Use of the ligand immunofunctional assay for human insulin-like growth factor ((IGF) binding protein-3 (IGFBP-3) to analyze IGFBP-3 proteolysis and igf-i bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics.

Abstract

The ligand immunofunctional assay for plasma insulin-like growth factor (IGF) binding protein (IGFBP)-3 developed in our laboratory provides for specific measurement of intact, as opposed to proteolyzed, IGFBP-3. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration; thereafter, intact and proteolyzed IGFBP-3 are captured by a monoclonal antibody in a solid-phase assay and incubated with (125)I-IGF-I, which detects the intact protein but not its proteolytic fragments. This assay was combined with assays for IGF-I (RIA of the ultrafiltrate) and total IGFBP-3 (immunoradiometric assay) to quantify the percentage of proteolyzed IGFBP-3 (percent proteolyzed IGFBP-3) and to calculate the IGF-I/intact IGFBP-3 ratio as an index of the fraction of exchangeable IGF-I bound to IGFBP-3. This fraction represents most of the IGF-I that is bioavailable. Because GH and insulin control the hepatic production and plasma concentrations of IGF-I and IGFBP-3, we set out to determine whether variations in the secretion of the two hormones are involved in the regulation of IGFBP-3 proteolysis. The study included adult populations of 36 healthy subjects, 23 hypopituitary patients untreated with GH, 43 acromegalics (13 untreated), 42 insulin-treated type 1 diabetics [insulin-dependent diabetes mellitus (IDDM)] patients, and 50 type 2 diabetics [non-IDDM (NIDDM)] patients, 22 of whom were insulin-treated and the remaining 28 treated with sulfonylurea and/or metformin). Unlike IGF-I and (to a lesser extent) total IGFBP-3 levels, which decline with age, percent proteolyzed IGFBP-3 seemed relatively stable. In healthy adults, the mean +/- SEM was 29.4 +/- 1.9 for subjects less than 45 yr old and was slightly (but not significantly) lower, 25.7 +/- 3, for those of more than 45 yr. There was no difference between male and female subjects. In GH-deficient patients, despite severely depressed IGF-I levels, percent proteolyzed IGFBP-3 and IGF-I/intact IGFBP-3 ratios were within the normal range. Among acromegalics, percent proteolyzed IGFBP-3 was elevated: 36.6 +/- 3.3 for patients of less than 45 yr, 33.3 +/- 3.2 for patients of more than 45 yr (P = 0.02 vs. healthy subjects). Consequently, the effects of excessive IGF-I synthesis are exacerbated by the enlarged exchangeable fraction of IGFBP-3-bound IGF-I. There was no significant difference in percent proteolyzed IGFBP-3 between GH-deficient patients before and after GH treatment or between treated and untreated acromegalics. In IDDM patients, the means for percent proteolyzed IGFBP-3 were higher than those in healthy adults: 36.7 +/- 3.7 (P = 0.03) and 31.3 +/- 3.3 for subjects of less than 45 and more than 45 yr, respectively. In NIDDM patients, all of whom were more than 45 yr old, the means were 35.2 +/- 2.5 (P = 0.02) for insulin-treated patients and 33 +/- 2.5 for the group treated orally. Among the diabetics, increased IGFBP-3 proteolysis resulted in an IGF-I/intact IGFBP-3 ratio that was normal for IDDM patients of less than 45 yr and above normal (P = 0.01) for the others. Percentage proteolyzed IGFBP-3 and the IGF-I/intact IGFBP-3 ratio were inversely related to body mass index in IDDM patients (r = -0.42, P = 0.008; and r = -0.31, P = 0.05, respectively) and to percentage glycosylated hemoglobin in all insulin-treated diabetics (r = -0.25, P = 0.05; and r = -0.33, P = 0.008, respectively). There was also an inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels in healthy adults (r = -0.39, P = 0.03) and orally treated NIDDM patients (r = -0.37, P = 0.05). Percentage proteolyzed IGFBP-3 was positively correlated to total IGFBP-3 in healthy adults (r = 0.65, P = 0.0001) and in all the groups of patients. It was negatively correlated to IGF-I/total IGFBP-3 in healthy subjects (r = -0.40, P = 0.02) and diabetics (r = -0.30, P = 0.005). This suggests an autoregulatory mechanism controlling the bioavailability of IGFBP-3-bound IGF-I in the 140-kDa complexes. In the pathological conditions studied here, regulation of IGF-I bioavailability by limited proteolysis of IGFBP-3 contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion.