A novel function of geranylgeraniol in regulating testosterone production

Abstract

Isoprenoids play widely differing roles in various physiological processes in animals and plants. Geranylgeraniol (GGOH) is an isoprenoid found in plants, and is an important metabolic derivative in the isoprenoid/cholesterol synthesis pathway. Earlier studies focused on GGOH’s ability to improve the side effects of bisphosphonate therapy by regulating the mevalonate pathway. More recently, the mevalonate pathway-independent effects of GGOH have been described, including anti-inflammatory, anti-tumorigenic, and neuroprotective activities. It is noteworthy that GGOH regulates the steroidogenesis pathway in testis-derived I-10 tumor cells. Testosterone is a hormone produced via steroidogenesis in testicles and plays a role in fetal development and the male reproductive system. GGOH enhanced testosterone and progesterone (its precursor) levels in I-10 cells by activating adenylate cyclase via cAMP/PKA signaling, without altering phosphodiesterase activity. These findings highlight the potential benefits of GGOH as a therapeutic agent for low testosterone levels, such as late-onset hypogonadism in men.

Geranylgeraniol enhances testosterone production.

Keywords:

• Geranylgeraniol
• cAMP/PKA
• steroidogenesis
• testosterone
• progesterone

Abbreviations:

• AC: adenylate cyclase
• FOH: farnesol
• GGOH: geranylgeraniol
• GGPP: geranylgeranyl diphosphate
• GOH: geraniol
• IBMX: 3-isobutyl-1-methylxanthine
• MDL: MDL-12,330A
• PDE: phosphodiesterase
• PKA: protein kinase A
• StAR: steroidogenic acute regulatory protein

Isoprenoids (also referred to as terpenoids) are the largest and most structurally diverse family consisting of isoprene (C5) units in nature, with approximately 55,000 compounds [1], and classified based on the number of isoprene units. The major groups are monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and triterpenes (C30). As far as we know, isoprenoids play numerous functional roles in the physiological processes that are required for cell growth in plants and animals [2–4], and are currently utilized as commercial flavors, fragrances, dietary supplements, and even as anticancer drugs [5,6]. Two distinct and independent isoprenoid biosynthetic pathways occur in nature. They both synthesize isopentenyl diphosphate and its isomer dimethylallyl diphosphate, which are the central intermediate of isoprenoids [7]. Although certain specialized isoprenoids are cataloged with well-characterized biochemical functions (e.g. sterols, carotenoids, and quinones), the functions of numerous other isoprenoids are still unknown [8] Diterpenes geranylgeraniol (GGOH) can be ingested from vegetables and grains, including rice. Geranylgeranyl diphosphate (GGPP) may also be hydrolyzed into GGOH by intestinal phosphatase [9] and then absorbed into the body. In the present article, we highlight the effects of exogenous GGOH on testosterone production.

Current knowledge on the potential benefits of GGOH

GGOH is an intermediate in the biosynthesis of vitamins E and K, as well as an important metabolic derivative in the isoprenoid/cholesterol synthesis pathway. Most previous studies on the effects of GGOH have focused on the mevalonate pathway (MVP), showing that GGOH improves the side effects of bisphosphonate therapy by regulating the MVP and inhibiting osteoclast formation [10–17] Other studies have found that GGOH restores the function of cells which were treated with statin or are deficient in mevalonate kinase [18–20], maintains the beneficial effects of cholesterol synthesis in human THP-1 cells treated with a MVP inhibitor [21], and exerts endotoxin tolerance in statin-treated murine macrophages [22]. These also suggest that GGOH can prevent the cytotoxicity of statin treatments by regulating MVP. Recent studies, however, indicate that GGOH also possesses beneficial effects independently from the regulation of the MVP. These include antibacterial [23], anti-inflammatory [24,25], anti-tumorigenic [26–29], and neuroprotective activities [30,31]. According to these results, GGOH is required for the protein isoprenylation that controls cytoskeletal reorganization, vesicular fusion, and further regulates apoptosis and cell survival. We were the first to report that GGOH enhanced testosterone levels in murine Leydig tumor cells [32]. These findings highlighted the need for further research into the potential benefits of GGOH as a local therapeutic agent in several diseases.

