Differential regulation of insulin-like growth factor I by growth hormone and thyroid hormone in the heart of juvenile hypophysectomized rats.

Recent data suggest that the heart can act as both a source and target for the actions of polypeptide growth factors. Insulin-like growth factor I (IGF-I) is a polypeptide that has both mitogenic and differentiation properties that function at the autocrine/paracrine level, and has recently been demonstrated to be expressed in the heart. This knowledge, coupled with the observation that thyroid hormone (T3) promotes relative cardiac growth compared to the proportional increases in body and heart growth evoked by growth hormone (GH), lead us to speculate whether differential induction of cardiac IGF-I may account for the specialized trophic effects of T3 on the heart. Cardiac IGF-I gene expression was studied in an in vivo model in which cardiac growth in the hypophysectomized juvenile rat was stimulated with either GH, T3 or GH + T3. Two week infusions of T3 that resulted in cardiac growth, but no gain in body weight, resulted in a 4.6-fold increase in cardiac IGF-I mRNA levels compared to hypophysectomized controls. GH infusions that resulted in similar cardiac growth, but were accompanied by proportional body growth, had no effect on cardiac IGF-I mRNA levels. These data are the first to demonstrate stimulation of cardiac IGF-I mRNA levels by T3 and further support cardiac autocrine/paracrine actions for this polypeptide growth factor.     

Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I

The potential role of satellite cells in mediating the effect of trenbolone [17 beta-hydroxyestra-4,9-11-trien-3-one (TBOH)] on skeletal muscle hypertrophy was examined. Young female Sprague-Dawley rats received TBOH injections daily for 2 weeks; growth, body composition, and the composition of selected muscles were assessed. Treated rats grew more rapidly and deposited less body lipid and more protein. The semimembranosus muscle from treated rats was larger and had approximately 60% more DNA per muscle than muscles from control rats. The addition of trenbolone directly to the medium of cultured satellite cells did not stimulate cell proliferation, nor did it augment the stimulatory response of these cells to fibroblast growth factor (FGF) or insulin-like growth factor I (IGF-I). In contrast, satellite cells cultured from TBOH-treated rats exhibited greater proliferative responses to FGF and IGF-I than satellite cells from control rats. In addition, serum from TBOH-treated rats stimulated greater cell proliferation in satellite cell cultures than serum from control rats. These experiments suggest that one possible mechanism responsible for the ability of TBOH to stimulate skeletal muscle hypertrophy may be through enhanced proliferation and differentiation of satellite cells as a result of the increased sensitivity of these cells to IGF-I and FGF.     

Induction of insulin-like growth factor I messenger ribonucleic acid during regeneration of rat skeletal muscle

Expression of insulin-like growth factor I (IGF-I) was studied in regenerating skeletal muscle. Irreversible damage to muscle cells was induced in the extensor digitorum longus muscle of adult rats by ischemia, preceded by glycogen depletion. IGF-I mRNA levels during the regeneration process were studied for periods up to 10 days after injury using a solution hybridization assay. Increased IGF-I mRNA levels could be demonstrated within 24 h after injury; maximum levels were achieved in 3 days and decreased to approximately normal levels by 10 days. Changes in IGF-I mRNA levels could not be seen in undamaged contralateral extensor digitorum longus muscles during the experimental period. An increase in IGF-I mRNA was also evident in injured muscles of hypophysectomized animals. In situ hybridization at the time of maximum induction showed the presence of IGF-I mRNA in proliferating myoblasts and in satellite cells. IGF-I, thus, may act as a locally produced non-GH dependent trophic factor during regeneration of skeletal muscle after injury.     

Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth.

Melanocytes, highly differentiated neural crest-derived cells, are located in the basal layer of the epidermis, where they play a role in protecting against UV damage in the skin. Previous studies suggest that both growth hormone (GH) and the insulin-like growth factor I (GH/IGF-I) system may be important for melanocyte growth and function. We have therefore characterized the role of the GH/IGF system in melanocyte growth in vitro and its interaction with the local growth factor basic fibroblast growth factor (bFGF). Analysis of the effects of GH, IGF-I, and bFGF and combinations of these growth factors on melanocyte growth in vitro revealed that 1) GH stimulates the growth of melanocytes when combined with IGF-I, des(1-3)IGF-I [an analog of IGF-I that has a reduced binding affinity for IGF-binding proteins (IGFBPs)], or bFGF, either separately or in combination; 2) in contrast to the lack of effect of GH or bFGF alone, both IGF-I and des(1-3)IGF-I enhance melanocyte growth in a dose-dependent manner; and 3) IGF-I is more efficacious in eliciting a growth response at low concentrations compared to des(1-3)IGF-I. Using Western ligand blotting, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we show that cultured human melanocytes synthesize and secrete minimal amounts of IGFBP. IGFBP-4 is the major IGFBP produced by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1-3)IGF-I alone. In conclusion, these studies provide support for a role for both GH and IGF-I in the growth of human melanocytes in vitro, involving synergy with bFGF. Low levels of melanocyte-derived IGFBP-4 may play a role in enhancing the modulation of IGF action.     

