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.     

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.     

Low serum levels of free and total insulin-like growth factor I (IGF-I) in patients with anorexia nervosa are not associated with increased IGF-binding protein-3 proteolysis

Patients with anorexia nervosa (AN) are GH resistant, with elevated GH levels and low serum levels of total insulin-like growth factor I (IGF-I). IGF-I action is modulated by IGF-binding proteins (IGFBPs), and a variety of catabolic states has been characterized by the presence of increased IGFBP-3 proteolysis. The present study was performed to examine the levels of free IGFs in AN and to clarify whether AN is associated with increased IGFBP-3 proteolytic activity. In 24 patients and 10 age-matched controls, the fasting serum concentrations of free IGF-I and -II were measured using ultrafiltration by centrifugation. In addition, GH, GH-binding protein, total IGFs, IGFBP-1 to -4, and IGFBP-3 proteolytic activity were measured. The IGFBPs were measured by both immunoassays and Western ligand blotting. Twelve of the patients were restudied 3 months after a minor increase in body mass index. In AN, the levels of GH-binding protein, free and total IGF-I, free IGF-II, and IGFBP-3 were significantly reduced; total IGF-II, IGFBP-2, and IGFBP-4 levels were unchanged; and IGFBP-1 was increased. No increased IGFBP-3 proteolytic activity could be detected in AN. In conclusion, the mechanisms responsible for the adaption of the GH-IGF-IGFBP axis in AN may be different from other catabolic conditions, because the low levels of free and total IGF-I in AN are not associated with increased IGFBP-3 proteolysis.     

Not all insulin-like growth factor-binding proteins (IGFBPs) are detectable by western ligand blotting: case studies of PC12 pheochromocytoma and rat anterior pituitary IGFBPs and proteolyzed IGFBP-3.

We studied the limitations of the Western ligand blot (WLB) for detecting insulin-like growth factor-binding proteins (IGFBPs). PC12 rat pheochromocytoma cells and rat anterior pituitary cells (AP) secrete IGFBPs that cannot be detected by WLB. We used affinity labeling, WLB, dot blotting, competitive binding, ion exchange chromatography, and deglycosylation to characterize these IGFBPs. These IGFBPs were compared with pregnancy protease-derived IGFBP-3 fragments that also bind insulin-like growth factors (IGFs), but are not detectable by WLB. We showed that PC12 IGFBP is cationic, not glycosylated, with 25,500 mol wt reduced (18,500 unreduced), with high affinity for IGF-II and low affinity for IGF-I. It cannot be detected by WLB and is not a proteolytic derivative of other IGFBPs or IGF-II receptors. Its binding activity is not destroyed by sodium dodecyl sulfate (SDS) and heating. It binds to nitrocellulose and IGF-II after dot blotting, but not to IGF-II during WLB. AP also secrete an IGFBP(s) that was not detectable by WLB. AP IGFBPs, unlike those of PC12, have a higher mol wt, and at least one component is glycosylated. The failure of WLB to detect these proteins remains unexplained. Pregnancy protease-derived IGFBP-3 fragments also bind IGFs and are not detectable by WLB. However, they do electrotransfer to nitrocellulose. The failure of WLB to detect these fragments is probably due to proteolysis rendering the binding site susceptible to irreversible denaturation (under conditions of WLB) during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that WLB, while valuable, may have significant limitations in specific cases. Other techniques must complement WLB for detection of IGFBPs in conditioned media and other biological specimens.     

Short-term changes in serum insulin-like growth factors (IGF) and IGF binding protein 3 after different modes of intravenous growth hormone (GH) exposure in GH-deficient patients.

