Growth factor-I Binding Research Articles

The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.

Variations in insulin-like growth factor-I binding in plasma and the ovary of the domestic hen during the ovulation cycle

Variations in insulin-like growth factor-I binding in plasma and the ovary of the domestic hen during the ovulation cycle

Purification of Rat C6 Glioma Plasma Membranes by Affinity Partitioning

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.

Impact of neonatal kainate treatment on hippocampal insulin-like growth factor receptors

The insulin-like growth factors-I and -II have neurotrophic properties and act through specific membrane receptors. High levels of binding sites for these growth factors are distributed discretely throughout the brain, being concentrated in the hippocampal formation. Functionally, the insulin-like growth factors, in addition to their growth-promoting actions, are considered to play important roles in normal cell functions, as well as in response to pharmacological or surgical manipulations. In adult rats, we have previously shown that systemic injection of kainate produces an overall decrease, in a time-dependent manner, in insulin-like growth factor-I and -II receptor binding sites in the hippocampus [Kar S. et al. (1997) Neuroscience 80, 1041–1055]. Given the evidence that insulin-like growth factors play a critical role during the early stages of brain development, the present study is a logical extension of this earlier report and established the effect of neonatal kainate injection on the developmental profile of insulin-like growth factor receptors. We have evaluated the time-course alteration of these receptors following systemic injection of kainate to newborn rats. After injection of a sublethal dose of kainate (5 mg/kg, i.p.) to postnatal one-day-old pups, [ 125I]insulin-like growth factor-I, [ 125I]insulin-like growth factor-II and [ 125I]insulin binding sites were studied at different postnatal days (7, 14, 21, 28 and 35) using receptor autoradiography. In the developing hippocampus, insulin-like growth factor-I and insulin binding sites are concentrated primarily in the dentate gyrus and the CA2/CA3 subfields, whereas insulin-like growth factor-II binding is discretely localized to the pyramidal layer and the granular layer of the dentate gyrus. Following kainate injection, we observed a slight increase in insulin-like growth factor-I binding sites in given hippocampal subfields starting at postnatal day 14, being significant at day 21. At later days, a progressive decrease was noted. This transient increase may represent an attempt for neuronal plasticity by up-regulating receptor levels. In contrast, insulin-like growth factor-II and insulin receptor binding sites are found to be decreased in various regions of the hippocampus in kainate-treated pups. Taken together, these results provide further evidence for the existence and differential alterations of insulin-like growth factor-I, insulin-like growth factor-II and insulin receptors in the developing rat hippocampus following kainate-induced lesion, suggesting possible involvement of these growth factors in brain plasticity.

Pharmacological Characterization and Cellular Localization of Insulin-Like Growth Factor-I (IGF-I) Binding to Human Placenta during the Course of Pregnancy and at Term

Pharmacological Characterization and Cellular Localization of Insulin-Like Growth Factor-I (IGF-I) Binding to Human Placenta during the Course of Pregnancy and at Term

The effect of Insulin like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) on cellular proliferation in mouse 3T3 fibroblast cells

