Insulin‐like growth factor binding protein secretion by muscle cells: Effect of cellular differentiation and proliferation

Abstract

Several cell types have been shown to secrete insulin-like growth factor binding proteins (IGF-BP) in vitro. Since IGF-BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF-BP and to identify factors that regulate IGF-BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H-1 myocytes were used. IGF-BP activity in serum-free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF-BP activity into the medium. Insulin was a potent stimulant of IGF-BP secretion for each cell type. Specifically, 1 microgram/ml insulin increased the IGF-BP concentration in conditioned media from 10.5 +/- 1.3 to 15.0 +/- 1.5 ng/ml in confluent L6 myotubes, from 42.5 +/- 11.1 to 90.5 +/- 9.8 ng/ml in confluent BC3H-1 cells, and from 2.1 +/- 0.1 to 3.8 +/- 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 micrograms/ml) to achieve a half-maximal stimulation of IGF-BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H-1 cells, where half-maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40-fold more sensitive to insulin stimulation of IGF-BP secretion than L6 myotubes. IGF-I, although it interferes with the assay and thereby lowers the amount of detectable IGF-BP, stimulated the secretion of IGF-BP from all three cell types. Dexamethasone, (10(-7) M) decreased IGF-BP secretion into the media by approximately 50% for all three cell types. Affinity cross-linking and ligand blotting of 125I-IGF-I to conditioned media from each cell type showed (IGF-BP)-(IGF-I) complexes with molecular weights ranging 32-40 kDa (24-32 kDa for IGF-BP and 7.5 kDa for IGF-I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H-1 myocytes. This cell proliferative response was associated with an increase in IGF-BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF-BP secretion/cell. We conclude that IGF-BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin-stimulated secretion. Insulin and IGF-I are stimulators of IGF-BP secretion, whereas dexamethasone inhibits IGF-BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF-BP may play an important regulatory role in these processes.