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. 2018 Oct 18;100(3):824–832. doi: 10.1093/biolre/ioy220

Acute effects of the translocator protein drug ligand FGIN-1-27 on serum testosterone and luteinizing hormone levels in male Sprague-Dawley rats†

Fenfen Chen 1,2,#, Hemin Lu 3, Panpan Chen 1, Xingxing Zhao 2,4, Xiaojui Guan 2,4, Qingquan Liang 2,4, Barry R Zirkin 5, Leping Ye 3,✉, Haolin Chen 1,2,4,5,✉
PMCID: PMC6437259  PMID: 30299464

Abstract

We reported that FGIN-1-27 (N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide, FGIN), a synthetic ligand for translocator protein (TSPO, 18 kDa), increased serum testosterone levels in young and aged Brown Norway rats after its administration daily for 10 days. It is not known, however, how soon after treatment with FGIN serum testosterone rises, how long levels remain elevated after cessation of treatment, or whether the drug acts solely through TSPO. Adult Sprague-Dawley male rats received a single ip dose of FGIN (1 mg/kg BW). Serial blood samples were collected, and serum testosterone and luteinizing hormone (LH) were assessed hourly throughout 24 h. Testosterone concentration was maximal by 3 h, remained significantly higher than the controls at 10 h, and returned to the control level by 24 h. Consistent with the in vivo study, culturing isolated Leydig cells with either FGIN (40 μM) or LH (0.1 ng/ml) resulted in significantly increased testosterone production by 30 min, and the stimulatory effects persisted through 48 h. At a very early (15 min) treatment time, however, FGIN significantly increased testosterone production but LH had not yet done so. Surprisingly, in vivo treatment with FGIN not only increased serum testosterone but also serum LH concentration, raising the possibility that FGIN may increase serum testosterone concentration by dual mechanisms.

Keywords: FGIN-1-27, testosterone, LH, Leydig cell

FGIN-1-27, a TSPO drug ligand, can acutely increase serum testosterone concentration by acting directly on the Leydig cells and indirectly through luteinizing hormone.

Abbreviations

Cyp11a1

C27 cholesterol side-chain cleavage cytochrome P450 enzyme

Cyp17a1

Cytochrome P450 17a1 (17α-hydroxylase/17,20 lyase/17,20 desmolase)

Lipe

hormone-sensitive lipase

Nr5a1

nuclear receptor subfamily 5 group A member 1 (Sf1)

FGIN

N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide

HSD3B

hydroxysteroid dehydrogenase 3B

22HC

22R-hydroxycholesterol

STAR

steroidogenic acute regulatory protein

TSPO

translocator protein

dbcAMP

dibutyryladenosine 3′, 5′-cyclic monophosphate

LH

luteinizing hormone

P5

pregnenolone

BAPTA

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

H89

N-[2-[[3-(4-Bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide

Introduction

About 20%–50% of men over age 60 years have been reported to have serum testosterone levels significantly below those of young men [1, 2]. Aging-related decline in serum testosterone levels typically does not result from reduced luteinizing hormone (LH), but rather from the Leydig cells becoming less responsive to LH [3, 4]. This condition, referred to as primary hypogonadism, also occurs in some young men. For example, it has been reported that 40%–50% of the male partners of couples seeking infertility-related medical appointments are hypogonadal [5, 6]. These are alarming numbers in that reduced serum testosterone has been shown to be associated with decreases in lean body mass, bone mineral density, muscle mass and strength, and libido, as well as with adiposity, cardiovascular disorders, altered mood, and fatigue [3, 7, 8].

Administering exogenous testosterone, known as testosterone replacement therapy, reverses many of the symptoms of low testosterone. Often, therefore, testosterone is prescribed to men diagnosed with low circulating testosterone levels (or with symptoms). Administering testosterone raises serum testosterone levels into the eugonadal range. However, there are recent studies indicating increased risk of cardiovascular disease in men administered exogenous testosterone [9–11]. Moreover, it has been suggested that exogenous testosterone administration might increase the risk of prostate cancer [12]. In addition, exogenous testosterone will suppress LH and therefore Leydig cell testosterone production. This can cause reduced intratesticular testosterone and thus contraception. This makes the administration of testosterone inappropriate for men who wish to father children [5, 6, 13, 14]. It is apparent that alternative or additional means by which to increase serum testosterone levels, preferably through increasing endogenous testosterone production, would be highly desirable.

Knowledge of how steroids are formed and regulated has suggested possible avenues for increasing serum testosterone without exogenous testosterone administration. The rate-determining step in Leydig cell testosterone production is cholesterol translocation from intracellular stores into the mitochondria. This is followed by the conversion of cholesterol to pregnenolone within the mitochondria, and then the formation of testosterone from pregnenolone in the smooth endoplasmic reticulum [15–17]. Translocator protein (TSPO), a mitochondrial protein with high affinity for cholesterol, is among the proteins in Leydig and other steroidogenic cells that have been identified as playing important roles in cholesterol translocation to the inner mitochondrial membrane [18–20]. There are TSPO-specific drug ligands that have been shown to be capable of increasing Leydig cell testosterone production in the presence or absence of LH [21, 22]. In our previous studies, we reported that the in vivo administration of one such drug, FGIN, for 10 days resulted in significant increases in serum testosterone concentrations in both adult and aged rats [23]. In the present study, we demonstrate that FGIN administration in adult Sprague-Dawley rats resulted in a significant increase in serum testosterone levels as soon as 1 h and thereafter, that the effect lasted for up to 24 h, and, unexpectedly, that although testosterone increased, there was an increase in serum LH levels. Thus, FGIN may increase testosterone not only via direct effects on the Leydig cells, but also indirectly through effects on LH synthesis and/or release.

Materials and methods

Reagents

FGIN, HEPES, Hanks balanced salt solution, Percoll, dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP), 22R-hydroxycholesterol (22HC), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), N-[2-[[3-(4-Bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide (H89), cycloheximide, and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, Missouri). The enzyme-linked immunosorbent assay (ELISA) kits for LH and follicle-stimulating hormone (FSH) were from Shanghai Westang Bio-Tech Co., Ltd (Shanghai, China). The Immulite 2000 Total Testosterone assay kit was from Siemens (Germany). All other unlabeled steroids, including pregnenolone, were obtained from Steraloids Inc. (Wilton, NY). M-199 was from Gibco BRL (Grand Island, New York). BSA was from ICN Biomedicals, Inc (Aurora, Ohio). Bovine LH (USDA-bLH-B-6) was provided by the US Department of Agriculture Animal Hormone Program (Beltsville, Maryland).

Treatment of male rats with FGIN

Adult male Sprague-Dawley rats of 90 days of age were purchased from Shanghai Animal Centre (Shanghai, China). Animals were housed in the animal facilities of the Second Affiliated Hospital of Wenzhou Medical University at 22°C, 12-h light, 12-h dark with free access to feed and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of NIH (NIH publication #85-23, revised in 1985).

Rats (n = 8) received a single dose of FGIN (1 μg/g BW) administered by ip injection. Control rats (n = 8) received vehicle (1 μl of 10% DMSO in saline/g BW). Blood samples (about 200 μl) were collected from the facial veins of each rat periodically for up to 24 h, according to a well-established method for collecting blood from rodents without the use of anesthesia [24]. In brief, blood was drawn from facial veins punctured with a Goldenrod Animal Lancet (manufactured by Bioseb, USA). After collection of about 200 μl of blood, pressure was applied to the area of the puncture with gauze. For assays of hormones other than testosterone, additional rats (5 rats per group) were treated with FGIN, and trunk blood was collected 2 h after the treatment. Serum was collected after centrifugation of the blood samples for 15 min at 4°C and stored at –80°C before hormone assays.

Collection of testicular fluid

Testicular fluid was collected by a well-established procedure [25]. In brief, dissected whole testes were first dried with a piece of gauze, and then were decapsulated. The tissue was extruded through the hub of a 3 ml syringe into a 50 ml centrifuge tube. This procedure disrupts the integrity of the seminiferous tubules without damage to the cells. After centrifugation at 6000 × g for 15 min (4°C), the supernatant above the solid tissue was collected. The collected testis fluid is a mixture of intersititial and seminiferous tubular fluids. The fluid was then stored at –80°C.