Background: Cell growth is a balance between cell proliferation and cell death. Insulin-like growth factor-I(IGF-I), which binds IGF-I receptor(IGF-IR), mediates cellular proliferation as a potent mitogen. IGF binding protein-3(IGFBP-3) as a circulating major IGFBP can inhibit or enhance the effects of IGF-I on cellular growth by binding IGFs. Methods: We investigated the expressions of mRNA of IGF-I and IGF-IR by northern blot and phosphorylation of IGF-IR with the treatment of IGF-I by western blot in 3T3 fibroblast cells. The cellular proliferations of 3T3 cells with the treatments of IGF-I were evaluated using -thymidine incorporation and MTT assay. Also to observe the effect of IGFBP-3 on cellular proliferation, 3T3 cells were treated with anti-IGFBP-3 and (monoclonal antibody to IGF-IR) alone or in combination. Results: Our results demonstrated that 3T3 cells showed mRNA expressions of IGF-I and IGF-IR and the IGF-I increased phosphorylation of IGF-IR. The treatments of 3T3 cells with IGF-I increased cellular proliferation in 5 % and 1 % seruma-containing media, not in serum-free media. The addition of anti-IGFBP-3 to neutralize IGFBP-3 showed 2-fold increase of cellular proliferation, and also co-incubation of anti-IGFBP-3 and together showed similar increase of cellular proliferation in 3T3 cells. Interestingly, when the cells were pretreated with for 4 hr, prior to the simultaneous addition of and anti-IGFBP-3, anti-IGFBP-3-mediated cellular proliferation was decreased to control level. All of these results suggest that free IGF-I released from IGF-I/IGFBP-3 complex would be involved in the cellular proliferation. Conclusion: IGF-I is a mitogen through the activation of IGF-IR in 3T3 cells, and IGFBP-3 could be a potent inhibitor for IGF-I action by binding IGF-I.

Influence of high-carbohydrate enriched diets on plasma insulin levels and insulin and IGF-I receptors in trout

Insulin and insulin growth factor-I (IGF-I) binding to skeletal muscle semipurified receptors were assessed in rainbow trout ( Oncorhynchus mykiss) fed with different enriched carbohydrate diets. The animals were fed for 2 months, either in spring, summer or autumn with a control diet (C, commercial diet containing 21% raw carbohydrates) or with two diets supplied with highly digestible carbohydrates (E1, 22% expanded wheat; and E2, 37% expanded wheat). Insulin and IGF-I receptors were semipurified by affinity chromatography (WGA-agarose). Fish fed with a carbohydrate enriched diet did not show lower growth rates than those fed with the control diet. Independently of the season, rainbow trout fed E1 and E2 presented higher insulin and glucose plasma levels as well as higher tissue glycogen reserves than fish fed C. An increase in the number of insulin receptors during the diet adaptation was observed especially in fish fed with E2. No differences in the affinity of receptors were observed. IGF-I specific binding in skeletal muscle was higher than that of insulin in all groups and in all seasons. Furthermore, IGF-I receptors showed the same tendency as insulin receptors, with increases in their number in experimentally fed fish, especially those fed with E2. Insulin and IGF-I receptors TKA increased only slightly, as a consequence of E1 and E2 diet adaptation. In conclusion, rainbow trout can be fed high-carbohydrate levels and show good rates of growth. This adaptation determines increases in circulating glucose and insulin, and muscle insulin receptors, which indicate an adaptation of the fish to higher levels of glucose supply. The response of IGF-I receptors also suggests a possible role in the regulation of metabolism.

Insulin-like growth factor-binding protein-3 (IGFBP-3) but not insulin-like growth factor-I (IGF-I) remains elevated in euthyroid TSH-suppressed Graves' disease.

