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Administration of insulin-like growth factor-I to patients with diabetes enhances insulin action and reduces the degree of hyperglycemia but it is associated with a high rate of adverse events. Infusion of the combination of rhIGFBP-3 (the principal binding protein for IGF-I in plasma) with rhIGF-I to patients with type I diabetes improved insulin sensitivity and was associated with a low incidence in side effects. In this study, 52 patients with insulin-treated type 2 diabetes received recombinant human IGF-I plus rhIGFBP-3 in one of four dosage regimens for 14 days. The four groups were: (1) continuous subcutaneous infusion of 2 mg/kg/day; (2) the same 2 mg/kg dose infused subcutaneously over 6 h between 2000 and 0200 h; (3) 1 mg/kg twice a day by bolus subcutaneous injection; (4) a single bedtime subcutaneous injection of 1 mg/kg. Across these four groups rhIGF-I/rhIGFBP-3 decreased insulin requirements between 54% and 82%. Fasting glucose decreased by 32-37%. Mean daily blood glucose (4 determinations per day) declined in all 4 groups (range 9-23% decrease). Frequent sampling for total IGF-I, free IGF-I and IGFBP-3 was performed on days 0,1,7,14 and 15. The peak total IGF-I values were increased to 4.0-4.8-fold at 16-24 h. For free IGF-I the increase varied between 7.1 and 8.2-fold and peak values were attained at 16-20 h after administration. Both the time to maximum concentration (Tmax) and the maximum free IGF-I levels (Cmax) on day 1 for all groups were substantially less than previously published studies, wherein lower doses of rhIGF-I were given without IGFBP-3. The improvement in glucose values and the degree of reduction in insulin requirement were the greatest in groups 2 and 3 and the patients in those groups had the highest free IGF-I levels. The frequency of side effects such as edema, jaw pain and arthralgias was 4% which is less than that has been reported in previous studies wherein IGF-I was administered without IGFBP-3. We conclude that rhIGF-I/rhIGFBP-3 significantly lowers insulin requirements yet improves glucose values and these changes may reflect improvement in insulin sensitivity. Coadministration of IGFBP-3 with IGF-I produces lower free IGF-I (Tmax and Cmax) levels compared to administration of IGF-I alone and is associated with relatively low incidence of side effects during 2 weeks of administration.

Severe insulin resistance represents a heterogeneous set of conditions with diverse underlying pathophysiology and limited therapeutic options. While recent studies have identified some of the molecular mechanisms resulting in extreme insulin resistance in peripheral tissues and liver, these studies have resulted in relatively few advances in therapy. Insulin like-growth factor 1 (IGF-1) lowers blood glucose while at the same time lowering serum insulin levels in normal volunteers. Its mechanism of action appears to be independent of activation of the insulin receptor although the role of IGF-1 in normal carbohydrate metabolism remains incompletely defined. IGF-1 also improves insulin resistance both in type 2 diabetes and in subjects with more severe insulin resistance. Small-scale clinical trials have demonstrated the potential utility of rhIGF-1 in selected cases of severe insulin resistance and, in these cases, the risk-benefit ratio appears to favor the use of this drug to ameliorate biochemical abnormalities and clinical symptoms.