Hormone assays by ELISA

Serum testosterone, LH, and FSH were assayed using ELISA kits according to manufacturer’s instructions. Testosterone was assayed using the Immulite 2000 Total Testosterone assay kit, which has a detection sensitivity of 0.15 ng/ml. The intra- and interassay coefficients of variation were 8.3% and 9.1%, respectively. For LH and FSH assays, the kits used were from Westang Bio-Tech Co., Ltd (Shanghai, China). To avoid interassay variations, all samples of LH and FSH were assayed in one run for each hormone. The LH kit has detection sensitivity of 0.1 ng/ml, with intra-assay coefficient of variation of 9.7%. The FSH kit has detection sensitivity of 0.2 ng/ml, with intra-assay coefficient of variation of less than 10%.

Primary Leydig cell isolation

Primary Leydig cells were isolated from Sprague-Dawley rats of 90 days of age by a combination of Percoll and bovine serum albumin (BSA) density gradient centrifugations, as previously described [26]. In brief, the testes were decapsulated and digested in dissociation buffer (M-199 medium with 2.2 g/L HEPES, 1.0 g/L BSA, 2.2 g/L sodium bicarbonate) containing collagenase I (0.5 mg/mL) at 34°C, with slow shaking (90 cycles/min, 30 min). To separate the interstitial cells from the seminiferous tubules, digested testes were placed in a solution containing 1% BSA for 1 min. The supernatants were collected and the interstitial cells were pelleted by centrifugation (1500× g, 5 min). Leydig cells were purified by Percoll gradient separation (55% Percoll, 27 000 g, 1 h) and then by BSA gradient centrifugation. After Percoll centrifugation, Leydig cells were collected at the density layer between 1.070 and 1.088 g/ml. Leydig cells were further purified by BSA gradients (2.5%/5%/10%) for 10 min (×45 g). The final purity of the Leydig cells, determined by staining the cells for hydroxysteroid dehydrogenase 3B (HSD3B) activity, was consistently about 95%.

Effect of FGIN on steroid production in vitro

Freshly isolated Leydig cells were suspended in M-199 culture media and plated at a density of about 106 cells/well in 24-well culture plates, or 105 cells/well in 96-well culture plates (Becton Dickson, Franklin Lakes, New Jersey). Cells were incubated for up to 48 h at 34°C with LH (0.1 ng/mL), FGIN (40 μM), or a combinations of LH plus FGIN. For the wells that did not receive FGIN, the same concentration of DMSO (0.2%) was included as vehicle control. DMSO of 0.2% was not found to have any effect on cell viability or testosterone production. Preliminary dose-dependent experiments established that 40 μM FGIN was the concentration at which maximal stimulation of testosterone production was achieved without significant cell toxicity. In some experiments, the medium was removed after 4 h treatment, and the cells were incubated with or without LH for another 4 h.

Cells in 96-well plates (105 cells/200μl/well) were treated for 2 h with stimulatory or inhibitory factors (6 × 4 cross-treatment with controls). The stimulatory factors were LH (0.1 ng/ml), dbcAMP (0.1 mM), 22R-hydrocholesterol (22HC, 5 μM), pregnenolone (P5, 5 μM), and FGIN (40 μM). The inhibitory factors were protein kinase A (PKA) inhibitor (H89, 5 μM), calcium mobilization inhibitor (BAPTA, 5 μM), and protein synthesis inhibitor cycloheximide (CHX, 25 μM). The cells were pretreated with inhibitors for 30 min and this was followed by treatment with stimulators for another 2 h. The media were frozen for testosterone assay. The cells were frozen for DNA assay.

Extraction of RNA and real-time PCR

For real-time PCR (qPCR) experiments, the cells were plated in a 24-well plate (106 cells/well) and treated with or without LH (10 ng/ml) and FGIN (40 μM) for 2 h. Total RNA was extracted from cells with Triazol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. RNA was isolated at high purity (260/280 ratio above 2.00 and 260/230 ratio above 1.80). Steady-state mRNA levels of key steroidogenic genes were assayed by qPCR with TaqMan reaction system. Compared to the traditional qPCR systems that use double-strand DNA-binding dye, the TaqMan reaction system, which uses dye-labeled probes, has higher specificity and reproducibility, and fewer off-target effects. The genes that were analyzed included hormone-sensitive lipase (Lipe), steroidogenic acute regulatory protein (Star), translocator protein (Tspo), P450scc (Cyp11a1), P450c17 (Cyp17a1), and Sf-1 (Nr5a1). The Taqman primer probes used were Rn00689222_m1 (Lipe), Rn00580695_m1 (Star), Rn00560892_m1 (Tspo), Rn00568733_m1 (Cyp11a1), Rn00562601_m1 (Cyp17a1), and Rn00584298_m1 (Nr5a1). The mRNA levels were expressed in relationship to an internal control, β-actin (Rn00667869_m1). Complementary DNAs were adjusted for each gene so that the Ct values were always below 35 cycles.

