The physiology of circulating insulin-like growth factor binding proteins: studies in the sheep

Abstract

Fetal growth and development is primarily limited by maternal substrate supply. Recent studies also suggest that IGFs and their binding proteins have a significant influence on fetal growth, and that maternal nutritional status is a major factor in the regulation of the IGF/IGFBP axis in fetal plasma. The aim of these studies was a) to develop specific homologous radioimmunoassays (RIAs) for ovine IGFBP-2 and IGFBP-3 for subsequent studies, b) to further define the interactions between maternal nutritional status and circulating IGFs and the IGFBPs in the sheep fetus and c) to characterise aspects of the fetal IGF/IGFBP axis that are temporarily or permanently altered (reprogrammed) in response to limited substrate supply. Such changes might provide mechanistic explanations for epidemiological data linking impaired growth in utero with increased susceptibility to specific diseases in adulthood. IGFBP-2 and IGFBP-3 were isolated from fetal and postnatal sheep serum respectively following 1) cation exchange chromatography to remove endogenous ligand, 2) IGF-2 affinity chromatography and 3) C8 and C18 reverse phase chromatography. N-terminal amino acid sequence analysis revealed strong homology for each peptide with data from several other species. Ovine IGFBP-2 had a 3 amino acid deletion at the N-terminal when compared to human or bovine IGFBP-2, the significance of which is unknown. Specific antisera were raised against both peptides and homologous RIAs were generated. While IGFs did not interfere in either RIA, IGF-1 addition was required in the oIGFBP-3 RIA to overcome a potency difference for the antiserum between purified oIGFBP-3 and plasma IGFBP-3:IGF-1 complex. Both RIAs were validated for analysis of fetal and postnatal sheep plasma. We characterised the roles of insulin and glucose in mediating the effects of substrate supply on plasma IGFs and IGFBPs in the fetal sheep. Pregnant ewes were starved for 72 hours and refed for 48 hours. Fetuses were infused with either glucose or insulin during the final 24hours of maternal starvation. Glucose, insulin, IGF-1 and IGF-2 were reduced by maternal starvation. While glucose infusion increased these parameters to near control values, insulin infusion was only associated with elevations in insulin and IGF-1 concentrations. These data suggest that insulin regulates the fetal plasma levels of IGF-1 but that IGF-2 concentrations were regulated by glucose in an insulin-independent fashion. Fetal plasma IGFBP-1 and -2 were increased, and IGFBP-3, IGFBP-4 and the circulating IGF-2/mannose-6-phosphate receptor (IGF-2/M6PR) were decreased, by maternal starvation. IGFBP-1 and IGFBP-4 levels were restored to normal by glucose and insulin infusions while the IGF-2/M6PR was increased by glucose infusion only (not measured in insulin-infused fetuses). These results are consistent with data regarding firstly the insulin dependency of IGFBP-1 and its role as a glucose counter-regulatory peptide, and secondly the role of insulin in determining IGFBP-4 and IGF-2/M6PR distribution between plasma and tissues. Fetal plasma IGFBP-2 and -3 levels were unaffected by either infusion suggesting that they are regulated by factors other than acute changes in plasma glucose or insulin. We have subsequently examined the hypothesis that exposure to undernutrition around the time of conception would result in a reduced growth rate and reprogramming of the plasma IGF/IGFBP axis in the late gestation fetus. Pregnant ewes were either well fed or undernourished from 60 days prior to mating to d30 of gestation and then subjected to a second period of undernutrition between d105 and d115 of gestation. Periconceptual undernutrition was associated in the late gestation fetus with reduced growth rate and a reduction in the circulating concentrations of IGFBP-1 and IGFBP-3. Furthermore the response of plasma IGF-1, IGFBP-1 and IGFBP-3 in fetuses of the periconceptual undernutrition group to severe maternal undernutrition in late gestation was significantly greater than that measured in fetuses from mothers well fed at the time of conception. The hypothesis that a severe nutritional insult only during late gestation would alter the postnatal regulation of plasma IGF-1 and IGFBP-3 in the lamb was also addressed. While birth weight was reduced in fetuses of mothers undernourished for 20 days, no differential effects of undernutrition in utero on the pre-pubertal ontogeny of IGF-1 and IGFBP-3 or on their response to a GH bolus could be found between the treatment groups. In summary, these studies indicate that IGFs and IGFBPs play an important role in mediating the effects of fetal substrate supply on fetal growth. Reprogramming of the responses by the plasma IGF/IGFBP axis to undernutrition in the late gestation sheep fetus could be induced by a period of periconceptual undernutrition. However, the lack of reprogramming effects on IGF-1 and IGFBP-3 in postnatal plasma following a severe nutritional insult only in late gestation suggests either that reprogramming of the IGF/IGFBP axis is dependent on exposure to undernutrition at earlier timepoints in gestation or that reprogramming of the fetal IGF/IGFBP axis does not persist postnatally.