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

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.

Leydig cells are the T-producing cells of the mammalian testis. The acute stimulation of T production involves LH stimulation, cAMP-activated cholesterol transfer from intracellular stores into mitochondria, the conversion of cholesterol to pregnenolone by the C27 cholesterol side-chain cleavage cytochrome P450 enzyme (CYP11A1) at the inner mitochondrial membrane (IMM), and the enzymatic transformation of pregnenolone in the smooth endoplasmic reticulum (1–3). Two proteins have been identified as playing particularly important roles in cholesterol transfer to the IMM, the rate-determining step in steroid formation: steroidogenic acute regulatory protein (STAR) (4, 5) and peripheral benzodiazepine receptor (6), now renamed translocator protein (TSPO; 18 kDa) (7). STAR is a hormone-responsive 37-kDa protein, the synthesis of which parallels hormone-induced steroid formation in some steroidogenic cells (8, 9). STAR binds cholesterol (10, 11) and acts at the outer mitochondrial membrane (OMM) to initiate cholesterol transfer to the IMM (12–14). TSPO is an OMM cholesterol-binding protein that takes up free cholesterol from a cytosolic donor and, in response to ligand induction, transfers it to the IMM for cleavage to pregnenolone by CYP11A1 (15–17). Site-directed mutagenesis and in vitro reconstitution studies showed that a region of the cytosolic TSPO C terminus contains a cholesterol-recognition amino acid consensus (CRAC) domain (18, 19) in which TSPO binds cholesterol (20–22), suggesting that the TSPO C terminus plays an important role in the uptake of cholesterol from a cytosolic donor and its import into the mitochondria. Disruption of TSPO in steroidogenic cells has been shown to arrest cholesterol transport into the mitochondria, resulting in decreased steroid formation, and transfection of TSPO-disrupted cells with a Tspo cDNA has been shown to rescue steroidogenesis (17, 23). In vivo studies have demonstrated a correlation between TSPO levels and steroidogenesis (24–29).

A decline in circulating T typically accompanies aging in men and rodents (29–33). Our studies of the Brown Norway rat have shown that this decline is not a consequence of reduced Leydig cell numbers or reduced LH but rather results from the reduced ability of Leydig cells to produce T in response to LH (30, 31). In a previous study in which the effect of age on the transport of cholesterol into mitochondria was examined, we reported that the mitochondria isolated from old cells produced significantly less steroid (pregnenolone) than mitochondria isolated from young cells and that only a fraction of the decrease could be accounted for by a decrease in CYP11A1 activity (27). These results suggested that the accumulation of hormonally recruited cholesterol into mitochondria may be defective in old Leydig cells. Additionally, we and others found that the expressions of TSPO mRNA and protein were decreased in old cells (27, 29), suggesting that age-related alteration in cholesterol transport may be related to reduced TSPO.

TSPO drug ligands have been shown to stimulate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells (6, 27, 34). The results of such studies have indicated that increased steroid formation in response to TSPO drug ligands is associated with increased cholesterol import into the inner mitochondrial membrane. In vivo, high-affinity TSPO-specific drug ligands have been shown to increase glucocorticoid and neurosteroid (7, 35, 36) levels. Based on these previous studies, we hypothesized herein that although there is reduced TSPO and STAR in aged Leydig cells, the pharmacological activation of TSPO by TSPO drug ligands increase T production by these cells.

To address this hypothesis, we determined the in vitro effects of the selective, high-affinity TSPO drug ligands N,N-dihexyl-2-(4-fluorophenyl) indole-3-acetamide (FGIN-1-27) and benzodiazepine 4′-chlorodiazepam (Ro5-4864) (37, 38) on steroidogenesis by primary Leydig cells isolated from the testes of aged (21 months old) in comparison with young adult (3 months old) Brown Norway rats and the in vivo effects of administered FGIN-1-27 on serum T levels in young and old animals. We show that young and aged cells stimulated with TSPO drug ligand produced T at equivalent levels and that administering FGIN-1-27 to aged rats resulted in significantly increased serum T levels to those of young rats. These results suggest that the direct activation of TSPO might serve as a novel therapeutic approach to increase the serum steroid levels in cases of primary hypogonadism.

Materials and Methods

Reagents

Methyl-thiazolyldiphenyl-tetrazolium, Ro5-4864, FGIN-1-27, HEPES, Hanks' balanced salt solution, Percoll, 22R-hydroxycholesterol (22R-HC), and dimethylsulfoxide were obtained from Sigma-Aldrich (St Louis, Missouri). 5-Androsten-3,17,19-triol (19-Atriol) was synthesized as previously described (39). M-199 was from Gibco BRL (Grand Island, New York). BSA was from ICN Biomedicals, Inc (Aurora, Ohio). [1,2,6,7,16,17-³H(N)]-testosterone (115.3 Ci/mmol) and [1,2,6,7-3H(N)]progesterone (96.6 Ci/mmol) were from PerkinElmer Life Sciences, Inc (Boston, Massachusetts). T and progesterone antibodies were obtained from MP Biomedical (Solon, Ohio). Bovine LH (USDA-bLH-B-6) was provided by the US Department of Agriculture Animal Hormone Program (Beltsville, Maryland).

Primary Leydig cell isolation

Brown Norway rats at an age of 3 months (young) and 21 months (aged) were obtained from Harlan Sprague Dawley (Indianapolis, Indiana) through the National Institute on Aging (Bethesda, Maryland) and were housed in the animal facilities of the Johns Hopkins Bloomberg School of Public Health (Baltimore, Maryland) at 22°C, 14-hour light, 10-hour dark with free access to feed and water ad libitum. Animal handling and care were in accordance with protocols approved by the institutional Animal Care and Use Committee of the Johns Hopkins University.

Primary Leydig cells were isolated from the testes of young and aged rats following established procedures (40). In brief, rats were euthanized by decapitation, and the testes were immediately placed in cold dissociation buffer (M-199 medium with 2.2 g/L HEPES, 1.0 g/L BSA, 2.2 g/L sodium bicarbonate, and 25 mg/L trypsin inhibitor). The testicular artery was cannulated and testes were perfused with type III collagenase (1 mg/mL) in dissociation buffer to clear the testicular blood. The testes then were decapsulated and placed in the dissociation buffer containing collagenase (0.25 mg/mL at 34°C with shaking for 30 minutes). Digested testes were passed through a 100-μm nylon mesh to remove the tissue clumps. The interstitial cells were pelleted by centrifugation at 1500 rpm for 5 minutes. Leydig cells were purified by Percoll gradient separation. After centrifugation, the supernatant was discarded and cell pellets were washed with M-199 culture media. The final purity of the Leydig cells, determined by staining the cells for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, was consistently about 90%.

Effect of TSPO drug ligands on steroid production in vitro

Freshly isolated Leydig cells were suspended in M-199 culture media and plated at a density of 3-5 × 10⁵ cells/well in 12-well culture plates (Becton Dickson, Franklin Lakes, New Jersey). Cells were incubated for 2 hours at 34°C with LH (0.1 ng/mL), Ro5-4864 (1-200 μM), or FGIN-1-27 (1-200 μM). The effects of the TSPO drug ligands on steroid production by MA-10 Leydig tumor cells served as a control for the primary Leydig cells. The MA-10 cells, a gift from Dr Mario Ascoli (University of Iowa, Iowa City, Iowa), were grown in Waymouth MB 752/1 medium (20 mM HEPES and 1.2 g NaHCO₃ per liter) containing 15% horse serum. As with the primary cells, the MA-10 cells were incubated for 2 hours in the presence or absence of LH, Ro5-4864, or FGIN-1-27.

To determine whether TSPO ligands affected TSPO, Leydig cells were preincubated for 30 minutes with 19-Atriol, a specific inhibitor of the TSPO CRAC domain (39, 41), dissolved in dimethylsulfoxide (final concentration in culture medium less than 0.5%), and then further incubated for 2 h with 19-Atriol plus LH, Ro5-4864, or FGIN-1-27. T was measured by RIA after HPLC separation of steroids (42). HPLC separation was necessary because we found that 19-Atriol cross-reacted with the T antibody. In brief, samples were mixed with 11β-hydroxyandrostenedione (100 ng/tube) as an internal standard. After evaporating the ether by placing samples in a 45°C water bath, the residual contents in each tube were dissolved in 50 μL absolute methanol and then injected into a reverse-phase HPLC C18 column packed with 5-μM particles (4.60 nm internal diameter, 15 cm long; Phenomenex, Torrance, California), with acetonitrile-water [60:40 (vol/vol)] as the mobile phase and at a constant flow rate of 1 mL/min. Peaks of the T were detected by a UV absorbance detecting system (GM 770 monochromator; Schoeffel Instrument Corp, Westwood, New Jersey). HPLC fractions corresponding to T were assayed by RIA.

