Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the activity of IGF-I. It exists in serum as phosphorylated and less phosphorylated forms. We wished to measure serum levels of both these forms of IGFBP-1, using a novel assay, in subjects with, or without ischaemic heart disease (IHD).
We measured serum concentrations of the phosphorylated and less phosphorylated forms of IGFBP-1 in 75 subjects (36 with and 39 without IHD). Two immunoassays were used, one which detects non-, and less-phosphorylated forms (LpIGFBP-1), and another which specifically detects the serine phosphorylated form of IGFBP-1 (pIGFBP-1).
LpIGFBP-1 concentrations were significantly higher in subjects without IHD than in those with IHD (5.3+/-0.5 microg/L vs. 2.7+/-0.4 microg/L, p<0.001). pIGFBP-1 levels were also significantly higher in subjects without IHD compared to those with IHD (33.3+/-2.0 microg/L vs. 25.3+/-2.2 microg/L, p<0.01). The correlation between LpIGFBP-1 and pIGFBP-1 for all subjects was (r=0.71, p<0.001). This association was stronger in subjects without IHD (r=0.76, p<0.001) than for those with IHD (r=0.60, p<0.001). A significant negative association was observed between IGF-I and the ratio between the two forms (r=-0.45, p<0.0001). Receiver-Operating Characteristic (ROC) curve showed the highest area under the curve for LpIGFBP-1 (0.75) [95% CI: 0.63-0.86] and optimum cut-off value of 2.83 microg/L with 75% sensitivity and 74% specificity.
We propose that low serum concentrations of IGFBP-1 forms could be a marker of coronary risk, and the LpIGFBP-1:pIGFBP-1 ratio may be an index of biologically active IGF-I.
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factors (IGFs) are peptide hormones that have significant structural homology with insulin. IGF binding proteins (IGFBPs), in particular IGFBP-1, are important determinants of IGF activity such as enhancing peripheral glucose uptake, decreasing hepatic glucose output and modifying lipid metabolism. Herein factors which alter IGFBP-1 and the utility of measuring IGFBP-1 are considered as the role of IGFBP-1 is explored within the context of insulin resistance and the development of cardiovascular disease.
The British Journal of Diabetes & Vascular Disease 03/2012; 12(1):17-25. DOI:10.1177/1474651412436701
[Show abstract][Hide abstract] ABSTRACT: Wepreviously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinityandinhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P<.0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IRoverexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (̃2- fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P<.05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our resultsprovidenoveldemonstrationthatIGFBP- 1phosphorylationatS119canenhanceaffinityforIGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.
[Show abstract][Hide abstract] ABSTRACT: Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at micro-scale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues (such as 2-mercaptoethanol (2ME) and 3-mercapto-1-propanol (3MP)), to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time and NH4HCO3 concentration) and derivatization efficiency parameters (e.g., reaction time and labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved effective for quantifying phosphopeptides in biological samples.
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