Synthesis of the acid-labile subunit of the growth-hormone-dependent insulin-like-growth-factor-binding protein complex by rat hepatocytes in culture

Abstract

Insulin-like growth factors (IGFs) circulate predominantly in a growth-hormone-dependent ternary complex of 125-150 kDa. This study investigates the production of the alpha-subunit of this complex, an acid-labile glycoprotein without intrinsic IGF-binding activity, which binds to the IGF-binding protein IGFBP-3 in the presence of IGFs. Medium conditioned by primary cultures of rat hepatocytes produced alpha-subunit with similar complex-forming activity to purified rat serum alpha-subunit. Bovine growth hormone stimulated hepatocyte production of both IGF-I and alpha-subunit. IGF-I tracer bound to pure rat IGFBP-3 was converted from approx. 60 kDa to 150 kDa by serum alpha-subunit, whole rat serum or rat hepatocyte culture medium; this converting activity was destroyed by transient acidification. In contrast, IGF-I bound to hepatocyte-medium IGF-binding proteins could not be converted into a high-molecular-mass from by purified rat serum alpha-subunit. Rat serum and hepatocyte-medium alpha-subunit appeared identical by electrophoretic analysis, since reaction of either with cross-linked IGF-I.IGFBP-3 tracer resulted in bands of molecular mass 130 kDa and 160 kDa, probably representing intact and partially deglycosylated complexes. However, IGF-binding proteins in rat serum and hepatocyte medium were different, in that affinity labelling of medium binding proteins, depleted of endogenous IGFs, showed no evidence of the 50-60 kDa cluster of bands characteristic of rat serum IGFBP-3. We conclude that rat hepatocytes in primary culture produce alpha-subunit similar to that in rat serum; however, alpha-subunit is unable to form ternary complexes with hepatocyte IGF-binding proteins, since cultured hepatocytes do not secrete IGFBP-3.