Statistical analyses

Data are expressed as the mean ± standard error of the mean (SEM) of four experiments unless indicated otherwise. For the experiments with 2 groups, the means were evaluated by unpaired t-test. For experiments with more than 2 groups, the means were evaluated by one-way ANOVA. If group differences were revealed by ANOVA (P < 0.05), differences between individual groups were determined with the SNK test, using SigmaStat software (Systat Software Inc., Richmond, CA). Values were considered significant at P < 0.05.

Results

Acute effects of FGIN on serum testosterone

We reported previously that the administration of FGIN for 10 days can significantly increase serum testosterone in both adult and aged rats [23]. To test the possibility that FGIN affects testosterone production acutely, we administered one dose of FGIN and then collected a series of blood samples from the facial veins of the rats over the course of 24 h. As shown in Figure 1, FGIN injection resulted in a rapid increase in serum testosterone concentration, by 1 h. The testosterone concentration continued to increase through 3 h, after which there were gradual decreases. By 10 h, the concentration was still twice that of controls. However, by 24 h, serum testosterone had returned to the control level.

Figure 1.

Figure 1.

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Acute effect of FGIN on serum testosterone levels. Adult (3-months-old) male rats (n = 8) received one dose of FGIN (1 mg/kg BW) administration by ip injection. Control rats (n = 8) received vehicle. Blood samples (about 200 μl) were collected from facial veins of each rat periodically for up to 24 h. Testosterone levels were assayed in the serum. *Significantly different from the time-matched controls at P < 0.05.

Effects of FGIN on hormones other than testosterone

Increased serum testosterone levels in response to FGIN would be expected to result in reduced LH via negative feedback effects of testosterone on the hypothalamus and pituitary. To test this, rats were treated with FGIN (1 μg/g BW), and serum and testicular fluid were collected at 2 h. Serum and testicular fluid testosterone concentrations were increased by FGIN at 2 h (Figures 2A and B). Surprisingly, however, we did not find a decrease in serum LH, but rather a significant increase 2 h after FGIN treatment (Figure 2C). In contrast, serum FSH did not change in response to FGIN treatment (Figure 2D). These unexpected results suggest the possibility that FGIN treatment may increase serum testosterone in part by increasing serum LH.

Figure 2.

Figure 2.

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Effects of FGIN administration on serum and testicular hormone levels. Serum (A) and intratesticular fluid (B) testosterone, serum LH (C), and serum FSH (D) concentrations were assayed 2 h after rats received vehicle (Vehicle) or FGIN (n = 5 each group). *Significantly different from the vehicle controls, P < 0.05.

Effect of FGIN on Leydig cell testosterone production in vitro

To examine whether FGIN also increases serum testosterone by directly affecting testicular Leydig cells, we isolated Leydig cells and assayed the effects on testosterone production of culturing the cells with FGIN. Both FGIN (40 μM) and low-dose LH (0.1 ng/ml) significantly increased testosterone production by 2 h (Figure 3A). An additive effect was noted when FGIN and LH were used together. A clear difference was seen between FGIN and LH in increasing testosterone production at early treatment time (15 min); FGIN significantly increased testosterone production at this time, whereas LH did not. We next examined FGIN treatment effects at times from 2 to 48 h. Interestingly, there was no difference between LH and FGIN in stimulating testosterone production beyond 2 h; both increased testosterone production up to 48 h. However, after 24 h, the slopes of both curves became flatter, suggesting a reduced efficiency after 24 h for both treatments (Figure 3B).

Figure 3.

Figure 3.

