Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary.

Somatomedin-C stimulates somatostatin release to a maximum of 390 percent of basal release during short-term (20-minute) incubation of rat hypothalamus. It has no effect on basal or stimulated growth hormone release from primary cultures of rat adenohypophyseal cells during a 4-hour incubation, but inhibits stimulated release by more that 90 percent after 24 hours. These findings suggest that somatomedin-C participates in the growth hormone negative feedback loop with an immediate effect on hypothalamic somatostatin and a delayed effect on the anterior pituitary.

Somatomedin: inhibiton of adenylate cyclase activity in subcellular membranes of various tissues.

Somatomedin in concentrations between 3 and 20 units per milliliter significantly inhibits the basal activity of adenylate cyclase in crude membrane preparations obtained from homogenates of fat cells, liver, and spleen lymphocytes of the rat, and from chondrocytes and cartilage of chick embryos. The enzyme activity measured in the presence of stimulating hormones (epinephrine, prostaglandin PGE(1), parathyroid hormone) is also inhibited in these preparations by somatomedin. These observations may be relevant in a general way to the mechanism of action of growth-prmoting substances and to the processes which normally regulate cell growth.

Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation.

Specific, high affinity insulin-like growth factor (IGF) binding proteins are secreted by human fibroblasts in culture. By multiple criteria, the species of IGF binding proteins produced by human fibroblasts are distinct from the HepG2/amniotic fluid IGF binding protein, but share many characteristics with the growth hormone-dependent IGF binding protein forms predominant in normal adult human plasma. Treatment of cultured human fibroblasts with growth hormone produced an increase in IGF binding protein activity in the medium, while addition of glucocorticoids markedly diminished IGF binding activity. Insulin, epidermal growth factor, platelet-derived growth factor, and progesterone had no effect on IGF binding activity in fibroblast media. In comparison, HepG2 IGF binding activity was enhanced by progesterone, decreased by insulin, and unaffected by growth hormone or glucocorticoid treatment. Five molecular forms of IGF binding proteins were identified by Western ligand blots in human fibroblast conditioned medium, with Mr = 41,500, 37,000, 32,000, 28,000, and 23,000. In human fibroblast conditioned medium, the Mr = 41,500 and 37,000 IGF binding protein species were abundant, as in normal human plasma, with a major Mr = 23,000 form which was a minor component in plasma.

Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults.

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.

Insulin-induced loss of insulin-like growth factor-I receptors on IM-9 lymphocytes.

Preexposure of IM-9 lymphocytes to the somatomedin peptide insulin-like growth factor-I (IGF-I) results in a time- and concentration-dependent reduction in specific receptors for IGF-I. Since insulin and proinsulin are structurally homologous to IGF-I, we investigated the ability of insulin analogues to compete for occupancy and to directly modulate IGF-I receptor concentrations. IGF-I binds rapidly and reversibly to IM-9 cells at 15 degrees C, with half-maximal displacement of 125I-I-IGF-I at IGF-I concentrations of 3.6 X 10(-9) M and insulin concentrations of 5 x 10(-7) M. Preexposure of cells at 37 degrees C to either IGF-I or insulin produced a concentration-dependent reduction in binding of 125I-IGF-I. A 50% decrease in binding was observed following preincubation of cells with IGF-I at 2.5 x 10(-9) M and insulin at 2 x 10(-7) M. At higher insulin concentrations (10(-6)-10(-5) M), up to 70% reduction in 125I-IGF-I binding occurred. Bovine proinsulin and guinea pig insulin competed less potently than porcine insulin for the IGF-I receptor, and produced receptor loss in proportion to their ability to occupy the IGF-I receptor. Scatchard analysis indicated that at all insulin concentrations, the decrease in binding was secondary to loss of available IGF-I receptors, with no change in affinity. Receptor loss was evident following 1-2 h preexposure to insulin, with a t1/2 of 4 h and maximal receptor loss within 10 h. Similarly, IGF-I and IGF-II competed for occupancy of the IM-9 insulin receptor, with 50% displacement of 125I-insulin occurring at peptide concentrations of 3.5 x 10(-9) M (insulin), 3.5 x 10(-8) M (IGF-II), and 3 x 10(-7) M (IGF-I). Preexposure of cells to these peptides at 37 degrees C for 20 h resulted in a concentration-dependent reduction in binding of 125I-insulin, with the order of analogue effectiveness being insulin greater than IGF-II greater than IGF-I. These data emphasize the structural and functional homology of insulin and the somatomedin peptides, IGF-I and II, as well as their respective receptors. Additionally, the data support the conclusion that the insulin and somatomedin peptides not only bind to both receptors, but downregulate each receptor in proportion to their ability to occupy that receptor.

