Insulin-like Growth factor-I Secretion Research Articles

Studies on the analysis of endocrinologic and metabolic factors controlling the concentration of plasma insulin-like growth factor-I (IGF-I) in maternal rats

The effects of the difference of nutritional intake on the kinetics of IGF-I secretion were investigated by 48 hours refeeding with standard, low-protein, low-fat, or low-sugar diet after 72 hours fasting in nonpergnant rats. The effects of hormonal and nutritional changes on the plasma level of IGF-I were also examined in pregnant rats by administration of hGH (100μg/day) for 3 days from day 12 or day 17 of pregnancy and by fasting for 48 hours from day 18. Plasma levels of GH and IGF-I were measured by radioimmunoassay, and the nitrogen balance was determined by the Plegl-Damas method.When nonpregnant rats were fed diets containing different nutrients after 72 hours of fasting, feeding with a low-protein diet, decreased plasma levels of total protein and albumin, and a decreased nitrogen balance were found to be closely related to a fall in the plasma level of IGF-I. In the nonpregnant rats, it appeared that nutrient intake and protein metabolism were important factors in the control of IGF-I.When the pregnant rats were administered 100μg/day of GH from day 12 to day 14 or day 17 to day 20 of gestation, the plasma levels of IGF-I were not significantly higher than in the control group given physiological saline intramuscularly.The IGF-I level in pregnant rats was 87.6±17.5 ng/ml on day 18. When the rats fasted for 48 hours from day 18, the IGF-I level fell to 60.6±10.7 ng/ml on day 20, a value significantry lower than that in rats allowed free access to food (118.6±24.8 ng/ml). A significant decrease in the plasma levels of total protein, albumin, triglycerides, and total cholesterol was seen after 48 hours of fasting from day 18. The nitrogen balance was 48.6±38.5 mg/day on day 18, and became markedly negative (-279.4±94.1 mg/day) on day 20 after fasting for 48 hours. The plasma level of IGF-I decreased as the nutritional status of the rats worsened, and changes in the IGF-I concentration were in close agreement with changes of the simultaneously measured nitrogen balance.These findings suggest that plasma levels of IGF-I in pregnant rats are influenced by changes of nutrient metabolism, especially protein metabolism.

Mutational Analysis of the Insulin-like Growth Factor I Prohormone Processing Site

Insulin-like growth factor I (IGF-I) is a mitogenic peptide that is produced in most tissues and cell lines and plays an important role in embryonic development and postnatal growth. IGF-I is initially synthesized as a prohormone precursor that is converted to mature IGF-I by endoproteolytic removal of the carboxyl-terminal E-domain. Regulation of the conversion of proIGF-I to mature IGF-I is a potential mechanism by which the biological activity of this growth factor might be modulated. Endoproteolysis of the IGF-I prohormone occurs at the unique pentabasic motif Lys-X-X-Lys-X-X-Arg71-X-X-Arg-X-X-Arg. Recently, a family of enzymes which cleave prohormone precursors at sites containing multiple basic residues has been discovered. The goals of this study were 1) to determine which basic residues in the pentabasic proIGF-I processing site were necessary for proper cleavage and 2) to examine the role that subtilisin-related proprotein convertase 1 (SPC1/furin) might play in proIGF-I processing. We have shown that an expression vector coding for an epitope-tagged proIGF-I directs synthesis and secretion of mature IGF-I-(1-70), extended IGF-I-(1-76), proIGF-I, and N-glycosylated proIGF-I in human embryonic kidney 293 cells. Extended IGF-I-(1-76) is produced by cleavage at Arg77 and requires both Arg74 (P4) and Arg77 (P1). Cleavage at Arg77 does not occur in the SPC1-deficient cell lines RPE.40 and LoVo, suggesting that processing at this site is mediated by SPC1. Mature IGF-I-(1-70) is produced by cleavage at Arg71 and requires both Lys68 (P4) and Arg71 (P1). Lys65 in the P7 position is important for efficient cleavage. SPC1 is not required for processing at Arg71 since this cleavage occurs in RPE.40 and LoVo cells. These data suggest the existence of a processing enzyme which is specific for the Lys-X-X-Arg motif of proIGF-I.

Multihormonal regulation of progesterone synthesis in cultured human midluteal cells.

