Conditioned medium from Reuber H-35 or Fao
hepatoma cells contains autocrine factors that both stimulate
DNA synthesis and activate
acetyl-coenzyme A (
CoA) carboxylase in serum-deprived Fao cells. The factor(s), which appears within 4 h of serum-free culture, also increases the cell number and the mitotic index. The effects of the
conditioned medium are insulinomimetic, both with respect to stimulation of
DNA synthesis and
acetyl-CoA carboxylase activity. However, no induction of
tyrosine aminotransferase activity or stimulation of aminoisobutyric
acid uptake is seen in response to the
conditioned medium.
Insulin over a 4-h period does not increase the concentration of
DNA synthesis stimulating activity that is observed in the medium. This activity is dialyzable and is resistant to
acid treatment or to heating to 60-100 degrees C and to
trypsin digestion; it is not extracted with
chloroform/
methanol nor adsorbed by
charcoal or by a C18 reverse-phase column. Fractionation of the
conditioned medium derived from Reuber H-35
hepatoma cells by gel filtration chromatography reveals two low molecular weight (less than 1000) compounds that both stimulate
DNA synthesis in Fao
hepatoma cells. The larger compound (peak I) also stimulates the activity of
acetyl-CoA carboxylase. The stimulatory effects of the peak I compound are destroyed by
nitrous acid deamination,
periodate oxidation, and methanolysis. Biosynthetic labeling studies indicate the probable presence of
glucosamine,
galactose, and perhaps
phosphate in the peak I-activating material. No significant incorporation of either
myoinositol or
mannose into the active material has been observed. These data, taken together, are consistent with a
glycan structure for this autocrine factor, which bears strong resemblance to similar insulinomimetic factors generated in BC3H1 myocytes and H-35
hepatoma cells in response to
insulin and on digestion of membranes with a
phosphatidylinositol-specific phospholipase C. Further characterization of this factor may provide insight into different pathways of
insulin action and could provide a strategy to check autocrine-stimulated
tumor growth.