Effect of exogenous insulin and fasting on growth hormone receptor and IGF-I expression in the pre-ovulatory follicle of ewes

The aim of this study was to investigate the effect of fasting and exogenous insulin administration on the expression of growth hormone receptor (GHR) and IGF-I mRNA in the pre-ovulatory follicle of ewes. Fifteen ewes received an intravaginal progesterone releasing device that was removed 6 days later (day of removal = day 0). On day -2, the ewes were divided into three groups: (i) fasting group (n = 5) that was fasted from day -2 to day 2; (ii) control group (n = 5) that received a maintenance diet; and (iii) insulin group (n = 5) that received insulin injections (0.25 IU/kg) every 12 h from day -2 to day 2 under the same diet as the control group. Follicular samples were obtained on day 2. Fasting increased plasma non-esterified fatty acids concentrations from day -1 to day 2 (P < 0.001). There was no difference (P > 0.05) in the number of follicles, although there was a tendency for an increase in the pre-ovulatory follicle diameter for the insulin group in comparison to the control group (P = 0.12). Thecal GHR mRNA expression was very low and was considered insignificant. Moreover, granulosa cells GHR mRNA expression increased (P < 0.05) in the insulin group. Expression of IGF-I mRNA was not different among groups in both tissues. In conclusion, insulin administration increases GHR mRNA but not IGF-I mRNA expression in granulosa cells of the pre-ovulatory follicle. However, fasting did not change the pattern of GHR/IGF-I mRNA expression in the pre-ovulatory follicle.     

Increased autophosphorylation of insulin-like growth factor-I and insulin receptors in placentas of diabetic women

Autophosphorylation of insulin and insulin-like growth factor (IGF)-I receptors were measured in lectin purified receptor preparations from placentas of normal and diabetic patients. The basal and insulin or IGF-I stimulated phosphorylation of the approximately 94 kD protein, corresponding to beta-subunit of the insulin and IGF-I receptors, were approximately 2 times greater (p less than 0.05) in placentas from diabetic patients with poor glycemic control (as judged by their serum HbA1c level) compared to the normals. The magnitude of IGF-I or insulin stimulation of the phosphorylation of the 94 kD protein was comparable in placentas from both diabetic and normal patients. Immunoprecipitation and immunodepletion of IGF-I receptor by alpha-IR3, a monoclonal antibody to IGF-I receptor, revealed the increased basal phosphorylation of the approximately 94 kD protein in placentas of diabetic patients to be associated with IGF-I and insulin receptors. The magnitude of IGF-I and insulin stimulated phosphorylation of the immunoprecipitated and immunodepleted IGF-I receptor, respectively, was the same in both normal and diabetic patients. These results suggested that the increased basal phosphorylation of the 94 kD protein in placentas from diabetic patients may be intrinsic to IGF-I and insulin receptor, however, the regulatory mechanisms effecting the increase may not be dependent on IGF-I or insulin.     

The effect of insulin-like growth factor-1 on adult rat cardiac contractility

There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-I on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca²⁺) handling in isolated cardiac myocytes. IGF-I had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 ± 4.6%, 18.5 ± 5.8% and 11.9 ± 4.9% (n = 12–20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 ± 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 ± 3.2%, 17.7 ± 4.1% and 19.7 ± 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16–20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca²⁺ concentration, the amplitude of the Ca²⁺ transient and the time to peak Ca²⁺ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca²⁺ in isolated cardiac myocytes. Increasing insulin concentrations (1–1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca²⁺. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca²⁺ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.     