The objectives of this investigation were to examine in vivo insulin like-growth factor-I (IGF-I) secretion by the human midcorpora lutea (mid-CL) and the effects of IGF-I, hCG, FSH, and human GH on progesterone (P) production by CL cells obtained from patients at laparotomy. We first examined whether the CL produces IGF-I by measuring IGF-I levels in the ovarian vein from the ovary bearing the CL. The IGF-I concentration in the ovarian vein bearing the CL (206 +/- 31 ng/mL) was significantly increased compared to the concentration in the contralateral ovarian vein (179.2 +/- 32 ng/mL; P < 0.05). Luteal cells isolated from mid-CL were cultured in serum-free medium 199 in the presence and absence of hCG, FSH, GH, and graded concentrations of IGF-I. At the end of the incubation period (24 h), P levels in the medium were measured by RIA. The treatment with IGF-I (0.1-10 ng/mL) showed a dose-dependent stimulatory action of IGF-I on P synthesis in the luteal cell system, being maximal between 5-10 ng/mL. The treatment with hCG (10 IU/mL), IGF-I (5 ng/mL), and GH (1000 ng/mL) increased basal P synthesis by 300%, 80%, and 30%, respectively (P < 0.001 and P < 0.05). FSH (100 ng/mL), either alone or in combination with IGF-I, failed to stimulate P synthesis. Treatment with IGF-I monoclonal antibody (1:5000) completely reduced P synthesis induced by 5 ng/mL IGF-I and slightly reduced basal P synthesis as well as GH-stimulated P synthesis by human midluteal cells. To further evaluate the specific role of IGF-I on luteal steroidogenesis, IGF-I receptor was identified by chemical cross-linking of [125I]IGF-I to mid-CL membranes. Experiments conducted in the absence and presence of unlabeled IGF-I (500 ng) revealed proteins with characteristics of the type I IGF receptor. These results are consistent with multihormonal regulation of P synthesis by the human mid-CL. hCG and IGF-I play a major role in the stimulation of P synthesis and, to a lesser extent, human GH. These in vivo and in vitro data suggest that the CL is a site of secretion, action, and reception of IGF-I during the midluteal phase.

N-terminal-truncated recombinant analogs of bovine placental lactogen: interaction with human and rat growth hormone receptors and insulin-like growth factor-I secretion mediated by somatogenic receptors in rat hepatocytes.

Bovine placental lactogen (bPL) was found to be as potent as human GH (hGH) in its ability to bind to soluble full-size recombinant hGH-binding protein (hGHBP) and to membrane-embedded hGH receptor in intact IM-9 human lymphocytes. bPL was also capable of forming a 1:2 complex with hGHBP, although the structure of this complex was probably more compact than that with hGH. Removal of 13 amino acids from the N-terminus of bPL did not affect its ability to bind to hGHBP or hGH receptors in intact IM-9 cells. Its ability to form a 1:2 complex with hGHBP was, however, impaired, unlike that of a corresponding analog in which an L28F mutation has been simultaneously introduced. Truncation of 17 amino acids decreased its affinity toward both hGHBP and GH receptors on intact IM-9 lymphocytes and in liver rat microsomal fraction and inhibited the formation of 1:2 complexes with hGHBP. Simultaneous L28F mutation did not affect affinity toward hGHBP, but increased affinity toward rat liver GH receptors and restored affinity toward membrane-embedded hGH receptors in IM-9 lymphocytes and the ability to form a 1:2 complex with hGHBP. Truncation of 20 amino acids further decreased affinity toward both hGHBP and receptors in intact IM-9 lymphocytes and completely abolished formation of a 1:2 complex with hGHBP. Both des-13-bPLs and bPL-des-17 (L28F) retained their full ability to stimulate insulin-like growth factor-I secretion by rat hepatocytes compared to that of bPL. The insulin-like growth factor-I stimulatory activities of bPL-des-17 and bPL-des-20, however, were decreased to 1-5%. These results indicate that the stoichiometry of 1:2 complex formation with hGHBP may be preserved despite decreased receptor binding affinity, but the lower affinity of the putative site 1 or site 2 of the analog may account for the decrease in biological activity. Furthermore, the ability or inability of bPL or its truncated analogs to form 1:2 complexes with soluble hGHBP cannot predict their somatogen receptor-mediated biological activity in rat hepatocytes.

Growth hormone directly and indirectly stimulates articular chondrocyte cell growth

Although growth hormone (GH) is known to regulate cartilage growth and differentiation during development, it is still unclear whether the cell growth of articular chondrocytes is stimulated directly by GH or mediated by GH-induced insulin-like growth factor-I (IGF-I). In the present study, we focused on whether GH directly or indirectly stimulates articular chondrocyte proliferation. Monolayer articular chondrocytes from 5-week-old male Sprague-Dawley rats were cultured in Ham's F-12/Dulbecco's modified essential medium supplemented with 10% fetal bovine serum. Stimulation of DNA synthesis by GH was dose-dependent between 0.1 and 1 microg/ml, and the maximum active concentration of GH was 500 ng/ml, which induced a 3.5-fold increase over control values. Anti-IGF-I antiserum neutralized about 80% of GH-induced DNA synthesis. GH stimulated the secretion of IGF-I into the conditioned medium in a dose-responsive manner. To determine whether GH stimulated DNA synthesis directly, we investigated the time-course changes in mRNA expression of IGF-I and the proto-oncogene c-myc. Induction of IGF-I mRNA occurred at 4 h, and reached a maximum level at 12 h, whereas the expression of c-myc mRNA was induced within 4 h, and continued to increase until 72 h after GH treatment. Furthermore, administration of cycloheximide, an inhibitor of protein synthesis, resulted in the superinduction of both IGF-I and c-myc mRNAs. These results suggest that early induction of c-myc is due to a direct stimulatory effect of GH, and that long-term induction of c-myc was attributable to an indirect effect of GH in which GH-induced secondary proliferative factors may act in an autocrine/paracrine manner. The superinduction of c-myc gene by cycloheximide also indicates that fresh protein synthesis of an intermediate protein was not required for GH-induced c-myc expression. Western ligand blot analysis of IGF-binding proteins revealed that cultured rat articular chondrocytes produced a predominant 41 kDa and a faint 32 kDa form, and that GH significantly stimulated the secretion of the 41 kDa form without affecting expression of the 32 kDa form. Furthermore, a specific IGF-I binding study suggested that the increase in DNA synthesis induced by GH was not associated with changes in affinity or in the number of IGF-I binding sites. These results support the conclusion that the stimulatory effect of GH was mainly mediated by GH-induced IGF-I production in monolayer rat articular chondrocytes. However, it is likely that GH may also have a direct stimulatory effect by inducing c-myc proto-oncogene expression.

Disturbances in growth hormone secretion and action in adolescents with anorexia nervosa

Women in whom anorexia nervosa develops during adolescence have failure of linear growth associated with low levels of insulin-like growth factor I (IGF-I). To investigate the pathophysiology of growth retardation in adolescents with anorexia nervosa, we measured basal growth hormone (GH), growth hormone-binding protein (GHBP), IGF-I, and insulin-like growth factor binding protein-3 (IGFBP-3) in three groups of patients: (1) 28 recently hospitalized female adolescents with anorexia nervosa, (2) 23 of the same patients after partial weight restoration, and (3) 28 healthy control subjects matched for age, sex, and pubertal stage. Fasting GH levels in group 1 did not differ significantly from those in group 3. In contrast, serum GHBP ( p <0.001), IGF-I ( p<0.001), and IGFBP-3 ( p <0.01) were significantly lower in group 1 than in group 3. Serum GHBP and IGFBP-3 levels were positively correlated with body mass index. Serum GHBP levels were low in patients in all five pubertal stages and even in those shown to have adequate GH secretion. In group 2 (after refeeding) the serum IGF-I concentration increased significantly and GHBP and IGFBP-3 returned to normal. We conclude that patients with anorexia nervosa have diminished GH action resulting in decreased secretion of IGF-I. The positive correlation with body mass index and the reversibility with refeeding suggest that these changes are secondary to malnutrition. Altered GH function that occurs during the years of active growth can explain the growth retardation seen in anorexia nervosa. (J P EDIATR 1994;125:655-60)

