Insulin-like growth factor-binding proteins in tissue fluids from the lamb

1991 ◽  
Vol 129 (1) ◽  
pp. 59-68 ◽  
Author(s):  
A. P. D. Lord ◽  
A. A. Martin ◽  
P. E. Walton ◽  
F. J. Ballard ◽  
L. C. Read
Keyword(s):  
Growth Factor ◽  
Binding Proteins ◽  
Specific Binding ◽  
Binding Protein ◽  
Extracellular Fluid ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Igf I ◽  
Acid Labile Subunit

ABSTRACT Heparinized samples of plasma, cerebrospinal fluid (CSF) and lymph from intestinal, prescapular and popliteal lymph nodes were collected from young lambs in order to characterize and compare the insulin-like growth factor-binding proteins (IGFBPs) in extracellular fluids with those from the circulation. Prior to collection and analysis, the superiority of heparin for plasma preparation was established over EDTA and citrate or the use of serum, according to the extent of IGF-I and IGF-II binding achieved in the high molecular mass IGFBP region in vitro. The IGFBPs were characterized by ligand blotting and competitive binding techniques using radiolabelled IGF-I, IGF-II and the truncated IGF analogue, des(1–3)IGF-I, as well as by ligand blotting of fractions after Superose 6 chromatography of incubations of fluids with labelled factors. This combined analysis demonstrated an IGF-II-specific binding protein at approximately 250 kDa that was present in plasma and each lymph type and presumably represented the soluble type-2 IGF receptor; a complex of 130 kDa containing 52 kDa and 46 kDa binding proteins that was labelled by all three IGF peptides was particularly evident in plasma and intestinal lymph and was probably a complex between IGFBP-3 and the acid-labile subunit; and a group of binding proteins that chromatographed as IGF complexes at approximately 50 kDa. This last group contained IGFBP bands of 52, 46, 35, 28 and 23·5 kDa in plasma and all lymphs as well as an IGF-II-specific band of 22 kDa in prescapular and popliteal lymphs. CSF differed qualitatively from plasma and lymph in that the 52/46 kDa IGFBP bands were undetectable in this fluid; the 35 kDa band was the predominant binding protein, and neither this nor the 28, 23·5 and 22 kDa proteins bound des(1–3)IGF-I to any significant extent. The 52,46 and 28 kDa bands in plasma and lymph did bind this ligand. Immunoblots using antisera against bovine IGFBP-2 showed binding at 35 kDa in all fluids as well as several bands at lower molecular masses. Taken together these results show not only marked differences in the binding protein profiles of sheep plasma, lymph and CSF, but both qualitative and quantitative differences between intestinal, prescapular and popliteal lymphs. We speculate that the differences between lymphs may result from tissuespecific release of binding proteins into extracellular fluid. Journal of Endocrinology (1991) 129, 59–68

Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells

1990 ◽  
Vol 125 (3) ◽  
pp. 439-448 ◽  
Author(s):  
J.-F. Wang ◽  
G. P. Becks ◽  
K. D. Buckingham ◽  
D. J. Hill
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Concanavalin A ◽  
Binding Proteins ◽  
Binding Capacity ◽  
Binding Protein ◽  
Hormonal Control ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Igf I

ABSTRACT We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (OH medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned OH medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (OH) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of OH medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between OH, 5H and 6H media suggest acute hormonal control of release. Journal of Endocrinology (1990) 125, 439–448

Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats

1996 ◽  
Vol 150 (1) ◽  
pp. 121-127 ◽  
Author(s):  
C G Prosser ◽  
J Schwander
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Plasma Clearance ◽  
Binding Protein ◽  
Plasma Concentrations ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Lactating Goats ◽  
Igf I ◽  
Insulin Like Growth Factors

Abstract Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n=4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0·37 ± 0·06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0·11 ± and 0·12 ± 0·01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 ± 6 min) and -II (254 ± 8 min) compared with IGFBP-2 (110 ± 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1·54 ±0·04, 3·3 ±0·6 and 4·1 ±0·4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 ± 8 and 198 ±7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2. Journal of Endocrinology (1996) 150, 121–127

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