Thyroid hormones have been shown to be involved in the regulation of insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) expression. This is a cross-sectional study to look at the effects of thyroid hormone status on the circulating levels of IGF-I and IGFBP-3 in a group of 127 patients, aged 20-80 years, who were hyperthyroid, hypothyroid, rendered euthyroid and clinically euthyroid with normal free thyroxine (fT4), but suppressed thyroid stimulating hormone (TSH) levels. TSH was measured by the IMx (Abbott) ultrasensitive assay, while radioimmunoassays for total T3 and T4 were performed using kits from ICN, USA; fT4 and fT3 using kits from DPC USA; IGF-I and IGFBP-3 using kits from Nichols Institute Diagnostics B.V., Netherlands. Differences in the levels of IGF-I between the 4 groups of patients were significant only in the patients aged 20-40. Mean (+/-SEM) IGF-I levels of hypothyroid patients (169+/-19ng/ml) was significantly lower than hyperthyroid (315+/-26 ng/ml, p=0.003), euthyroid patients (241+/-19 ng/ml, p=0.002) and patients with suppressed TSH (308+/-29 ng/ml, p=0.02). The IGF-I levels of the hyperthyroid and suppressed TSH patients were, however, comparable to age-matched normal subjects (281+/-86 ng/ml). Although there was no difference in mean IGFBP-3 levels between the 4 groups of patients, the levels in the patients aged 20-40 with hyperthyroidism (3.7+/-0.9 microg/ml) and suppressed TSH (3.9+/-1.2 microg/ml) were significantly higher (p=0.02) than age-matched normal subjects (3.1+/-0.8 microg/ml). The IGF-I levels of the thyroid patients aged 20-40 showed significant negative correlation to TSH and positive correlations to the thyroid hormones. Hence, whilst low IGF-I is associated with hypothyroidism, high IGFBP-3 is associated with hyperthyroidism. Our finding that IGFBP-3 remained significantly elevated in patients with suppressed TSH but normalised fT4 and fT3 is important as it suggests a prolonged tissue effect of thyroid hormones on IFGBP-3. As such patients have been shown to have higher risk for atrial fibrillation, the significance and possible role of IGFBP-3 in these conditions should be further elucidated in future studies.

Age-related decline in chondrocyte response to insulin-like growth factor-I: the role of growth factor binding proteins.

The synthetic activity of chondrocytes in articular cartilage declines with age, possibly as a result of decreased sensitivity to anabolic growth factors such as insulin-like growth factor-I. The sensitivity of these cells to insulin-like growth factor-I is regulated, in part, by the synthesis of insulin-like growth factor-I binding proteins. We hypothesized that, as cartilage ages, an increase in the expression of these binding proteins suppresses the synthetic response of chondrocytes to insulin-like growth factor-I. To test this hypothesis, we measured proteoglycan synthesis (incorporation of [35S]sulfate per cell) in alginate cultures of chondrocytes from the articular cartilage of 1, 3, 12, and 24-month-old rats. A dose-response to insulin-like growth factor-I was determined for cells from each age group; incorporation of [35S]sulfate per cell declined with age, regardless of the dose. The sharpest decline was found between cells from the 1 and 3-month-old groups. Using the Western ligand blot technique, we then compared the expression of insulin-like growth factor-I binding protein in chondrocytes from the 1 and 3-month-old rats and found that it was increased in the cells from the older animals. Recombinant insulin-like growth factor-3, when added to the cell cultures of the 1-month-old rats, inhibited incorporation of [35S]sulfate and blocked responses to insulin-like growth factor-I. These findings suggest that the age-related decline in the synthetic response of chondrocytes to insulin-like growth factor-I results, at least in part, from increased expression of insulin-like growth factor binding protein.

Characterization of Two Forms of Recombinant Salmon Insulin-Like Growth Factor-I: Activities and Complexing with Insulin-Like Growth Factor-I Binding Proteins

Coho salmon insulin-like growth factor I (IGF-I) with N-terminal glycine residue ([Gly]-rsIGF-I) was derived from methionine-extended recombinant salmon IGF-I ([Met]-rsIGF-I) produced in Escherichia coli according to [18]. Purified [Met]-rsIGF-I was treated with methionine aminopeptidase (MAP) to remove the N-terminal Met residue and resultant [Gly]-rsIGF-I was further purified by HPLC on a reverse-phase C4 column. The partial N-terminal amino acid sequence (residues 1–25) of [Gly]-rsIGF-I was identical to that of the native salmon IGF-I. [Gly]-rsIGF-I appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as a single band with a molecular weight of 7 kDa and cross-reacted with an antibody against [Met]-rsIGF-I in immunoblot and radioimmunoassay (RIA). In the salmon IGF-I RIA, the dilution curves of [Gly]-rsIGF-I were parallel to the [Met]-rsIGF-I standard curves and the two peptides appeared to be equally potent in displacing label. At concentrations 50 and 100 ng/ml, [Gly]rsIGF-I significantly stimulated the sulfate uptake by the cultured salmon branchial cartilage in a dose-dependent manner. The stimulatory effect of [Gly]-rsIGF-I was equipment to that of [Met]-rsIGF-I. Used as probes in a Western ligand blotting, both [Gly]-rsIGF-I and [Met]-rsIGF-I were able to bind to two IGF binding proteins (IGFBPs) of 23 kDa and 28 kDa from salmon hepatocyte culture medium. The binding could be prevented by preincubation with either unlabeled [Gly]-or [Met]-rsIGF-I. These results indicate that [Gly]-rsIGF-I, which is identical to the native form of IGF-I, and the Met-extended form of recombinant salmon IGF-I, are equally biologically and immunologically active and may substitute for each other in physiological experiments.

