Expression of TR Isoforms in Failing Human Heart (2001)

To the editor:Lassarre and Binoux (1) recently described an assay for intact IGFbinding protein-3 (IGFBP-3) in which serum is acidified to destroy the acid-labile subunit (ALS) and dissociate IGF-IGFBP complexes, IGFs are removed by ultrafiltration, and active IGFBP-3 is quantitated by radiolabeled IGF-I binding.This assay is described as opening new perspectives in investigating IGFBP-3 proteolysis and IGF bioavailability.The authors state that proteolyzed IGFBP-3 loses affinity for IGFs, which are then "redistributed towards the 40-kDa binary IGFBP-IGF complexes" (1), and point out that in human pregnancy, "virtually all circulating IGFBP-3 is degraded."There is some disagreement as to the changes in total serum IGFs through pregnancy, with increases in both IGF-I and IGF-II (2), an increase in IGF-I but not IGF-II (3), and a 15% decrease in IGF-I (4) reported.The proportion of total IGF-I detectable in "free IGF-I" assays approximately doubles in pregnancy from the value of about 1% reported for nonpregnant women (4, 5), although the biological significance of these measurements is unclear.Abundant published evidence refutes the concept that there is a substantial redistribution of IGFs in pregnancy serum away from IGFBP-3-ALS complexes; direct measurement indicates that the proportion of total IGFs in these complexes is unchanged or even slightly increased in pregnancy (6, 7).The size distribution of IGFBP-3 is also unchanged throughout the course of pregnancy, with 85-90% remaining in high molecular weight fractions (8), whereas the total immunoreactive IGFBP-3 concentration is reported to increase ( 8).This provides important information about the distribution of IGFs, since human IGFBP-3 has an unmeasurably low affinity for ALS unless IGF-I or IGF-II is bound to the IGFBP-3 (9).Thus, the unaltered proportion of pregnancy serum IGFBP-3 in high molecular weight complexes indicates that IGFs are also in these complexes.It can be demonstrated that, if there is a substantial loss of affinity in the IGF-IGFBP interaction, there would be a decreased binding of IGFs, and consequently a loss of ALS binding (10).In contrast, IGFBP-3 isolated from nonpregnancy or pregnancy serum by immunoaffinity chromatography, and not depleted of its endogenous IGFs, shows normal affinity for ALS (11).Although IGFBP-3 in pregnancy serum, or isolated from pregnancy serum in the presence of endogenous IGFs, seems to bind and transport IGFs normally, depletion of endogenous IGFs under acidic conditions leads to a loss of affinity as determined by radioligand binding (12)-indeed, this is the basis of the new assay of Lassarre and Binoux (1).While this may in part be explained by the selective effect of altering tyrosine residues 24 and 60 of IGF-I during the iodination process (13), it has been clearly demonstrated that IGF-I affinity is decreased by up to 8-fold, and IGF-II affinity by 2.5-fold, in IGFdepleted pregnancy serum compared with nonpregnancy serum (12,13).Similarly, IGFBP-3 in pregnancy serum is undetectable by ligand blotting after SDS-PAGE, as first demonstrated over a decade ago (14,15).Because an 8-fold loss of affinity from the intact IGFBP-3 affinity of 20 liters/nmol would be insufficient to abolish IGF binding on a ligand blot, it must be assumed that further damage to the proteolyzed protein during electrophoresis, or to the IGF-I during iodination ( 16), contributes to this observation.What is the biological consequence of this limited proteolysis of IGFBP-3, detectable by radioligand binding in vitro after depletion of bound IGFs, but apparently without effect on IGF transport in the circulation?It is certainly possible that this could alter the bioavailability of IGFs to the tissues, and this may eventually turn out to be true, but there has been no definitive experimental proof of this to date, in which pregnancy-proteolyzed IGFBP-3, not subjected to destructive isolation and processing, has been shown to allow IGFs to pass from circulating complexes to their target tissues more readily than unproteolyzed IGFBP-3.Indeed, the definitive experiment is difficult to envisage.Even if this accelerated release of IGFs does occur following proteolysis, it is unclear whether the released IGFs would have increased availability to tissue receptors (local IGFBPs might rapidly re-bind them), or would simply be lost through degradative clearance, for example through the kidneys.Therefore, it appears that there is insufficient evidence at present to state, as Lassarre and Binoux (1) do, that "limited proteolysis of IGFBP-3 is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream."