Serum insulin-like growth factor type 1, insulin-like growth factor-binding protein-1, and insulin-like growth factor-binding protein-3 concentrations in patients with thyroid dysfunction.

Thyroid hormones play a role in the regulation of insulin-like growth factor type 1 (IGF-1) and insulin-like growth factor-binding protein-3 (IGFBP-3) expression, and both IGF-1 and IGFBPs have been shown to be related to the function and growth of the thyroid. Our aim was to evaluate serum concentrations of IGF-1, IGFBP-1, and IGFBP-3 in patients with thyroid dysfunction before and after normalization of thyroid function. The study was performed in 86 patients with thyroid dysfunction (43 hyperthyroid and 43 hypothyroid patients) and 17 euthyroid subjects. Serum growth hormone (GH), insulin, IGF-1, IGFBP-1, and IGFBP-3 were measured in all patients before and after normalizing serum thyroid hormone concentrations. Hyperthyroid patients showed IGF-1 (198.8 +/- 17.0 microg/L) and IGFBP-3 levels (4.2 +/- 0.2 mg/L) similar to those found in the control group (217.9 +/- 20.3 microg/L and 4.2 +/- 0.3 mg/L, respectively). After therapy these levels significantly decreased to 156.6 + 11.1 microg/L (p < 0.01) and 3.3 +/- 0.1 mg/L (p < 0.001), respectively. IGFBP-1 concentrations were clearly higher than those found in controls (22.7+/- 2.6 vs. 5.7 +/- 1.5 microg/L, p < 0.001) and exhibited a significant reduction after therapy for thyroid hyperfunction (11.0 +/- 1.7 microg/L, p < 0.001). Patients with hypothyroidism showed serum concentrations of IGF-1 (161.5 +/- 13.1 microg/L, p < 0.05) and IGFBP-3 (3.2 +/- 0.3 microg/L, p < 0.05) significantly lower than those found in healthy volunteers. However, replacement therapy with levothyroxine did not induce any significant modification of these concentrations (152.6 +/- 10.6 microg/L and 3.2 +/- 0.2 mg/L, respectively). Similarly, patients with thyroid hypofunction exhibited raised levels of IGFBP-1 (15.5 +/- 0.9 microg/L, p < 0.05 vs. control group) that were significantly decreased after therapy (8.8 +/- 1.4 microg/L, p < 0.01). The results of the present study show that thyroid status affects GH/IGF axis. Hypothyroidism is associated with significant reductions of IGF-1 and IGFBP-3, and IGFBP-1 is elevated in both hypothyroidism and hyperthyroidism.     

Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV-lipodystrophy. These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin concentration were increased (>90%, P < .01) and insulin sensitivity (Log10ISI(composite)) was decreased (-50%, P < .001). Total and free IGF-I, IGF-II, IGFBP-3, and IGFBP-3 protease were similar between groups (all P > .5), whereas, in LIPO, IGFBP-1 and IGFBP-2 were reduced (-36%, P < .05 and -50%, P < .01). In pooled groups, total IGF-I, free IGF-I, total IGF-II, and IGFBP-3, respectively, correlated inversely with age (all P < .01). In pooled groups, IGFBP-1 and IGFBP-2 correlated positively with insulin sensitivity (age-adjusted all P < .05). IGFBP-3 protease correlated with free IGF-I in pooled groups (r(p) = 0.47, P < .02), and in LIPO (r(p) = 0.71, P < .007) controlling for age, total IGF-I, and IGFBP-3. GHBP was increased, whereas GH was decreased in LIPO (all P < .05). GH correlated inversely with GHBP in pooled groups (P < .05). Taken together the similar IGFs and IGFBP-3 concentrations between study groups, including suppressed GH, and increased GHBP in LIPO, argue against GH resistance of GH-sensitive tissues in LIPO compared with NONLIPO; however, this notion awaits examination in dose-response studies. Furthermore, our data suggest that IGFBP-3 protease is a significant regulator of bioactive IGF-I in HIV-lipodystrophy.     

Expression of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in the rat uterus during decidualization.

