). The most important regions of the primary structure, determining the functional activity of receptors, were shown to have the symmetrical structure. The regions are involved in receptor autophosphorylation and kinase activity form the receptor ATP-binding site, contain N-glycosylation sites, participate in the formation of intermolecular disulfide bridges with receptor alpha-subunits, and interact with other components of the signal transduction system (in particular, with G-proteins). The functionally important amino acid residues or their clusters either appear themselves the centres of internal symmetry, or are spaced in the immediate vicinity of these centres.     

The functional role of CrkII in actin cytoskeleton organization and mitogenesis

Crk is a member of a family of adapter proteins predominantly composed of Src homology 2 and 3 domains, whose role in signaling pathways is presently unclear. Using an in situ electroporation system which permits the introduction of glutathione S-transferase (GST) fusion proteins into cells, we found that c-CrkII bound to p130(cas), but not to paxillin in serum-starved rat-1 fibroblasts overexpressing the human insulin receptor (HIRc cells) in vivo. 17 nM insulin stimulation dissociated the binding of c-CrkII to p130(cas), whereas 13 nM insulin-like growth factor-I, 16 nM epidermal growth factor (EGF), and 10% serum each showed little or no effect. We found that stress fiber formation is consistent with a change in the p130(cas).c-CrkII interactions before and after growth factor stimulation. Microinjection of either GST-Crk-SH2 or -Crk-(N)SH3 domains, or anti-Crk antibody each inhibited stress fiber formation before and after insulin-like growth factor-I, EGF, and serum stimulation. Insulin stimulation by itself caused stress fiber breakdown and there was no additive effect of microinjection. Microinjection of anti-p130(cas) antibody also blocked stress fiber formation in quiescent cells. Microinjection of the Crk-inhibitory reagents also inhibited DNA synthesis after insulin-like growth factor-I, EGF, and serum stimulation, but not after insulin. These data suggest that the complex containing p130(cas).c-CrkII may play a crucial role in actin cytoskeleton organization and in anchorage-dependent DNA synthesis.     

Cys860 in the extracellular domain of insulin receptor beta-subunit is critical for internalization and signal transduction

The C860S mutation (IRC860S) in the extracellular domain of the insulin receptor beta-subunit has previously been shown to result in an inhibition of insulin receptor internalization. The present work aims at further dissecting the consequences of this mutation not only on insulin receptor internalization, but also on the signaling of the receptor. Following transfection of Chinese hamster ovary (CHO) cells with insulin receptors with the C860S mutation (CHO-IRC860S) and quantitative electron microscopic analysis of [125I]insulin localization in these cells, the inhibition of receptor internalization appears to be due to an inhibition of the lateral translocation of the receptor from microvilli to nonvillous domains of the cell surface. At 37 C, insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation is inhibited by 50% in CHO-IRC860S, whereas Shc phosphorylation remains unaffected. The inhibition of IRS-1 phosphorylation is still present when experiments are conducted at 4 C, a temperature at which insulin receptor internalization is prevented, suggesting that the defect in IRS-1 phosphorylation is not due to the reduced internalization of the receptor. In terms of biological effects, the mutation has negative consequences on insulin-stimulated c-fos expression and DNA synthesis as well as on glycogen synthase activity. Eventually, the events observed are specific for Cys860, as individual substitution of the two more proximal Cys residues (795 and 872) to Ser is not accompanied by any change in either insulin-induced insulin receptor internalization or IRS-1 phosphorylation. Thus, the present analysis of CHO-IRC860S 1) reveals that insulin receptor surface redistribution is not solely dependent on receptor autophosphorylation, 2) emphasizes that IRS-1 phosphorylation is not dependent on receptor internalization and can be triggered from microvilli, and 3) stresses divergent aspects between two of the major signaling pathways of the insulin receptor.     

Insulin signal transduction by a mutant human insulin receptor lacking the NPEY sequence. Evidence for an alternate mitogenic signaling pathway that is independent of Shc phosphorylation

The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.     

Autoradiographic mapping and characterization of insulin-like growth factor-I receptor binding in human greater saphenous vein

PURPOSE: The proliferation of vascular smooth muscle cells is an important step in the process of intimal hyperplasia. Veins exposed to arterial pressure develop intimal hyperplastic lesions that lead to failure of vein bypasses. Insulin-like growth factor-I is a polypeptide hormone structurally related to insulin with insulin-like metabolic effects. Insulin-like growth factor-I has been found to work in concert with other growth factors, including platelet-derived growth factor, to promote the growth of vascular smooth muscle cells in culture. Insulin-like growth factor-I exerts its effects via specific receptors located on the cell surface. We studied the in situ distribution of insulin-like growth factor-I receptor binding using autoradiography and examined insulin-like growth factor-I binding characteristics in normal human greater saphenous vein. METHODS: Frozen sections 20 microns thick were prepared from the greater saphenous vein specimens. The sections were incubated in a buffer containing 125I-insulin-like growth factor-I in the presence of increasing concentrations of the unlabeled peptide. Autoradiograms were obtained by apposing the treated sections to autoradiography film. RESULTS: Analysis of the autoradiographs showed that insulin-like growth factor-I binding was consistently present in the wall of human greater saphenous vein. To characterize these binding sites binding inhibition studies were performed. High-affinity insulin-like growth factor-I receptor binding was found with dissociation constant of 1.0 +/- 0.32 nmol/L and maximum binding capacity of 0.46 +/- 0.23 pmol/mg protein. These values are consistent with a physiologic role for insulin-like growth factor-I in the tissue examined. CONCLUSIONS: The presence of high-affinity (dissociation constant = 1.0 +/- 0.32) insulin-like growth factor-I binding sites in the wall of saphenous vein suggests that insulin-like growth factor-I plays an important role in regulating the proliferation of venous wall cellular components, an essential step in the process of venous intimal hyperplasia.     

Personal viewpoint: new developments in the therapy of diabetic hypertensive disease. The ABCD study adds to strong evidence favoring the prime use of ACE inhibitors. Appropriate Blood Pressure Control in Diabetes

The effects of chronic ethanol feeding on the binding of transforming growth factor-alpha (TGF-alpha) and TGF-alpha-stimulated receptor autophosphorylation were investigated in isolated rat hepatocytes. When hepatocytes were isolated from rats that were fed an ethanol liquid diet for 6-8 weeks, these cells exhibited a marked impairment of TGF-alpha-stimulated autophosphorylation of the receptor that binds this growth factor compared with hepatocytes from the pair-fed controls. This impaired autophosphorylation of receptor tyrosine residues was accompanied by significant decreases in the amount of surface-bound TGF-alpha. Immunoanalysis indicated no changes in receptor number, indicating that decreased receptor content was not responsible for decreased TGF-alpha binding in the hepatocytes from the ethanol-fed rats. In conclusion, chronic ethanol feeding reduced TGF-alpha binding to hepatocytes with a concomitant decrease in the ability of the receptor tyrosine kinase to autophosphorylate its tyrosine residues. These changes were not accompanied by decreased receptor protein content. These defects could lead to altered signal transduction and to impaired reparative and regenerative processes in the liver.