Virtually all circulating insulin-like growth factors I and II (IGF-I and IGF-II) are bound to specific binding proteins (IGFBP), of which IGFBP-3 is the quantitatively most important. The mechanisms regulating the close coordination between serum levels of IGFs and IGFBP-3 is poorly understood. We therefore evaluated the temporal association of serum IGF-I, IGF-II, and IGFBP-3 measured by RIAs after well defined short-term GH exposure in GH-deficient patients. Six patients (mean +/- SE age: 20.5 +/- 1.1 yr) each underwent three GH study protocols in random order. Each study was preceded by 4 weeks without GH therapy. Two units of GH were administered iv as either: 1) two boluses, 2) eight boluses, or 3) a constant infusion. The duration of each study was 44 h including at least 16 h after termination of GH administration. Increments in serum IGF-I occurred 4-6 h after initiated GH exposure in all studies. In the two-bolus study the IGF-I increase was modest with mean +/- SE peak values of 12.4 +/- 2.1 nmol x L-1 after GH administration. In the eight bolus and constant infusion studies significantly higher IGF-I levels were generated: 17.0 +/- 2.2 nmol x L-1 (8 bolus) and 18.8 +/- 1.1 h nmol x L-1 (infusion). In contrast the time course change in serum IGF-II did not differ in the three studies, and it was characterised by a sluggish increase of approximately 30% evidenced after 16-20 h. The changes in IGFBP-3 were almost identical in the three studies. After a lag phase of approximately 18-20 h a gradual increase of approximately 40%, which had not ceased at the end of the study period, was observed. The molar ratio of serum IGF-I plus IGF-II:serum IGFBP-3 remained constant with values between 0.8-0.9 except in the constant infusion experiment, in which the ratio increased significantly with time reaching a mean peak value, which exceeded 1.0, after 24 h. Our data suggest that a pulsatile GH pattern is not superior to constant GH levels as regards generation of IGFs and IGFBP. The earlier increase in serum IGF-I compared to IGF-II and IGFBP-3 suggests that IGF-I may be the main regulator of IGFBP-3 production. Accordingly, the slow increase in serum IGF-II, which paralleled that of IGFBP-3, could indicate that serum IGF-II levels mainly depend on the concentration or binding site availability of IGFBP-3.     

Expression of serum insulin-like growth factors, insulin-like growth factor-binding proteins, and the growth hormone-binding protein in heterozygote relatives of Ecuadorian growth hormone receptor deficient patients.

Recently, an isolated population of apparent GH-receptor deficient (GHRD) patients has been identified in the Loja province of southern Ecuador. These individuals presented many of the physical and biochemical phenotypes characteristic of Laron-Syndrome and are believed to have a defect in the GH-receptor gene. In this study, we have compared the biochemical phenotypes between the affected individuals and their parents, considered to be obligate heterozygotes for the disorder. Serum GH, insulin-like growth factor I and II (IGF-I and IGF-II) levels were measured by RIA Insulin-like growth factor binding proteins. (IGFBPs) were measured by Western ligand blotting (WLB) of serum samples, following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and relative quantitation of serum IGFBPs was performed with a scanning laser densitometer. Serum GH-binding protein (GHBP) levels were measured with a ligand-mediated immunofunctional assay using a monoclonal antibody raised against the GHBP. These values were then compared to values obtained from normal, sex-matched adult Ecuadorian controls, to determine if the above parameters were abnormal in the heterozygotes. The serum IGF-I levels of the GHRD patients were less than 13% of control values for adults and 2% for children. However, the IGF-I levels of both the mothers and fathers were not significantly different from that of the control population. The serum IGF-II levels of the GHRD patients were approximately 20% of control values for adults and 12% for the children. The IGF-II levels of the mothers were reduced, but were not significantly different from that of the control population. However, IGF-II levels of the fathers were significantly lower than those of controls (64% of control male levels). WLB analysis of serum IGFBP levels of the affected subjects demonstrated increased IGFBP-2 and decreased IGFBP-3, suggesting an inverse relationship between these IGFBPs. The GHRD patients who had the lowest serum IGFBP-3 levels (as measured by WLB) demonstrated a serum protease activity that could proteolyze 125I-IGFBP-3. GHRD patients who had higher serum IGFBP-3 levels lacked this serum protease activity. There were no differences in the serum IGFBP profiles of the mothers or the fathers for either IGFBP-2 or IGFBP-3, and serum from both groups lacked the ability to significantly proteolyze 125I-IGFBP-3. While GHRD patients had very low levels of serum GHBP, some patients did have measurable GHBP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationship between the circulating IGF system and the presence of retinopathy in Type 1 diabetic patients.

