Recent Topics in the Studies of Laboratory of Nutrition, Tohoku University : Newly Clarified Function of Vitamin K

Vitamin K (VK) is essential for blood coagulation and bone metabolism in mammals as a Gla-protein activating factor, i.e., VK acts as a cofactor in the posttranslational synthesis of γ-carboxyglutamic acid (Gla) from glutamic acid (Glu) residues in the nascent proteins. Menaquinone-4 (MK-4) is one of the VK_2 analogues, and is well known to have bioactivity in the suppression of bone resorption through apoptosis of osteoclast cells, thus MK-4 is now also used clinically as a therapeutic drug for the osteoporosis. Besides of these well-known functions, MK-4 is strongly presumed to have other novel functions because we have gradually recognized that MK-4 accumulates in various tissues of germfree animals fed an MK-4-free diet. Accordingly, we have focused on clarification of the mechanism of MK-4 formation in several tissues, using both in vitro tissue homogenates (bovine, rats, mice, chicken) and in vivo experiments with rats and mice. To elucidate the biological role of MK-4 production, we used germfree rats to eliminate MK-n synthesized by intestinal flora. Our in vivo results indicate that MK-4 is produced in diverse tissues from ingested vitamin K analogues, including vitamin K_1, MK-n (MK-6, MK-7, and MK-10); and VK_3 without enzymatic participation of microorganisms in the intestine. In addition to the liver and bone, VK is found in the brain, heart, testis, kidney, pancreas and salivary glands mainly as menaquinone-4 (MK-4). However, the physiological role of MK-4 in these various organs has not been fully understood yet. In the present study we identified genes of which expression is changed in testis under vitamin K deficient condition using DNA microarray. The genes involved in the biosynthesis pathway of cholesterol and steroid hormone were decreased in vitamin K deficient group. The amount of Cyp11a (P450scc) mRNA, rate-limiting enzyme for testosterone synthesis, was positively correlated with the concentration of MK-4 in testis. Moreover, the concentration of testosterone in plasma and testis was decreased in vitamin K deficient group compared with the control and vitamin K supplemented groups. These results suggests that vitamin K is involved in steroid production in testis through the regulation of Cyplla

Menaquinone-4 enhances testosterone production in rats and testis-derived tumor cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K₂vitamins present in the testis, for 5 weeks. <it>In vivo </it>testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and <it>in vitro </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Combined Supplementation of Pre-Exercise Carbohydrate, Alanine, and Proline and Continuous Intake of Green Tea Catechins Effectively Boost Endurance Performance in Mice

Continuous intake of green tea catechins (GTC) increases fatty acid utilization as an energy source and improves endurance capacity. Conversely, the single pre-exercise intake of maltodextrin (MD) as a carbohydrate source and the gluconeogenic amino acids alanine (Ala) and proline (Pro) effectively maintain blood glucose levels and increase endurance performance. In this study, we investigated the synergistic combinational effect of these interventions on endurance performance in mice. Male BALB/c mice were fed a 0.5% GTC diet or Control diet for 8 weeks. Maximum running time was measured every 2 weeks. MD (2 g/kg body weight (B.W.)), MD (1 g/kg B.W.) + AlaPro (9:1, 1 g/kg B.W.), and vehicle were orally administrated 60 mins before measurements in each diet group. The GTC + MD + AlaPro group showed significantly higher endurance performance than the Control-Vehicle group at all measurements. Indirect calorimetry analysis during running exercise at 4 weeks in the Control and GTC groups supplemented with pre-exercise MD + AlaPro administration revealed significantly higher fat oxidation in the GTC groups compared to the Control group. The combined increase in fatty acid utilization through continuous GTC intake and pre-exercise MD + AlaPro carbohydrate energy supplementation synergistically improves endurance capacity

The Impact of Milk Fat Globule Membrane with Exercise on Age-Related Degeneration of Neuromuscular Junctions

Morphological changes in neuromuscular junctions (NMJs), which are synapses formed between α-motor neurons and skeletal muscle fibers, are considered to be important in age-related motor dysfunction. We have previously shown that the intake of dietary milk fat globule membrane (MFGM) combined with exercise attenuates age-related NMJ alterations in the early phase of aging. However, it is unclear whether the effect of MFGM with exercise on age-related NMJ alterations persists into old age, and whether intervention from old age is still effective when age-related changes in NMJs have already occurred. In this study, 6- or 18-month-old mice were treated with a 1% MFGM diet and daily running wheel exercise until 23 or 24 months of age, respectively. MFGM treatment with exercise was effective in suppressing the progression of age-related NMJ alterations in old age, and even after age-related changes in NMJs had already occurred. Moreover, the effect of MFGM intake with exercise was not restricted to NMJs but extended to the structure and function of peripheral nerves. This study demonstrates that MFGM intake with exercise may be a novel approach for improving motor function in the elderly by suppressing age-related NMJ alterations