Sharron E. Gargosky

Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics.

The IGFBPs are a family of homologous proteins that have co-evolved with the IGFs and that confer upon the IGF regulatory system both functional and tissue specificity. IGFBPs are not merely carrier proteins for IGFs, but hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, IGFBPs appear to have intrinsic biological activity independent of IGFs. The current status of research on IGFBPs is reviewed herein. Following a brief introduction to the entire IGF/IGFBP system, separate sections for each of the six cloned mammalian IGFBPs, the most extensive for IGFBP3, cover selected topics that emphasize the dynamics of IGFBPs--that is, their regulation in cells, their functionally important post-translational modifications, and their interactions in the cellular microenvironment--and how these dynamics influence physiological function.

Immunoblot studies of the acid‐labile subunit (ALS) in biological fluids, normal human serum and in children with GH deficiency and GH receptor deficiency before and after long‐term therapy with GH or IGF‐I respectively

The aims of this investigation were (a) to study the presence of immunoreactive forms of the acid‐labile subunit (ALS) in different human biological fluids, (b) to define the age dependence of serum ALS in normal children and adults and (c) to compare the regulation of ALS by GH or IGF‐I in children with GH deficiency (GHD) and GH receptor deficiency (GHRD) before and after 1 year of therapy with GH or IGF‐I, respectively.

Assays for insulin-like growth factors and their binding proteins: Practicalities and pitfalls

Insulin-like growth factors (IGFs) are growth hormone-dependent anabolic peptides that circulate in biologic fluids complexed to a family of IGF binding proteins. Measurement of the serum concentrations of IGF peptides and IGF binding proteins has proved to be an effective means of evaluating functional growth hormone status and makes it possible to establish a diagnosis of IGF deficiency.

The effect of GH therapy on the immunoreactive forms and distribution of IGFBP-3, IGF-I, the acid-labile subunit, and growth rate in GH-deficient children

We have previously shown that the major correlates of growth following growth hormone (GH) therapy in growth hormone-deficient (GHD) children are changes in circulating insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3), suggesting a synergistic interaction between IGF-I and IGFBP-3 (1). The first aim of this project was to examine the molecular forms of IGFBP-3 and the acid-labile subunit (ALS), and to assess the changes in these molecular forms during GH administration to GHD children. Plasma samples from prepubertal GHD patients, prior to therapy and during the first year of GH treatment, were subjected to Western ligand and immunoblot analysis. Densitometric analysis of Western ligand blotting (WLB) showed a 76% increase in IGFBP-3 (p=0.02), but 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- to 45-kDa doublet) as well as a proteolyzed (28-kDa) form of IGFBP-3 in the serum of GHD and healthy children. Both immunoreactive forms of IGFBP-3 increased by 64% during GH therapy (intactp=0.003; proteolyzedp=0.0001). WIB analysis of the ALS showed an 84-to 86-kDa doublet, which increased by 41% with GH therapy (p=0.01). The response to GH therapy, as measured by the height velocity standard deviation score (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 proteolyzed IGFBP-3 (r=0.703,p=0.007), as well as with the percent change in ALS (r=0.813,p=0.014).The second aim of this project was to assess the changes in distribution of the immunoreactive forms of IGFBP-3 and IGF-I among the ternary (ALS/IGFBP-3/IGF) complex, the binary (IGFBP-3/IGF) complex, and uncomplexed IGF during the first year of GH therapy, and to explore further the correlation with growth response to GH. Plasma samples, prior to therapy and after the first year of GH treatment, were separated by neutral size-exclusion chromatography and then subjected to IGFBP-3 immunoradiometric assay (IRMA), IGFBP-3 WIB, and IGF-I IRMA analysis. IGFBP-3 increased in both the ternary (p<0.0001) and binary (p=0.01) complexes, but there was a shift in the percentage of IGFBP-3 from the binary to the ternary complex during GH therapy. Both intact and proteolyzed forms of IGFBP-3 were found in both the ternary and binary complexes, but the shift occurred primarily for the proteolyzed (28-kDa) form (p=0.001). There was a significant increase in IGF-I in the ternary (p=0.001) and binary (p=0.005) complexes, but not in uncomplexed IGF-I. The percentage of IGF-I in the ternary complex increased (p=0.006), whereas the percentage of uncomplexed IGF-I decreased (p=0.02), during GH therapy. Growth rate, assessed by the height velocity SDS for bone age, correlated best with the changes in ternary complex IGFBP-3 (r=0.72,p=0.01) and ternary complex IGF-I (r=0.56,p=0.10).In conclusion, GH treatment of GHD children results in significant increases of intact, proteolyzed, and total IGFBP-3, as well as an increase in ALS, which all correlate with the growth response to GH therapy. In addition, GH treatment results in increases in ternary complex IGFBP-3 and IGF-I, which also correlate with the response to therapy. We suggest that the formation of the ternary complex may be a determining factor in the somatic growth response.

Collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF.

The major serum carrier of the insulin-like growth factors (IGFs) is IGF-binding protein-3 (IGFBP-3) that exists in the circulation associated with IGF and an acid labile subunit to form a ternary (158-kDa) complex. It has been reported that heparin disrupts the IGF carrying capacity of the ternary complex and is a potent inhibitor of ternary complex reformation (Clemmons et al., 1983; Baxter, 1990). Thus, the aim of this study was to determine if, in a clinical setting where blood may be collected in both nonheparinized and heparinized tubes, heparin alters the molecular distribution or immunoreactive measurement of IGFBP-3 and IGF-I. Two different collection modalities were examined: protocol 1, blood was drawn and immediately centrifuged and aliquotted; and protocol 2, blood was drawn, left at room temperature for 2 h and then at 4°C overnight prior to centrifugation. Samples were drawn from a normal adult and from a growth hormone-deficient (GHD) child and subjected to neutral size-exclusion chromatography to separate the ternary 158-kDa complex from the binary IGFBP-3-IGF (approx 50 kDa) complex. Fractions were then subjected to Western ligand blot (WLB), western immunoblot (WIB), and measurement of IGFBP-3 by immunoradiometric assay (IRMA), while the IGF distribution was measured by radioimmunoassay (RIA) following acidic size-exclusion chromatography. In both serum and plasma of a normal adult, WLB detected a 45–40-kDa IGFBP-3 doublet eluting primarily within the 158-kDa IGFBP region (i.e., ternary complex). Similarly, assessment of immunoreactive IGFBP-3 by WIB showed a 45–40-kDa IGFBP-3 doublet, as well as a 29 kDa immunoreactive form primarily eluting in the 158-kDa IGFBP region of the chromatography. Measurement of IGFBP-3 by IRMA confirmed these findings. No difference between serum and plasma was detected in either collection protocol. RIA of IGF-I revealed that the ternary complex carried the majority of the circulating IGF-I and that there was no difference between serum and plasma. Assessment of serum and plasma of a GHD child showed reduced serum concentrations of IGFBP-3 but no difference in the IGFBP profiles between serum and plasma. These data demonstrate that the collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF-I.

Upregulation of GH receptor and GH binding protein during pregnancy in the GH deficient rat.

During pregnancy there are dramatic changes in the endocrine and metabolic status of the mother: growth hormone (GH) is an important regulator of growth and development. A proportion of GH is bound by specific GH binding proteins (GHBP) that closely resemble the GH receptor (GHR). In the rodent both GHBP and the GHR are considered to be GH dependent, and consequently during pregnancy the increase in serum GH is associated with an increase in GHBP. To examine whether an increase in maternal GH is obligatory for elevation of maternal GHBP or GHR during pregnancy, we used a unique GH-deficient (GHD) strain of rats, to avoid the methodological complications of hypophysectomy and assessed serum GH, GHBP and hepatic GHR binding during the course of pregnancy. In GH normal rats, serum GH concentrations increased twofold and GHBP levels increased threefold; there was no change in hepatic GHR binding. In GHD rats, serum GH concentrations were low and did not increase during pregnancy. Nonetheless, levels of both serum GHBP and hepatic GHR binding increased to that measured in normal rats. Thus, an increase in maternal GH concentration is not required for the gestational upregulation of maternal GHBP or hepatic GHR binding, suggesting that other hormones may be essential in modulating the GH axis during pregnancy.