title: Energized, polarized, and actively respiring mitochondria are required for acute Leydig cell steroidogenesis.
abstract: 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 cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3',5'-cyclic 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 (Deltapsim), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Deltapsim. Dissipating Deltapsim with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized 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 Deltapsim. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Deltapsim. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3beta-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Deltapsim, 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.
authors.affiliation: Department of Physiology and Biophysics (MC 901), University of Illinois at Chicago, Chicago, Illinois 60612-7342, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Allen
authors.forename: Tristan
authors.initials: T
authors.lastname: Shankara
authors.forename: Paul
authors.initials: P
authors.lastname: Janus
authors.forename: Steve
authors.initials: S
authors.lastname: Buck
authors.forename: Thorsten
authors.initials: T
authors.lastname: Diemer
authors.forename: Karen Held
authors.initials: KH
authors.lastname: Hales
authors.forename: Dale B
authors.initials: DB
authors.lastname: Hales
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006888:Hydroxycholesterols / Q000494:pharmacology; D007985:Leydig Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D009942:Organometallic Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D013256:Steroids / Q000096:biosynthesis* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006888:Hydroxycholesterols; D008565:Membrane Proteins; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D009942:Organometallic Compounds; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D013256:Steroids; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; C110932:tetramethyl rhodamine ethyl ester; C003585:22-hydroxycholesterol; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D002786:Cholesterol Side-Chain Cleavage Enzyme; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D014450:Electron Transport Complex III; C020439:oligomycin sensitivity-conferring protein