Effect of tumor necrosis factor-alpha on the expression of insulin-like growth factor I and insulin-like growth factor binding protein 4 in mouse osteoblasts.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine produced by immune cells, which has multiple effects on bone cells and is therefore thought to mediate changes in bone metabolism occurring during inflammation. In the present study we have investigated the effect of TNF-alpha on the secretion of insulin-like growth factor I (IGF-I) and IGF binding protein 4 (IGFBP-4) by clonal mouse osteoblasts (MC3T3-E1 cells) using subconfluent in vitro cultures and serum-free conditions. The IGF-I was determined by radioimmunoassay under conditions eliminating the interference of IGFBPs. Treatment of MC3T3-E1 cultures with TNF-alpha for 24 h resulted in a dose-dependent decrease in IGF-I secretion (maximally to 34 +/- 9.7% of control with 60 pmol/l TNF-alpha; mean +/- SD). The TNF-alpha treatment also resulted in decreased messenger ribonucleic acid (mRNA) levels of IGF-I at 4 and 24 h, as detected by Northern analysis. Because basal secretion of IGFBPs is very low in MC3T3-E1 cells, effects of TNF-alpha on IGFBP secretion were studied in cultures in which IGFBP-4 expression was increased by calcitriol (1,25(OH)2D3) treatment. The presence of TNF-alpha (600 pmol/l) inhibited this calcitriol-induced stimulation of IGFBP-4 mRNA levels from 4 h onwards, with complete inhibition of the calcitriol effect occurring at 24 h. We also observed a dose-dependent inhibition of calcitriol-stimulated IGFBP-4 secretion into the culture medium (as detected by Western ligand blot), with the maximal inhibition occurring with 600 pmol/l TFN-alpha to 25 +/- 7% of control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic hormones differentially regulate insulin-like growth factor (IGF)-I and IGF-binding protein production by primary rat hepatocytes.

We investigated the influence of and interactions among pancreatic hormones on the secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IG-FBPs) by treating primary hepatocytes from young male Long-Evans rats with insulin or glucagon in combination with rat GH (rGH). The concentration of IGF-I secreted into the medium was estimated by radioimmunoassay after formic acid-acetone cryoextraction, and secreted IGFBPs were analysed by Western ligand blot and immunoblot; accumulation of IGF-I mRNA was analysed by Northern blot. Both insulin (0.1-100 nmol/l) and rGH (0.5, 5 and 50 pmol/l) produced a dose-dependent stimulation of IGF-I secretion over a 24-h incubation period. In contrast, glucagon (0.1-100 nmol/l) inhibited IGF-I production in a dose-related manner. Glucagon (10 nmol/l) also inhibited IGF-I secretion stimulated by rGH (5 pmol/l) and insulin (10 nmol/l). Northern blot analysis of total RNA isolated from rat hepatocytes revealed that rGH (5 pmol/l) elevated IGF-I mRNA levels, glucagon (10 nmol/l) alone had no effect on this parameter, but glucagon significantly reduced IGF-I transcript accumulation in response to rGH. IGFBPs secreted by rat hepatocytes run in two molecular weight ranges on SDS-PAGE: approximately 25 kDa (IGFBP-4) and approximately 29-31 kDa (IGFBP-1 and -2); the predominant hormonally regulated IGFBP was identified as IGFBP-1. Insulin produced a dose-dependent inhibition of production of IGFBP-1, while glucagon was stimulatory; when given together at an equivalent concentration (1 nmol/l), the effects of insulin were dominant to glucagon on IGFBP-1. These observations provide support for significant opposite roles for the pancreatic hormones, insulin and glucagon, in the regulation of liver IGF-I and IGFBP-1 production. As the production of pancreatic hormones is influenced by nutritional status, these polypeptides may mediate the effects of changing nutritional state on the hormonal control of protein anabolism and glucose homeostasis by directly influencing the circulating level of liver-derived IGF-I and its binding proteins.

Low dose continuously infused growth hormone results in increased lipoprotein(a) and decreased low density lipoprotein cholesterol concentrations in middle-aged men.

