Low-dose vitamin K2 (MK-4) supplementation for 12 months improves bone metabolism and prevents forearm bone loss in postmenopausal Japanese women.

Menaquinone-4 (MK-4) administered at a pharmacological dosage of 45 mg/day has been used for the treatment of osteoporosis in Japan. However, it is not known whether a lower dose of MK-4 supplementation is beneficial for bone health in healthy postmenopausal women. The aim of this study was to examine the long-term effects of 1.5-mg daily supplementation of MK-4 on the various markers of bone turnover and bone mineral density (BMD). The study was performed as a randomized, double-blind, placebo-controlled trial. The participants (aged 50-65 years) were randomly assigned to one of two groups according to the MK-4 dose received: the placebo-control group (n = 24) and the 1.5-mg MK-4 group (n = 24). The baseline concentrations of undercarboxylated osteocalcin (ucOC) were high in both groups (>5.1 ng/ml). After 6 and 12 months, the serum ucOC concentrations were significantly lower in the MK-4 group than in the control group. In the control group, there was no significant change in serum pentosidine concentrations. However, in the MK-4 group, the concentration of pentosidine at 6 and 12 months was significantly lower than that at baseline. The forearm BMD was significantly lower after 12 months than at 6 months in the control group. However, there was no significant decrease in BMD in the MK-4 group during the study period. These results suggest that low-dose MK-4 supplementation for 6-12 months improved bone quality in the postmenopausal Japanese women by decreasing the serum ucOC and pentosidine concentrations, without any substantial adverse effects.

Low-dose menaquinone-4 improves γ-carboxylation of osteocalcin in young males: a non-placebo-controlled dose-response study.

BACKGROUND: Menaquinone-4 is a type of vitamin K that has a physiological function in maintaining bone quality via γ-carboxylation of osteocalcin. However, little is known about the beneficial effect of intake of dosages below1500 µg/day. FINDINGS: Fifteen healthy males aged 25.0 years (median) participated in a non-placebo-controlled dose-examination study. They received menaquinone-4 daily for 5 weeks at 0, 300, 600, 900, and 1500 µg/day in weeks 1, 2, 3, 4, and 5, respectively. Compared with baseline, serum γ-carboxylated osteocalcin levels were significantly greater at an intake of 900 µg/day or more; serum undercarboxylated osteocalcin levels and the ratio of serum undercarboxylated osteocalcin to γ-carboxylated osteocalcin were significantly lower than baseline at doses of 600 µg/day or more. CONCLUSIONS: This preliminary graded-dose study suggested that menaquinone-4 supplementation at 600 µg/day or more is likely to be important in terms of vitamin K requirements for bone health.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

Increasing effect of an oral intake of L-hydroxyproline on the soluble collagen content of skin and collagen fragments in rat serum.

We examined the effects of oral L-hydroxyproline (Hyp) on collagen in the body. After 2 weeks' administration of Hyp (0.5 or 1 g/kg) to F344 male rats, the soluble collagen content of the skin had increased, and the serum concentration of collagen peptides was correlated with the skin content of soluble collagen. This result suggests that oral Hyp augmented collagen metabolism.

Orally administered L-ornithine elevates brain L-ornithine levels and has an anxiolytic-like effect in mice.

Intracerebroventricular injection of L-ornithine has demonstrated sedative and hypnotic effects in neonatal chicks exposed to acute stressful conditions. However, whether orally administered L-ornithine can reduce acute mental stress remains to be defined. To clarify the nutritional importance of L-ornithine in controlling the stress response, in Experiment 1 we first investigated whether orally administered L-ornithine can be transported into the brain of mice. Mice were orally administered L-ornithine (3 mmol/water 10 ml/kg, per os). L-Ornithine levels were significantly elevated in the cerebral cortex and hippocampus at 30 and 60 minutes post-administration. In Experiment 2, the effect of orally administered L-ornithine (0, 0.1875, 0.75 and 3 mmol/water 10 ml/kg, per os) on anxiety-like behavior in mice exposed to the elevated plus-maze test was examined at 30 minutes post-administration. There was a significant increase in the percentage of time spent and entries in the open arms in the group receiving 0.75 mmol of L-ornithine compared to the control group. Furthermore, locomotion activity in a novel environment was not significantly changed between the control group and 0.75 mmol of L-ornithine group in Experiment 3. Therefore, it appears that orally administrated L-ornithine is bioavailable to the rodent brain and reduces anxiety-like behavior as demonstrated by the elevated plus-maze test.

