Energized, Polarized, and Actively Respiring Mitochondria Are Required for Acute Leydig Cell Steroidogenesis

John A. Allen, Tristan Shankara, Paul Janus, Steve Buck, Thorsten Diemer, Karen Held Hales, Dale B. Hales
2006 Endocrinology  
The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute
more » ... MP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3,5cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (⌬ m ), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and ⌬ m . Dissipating ⌬ m with carbonyl cyanide mchlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly syn-thesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on ⌬ m . Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on ⌬ m . 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3␤-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that ⌬ m , mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid biosynthesis. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis, and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis. (Endocrinology 147: 3924 -3935, 2006) First Published Online May 11, 2006 Abbreviations: BCA, Bicinchoninic acid; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; ⌬⌿ m , mitochondrial membrane potential; ⌬pH, mitochondrial pH gradient; P450scc, P450 side-chain cleavage enzyme; R22, 22(R)-hydroxycholesterol; SDS, sodium dodecyl sulfate; SFM, serum-free medium; StAR, steroidogenic acute regulatory protein; TCA, trichloracetic acid; TMRE, tetramethylrhodamine ethyl ester dye. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
doi:10.1210/en.2005-1204 pmid:16690799 fatcat:4tkjjuimbrhlhimu6h2ku26m4a