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.

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

“…5, inhibition of electron transport and ATP synthesis does not allow cAMP to increase StAR protein mitochondrial levels (Fig. 5, lanes 3 and 4 vs. lane 2), in accordance with Allen et al [13]. Exogenous AA was able to overcome the inhibition produced by antimycin A and oligomycin on cAMP-induced StAR protein levels ( Fig.…”

“…Then, the interpretation of the results presented in the present paper is on the basis that the inhibitors have equal effects on both rat interstitial testicular cells and MA-10 Leydig cells. This is also supported by several previous studies performed using similar compounds in adrenal cells in vivo and in MA-10 Leydig cells, an accepted model of analysis of the regulation of Leydig cell steroidogenesis [10][11][12][13]. These results suggest that the inhibition of steroidogenesis by antimycin A and oligomycin is due to their indirect action on AA export and that ATP synthesis is necessary in an event located upstream of AA release/metabolism, while DW m is necessary in an event located downstream of AA release/metabolism.…”

“…It has been suggested that mitochondrial production of ATP is necessary for StAR translation [13]. The results presented here demonstrate that mitochondrial ATP is necessary for AA export as it was demonstrated for rat heart mitochondria [9], since AA can restore StAR induction and steroidogenesis in the presence of an ATP synthase inhibitor.…”

“…Due to their mechanism of action (inhibition of the respiratory chain, ATP synthesis or electrochemical gradient) no evidence exist showing that they exert a different effect in tumour as compared to normal cells. The reported concentrations used in tumour and normal cells are similar and correspond to the concentration used in our study [11][12][13][16][17][18].…”

“…Several studies have focused on the importance of the mitochondrial function in the regulation of steroid synthesis [10][11][12][13]. The latest report [13] indicated that antimycin A (inhibitor of electron transport) or oligomycin (inhibitor of ATP synthesis) significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels.…”