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, 154 (6), 2156-65

Drug Ligand-Induced Activation of Translocator Protein (TSPO) Stimulates Steroid Production by Aged Brown Norway Rat Leydig Cells

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Drug Ligand-Induced Activation of Translocator Protein (TSPO) Stimulates Steroid Production by Aged Brown Norway Rat Leydig Cells

J Y Chung et al. Endocrinology.

Abstract

Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4'-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

Figures

Figure 1.
Figure 1.
Effect of TSPO ligands on Leydig cell viability and steroid production. A and B, 3β-HSD staining of Leydig cells isolated from the testes of young adult (A; 3 months old) and aged (B; 21 months old) Brown Norway rats. The isolated cells consistently were greater than 90% pure. C and D, Viability of Leydig cells isolated from young and aged Brown Norway rats in response to increasing concentrations of Ro5-4864 (A) or FGIN-1-27 (B), determined by the Methyl-thiazolyldiphenyl-tetrazolium assay. E and F, T production by Leydig cells freshly isolated from the testes of young and aged rats and cultured with increasing concentrations of Ro5-4864 (E) or FGIN-1-27 (F) for 2 hours. G, Comparison of the effect of FGIN-1-27 alone and in combination with LH (0.1 ng/mL) on T production by young Leydig cells. In each case, at least 3 independent experiments were performed. Data shown are the mean ± SEM. *P < .05, ***P < .001 compared with controls.
Figure 2.
Figure 2.
Comparison of the effects of LH and TSPO ligands on T production by Leydig cells from young adult and aged rats. A–C, T production by young (closed circles) and aged (open circles) Leydig cells incubated with LH (A), Ro5-4864 (B), or FGIN-1-22 (C) for 0-120 minutes. D, Comparison of T production by young vs aged cells in response to LH, Ro5-4864, and FGIN-1-27. In each case, at least 3 independent experiments were performed (mean ± SEM). ***P < .001 compared with value of young rats.
Figure 3.
Figure 3.
Effects of TSPO-specific inhibitor 19-Atriol on steroid production by primary and MA-10 tumor Leydig cells in response to LH and to TSPO ligands. A, Effect of 19-Atriol on T production by young Leydig cells in response to LH, Ro5-4864, and FGIN-1-27 (mean ± SEM). Cont, control. *P < .05, **P < .01, ***P < .001 compared with control (without 19-Atriol). B, 19-Atriol effects on progesterone production by MA-10 cells treated with LH (2 hours) (mean ± SEM). ***P < .001 compared with control (without 19-Atriol). C, 19-Atriol effects on progesterone production by MA-10 cells cultured with Ro5-4864 or FGIN-1-27 (mean ± SEM). ***P < .001 compared with control. D, Effect of providing MA-10 cells with 22R-HC (5 μM) on progesterone production in response to TSPO ligand in the presence or absence of 19-Atriol.
Figure 4.
Figure 4.
In vivo effects of FGIN-1-27 on serum T levels and body weights in young and old rats. A, Young and aged rats received FGIN-1-27 [low (L): 0.1 mg/kg body weight; high (H): 1 mg/kg body weight] by ip injection for 10 days. Then serum T was measured by RIA. Data shown are the mean ± SEM. C, control. ***P < .001 compared with controls (vehicle treated). B, Body weights before and after FGIN-1-27 injections for 10 days.

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