Insulin-like growth factor-I (IGF-I) enhances insulin action in normal subjects and in patients with both type 1 and 2 diabetes; however, its administration is associated with significant side effects in a high percentage of patients. The coadministration of IGF binding protein-3 (IGFBP-3, the predominant IGF binding protein in serum) with IGF-I limits IGF-I inducible side effects, but it does not attenuate the ability of IGF-I to enhance protein synthesis and bone accretion; therefore, we determined whether IGF-I/IGFBP-3 would retain biological activity in type 1 DM and limit side effects associated with free IGF-I administration. Twelve patients received recombinant human IGF-I plus IGFBP-3 (2 mg/kg-day) by continuous sc infusion for 2 weeks. Each subject served as his own control; and, during a paired 2-week period, each received a placebo infusion. The order of the treatments was randomized. Subjects were placed on a constant caloric intake but were allowed to adjust insulin doses to maintain appropriate levels of glycemic control. Subjects measured blood glucose four times per day at home and kept a log of their insulin use. Frequent sampling for glucose, insulin, and GH was conducted during four inpatient study periods, one at the beginning and one at the end of each 2-week study interval. During IGF-I/IGFBP-3, insulin doses were reduced by 49%, and mean serum glucose was reduced by 23%. Free insulin levels obtained during frequent sampling in hospital fell 47% on IGF-I/IGFBP-3, compared with control, but showed no change with placebo. Concomitant glucose measurements did not differ in the two treatment groups. There was no change in body weight. Fructosamine levels decreased by 12%, but this was not significant (P < 0.1). Fasting triglyceride was unchanged, but cholesterol declined from 170 +/- 24 to 149 +/- 31 mg/dL (P < 0.05). IGFBP-2 (an IGF-I-dependent responsive variable) rose from 141 +/- 56 to 251 +/- 98 ng/mL (P < 0.01) on IGF-I/IGFBP-3. To analyze the mechanism by which IGF-I/IGFBP-3 might reduce insulin requirements, the change in serum GH was quantified. Mean GH levels were reduced by 72%, from 2.48 to 0.55 ng/mL (P < 0.001). An equal number (40%) of drug- and placebo-treated subjects had minor hypoglycemic episodes at home that required adjustment of insulin doses. No episode was classified as severe. In contrast to previous studies with free IGF-I, there were no cases of edema, headache, jaw pain, retinal edema, or Bell's palsy. No subject withdrew because of drug complications. These findings indicate that IGF-I/IGFBP-3 is biologically active on carbohydrate metabolism, as measured by a decrease in insulin requirements in patients with type 1 diabetes. Further studies will be required to determine the long-term safety and efficacy of this combination in patients with insulin resistance and diabetes.

A growing body of evidence has been accumulated recently suggesting that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) affect cardiac function, but their mechanism(s) of action is unclear. In the present study, GH and IGF-1 were administered to isolated isovolumic aequorin-loaded rat whole hearts and ferret papillary muscles. Although GH had no effect on the indices of cardiac function, IGF-1 increased isovolumic developed pressure by 24% above baseline. The aequorin transients were abbreviated and demonstrated decreased amplitude. The positive inotropic effects of IGF-1 were not associated with increased intracellular Ca2+ availability to the contractile machinery but to a significant increase of myofilament Ca2+ sensitivity. Accordingly, the Ca2+-force relationship obtained under steady-state conditions in tetanized muscle was shifted significantly to the left (EC50, 0.44+/-0.02 versus 0.52+/-0.03 micromol/L with and without IGF-1 in the perfusate, respectively; P<0.05); maximal Ca2+-activated tetanic pressure was increased significantly by 12% (211+/-3 versus 235+/-2 mm Hg in controls and IGF-1-treated hearts, respectively; P<0.01). The positive inotropic actions of IGF-1 were not associated with changes in either pHi or high-energy phosphate content, as assessed by 31P nuclear magnetic resonance spectroscopy, and were blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Concomitant administration of IGF binding protein-3 blocked IGF-1-positive inotropic action in ferret papillary muscles. In conclusion, IGF-1 is an endogenous peptide that through a wortmannin-sensitive pathway displays distinct positive inotropic properties by sensitizing the myofilaments to Ca2+ without increasing myocyte [Ca2+]i.

Background: Despite their increasing clinical use and recent evidence that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) target the heart, there has been no systematic investigation of the effects of GH and IGF-1 on the cardiovascular system., Methods and Results: Sixty normal but growing adult female rats were randomized to receive 4 weeks of treatment with GH (3.5 mg.kg-1.d-1), IGF-1 (3 mg.kg-1.d-1), a combination of the two, or placebo. Transthoracic echocardiograms were performed at baseline and at 2 weeks and 4 weeks of treatment. After the final echocardiography, rats underwent either closed-chest left ventricular (LV) catheterization or Langendorff perfusion studies. Myocyte diameter and interstitial tissue fraction were assessed by morphometric histology. Echocardiographic and ex vivo data demonstrated a LV hypertrophic response in all three groups of treated animals that was most marked in the GH group, which alone exhibited a concentric growth pattern (relative wall thickness, 0.52 versus 0.42 to 0.44 in the other groups; P < .001). At 4 weeks, cardiac index was significantly higher and total systemic vascular resistance was lower in all groups of treated animals than in control animals (both P < .001), whereas arterial blood pressure did not differ significantly. All indexes of in vivo and in vitro cardiac function were higher in GH- and IGF-1-treated rats than in control animals, whereas combination therapy yielded a blunted effect. Myocyte diameter was increased in all three treated groups without an increase in interstitial tissue., Conclusions: Exogenous administration of GH and IGF-1 in the normal adult rat induces a cardiac hypertrophic response without development of significant fibrosis. Cardiac performance is increased both in vivo and in the isolated heart.