BACKGROUND: Patients with proliferative diabetic retinopathy (PDR) have increased vitreous levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding proteins (IGFBPs). This accumulation is probably caused by increased leakiness of the blood-retina barrier and influx of circulating IGFs and IGFBPs. To date, interest has focused on the role of circulating total IGF-I in the development of PDR, and there are only sparse data on circulating levels of free IGF-I and IGFBPs. METHODS: We compared fasting serum samples from matched groups of Type 1 diabetic patients with no retinopathy (n = 29), non-PDR (n = 13) and PDR (n = 16). We also included matched controls (n = 26). Serum was analysed for free and total IGF-I and -II, free plus dissociable IGF-I, IGFBP-1, -2 and -3, IGFBP-1-bound IGF-I as well as IGFBP-3 proteolysis. RESULTS: When compared with controls, diabetic patients (n = 58) showed reduced (P < 0.0005) levels of free and total IGFs, free plus dissociable IGF-I and IGFBP-3, whereas levels of IGFBP-1, IGFBP-1-bound IGF-I and IGFBP-2 were elevated. IGFBP-3 proteolysis remained unaltered. When comparing diabetic patients with different degrees of retinopathy, IGFBP-2 and IGFBP-1-bound IGF-I were the only parameters that differed significantly. Patients with retinopathy (non-PDR as well as PDR) had elevated IGFBP-2 (P < 0.03) and reduced IGFBP-1-bound IGF-I (P < 0.03), when compared with patients without retinopathy. Noteworthy, both parameters correlated (0.11 < r2 < 0.14, P < 0.02) with the severity of retinopathy. CONCLUSION: Our study gives no evidence of a direct role of circulating free IGF-I in the development of PDR, whereas IGFBP-2 and IGFBP-1-bound IGF-I showed a relationship with the degree of retinopathy. However, further investigations are needed in order to clarify the basis and clinical relevance of this finding.     

Age-related changes in cortical bone content of insulin-like growth factor binding protein (IGFBP)-3, IGFBP-5, osteoprotegerin,and calcium in postmenopausal osteoporosis: a cross-sectional study

Serum GH and IGF-I levels decline with increasing age, whereas osteoprotegerin (OPG) increases. IGFs as well as OPG are present in bone matrix and mediate the effects of many upstream hormones (e.g. estrogen). To evaluate whether changes in these proteins may to some extent explain the decrease in bone mass in postmenopausal or senile osteoporosis, we measured bone contents of IGF-I, IGF-II, IGF binding protein (IGFBP)-3, IGFBP-5, and OPG in combined extracts obtained after EDTA and guanidine hydrochloride extraction in 60 postmenopausal women aged 47-74 (mean, 63) yr with a previous distal forearm fracture and a hip or spine Z-score less than 0. We found age-related increases in IGFBP-3 (r = 0.35; P < 0.01), IGFBP-5 (r = 0.59; P < 0.001), and OPG (r = 0.36; P < 0.01) in cortical bone, significantly inversely correlated with femoral neck and lumbar spine BMD. A correlation between age and OPG was also detected in trabecular bone (r = 0.27; P < 0.05). A pronounced age-related decrease in cortical calcium contents (r = -0.60; P < 0.001), positively correlated with femoral neck and lumbar spine BMD, was also found. No age-related changes were detected for IGF-I or IGF-II. The present study demonstrates age-related changes in cortical bone contents of IGFBPs, calcium, and OPG, possibly related to the pathophysiology of postmenopausal osteoporosis. As for OPG, our findings probably represent compensatory responses to increased osteoclastic resorption.     

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.     

Growth hormone-binding protein is directly and IGFBP-3 is inversely associated with risk of female breast cancer

OBJECTIVE: Several components of the GH and IGF systems have been implicated in the development of malignancies. All components of these hormonal systems have never been jointly evaluated in female breast cancer, and previous studies have not examined the role of IGF-binding proteins (IGFBP-4, IGFBP-6) or GH-binding protein (GHBP). DESIGN: Hospital-based case-control study. METHODS: In this sample of primarily postmenopausal women, we obtained serum measures of IGF-I, IGF-II, and binding proteins IGFBP-1, IGFBP-3, IGFBP-4, IGFBP-6, as well as GHBP, insulin, and leptin from 74 breast cancer cases and 76 control subjects. RESULTS: In crude analyses, we found lower age-standardized mean IGF-I, IGFBP-3, IGFBP-4, IGFBP-6, and higher IGFBP-1 and GHBP in breast cancer cases when compared with controls. Multivariate models mutually adjusted for other GH-IGF system components and classical breast cancer risk factors demonstrated an inverse association between IGFBP-3 and risk of breast cancer (odds ratio (OR) = 0.2, P < 0.01) and a direct association between GHBP and disease risk (OR = 3.3, P < 0.01). No significant associations were detected in multivariate analyses among IGF-I, IGF-II or IGFBP-1, IGFBP-4, IGFBP-6 with risk of breast cancer, indicating that these factors may not have effects independent of and/or comparable with IGFBP-3 and GHBP. CONCLUSIONS: These results support a protective role of IGFBP-3 and demonstrate for the first time an increased risk of breast cancer with higher GHBP, after accounting for variation in IGFs, IGFBPs, and classical breast cancer risk factors.     

