Intramammary infusion of Panax ginseng extract in the bovine mammary gland at cessation of milking modifies components of the insulin-like growth factor system during involution.

The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng extract (GS) on insulin-like growth factors (IGF) in bovine mammary gland during early involution. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of ginseng extract solution (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Concentrations of IGF1 in mammary secretions were higher in GS-treated quarters than in placebo and uninoculated control quarters at 24, 48 and 72 h post-treatment (p<0.05). Treatment with GS did not affect mammary secretion of IGF2 (p=0.942). At 7 d of post-lactational involution, a decrease of immunostained area and mRNA expression for IGF1 was observed in mammary tissue of GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). The IGF2 immunostained area and mRNA expression for this growth factor were not affected by GS treatment (p=0.216 and p=0.785, respectively). An increase in protein levels and mRNA expression in mammary tissue of IGFBP3, IGFBP4 and IGFBP5 was observed in GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). These results provide evidence that intramammary inoculation of GS extract at cessation of milking may promote early mammary involution through the inhibition of IGF1 local production and bioavailability.

Development of a scintillation proximity assay for human insulin-like growth factor-binding protein 4 compatible with inhibitor high-throughput screening.

The insulin-like growth factor-binding protein 4 (IGFBP-4), which exists in many different tissues and biological fluids, modulates insulin-like growth factor 1 (IGF-1) bioavailability in part by competitive sequestration and prevention of interaction with cell membrane IGF-1 receptors. Accordingly, small molecules that inhibit the ability of IGF-1 to associate with IGFBP-4 may have clinical utility as regulators of cellular proliferation, survival, and differentiation. Currently, a polyethylene glycol-based precipitation of [(125)I]IGF-1 bound to IGFBP-4 is used to quantify selective IGFBP-4 ligand interactions. We have developed a novel 96-well plate scintillation proximity assay (SPA) for measuring small molecule interactions at IGFBP-4 using a biotinylated form of IGFBP-4 coupled to streptavidin-coated polyvinyltoluene (PVT) SPA microbeads and using [(125)I]IGF-1 as the endogenous ligand. Dose-displacement curves with unlabeled IGF-1 exhibited a mean K(d) value of 0.46 nM. Parallel studies using the nonselective IGFBP inhibitor, NBI-31772, generated a K(i) value of 47 nM. Under optimized conditions, the IGFBP-4 SPA was stable for up to 24h at room temperature and was unaffected by dimethyl sulfoxide (DMSO,<0.5%). This homogeneous binding assay is simple, stable, sensitive, and amenable to automation. The good signal/noise ratio (10:1) and Z' factor (0.7-0.8) make it compatible with high-throughput screening platforms for the identification of IGFBP-4 inhibitors. The IGFBP-4 binding assay may be expanded to other IGFBP members, in biotinylated form, to provide a powerful tool amenable to drug screening and the design of therapeutics to treat a variety of IGF-responsive diseases.

Insulin-like growth factor-I and its binding proteins in colostrum compared to measures in serum of Holstein neonates.

Colostral insulin-like growth factor-I (IGF-I) may be beneficial in the development of gastrointestinal tracts of bovine neonates. Thus, the purpose of this study was to examine relationships among concentrations of IGF-I and IGF-binding proteins (IGFBP) in colostrum used at two initial feedings and serum concentrations of IGF-I, IGFBP, total protein, gamma glutamyltransferase (GGT), and immunoglobulin G at 0 and 48 h after birth in Holstein neonates. Calves (n = 22) were separated from dams immediately after birth. Blood samples were taken before initial feeding and at 48 h after birth. Calves were fed 2 L of colostrum twice and milk replacer thereafter. Linear regression of serum IGF-I at 48 h and colostral IGF-I revealed a significant positive relationship (R2 = 0.204). Serum IGFBP-3 at 48 h and colostral IGFBP-3 also had a positive relationship (R2 = 0.143). However, linear regression of colostral IGF-I on the difference in serum IGF-I at 48 and 0 h was not significant. Calves were assigned to group 1 (0-h serum IGF-I < 10 ng/ml; n = 11) or group 2 (0-h serum IGF-I > or = 10 ng/ml; n = 11) for further analysis. There were no differences in serum IGF-I or IGFBP-2, -3, -4, and -5 concentrations at 48 h between groups 1 and 2. Correlation coefficients revealed negative relationships of serum IGF-I at 0 h to the difference between serum IGF-I at 48 and 0 h (r = -0.824), as well as birth weight of the calf to the amount of GGT at 48 h (r = -0.604). Females had lower birth weights than males, but sex of calf did not affect serum measures. At 0 h, but not 48 h, total serum protein was correlated to serum GGT concentrations (r = 0.573). From indirect evidence, absorption of colostral IGF-I and IGFBP-3 into systemic circulation may occur, but relative importance compared to endogenous sources is uncertain.

Osteogenic protein-1 and insulin-like growth factor I synergistically stimulate rat osteoblastic cell differentiation and proliferation.

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) alters the steady state levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) in primary cultures of fetal rat calvaria (FRC) cells. In the present study, the effects of exogenous IGF-I on bone cell differentiation and mineralized bone nodule formation induced by OP-1 were examined. Exogenous IGF-I synergistically and dose dependently enhanced OP-1 action in stimulating [3H]thymidine incorporation, alkaline phosphatase activity, PTH-dependent cAMP level, and bone nodule formation. Maximal synergism between OP-1 and IGF-I was observed when both factors were added simultaneously. Synergism was not observed when FRC cells were pretreated with IGF-I for 24 h, followed by OP-1 treatment. These findings suggest that IGF-I acted on OP-1-sensitized FRC cells. To examine the mechanism(s) by which this sensitization may occur, levels of mRNA encoding OP-1 receptor, IGF-I receptor, and IGFBPs were measured. The mRNA levels of both type I and II OP-1 receptors were elevated by OP-1, but were not changed further by combined OP-1 and IGF-I treatment. IGF-I receptor gene expression was not changed by OP-1, IGF-I, or a combination of both factors. OP-1 alone or together with IGF-I increased the steady state IGFBP-3 mRNA level and reduced the steady state mRNA levels of IGFBP-4, -5, and -6. IGF-I alone did not change the steady state mRNA levels of IGFBP-3, -4, and -6, but elevated that of IGFBP-5. Des(1-3)-IGF-I, which has a lower affinity for IGFBPs, was more effective than the full-length IGF-I in enhancing the OP-1-induced alkaline phosphatase activity. Exogenous IGFBP-5 inhibited the OP-1-induced alkaline phosphatase activity and reduced the synergistic stimulatory effect of IGF-I and OP-1. These findings strongly suggest that the OP-1-induced down-regulation of IGFBPs, especially that of IGFBP-5, is an important mechanism by which OP-1 and IGF-I synergize to stimulate FRC cell differentiation.