Insulin resistance is a major factor in the pathophysiology of type II diabetes and a major impediment to successful therapy. The identification of treatments that specifically target insulin resistance could improve diabetes management significantly. Since IGFs exert insulin-like actions and increase insulin sensitivity when administered at supraphysiological doses, we determined the effect of 6 weeks of recombinant human IGF-I (rhIGF-I) administration on insulin resistance and glycemic control in obese insulin-resistant patients with type II diabetes. A total of 12 patients with type II diabetes were recruited for the study. Subcutaneous administration of rhIGF-I (100 micrograms/kg b.i.d.) significantly lowered blood glucose. Fructosamine declined from 369 to 299 mumol/l by 3 weeks of administration and then declined further to 271 at the end of 5 weeks. Glycosylated hemoglobin, which was 10.4% pretreatment, declined to 8.1% at the end of therapy. Mean 24-h blood glucose during a modal day was 14.71 +/- 4.5 mmol/l pretreatment and declined to 9.1 +/- 3.21 mmol/l by the end of treatment. These improvements in glycemia were associated with a decrease in serum insulin levels. Mean insulin concentrations declined from 108.0 to 57.0 pmol/l during the modal day measurements and from 97.2 to 72.0 pmol/l during the mixed-meal tolerance test. Changes in glycemia were accompanied by a marked increase in insulin sensitivity. The insulin sensitivity index (SI) calculated from a frequently sampled intravenous glucose tolerance test (FSIVGTT) after the method of Bergman et al. (Bergman RN, Finegold DT, Ader M: Assessment of insulin sensitivity in vivo. Endocr Rev 6:45-86, 1985) increased 3.4-fold. Furthermore, the improvement in glycemic control was accompanied by a change in body composition with a 2.1% loss in body fat as calculated by dual energy x-ray absorptiometry without change in total body weight. Significant side effects were present in some subjects, although nine subjects were able to complete at least 4.5 weeks of the protocol and six subjects completed the entire 6 weeks. Supraphysiological IGF-I concentrations were maintained throughout the study, increasing from 206 micrograms/l in the control period to 849 micrograms/l at the end of 6 weeks of rhIGF-I treatment. The increase in IGF-I levels was accompanied by a significant increase in IGF binding protein-2 levels, a slight reduction in IGF binding protein-3 levels, and an increase in levels of IGF binding protein-1. In summary, IGF-I significantly lowered blood glucose as reflected by short-term and long-term indexes of glycemic control and increased insulin sensitivity. It remains to be determined whether a dosage can be administered that avoids significant side effects and still achieves reasonable glycemic control.

Human growth hormone (hGH) and insulin-like growth factor I (IGF-I) both stimulate bone formation and have been proposed as therapeutic agents for osteoporosis. We examined the effect of hGH and IGF-I alone and in combination on bone size, bone mineral content (BMC), and bone mineral density (BMD) in 10- to 12-week old growing female Sprague-Dawley rats. Sixty rats were assigned to treatment with either placebo, hGH, IGF-I, or both for 4 weeks. After 4 weeks, the right femurs and tibias were excised, and ex vivo BMC and the area of the tibia and femur were measured by dual-energy X-ray absorptiometry (DXA); volume of these bones was measured by Archimedes' principle. In addition, proximal tibial bone density was measured directly by peripheral quantitative computerized tomography (pQCT). Bone length, area, and volume in all treated groups was greater than controls. Areal bone density by DXA (BMC/area) was higher in IGF-treated rats and lower in GH-treated rats than in controls. Volumetric bone density (BMC/volume) was lower in treated groups than in controls. Measurements by pQCT confirmed that true bone density was lower in all treated groups than in controls. We conclude that treatment with hGH or IGF-I increased bone size and mineral content but decreased bone density in growing rats. Because areal correction of BMC did not adequately correct for the increased bone volume in IGF-treated rats, results of areal bone density by DXA should be interpreted with caution when treatment causes a disparity in bone size between groups.

