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

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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 ug/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 ug/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.

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