Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol, antioxidants, and oxygen: effects on cell proliferation.

The regulation of CO2 production from [U-14C]glutamine and C2 of [2-14C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. 14CO2 production from 2 mM [U-14C]glutamine and 2 mM [2-14C]pyruvate was measured in the presence of 100 microM 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures of 24 h with 50 microM cortisol increased the oxidation of [14C]glutamine relative to that of [14C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 microM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11 beta-hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O2 rather than the standard 19% O2, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol-treated cells, control cells (19% O2) did proliferate, although growth was limited, whereas cells at 2% O2 proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.