Insulin resistance is one of the major underlying abnormalities in NIDDM, however, its pathophysiologic mechanisms are not well understood. Many clues about the mechanisms of insulin action have come from patients with the most severe forms of insulin resistance, including those with genetic abnormalities in the insulin signal transduction cascade. We used rhIGF-I as a probe to differentiate insulin and IGF-I action and to study the therapeutic potential of IGF in states of insulin resistance. To date, we have studied six subjects with varying phenotypes of severe insulin resistance but without mutations in the insulin receptor itself. All subjects underwent baseline physiologic monitoring to quantitate carbohydrate tolerance, insulin secretion, and insulin action prior to receiving rhIGF-I at 100 micrograms/kg body wt s.c. bid for 1 month with interval testing of glycemic control and insulin sensitivity. None of the six subjects noted significant side effects from the rhIGF-I. Four of the six subjects had overt diabetes during control testing; three of these subjects demonstrated normalization of fasting and postprandial blood glucose concentrations during rhIGF-I administration on no other therapy. In the fourth patient, insulin requirements and fasting hypertriglyceridemia decreased without improvement in glycemic control. The two subjects with normal glucose tolerance (two sisters with congenital lipodystrophy) maintained normal glucose tolerance at dramatically lower insulin levels and had a dramatic reduction in triglyceride levels. The efficacy of IGF-I continued to increase over the duration of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human (rh) insulin-like growth factor-I (IGF-I) is a potential therapy for individuals with severe insulin resistance, but its efficacy, mechanism of action, or duration of effect for these patients have not been explored fully. Two subjects with the type A phenotype of severe insulin resistance without insulin receptor mutations were investigated to assess insulin secretion, insulin action, and carbohydrate tolerance before and after 3-4 weeks of rhIGF-I treatment (100 micrograms/kg, sc, twice daily). Tests included 24-h glucose and insulin profile (modal day), standardized liquid meal with Sustacal, insulin tolerance test, insulin suppression test, and iv glucose tolerance test. In subject 1, the 24-h mean blood glucose level was 8.1 +/- 2.7 mmol/L before rhIGF-I treatment and fell to 4.2 +/- 0.9 mmol/L during rhIGF-I treatment. The pretreatment 24-h mean serum insulin level was 10,251 +/- 8,849 pmol/L and fell to 1533 +/- 1198 pmol/L. Fasting blood glucose fell from 4.4 to 3.4 mmol/L, and 2-h blood glucose after Sustacal administration fell from 10.3 to 5.3 mmol/L. Fasting serum insulin declined from 808 to 246 pmol/L, and the 2-h serum insulin level fell from 5,491 to 3,443 pmol/L. After bolus iv insulin injection (0.15 U/kg), glucose fell by 20% before rhIGF-I treatment and by 67% during rhIGF-I treatment. The steady state plasma glucose level was 18.2 +/- 0.7 before rhIGF-I and 10.8 +/- 0.1 mmol/L during rhIGF-I. In subject 2, fasting blood glucose fell from 12.0 to 7.4 mmol/L and 24-h mean blood glucose fell from 12.7 +/- 1.9 to 6.6 +/- 1.3 mmol/L. Twenty-four-hour mean serum insulin fell from 892 +/- 635 to 521 +/- 293 pmol/L, and first phase insulin secretion was restored during the iv glucose tolerance test. We conclude that sc rhIGF-I can reduce blood glucose effectively in selected patients with the type A phenotype of severe insulin resistance who have diabetes mellitus. rhIGF-I also can enhance insulin sensitivity, as assessed by a decrease in endogenous insulin levels, normalization of response to iv insulin, and a reduced steady state plasma glucose. The cellular mechanisms for these effects remain undefined.