Effect of TSPO drug ligands on steroid production in vivo

To determine the in vivo effect of TSPO ligand on steroidogenesis, young and aged rats received FGIN-1-27 (0.1 or 1 mg/kg body weight) by daily ip injection. After a 10-day injection period, serum was collected for T measurement by RIA.

Statistical analysis

Data are expressed as means ± SEM of at least 3 independent experiments. For group comparisons, 1-way ANOVA followed by a Duncan's post hoc test were performed by GraphPad Prism software (version 4.0; GraphPad Inc, San Diego, California. Values were considered significant at P < .05.

Results

In vitro effects of TSPO drug ligands Ro5-4864 and FGIN-1-27 on Leydig cells from young and aged rats

Leydig cells were isolated from the testes of young adult (3 months old) and aged (21 months old) Brown Norway rats. The isolated cells consistently were over 90% pure, as assessed by 3β-HSD staining (Figure 1, A and B). Cells from young and aged rats were incubated for 2 hours with Ro5-4864 or FGIN-1-27 (0-200 μM). There was no change in the viability of aged cells exposed to increasing concentrations of either Ro5-4864 (Figure 1C) or FGIN-1-27 (Figure 1D), even at 200 μM. In the case of young cells incubated with either Ro5-4864 (Figure 1C) or FGIN-1-27 (Figure 1D), there was reduced viability with increasing drug ligand concentration, with the first evidence of decline (10%–15%) seen at 50 μM with both ligands.

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.

In light of these results, we used concentrations of TSPO drug ligands no higher than 50 μM for studies of both aged and young cells. With increasing Ro5-4864 concentrations from 0 to 50 μM, relatively modest increases in T production were seen by both aged and young cells (Figure 1E). With aged cells, there was a significant, approximately 4-fold, increase at 50 μM; and with young cells, there was a significant, approximately 2.5-fold, increase at that concentration. The responses of aged and young cells to FGIN-1-27 were more dramatic, with increases in T production of about 6-fold by young cells at 10 and 50 μM, respectively, and of 4- to 5-fold by aged cells at those concentrations (Figure 1F). Incubation of Leydig cells with a combination of LH (0.1 ng/ml) plus FGIN-1-27 resulted in significantly higher T production than when the cells were incubated with FGIN-1-27 alone (Figure 1G). Additionally, as is consistent with previous reports (37, 38, 43), Ro5-4864 and FGIN-1-27 stimulated steroid (progesterone) production by MA-10 tumor cells (not shown). In contrast to primary cells, however, the response of MA-10 cells to Ro5-4864 was greater than that to FGIN-1-27.

Figure 2 compares the responses of young and aged Leydig cells, over time (0–120 minutes), to LH, Ro5-4864, and FGIN-1-27. For these studies, the LH concentration used, 0.1 ng/mL, was well below the concentration that is maximally stimulating (30). In response to LH, T production increased in a time-dependent manner in cells of both ages, with production at 120 minutes significantly reduced in the aged as compared with the young cells (Figure 2A). Increases in T production by both young and aged cells also were seen in response to Ro5-4864 (Figure 2B) and FGIN-1-27 (Figure 2C). However, in contrast to the reduced T production by aged cells in response to LH, T production by aged and young cells in response to the TSPO drug ligands did not differ significantly (Figure 2D). Moreover, aged cells that were stimulated with FGIN-1-27 or LH produced T at about the same levels (Figure 2D). This was the case despite the fact that TSPO expression is reduced in the aged cells by about 70% (27, 29).

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.

To assess the mechanism of action and specificity by which treatment of primary Leydig cells with TSPO ligands resulted in increased steroid production, young cells were incubated for an initial 30-minute period with 19-Atriol (100 μM), which inhibits cholesterol binding at the TSPO CRAC domain (19, 41), and then were incubated with 19-Atriol plus LH, Ro5-4864, or FGIN-1-27 for an additional 2 hours. T then was measured by RIA after HPLC separation of steroids. As seen in Figure 3A, the increased T produced in response to each of LH, Ro5-4864, and FGIN-1-27 was significantly inhibited by 19-Atriol. There was no effect of the inhibitor on cell viability (not shown). The results obtained with the primary cells was supported by comparable studies with MA-10 cells; LH-induced progesterone production was significantly inhibited by 19-Atriol (Figure 3B), as was progesterone production in response to Ro5-4864 and FGIN-1-27 (Figure 3C). However, providing the MA-10 cells with 22R-HC (5 μM) to bypass the TSPO-mediated cholesterol transport resulted in the same level of progesterone production in the presence or absence of 19-Atriol (Figure 3D). These results, taken together, indicate that Ro5-4864 and FGIN-1-27 act by stimulating cholesterol transport into the mitochondria via TSPO, thus providing the substrate cholesterol for side-chain cleavage by CYP11A1.