Growth promotion by insulin-like growth factor I in hypophysectomized and diabetic rats

Two animal models, the hypophysectomized (hypox) and the streptozotocin-diabetic rat, both of which are GH-deficient, were used to study the effects of infused IGF I and its molecular size distribution in serum, and to investigate whether GH and IGF I act identically on growth plate chondrocyte maturation. In hypox rats, IGF I (300 μg/rat per day) stimulated body weight gain, total growth plate height and longitudinal growth rate, although less than GH (200 mU/rat per day), and mimicked the effect of GH at all stages of chondrocyte differentiation, including stem cells. Infused IGF I was nearly exclusively found in a <100 kDa IGF binding protein (IGFBP) complex, whereas free IGF I was barely detectable. These findings argue against the ‘dual effector theory’ in vivo which postulates priming of the stem cells by GH before IGF I stimulates proliferation. They also suggest that IGF I bound to the <100 kDa IGFBP complex is bioavailable for growth. In diabetic rats infused with 2.5 mg/rat per day of IGF I, body weight, tibial epiphyseal width and accumulated bone growth increased dramatically despite persisting hyperglycemia. Insulin infusion (2.5 U/rat per day), which nearly normalized elevated blood sugar values, raised endogenous IGF I serum levels and stimulated growth parameters to a similar extent as IGF I, in line with a similar distribution of the infused exogenous and the insulin-induced endogenous IGF I between the free and the <100 kDa-bound form. Since GH secretion is inhibited in diabetic rats and the animals are resistant to GH action, these results, like those in hypox rats, demonstrate that IGF I can act on growth independently of GH. Because insulin restores GH secretion and the responsiveness of the liver to GH in diabetic rats, one may conclude that insulin acts on growth of diabetic rats mainly via restoration of the GH/IGF I axis.     

Repopulation of the atrophied thymus in diabetic rats by insulin-like growth factor I.

Atrophy of the thymus is one of the consequences of severe insulin deficiency. We describe here that the weight and the architecture of the thymus of diabetic rats is restored towards normal not only by insulin but also by insulin-like growth factor I (IGF-I) treatment. In contrast to insulin, this effect of IGF-I occurs despite persisting hyperglycemia and adrenal hyperplasia. We also investigated the in vivo effect of IGF-I on replication and differentiation of thymocytes from streptozotocin-induced diabetic rats. Thymocytes from diabetic rats incorporated less [3H]thymidine than did thymocytes from healthy rats. Insulin, as well as IGF-I treatment of diabetic rats increased [3H]thymidine incorporation by thymocytes. Flow cytometry of thymocytes labeled with monoclonal antibodies revealed a decreased expression of the Thy-1 antigen in diabetic rats compared with control rats. In addition, a major deficiency of thymocytes expressing simultaneously the W3/25 and the Ox8 antigens (corresponding to immature human CD4+/CD8+ thymocytes) was observed. These changes were restored towards normal by insulin as well as by IGF-I treatment. The antibody response to a T cell-dependent antigen (bovine serum albumin) was comparable in normal and diabetic rats. We conclude that IGF-I has important effects on the thymocyte number and the presence of CD4+/CD8+ immature cells in the thymus of diabetic rats despite persisting hyperglycemia. However, helper T-cell function for antibody production appears to be preserved even in the severely diabetic state.     

Divergent effect of endogenous and exogenous sex steroids on the insulin-like growth factor I response to growth hormone in short normal adolescents

The lower responsiveness to GH in women than in men is probably due to a divergent effect of gonadal steroids. It is unknown, however, how the progressive increase in sex steroid production that occurs during puberty affects this responsiveness. To compare the effects of puberty and sex steroid administration on responsiveness to GH, we used the IGF-I generation test, in which the peak IGF-I level 24 h after a single injection of GH (2 mg/m2) was studied in 117 healthy short subjects (56 females and 61 males). The subjects, aged 8-16 yr, were divided into four groups: prepuberty, early puberty, midpuberty, or pubertal delay. In the latter group, the IGF-I response was determined before and after priming with oral 17beta-estradiol in girls and im testosterone in boys. We also tested for an association between body composition (by dual energy x-ray absorptiometry) and the IGF-I response to GH. The IGF-I increment in response to GH (change in IGF-I from baseline) was correlated with the growth velocity sd score (P < 0.05). Progression throughout puberty was associated with an increase in both baseline IGF-I (P < 0.05) and the IGF-I increment in response to GH (P < 0.05), with no gender difference. Pubertal category (pre-, early, and midpuberty; P < 0.05) and fat percentage (P < 0.05) were the main positive predictors of the IGF-I increment in response to GH, expressed as micrograms per liter as well as sd score, independently of baseline IGF-I. After sex steroid priming, both the GH peak in response to insulin-induced hypoglycemia and baseline IGF-I were increased (P < 0.05, after vs. before sex steroid). However, the IGF-I increment in response to GH decreased after oral 17beta-estradiol (P < 0.05), whereas it was unchanged after testosterone administration. Endogenous gonadal steroid secretion appears to result in increased responsiveness to GH in peripubertal girls and boys. By contrast, exogenous estrogen and testosterone, respectively, produce a relative decrease and no change in responsiveness to GH in similar populations, possibly through the achievement of sex steroid concentrations exceeding physiological ranges for age. Fat percentage was a positive determinant of the responsiveness to GH, suggesting a link between the energy stores and the anabolic action of GH.     