Somatostatin, a cyclic tetradecapeptide, is both a hypothalamic hormone and a paracrine peptide, with effects on many tissues. Despite the fact that somatostatin can inhibit various cellular events in a number of cell lines, somatostatin is a constituent of medium defined for optimal growth of FRTL5, a line of differentiated and nontransformed rat thyroid follicular cells. In the present study we have evaluated the role of somatostatin in the control of DNA synthesis in FRTL5 cells and have investigated the mechanisms of somatostatin interaction with pathways stimulated by TSH and insulin-like growth factor-I (IGF-I). Somatostatin inhibits TSH-stimulated DNA synthesis and cell proliferation in FRTL5 cells. Maximal effects are observed at somatostatin concentrations of 0.1-10 ng/ml, and the effects are diminished at somatostatin concentrations above 10 ng/ml. Somatostatin also inhibits (Bu)2cAMP-stimulated DNA synthesis, suggesting that the loci of somatostatin action are both proximal and distal to activation of adenylate cyclase. Somatostatin also inhibits DNA synthesis stimulated by insulin-like growth factor-I (IGF-I), a pleiotropic growth factor that works through non-cAMP-dependent pathways. The somatostatin analog octreotide is more potent than native somatostatin in inhibiting DNA synthesis stimulated by either TSH or IGF-I. Somatostatin does not alter TSH or IGF-I binding to FRTL5, demonstrating that somatostatin affects the postreceptor signal transduction pathways stimulated by these factors. We conclude that 1) the use of somatostatin in hormone-supplemented medium for FRTL5 is unnecessary and may inhibit cell growth; 2) somatostatin can inhibit the direct effects of IGF-I on peripheral tissues in addition to its ability to interfere with IGF-I synthesis by inhibiting the synthesis and release of pituitary GH; and 3) somatostatin is a useful tool for dissecting the pathways involved in mediating differentiated function and growth of FRTL5 cells.

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

The binding of [125I]insulin-like growth factor-I ([125I]IGF-I) to human skin fibroblasts (HSF) is regulated by multiple factors. In monolayers of HSF, IGF-I binds to both the type I IGF receptor and IGF-binding proteins (BPs) associated with the cell surface. [125I]IGF-I binding to both of these proteins depends markedly on the sodium chloride concentration of the binding buffer. In monolayers of HSF, replacing 120 mM NaCl with isoosmotic concentrations of sucrose increases binding of [125I]IGF-I by 2- to 6-fold. Enhancement of [125I]IGF-I binding in the absence of sodium chloride is also seen in HSF in suspension, in human erythrocytes, in monolayers of HEP G2 cells and FRTL5 cells, and in membranes prepared from human placentae. Kinetic analysis of [125I]IGF-I binding to HSF monolayers reveals that association rates are increased and dissociation rates are decreased in the absence of sodium chloride. The binding of [125I]alpha IR-3, a monoclonal antibody to the human type I IGF receptor, to monolayers and suspensions of HSF also depends on the sodium ion concentration; it is 5- to 7-fold higher in the absence of sodium chloride. Binding of [125I]IGF-I to monolayers of HSF also depends on NaCl under conditions where alpha IR-3 saturates the type I IGF receptor but does not affect IGF-BPs. These findings demonstrate that sodium chloride has a marked effect on the interaction of IGF-I with the type I IGF receptor in the plasma membrane and with BPs associated with the surface of intact HSFs. Since an effect is also evident in membranes prepared from intact tissues (human placenta), occurs at 4 C, and occurs with cells devoid of BPs, a mechanism involving receptor or BP translocation seems unlikely, at least as the sole explanation for these findings. Sodium ions (and other ions) may induce a conformational change in the receptor and BPs and cause decreased availability of both the IGF-I-binding site and the alpha IR-3 epitope on the receptor and the IGF-binding site on the BP.

Insulin and insulin-like growth factor-I (IGF-I) effects on protein phosphorylation were investigated in intact skeletal muscle cells at different stages of differentiation. In undifferentiated L6 myoblasts, stimulation by either insulin or IGF-I, but not IGF-II, led to a 3- to 5-fold increase in phosphorylation of insulin and IGF receptor beta-subunits and the appearance of a 175,000 mol wt (Mr) phosphoprotein (pp175). These effects reached a maximum within 3 min, were maintained for 12 min, and then declined. Dose-response curves for pp175 phosphorylation in response to insulin (ED50 = 2 nM) and IGF-I (ED50 = 0.2 nM) were consistent with occupancy and stimulation of each receptor kinase by its specific hormone. The 175,000 Mr phosphoprotein was not precipitated by antireceptor antibodies, and the phosphoamino acid composition differed markedly from that of insulin and IGF-I receptors, with a 10-fold lower phosphotyrosine/phosphoserine ratio after insulin stimulation. In contrast to insulin and IGF-I receptors, pp175 was not extracted by the nonionic detergent Triton X-100, but required sodium dodecyl sulfate for solubilization. When experiments were carried out with L6 cells after differentiation into skeletal muscle myotubes, hormone-induced phosphorylation of pp175 was almost undetectable. We conclude that pp175 is a phosphoprotein distinct from insulin and IGF-I receptors that is involved in the early phosphorylation events that follow the activation of the insulin and IGF-I receptor kinases. Its disappearance after terminal differentiation of the L6 cells is consistent with a role in hormonal stimulation of cell proliferation.