Animal studies have shown that slight increases in basal GH concentrations may result in changes in lipoprotein metabolism. Such changes in GH secretion have been observed in physiological and pathophysiological states such as fasting, uncontrolled diabetes and during oestrogen treatment. The aim of this study was to investigate the possible effects of increases in basal plasma GH concentrations on lipoprotein concentrations. Recombinant human growth hormone (rhGH) was given as a continuous subcutaneous infusion in a low dose (0.02 U/kg/day) in an open study. Eight middle-aged (42-59 years) overweight (body mass index: 26.1-33.8 kg/m2) but otherwise healthy men were studied over a period of 14 days. Blood samples were obtained after an over-night fast before and after 2, 7 and 14 days of treatment. Plasma and serum were separated and used for subsequent measurements of hormone and lipoprotein concentrations. On days 0, 7 and 14 of treatment, post-heparin plasma was also obtained for determinations of plasma lipoprotein lipase and hepatic lipase activities. In addition, a hyperinsulinaemic euglycaemic glucose clamp was performed on days 0 and 13 of the study. Fat biopsies from abdominal and gluteal fat depots were obtained for measurement of lipoprotein lipase activities on days 0 and 14 of the study. Serum GH concentrations increased to a steady level of 2-4 mU/l during treatment. Serum insulin-like growth factor-I (IGF-I) concentrations increased throughout the treatment period to twice the pretreatment levels. Plasma insulin and blood glucose concentrations increased on day 2 of treatment. After 7 and 14 days of treatment blood glucose concentrations were not different from pretreatment levels, but plasma insulin concentrations were still elevated. Serum cholesterol and low density lipoprotein (LDL) cholesterol concentrations had decreased after 7 and 14 days of treatment. High density lipoprotein (HDL) cholesterol concentrations were not affected, but very low density lipoprotein (VLDL) cholesterol and triglyceride concentrations increased transiently at day 2 of treatment. Serum apolipoprotein (apo) A-I, apoB and apoE concentrations were not significantly affected. Serum lipoprotein(a) concentrations had increased by days 7 and 14 to 147 and 142% of pretreatment concentrations, respectively. Lipoprotein lipase and hepatic lipase activities in post-heparin plasma, as well as abdominal and gluteal adipose tissue lipoprotein lipase activities, were not affected. There was no significant change in glucose disposal rate estimated from the glucose clamp studies. A low dose infusion of GH results in marked changes in lipoprotein concentrations with a transient increase in VLDL cholesterol and thereafter in a decrease in LDL cholesterol. In addition, this low dose of GH resulted in marked increases in lipoprotein(a) concentrations. The observed effects of GH may partly involve changes in IGF-I and insulin secretion.

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.

IGF-I mediates the stimulatory effect of high calcium concentration on osteoblastic cell proliferation

Since our recent study revealed that an increase in extracellular calcium ([Ca2+]e) but not magnesium enormously stimulated DNA synthesis in osteoblastic MC3T3-E1 cells at the minimal and maximal effective concentration of 3 and 5 mM, respectively, the present study was performed to clarify how an increase in [Ca2+]e caused a stimulation of DNA synthesis of these cells. Neither calcium channel blockers (verapamil, diltiazem, and nifedipine) and dantrolene, an inhibitor of Ca release from intracellular Ca pool, nor indomethacin, an inhibitor of prostaglandin synthesis, affected the high [Ca2+]e-induced increase in DNA synthesis. DNA synthesis first increased after a 12-h exposure to 5 mM [Ca2+]e, and cycloheximide eliminated the stimulatory effect of high [Ca2+]e on DNA synthesis, suggesting that this stimulatory effect of high [Ca2+]e was dependent on new protein synthesis. There is recent evidence that MC3T3-E1 cells constitutively produce and secrete insulin-like growth factor I (IGF-I) and possess IGF-I receptors. IGF-I antiserum (1:10,000 to 1:100) blocked the high [Ca2+]e-induced increase in DNA synthesis in a concentration-dependent manner. A neutralizing abolished DNA synthesis stimulated by high [Ca2+]e, indicating that IGF-I mediated the high [Ca2+]e-induced effect. Furthermore, high [Ca2+]e significantly increased the secretion of immunoreactive IGF-I into the medium as well as the expression of IGF-I mRNA. Present findings indicate that an increase in [Ca2+]e stimulated DNA synthesis of osteoblasts through the mechanism of an increase in the production and secretion of IGF-I.

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells.

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt2cAMP) augmented the secretion. In contrast, TPA increased the level of type I IGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt2cAMP affected neither IGF-I receptor number nor the IGFBP secretion. These results indicate that the production of IGF-I and the expression of IGF-I receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells.

Effect of transforming growth factor-β 1 on the insulin-like growth factor system in cultured porcine Leydig cells

Using as a model system, primary cultures of porcine Leydig cells, we have shown that transforming growth factor-beta 1 (TGF-β 1) (2 ng/ml, 72 h) antagonizes the stimulatory action of insulin-like growth factor-I (IGF-I) on luteinizing hormone (LH/hCG) receptors. We therefore investigated the action of TGF-β 1 on the different components of the IGF system, namely, IGF-I, II, IGF binding proteins (IGFBPs) and IGF-I receptor present in testicular Leydig cells. TGF-β 1 was shown to decrease in a dose and time dependent manner the binding of 125I-IGF-I to Leydig cells. The maximal (40% decrease) effect was obtained with 1.3 ng/ml (0.05 nM) after 72 h of treatment. Such a decrease in IGF-I binding by TGF-β 1 treatment was shown to be related to the number of receptor but not to their affinity. Affinity labeling of these receptors by covalently binding them to 125I-IGF-I with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complex revealed that the inhibitory action of TGF-β 1 (2 ng/ml, 72 h) occurs at the level of a 135 kDa protein which represents the classical form of the binding subunit of the IGF-I receptor. Moreover, our study indicates that TGF-β 1 was unable to affect the other components of the IGF system in cultured porcine Leydig cells. Indeed, TGF-β 1 (2 ng/ml, 72 h) was without effect on immunoreactive IGF-I and IGF-II secretion and also on the different IGF binding proteins (IGFBPs) (44, 40, 34, 29, and 24 kDa) as evaluated by ligand blotting analysis. Together, our findings clearly suggest that in Leydig cells TGF-β 1 reduces IGF-I action probably by decreasing IGF-I receptor levels but not by affecting the other components of the IGF system. Such a decrease in IGF-I receptors may provide an explanation for the antagonistic effect of TGF-β 1 on IGF-I action in Leydig cells.

PATHOGENESIS OF DISPROPORTIONATE SEPTAL HYPERTROPHY IN INFANTS OF STREPTOZOTOCIN-INDUCED DIABETIC MOTHER RATS

Transient hypertrophic cardiomyopathy in human infants born of diabetic mothers is well known. In order to elucidate the pathogenesis of this unique type of myocardial disease, morphological and endocrinological studies were performed on fetuses and infants of streptozotocin-induced diabetic rats in the present study.Disproportionate ventricular septal thickening (DVST) was observed in 76% of the fetuses of diabetic mothers (FDM) on day 21 of gestation and in 60% of 2-day-old infants born of diabetic mothers (IDM). Although DVST was a usual finding in fetuses on day 18 of gestation or before, it was infrequent in control fetuses on day 21 of gestation. Immature side-to-side or side-to-end type intercellular connections were more frequently seen in the ventricular cardiac muscles of FDM on day 21 of gestation and in 2-day-old IDM than in the controls. Serum insulin-like growth factor I (IGF-I) and insulin levels and pancreatic insulin content were significantly lower in FDM and IDM than in the controls, and significant negative correlations were found between serum IGF-I and insulin levels and interventricular septal to left ventricular free wall thickness ratios.The above findings indicate that DVST in FDM and IDM represents immaturity of cardiac architecture. It is also quite conceivable that insufficient secretion of IGF-I and insulin play significant roles in this immaturity.

Triiodothyronine stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

We examined the effects of triiodothyronine (T3) on45Ca-accumulation into extracellular matrix in osteoblast-like MC3T3-El cells cultured for 7-week period, which was considered to be long enough to develop calcification. T3 increased45Ca-accumulation into sodium dodecyl sulfate-insoluble, EDTA-extractable materials cultures of MC3T3-El cells in a dose-dependent manner in the range between 10 pM and 10 nM. Contrary to the stimulatory effect on45Ca-accumulation, T3 inhibited DNA synthesis in MC3T3-El cells dose dependently between 10 pM and 10 nM. T3 stimulated the secretion of insulin-like growth factor-I (IGF-I) dose dependently between 0.1 nM and 10 nM in MC3T3-El cells cultured for 7 weeks. Antibodies to IGF-I suppressed the45Ca-accumulation induced by T3 almost to the control level. These results strongly suggest that T3 stimulates45Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.

Regulation of maternal IGF-I by placental GH in normal and abnormal human pregnancies.

Throughout gestation, maternal insulin-like growth factor I (IGF-I) increases progressively despite suppressed pituitary growth hormone (GH) secretion. We have previously shown that in normal pregnancy, a specific placental GH variant, rather than human placental lactogen (hPL), substitutes for pituitary GH in the regulation of maternal IGF-I. We studied the maternal IGF-I secretion in a cohort of 286 normal and abnormal pregnancies (617 blood samples). Regardless of pathology and gestational age, IGF-I values correlated with corresponding placental GH but not with hPL values. Similar correlations were evidenced for each 2-wk gestational period between 32 and 39 wk. In pathological pregnancies, when only those hormonal results that are obtained before any treatment are considered and diabetes is excluded, IGF-I levels were closely related to corresponding placental GH, but not to hPL. In women with a fetoplacental unit disorder, low placental GH levels resulted in low IGF-I and in a secondary pituitary GH increase, whereas in patients without detectable impairment of the fetoplacental unit normal placental GH corresponded to normal IGF-I. These results suggest that in pathological as well as in normal pregnancy, placental GH, and not hPL, substitutes for pituitary GH to regulate the maternal IGF-I secretion.

Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells.

The repair process of the vascular endothelium is modulated by growth factors from both endogenous (within the vessel wall) and exogenous (blood borne) sources. We utilized a tissue culture model of endothelial wounding to gain further insight into the potential autocrine control of proliferation during wound repair. Cultured porcine aortic endothelial monolayers were mechanically wounded by passing a 7-mm sterile glass rod over the surface of the culture. Proliferation at the wound edge was quantified using [3H]thymidine autoradiography. In wounded cultures incubated in media supplemented with 10% fetal calf serum, 81 +/- 2% of the nuclei at the wound edge were labeled. When the cultures were incubated in serum-free media, proliferation at the wound edge was only slightly diminished with 65 +/- 3% (P < 0.05) of the cells labeled. These findings raise the possibility that there is a significant contribution from autocrine growth factors to endothelial wound repair. To evaluate the potential role of insulin-like growth factor I (IGF-I) in the wound repair process, we used a radioimmunoassay to measure IGF-I secretion. Wounded cultures exhibited a 187 +/- 58% increase in IGF-I production when compared with nonwounded cultures (P < 0.05). To determine the extent to which endogenous IGF-I mediates the proliferative response of endothelial cell monolayers to wounding, wounded cultures were incubated with inactivating concentrations of IGF-I antibody. When IGF-I antibody was present in the culture media, only 26 +/- 3% of the nuclei at the wound edge were labeled with [3H]thymidine (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Tumour necrosis factor-α modulates oestradiol responsiveness of MCF-7 breast cancer cells in vitro

We studied the effect of tumour necrosis factor-alpha (TNF) on oestradiol regulation of growth and metabolism of MCF-7 breast cancer cells to determine whether TNF altered the oestradiol responsiveness of these cells. We found that TNF antagonized oestradiol stimulation of cell growth in a dose-dependent manner, with partial inhibition at 1.0 U/ml and complete inhibition at 1000 U/ml. TNF inhibited cell cycle progression, increasing cells in the G0G1 phase and blocking oestradiol-stimulated progression into the S phase. We examined the effect of TNF on three oestrogen-regulated proteins, the oestrogen receptor (ER), the progesterone receptor (PR) and insulin-like growth factor-I (IGF-I). TNF down-regulated the ER and up-regulated the PR. Both of these processes were enhanced by the addition of oestradiol. The effects of TNF on the ER and PR were dose-dependent and occurred without a change in the Kd of the receptor. TNF did not change the respective steady-state mRNA levels. In addition, TNF did not alter secretion of IGF-I either in the absence or presence of oestradiol, indicating that the effects of TNF on oestrogen-regulated proteins in selective. These findings indicate an important interaction between the immune and endocrine systems. The cytokine TNF has a prominent effect on oestradiol stimulation of MCF-7 cells, blocking its proliferative response and enhancing certain metabolic effects. These actions may be mediated in part through modulation of the ER, although other pathways appear to be involved.

