Kristin Wilson

Maternal Hypoxia as a Model for Intrauterine Growth Retardation: Effects on Insulin-Like Growth Factors and Their Binding Proteins

ABSTRACT: Evidence suggests that IGF and their binding proteins play a role in fetal growth, but more knowledge concerning their regulation is essential. We examined the expression of IGF and their binding proteins in experimental intrauterine growth-retarded (lUGR) rat fetuses of hypoxic dams (13–14% oxygen, d 14–21 of gestation). The mean body weight of the fetuses (d 21 of gestation, n = 72) of the six hypoxic dams was 24% lower (p < 0.0001) than the mean weight of the fetuses of six control dams (n = 82). Wet liver weights demonstrated a 20% decrease (p < 0.0001) and placentas a 10% decrease (p < 0.01) compared with control fetuses. The mean serum concentrations of immunoreactive IGF-I in both groups were low but did not differ significantly. The mean serum concentrations of immunoreactive IGF-II, however, were higher in IUGR fetuses. As assessed by Northern blot analysis, there was a 4-fold increase in insulin-like growth factor binding protein-1 (IGFBP-1) mRNA expression in the livers of the IUGR fetuses compared with controls. IGFBP-2 mRNA expression was 6-fold increased in IUGR fetal livers. No difference was found in IGFBP-4 mRNA. An increase in IGFBP-1, −2, and −4 concentrations could be seen by Western ligand blotting in the serum of growth-retarded fetuses compared with control fetuses. This finding was verified by immunoprecipitation with specific antibodies, which demonstrated increases in IGFBP-1 and IGFBP-2. Our results validate the use of maternal hypoxia as an experimental model of intrauterine growth retardation and indicate that increased IGFBP-1 and −2 expression may be of importance in the etiology of fetal growth retardation caused by maternal hypoxia.

Insulin-like growth factor binding protein-3 concentrations and insulin-like growth factor binding protein-3 protease activity in sera of patients with malignant solid tumors or leukemia.

ABSTRACT: IGF binding proteins (IGFBP) regulate the bioavailability and bioactivity of IGF. The major IGFBP in serum is IGFBP-3. We investigated whether sera from children with malignancies show alterations in levels of IGFBP-3 as measured by Western ligand blot analysis (WLB) and RIA with αIGFBP-3gl, a specific rabbit polyclonal antibody. Furthermore, IGFBP-3 proteolysis was quantified by densitometric analysis of [125I]IGFBP-3 protease assays, and IGFBP-3 fragments were visualized by Western immunoblot with αIGFBP-3gl. We examined sera from 21 children with solid tumors, five patients with sarcoma who had reached complete remission, and 13 children with acute leukemia. Serum samples were collected at diagnosis, before initiation of therapy. Sera of 10 healthy children served as normal controls. Children with solid tumor or leukemia had significantly higher (p < 0.001) IGFBP-3 protease activity in serum than did normal controls or patients with sarcoma in complete remission. Corresponding to this finding, densitometry of WLB showed lower IGFBP-3 levels in sera of children with malignancies in comparison with normal controls. The negative correlation (p < 0.001, r =-0.80) between IGFBP-3 proteolysis, as measured by [125I]IGFBP-3 protease assay, and IGFBP-3 band density on WLB indicates that proteolysis is the probable reason for reduction of IGFBP-3 on WLB. IGFBP-3 concentrations measured by RIA were in the normal range for most patients, further indicating that differences in serum IGFBP-3 levels measured by WLB reflect protease activity. We conclude that sera from children with malignancies frequently contain protease activity that causes IGFBP-3 cleavage in a way that might affect the tissue availability of IGF. Because many tumor cells are responsive to the mitogenic actions of IGF in vitro, IGFBP-3 proteases in serum could also play a role in tumor progression and metastasis in vivo.

Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency

OBJECTIVE The aim of this investigation was to study the effect of relatively high dose IGF‐I therapy given for several months, on serum levels of IGF‐I, IGF‐II and IGFBP‐3, and on IGF‐I pharmacokinetics In patients with growth hormone insensitivity due to GH receptor dysfunction.

HYPOXIA-INDUCED CHANGES IN INSULIN-LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS IN PREGNANT RATS

Pregnancy is associated with important changes in the insulin-like growth factor (IGF)-insulin-like growth factor binding protein (IGFBP) axis, but the importance of these growth factors for fetal growth is not well understood. We have recently established a maternal hypoxia model that results in significant intrauterine growth retardation in the fetus, and characterized the IGF-IGFBP axis in growth-retarded fetuses. To determine if maternal IGFs and their binding proteins are similarly regulated by hypoxia, we examined their expression in 6 hypoxic dams (13% oxygen, days 14-21 of gestation) and 6 control dams (21% oxygen). There was no significant difference in the food intake between the groups. The mean body weight of hypoxic dams, however, was 20% less than that of controls. Of all the organs, the lungs were most affected by hypoxia, weighing 17% more in the hypoxic dams than in the control dams; placental weight was reduced by 10% in the hypoxic dams. Liver and brain weights were not changed significantly by hypoxia. The mean concentration ofimmunoreactive IGF-I was 123 +/- 11 ng/ml in the hypoxic dams and 130 +/- 18 ng/ml in the control dams (nonsignificant). Similarly, there was no significant difference in hepatic IGF-I mRNA levels determined by solution hybridization nuclease-protection assay. An increase in IGFBP-1, IGFBP-2 and IGFBP-4 concentrations, however, could be observed by Western ligand blotting of the sera of hypoxic dams, compared to control dams. As assessed by Northern blot analysis, there was a 2.8-fold increase in IGFBP-1 mRNA expression in the livers of hypoxic dams compared to controls. Hepatic IGFBP-4 expression was also slightly increased (1.25-fold) in the hypoxic dams. No difference in hepatic IGFBP-2 or IGFBP-3 mRNA was found. Our results show parallel patterns in fetal and maternal IGF and IGFBP responses to hypoxia. This suggests that hypoxia may inhibit fetal growth by both directly affecting the fetus and via inhibition of placental growth.

Serum profiles of insulin-like growth factors and their binding proteins in adults with growth hormone receptor deficiency treated with insulin-like growth factor I.

Six adult patients with growth hormone receptor deficiency (GHRD) (2 men, 4 women) with an identical defect in the growth hormone receptor (GHR) gene, were treated with recombinant human insulin‐like growth factor I (IGF‐I), 40 μgikg S.C. twice daily, for 7 days. Serum concentrations of IGF peptide and IGF binding protein‐3 (IGFBP‐3) were measured by specific radioimmunoassays; serum IGFBPs were also measured by Western ligand blotting. The size distribution of both IGF‐I and IGF‐II was measured in serum following size‐exclusion fast‐performance liquid chromatography. IGF‐I treatment resulted in a normalization of serum IGF‐I levels on days 1–7 of treatment and a decrease in serum IGF‐II levels. The fall in IGF‐II levels and the simultaneous rise in IGF‐I levels, however, resulted in an unchanged total serum IGF level. The low IGFBP‐3 values did not significantly change during treatment, whereas there was a slight increase in IGFBP‐2 levels. Preliminary analysis of size‐fractionated sera suggested an increase in IGF‐I levels in the 40 and 150 kDa regions at the expense of IGF‐II levels. The results suggest that despite the failure of IGF‐I treatment to increase IGFBPs significantly, serum IGFBP concentrations were sufficient to maintain normal levels of IGF‐I. 0 Laron syndrome, growth hormone receptor deficiency, insulin‐like growth factors, insulin‐like growth factor binding protein

Effects of insulin-like growth factor I treatment on the molecular distribution of insulin-like growth factors among different binding proteins