Decidualization of the uterus involves proliferation and differentiation of uterine cells. The effects of decidualization on uterine expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) have been examined in the hypophysectomized-ovariectomized (hypox-ovx) rat and the pituitary-intact (ovx) rat. Decidualization was induced by uterine stimulation of animals treated with a combination of 17 beta-estradiol and progesterone. The patterns of change in uterine IGF-I mRNA and IGFBP-1 mRNA abundance were similar to hypox-ovx rats, hypox-ovx rats replaced with GH and T4, and ovx rats. The changes in IGF-I mRNA abundance were temporally related to 17 beta-estradiol injections. IGFBP-1 mRNA was undetectable early in the decidualization process and reached maximal levels on day 6. Mechanical separation of the deciduoma tissue from the underlying myometrium revealed that the deciduoma tissue was depleted in IGF-I mRNA, while the majority of the IGFBP-1 was located in the deciduoma tissue. The in situ hybridization technique was used to localize IGF-I and IGFBP-1 mRNA in the decidualized uterus. The majority of the IGF-I expression was localized to the outer stroma and smooth muscle cell layer, whereas IGFBP-1 mRNA was detected in uterine epithelial cells and stromal glands. These experiments demonstrated that uterine IGF-I and IGFBP-1 expression during the process of decidualization are pituitary independent. Furthermore, our observations support the hypothesis that the expression of IGFBP-1, a protein capable of inhibiting the mitogenic activity of IGF-I, in deciduoma tissue may inhibit paracrine IGF-1 actin and allow for the differentiation of stromal tissue.     

Role of protein kinase-C in regulation of insulin-like growth factor-binding protein-1 production by HepG2 cells.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.     

Cytochalasin B stimulates insulin-like growth factor-binding protein-1 production by Hep G2 cells.

Hep G2 cells were used to study the early sequence of events regulating production of insulin-like growth factor-binding protein-1 (IGFBP-1). Cytochalasin B (100 microM) specifically inhibited 2-deoxyglucose uptake by Hep G2 cells and stimulated IGFBP-1 production 2-fold. Insulin (300 nM) did not stimulate hexose uptake but inhibited IGFBP-1 production more than 50%. A change in IGFBP-1 secretion was observed as early as 2 h after a 15-min or 2-h pulse exposure to either effector. In contrast to IGFBP-1, albumin production was diminished in the presence of cytochalasin B and increased by insulin. From these results we conclude that IGFBP-1 synthesis is (i) stimulated by transient inhibition of cellular glucose uptake and further stimulated by long-term glucose deprivation, and (ii) inhibited by transient exposure to insulin with further inhibition on long-term exposure. These effects are consistent with the dynamic regulation of IGFBP-1 by nutritional status.     

Intrauterine progestin induces continuous insulin-like growth factor-binding protein-1 production in the human endometrium.

The effect of local intrauterine progestin on endometrial insulin-like growth factor-binding protein-1 (IGFBP-1) production was studied in 60 women using a levonorgestrel-releasing intrauterine device (IUD). Intrauterine progestin was a potent stimulator of stromal cell IGFBP-1 production, with 97% of endometrial specimens showing positive staining by immunohistological methods. After 5 yr or more of intrauterine progestin exposure, 100% (n = 20) of the tissues remained strongly positive for IGFBP-1. The IGFBP-1 content in endometrial tissue homogenates reached values as high as 10 micrograms/mg protein when measured by immunoradiometric assay. In contrast to the continuous endometrial IGFBP-1 production induced by local progestin, no such effect could be found in endometria from subjects with sc progestin-releasing implants or copper IUDs. Although the levonorgestrel-releasing IUD had a striking effect on local endometrial IGFBP-1 production, it had no effect on serum IGFBP-1 levels. By Western ligand blot analysis, the domainating IGF-binding species in endometria exposed to intrauterine progestin was of 28K mol wt, corresponding to IGFBP-1, whereas no IGFBP species of 31-43K, corresponding to IGFBP-2 or IGFBP-3, were detected.     

Lithium chloride inhibits the expression and secretion of insulin-like growth factor-binding protein-1.

Insulin-like growth factor-binding protein-1 (IGFBP-1) regulates IGF availability for glucose homeostasis. The IGFBP-1 promoter shares common regulatory response elements with phosphoenol pyruvate carboxykinase (PEPCK), the expression and activity of which is inhibited by lithium chloride, associated with an inhibition of glycogen synthase kinase (GSK)-3 activity, in the rat hepatoma cell line H4-II-E. We therefore determined the effect of lithium chloride on IGFBP-1 expression and secretion in H4-II-E cells. Lithium chloride inhibited IGFBP-1 secretion in a dose response and reversible manner by approx 80% during 5-h and 16-h incubations. An inhibitory effect on IGFBP-1 mRNA expression was observed at 2 h. The inhibitory effect of lithium and insulin were not additive when used alone, but inhibition by lithium occurred when insulin action was blocked by activating AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide-riboside (AICAR). These findings suggest that GSK-3 inhibition, or another pathway activated by lithium, may be involved in a pathway controlling IGFBP-1, inhibiting synthesis when insulin activity is absent or impaired.     

Epidermal growth factor stimulates production of insulin-like growth factor-binding protein-1 in human granulosa-luteal cells.

Insulin-like growth factor-I (IGF-I) enhances and epidermal growth factor (EGF) inhibits gonadotrophin-induced aromatization in granulosa cells. Our previous studies have shown that human ovarian granulosa cells synthesize insulin-like growth factor-binding protein-1 (IGFBP-1) which inhibits IGF-stimulated DNA synthesis. The present study addresses the effect of EGF and gonadotrophins in the regulation of IGFBP-1 release by human granulosa cells cultured in serum-free medium. At concentrations of 1-100 micrograms/l EGF was found to stimulate IGFBP-1 secretion. This was not due to cell proliferation, as the viable cell count remained unaffected. Growth hormone and gonadotrophins had no effect on IGFBP-1 secretion when added alone to culture medium. These results suggest that EGF regulates IGFBP-1 secretion in human granulosa-luteal cells.     

Regulation of rat insulin-like growth factor-binding protein-1: the effect of insulin-induced hypoglycemia.

Rat insulin-like growth factor-binding protein-1 (rIGFBP-1) was purified from H4IIE rat hepatoma cells by IGF-I affinity chromatography and reverse-phase HPLC. A rabbit antiserum (B2) was raised to rIGFBP-1 and a RIA established. Immunoreactive IGFBP-1 was present in rat amniotic fluid and in the medium conditioned by isolated rat hepatocytes and HTC rat hepatoma cells. To study the effect of hypoglycemia, fasting female Wistar rats were anesthetized and cannulated for multiple venous sampling after the administration of insulin or saline. Serum IGFBP-1 rose in adrenal intact rats from < 0.1 micrograms/ml to a maximum of 1.41 +/- 0.23 micrograms/ml approximately 120 min after insulin administration. Compared to adrenal-intact rats, adrenalectomized animals demonstrated a delayed rIGFBP-1 response to hypoglycemia and did not appear to have reached a maximum at 180 min. A slow rise in rIGFBP-1 levels throughout the sampling period was seen after saline injection in both adrenal-intact and adrenalectomized animals. Glucose, corticosterone, rat insulin, and human insulin levels were measured and none, alone, appeared responsible for the observed rIGFBP-1 responses. We conclude that 1) rIGFBP-1 is stimulated in response to hypoglycemia in a similar manner to glucose counterregulatory hormones, 2) an adrenal factor is required for an early rIGFBP-1 response to hypoglycemia, and 3) neither circulating glucose nor insulin levels, alone, are responsible for the observed patterns of response.     

Phosphorylation of insulin-like growth factor binding protein-3 by deoxyribonucleic acid-dependent protein kinase reduces ligand binding and enhances nuclear accumulation

The IGF binding proteins (IGFBPs) regulate the mitogenic effects of IGFs in the extracellular environment. Several members of this family, including IGFBP-3, also appear to have IGF-independent effects on cell function. For IGFBP-3 and IGFBP-5, both of which are translocated to the cell nuclei, these effects may be related to their putative nuclear actions. Because reversible phosphorylation is an important mechanism for controlling nuclear protein import, we have examined the effect of phosphorylating IGFBP-3 with a number of serine/threonine protein kinases on its nuclear import. Phosphorylation of IGFBP-3 by the double-stranded DNA-dependent protein kinase (DNA-PK) increased both the nuclear import of IGFBP-3 and the binding of IGFBP-3 to components within the nucleus compared with nonphosphorylated IGFBP-3. However, there was no difference in the binding of the nuclear transport factor, importin beta, to nonphosphorylated and phosphorylated IGFBP-3. The ability of the DNA-PK phosphoform of IGFBP-3 to bind IGFs was severely attenuated, and in contrast to nonphosphorylated IGFBP-3, the DNA-PK phosphoform was unable to transport IGF-I to the nucleus. Furthermore, IGFBP-3 was phosphorylated by DNA-PK when complexed to IGF-I causing the phosphoform to release IGF-I. Together, these results suggest that when IGF-I is cotransported into the nucleus by IGFBP-3, phosphorylation of IGFBP-3 by nuclear DNA-PK provides a means for releasing bound IGF-I and creating a phosphoform of IGFBP-3 with increased affinity for nuclear components.     

