Evaluation of the developmental and nutritional changes in porcine insulin-like growth factor-binding protein-1 and -2 serum levels by immunoassay.
Porcine serum contains five insulin-like growth factor-binding proteins (IGFBPs), whose regulation has been studied by ligand blotting. To more accurately quantify changes in two specific forms of IGFBP a heterologous RIA for porcine (p) IGFBP-2 was developed, and IGFBP-1 levels were analyzed by immunoblotting. By RIA, postnatal hypophysectomy caused a 7-fold increase in serum pIGFBP-2 levels compared to controls (2,622 +/- 378 vs. 382 +/- 10 ng/ml, respectively). Fetal pIGFBP-2 levels were higher at 110 vs. 45 days gestation (1,074 +/- 214 vs. 418 +/- 30 ng/ml, respectively), rose to 1,905 +/- 167 ng/ml within 12 h after birth, then decreased to 1,010 +/- 10 ng/ml at 48 h. By immunoblot analysis, bovine IGFBP-2 antiserum reacted with a 34,000 mol wt (Mr) IGFBP and did not react with other forms of IGFBP detected by ligand blotting. Serum levels of the 34,000 Mr IGFBP, as detected by ligand blot analysis, are decreased when neonatal pigs are fasted for 48 h. In contrast, by RIA, pIGFBP-2 concentrations increased 4-fold. Immunoblots of these sera showed two lower Mr (22,000 and 14,000 Mr) bands that did not bind either [125I]IGF-I or [125I]IGF-II and were distinct from a smaller (20,000 Mr) IGFBP which bound only [125I]IGF-II. These two bands were increased in serum of 48-h fasted compared to fed piglets, suggesting that they are proteolytic fragments of pIGFBP-2. In vitro incubation of 48-h fasted pig serum with intact IGFBP-2 failed to reveal proteolytic fragments, indicating that the IGFBP-2 fragments were not generated by a protease that was released into the serum. Analysis performed with human IGFBP-1 antiserum revealed a 29,000 Mr immunoreactive band whose abundance was increased by either postnatal hypophysectomy or fasting. No fragments of IGFBP-1 were found in any serum tested. We conclude that heterologous antibodies can be used to identify and quantify IGFBP-1 and IGFBP-2 in porcine serum. Changes in pIGFBP-2 levels measured during fasting are due to the combination of changes in intact 34,000 Mr IGFBP-2 and smaller non-IGF-binding fragments. Changes in levels of specific forms of IGFBP as well as the presence of fragments have the potential to modulate the transport of IGF-I and -II out of the vasculature.
McCusker
R H
RH
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Cohick
W S
WS
Busby
W H
WH
Clemmons
D R
DR
D000824:Animal Nutritional Physiological Phenomena; D000818:Animals; D000831:Animals, Newborn / Q000097:blood*; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry; D005314:Embryonic and Fetal Development; D010450:Endopeptidases / Q000097:blood; D005312:Fetal Blood; D007118:Immunoassay; D015151:Immunoblotting; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008024:Ligands; D008970:Molecular Weight; D010902:Pituitary Gland / Q000502:physiology; D011863:Radioimmunoassay; D013552:Swine / Q000097:blood*
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D008024:Ligands; D007334:Insulin-Like Growth Factor I; D010450:Endopeptidases