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

Preterm human placentae produce insulin-like growth factor- (IGF-)specific binding protein(s). Competitive binding experiments revealed at least two distinct, high-affinity binding sites for IGF-I and IGF-II. Gel permeation chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the binding protein(s) suggested that they possess a molecular weight in the 45 to 50 kD range. In contrast, term placental tissue produced almost no detectable binding protein activity.

Adenosine inhibits TSH-stimulated [3H]thymidine incorporation into DNA in FRTL5 thyroid follicular cells by both inhibiting cAMP generation and acting at a locus beyond adenylate cyclase. On the other hand, adenosine markedly potentiates DNA synthesis in FRTL5 stimulated by insulin-like growth factor-I (IGF-I). The mechanisms of this latter effect are unknown, but require the coincubation of adenosine and IGF-I and not mediated by an increase in intracellular cAMP concentration. Adenosine increases the maximal response of FRTL5 to [3H]thymidine incorporation stimulated by IGF-I and increases the sensitivity of FRTL5 to IGF-I. These effects of adenosine are reflected by an increase in nuclear labeling as well as by an increase in [3H]thymidine incorporation into DNA. Adenosine also plays a role as an autocrine growth factor in FRTL5, since adenosine deaminase increases the response of these cells to TSH. The effects of adenosine on both TSH- and IGF-I-stimulated DNA synthesis are shared by guanosine and inosine, although with different potencies for the various guanine nucleosides. Inosine potentiates IGF-I-stimulated DNA synthesis, but inhibits TSH-stimulated DNA synthesis only weakly. Adenosine interacts with multiple receptors and with multiple postreceptor pathways in FRTL5 to produce divergent effects on the control of cell replication by two growth factors (TSH and IGF-I) that act through different postreceptor pathways.

IGF-I concentrations were determined by RIA in eluates of dried blood collected on filter paper from infants who ranged in age from 3 to 21 d. The infants were separated into normal (greater than 2.5 kg) and low (less than 2.5 kg) birth wt groups and further subdivided on the basis of normal (greater than 83.7 nmol/liter) or low (less than 64.4 nmol/liter) levels of thyroxine. Both the normal and low birth wt groups whose blood thyroxine was in the normal range had similar mean IGF-I values during the 1st wk of life that were significantly higher (p less than 0.05) than those of either the normal or low birth wt groups whose thyroxine concentrations were below normal. Infants older than 1 wk of age with normal birth wt and normal thyroxine levels had significantly greater (p less than 0.05) mean IGF-I values than those of any of the other groups. Infants in the low birth wt-normal thyroxine group exhibited modest increases in IGF-I levels after the 1st wk of life that were significantly greater (p less than 0.05) than those found in the low birth wt-low thyroxine infants. These studies have demonstrated that IGF-I concentration is correlated positively with total thyroxine and with birth wt and that the latter is confounded by the former.

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

Although regulatory effects on their own receptors are among the major responses to peptide hormones and growth factors, little is known of the mechanism of these effects. For example, a wealth of evidence indicates that cAMP is the mediator of many, if not most, of the actions of TSH on the growth and function of the thyroid cell, but evidently no information has been available concerning the possibility that cAMP may also mediate the effect of TSH to down-regulate its own receptors. Therefore, we examined this question using FRTL5 cells as a model, since we had previously shown that withdrawal of TSH from their culture medium and its subsequent readdition lead to increases and decreases, respectively, in the number of their TSH receptors. Because the growth of FRTL5 cells in response to TSH is modified by insulin-like growth factor I (IGF-I), we also examined the independent effect of TSH on receptors for IGF-I, the effects of IGF-I alone on both its own receptors and those for TSH, the effects of the two agents when added together, and the role of cAMP in the independent and conjoint effects of TSH and IGF-I on their own and each other's receptors. Growth of FRTL5 cells in the presence of bovine TSH (bTSH) resulted in a dose-dependent down-regulation of receptors for both bTSH and IGF-I. In contrast, IGF-I alone produced only a modest down-regulation of its own receptors and had no effect on the binding of bTSH. IGF-I did, however, enhance the effect of bTSH on the binding of both ligands. All of the foregoing down-regulatory effects of bTSH, acting both alone and together with IGF-I, were closely mimicked when either (Bu)2cAMP or forskolin was substituted for bTSH. Therefore, cAMP appears to mediate, at least in part, the effects of both bTSH alone and bTSH acting in concert with IGF-I to down-regulate receptors for both mitogens.

