Hornsby P J, Gill G N
J Cell Physiol. 1981 Oct;109(1):111-20. doi: 10.1002/jcp.1041090113.
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.
在培养的牛肾上腺皮质细胞中研究了[U-14C]谷氨酰胺和[2-14C]丙酮酸C2产生二氧化碳的调节情况，并检测了这些底物氧化相对速率的改变对细胞增殖的影响，特别是在存在转氨反应抑制剂的情况下。在存在100微摩尔2,4-二硝基苯酚（一种氧化磷酸化解偶联剂）的情况下，测定了2毫摩尔[U-14C]谷氨酰胺和2毫摩尔[2-14C]丙酮酸产生14CO2的情况。用50微摩尔皮质醇处理原代培养物24小时，相对于[14C]丙酮酸，增加了[14C]谷氨酰胺的氧化，这种效应取决于先前的低细胞密度。皮质醇处理还导致低密度下增殖开始的延迟延长，并且在存在2毫摩尔氨基氧乙酸（其降低谷氨酰胺的线粒体利用率）的情况下完全抑制生长。同时用抗氧化剂二甲基亚砜（10毫摩尔）和丁基化羟基茴香醚（100微摩尔）处理可防止对谷氨酰胺和丙酮酸代谢以及对细胞生长的影响（无论有无氨基氧乙酸），这表明脂质过氧化参与了皮质醇的作用，正如先前证明其对11β-羟化酶的作用一样。在没有皮质醇的情况下，肾上腺皮质细胞持续增殖期间，相对于[14C]丙酮酸，[14C]谷氨酰胺氧化的增加也随着群体倍增水平而缓慢增加。在2%氧气而非标准的19%氧气中培养细胞会减缓这种增加的速率，而在存在皮质醇的情况下细胞持续生长会加速这种速率。在这三种条件下，相对于[14C]丙酮酸，[14C]谷氨酰胺氧化增加的速率与氨基氧乙酸对细胞生长的抑制相关。与皮质醇处理的细胞在氨基氧乙酸中完全抑制生长相反，对照细胞（19%氧气）确实增殖，尽管生长受限，而在2%氧气中的细胞增殖程度更大。在没有氨基氧乙酸的情况下，这三种条件下原代肾上腺皮质细胞培养物中的生长速率相似。脂质过氧化似乎通过抑制正常底物丙酮酸的利用，使培养的肾上腺皮质细胞依赖谷氨酰胺来维持线粒体功能和增殖。