Hormonal regulation of type I insulin-like growth factor receptors of Leydig cells in hypophysectomized rats

The effects of hCG and various pituitary hormones on type I insulin-like growth factor (IGF) receptors of purified Leydig cells of hypophysectomized rats were studied. The number of type I IGF receptors of Leydig cells obtained from hypophysectomized rats (18.0 +/- 1.5 fmol/10(6) cells) was lower than that in normal rats (54.6 +/- 5.3 fmol/10(6) cells; P less than 0.05). After a single administration of hCG (10 U, ip), specific binding of [125I]IGF-I to purified Leydig cells increased 3-fold. Scatchard analyses of the binding data suggested that increased binding was the result of an increase in receptor number, whereas binding affinity remained unaltered. Type I IGF receptor increased within 12 h and remained persistently elevated 96 h after hCG treatment. Administration of hCG (10 U, ip) daily for 5 days increased type I IGF receptor levels to 73.2 +/- 8 fmol/10(6) cells (P less than 0.001). FSH caused a small but significant increase in type I IGF receptors. Concomitant administration of FSH and hCG further enhanced IGF-I-binding capacity. IGF-I-binding affinity of Leydig cells treated with FSH or FSH plus LH was not significantly different from that in the control hypophysectomized rats. Daily administration of GH for 5 days also upregulated type I IGF receptors, whereas PRL had no effect. FSH, GH, and PRL administration had no effect on serum testosterone levels. Serum testosterone levels increased to 3.99 +/- 0.35 ng/ml after 5 days of treatment with hCG. Concomitant administration of FSH and hCG caused a further increased in serum testosterone levels (6.13 +/- 0.46 ng/ml; P less than 0.01). The present study suggests that type I IGF receptors of Leydig cells can be up-regulated by LH, FSH, and GH. However, hCG/LH seems to be the most important factor in maintaining and regulating type I IGF receptors of Leydig cells. Steroidogenic and growth-promoting effects of hCG and pituitary hormones on Leydig cells may be mediated by increased type I IGF receptors.     

Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is the presumed paracrine or autocrine growth-promoting mediator of growth hormone in peripheral tissues. In order to evaluate the role of IGF-I as mediator of human growth hormone (hGH) in erythropoiesis, we compared the effects of both peptides upon in vitro colony formation by primitive (BFU-E) and relatively mature (CFU-E) human erythroid precursors. Biosynthetic IGF-I (2 ng/ml) and hGH (25 ng/ml) induced a significant increase in the growth of both BFU-E and CFU-E. BFU-E growth was maximally enhanced by 6 ng/ml IGF-I and by 50 ng/ml hGH, resulting in an increase in burst numbers of 62 ± 12% and 52 ± 12%, respectively. Maximal enhancement of CFU-E growth was detected at higher concentrations of IGF-I (20 ng/ml) and hGH (150 ng/ml), with respective increases of 121 ± 35% and 137 ± 18% in colony numbers. Enhancement of bone marrow and peripheral blood erythroid progenitor cell growth by hGH required the presence of monocytes and was abrogated by specific monoclonal antibodies directed against IGF-I membrane receptors. The in vitro growth-promoting effect of hGH upon human erythroid precursors thus appears to be mediated by paracrine IGF-I.     