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

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

Thyroid hormones influence growth in part by altering the secretion and effects of GH. GH, in turn, mediates its effects by regulating the synthesis and secretion of insulin-like growth factor-I (IGF-I). IGF-I is a pleiotropic growth factor that is synthesized by many tissues and acts on many tissues to regulate both cellular replication and differentiated function. We have studied the direct effects of thyroid hormones and the combined effects of thyroid hormones and GH on the regulation of IGF-I synthesis and secretion in hypophysectomized (hypox) rats in vivo. All rats, except normal littermates and a hypox control group, received 100 micrograms hydrocortisone/100 g BW for 10 days. Circulating IGF-I was measured by specific RIA (normal rats, 1 U/ml), and hepatic IGF-I mRNA was measured by Northern blot hybridization with an antisense cRNA probe. 1) Hypox rats treated with hGH (75 micrograms, ip, twice daily) for 10 days gained 17 g BW vs. 70 g for normal littermates. GH markedly increased hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 U/ml 12 h after the last GH injection vs. 0.03 +/- 0.02 for hypox controls). 2) T4 (1 micrograms/100 g BW, ip) for 10 days increased neither weight, hepatic IGF-I mRNA, nor circulating IGF-I. 3) Rats treated with T4 for 10 days followed by a single injection of 1 mg GH, ip, increased hepatic IGF-I mRNA and circulating IGF-I levels comparably as in rats receiving acute GH alone (IGF-I, 12 h, 0.31 +/- 0.09 vs. 0.36 +/- 0.06 U/ml). 4) Hypox rats treated with a single injection of T3 (1.5 micrograms/100 g BW, ip) had slightly increased hepatic IGF-I mRNA, but showed no significant change in circulating IGF-I levels. 5) A single injection of T3 plus GH to hypox rats increased IGF-I mRNA levels above those in rats injected with GH alone and increased serum IGF-I levels to 0.48 +/- 0.12 U/ml compared to 0.36 +/- 0.06 U/ml for GH alone. 6) After 10 days of GH treatment, a single injection of T3 lowered both hepatic IGF-I mRNA and circulating IGF-I (0.52 +/- 0.14 to 0.16 +/- 0.06 U/ml, 6 h after T3). These studies demonstrate that thyroid hormones have relatively little direct effect on IGF-I synthesis but can have major effects on GH-stimulated IGF-I synthesis and secretion. The pattern of these effects depends on the integrity of the pituitary gland, prior exposure of the liver to GH and/or thyroid hormones, and the temporal relationship between GH and thyroid hormone administration.