The differential regulation of the circulating levels of the insulin-like growth factors and their binding proteins (IGFBP) 1, 2 and 3 after elective abdominal surgery

OBJECTIVES Patients undergoing abdominal surgery often suffer from morbidity associated with increased protein catabolism. Therapeutic recombinant human insulin-like growth factor (rhIGF)-I has been proposed as a means of reversing this process. As IGFBPs modulate the bioavailability of the IGFs, we have studied the changes in the circulating levels of these peptides during surgery. DESIGN Patients undergoing elective intestinal surgery were recruited prospectively. Blood samples were taken before, during and after surgery. Standard anaesthetic techniques were used. METHODS Twelve adults (aged 30–70 years; 9 female, 3 male) undergoing surgery were studied. Serum was taken before premedication (preop), end of surgery (end surg), 2 h, 6 h post surgery, on days 1–4, 7, 10 and 14, and on recovery at 6 weeks. MEASUREMENTS Serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, insulin and C-peptide were measured by radioimmunoassay. IGFBP profiles were also assessed by Western ligand blot (WLB). Samples taken preop and at 2 days were separated by fast-phase liquid chromatography (FPLC) using a Superose 12 column under neutral conditions (pH 7.4), and the fractions were analysed subsequently by WLB and immunoblot using a specific IGFBP-3 antiserum. RESULTS IGF-I fell rapidly during surgery from 170 ± 21 (preop) to 133 ± 14 μg/ll (end surg) (P < 0.05). The magnitude of this fall could not be explained by haemodilution. IGF-I levels then fell further to a nadir of 103 ± 10 μg/l at day 4 (P < 0.05). IGF-II fell from 580 ± 46 (preop) to 397 ± 38g/l (day 2). Both IGF-I and IGF-II recovered to preop levels at 6 weeks (205 ± 14 μg/l and 623 ± 30 μg/l respectively). IGFBP-3 levels fell similarly from 4.46 ± 0.45 to 3.2 ± 0.3 mg/l (end surg) and to a nadir of 2.66 ± 0.19 mg/l at day 2. There was a close correlation between IGFBP-3 levels and the sum of IGF-I and IGF-II levels before surgery (r = 0.9, P < 0.01) and this was maintained throughout the post-operative period (mean correlation coefficient of 0.86 ± 0.02, P < 0.05). On days 2 and 3 there was a small but significant increase in the ratio between serum IGF-I and IGFBP-3 levels compared with the preop ratio (P < 0.05 and < 0.005, respectively). WLB demonstrated almost complete absence of IGFBP-3 by day 2. This discrepancy between RIA and WLB analysis of IGFBP-3 suggested the presence of IGFBP-3 protease activity between days 1 and 4. This was confirmed by WLB and immunoblot analyses of samples taken 2 days after surgery. The decrease in IGFBP-3 on WLB was shown to be associated with an increase in the proteolytically cleaved fragments of IGFBP-3. These fragments following FPLC were detected in the high molecular weight fractions, suggesting that the fragments were still able to form the high molecular weight IGFBP-3/ALS complex which is thought to form only when IGF is bound by IGFBP-3. IGFBP-1 levels rose during surgery (mean duration of surgery was 125 minute) from 18 ± 3 (preop) to 51 ± 12 μg/l (end surg) (P < 0.05). This rise in IGFBP-1 paralleled increases in insulin from 7.3 ± 1.0 to 20.8 ± 7.5mU/l and glucose from 4.6 ± 0.3 to 8.7 ± 1.2 mmol/l. IGFBP-1 levels then fell to basal values by 6 hours. IGFBP-2, in contrast, fell slightly during surgery from 636 ± 14 to 599 ± 96 mg/l and then returned to basal levels by 6 hours. CONCLUSION After major surgery there are complex and diverse changes in the IGFs and IGFBPs. The effect of these changes on IGF bioavailability may significantly affect the therapeutic potential of IGF-I in this setting.     