Sex differences in behavioral and corticosterone responses to mild stressors in ICR mice are altered by ovariectomy in peripubertal period.

Among rodents, females are generally considered to be highly responsive in terms of emotionality under stressful conditions, and have higher corticosterone levels and activity. In this study, we examined sex differences in mice by evaluating anxiety behaviors and corticosterone responses to mild stressors. In our first experiment, we analyzed the behavioral and corticosterone responses to the elevated plus-maze test and open-field test in male and female mice, and compared sex differences. Principal component analysis (PCA) was used to investigate the correlation of these responses between males and females. The corticosterone level was higher in females under both basal and stressed conditions. In the behavioral response, higher locomotor activity was seen in females in the elevated plus-maze test. PCA showed little association among anxiety behavior, locomotor activity, and corticosterone secretion. In our second experiment, we examined the activational effects of sex steroids on the corticosterone response to the elevated plus-maze test by gonadectomizing male and female mice and using testosterone or estrogen capsules as hormonal replacements. Sex differences at the basal corticosterone level were not altered by the hormonal milieu in adults, however the higher corticosterone level of females in response to stress was diminished by ovariectomy, although replacement with neither testosterone nor estrogen had any effect. These results suggest that the sex difference in novelty exposure observed in the form of a greater hypothalamic-pituitary-adrenal (HPA) axis response in female ICR mice is controlled by ovary-derived factors in adults.

Central L-ornithine, but not polyamines, induces a hypnotic effect in neonatal chicks under acute stress.

To clarify whether L-ornithine and/or its metabolite involves sedative and hypnotic effects under social separation stress, the effects of intracerebroventricular (i.c.v.) injection of L-ornithine and polyamines (putrescine, spermidine and spermine) were compared in chicks. Birds were injected i.c.v. with 0.5 mumol of L-ornithine, putrescine, spermidine, spermine or saline (control). After injection, chicks were immediately separated from the flock and monitored for the number of distress vocalizations and various postures. L-Ornithine greatly attenuated the stress response and caused sedative and hypnotic effects. Among the polyamines, only putrescine attenuated distress vocalizations but did not induce sleep. In conclusion, the sedative and hypnotic effect of L-ornithine was mainly induced by L-ornithine itself, while the polyamines contributed to the sedative, but not hypnotic, effect under social separation stress.

Wheel-running activity increases with social stress in male DBA mice.

Social affiliation-avoidance behaviors are essential indices of sociality. We examined changes in social affiliation-avoidance behaviors in an open-field apparatus while simultaneously measuring wheel-running activity. Recent studies suggest that mice increase wheel-running activity in stressful situations; thus, we hypothesized that wheel-running activity would reflect a state of social stress and avoidance. Mean duration of wheel-running increased significantly when mice were confronted with unfamiliar mice compared to cage mates. There were negative correlations between the amount of wheel-running and social affiliation indices. We also examined the effect of social defeat on wheel-running activity. Mice that had experienced social defeat significantly increased their wheel-running when an aggressor mouse was present. This social defeat-induced wheel-running activity was ameliorated by the administration of diazepam. Our results indicate that wheel-running activity is relevant to social affiliation-avoidance behaviors and may be a reliable index of anxiety induced by social stress.

Effects of time of L-ornithine administration on the diurnal rhythms of plasma growth hormone, melatonin, and corticosterone levels in mice.

The synthesis and secretion of many hormones such as growth hormone (GH), melatonin, and corticosterone, exhibit temporal variations over each day and night. Oral administration of several nutritional factors, including L-ornithine, modulates these hormonal secretions and induces an acute increase in plasma GH levels. However, the impact of L-ornithine on the diurnal rhythms of hormone secretion remains unclear. In this study, we evaluated whether the diurnal rhythms of plasma GH, melatonin, and corticosterone secretion were altered by the daily administration of L-ornithine as well as the timing of the administration, in CBA/N mice. Our results showed that the plasma GH levels that peaked at light phase were amplified by L-ornithine (500 mg/kg) administered at Zeitgeber time (ZT) 22, but not at ZT10. Additionally, L-ornithine (1000 mg/kg) administered at ZT22 advanced the onset of the nocturnal rise of melatonin, which resulted in the elongation of the melatonin peak. On the other hand, L-ornithine (500 and 1000 mg/kg) administered at ZT10, but not at ZT22, suppressed the diurnal rhythm peaks of plasma corticosterone. The effects of L-ornithine on plasma GH rhythms lasted for at least 2 days after cessation of the daily administration. Running wheel activity during the active phase was slightly elevated by L-ornithine administration at ZT22, but the overall patterns were only slightly affected. L-Ornithine levels in the plasma and hypophysis after a single administration of L-ornithine at ZT22 were lower than those after administration at ZT10, suggesting that the metabolic rate of L-ornithine differs between day and night. In conclusion, our data suggest that a daily administration of L-ornithine regulates the diurnal rhythms of GH, melatonin, and corticosterone in a manner dependent on administration time, which might be related to the diurnal rhythms of L-ornithine metabolism.

