Genetic and environmental components of interindividual variation in circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3.

We assessed the magnitude of the genetic component in the variation of circulating levels of insulin-like growth factors I and II (IGF-I and IGF-II), and their binding proteins IGFBP-1 and IGFBP-3 by measuring their serum concentrations in 32 monozygotic and 47 dizygotic adult twin pairs of the same sex. The intrapair correlation for the IGF-I levels was r = 0.41 (P < 0.009) for monozygotic twins and r = 0.12 (P < 0.22) for dizygotic twins. For the IGF-II concentration the intrapair correlations were r = 0.66 (P < 0.0001) for the monozygotic and r = 0.34 (P < 0.01) for the dizygotic twins. No significant intrapair correlation was found for IGFBP-1 levels in either group. The correlations for IGFBP-3 concentration were r = 0.65 (P < 0.0001) and r = 0.23 (P < 0.06) for monozygotic and dizygotic twins, respectively. Women had higher IGF-II levels than men (635+/-175 vs. 522+/-144 microg/liter; P < 0.0001) and IGFBP-3 levels were also higher in women compared with men (5441+/-1018 vs. 4496+/-1084 microg/liter; P < 0.001). The proportion of variance attributable to genetic effects was 38% for the IGF-I concentration, 66% for the IGF-II concentration, and 60% for the IGFBP-3 concentration. No significant heritability was found for the IGFBP-1 concentrations. Our results show that, in adults, there is a substantial genetic contribution responsible for interindividual variation of the circulating levels of IGF-I, IGF-II, and IGFBP-3, but not for the IGFBP-1 levels.

Effect of dexamethasone on the activity and expression of ornithine decarboxylase in rat liver and thymus.

A single intraperitoneal injection of the synthetic glucocorticoid dexamethasone into rats resulted in a marked stimulation (more than 60-fold) of hepatic ornithine decarboxylase (ODC) at 4 h after the injection, whereas the enzyme activity in thymus was almost totally (about 95%) depressed at the same time. The stimulation of ODC activity in liver was in all likelihood attributable to a greatly enhanced accumulation of mRNA species for the enzyme as revealed by Northern blot and dot-blot hybridization analyses. ODC activity in thymus, in response to dexamethasone, was only 5% of that found in control animals, but this decrease was apparently not accompanied by similar reductions of the levels of ODC message, which was in fact decreased only by 50% at the maximum. In addition to two mRNA species (2.1 and 2.6 kilobases; kb), typical to mouse cells, rat tissues seemed to contain a third hybridizable message for ODC, smaller (1.6 kb) than the above-mentioned species and not seen in samples obtained from mouse or human cells. Interestingly, these smaller poly(A)+ RNA sequences, hybridizable with cDNA complementary to mouse ODC mRNA, were apparently constitutively expressed, as the treatment with glucocorticoid altered the amount of these sequences only slightly.

Leydig cell desensitization by human chorionic gonadotropin does not occur in the human fetal testis.

In vivo data concerning human fetal testicular testosterone production as well as in vitro findings in fetal and neonatal rats suggest that fetal Leydig cells may be capable of responding to gonadotropins and secreting testosterone at high levels for prolonged periods, in contrast to adult testes which reportedly become desensitized after high dose gonadotropin administration. To evaluate fetal testicular testosterone production during long term, high dose gonadotropic stimulation, we cultured human, rhesus monkey, and rabbit fetal testes in organ and cell cultures. After 24 h of culture with different concentrations of hCG (0-100 ng/ml, physiological fetal concentrations during human gestation), the fetal testes were still able to respond to a second hCG stimulus (no desensitization). The 24-h incubation with hCG (0-100 ng/ml) also increased the capacity of the cultures to secrete testosterone during a second incubation in a dose-dependent manner even in the absence of hCG (steroidogenic enzyme induction). Furthermore, hCG increased thymidine incorporation into DNA by the human fetal testis. The results of this study substantiate the role of hCG in the regulation of fetal Leydig cells. They suggest that long term effects via nuclear mechanisms (RNA and DNA synthesis) may be important aspects of this regulation, and that fetal Leydig cells are able to respond to sustained concentrations of gonadotropin without being desensitized.

Testosterone and some of its precursors and metabolites in the human epididymis.

The concentrations of testosterone, four of its precursors (pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione), and three of its metabolites (5 alpha-dihydrotesterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and androsterone) were measured in the epididymis and proximal ductus deferens of elderly men with prostatic carcinoma. In addition, they were measured in testis tissue and spermatic and p eripheral blood sera.l The main androgen in the epididymis was testosterone [37.3 +/- 22.0 (SD) ng/g wet tissue]; 5 alpha-dihydrotestosterone was also present in a relatively high concentration [9.7 +/- 6.5 (SD) ng/g wet tissue]. There were no steroid concentration gradients along the epididymis. The actual and relative concentrations of the steroids measured strongly suggest that they are transferred from testes to epididymides in the testicular lymph or rete testis fluid. Estrogen administration led to significant decreases in epididymal androstenedione, testosterone, and 5 alpha-dihydrotestosterone concentrations. Thus, one facet of the deleterious effects of estrogen on male reproductive functions may be interference with normal epididymal function, essential for undisturbed sperm maturation.