Inhibition of HCG production by retinoids (acitretin) using cultured trophoblast cells

Prenatal exposure to excessive glucocorticoids (GCs) leads to intrauterine growth retardation and fetal programming of adult health and disease through deregulation of placental functions. Placental secretion of insulin-like growth factor-I (IGF-I) plays a critical role in the regulation of placental development and function. However, it remains elusive whether GCs affect placental functions through glucocorticoid receptor (GR)-mediated transcriptional regulation of IGF-I gene. In this study, human placental choriocarcinoma (BeWo) cells before and after syncytialization were used as cytotrophoblast and syncytiotrophoblast models, respectively, to explore the effects of dexamethasone (Dex) on transcriptional regulation of IGF-I gene at both stages. Dex significantly inhibited (P < 0.05) cell proliferation in cytotrophoblasts and down-regulated amino acid transporter SLC7A5 in syncytiotrophoblasts. Concurrently, the abundance of IGF-I mRNA and its transcript variants, together with IGF-I level in culture media, were significantly reduced, in association with significantly enhanced (P < 0.05) GR phosphorylation. GR antagonist RU486 was able to abolish all these effects. Two glucocorticoid response elements (GREs) were predicted in the promoter regions of IGF-I gene. GR binding to GRE1 was significantly enriched (P < 0.05) in both cytotrophoblasts and syncytiotrophoblasts, but that to GRE2 was significantly diminished (P < 0.05) in cytotrophoblasts but not in syncytiotrophoblasts, in response to Dex treatment. IGF-I supplementation completely rescued Dex-induced cell cycle arrest but not SLC7A5 down-regulation, indicating different regulatory mechanisms. Taken together, our results suggest that GR-mediated transcriptional regulation of IGF-I is involved in Dex-induced inhibition of placental cell proliferation and function.

The effect of recombinant human growth hormone on regulation of growth hormone secretion and blood glucose in insulin-dependent diabetes.

GH hypersecretion in insulin-dependent diabetes (IDDM) is well documented. Although it has recently been shown that residual insulin secretion determines the magnitude of this GH hypersecretion, the underlying mechanisms of the disorder have not yet been clarified. The 24-h GH and blood glucose profiles, insulin-like growth factor I (IGF-I) concentrations and GH responses to GRF were analyzed in 21 insulin-dependent diabetics and 4 healthy subjects before and after 7 days treatment with recombinant human GH (rhGH) (4 IU given sc at 0800 h). According to C-peptide response to glucagon IDDM patients were subdivided into C-peptide negative (CpN, n = 12) patients without endogenous pancreatic beta-cell activity and C-peptide positive (CpP, (n = 9) patients with endogenous insulin secretion. No significant difference could be observed between the mean 24-h blood glucose profile before and after rhGH treatment in any treated group. Before and on rhGH treatment the highest 24-h GH values were observed in CpN patients when compared to CpP and controls. The rhGH treatment induced a similar increase in the mean 24-h GH concentrations in all groups studied which was statistically significant only in CpP diabetics. Mean pretreatment serum IGF-I concentrations were not significantly different between CpN, CpP patients and controls. The net increase in IGF-I concentrations after rhGH treatment was however, significantly lower in CpN patients than in CpP and control subjects. GRF-induced GH response before and after rhGH treatment was significantly greater in diabetics than in controls. The response of GH to GRF in CpN diabetics was however, almost unchanged after treatment whereas it became lower in CpP diabetics and controls. The dose of 4 IU of rhGH increased significantly GH levels in diabetics with preserved beta-cell function with consequent increase in IGF-I levels and attenuation of GRF induced GH response. In contrast, the same dose of rhGH failed to induce significant increase in GH levels in diabetics without residual beta-cell activity, most probably due to already high pretreatment levels. In addition, neither increase in IGF-I levels nor suppression of GH response to GRF on rhGH treatment was observed in CpN diabetics. The results are in keeping with an important role of portal insulin in GH-induced hepatic IGF-I secretion.