Increased insulin receptor binding in erythrocytes from growth hormone-deficient children

Erythrocytes from growth hormone-deficient children (GHd-children) (n=10) showed a statistically significant increase in insulin binding at low unlabeled insulin concentrations, together with a threefold decrease in apparent receptor affinity, as compared to control children (C) (n=11). Scatchard analysis of the binding data using the two-site model revealed that both the receptor concentration R₁ [GHd-children 0.10±0.01 ng/ml and C 0.03±0.002 ng/ml] and the dissociation constant K_D1 [GHd-children (0.48±0.05)×10^–9M and C (0.19±0.01)×10^–9M] for high affinitylow capacity sites were significantly increased in erythrocytes from GHd-children, while neither receptor concentrations (R₂) nor the dissociation constant (K_D2) for low affinity-high capacity sites proved to be altered. These events were accompanied by a normal sensitivity to insulin as well as glucose tolerance in the GHd-group. The meaning of the increased insulin binding with normal insulin sensitivity in GH-deficiency is discussed.     

Insulin receptor binding from mid-term and full-term placentas of patients with gestational diabetes mellitus and normal pregnant women

Insulin receptor binding was examined in the microvillous membranes of mid-term (20–22 weeks of gestation, MT) and full-term (FT) placentas from patients with gestational diabetes mellitus (GDM) and in normal pregnant control (N). Mid-term placentas were obtained from patients who have had spontaneous abortion. The maximum per cent specific binding (%SB) in MT placenta for GDM was significantly lower (4.8%) compared with the FT placenta (22%, p<0.001), while in the N group the maximum per cent specific binding for MT placenta was 14.1% compared with 26% for the FT placneta (p<0.001). Binding data from FT placenta of well-controlled GDM patients were similar with the FT placenta from N group (22%SB for GDM VS 26% SB for N). Even as there were similarities in the binding characteristics of FT placentas from both groups the placental membrane protein content in the GDM group was lower by 50% compared with the N control (2.5±0.11 VS 4.8±0.15 mg protein/g placenta respectively, p<0.001) suggesting that in the GDM group achieving a tight glycemic control could improve receptor affinities. Data from the competitive binding assay of GDM patients showed that the insulin necessary to achieve 50% inhibition (ID₅₀) was significantly lower in MT compared with the FT placenta (0.9×10^–9 M VS 3.8×10^–9 M, p<0.001) but in the N placenta there was no alteration in the ID₅₀ of MT and FT placentas (3.1×10^–9 M VS 4×10^–9 M, p<0.01, respectively). The present study demonstrated that in GDM the placental insulin receptor binding was significantly lower in spontaneously aborted placenta compared with placentas collected at full-term. Furthermore, these data suggest that the objective to achieve a tight glycemic control in GDM patients could optimize insulin receptor function similar to that of a normal pregnancy. Thus a full term placenta from GDM patients under a well managed glycemic control throughout the entire duration of pregnancy would result in an optimum insulin receptor function.     

An insulin-like hybrid consisting of a modified A-domain of human insulin-like growth factor I and the B-chain of insulin

We have synthesized an insulin-like compound, consisting of the B-chain of bovine insulin and an A-chain corresponding to the A-domain of human insulin-like growth factor-I (IGF-I), in which the isoleucine residue normally present in position 2 of the A-domain of IGF-I has been replaced with glycine. Biological evaluation of the compound indicated that its insulin-like activity (insulin receptor-binding and stimulation of lipogenesis) was 0.2%, and its growth-factor activity (stimulation of thymidine incorporation) was less than 1%, both relative to natural insulin. We conclude that interactions between Ile^A2 and Tyr^A19, which are crucial to high biological activity in insulin, are also present in IGF-I, and are equally critical for its biological activity.     

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

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

Nutritional insult and recovery in the neonatal rat cerebellum: Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs)