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Effects of FGIN (40 μM), LH (0.1 ng/ml), or a combination on testosterone production by isolated Leydig cells in vitro. (A) FGIN, LH, and FGIN + LH increased testosterone production linearly over the course of 2 h. However, at 15 min, FGIN but not LH significantly increased testosterone production. (B) FGIN and LH effects on testosterone production in vitro over the course of 48 h. Data are expressed as mean ± SEM of four repeats. aSignificantly different from FGIN or LH + FGIN at P < 0.05 (A). bSignificantly different from FGIN or LH at P < 0.05 (A). Under all other circumstances (A and B), FGIN, LH, or LH + FGIN were significantly different from basal controls (C) at P < 0.05 except at 15 min (A).

When administered in vivo, FGIN stimulation of serum testosterone concentration was maximal by 3 h, after which the stimulation began to diminish slowly. We asked whether the decrease in testosterone production is due to the dilution or loss of FGIN, or alternatively, to the reduced ability of the Leydig cells to produce testosterone in response to a stimulus. Isolated Leydig cells were used to explore these alternative possibilities. As seen in Figure 4A, when the cells were cultured without either FGIN or LH (C+C), testosterone production was minimal. When cultured with FGIN, the isolated cells produced increasingly high levels of testosterone over the course of 4 h (FGIN+C). After this 4-h period, the FGIN-containing medium was replaced with fresh medium in which there was no FGIN. Within 2 h of the removal of the FGIN-containing culture medium, testosterone production by the cells was reduced significantly (FGIN+C). However, the cells remained capable of producing high levels of testosterone. This was seen by continuing the treatment of the cells with FGIN after the initial 4 h (Figure 3B), or by treating the cells with LH for another 4 h (FGIN+LH) (Figure 4A). In both cases, testosterone production rose significantly. These results support the conclusion that the reduced testosterone production that occurs after its significant rise following a single dose of FGIN in vivo results from reduced exposure of the cells to FGIN over time rather than from the reduced ability of the cells to respond to a stimulus.

Figure 4.

Figure 4.

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(A) Effects of FGIN and its removal or its replacement by LH on Leydig cell testosterone production. Leydig cells were incubated without or with FGIN (40 μM) for 4 h, after which the FGIN-containing medium was replaced with fresh medium with or without LH (10 ng/ml). *Significantly different from the basal control after 4 h at P < 0.05. #Significantly different from the respective controls at 4 h at P < 0.05. (B) Comparison of the effects on Leydig cell testosterone production in vitro of incubation with FGIN, the steroidogenic stimulators LH, dbcAMP, 22HC or P5, or the steroidogenic inhibitors cycloheximide, BAPTA or H89. Data are expressed as mean ± SEM. *Significantly different from the treatments without inhibitors (C, control) within each group at P < 0.05.

Where does FGIN work in the steroidogenic pathway?

To examine the mechanism by which FGIN may affect Leydig cell testosterone production, we compared the effect of FGIN with that of other acute stimulators or substrates of steroidogenesis, including LH, dbcAMP, 22R-hydrocholesterol (22HC), and pregnenolone (P5). We also tested the effects of specific inhibitors of PKA (5 μM H89), calcium mobilization (5 μM BAPTA), and protein synthesis (25 μM CHX). As shown in Figure 4B, the stimulatory effect of LH was affected by all three inhibitors. The dbcAMP effect on steroidogenesis was affected by PKA and protein synthesis inhibitors but not the calcium signaling inhibitor. The effects of 22HC and P5 were only sensitive to PKA inhibitor. Interestingly, the effects of inhibitors on FGIN stimulation were similar to those of 22HC and P5, only sensitive to PKA inhibitor but not sensitive to calcium signaling and protein synthesis inhibitors.

To examine the effects of short-term FGIN treatment on Leydig cell steroidogenic gene expression, we examined the levels of six genes, including the cholesterol mobilization and transport genes Lipe, Star and Tspo, and steroidogenic enzymes Cyp11a1 and Cyp17a1, as well as nuclear receptor Nr5a1. Two-hour treatment with LH resulted in significant upregulation of Lipe and Star, with no change in other genes (Figure 5). FGIN did not affect the expressions of any of these genes.

Figure 5.

Figure 5.