The molecular distribution of insulin‐like growth factor I (IGF‐I) and IGF‐II among the IGF binding proteins (IGFBPs) was studied before and during IGF‐I therapy in Ecuadorean adults with growth hormone receptor deficiency (GHRD). Of the total circulating IGF‐I and IGF‐II, 70% was carried by the 150 kDa complex in normal subjects, while in patients with GHRD, 50% of serum IGF‐I, but only 30–35% of serum IGF‐II, was measured within the 150 kDa IGFBP‐3 region. Administration of IGF‐I altered the concentration of IGF‐I and IGF‐II, although the percentage of total IGF measured within each IGFBP region was not affected, as the increase in IGF‐I and the decrease in IGF‐II were proportional. Similarly, serum concentrations of IGFBP‐3 and the acid‐labile subunit, measured by radioimmunoassay, were unaltered. Thus, administration of IGF‐I to patients with GHRD was unable to correct the aberrant distribution of IGFs among the IGFBPs.

Medical and Scientific Aspects of Growth Hormone Receptor Deficiency (Laron Syndrome) in Ecuador

In 1966, Laron et al., in Israel, described a genetic form of dwarfism in three siblings with physical features similar to growth hormone (GH) deficiency, but with abnormally high serum levels of immunoreactive GH (1). In subsequent reports, they described 29 patients in Israel, primarily of Oriental Jewish ancestry from North Africa and the Persian Gulf (2, 3). An additional 70 subjects have been reported (4), many of Mediterranean ancestry, including individuals from Iran, Iraq, Yemen, Tunisia, Lebanon, Saudi Arabia, Algeria, France, Italy, Sardinia, Spain, Brazil, Argentina, Mexico, Pakistan, Bangladesh, northern Europe, Japan, South Africa, Canada, and the United States.

IS IMMUNOREACTIVE IGFBP-3 IN THE URINE AN INDICATOR OF GROWTH HORMONE DEFICIENCY (GHD)?

The insulin-like growth factors (IGF) arc potent polypeptide mitogens that are carried in the blood, primarily by a GH-dependent protein, IGF binding protein-3 (IGFBP-3). This IGFBP can be fragmented by IGFBP-proteases, subsequently altering the affinity for IGFs. We have characterised the serum and urine IGFBP profiles of children both pre- and post GH treatment. Techniques used were an IGFBP-3 RIA, Western ligand blot (WLB) and an IGFBP-3 protease assay. In the sera of pretreatment GHD children, IGFBP-3 levels were low by both RIA and WLB analysis, and there was no protease activity. Serum concentrations of IGFBP-3 were corrected following GH administration. In the urine, we have established a normal range of IGFBP-3 and IGFBP-3 protease activity in subjects between 5-44 years. Urinary IGFBP-3 (uIGFBP-3) is age-dependent, increasing from 40 ng/mg Cr to 60 ng/mg Cr at age 11-15, after which levels decline to 18 ng/mg Cr by 25 years. Furthermore, little uIGFBP-3 protease activity is delected. In pretreatment GHD subjects, immunoreactive uIGFBP-3 as determined by RIA were generally higher than age-matched controls, although there was some overlap with the normal range. Following GH therapy, uIGFBP-3 levels declined to within the normal range. This was in striking contrast to serum, where serum IGFBP-3 levels increased with GH administration. Although immunoreactive uIGFBP-3 were high, IGFBP-3 was undetectable by WLB. This difference was due to significant protease activity in these subjects, especially in the younger age groups. Following GH therapy, uIGFBP-3 became detectable by WLB and the protease activity diminished. Thus, in the urine of GHD children, an IGFBP-3 protease activity was delected that was apparently GH dependent. Furthermore, the urinary IGFBP-3 protease activity is a serine-type protease which is not metal ion dependent. The source of the IGFBP-3 protease is uncertain, although its appearance solely in the urine of GHD patients is a unique finding. Further assessment of this proteolytic activity may provide insight as to the renal function and clearance in GHD patients.