Simultaneous inhibition of insulin and somatomedin-C binding to cultured IM-9 lymphocytes by naturally occurring antireceptor antibodies.

A 53-y4-old male patient with insulin-resistant diabetes was found to have circulating inhibitors of both insulin and somatomedin-C binding. Serum obtained from the patient at the time of initial presentation inhibited 50% of both 125I-insulin and 125I-SM-C binding to IM-9 lymphocytes at dilutions of 1:150. Spontaneous improvement in the patient's diabetic state was associated with a simultaneous and equal decrease in the serum inhibitory titers for both radioligands. Scatchard analysis indicated that the observed serum-induced decrease in both insulin and SM-C binding was due to decreased receptor affinity, with no alteration in receptor number. The serum inhibitors of both insulin and SM-C binding were precipitated equally by Staph-A and also by 40% ammonium sulfate, suggesting they were immunoglobulins. The observation of naturally occurring autoantibodies against both the insulin and SM-C receptors suggests a structural homology between the two receptors.

Effect of diabetes and its control on insulin-like growth factors in the young subject with type I diabetes.

The influence of diabetes and its control on circulating levels of growth hormone and growth hormone-dependent, insulin-like growth factors (IGF) remains controversial. In the present study, the effect of a 1-wk period of intensive insulin therapy on growth hormone and IGF I and II has been determined in 19 young (age 13-22 yr), insulin-dependent (type I) subjects with diabetes mellitus. IGF I was low during conventional insulin therapy (198 +/- 20 versus 438 +/- 38 ng/ml in nondiabetic subjects, P less than 0.001), and rose within the week of intensified treatment (to 255 +/- 15 ng/ml, P less than 0.005), concomitant with a reduction in plasma glucose from 233 +/- 16 to 110 +/- 5 mg/dl. IGF I rose despite a significant fall in mean 24-h growth hormone levels from 14.1 +/- 2.2 to 9.0 +/- 1.2 ng/ml (P less than 0.02). The mean IGF II value for the diabetic subjects (504 +/- 39 ng/ml) was not significantly different from that of the nondiabetic control group (506 +/- 30 ng/ml, P greater than 0.3) and was not altered by intensified therapy. However, four individual patients with very low IGF I also had depressed IGF II (248 +/- 16 ng/ml), which was corrected (to 377 +/- 35 ng/ml) with improved metabolic control. These data suggest that elevated growth hormone levels in poorly controlled diabetes are ineffective in IGF I generation and that this defect is at least partially corrected by acute improvement in control. The rise in IGF I levels accompanying intensive insulin treatment may suppress the excessive secretion of growth hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of insulin-like growth factor binding proteins from human breast cancer cells.

The insulin-like growth factor binding proteins (IGF-BPs) are structurally and immunologically distinct from the IGF type 1 or type 2 receptors and are characterized by two major forms: a large, GH-dependent BP found in human plasma (Mr = 150 k) and a small GH-independent BP (Mr = 28-42 k) present in human plasma, amniotic fluid, and HEP G2 cells. Using affinity cross-linking techniques, we have identified several binding proteins secreted by human breast cancer cell lines (Hs578T, MDA-231, T-47D, and MCF-7). Under nonreducing conditions these proteins migrated at an apparent Mr = 35, 28, 27, and 24 k, while reducing conditions revealed bands of apparent Mr = 35, 32, 27, and 24 k. Competitive binding studies in T-47D-conditioned media demonstrated that these BPs bound more IGF-II than IGF-I, and that IGF-II potently inhibited binding of either IGF-I or -II. Immunological studies using a polyclonal antibody against the HEP G2 small BP revealed no immunoreactive BP in conditioned media from MCF-7 and T-47D and only slight immunoreactivity in conditioned media from Hs578T and MDA 231. Analysis by Northern blot, using a probe from the cDNA sequence of the HEP G2 BP, demonstrated that Hs578T and MDA-231 cell lines contained small amounts of the 1.65 kilobase mRNA characteristic of the HEP G2 BP, while MCF-7 and T-47D tested negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells: predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors.