Protein-caloric food restriction affects insulin-like growth factor system in fetal Wistar rat

We have previously shown that fetuses from protein-caloric undernourished pregnant rats (35% of control diet during the last week of pregnancy) at 21.5 d post coitum exhibit increased beta-cell mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern similar to that reported in infants of mild diabetic mothers. In this work, we investigated in undernourished fetuses: 1) whether availability of growth factors such as insulin, GH, and IGFs and their binding proteins (IGFBPs) could be implicated in this alteration, and 2) the beta-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished in undernourished pancreases and unchanged in the liver, compared with control fetuses. Serum levels and liver and pancreatic mRNA expression of IGFBP-1 were found to be normal in undernourished fetuses, whereas the serum concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the beta-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased beta-cell mass can be related to the stimulation of replicative beta-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.     

Prolonged hypoxia induced by the reduction of maternal uterine blood flow alters insulin-like growth factor-binding protein-1 (IGFBP-1) and IGFBP-2 gene expression in the ovine fetus.

Insulin-like growth factors (IGF-I and IGF-II) are potent mitogenic and differentiating peptides which are synthesized by many fetal tissues. In the circulation and tissue fluids, IGFs are bound to binding proteins (BPs) which not only function as carrier proteins, but also inhibit or modulate the biological actions of IGFs. We have previously shown that prolonged hypoxia in the ovine fetus induced by the reduction of maternal uterine blood flow for 24 h causes a reduction in the DNA synthesis rate in selected fetal tissues. To determine if this effect is due to alterations in the local synthesis of tissue IGFs and their binding proteins or to changes in systemic concentrations of IGFs and IGFBPs, we have investigated the abundance of mRNAs encoding IGFs and IGFBPs in selected tissues and changes in plasma IGFs and IGFBPs. Ovine fetuses (115-120 days gestation; n = 6) underwent 24 h of hypoxia by the reduction of maternal uterine blood flow (RUBF). Controls (n = 6) underwent the same surgical procedure without RUBF. Serial plasma samples were collected before, during, and after the experiment, and tissues were collected at the end of 24 h. Mean plasma IGF-I and IGF-II concentrations tended to be lower in hypoxic fetuses than in controls during the course of hypoxia, but these differences were not statistically significant. Tissue mRNA levels for IGF-I and IGF-II in lung, muscle, thymus, and kidney were similar in control and hypoxic fetuses after 24 h of hypoxia. The relative abundance of liver IGF-I and IGF-II mRNAs was lower in hypoxic fetuses, but only IGF-I mRNA levels were significantly different from the control values (P < 0.05). Compared to control fetuses, IGFBP-1 mRNA levels in the liver of hypoxic fetuses were increased 3- to 7-fold, and IGFBP-1 mRNA expression was induced in kidneys of some hypoxic fetuses (two of six). In addition, IGFBP-2 mRNA levels were decreased in the liver (50%) and kidney (30%) of hypoxic fetuses. The increase in liver IGFBP-1 mRNA abundance and the decrease in liver and kidney IGFBP-2 mRNA abundance were accompanied by an increase in IGFBP-1 levels and a decrease in IGFBP-2 levels in fetal plasma. No changes were observed in either plasma levels or tissue mRNA abundance for IGFBP-3. Analysis of the time course of changes in plasma revealed that the changes in IGFBP-1 and IGFBP-2 occurred within 4 h of hypoxia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of insulin-like growth factor binding proteins (IGFBPs) during gestation in mice: effects of hypophysectomy and an IGFBP-specific serum protease activity.