Presence and differential internalization of two distinct insulin-like growth factor receptors in rat hippocampal neurons

The pharmacological characteristics, localization and process of internalization of the insulin-like growth factor I and II receptors were studied in rat primary hippocampal cultured neurons grown under serum-free conditions. [ 125I]insulin-like growth factor-I binding was specific with an apparent affinity (K d) of 0.1 nM and IC 50 values of 0.1, 2.9 and 99.7 nM for insulin-like growth factor-I, insulin-like growth factor-II and insulin, respectively. The competition by insulin suggests the presence of genuine insulin-like growth factor-I receptors and not insulin-like growth factor binding proteins. In contrast, [ 125I]insulin-like growth factor-II binding showed a K d of 0.1 nM and IC 50 values of 0.2 and 20.5 nM for insulin-like growth factor-II and insulin-like growth factor-I while insulin was inactive, a well established characteristic of the insulin-like growth factor-II receptor. Using emulsion autoradiography, specific binding sites for [ 125I]insulin-like growth factor-I and -II were over the whole cultured neurons. The use of selective insulin-like growth factor-I and -II receptor antibodies further confirmed the existence of these receptors in rat hippocampal cultured neurons. To investigate the respective internalization profile of [ 125I]insulin-like growth factor-I and [ 125I]insulin-like growth factor-II receptor-ligand complexes in neurons, a technique of acid stripping was used. The apparent rate of endocytosis was found to be greater for the insulin-like growth factor-II than for the insulin-like growth factor-I receptor complexes. The internalization of [ 125I]insulin-like growth factor-I and [ 125I]insulin-like growth factor-II ligand-receptor complexes was confirmed using phenylarsine oxide which significantly blocked both internalization processes. In order to eliminate possible receptor recycling, monensin was used and shown to have no effect on the internalization of either ligand. Since the insulin-like growth factor-I receptor is coupled to tyrosine kinase activity, tyrphostin 47, a specific tyrosine kinase inhibitor, was used and shown to decrease [ 125I]insulin-like growth factor-I but not the [ 125I]insulin-like growth factor-II receptor internalization profile. Accordingly, insulin-like growth factor-I is apparently internalized mostly via the insulin-like growth factor-I tyrosine kinase type receptor, while insulin-like growth factor-II is not. The insulin-like growth factor-II receptor ligand complex is likely internalized via a pathway possibly related to mannose-phosphorylated residues as the insulin-like growth factor-II/mannose-6-phosphate receptor has been implicated in the intracellular targeting of lysosomal proteins containing glycosylated residues. Taken together, our results indicate that primary hippocampal cultured neurons represent a unique model for investigating the differential role and intracellular trafficking of both insulin-like growth factor-I and insulin-like growth factor-II receptor ligand complexes and their relevance to the respective functional role of these two-related trophic factors in the central nervous system.

Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: regulation by high insulin levels

Insulin and IGF-I binding to semi-purified red muscle receptors was characterized in brown trout, Salmo trutta and the common carp, Cyprinus carpio. The yield of glycoprotein obtained after semipurification of receptors with WGA-agarose affinity chromatography in μg g −1 initial tissue was 210.6±21 μg g −1 in trout and 108.5±2.5 μg g −1 in carp. IGF-I specific binding (4.72±0.64%/10 μg glycoprotein) was 4–5-times higher than insulin binding (1.04±0.12%/10 μg glycoprotein) in trout red muscle. This difference in binding was due to a higher number and a greater affinity of the IGF-I ( K d, 0.21±0.03 nM) compared with the insulin ( K d, 0.67±0.06 nM) receptors in this tissue. Carp red muscle IGF-I binding (9.14±0.55%/10 μg glycoprotein) surpassed insulin binding (2.59±0.094%/10 μg glycoprotein) mainly because of a greater affinity of the IGF-I ( K d, 0.092±0.027 nM) compared with the insulin ( K d, 0.1515±0.0285 nM) receptor. IGF-I and insulin binding in carp red muscle were higher than in trout, as a consequence of a higher affinity of carp red muscle receptors. Arginine injection provoked acute hyperinsulinemia in both trout (23.3±1.01 ng ml −1) and carp (24.3±1.34 ng ml −1). Specific binding of insulin and IGF-I to the red muscle decreased 4 h after injection. In trout, a decrease of insulin and IGF-I binding of 47.0% and 63.3%, respectively was observed compared with controls; in carp, these values were 44.0% and 45.0%. The number of insulin and IGF-I receptors decreased (42–55%) but affinities did not change suggesting that receptor down-regulation is a consequence of high insulin levels.

Serum insulin-like growth factor-I (IGF-I) and IGF binding protein-3 levels in children with precocious puberty treated with gonadotropin-releasing hormone analog without or in combination with cyproterone acetate

In order to assess the behavior of growth hormone, insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) in girls with central precocious puberty treated with gonadotropin-releasing hormone (GnRH) analog therapy, we studied 14 girls with this condition; the patients were subdivided into two groups, according to the therapy followed. Group A (n= 7; age 4.2-7.1 years) received GnRH analog in combination with cyproterone acetate, and Group B (n = 7; age 4.4-6.9 years) received long-acting analog alone.Before treatment, IGF-I levels were significantly increased compared to healthy age-matched children in the two groups (447 ± 33μg/l for Group A and 435 ± 38 μg/lfor Group B vs. control 115 ± 78 μg/l; p < > 0.01). Moreover, serum IGFBP-3 levels were significantly higher than the age-related reference range for IGFBP-3 (4478.2 ± 178μg/lfor Group A and 4532.3 ± 167 μg/l for Group B vs. control 2905 ± 641 μg/l; p< 0.01).During the two years of gonadal suppression, Group A patients showed a significant decrease in IGF-I and IGFBP-3 levels, while in Group B there was no significant change in IGF-I; moreover, in Group B, IGFBP-3 levels increased significantly compared to baseline values during the first year of treatment (4532.3 ± 167 μg/l vs. 5410.3 ± 169 μg/l; p < 0.05) and decreased significantly at the end of the second year of treatment (3816.1 ± 189 μg/l vs. 5410.3 ± 169 μg/l; p < 0.01).Our study shows that the two different treatments of precocious puberty (with and without cyproterone acetate) have different effects on IGF-I and IGFBP-3, and suggests that these growth factors are under different metabolic regulation.

The changes in serum type I and III procollagen, insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels in children with chronic renal failure.