Sensitization of human fibroblasts to insulin-like growth factor I by serum deprivation and dexamethasone pretreatment

Pretreatment of confluent human fibroblast cultures for two days in dexamethasone, serum free medium increased 10-20 fold the sensitivity of the cells to insulin-like growth factor I (IGF I) stimulation of amino acid uptake using the amino acid analog alpha-aminoisobutyric acid (AIB). This increased sensitivity resulted both from the use of serum free medium and the addition of dexamethasone to the serum free media. Pretreatment of the cells for 1, 2, or 3 days before assay showed that the maximum increase in sensitivity was obtained after a two day pretreatment. Pretreatment of the cells also increased their sensitivity to insulin and bovine insulin-like growth factor II stimulation of AIB uptake similar to that seen for IGF I. No consistent effect of the pretreatment was observed on either the basal level of AIB uptake or the maximal hormonal stimulation of AIB uptake. Nor was any change noted in the shape of the dose response curves. Addition of IGF I to the pretreatment medium greatly reduced the sensitivity of pretreated cells. [125I]IGF I binding studies done on suspended fibroblasts indicated that there was up to a two fold increase in the number of receptors with no increase in their affinity for IGF I. Thus, pretreatment of fibroblasts with dexamethasone and serum free medium greatly enhances their sensitivity to IGF I stimulation of AIB uptake and makes this an excellent in vitro bioassay system for IGF I.     

Growth hormone exerts acute vascular effects independent of systemic or muscle insulin-like growth factor I

CONTEXT: Endothelial dysfunction is common in patients with GH deficiency who are at increased risk for premature cardiovascular death. GH regulates vascular tone and reactivity in humans. OBJECTIVE: Our objective was to explore the mechanisms underlying the GH's acute vascular effects. DESIGN AND STUDY SETTING: There were 10 healthy, lean and young, volunteers studied after an overnight fast. GH was infused systemically for 6 h at 0.06 microg/kg.min. Biopsy of the vastus lateralis muscle was done in seven subjects before and after GH infusion. Human aortic endothelial cells (HAECs) were incubated with GH in vitro. RESULTS: GH infusion increased plasma GH to 32.9 +/- 1.5 ng/ml and forearm blood flow by 66% (P < 0.001). GH infusion did not significantly change plasma IGF-I concentrations, muscle IGF-I mRNA expression, and muscle Akt phosphorylation, suggesting a lack of IGF-I action in muscle. Because it was reported that GH exerts an acute vascular effect via a nitric oxide (NO)-dependent mechanism, we performed additional in vitro experiments using HAECs. HAECs express abundant GH receptors. Incubating HAECs with GH at 30 ng/ml for 3 or 6 h did not alter endothelial NO synthase (eNOS) protein content but time dependently increased the phosphorylation and activity of eNOS, thus demonstrating a direct effect of GH on endothelial cells. CONCLUSIONS: GH exerts an acute vascular effect independent of both systemic and local IGF-I production, and this effect is likely via direct action on GH receptors and eNOS in the vascular endothelium.     

Regulation of insulin-like growth factor binding protein-3 expression by dexamethasone.

Dexamethasone (DXM), a potent long acting glucocorticoid results in growth retardation when administered to children and experimental animals. We have used ligand blotting and RNA blotting techniques to examine the effects of DXM on serum insulin-like growth factor binding protein 3 (IGFBP-3) levels and hepatic IGFBP-3 mRNA abundance. A time- and dose-dependent increase in the 39-42 kDa serum IGF binding proteins and in IGFBP-3 mRNA abundance was observed in DXM-treated rats. A significant increase in serum IGF binding capacity was seen with as little as 0.1 microgram/100 g body weight. A significant increase in IGFBP-3 mRNA abundance was apparent as early as 1 h following DXM administration. IGFBP-3 mRNA levels reached a peak at 3 h (1.9 +/- 0.2 fold, p less than 0.005) and declined to normal levels in 6-12 h after DXM administration. Since the IGF binding proteins may be able to inhibit the action of the IGFs, enhanced expression of IGFBP-3 may be one of the mechanisms involved in DXM-induced growth retardation.     

Interactive regulation of postmenopausal growth hormone insulin-like growth factor axis by estrogen and growth hormone-releasing peptide-2

Estrogen is the proximate sex steroid sustaining GH secretion throughout the human life span in both sexes. However, very little is known about the specific neuroendocrine mechanisms by which estrogen activates and maintains GH secretion in the young or aging human. The identification of somatostatin in 1973 as a key negative peptidyl regulator of the GH axis and the discovery of GH-releasing hormone (GHRH) in 1982 as a dominant feedforward agonist of GH secretion provided an initial basic science foundation for exploring sex-steroid control of the GH-IGF-1 axis. Although GH-releasing peptides (GHRPs) were first recognized in 1977-1981, subsequent cloning of hypothalamopituitary receptors transducing potent secretagogue actions of GHRPs in 1996 and of an endogenous ligand for this effector pathway in 1999 now extend the framework for examining the mechanisms of estrogen-driven GH secretion in aging. Herein, we review several novel and multifaceted interactions in postmenopausal women between estrogen and GHRP-2. We combine these observations into a simplified construct of GH-axis neuroregulation comprising the somatostatin, GHRH, and GHRP effector pathways, as well as GH and IGF-1 autofeedback. We suggest the thesis that estrogen controls the interfaces among these pivotal regulatory peptides in hyposomatotropic postmenopausal individuals.     

Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.