The effect of GH therapy on the molecular forms of IGFBP in GHD children

We have previously shown that the major correlates of growth following GH therapy in GH deficient children are changes in circulating IGF-I and IGFBP-3, suggesting a synergistic interaction between IGF-I and IGFBP-3. The aim of this project was to examine the change in the molecular forms of the IGFBPs, particularly IGFBP-3 and the acid-labile subunit (ALS), during GH administration in GHD children. To asses the changes in the IGFBPs as well as the ALS, samples from prepubertal GHD patients, prior to therapy and during the first year of GH treatment, were subjected to Western analysis. Densitometric analysis of Western ligand blotting (WLB) showed a 76% increase in IGFBP-3 (p=0.02) and a 56% decrease in 36 kDa IGFBP-2 (p=0.03) during GH therapy. Western immunoblot (WIB) analysis of IGFBP-3 revealed the presence of intact (40-50 kDa doublet) as well as alternative (28 kDa) forms of IGFBP-3 in the serum of GHD and healthy children. Both forms of IGFBP-3 increased by 64% during GH therapy (intact p=0.003; alternative p=0.0001). WIB analysis of the ALS showed an 85 kDa doublet which was diminished in GHD and increased 41% with GH therapy (p=0.01). The response to GH therapy as measured by the height velocity SDS adjusted for bone age correlated with the percent change in total IGFBP-3 (r=0.772, p=0.002 by WIB), intact IGFBP-3 (r=0.845, p=0.0005 by WLB; r=0.541, p=0.05 by WIB), and alternative IGFBP-3 (r=0.772, p=0.002), as well as with the percent change in ALS (r=0.813, p=0.014). To define the molecular forms of IGFBP-3, IGFBP regions were separated by neutral size-exclusion chromatography and subjected to WLB and WIB analysis. Both intact and alternative forms of IGFBP-3 were found in both the 150/130 kDa and 44 kDa regions. A shift was seen in both the intact and alternative forms of IGFBP-3 from the 44 kDa to the 150/130 kDa (ternary) complex during GH therapy. GH treatment of GHD children results in increases of intact, alternative, and total IGFBP-3, as well as an increase in ALS, and a decrease in IGFBP-2. The growth response to GH therapy correlates with the percent increase in the intact, alternative, and total IGFBP-3, as well as with the percent increase in ALS, suggesting that the formation of the ternary complex may be a determining factor in the somatic growth response.