Recombinant human insulin-like growth factor-binding protein-5 stimulates bone formation parameters in vitro and in vivo.

Insulin-like growth factor-binding protein-5 (rhIGFBP-5) is stored in bone and stimulates osteoblast cell proliferation in vitro. Bone formation is dependent on the number and activity of osteoblasts. We therefore evaluated the ability of recombinant human (rh) IGFBP-5 to increase osteoblast activity in vitro; both alkaline phosphatase (ALP) activity and osteocalcin levels showed a dose-dependent increase. In in vivo time-course studies, daily s.c. administration of 50 microg rhIGFBP-5/day/mouse significantly increased serum osteocalcin levels by day 7, and these levels were sustained through day 21. We further evaluated whether rhIGFBP-5 was as effective as IGF-I. Daily s.c. administration of rhIGFBP-5 (50 microg/day), IGF-I (13 microg/day), or IGF-I plus rhIGFBP-5 complex for 9 days increased serum osteocalcin levels by 58%, 65%, and 81% (P < 0.001 in all) and femoral bone extract ALP activity by 85% (P < 0.001), 29% (P < 0.05), and 13% (P = NS), respectively, and decreased carboxyl-terminal cross-linked telopeptide of type I collagen by 29% (P < 0.05), 20% (P = NS), and 12.5% (P = NS), respectively. One s.c. injection of rhIGFBP-5 (50 microg/mouse) increased serum osteocalcin and bone ALP activity by 21% (P < 0.05) and 27% (P < 0.02), respectively, after 5 days, but did not significantly increase serum IGF-I (1, 6, or 24 h/postinjection), suggesting that the effects of rhIGFBP-5 on bone are not mediated by increasing circulating IGF-I. Our data demonstrate that systemic administration of rhIGFBP-5, either alone or in combination with IGF-I, increases bone formation parameters in vivo.     

Growth hormone and insulin-like growth factor regulate insulin-like growth factor-binding protein-1 in Laron type dwarfism, growth hormone deficiency and constitutional short stature.

Insulin-like growth factors (IGFs) mediate the effects of growth hormone (GH), and the insulin-like growth factor-binding proteins (IGFBPs) modulate the actions of IGFs in tissues. We studied the circulating levels of IGFBP-1 in 6 children and 9 adults with Laron type dwarfism (LTD), in 11 children and 21 adults with growth hormone deficiency (GHD), and in 8 children with constitutional short stature. Compared with the situation in healthy children, the basal serum IGFBP-1 concentration was 5.4-fold higher in LTD children, 4.1-fold higher in GHD children, and 3.8-fold higher in children with short stature (p < 0.02 vs controls in all groups). In adult patients with multiple pituitary hormone deficiency (MPHD), the IGFBP-1 concentration was 2-fold elevated, but it was normal in adult LTD patients. Intravenous (N = 10) or subcutaneous (N = 9) administration of IGF-I (75 micrograms.kg-1 and 150 micrograms.kg-1, respectively) in LTD children resulted in a rapid 50-60% fall in serum insulin (p < 0.02), a decline in blood glucose and a concomitant 40-60% rise of IGFBP-1 levels (p < 0.05). Treatment for seven days with IGF-I (150 micrograms.kg-1 x d-1) resulted in a decrease by 34% and 44% of serum IGFBP-1 level in two out of three children with LTD. After prolonged GH therapy, the IGFBP-1 level fell in GHD children by 29% (p < 0.05), in GHD adults by 52% (p < 0.02) and in children with constitutional short stature by 17% (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)