Characterization of insulin-like growth factor binding proteins (IGFBPs) and their pituitary regulation were investigated in intact and hypophysectomized (HX) pregnant and nonpregnant mice. Serum samples were obtained from pregnant mice killed on days 5, 8, 10, 12, 14, and 18 of gestation and fetal mice killed on day 18 of gestation. Some animals were HX or sham operated (SH) on days 9, 11, and 14 of gestation; HX and SH control animals were killed 3 days post surgery. Identification and relative quantities of IGFBPs were determined by Western ligand blotting (WLB) of whole serum, or immunoprecipitated and/or deglycosylated serum, using [125I]IGF-II as the ligand. Serum IGF-I and -II concentrations were determined in both HX and SH day 14 pregnant, nonpregnant, and day 18 fetal mouse sera. WLB analysis of nonpregnant mouse serum demonstrated IGFBPs with apparent Mr of approximately 45-40, 31-27, and 24 K. The 45-40 K IGFBPs appeared to be the mouse equivalent of IGFBP-3, and one of the 31-27 K IGFBPs appeared to be IGFBP-2. The other IGFBPs present are not yet fully identified. During pregnancy, the amount of IGFBP-3 present in serum (as measured by WLB) gradually decreased to 42%, 17%, and 10% of virgin levels on days 5, 8, and 10 of gestation. The amount of IGFBP-3 present in pregnant serum after day 10 of gestation was not measurable by scanning laser densitometry. Additionally, an IGFBP of approximately 27 K appeared during gestation; after treatment with Endoglycosidase F, this IGFBP decreased to 24 K. The major IGFBP in fetal serum appeared to be IGFBP-2, with lesser amounts of IGFBP-3 and the 27 and 24 K IGFBPs. Hypophysectomy significantly decreased IGFBP-3, IGF-I, and IGF-II in nonpregnant mouse serum, increased the amount of IGFBP2 found in pregnant mouse serum, and had no effect on IGF-I or -II concentration in day 14 pregnant mouse serum. Incubation of nonpregnant serum with late (day 17) pregnant serum for 5 h at 37 C, decreased the amount of IGFBP-3 (as measured by WLB) in the mixed sample to 93% of the amount present in the nonmixed sample. Iodinated recombinant IGFBP-3 ([125I]human (h)IGFBP-3) was incubated with different mouse serum samples, and proteolytic fragments were visualized after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography.(ABSTRACT TRUNCATED AT 400 WORDS)     

The presence of cation-dependent proteases for insulin-like growth factor binding proteins does not alter the size distribution of insulin-like growth factors in pregnancy

OBJECTIVE: The aim was to investigate the sera of pregnant women for the presence of specific proteases for insulin-like growth factor binding proteins (IGFBPs) and to determine the effect of these on the distribution of IGFs in the circulation. DESIGN: The method used was the chromatographic and electrophoretic analysis of patients' serum. PATIENTS: Sera were examined from normal women during pregnancy: first trimester (n = 4), second trimester (n = 4) and third trimester (n = 10). Eight women with Type I diabetes in the third trimester were also studied along with sera from ten normal adult volunteers. MEASUREMENTS: Circulating IGF-I and IGF-II levels were measured by RIA and their distribution examined by gel filtration. The pattern and stability of the IGFBPs was investigated by Western ligand blotting. RESULTS: A marked reduction in the serum levels of IGFBP-2, IGFBP-3 and IGFBP-4 on Western ligand blotting, which was associated with the presence of three independent, cation-dependent proteases that were specific for different IGFBPs, was found in late pregnancy. Gel filtration of third trimester serum revealed most of the IGF-I to be present in a complex larger than 130 kDa, with a similar distribution to that found in serum of non-pregnant women. The enzymatic modification of the binding proteins made apparent by the decrease in binding protein bands on Western ligand blotting of preincubated samples had no effect on the distribution of IGF-I following size fractionation. CONCLUSIONS: There appear to be at least three independent enzymes that are induced or activated during pregnancy to modify IGFBP-2, IGFBP-3 and IGFBP-4 sufficiently to prevent their detection by ligand blotting. However, this enzymatic processing does not alter the distribution of IGFs, suggesting that the altered binding proteins are still able to carry IGFs but with reduced affinity. Such an alteration in the carrying mechanism of IGFs may have profound effects upon the bioavailability of the IGFs to the maternal tissues and contribute to the altered metabolic demands of pregnancy.     

Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.     

Increased insulin-like growth factor (IGF)-II and IGF/IGF-binding protein ratio in prepubertal constitutionally tall children