Alterations in growth caused by neonatal malnutrition may be mediated in part by changes in insulin-like growth factor (IGF) and IGF binding protein (IGFBP) expression. Since the neonatal rat cerebellum undergoes a transient, proliferative growth phase in the first two weeks of life, this structure was used to determine whether alterations in circulating and tissue IGFs and IGFBPs may mediate effects of impaired nutrition on the developing central nervous system. Gravid rats were placed on a 4% (protein-calorie deprived, D) or 20% (control, C) protein diets one day prior to delivery and allowed to nurse their pups postpartum. Pups nursing from D mothers received a limited volume of milk and were calorically deprived. Some litters of D pups were foster fed by C mothers from day 8 to day 13 to constitute a recovery group (R). Cerebellar weight, protein, and DNA content in D pups were less than C, p<0.001. In R pups, DNA and protein returned to C levels by day 13. Between days 6 and 13, serum IGF-I levels rose from 158±18 to 210±18 ng/ml in C but remained low in D (47±6 ng/ml and 25±3 ng/ml), respectively. In R pups, serum IGF-I partially recovered during this time, and increased from 49±5 to 110±7 ng/ml. In cerebellar extracts, IGF-I levels in both C and D were lower at 13 days than at 6 days, p<0.05 and p<0.005, respectively. IGF-I levels in C were similar at day 9 and day 11 and were consistently higher than D (11.84±0.83 vs 8.56±0.92 ng/g, p<0.02 C vs D). In R, IGF-I was reduced on day 11, but was similar to C on day 13. Serum IGF-II in D was lower than C, p<0.01, and did not increase in the R group. Cerebellar IGF-II was virtually undetectable in either group. Immunoprecipitation and ligand blotting studies of serum demonstrated that circulating levels of 32–34 K IGFBPs were increased 3–4 fold in D vs C, reflecting high levels of IGFBP-1 and/or-2, while levels of 24 K IGFBP-4 were lower in D vs C. By contrast, immunoprecipitation and ligand blotting of cerebellar extracts detected IGFBP-2 and-4, but did not detect IGFBP-1. Further, tissue levels of IGFBP-2 were not increased in D vs C, and levels of IGFBP-4 also were not markedly affected by nutritional deprivation. These results suggest that alterations in tissue content and the availability of IGF-I only modestly contributed to the effects of impaired nutrition in the developing central nervous system.     

Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD₅₀ of 1.1×10^–8M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [¹²⁵I]-insulin with an ED₅₀ of 1.1×10^–8 M, which is identical to the ED₅₀ of insulin (1.1×10^–8M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [¹²⁵I]-IGF-1 with a DTaI concentration of 1.1×10^–8 M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0±2.0 fmoles/mg protein down to 3.6±0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2±0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [¹²⁵I]-insulin and no apparent difference in [¹²⁵I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.     

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

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

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Alternate signaling pathways selectively regulate binding of insulin-like growth factor I and II on fetal rat bone cells

Bone cells synthesize and respond to IGF-I and IGF-II which contribute to bone remodeling and linear growth. In osteoblasts, prostaglandin (PG)E₂ stimulates IGF-I but not IGF-II synthesis through a cAMP-dependent protein kinase A (PKA)-related event. However, protein kinase C (PKC) activation by PGE₂ enhances replication and protein synthesis by less differentiated periosteal cells more so than in osteoblast-enriched cultures from fetal rat bone. Using various PGs and other PKA and PKC pathway activators, the importance of these aspects of PGE₂ activity has now been examined on IGF receptors in these bone cell culture models. PGE₂ and other agents that activate PKA enhanced ¹²⁵I-IGF-II binding to type 2 IGF receptors on both cell populations. In contrast, agents that activate PKC enhanced ¹²⁵I-IGF-I binding to type 1 receptors on less differentiated bone cells, and of these, only phorbol myristate acetate (PMA), which activates PKC in a receptor-independent way, was effective in osteoblast-enriched cultures. No stimulator increased total type 1 receptor protein in either cell population. Consequently, ligand binding to type 1 and type 2 IGF receptors is differentially modulated by specific intracellular pathways in bone cells. Importantly, changes in apparent type 1 receptor number occur rapidly and may do so at least in part through post-translational effects. These results may help to predict new ways to manipulate autocrine or paracrine actions by IGFs in skeletal tissue. J. Cell. Biochem. 68:446–456, 1998. © 1998 Wiley-Liss, Inc.     

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37–40, 32, 30–31, 28, and 24 kDa. The 30–31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM-conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [¹²⁵I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [¹²⁵I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [¹²⁵I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an ∼400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the α2β2 form of the IGF-I receptor, and two β subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two β subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two β subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system. © 1996 Wiley-Liss, Inc.     

Purification and characterization of the receptor for insulin-like growth factor I

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.     

Effect of insulin-like growth factor I and its interaction with gonadotropins on in vitro maturation and embryonic development,cell proliferation,and biosynthetic activity of cumulus-oocyte complexes and granulosa cells in buffalo

In this study we have examined the effect of insulin like growth factor I (IGF-I) and its interaction with gonadotropins in the presence or absence of granulosa cell coculture on in vitro oocyte maturation (IVM) and their subsequent embryonic development in buffalo. We also have examined the role of IGF-I alone or in combination with gonadotropins on DNA synthesis, steroidogenesis, and protein synthesis of cumulus-oocytes complexes (COCs) and granulosa cells. Results showed that IGF-I stimulates oocytes maturation in a dose-dependent manner, with maximal effect at a dose of 100 ng/ml (P < 0.05). IGF-I showed positive interaction with follicle-stimulating hormone (FSH) in the presence or absence of granulosa cells on meiotic maturation and synergistically enhanced DNA synthesis, protein synthesis, and steroidogenesis in the presence of granulosa cells. This synergistic effect is mainly caused by the increase of IGF-I receptors in granulosa cells by FSH, as evident by [¹²⁵I]IGF-I binding study. Luteinizing hormone (LH), however, was found to suppress IGF-I and IGF-I + FSH stimulated oocyte maturation. Addition of LH to cultures containing IGF-I + FSH, on the contrary, caused a significant increase in oocyte maturation when cocultured with granulosa cells. Addition of IGF-I during IVM significantly improve cleavage and blastocyst development rate over the control group. However, there was no cumulative effect when IGF-I and gonadotropins were present together. Addition of granulosa cells during IVM, however, enhanced blastocyst development in the IGF-I + FSH and IGF-I + FSH + LH groups. Our results demonstrated that IGF-I is a major follicular factor responsible for stimulating oocyte maturation in the buffalo. Interaction between IGF-I and FSH suggests that they seem to act synergistically as an autocrine and paracrine regulator of granulosa cells and therefore together promote mitosis, steroidogenesis, and protein synthesis. Mol. Reprod. Dev. 49:277–285, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

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%/10⁵ 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 (αIR-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 αsubunit 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.     

Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats

To investigate the response of the growth retarded neonatal rat to insulin-like growth factor-I (IGF-I) we have measured the effect of IGF-I on in vitro muscle protein synthesis and degradation rates in growth retarded and control neonatal rat pups. The growth retarded pups were growth retarded in utero by ligation of the uterine blood supply at day 17 of gestation. Basal levels of muscle protein synthesis in vitro were significantly lower in growth retarded pups compared with controls. Protein degradation rate were not different in muscles taken from the two groups. IGF-I stimulated protein synthesis in muscle from control pups by 12% and 15% at 20 ng/ml and 200ng/ml respectively. Net protein degradation was inhibited by 20% in the presence of 20ng/ml IGF-I. IGF-I had no effect on net protein synthesis or degradation in muscle from growth retarded pups. Neither Multiplication Stimulating Activity (at 20ng/ml or 200ng/ml) nor insulin (at 40ng/ml or 800ng/ml) was able to increase synthesis or decrease degradation of protein. Specific receptors for IGF-I are present on muscle membranes from both groups. Unlabelled IGF-I was more effective than MSA or insulin in competing with 125I-IGF-I for binding to the receptor. The relative affinities are consistent with type I IGF receptors. The affinity of these receptors for IGF-I was similar (Kd approximately 5nM) in both groups and the receptor concentration in both cases was approximately 250 fmol/mg protein. The refractility of tissue from growth retarded pups to IGF-I may be partially responsible for the lack of catch up growth in growth retarded neonates.     

Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.