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.

In vivo effects of TSPO drug ligand FGIN-1-27 in young and aged rats

To determine whether the direct stimulation of TSPO also would affect T production in vivo, FGIN-1-27 was administered to young and aged rats by daily ip injection of 0.1 or 1 mg/kg body weight over a period of 10 days. Control rats received vehicle only. After the treatment period, serum was collected for T measurement. As seen in Figure 4A, serum T in both young and aged rats significantly increased relative to controls in response to the higher (1 mg/kg) concentration of FGIN-1-27. Importantly, serum T levels in aged rats administered 1 mg/kg FGIN-1-27 were equivalent to serum T levels in control young rats. Figure 4B shows that there were no changes in the body weights of groups of young and aged rats that received FGIN-1-27 for 10 days, an indication that there were no cytotoxic effects of the TSPO ligand.

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.

Discussion

In many species, including rat and man, aging is associated with reduced serum T concentration (29–33, 44, 45). Thus, 20%–50% of men over the age of 60 years are reported to have serum T levels significantly below those of young men (aged 20–30 years) (44–51). Age-related decline in serum T typically is not in response to reduced LH but rather is gonadal (46, 50, 52). Primary hypogonadism also occurs in many infertile men. Approximately 15% of couples seek infertility-related medical appointments, with male factor effects contributing to 40%-50% of these cases (46, 53, 54). For 30% of infertile men, no identifiable cause is found, and in greater than 50% of these men, there is both primary hypogonadism and reduced sperm production (53, 55). Whether in aging or young men, reduced serum T is linked to a number of metabolic and quality-of-life changes, including decreased lean body mass and bone mineral density, increased visceral fat, decreased libido and sexual function, altered mood, and fatigue (48, 50, 56, 57). Age-related reductions in serum T in Brown Norway rats also are not secondary to reduced serum LH levels and are not the result of reduced Leydig cell numbers (30, 58–60). Rather, as in men, there is a reduced ability of the Leydig cells to produce T despite normal or elevated levels of LH.

It is well established that the acute stimulation of Leydig cell T production by LH requires cAMP-activated cholesterol transfer from intracellular stores into mitochondria and that this is the rate-determining step in steroid biosynthesis. Recent data support the idea that STAR and TSPO act in concert to elicit cholesterol transfer. As summarized by Papadopoulos and Miller (61), cholesterol is targeted to the OMM, in which TSPO (18 kDa) is abundant; reduction of either STAR or TSPO expression in MA-10 Leydig cells resulted in reduced progesterone production in response to human chorionic gonadotropin, suggesting a functional STAR-TSPO interaction; and the 37-kDa STAR is not processed to the mature 30-kDa protein in TSPO-depleted cells or in the presence of a peptide antagonist of TSPO, suggesting that TSPO may play a direct role in the activity of STAR. Recently TSPO was shown to be part of a hormone-dependent mitochondrial protein complex that facilitates the import, segregation, and transfer of the steroidogenic pool of cholesterol to CYP11A1 at the IMM, and STAR was shown to act on this protein complex to induce cholesterol transfer to the CYP11A1 (62). These observations, taken together, suggest that STAR's primary function is the activation of the cholesterol transport mechanism from the OMM to CYP11A1 and that TSPO is part of the mechanism by which cholesterol remains segregated in OMM and is transferred to CYP11A1 in the IMM.

