Ligand-Induced Activation of Translocator Protein (TSPO) Induces Cholesterol Transport and Steroidogenesis in Aged Leydig Cells.

Previous studies have shown that the capacity of Leydig cells from aged testes to produce testosterone is significantly reduced compared to cells from young testes. Cholesterol transport to the inner mitochondrial membrane is the rate-determining step in Leydig cell testosterone production. Two proteins, steroidogenic acute regulatory protein and translocator protein (TSPO; aka peripheral benzodiazepine receptor), have been shown to be critically involved in cholesterol transport. We previously showed that reduced expression of TSPO in aged Leydig cells correlated with the reduced ability of the cells to form testosterone. In the present study, we examined the effect of the TSPO selective drug ligands indoleacetamide FGIN-1-27 and benzodiazepine Ro5-4864 on cholesterol transport and steroidogenesis in young and aged Leydig cells. Previous studies showed that FGIN-1-27 binds exclusively to the 18 kDa TSPO protein whereas other mitochondrial proteins are needed for maximal binding of Ro5-4864 to 18 kDa TSPO. Incubation of MA-10 mouse tumor Leydig cells with Ro5-4864 resulted in significantly increased progesterone production, whereas incubation with FGIN-1-27 had a lesser effect. Ro5-4864 had limited effect on testosterone production by Leydig cells isolated either from young (6-month-old) or old (21-month-old) Brown Norway rats. In contrast, incubation of young or old Leydig cells with FGIN-1-27 resulted in significant increases in testosterone production in both cases. Indeed, the old Leydig cells stimulated with FGIN-1-27 produced testosterone at the same level as LH (0.1 ng/ml)-incubated old cells, in both cases significantly higher than controls. Incubation of the cells with a NBD fluorescent cholesterol derivative revealed decreased cholesterol transport to the mitochondria of old compared to young Leydig cells in response to LH, but increased cholesterol transport in both young and old cells incubated with FGIN-1-27. These results point to a potential defect in cholesterol transport to the mitochondria in aged Leydig cells that can be overcome by activating TSPO-mediated cholesterol transport using high affinity TSPO drug ligands, and that doing so increases testosterone production even without LH stimulation and presumably without altering the downstream steroidogenic enzymes.

(poster)