Ethanol-induced inhibition of testosterone biosynthesis in rat leydig cells: Central role of mitochondrial NADH redox state

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonado-tropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by l-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD⁺ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD⁺ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD⁺ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD⁺ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.