Thyroxine withdrawal is accompanied by decreased circulating levels of insulin-like growth factor-binding protein-1 in thyroidectomized patients.

We carried out a study on the effect of T4 withdrawal on serum insulin-like growth factor-binding protein-1 (IGFBP-1) levels in 10 thyroidectomized patients with papillary thyroid cancer receiving T4 replacement therapy. As controls we also measured serum sex hormone-binding globulin (SHBG) levels, known to be regulated by T4. Each patient acted as his/her own control, studied both before and 30 days after T4 withdrawal. On the average, the IGFBP-1 level decreased by 36% from 66 +/- 9 to 44 +/- 8 micrograms/L (mean +/- SE; P = 0.0007) during T4 withdrawal. The SHBG level decreased by 47% from 78 +/- 17 to 46 +/- 13 nmol/L (P = 0.0001). The fasting insulin levels were unaffected by T4 withdrawal. There also was an 18% decline in the serum IGF-I concentration after T4 withdrawal (P = 0.011). It is concluded that in athyreotic patients receiving T4, withdrawal of the drug decreases serum IGFBP-1 and SHBG concentrations and IGF-I levels. In view of the possible role of IGFBP-1 in glucose counterregulation, these results indicate a novel mechanism by which T4 may exert its metabolic action on liver carbohydrate metabolism.

Chronic exposure to dexamethasone induces hypomethylation of ornithine decarboxylase genes in a human myeloma cell line.

Chronic exposure of a human myeloma cell line to dexamethasone resulted in a selection of cells resistant to the growth-inhibitory action of the glucocorticoid. Upon acute exposure of the parental myeloma cells to dexamethasone growth inhibition was associated with depression of ornithine decarboxylase (ODC, EC 4.1.1.17) activity. However, in cells adapted to grow in the presence of micromolar concentrations of dexamethasone, ODC activity was fully comparable to that in the parental cells. Restriction enzyme analyses with the two isoschizomers HpaII and MspI as well as with the methylation-sensitive CfoI, indicated that the otherwise heavily methylated ODC gene(s) were rendered hypomethylated in the myeloma cells resistant to dexamethasone. This hypomethylation within and/or around ODC genes was associated with a 2-4-fold enhancement of accumulation of ODC mRNA.

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway.

The concentrations of metabolites in the pregnenolone in equilibrium testosterone pathway were determined in freeze-stopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 g ethanol/kg body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63-74%), androstenedione (49-81%), 17-hydroxyprogesterone (60-76%), progesterone (29-67%) and pregnenolone (12-25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone----androstenedione----testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone----progesterone----17-hydroxyprogesterone steps.

Tri-iodothyronine and cycloheximide enhance insulin-like growth factor-binding protein-1 gene expression in human hepatoma cells.

The growth-regulating actions of IGFs are modulated by their binding proteins (IGFBPs). The serum concentration of IGFBP-1 is down-regulated by insulin, and in-vitro studies have demonstrated that IGFBP-1 secretion from various tissues and cells can be stimulated by theophylline, forskolin, oestrogen and progesterone. We have studied the effects and mechanisms of thyroid hormone action on IGFBP-1 gene expression and secretion by human hepatoma cells in vitro. Tri-iodothyronine dose-dependently enhanced IGFBP-1 secretion in serum-free HepG2 cell cultures after 24-48 h of exposure, as measured by a specific immunofluorometric assay. This was accompanied by an increase (+ 50%) in the amount of IGFBP-1 mRNA, which could be prevented by cycloheximide, a protein synthesis inhibitor. Cycloheximide transiently enhanced (+ 200%) the accumulation of IGFBP-1 mRNA at 3-12 h of incubation, when no effect of tri-iodothyronine was observed. It is concluded that thyroid hormone stimulates IGFBP-1 secretion slowly by enhancing IGFBP-1 gene expression by a protein mediator. The acute stimulation of IGFBP-1 gene transcription by cycloheximide associates this gene with a number of growth-related genes encoding growth- and tumour-associated peptides.

Comparative studies on the regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production by insulin and insulin-like growth factors in human hepatoma cells.

Production of insulin-like growth factor-binding protein-1 (IGFBP-1) by the liver is efficiently inhibited by insulin both in vivo and in vitro. Consequently, serum IGFBP-1 concentration reflects insulin bioactivity in portal vein. Sex hormone-binding globulin (SHBG) is another insulin-regulated liver-derived protein that has appeared promising in detecting individuals with portal hyperinsulinemia. We compared the regulation of IGFBP-1 and SHBG production by insulin and insulin-like growth factors (IGF-I and IGF-II) in human hepatoma cell cultures. Insulin equipotently inhibited IGFBP-1 and SHBG production, with maximal decrease in culture medium concentrations being about 35% for both proteins during 48 h of culture in serum-free medium. IGF-I and IGF-II also inhibited the IGFBP-1 and SHBG levels. We conclude that IGFBP-1 and SHBG are equally sensitive to ambient insulin concentrations in human hepatoma cell cultures, and the production of both proteins is also attenuated by the IGFs.