Dietary ornithine affects the tissue protein synthesis rate in young rats.

The purpose of this study was to determine whether ornithine affects the rate of tissue protein synthesis in male rats. Two experiments were done on five or two groups of young rats (5 wk) given diets containing 0.15, 0.3, 0.5 or 0.7% ornithine-HCl added to a 20% casein diet for 1 d (only one 3 h period) (Experiment 1), and given a diet containing 0 or 0.7% ornithine-HCl added to a 20% casein diet for 10 d (Experiment 2). The plasma concentration of growth hormone (GH) was the highest in rats fed 0.5 and 0.7% ornithine added to the 20% casein diet. The fractional rates of protein synthesis in brain regions, liver and gastrocnemius muscle increased significantly with the 20% casein+0.7% ornithine diet compared with the 20% casein diet. In brain regions, liver and gastrocnemius muscle, the RNA activity [g protein synthesized/(g RNA·d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The present results suggest that the treatment of young rats with ornithine is likely to increase the concentration of plasma GH and the rate of protein synthesis in the tissues, and that RNA activity is at least partly related to the fractional rate of tissue protein synthesis.

Effect of dietary ornithine on the brain protein synthesis rate in hypophysectomized aged rats.

The purpose of this study was to determine whether the regulation of brain protein synthesis is mediated through changes in the plasma concentration of growth hormone (GH) when dietary ornithine treatment is manipulated in the hypophysectomized or sham-operated aged rats. Experiments were done on four groups of hypophysectomized and sham-operated (24-wk-old) male rats given 0% or 0.7% ornithine-HCl added to a 20% casein diet. The concentrations of plasma GH and fractional rates of protein synthesis in the brains increased significantly with the 20% casein+0.7% ornithine compared with the 20% casein diet alone in the sham-operated rats. However ornithine supplementation to the basal diet did not affect the rates of protein synthesis in the hypophysectomized rats. In the cerebral cortex and cerebellum, the RNA activity [g protein synthesized/(g RNA•d)] significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was also related to the fractional rate of protein synthesis in these organs. The results suggest that the treatment with ornithine is likely to increase the concentration of GH and the rate of brain protein synthesis in the sham-operated rats only, not in the hypophysectomized rats, and that the ornithine-induced increase in the concentration of GH may be primarily responsible for changes in the brain protein synthesis. The RNA activity is at least partly related to the fractional rate of brain protein synthesis.

Orally administered L-ornithine reduces restraint stress-induced activation of the hypothalamic-pituitary-adrenal axis in mice.

In a previous study, we confirmed that orally administered L-ornithine can be transported into the brain of mice. In addition, orally administered L-ornithine, within a limited dose range, had an anxiolytic-like effect in the elevated plus-maze test. However, the mechanism by which orally administered L-ornithine reduced the stress response in mice is still unclear. Experiment 1 determined whether orally administered L-ornithine could reduce the stress-induced activation of hypothalamic-pituitary-adrenal axis. Mice were orally administered L-ornithine (0, 0.75, 1.5 and 3 mmol/10 ml/kg, p.o.), and restrained for 30 min from 30 min post administration. There was a significant decrease in the corticosterone levels in the group receiving 0.75 mmol of L-ornithine compared to the control group. In Experiment 2, the effect of orally administered L-ornithine (0 and 0.75 mmol/10 ml/kg, p.o.) on endogenous monoamine release was investigated using in vivo microdialysis. Only the monoamines metabolites 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and homovallinic acids (HVA) were detected in the present study. Dialysate concentrations of 5-HIAA, DOPAC and HVA were not significantly changed immediately after administration of L-ornithine and restraint stress. In conclusion, changes of corticosterone concentrations by orally administered L-ornithine were not related to alterations in brain monoamine metabolisms.

Expression of programmed death 1 ligands by murine T cells and APC.

Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.