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. 2011 Sep 13;10:158.
doi: 10.1186/1476-511X-10-158.

Menaquinone-4 Enhances Testosterone Production in Rats and Testis-Derived Tumor Cells

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Free PMC article

Menaquinone-4 Enhances Testosterone Production in Rats and Testis-Derived Tumor Cells

Asagi Ito et al. Lipids Health Dis. .
Free PMC article

Abstract

Background: Vitamin K is essential for the posttranslational modification of various Gla proteins. Although it is widespread in several organs, including the testis, the function of vitamin K in these organs is not well characterized. In this study, we investigated the function of vitamin K in the testis and analyzed its role in steroidogenesis.

Methods: Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K₂ vitamins present in the testis, for 5 weeks. In vivo testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and in vitro testosterone levels of testis-derived tumor cells (I-10 cells) maintained in Ham's F-10 medium with 10% fetal bovine serum were measured following treatment with MK-4 (0 to 100 μM) at several time points. Testosterone and cellular protein levels were analyzed with respect to their effects on steroidogenesis.

Results: Testosterone levels in the plasma and testes of MK-4-fed rats were significantly increased compared to those of control rats, with no obvious differences in plasma luteinizing hormone levels. Secreted testosterone levels from I-10 cells were elevated by MK-4, but not by vitamin K₁, in a dose-dependent manner independent of cAMP treatment. Western blot analysis revealed that expression of CYP11A, the rate-limiting enzyme in steroidogenesis, and phosphorylation levels of protein kinase A (PKA) and the cAMP response element-binding protein were all stimulated by the presence of MK-4. Enhancement of testosterone production was inhibited by H89, a specific inhibitor of PKA, but not by warfarin, an inhibitor of γ-glutamylcarboxylation.

Conclusions: MK-4 stimulates testosterone production in rats and testis-derived tumor cells via activation of PKA. MK-4 may be involved in steroidogenesis in the testis, and its supplementation could reverse the downregulation of testosterone production in elders.

Figures

Figure 1
Figure 1
Menaquinone-4 supplemented diet enhances testosterone production in rat testis. Male Wistar rats were fed with a control (Cont) or a MK-4-supplemented (MK-4 sup) diet for 5 weeks. Testosterone concentrations in the plasma from the tail vein (A) and testis (B) and luteinizing hormone levels in plasma from the abdominal aorta (C) were measured by ELISA. Data are represented as means ± SE (n = 7 or 8). Values are significantly different from those of the control group at *p < 0.05. †Plasma testosterone levels in the MK-4 sup group were significantly increased when compared with the control group (p < 0.05).
Figure 2
Figure 2
Menaquinone-4 enhances testosterone production in I-10 cells in the presence of cAMP. Mouse testis tumor-derived I-10 cells were treated with dibutyryl-cAMP (db-cAMP, 3 μM) and various concentrations of menaquinone-4 (MK-4) for 24 h. (A) Testosterone levels in cultured medium were determined by ELISA. Data are represented as means ± SE (n = 3). (B) The cell proliferation assay was performed using the WST-1 assay kit. Data are represented as means of relative absorbance values at 450 nm ± SE (n = 3). Values with different letters are significantly different at p < 0.05.
Figure 3
Figure 3
Menaquinone-4 enhances testosterone production in I-10 and R2C cells without cAMP stimulation. I-10 cells were treated with various concentrations of menaquinone-4 (MK-4) for 24 h (A) or 30 μM MK-4 for different time periods (B). Rat testis tumor-derived R2C cells were treated with various concentrations of MK-4 for 24 h (C). Testosterone levels in the culture medium were determined by ELISA. Data are represented as means ± SE (n = 3). Values indicated with different letters in A and C are significantly different at p < 0.05. Values indicated by an asterisk in B are significantly different at p < 0.05.
Figure 4
Figure 4
Warfarin does not inhibit enhanced testosterone production by menaquinone-4 in I-10 cells. I-10 cells were treated with warfarin and menaquinone-4 (MK-4) simultaneously for 24 h, and testosterone levels in the culture medium were then determined by ELISA. Data are represented as means ± SE (n = 3). Values indicated with different letters are significantly different at p < 0.05.
Figure 5
Figure 5
Menaquinone-4, but not vitamin K1, enhances testosterone production in I-10 cells. I-10 cells were treated with 10 μM vitamin K1 (K1), or menaquinone-4 (MK-4) for 24 h, and testosterone levels in the culture medium were determined by ELISA (A), or I-10 cells were treated with K1 or MK-4 for 3 h, and vitamin K levels in cells were determined by fluorescent-HPLC (B). Data are represented as means ± SE (n = 3). Values with different letters are significantly different at p < 0.05. ND, not detected.
Figure 6
Figure 6
Menaquinone-4 increases CYP11A protein levels in I-10 cells. I-10 cells were treated with menaquinone-4 (MK-4) for the indicated time. After the treatment, whole cell extract was prepared, and CYP11A and StAR levels were measured by Western blot analysis (A). Whole cell extracts from the testis of rats fed control (Cont) or MK-4 supplemented (MK-4 sup) diets for 5 weeks were prepared, and CYP11A levels were measured by Western blot analysis (B). Data are represented as mean ± SE (n = 3) normalized to α-tubulin (A) or β-actin levels (B) and expressed as a fold-increase/decrease of values compared to the MK-4 control cells or the control diet group. Values with different letters are significantly different at p < 0.05.
Figure 7
Figure 7
Menaquinone-4 stimulates the protein kinase A pathway in I-10 cells. I-10 cells were treated with menaquinone-4 (MK-4) or forskolin (Fsk) for 2 h. After the treatment, whole cell extracts were prepared, and the phosphorylation levels of protein kinase A (p-PKA) and CREB (p-CREB) were measured by Western blot analysis (A). I-10 cells were treated with MK-4 for the indicated time. After the treatment, p-PKA and p-CREB levels in the whole cell extract were measured by Western blot analysis (B). Data are represented as mean ± SE (n = 3), normalized to α-tubulin levels and expressed as a fold-increase/decrease compared to the control cell values (A: cells not treated with MK-4 or Fsk, B: cells treated with MK-4 for 0 min). Values with different letters are significantly different at p < 0.05.
Figure 8
Figure 8
Treatment with PKA inhibitor abolishes the enhancement of testosterone production by menaquinone-4 in I-10 cells. I-10 cells were treated with menaquinone-4 (MK-4) and protein kinase A inhibitor H89 simultaneously for 24 h, and then testosterone levels in the culture medium were determined by ELISA. Data are represented as means ± SE (n = 3). Values with different letters are significantly different at p < 0.05.

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