Dual effects of 2-deoxyglucose on synthesis of the glycoprotein hormone common alpha-subunit in butyrate-treated HeLa cells.

Sodium butyrate (Btr) (3 mM) causes a 10-fold increase in production of the glycoprotein hormone alpha-subunit in HeLa cells. The following report demonstrates that this response could be inhibited about 95% by 5 mM 2-deoxy-D-glucose (dGlc), whereas alpha-subunit production in uninduced cells was affected little or not at all. Addition of D-mannose restored the Btr induction of Hela-alpha in cultures that had been treated with dGlc. When the alpha-subunits secreted by cells cultured in Btr plus dGlc or in Btr alone were compared by gel filtration (Sephadex G-75) and lectin affinity (concanavalin A and ricin) chromatography, differences were noted that probably reflect changes in their carbohydrate moieties. Immunoprecipitation of [35S]methionine-labeled HeLa-alpha and incubation with endoglycosidase H indicated that the subunit secreted from cells in the presence of dGlc contained oligosaccharide side chains that were not processed to the complex type. Cells that were simultaneously treated with Btr plus dGlc showed no increase in alpha-subunit production over cells receiving Btr only; in contrast, cells that were preincubated with Btr for either 16 or 36 h before dGlc was added exhibited high levels of subunit synthesis. Measurement of alpha-mRNA levels at various times after Btr and dGlc were added to cultures indicated that Btr brought about a dramatic increase in alpha-specific mRNA about 24 h after being added to cultures. This increase could be prevented by dGlc when added simultaneously with Btr but not when added after a 24-h preincubation. Although dGlc prevented the induction of alpha-subunit and alpha-mRNA in response to Btr, it had no effect on histone hyperacetylation, suggesting that if this chromatin modification is necessary for the induction process, it is not in itself sufficient. Together, the data demonstrate that dGlc inhibits the accumulation of alpha-subunit mRNA normally produced in response to Btr and that the subunit produced contains altered oligosaccharide constituents.

Glucose requirement for induction by sodium butyrate of the glycoprotein hormone alpha subunit in HeLa cells.

Butyric acid produces multiple effects on mammalian cells in culture, including alterations in morphology, depression of growth rate, increased histone acetylation, and modified production of various proteins and enzymes. The latter effect is exemplified by the induction in HeLa cells of the glycoprotein hormone alpha subunit by millimolar concentrations of the fatty acid. This report demonstrates that increased subunit accumulation in response to sodium butyrate is strikingly dependent on the presence of glucose (or mannose) in the growth medium. In contrast, basal levels of subunit synthesis are only marginally affected when the culture medium is supplemented with one of a variety of hexoses. An increase in the accumulation of HeLa alpha does not occur in medium containing pyruvate as the energy source, and sustained induction requires the simultaneous and continued presence of both glucose and butyrate. The effects of butyrate on HeLa cell morphology and subunit induction can be separated, since the latter is glucose-dependent while the former is not. Failure of butyrate to induce alpha in medium containing pyruvate does not result from restricted subunit secretion, since the levels of intracellular alpha are not increased disproportionately relative to those in the medium. The hexoses which support induction of HeLa alpha (glucose greater than or equal to mannose greater than galactose greater than fructose) are identical to those which have been shown previously to stimulate the glucosylation of lipid-linked oligosaccharides and enhance the synthesis of certain glycoproteins. Labeling of various glycosylation intermediates with [3H]mannose indicates that in glucose medium there is a decrease in the level of radioactivity associated with both dolicholpyrophosphoryl oligosaccharide and cellular glycoproteins and a concomitant increase in the fraction of label recovered in secreted glycoproteins. Butyrate also causes a decrease in [3H]mannose-labeled cellular glycoproteins and an increase in tritiated extracellular glycoproteins, particularly in glucose medium. Likewise, glucose stimulates the incorporation of [3H]glucosamine into immunoprecipitable alpha subunit relative to the bulk of HeLa-secreted glycoproteins, and this is further enhanced by butyrate. However, as demonstrated by lectin chromatography of conditioned media, a nonglycosylated subunit does not accumulate in pyruvate medium, either in the absence or presence of butyrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of media composition on the induction of chorionic gonadotropin by sodium butyrate in HeLa cells.

Production of the glycoprotein hormone alpha-subunit by HeLa cells and its induction by sodium butyrate are dependent on the choice of culture medium. Under identical growth conditions it was found that subunit synthesis in the presence of butyrate was highest in RPMI 1640, lowest in Medium 199 (M199), and intermediate in minimum essential medium (MEM) and Waymouth's MB 752/1. Cell growth was similar in all media examined and was retarded in the presence of butyrate. Alkaline phosphatase activity was also lower in M199 than in RPMI 1640, although, in general, the magnitude of this difference was less than that for the hormone subunit. Incorporation of [1-14C]butyrate by HeLa cells was similar in both M199 and RPMI 1640, indicating that uptake and metabolism of the fatty acid were not significantly different under these conditions. In the presence of 3 mM butyrate, mixtures of RPMI 1640 and M199 gave intermediate levels of alpha-subunit and alkaline phosphatase compared to each medium alone. Intracellular levels of alpha-subunit as well as that of the culture medium were reduced in M199 compared to RPMI 1640 indicating that synthesis rather than secretion was altered.