Factors regulating insulin-like growth factor binding protein-3 secretion from human hepatoma (HepG2) cells.

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is a growth hormone (GH) dependent carrier of the IGFs in human serum. Apart from GH regulation the hormonal control of IGFBP-3 production is not well established and although the liver is considered to be the main source of circulating IGFBP-3, there are no in vitro studies of the effect of both insulin and IGFs on the IGFBP-3 produced in human hepatoma cells. The effect of sex hormones as well as cortisol has not been studied. To elucidate this we performed cell culture studies on HepG2 cells in the presence of various effectors. Insulin, IGF-I and IGF-II brought about a 1.5-2-fold enhancement of IGFBP-3 release at 7.5-30 nM concentrations. In contrast, cortisol decreased IGFBP-3 secretion by 30-40% whereas estradiol, tamoxifen and testosterone had no effect at physiological concentrations. We conclude that, in addition to GH, also insulin, IGF-I and IGF-II and glucocorticoids can modulate IGFBP-3 secretion by human hepatoma cells.

Regulation of human granulosa-luteal cell progesterone production and proliferation by gonadotropins and growth factors.

The effects of human chorionic gonadotropin (hCG), follicle-stimulating hormone (FSH), fibroblast growth factor (FGF), and epidermal growth factor (EGF) on human granulosa-luteal cell proliferation and progesterone (P) production were studied in vitro. The cells were obtained from an in vitro fertilization protocol and were cultured for 2 to 12 days on plastic culture dishes or on dishes coated with extracellular matrix (ECM). During the first 2 to 4 days of culture, basal P production was high and could not be further stimulated with gonadotropins. Thereafter, basal P production decreased and could be stimulated by both hCG and FSH. The cells growing on ECM produced less P than the cells growing on plastic. EGF and FGF significantly increased cell proliferation on both substrates. FGF did not influence P production, while EGF clearly increased basal P production of the cells cultured on plastic. The high P production in cultured human granulosa cells obtained from follicles stimulated in vivo indicates that at least some of the cells were luteinized. The present data also demonstrate that EGF and FGF are mitogenic for human granulosa-luteal cells, and EGF regulates their biosynthesis in vitro. These results suggest that growth factors may also regulate granulosa cell function in vivo.

Estradiol increases the production of sex hormone-binding globulin but not insulin-like growth factor binding protein-1 in cultured human hepatoma cells.

OBJECTIVE: To study the effects of E2 on insulin-like growth factor binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production with cultured human HepG2 hepatoma cells. DESIGN: Experimental cell culture. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): Addition of E2 to cell culture medium. MAIN OUTCOME MEASURE(S): Intracellular and released concentrations of IGFBP-1 and SHBG. RESULT(S): Estradiol did not affect the intracellular or extracellular IGFBP-1 concentration, whereas the intracellular SHBG concentration increased significantly in response to 0.5-2.5 microM of E2. CONCLUSION(S): Whereas the two binding proteins share a number of regulatory factors, their regulation by E2 is dissimilar in human hepatoma cells. Estradiol does not affect the intracellular or secreted IGFBP-1 concentration, but it does increase the production of SHBG.

Thromboprophylaxis with low molecular weight heparin (dalteparin) in pregnancy.

Venous thromboembolism remains an important cause of maternal mortality. For women at risk during pregnancy, the recommended venous thromboembolismprophylaxis is unfractionated heparin. Low molecular weight heparins, such as dalteparin, also may be suitable, but randomised trials have not been performed. Pregnant women (105) with confirmed previous or current thromboembolism were randomised to receive either unfractionated heparin twice daily (mean 20569 IU/day) or dalteparin once daily (mean 4631 IU anti-factor Xa units/day) subcutaneously for thromboprophylaxis during pregnancy and postpartum period. Recurrence of venous thromboembolism and safety of treatments were assessed. Dalteparin administered once daily was safe and effective in thromboprophylaxis during pregnancy and postpartum.

Phosphorylation of insulin-like growth factor-binding protein-1 increases in human amniotic fluid and decidua from early to late pregnancy.

Different fractions of insulin-like growth factor-binding protein-1 (IGFBP-1) from anion exchange chromatography represent differently phosphorylated forms as demonstrated by protein kinase and alkaline phosphatase treatments. The major fraction is non-phosphorylated. Three minor fractions are more phosphorylated and, in native polyacrylamide gel electrophoresis (PAGE), they migrate faster than the major fractions. We studied the changes in phosphorylation of decidual and amniotic fluid IGFBP-1 during pregnancy. Both in decidua and in amniotic fluid the degree of phosphorylation increased from early to late pregnancy, as indicated by faster mobility of IGFBP-1 in native PAGE and increased relative amount of the phosphorylated forms in anion exchange chromatography. The more phosphorylated forms had higher IGF-binding affinity than the less phosphorylated forms. As the degree of phosphorylation of IGFBP-1 is highest in full term decidua it is likely that the inhibitory role of IGFBP-1 is accentuated in the end of pregnancy.