The primary structure of an insulin-like growth factor (IGF) binding protein produced by human HEP G2 hepatoma cells has been deduced from the cDNA sequence. The 234 amino acid protein has a predicted molecular mass of 25,274 and contains a single, distinctive cysteine-rich region. The N-terminal sequence of this protein is quite similar to the limited sequence data available for a rat IGF binding protein produced by BRL-3A cells and suggests a common ancestral origin. In contrast, the HEP G2 IGF binding protein sequence bears no similarity to the N-terminal 15 amino acids of a 53 kilodalton binding protein purified from human plasma. Comparison of full-length protein sequences for the IGF-I and IGF-II receptors with that of the HEP G2 IGF binding protein also fails to demonstrate any significant similarities among these three proteins, and suggests that each contains a unique binding domain for the IGF peptides.

Effects of recombinant insulin-like growth factor-I and growth hormone on bone turnover in elderly women.

We evaluated the effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone (GH) on calciotropic hormones and bone turnover markers in 16 healthy elderly women 71.9 +/- 1.3 years of age (mean +/- SEM). Subjects consumed a fixed diet providing 1000 mg of calcium and 0.9 g/kg of protein for 10 days before starting baseline 24-h urine and blood collections. Specimens were collected for 6 consecutive days before initiating subcutaneous injections of GH (25 micrograms/kg/day, n = 5) and IGF-I at 60 micrograms/kg b.i.d. (high-dose, n = 5) or at 15 micrograms/kg b.i.d. (low-dose, n = 6) for 28 days. Resorption markers included urine hydroxyproline (OHP), total pyridinolines (PYD), and N-telopeptide; formation markers include osteocalcin, skeletal alkaline phosphatase (sALP), and type I procollagen carboxy-terminal extension peptide (CICP). For each subject, baseline daily turnover markers varied substantially (DV = 16-22%). With GH and high-dose IGF-I, resorption and formation markers increased progressively to maximum levels at day 21. For GH, the increase in day 21 PYD, N-telopeptide, osteocalcin, and CICP was 143, 111, 53, and 81%, respectively (p < 0.96-0.02). For high-dose IGF-I, these increases were 108, 81, 77, and 111% (p < 0.02-0.002). However, with low-dose IGF-I no change was observed in resorption markers while osteocalcin and CICP increased progressively (day 21, % increases = 88 +/- 51, 36 +/- 14). Twenty-four hour urine collections during the last days of baseline and of study drug were taken as six 4 h aliquots. When deoxyPYD was measured on these samples in the low-dose IGF-I group, a significant increase was observed only on the 0800-1200 h aliquot. Serum phosphorus concentrations increased with GH (21.2 +/- 3.3%) and high-dose IGF-I (8.8 +/- 3.6%) by day 21 but actually decreased by day 28 (-9.7 +/- 2.7, p < 0.02) with low-dose IGF-I. Urinary phosphorus excretion decreased with high-dose IGF-I only. Twenty-four hour calcium excretion increased with all treatments. These results indicate that both GH and high-dose IGF-I activate remodeling osteons. By contrast, low-dose IGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption and may therefore provide a useful means to increase bone mass. The results also suggest some of the GH action on renal phosphorus handling represents a direct action of GH on the nephron which does not involve the intermediacy of IGF-I. Finally, even under controlled conditions bone turnover markers exhibit substantial daily variation so that a very large treatment effect will be required for these markers to have clinical utility.

Insulin-like growth factors are mitogenic for human keratinocytes and a squamous cell carcinoma.