Steroidogenesis in testicular Leydig cells

As shown in Figure 1, luteinizing hormone (LH) from pituitary gland regulates testosterone production in testicular Leydig cells via the LH receptor, which then promotes the 3′,5′-cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway by modulating G proteins. Intracellular cAMP promotes the cAMP/PKA pathway, which is mediated by the activities of adenylate cyclases (ACs) and phosphodiesterases (PDEs). The elevation of cAMP regulates the activation of PKA to stimulate signaling in downstream steroidogenic proteins. Steroidogenic acute regulatory protein (StAR) regulates cholesterol transfer within the mitochondria in a rate-limiting step to initiate steroidogenesis in Leydig cells. Moreover, a cholesterol side-chain cleavage enzyme, cytochrome P450scc (also referred to as CYP11A), catalyzes a cascade of steroidogenic reactions that convert cholesterol to pregnenolone, the metabolic intermediate in the biosynthesis of most of the steroid hormones including testosterone. Testosterone inhibits the release of LH from the pituitary gland by negative feedback via the hypothalamus [33,34].

Figure 1. Steroidogenic pathway in testicular Leydig cells. AC, adenylate cyclase; CREB, cAMP response element-binding protein; CYP11A, cholesterol side-chain cleavage enzyme; G, G protein; LH, luteinizing hormone; LHR, LH receptor; Mito, mitochondria; PDE, phosphodiesterase; PKA, protein kinase A; SER, smooth endoplasmic reticulum; StAR, steroidogenic acute regulatory protein.

Testosterone level as a predictor of various diseases in men

Testosterone is crucial for the development of fetuses and male reproductive systems, as well as for sexual function. Increasing evidence shows that testosterone deficiencies cause infertility and sexual dysfunction in men, sometimes resulting in late-onset hypogonadism (LOH) [35–37]. In an aging population with LOH, the common symptoms include sexual dysfunction, muscle weakness and depression. It is noteworthy that low testosterone levels may predict the development of several diseases, such as type 2 diabetes mellitus and cardiovascular diseases [38–41]. The current treatment for LOH is testosterone replacement therapy. Although it may improve bodily health, quality of life, and sexual function in men, it can also increase mortality [42–44]. In our recent study, it was revealed that GGOH may boost testosterone levels in Leydig cells [32].

GGOH stimulates testosterone and progesterone production in testis-derived Leydig cells

The following important turning points in understanding the connection between GGOH and testosterone were previously reported by us. First, we found that menaquinone-4 (MK-4), one of vitamin K2, stimulated testosterone production in I-10 cells derived from a Leydig cell testicular tumor in a mouse [45]. Second, we found that both MK-4 and GGOH showed anti-inflammatory effect in a lipopolysaccharide-induced inflammation model via inhibition of nuclear factor-κB activation [25,46]. According to these results, we presumed that the geranylgeranyl side chain of MK-4 was important in expressing this activity and, subsequently, investigated the effects of GGOH on testosterone production [32].

GGOH enhanced the levels of testosterone and its precursor, progesterone, in I-10 cells in a dose- and time-dependent manner (as shown in Figure 2). The testosterone-enhancing abilities of other structurally related isoprenoids (geraniol, GOH, 2 isoprene units; and farnesol, FOH, 3 isoprene units), as well as those of chemicals structurally close to GGOH (phytol and GGPP), were also examined. Testosterone and progesterone levels in I-10 cell culture mediums markedly increased in the presence of phytol and GGPP, but not in the presence of GOH. Meanwhile, FOH enhanced progesterone, but not testosterone levels. These results indicated that phytol and GGOH have similar effects on testosterone and progesterone production although, unexpectedly, GOH did not affect steroid production in I-10 cells. Recent researches have revealed the biological activity of compounds derived from the isoprenoid/cholesterol synthesis pathway. These isoprenoids, including FOH, GOH, and phytol, regulate various biological processes [47–49]. The results indicated that not only GGOH, but other isoprenoid derivatives, can enhance testosterone and progesterone levels, although the mechanisms by which they exert these effects are yet to be clarified.

Figure 2. Testosterone and progesterone levels after GGOH treatment in I-10 cells. Cells were treated with of GGOH for 0–24 h, and testosterone (A) and progesterone (B, C) levels in cultured medium were measured. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 vs. 0 μM. Different letters indicate significant differences (p < 0.05). Data for panels A–C were taken from reference [32].