Serum levels of carboxyterminal propeptide of type I procollagen (PICP) and aminoterminal propeptide of type III procollagen (PIIINP) can be used as markers of bone formation and the evaluation of children with growth disorders. We measured the serum levels of these collagens by radioimmunoassay (RIA), insulin-like growth factor I (IGF-I) (by immunoradiometric assay) and insulin-like growth factor binding protein-3 (IGFBP-3) (by RIA) levels in 24 children aged between 4 and 14 years with chronic renal failure (CRF) (n = 12 dialysis, n = 12 non-dialysis) and 12 age-matched healthy controls, to find out whether these parameters have a prognostic or therapeutic value on monitoring of growth retardation in CRF. Mean serum IGFBP-3 and PIIINP levels in the dialysis patients were higher than the control group, the difference between the groups was statistically significant (P < 0.05). It seemed that the pubertal stage of the patients did not affect the levels of PICP, PIIINP, and IGFBP-3 while serum IGF-I levels in pubertal patients were significantly higher than those in prepubertal patients (P < 0.001). There was no significant correlation between PICP and PIIINP in any patients. Neither PICP nor PIIINP correlated with height z-score, bone age, IGF-I, or IGFBP-3. It was concluded that the increased serum IGFBP-3 and PIIINP levels in chronic renal disease are poor prognostic indicators leading to progressive renal failure and end-stage renal disease (ESRD). Further investigations into the effects of these collagens on growth failure associated with CRF are needed.

Insulin-like growth factor-I and IGF binding protein-3 remain high after GnRH analogue therapy in girls with central precocious puberty

Insulin-like growth factor-I and IGF binding protein-3 remain high after GnRH analogue therapy in girls with central precocious puberty

Changes in insulin-like growth factor-I receptor expression and binding protein secretion associated with tamoxifen resistance and estrogen independence in human breast cancer cells in vitro.

Conditioned medium from a series of human breast cancer cell lines representing the phenotypes of estrogen dependence ZR-75-1, MCF-7), tamoxifen resistance (Z(+)-75-9a1, LY2) and estrogen independence (ZR-PR-Lt) contained four detectable insulin-like growth factor binding proteins (IGFBPs) of MW 24, 34, 44 and 70 kDa. in the tamoxifen resistant lines secretion of the 24 kDA IGFBP was depressed in comparison to the parent lines whilst secretion of the 44 kDa IGFBP was unaffected. Secretion of this species by ZR-PR-LT cells was reduced. Following incubation in medium containing 1% serum both tamoxifen resistant cell lines secreted higher levels of the 44 kDa IGFBP than the respective parent lines. These changes in IGFBP secretion associated with the development of tamoxifen resistance and estrogen independence were accompanied by changes in the expression of receptors for IGF-1.

Serum levels of insulin-like growth factor-I, -II and insulin-like growth factor binding proteins -2 and -3 in children with acute lymphoblastic leukaemia

Serum levels of insulin-like growth factor-I, -II and insulin-like growth factor binding proteins -2 and -3 in children with acute lymphoblastic leukaemia

Increased specific binding of insulin‐like growth factor‐I in healing cutaneous wounds

Insulin-like growth factor-I is a polypeptide hormone structurally related to insulin. It is a potent mitogen that promotes growth and differentiation in many tissues. A role for insulin-like growth factor-I in wound healing is suggested by its rapid rise in levels and increased insulin-like growth factor-I messenger RNA expression in tissue after wounding. We designed our study to characterize possible changes in insulin-like growth factor-I receptor binding during wound healing. Surgical wounds created on the abdominal skin of anesthetized New Zealand White rabbits were either left open or closed primarily. Size- and weight-matched specimens were harvested at wounding time (day 0), and at 1, 4, 7, 38, and 50 days after wounding. Preliminary experiments showed that the greatest difference in specific binding occurred between day 0 and day 7. (125)I-insulin-like growth factor-I binding studies were performed on frozen tissue specimens and autoradiography was performed and analyzed by computerized densitometry. Scatchard analysis of the binding data showed a single class of insulin-like growth factor-I binding sites whose affinity that is, binding constant (K(d) = 0.6 x 10(-9)) did not change significantly over time; in contrast there was a threefold increase in the number of receptors per milligram tissue in day 7 wound tissue versus normal skin harvested at day 0 (17.3 +/- 2.6 x 10(10) versus 4.7 +/- 2.5 x 10(10), respectively, p < 0.05). Binding inhibition experiments showed that (125)I-insulin-like growth factor-I binding was most specific to insulin-like growth factor-I with insulin-like growth factor-I > insulin-like growth factor-II > insulin. This increase in binding was due to upregulation of insulin-like growth factor-I receptors rather than increased levels of insulin-like growth factor-I binding protein as less than 20% of the threefold increase in binding at day 7 could be attributed to insulin-like growth factor-I binding protein in membrane-free extracts. The presence of specific, high-affinity insulin-like growth factor-I receptors in the skin and their upregulation at day 7 after wounding suggest that insulin-like growth factor-I plays an important role during wound healing.