Normal adult human keratinocytes in monolayer culture and SCL-1, a skin-derived squamous-cell carcinoma cell line, were investigated for the expression of receptors for insulin-like growth factors (IGF) and insulin. As demonstrated by affinity crosslinking, radiolabeled IGF-1, IGF-2, and insulin bound specifically to both cell types. Each cell expressed type I IGF receptors, with affinity for IGF-1 greater than IGF-2 much greater than insulin. Insulin receptors, with highest affinity for insulin, were also present on both cells. However, keratinocytes and SCL-1 cells differed in 125I-IGF-2 binding. 125I-IGF-2-bound to both type I and type II IGF receptors in normal keratinocytes, but bound predominantly to membrane-associated IGF binding proteins in SCL-1. IGF-1 was slightly more potent than IGF-2 in stimulating growth of both keratinocytes and SCL-1 cells. In keratinocytes, concentrations of IGF-1 ranging from 5-100 ng/ml, and of IGF-2 from 50-100 ng/ml, resulted in a significant increase in cell number. At the maximum dose of 100 ng/ml, either IGF-1 or IGF-2 caused a 2.3-times increase in cell number. In SCL-1 cells, IGF-1 was more potent than IGF-2 or insulin at lower concentrations, but either IGF-1 or IGF-2 at the maximal concentration of 333 ng/ml stimulated a 4.7-times increase in thymidine incorporation. The stimulatory effect of insulin in SCL-1 was 10-50 times less potent than that of the IGF. The effect of either IGF on SCL-1 was completely inhibited by the type I IGF receptor antibody alpha IR-3, suggesting that both IGFs are mitogenic through the type I IGF receptor. Insulin action was partially blocked by alpha IR-3, suggesting that insulin can act through both the insulin and type I IGF receptors. It thus appears that IGF-1 and IGF-2 are mitogens for normal and transformed human keratinocytes and that their actions are primarily mediated through the type I IGF receptor, whereas insulin is a mitogen through both the IGF-1 receptor and the insulin receptor.

Characterization of insulin-like growth factor-I/somatomedin-C receptors on human keratinocyte monolayers.

A membrane receptor for insulin on cultured human keratinocyte monolayers has recently been reported. It has also been established by previous investigators that the receptors for insulin and the somatomedin peptide, insulin-like growth factor-I/somatomedin-C (IGF-I), have similar oligomeric composition. However, these 2 receptors can be distinguished by their high affinity for their respective peptides. The present study was undertaken to identify and characterize IGF-I receptors on human keratinocytes, using time courses at different temperatures, competitive displacement of [125I]IGF-I by unlabeled IGF-I and insulin, and comparative binding of IGF-I, IGF-II, and insulin. The binding of [125I]IGF-I was time and temperature dependent, achieving steady state after 6 h of incubation at 15 degrees C. Specific binding at 15 degrees C averaged 4.33% for 0.8-1.0 million cells. Competition for binding was observed at IGF-I concentrations as low as 1 ng/ml, with half maximal displacement of [125I]IGF-I at IGF-I concentration of 17 ng/ml. Insulin, on the other hand, was 100-fold less potent than IGF-I in displacing [125I]IGF-I. Scatchard analysis of the competitive binding data revealed a curvilinear plot, with a calculated Kd = 1.4 X 10(-9) mol/liter. When the binding of [125I]IGF-I, -IGF-II, and -insulin was compared in the same experiment, the specific binding of [125I]IGF-I was 3.43% as compared with 5.60% for [125I]insulin and 0.90% for [125I]IGF-II. The finding of specific IGF-I receptors on cultured human keratinocytes suggests a possible role for this mitogenic peptide in epidermal cell proliferation.

Characterization of a specific receptor for somatomedin C (SM-C) on cultured human lymphocytes: evidence that SM-C modulates homologous receptor concentration.