We hypothesized that although both STAR and TSPO expression are reduced in aged as compared with young Leydig cells (27, 29), ligand-induced activation of TSPO in vitro might provide a means by which to increase T production by aged Leydig cells, thus bypassing LH-dependent cAMP production. To test this hypothesis, we used 2 TSPO drug ligands, Ro5-4864, a benzodiazepine, and FGIN-1-27, an indole acetamide. These are structurally diverse, unrelated chemicals that both have the ability to bind with high affinity to highly enriched preparations of TSPO-containing membranes and thus affect steroid formation (63). We show that both drug ligands elicited increases in T production by young and aged Leydig cells in vitro. FGIN-1-27 had a more robust effect than Ro5-4864, perhaps due to the facilitation of FGIN-1-27 action by the mitochondrial membrane microenvironment. In contrast to LH-stimulated T production, which was reduced in aged as compared with young cells, T production by ligand-stimulated aged and young adult cells was equivalent. Moreover, aged cells stimulated with FGIN-1-27 produced T at levels equivalent to those achieved stimulating aged cells with LH. It should be noted that in most previous studies of the responsiveness of aged Leydig cells to LH, the concentration of LH to which the cells were exposed was maximally stimulating (10–100 ng/mL), whereas in the present studies, the LH concentration, 0.1 ng/mL, was well below maximally stimulating (30).

Midzak et al (41) identified a compound, 19-Atriol, that is capable of inhibiting cholesterol binding at the TSPO CRAC motif. The CRAC domain is responsible for binding cholesterol and its translocation into the IMM (19). 19-Atriol has been shown to inhibit hormone-induced steroidogenesis in MA-10 mouse Leydig tumor cells, constitutive steroidogenesis in R2C rat Leydig tumor cells, and TSPO ligand (PK 11195)-stimulated steroid formation in MA-10 cells (41). We used 19-Atriol in the present studies to confirm that the TSPO drug ligands, Ro5-4864 and FGIN-1-27, increased T formation in primary cells through the stimulation of TSPO. Indeed, 19-Atriol treatment resulted in the inhibition of T production in response to each of LH, Ro5-4864, and FGIN-1-27 but not in the presence of the membrane-permeable 22R-HC. Thus, although the mitochondrial CYP11A1 and the downstream steroidogenic enzymes in the smooth endoplasmic reticulum (3β-hydroxysteroid dehydrogenase, CYP17, and 17β-hydroxysteroid dehydrogenase) are reduced in aged cells (64), the activation of TSPO by drug ligands can result in increased T production.

These in vitro results have implications for understanding the mechanism(s) responsible for steroidogenic deficits in aged Leydig cells. The rate-determining step in steroidogenesis is the transport of cholesterol into the IMM via TSPO and STAR, which occurs in response to LH-stimulated cAMP synthesis (65, 66). The production of cAMP in response to LH is reduced in aged cells, presumably as a consequence of the relative insensitivity of the cells to LH (67). Our results indicate that bypassing cAMP with drug ligands that activate TSPO directly can result in significantly increased T formation by aged cells.

The objective of T therapy is to raise serum T levels to reduce symptoms of hypogonadism. Ideally, a steady serum T concentration within the physiological range of adult men should be produced. With T injections, serum T levels initially are supraphysiological, then reduced to physiological, and in time reduced further (50). High T levels may pose a risk for aging males due to possible prostate (benign prostatic hyperplasia, prostate cancer) and cardiovascular consequences (49, 50, 68). T administered by gels and other transdermal methods may produce relatively constant serum T concentrations but has the potential for T transfer via skin contact (49, 50, 68). Moreover, the administration of exogenous T by any means can suppress LH and thus result in contraception, making this therapy inappropriate for men wishing to father children (46, 53, 54, 69). These disadvantages notwithstanding, T therapy for hypogonadal men has been shown to provide benefits to muscle mass and strength, bone mineral density, adiposity, lipid abnormalities, glucose control, cardiovascular disorders, sexual function, mood, and cognitive function (49, 50, 68). The availability of new therapies that increase serum and intratesticular T levels without the need to administer T and without affecting the hypothalamic-pituitary axis would be of great benefit to the many men with primary hypogonadism, including those who wish to father children.

In the in vivo studies reported herein, we observed that FGIN-1-27 is able to exert a major stimulatory effect on the circulating T levels in aged rats; this despite the fact that there are reductions in the steroidogenic enzymes in the mitochondria and smooth endoplasm in aged vs young Leydig cells (31, 32, 64, 70). Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of untreated young rats. These results are consistent with the in vitro results that we obtained, discussed above.

Taken together, these results suggest the exciting possibility that the in vivo pharmacological activation of TSPO with TSPO drug ligands is an approach that might be used therapeutically for the gonadotropin-independent induction of T formation in cases of both primary and secondary hypogonadism.