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Effect of FGIN and LH treatments on steroidogenic gene expression. The mRNA levels of Lipe (A), Star (B), Tspo (C), Cyp11a1 (D), Cyp17a1 (E), and Nr5a1 (F) were analyzed by qPCR after cells were treated with either LH (10 ng/ml) or FGIN (40 μM) for 2 h. Data are expressed as mean ± SEM. *Significantly different from the control cells at P < 0.05.

Figure 6 summarizes the possible effects of FGIN along the hypothalamic–pituitary–testis axis and steroidogenic pathway. First, FGIN is capable of stimulating Leydig cell steroidogenesis by acting directly on Leydig cells. Second, FGIN may also be capable of stimulating LH synthesis/release by acting at the hypothalamic and/or pituitary levels.

Figure 6.

Figure 6.

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Diagram of proposed effects of FGIN along the hypothalamic–pituitary–testicular axis. First, FGIN is capable of stimulating Leydig cell steroidogenesis by directly acting on Leydig cells, presumably by its effects on cholesterol translocation to the inner mitochondrial membrane. Second, FGIN also is able to increase serum LH levels, perhaps by stimulating LH synthesis/release at the hypothalamus and/or pituitary level(s). LHR: luteinizing hormone receptor; Gs: Stimulatory G-proteins; AC: adenylyl cyclase.

Discussion

Proposed mechanism by which FGIN increases serum testosterone: stimulating Leydig cell testosterone production directly, and indirect stimulation via effects on serum LH

It is well established that the acute stimulation of Leydig cell testosterone production by LH requires cAMP-activated cholesterol transfer from intracellular stores into mitochondria, and that cholesterol translocation to the inner mitochondrial membrane is the rate-determining step in steroid biosynthesis. Over many years, and based on the studies of many labs, a great deal of data have been generated supporting the contention that steroidogenic acute regulatory protein (STAR) and TSPO act in concert, along with other proteins, to elicit cholesterol mobilization from intracellular stores and its translocation to the inner mitochondrial membrane [18, 19]. However, recent studies have challenged the involvement of TSPO in steroidogenesis [27, 28]. Thus, in striking contrast to the results of studies showing significant reduction of steroid production after TSPO knockdown [29, 30], there have been reports of no effect on steroid formation after Tspo deletion [28]. Although the matter is being debated [31, 32], several TSPO-specific ligands have been shown to stimulate cholesterol import into the mitochondria of MA-10 and primary Leydig cells in vitro, and to result in elevated testosterone production when administered in vivo [18, 23, 33, 34].

We reported previously that daily treatment of rats with a synthetic TSPO ligand, FGIN, for 10 days, resulted in significantly increased serum testosterone in both adult and aged Brown Norway rats [23]. In the present study, we have confirmed these early studies, and extended these observations by showing that treatment of adult Sprague-Dawley rats for as long as 10 days is not required to elicit a significant increase in serum testosterone. Instead, we show that FGIN increased serum testosterone levels as soon as an hour after exposure of rats to an appropriate, single dose of FGIN, with maximal effect reached by 3 h. By 10 h, testosterone concentration was still significantly higher in the treated than the control animals. By 24 h, serum testosterone had returned to the untreated control level. The rapid increase in serum testosterone in response to FGIN may be a consequence of its reported effects on stimulating Leydig cell testosterone production acutely through translocation of cholesterol to the inner mitochondrial membrane [18].

A wholly unanticipated finding of the present study is that there was an increase in serum LH levels in response to FGIN treatment. We had assumed that with an increase in serum testosterone, pituitary LH would be downregulated via the negative feedback of testosterone on the hypothalamus and/or pituitary. However, we found a significant increase in serum LH despite increased serum testosterone. This observation raises the possibility that one of the mechanisms by which FGIN increases serum testosterone could be through increasing LH synthesis and/or release (see Figure 6). The mechanism by which FGIN affects LH deserves further study.