A membrane receptor for somatomedin C (SM-C) on cultured IM-9 cells has recently been reported. The current studies were undertaken to further characterize this intact cellular receptor in terms of both kinetics and specificity and to investigate the ability of SM-C to induce homologous receptor loss. The binding of [125I]iodo-SM-C was rapid, achieving a steady state within 90 min and was greater than 95% reversible. Specific binding at 15C averaged 25-30% for 20 X 10(6) cells/ml. Competition for binding was observed at SM concentrations as low as 2 mU/ml, with half-maximal displacement of [125I]iodo-SM-C at SM concentrations of 33 mU/ml (3.33 X 10(-9) M). Insulin-like growth factor I (IGF-I) and a purified SM-C preparation were approximately equipotent in their ability to displace [125I]iodo-SM-C from the IM-9 receptor. The relative potencies of other growth-related peptides, in comparison with SM-C/IGF-I, were IGF-II (1:10), multiplication stimulating activity (1:10), insulin (1:100), and hGH (nonreactive). Preexposure of IM-9 cells to SM-C at 37 C resulted in a time- and concentration-dependent reduction in [125I]iodo-SM-C. A 10-15% decrease in binding was observed after preincubation with SM in concentrations of 7 mU/ml. Preincubation with 100 mU/ml SM resulted in 50% reduction in binding, but no further decreases were observed after preincubation with higher concentrations of SM (up to 1 U/ml). Scatchard analysis indicated that the reduction in binding was due to a loss of available specific receptors on the cell membrane. These data indicate that the IM-9 cell possesses a specific SM receptor and that SM-C and IGF-I compete equally for occupancy. Furthermore, the data provide a direct demonstration in cell culture that SM-C, like insulin and growth hormone, can modulate homologous receptor concentrations and potentially alter target-cell sensitivity.

The action of somatomedin on glycosaminoglycan synthesis in cultured chick chondrocytes.

A basic somatomedin preparation results in a 2-fold stimulation of incorporation of both [3H]leucine into protein and 35SO4 into glycosaminoglycans by cultured chick sternal chondrocytes. The stimulation is seen in the presence of 20 mM 4-methylumbelliferyl-beta-D-xyloside alone but is not observable in the presence of both xyloside and cycloheximide, indicating that the stimulation by somatomedin is dependent on protein synthesis. Somatomedin stimulates incorporation of leucine into glycosaminoglycans to the same extent to which it stimulates incorporation of sulfate into glycosaminoglycans, suggesting involvement of core protein synthesis. In the presence of the xyloside, however, there is stimulation of synthesis of glycosaminoglycan chains initiated on the xyloside, with a shift in molecular weight distribution to larger molecular weights. Thus, when core protein is not rate limiting, a more general stimulatory effect of somatomedin is observed.

An improved method for the purification of human insulin-like growth factors I and II.

Insulin-like growth factors (IGFs) I and II have been purified from Cohn fraction IV-1 of human plasma. After acid-ethanol extraction, the consecutive use of conventional gel filtration and reverse phase liquid chromatography has permitted the rapid isolation of these polypeptides. Purification was monitored by the use of specific RIAs. In both chromatography systems, separation was optimized by performing it on the same stationary phase but successively with mobile phases of different pH or different solute selectivity. The two polypeptides were shown to be pure by their unique amino acid composition, particularly by the absence of specific amino acids (histidine, tryptophan, and methionine), and their unique amino-terminal sequences. In addition, the lack of cross-contamination of the two growth factors with each other was established by the unique isoelectric focusing patterns of IGF-I at pI 8.25 and IGF-II at pI 6.5. From 900 g Cohn fraction IV-1, which is equivalent to 66 liters human plasma, approximately 100 micrograms of each IGF can be obtained by our procedure, which can easily be carried out in a clinical research laboratory.

Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone.

Insulin-like growth factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism. Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions. Thus, IGFBPs may act as modulators for the biological functions of IGFs. We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures. ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures. [125I]IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons. This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB. A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4. The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable. When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M. These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level. rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range. hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion. The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins.

Maternal insulin-like growth factor-binding protein messenger ribonucleic acid during rat pregnancy.

