Essential role of adenosine triphosphate in activation of 17beta-hydroxysteroid dehydrogenase in the rat Leydig cell.

The forskolin-induced steroidogenic block of testosterone production residing beyond pregnenolone synthesis in rat Leydig cells was localized to the level of the 17beta-hydroxysteroid dehydrogenase (17betaHSD) reaction in this study. The use of forskolin analogs that discriminate between the diterpene's inhibitory effect on the glucose transporter(s) (1,9-dideoxyforskolin) and its activation of adenylate cyclase (6-aminoethyl carbamyl forskolin) revealed that the block is related to inhibition of glucose transporter(s). 1,9-Dideoxyforskolin, but not 6-aminoethyl carbamyl forskolin, caused a significant inhibition of basal and hCG-stimulated testosterone production with accumulation of androstenedione. Glucose-deficient media produced the same metabolic block in the absence of forskolin, with a significant reduction in 17betaHSD activity and increases in the apparent Km for androstenedione. In contrast, metabolic steps before testosterone formation were not affected. Glucose-induced 17betaHSD activation was mimicked by the addition of ATP or GTP in glucose-deficient media, but not by nonhydrolyzable triphosphate analogs or NADPH. A decrease in 17betaHSD activity caused by KT-5720, a specific inhibitor of protein kinase A and the calmodulin antagonist W-7, indicates that the ATP requirement may be related to the participation of protein kinases in the activation of 17betaHSD. ATP levels derived from alternative (nonglycolytic) pathways are adequate to support basal and hormone-stimulated enzymatic activities in the metabolism of cholesterol to androstenedione. However, the integrity of the glucose transport system with subsequent ATP generation is required for activation of 17betaHSD in the final step of androgen biosynthesis. In conclusion, the conversion of androstenedione to testosterone requires the contribution of the glycolytic pathway to meet ATP requirements for 17betaHSD activity.