Having shown that FGIN can increase testosterone but also serum LH, we asked whether, as in Brown Norway rats [23], FGIN is capable of affecting Leydig cell testosterone production directly in Sprague-Dawley rats. When isolated Leydig cells were incubated with FGIN and/or LH, both were able to increase testosterone production through 48 h. An additive effect of the two was seen when LH was used at submaximal concentration. At the maximally stimulating concentration of LH, the additive effect was no longer apparent (unpublished observations). These observations suggest overlapping effects of FGIN and LH. However, significant differences were seen short-term. Thus, treatment of cells with FGIN resulted in significantly increased testosterone production by 15 min, a time at which there was no effect of LH alone. This observation is also consistent with the proposed effects of FGIN on steroidogenesis, namely to increase cholesterol transport to the inner mitochondrial membrane by directly targeting mitochondria [22, 23]. Although LH also eventually targets events at the mitochondrial membrane, more time is required for it to do so. In sum, the in vitro results support the contention that FGIN is capable of increasing serum testosterone by directly affecting the Leydig cells. The in vivo results suggest strongly that FGIN may be able to increase serum testosterone by dual effects: indirect effects on LH and direct effects on Leydig cell steroidogenesis. Further work is needed to elucidate how, and to what extent, these two mechanisms may work together to contribute to the increase in serum testosterone.

The time course study that we conducted in vivo indicated that the effect of a single dose of FGIN on testosterone production lasts over 10 h. The loss of effect might be due to loss in the cell's response to FGIN stimulation over time, or to FGIN metabolism and/or inactivation in the circulation. The in vitro results support the latter possibility. Thus, the culture of isolated Leydig cells with FGIN did not result in desensitization of the cells to being further stimulation by FGIN or by LH to produce testosterone, indicating the likelihood that the reduced effect of FGIN after its in vivo administration could be largely due to the progressive loss of the drug from the circulation.

Mechanism by which FGIN affects steroidogenesis: responses of FGIN to agents that stimulate or inhibit steroidogenesis

To further elucidate the mechanism by which FGIN affects steroidogenesis, we compared FGIN effects with the effects of agents known to increase steroidogenesis by acting at well-defined sites of the steroidogenic pathway, and with established steroidogenic inhibitors. When the cells were stimulated with LH, the effects were sensitive to inhibitors of protein synthesis, PKA activity, and calcium signaling. When the cells were treated with dbcAMP, which bypasses the LH receptor-G protein-adenlyly cyclase signaling cascade, the effects were only sensitive to inhibitors of protein synthesis and PKA activity. The loss in the sensitivity to calcium signaling by dbcAMP suggests that LH stimulation may require both cAMP and calcium signaling, while dbcAMP does not affect steroidogenesis through calcium signaling. When the cells were supplied with 22HC and P5, the effects were only sensitive to PKA inhibitor, suggesting that the calcium signaling and new protein synthesis processes are no longer necessary if appropriate steroid substrates are provided to CYP11A1 or HSD3B directly. Interestingly, the stimulatory effect of FGIN on testosterone production was also only sensitive to PKA inhibitor, suggesting that FGIN actions on steroidogenesis are at the mitochondria or beyond. As expected, our studies of isolated rat Leydig cells showed that Star and Lipe gene expression were increased in response to LH treatment. However, FGIN did not affect any of the steroidogenic genes examined, including Star or Lipe, suggesting that the effect of FGIN may not be mediated by increasing the expressions of the key steroidogenic genes, such as Star or Lipe that are critical for the acute regulation of steroidogenesis.

Taken together, these results confirmed that the TSPO drug ligand, FGIN, is a potent stimulator of rat steroidogenesis. Interestingly, however, FGIN may increase serum testosterone by acting at both hypothalamus–pituitary and gonad levels. These dual mechanisms could work in favor of using a ligand such as this to increase serum steroid hormone levels since such an approach would be expected to be more potent if actions were at the level of multiple targets. These results suggest that despite of the debate on TSPO itself, some of the small molecules originally synthesized as its ligands should not be neglected for their potential roles in steroidogenesis. FGIN, for example, could be an effective stimulator to induce gonadotropin-dependent and gonadotropin-independent production of testosterone in cases of both primary and secondary hypogonadism.

Notes

Edited by Dr. T. Rajendra Kumar, PhD, University of Colorado Anschutz Medical Campus

Footnotes

†

Grant Support: This work was supported by Natural Science Foundation of China Grants 81771635 (LP), 81471411(HC), and 81741041(HC); Natural Science Foundation of Zhejiang Province grant LY17H040012 (HC); Wenzhou City Public Welfare Science and Technology Project Y20150012 (HC); and NIH grant R01 AG021092 (BZ).

Conflict of Interest: The authors have declared that no conflict of interest exists.

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