Pregnancy is associated with rapid growth of maternal and fetal tissues. Insulin-like growth factors (IGF-I and -II) have roles in mediating both fetal and placental growth. In this study serum IGFs and IGF-binding proteins (IGFBPs) were characterized, IGFBP protease activity was quantified, and hepatic IGFBP-1, -2, -3, and -4 mRNA were investigated throughout rat pregnancy. IGF-I in maternal serum was elevated (P less than or equal to 0.001) on days 5 and 10 (d5 and d10) of gestation compared to levels in nonpregnant controls (NP), but was significantly decreased below NP levels (P less than or equal to 0.001) after d10 of pregnancy. Serum IGF-II levels were unaffected by pregnancy. Using Western ligand blotting (WLB), six IGFBP bands were visualized in NP, d5, and d10 pregnancy rat sera. At 15 and 20 days gestation, the IGFBP-3 bands were no longer detectable by WLB. Using an in vitro IGFBP protease assay, sera from rats at 15 and 20 days gestation proteolyzed 63 +/- 4% and 81 +/- 5% of recombinant human IGFBP-3, respectively. Regression analyses demonstrated that serum IGF-I was positively correlated with serum IGFBP-3 (r2 = 0.73; P = 0.001), whereas serum IGFBP-3 (r2 = -0.85; P = 0.001) and serum IGF-I (r2 = -0.78; P = 0.001) were negatively correlated with serum protease activity. In addition, no change was observed in liver IGFBP-3 mRNA during pregnancy, further suggesting that protease activity is primarily responsible for the decrease in serum IGFBP-3. However, IGFBP-1 and -4 mRNA levels were increased 3- to 11-fold after d5 of gestation. The hormonal and/or metabolic regulators of hepatic IGFBP-1 and -4 expression during rat pregnancy remain to be determined.

Effects of continuous infusion of insulin-like growth factor I and II, alone and in combination with thyroxine or growth hormone, on the neonatal hypophysectomized rat.

In this study, we examined the effects of systemically administered insulin-like growth factor (IGF)-I and -II on growth of the hypophysectomized (Hx) neonatal rat. Neonatal Wistar rats were Hx or sham Hx on postnatal day (PND) 6 and implanted sc with Alzet pumps on PND 10. Recombinant human IGF-I or -II were infused between PND 10 and 18 at an average dose of 1.9 micrograms/g body weight (BW) per day. In addition, some groups received daily sc injections of recombinant human GH or thyroxine (T4) at 2.5 micrograms and 25 ng/g BW per day, respectively. Pups were sacrificed on PND 18 and serum IGF levels determined. Despite restoration of serum IGF-I levels to sham control values in the Hx pups infused with IGF-I, no significant increase in BW occurred, although some increase in individual organ growth was observed (spleen, kidney, lung). Similarly, administration of IGF-II proved ineffective as a growth promoter in the neonatal Hx rat. In contrast, GH alone stimulated BW gain (P less than 0.001). T4 proved most potent in increasing skeletal growth (50% increase over Hx controls, P less than 0.001), without increasing serum IGF-I or -II levels. IGF-I and GH were equally effective in promoting a small yet statistically significant (17% over Hx controls, P less than 0.05) increase in skeletal growth. A synergistic effect on BW was observed with combined administration of T4 plus IGF-I to the Hx pups (P less than 0.05). The effects of hormonal therapy on serum IGF binding proteins (IGFBPs) was assessed by Western ligand blots. Administration of IGF-I, but not GH, resulted in increased levels of IGFBP-3, the predominant IGFBP of the adult rat. We conclude that systemically administered IGFs in doses that result in normalization of serum levels are ineffective promoters of somatic growth in neonatal rats. While normalization of serum IGF-I levels does result in modest skeletal growth, selective organ growth and increased serum IGFBP-3, growth stimulation does not equal that seen with GH (body weight) or thyroid hormone (skeletal growth). Differences in IGFBP profiles fail to account for the increased potency of GH as a promoter of BW gain. Thus, our data do not support a major endocrine role for IGF-I or -II in neonatal growth, but are consistent with an autocrine/paracrine action of IGF in the mediation of neonatal mammalian growth.

Characterization of insulin-like growth factor I receptors on cultured rat bone cells: regulation of receptor concentration by glucocorticoids.

A specific receptor for insulin-like growth factor I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at 15 C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human fibroblasts. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human fibroblasts, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.