Erythrocyte insulin and insulin-like growth factor-I receptor tyrosine kinase activity in hypertension in pregnancy

We have shown that preeclampsia is associated with insulin resistance. In the present study, we examined young normal, preeclamptic (PE), and gestational hypertensive (GH) nulliparous African-American women at term to investigate cellular determinants of this resistance and insulin and insulin-like growth factor-I (IGF-I) binding to partially purified erythrocyte receptors and receptor tyrosine kinase activity (TKA). Blood pressure was significantly elevated in PE and GH subjects as compared with controls. Insulin binding was similar in number and affinity in the three groups (femtomoles per microgram). IGF-I binding was increased in PE subjects as compared with either normals or GH subjects (0.2 ± 0.02, 0.15 ± 0.01, and 0.14 ± 0.02 fmol/μg protein). Insulin receptor TKA was increased in PE subjects as compared with normals when assessed either per microgram protein or per femtomole insulin binding ( P < .01). In contrast, IGF-I-potentiated TKA was elevated in PE subjects only when assessed per microgram protein ( P < .03). Thus, the increased number of IGF-I receptors in erythrocytes of PE subjects yields a net increase in receptor tyrosine kinase. Also, there is an augmentation of insulin receptor TKA in PE subjects. Together, these two alterations may be a compensatory mechanism for the insulin resistance associated with hypertensive diseases of pregnancy.

IGF-I-Induced IGFBP-3 potentiates the mitogenic actions of IGF-I in mammary epithelial MD-IGF-I cells

Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs) in bovine mammary epithelial cells. Here, we report on the autocrine mechanisms of action of IGF-I and hormonal regulation of expression of IGFBPs in bovine mammary epithelial MD-IGF-I cells which express recombinant IGF-I under the control of the glucocorticoid-inducible mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Levels of IGFBP-3 mRNA and secretion of IGFBP-3 by MD-IGF-I cells were stimulated by IGF-I, insulin (INS), and IGF-I analogs but not prolactin (PRL). Conversely, parental MAC-T cells expressed little IGF-I and secreted primarily IGFBP-2 (29–32 kDa) in response to stimulation with INS, dexamethasone (DEX), or IGF-I analogs. Secretion of recombinant IGF-I caused a 26.5-fold increase in secretion of IGFBP-3, as measured by densitometric analysis of ligand blots, which was associated with a 1.7-fold increase in total DNA. Conditioned media (CM) from MD-IGF-I cells induced with DEX stimulated a 2.8-fold increase in [ 3H]thymidine incorporation into DNA of parental MAC-T cells, compared with uninduced cells. Moreover, inclusion of exogenous IGF-I with CM from MD-IGF-I cells triggered an additional 3.0-fold increase in label incorporation, but only a 1.6-fold increase in the presence of IGFBP-2-containing media conditioned by MAC-T cells. Des(l-3)IGF-I added to CM from both MAC-T and MD-IGF-I cells respectively, stimulated a 10.2 and 6.9-fold increase in [ 3H]thymidine incorporation into DNA of MAC-T cells. We suggest that expression of IGF-I-induced IGFBP-3 is an important component of an autocrine loop which potentiates the local mitogenic actions of IGF-I in bovine mammary epithelial cells.