GGOH regulates the cAMP/PKA signaling pathway by modulating AC activity

Our findings indicated that GGOH could enhance progesterone and testosterone production via the regulation of PKA activity in a cAMP-dependent pathway (Figure 3(A) and (B)). Moreover, as shown in Figure 3(C) and (D), the enhancement of progesterone production in GGOH supplementation was suppressed by AC inhibitor, MDL-12,330A (MDL); on the other hand, progesterone level did not rise significantly with GGOH and addition of PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). These results suggested that GGOH regulates cAMP levels by inducing AC activity, and not by suppressing PDE. To further confirm that GGOH stimulates steroidogenesis via the induction of AC activity, we downregulated AC mRNA expression using small interfering RNA. The results met our expectations, indicating that GGOH stimulated steroidogenesis via the regulation of AC, especially the activation of AC9 [32]. There are 10 types of AC isoforms (AC1–10) in mammals; AC1 to AC9 are transmembrane enzymes, while AC10 is soluble. The transmembrane isoforms are mainly regulated via G-protein-coupled receptors [50,51]. Human AC9 mRNA is abundant in the brain, heart, and skeletal muscle [52]. AC9 is highly expressed in I-10 cells and our findings by using AC9 siRNA (Figure 3(E)) showed that AC9 may regulate cAMP levels after GGOH stimulation. However, among the gene expression of steroidogenesis-related proteins, both the mRNA and protein levels of StAR increased in the presence of GGOH. StAR functions in the rate-limiting step of steroid production in Leydig cells and is required for the transport of cholesterol to the inner mitochondrial membrane [53]. We showed that GGOH treatment increases the expression of StAR, which acts downstream in the cAMP/PKA pathway. Taken together, our results demonstrate that GGOH enhances testosterone and progesterone production in I-10 cells via the induction of cAMP/PKA signaling (Figure 3).

Figure 3. GGOH regulates cAMP/PKA signaling pathway by modulating AC activity in I-10 cells. (A) Intracellular cAMP levels were enhanced after GGOH treatment (30 μM) in I-10. (B–D) GGOH (30 μM) stimulated cAMP/PKA signaling pathway via regulation of AC but not PDE activity in I-10 cells. H89: PKA inhibitor, MDL: AC inhibitor, IBMX: PDE inhibitor. (E) Enhanced progesterone production by GGOH (30 μM) was diminished by AC9 siRNA treatment. Data are presented as mean ± SD (n = 3). **p < 0.01 vs. Ctrl group. Different letters indicate significant differences (p < 0.05). Data for panels A–D were reproduced from reference [32].

Dietary supplementation of GGOH enhances plasma testosterone levels in rats

We also found that dietary supplementation of MK-4 enhanced plasma testosterone levels in rats, without any alternation of plasma LH levels [45]. Based on the results of our cell-based experiments [32], we conducted further experiments on the effects of GGOH on testosterone production in animals. Eight-week-old Wistar male rats were purchased from SLC Japan (Shizuoka, Japan) and fed either GGOH supplemented (48.3 mg/kg of diet) or control diet for 10 days. Growth performance did not differ between both diets, but plasma testosterone levels in GGOH supplemented group were found to be elevated compared to that of control group, indicating that dietary supplementation of GGOH significantly elevates plasma testosterone levels (unpublished data, Figure 4). These findings provide novel mechanistic insights into the process of testosterone production and may be useful for the development of therapeutic strategies to counter age-associated declines in testosterone levels in men.

Figure 4. Dietary supplementation of GGOH enhances plasma testosterone levels in male Wistar rats. Data are presented as mean ± SD (n = 7 − 8). *p < 0.05 vs. Ctrl group.

In summary, the novel role of GGOH in steroidogenesis may bring new possibilities and could be useful in the development of therapeutics for the treatment of men with LOH.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was partially supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) to HS [grant number #17H03814], and by the JSPS Core-to-Core Program A (Advanced Research Networks) entitled: “Establishment of international agricultural immunology research-core for a quantum improvement in food safety.”

Acknowledgement

We are deeply grateful to Tama Biochemical Co. Ltd. (Tokyo, Japan) for providing all trans-GGOH used in animal experiments.