In previous studies we have demonstrated that bovine TSH (bTSH) and insulin-like growth factor I (IGF-I) independently stimulate both the incorporation of [3H]thymidine into DNA and replication in quiescent FRTL5 cells. In the case of TSH, evidence was presented that these responses are cAMP mediated. In addition, responses of thymidine incorporation are greatly amplified when particular concentrations of the two agents are added together, but this effect diminishes as the concentration of either bTSH or IGF-I is increased. The present experiments were undertaken to obtain further information concerning the mechanism of the independent mitogenic effects of bTSH and IGF-I and to explore the nature of the biphasic synergistic interaction with respect to thymidine incorporation that occurs when bTSH and IGF-I are added together. Verification that the increases in [3H] thymidine incorporation induced by bTSH and IGF-I, alone and together, are truly reflective of increases in DNA synthesis was obtained in experiments in which labeled nuclei were counted in cultures of cells grown in the presence of one or both mitogenic agents to which [3H]thymidine had been added. In these studies the number of cells with labeled nuclei was increased markedly by each of the two agents, and the response when the two mitogens were added together was far greater than the sum of their individual effects. Over a range of concentrations which included those that elicit a mitogenic response in FRTL5 cells, IGF-I, unlike bTSH, failed to increase cAMP generation when added alone. Moreover, IGF-I did not significantly enhance the cAMP response to varying concentrations of bTSH. A concentration-dependent increase in the incorporation of [3H]thymidine into DNA was induced by culturing cells in the presence of the cAMP analog (Bu)2cAMP (Bt2cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, and the stimulator of adenylate cyclase forskolin. When increasing concentrations of these agents were added together with IGF-I, a biphasic pattern of response of DNA synthesis, mimicking that produced by the combination of IGF-I and increasing concentrations of bTSH, was observed. Further evidence that cAMP mediates the mitogenic response to bTSH was the observation that adenosine inhibited the stimulation of both cAMP generation and DNA synthesis that bTSH produced. Although preincubation of quiescent FRTL5 cells for 24 h in the presence of bTSH resulted in only a small increase in DNA synthesis, measured during the last 3 h of a subsequent 24-h incubation carried out in the absence of bTSH, it greatly amplified the response to IGF-I added alone during the second incubation.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Insulin and insulin-like growth factors (IGFs) stimulate responses in skeletal muscle that include effects on carbohydrate and fat metabolism, protein turnover, growth, and differentiation. To gain insight into the relative importance of insulin and IGFs at different stages of development, the expression of their specific receptors was evaluated in skeletal muscle of rats from the late fetal period through 40 weeks of age. Distinct receptors for insulin. IGF-I and IGF-II are present in crude membrane preparations and wheat germ agglutinin-purified extracts of hindlimb muscle from rats at all ages, but each of the three receptors follows a different pattern of expression during development. There is a marked predominance of IGF-II receptors in fetal muscle (80- and 55-fold more abundant than insulin and IGF-I receptors, respectively) and a rapid decline in IGF-II receptors in early postnatal life. IGF-I receptors are more abundant than insulin receptors in the term fetus, remain constant in number until approximately 4 weeks of age, and then gradually decline to adult levels. Insulin receptor number rises 2- to 3-fold postnatally, peaks at approximately 4 weeks, and decreases to levels in the adult that are slightly lower than those in the term fetus. Although binding affinities and receptor specificity did not change during development, the relatively large number of IGF-II receptors in the fetus resulted in significant binding of IGF-I to receptors for both IGF-II and IGF-I. There was a modest increase in apparent mol wt of all three receptor types during development, suggesting a change in a common pathway, such as posttranslational glycosylation. The marked changes in number and distinct patterns of expression of the insulin, IGF-I, and IGF-II receptors in muscle during development are consistent with evolving functions of the three hormones determined by alterations in both receptor number and hormone concentrations.

We have studied the binding of recombinant human insulin-like growth factor I (hIGF-I), and hIGF-II, and rat IGF-II [rIGF-II (multiplication-stimulating activity)] to the human amniotic fluid IGF-binding protein placental protein-12 (PP12). PP12 displayed a 5- to 10-fold higher affinity for IGF-I compared to hIGF-II or rIGF-II. These differences in binding affinity were confirmed by both saturation binding analysis and competitive binding analysis using 125I-labeled IGF-I, hIGF-II, and rIGF-II and each of the unlabeled ligands. PP12 produced dose-dependent inhibition of IGF-I-stimulated [3H]thymidine incorporation in the rat thyroid follicular cell line FRTL5. Inhibition of IGF-I-stimulated thymidine incorporation paralleled the ability of PP12 to inhibit IGF-I binding to the surface of FRTL5. At a high concentration, PP12 also inhibited TSH-stimulated DNA synthesis but did not inhibit the binding of 125I-labeled TSH to FRTL5. Insulin did not inhibit the binding of 125I-labeled IGFs to PP12, and PP12 did not inhibit the ability of insulin to stimulate DNA synthesis. These data suggest that the ability of PP12 to inhibit TSH-stimulated DNA synthesis is through the inactivation of IGF produced endogenously by FRTL5. Low concentrations of PP12 produced a statistically significant enhancement of TSH-stimulated DNA synthesis; the mechanism by which this occurs remains unclear.