The height of subjects with constitutionally tall stature (CTS) is at least 2 SD above the mean of subjects of the same age and sex. Apart from a few discordant data on the role of GH and its direct mediator, IGF-I, no studies have been conducted on other components of the IGF system, which also condition the bioavailability and activity of IGF-I. We, therefore, investigated the possibility that other components of the IGF system might play a role in determining the increased growth velocity seen in CTS. To this end, we evaluated the behavior not only of IGF-I but also of IGF-II, IGF-binding protein (IGFBP)-3, and acid-labile subunit, the subunits that constitute the main IGF complex in circulation (150-kDa complex), as well as of IGFBP-1 and IGFBP-2, which are negatively regulated by GH and, like IGFBP-3, able to influence the bioavailability of the IGFs. The study was performed on 22 prepubertal subjects affected by CTS (16 males and 6 females), aged 2.8-13.3 yr (6.8 +/- 0.5 yr, mean +/- SEM). Thirty-seven normal prepubertal subjects (16 males and 21 females) aged between 2.2 and 13.3 yr (6.7 +/- 0.5 yr), who were comparable in socioeconomic and nutritional terms, served as controls. From the auxological point of view, subjects with CTS differed significantly from controls only in terms of growth velocity (HV-SD score; CTS, 1.8 +/- 0.3; controls, 0.4 +/- 0.2; P < 0.0001) and height (H-SD score; CTS, 3.1 +/- 0.1; controls, 0.4 +/- 0.2; P < 0.0001). The results demonstrated that the concentrations of IGF-I (27.3 +/- 2.0 nmol/liter), IGFBP-3 (66.9 +/- 3.8), and acid-labile subunit (216.8 +/- 13.6) in CTS-affected subjects were not significantly different from those determined in controls (25.0 +/- 2.9, 74.4 +/- 4.1, and 241.0 +/- 11.9, respectively). By contrast, IGF-II levels proved significantly higher in CTS subjects (IGF-II: 87.2 +/- 3.4 vs. 52.4 +/- 2.3, P < 0.0001). Chromatographic analysis, performed after acid treatment of pooled sera, showed only the presence of normal 7.5-kDa IGF-II in both CTS subjects and controls. In comparison with controls, CTS children showed a lower concentration of IGFBP-1 (1.6 +/- 0.3 vs. 4.1 +/- 0.7, P = 0.03) and a higher concentration of IGFBP-2 (14.3 +/- 1.8 vs. 9.6 +/- 1.1, P = 0.03). The IGFs (IGF-I and -II)/IGFBPs (-1 + -2 + -3) molar ratio was significantly higher (P < 0.0001) in CTS children than in controls. In particular, the IGF-II/IGFBP ratio (P < 0.0001) was responsible for the excess of the IGF peptide in relation to the concentrations of IGFBPs and, therefore, for the increase in the potentially bioactive free form of the IGFs. Moreover, the IGFBP-3/IGF molar ratio was significantly reduced, being less than 1 in CTS subjects (0.6 +/- 0.1 vs. 1.1 +/- 0.1), so that a quantity of IGF peptides lack sufficient IGFBP-3 to form the 150-kDa complex with which are normally sequestered in the vascular compartment. The data show that in CTS: 1) the most GH-dependent components of the IGF system are normal, consistent with the finding of a normal GH secretory state; 2) the less GH-dependent IGF-II is significantly increased, in agreement with the finding of a relationship between high levels of IGF-II and overgrowth in some syndromes; and 3) the IGF/IGFBP molar ratio is increased, and, therefore, a greater availability of free IGF for target tissues may be responsible for overgrowth in CTS.     

Coordinated control of fetal gastric epithelial functions by insulin-like growth factors and their binding proteins

The influence of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) on human gastric functions are unknown. This study was undertaken to evaluate the ability of fetal gastric mucosa to produce IGFBPs and to test the effects of IGF-I, IGF-II, and synthetic truncated IGFs that do not interact with IGFBPs on epithelial cell proliferation and glandular zymogenic function. Western blots, Far Western blots, and immunohistochemistry were performed to characterize the expression of IGFBP-1 to -6 and IGF-I receptor. The effects of growth factors on DNA synthesis and lipase and pepsin activities were determined in gastric explants maintained in serum-free organ culture. All gastric epithelial cells expressed the IGF-I receptor. IGFBP-2 to -6 were produced endogenously, and they were differentially localized along the foveolus-gland axis and modulated in culture. Exogenous IGF-I and IGF-II were able to reduce lipase activity without affecting pepsin, whereas they exerted different effects on cellular proliferation: IGF-I was stimulatory and IGF-II had no influence. Illustrating the complex regulatory effect that IGFBPs exert on IGF bioactivity, both truncated IGF-I and IGF-II stimulated DNA synthesis more than IGF-I. Moreover, the striking difference in mitogenic activity between truncated and native forms of IGF-II probably reflects the abundance of IGFBP-2 and IGFBP-6, two IGF-II carriers, in the foveolus/neck region, including the proliferative compartment. This study provides new evidence for the involvement of an intragastric IGF/IGFBP system in the fine regulation of epithelial cell division and also in the control of zymogen synthesis. Moreover, the specific influence of IGF-II as a mitogen appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells.