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1. Introduction

A dose-finding study of menatetrenone in Japan [] administered daily doses of 15, 45, 90, and 135 mg and revealed that 45 mg was the minimum effective dose for improving bone mass parameters evaluated by microdensitometry and/or single photon absorptiometry in postmenopausal women with osteoporosis. This optimal dose (45 mg/day) for the treatment of osteoporosis is about 150–180 times greater than the recommended daily dietary intake of vitamin K (250–300 μg) []. No toxic effects of menatetrenone (45 mg/day) have been reported []. High-dose vitamin K is needed to prevent fractures in postmenopausal women with osteoporosis []. However, the effect of menatetrenone on the skeleton remains a matter of controversy [,,,,,,,], and the role of menatetrenone in the treatment of osteoporosis therefore needs to be clarified. In clinical practice, menatetrenone is frequently used in combination with bisphosphonates.

3. Menatetrenone Monotherapy

Menatetrenone reduces serum ucOC concentrations [,]. The anti-fracture efficacy of menatetreone in postmenopausal osteoporosis has been shown in eight RCTs () [,,,,,,,]. Of the eight RCTs, six were performed in Japan, one in Indonesia, and one in China. One Japanese RCT was a phase IV trial (Osteoporotic Fracture (OF) study) conducted in a large sample of postmenopausal women with osteoporosis (n = 4378) []. The dose of menatetrenone was 45 mg/day in all these studies. Placebo controls were used in one RCT, non-treatment controls in four RCTs, calcium supplementation controls in two RCTs, and active controls (alfacalcidol) in one RCT. The number of study subjects ranged from 44–4378 in the menatetrenone and control groups, and the study periods ranged from 1–3 years.

5. Discussion

The present review revealed positive evidence for the effect of menatetrenone monotherapy on BMD and the incidence of fractures (mainly vertebral fractures) in postmenopausal women with osteoporosis. However, this evidence was derived from RCTs with small sample sizes. Thus, menatetrenone is used as a second-line medicine for the treatment of postmenopausal osteoporosis in Japan []. Knapen et al. [] reported that menatetrenone (45 mg/day) improved hip bone geometry, bone strength indices, and bone mineral content in healthy postmenopausal women, supporting the effect of menatetrenone on bone health.

4. Combined Menatetrenone and Alendronate Therapy

Hirao et al. [] conducted a RCT to examine the benefit of combined menatetrenone and alendronate therapy in postmenopausal women with osteoporosis. The increase in femoral neck BMD and the decrease in serum ucOC concentrations were greater in the alendronate plus menatetrenone group compared with the alendronate alone group.

Abstract

A novel application of the liquid chromatography method combined with the triple quadrupole tandem mass spectrometry method was developed for the quantification of vitamin K1 and two forms of vitamin K2 (menaquinone-4, menaquinone-7) in human serum. Total chromatography time for each run was 9 min. Time required for the sample pretreatment procedures was approximately 4 h. The coefficients of variation (CVs) of intra-assay were 10.4%, 3.2 % and 2.3% for vitamin K1 in three levels of quality control samples; were 14.3%, 3.2% and 6.7% for menaquinone-4; and were 11.1%, 6.0% and 7.0% for menaquinone-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for vitamin K1; were 15.2%, 9.2% and 8.7% for menaquinone-4; and were 13.2%,11.1% and 7.2% for menaquinone-7. No interference was found between K1, menaquinone-4 and menaquinone-7, nor any deuterated internal standards. This method was then used to determine reference values for Caucasian populations of central European origin. Samples were measured from 191 healthy volunteers (51.2 ± 16.2 years (mean ± SD)) and the values concerning K1 were 0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. The values for menaquinone-4 and menaquinone-7 did not exhibit any differences between women and men, and were 0.050–1.598 and 0.074–0.759 ng/mL, respectively.

Subject and study design

Volunteers were determined to be healthy according to their medical history, physical examination and standard laboratory procedures. Samples were measured from 191 volunteers (51.2 ± 16.2 years (mean ± SD)), with 149 participants being female and 42 male. Ethical approval for this study was obtained from Ethics committee of University Hospital Motol (Ref.No.: EK-1422/13). Written informed consent was obtained from the participants at recruitment prior to their inclusion in the study.

Results

Our LC–MS/MS method for analysis of K1, MK-4 and MK-7 in human serum has been successfully validated following the criteria of the . Calibration curves were established by plotting the peak area ratios K1, MK-4 and MK-7 against IS at 10-point concentrations. Pooled serum samples exposed to UV light were spiked with 0.03, 0.05, 0.1, 0.3, 0.6, 1.0, 1.5, 2.0, 4.0 and 10.0 ng/mL to obtain calibration curves. The assays were linear across the whole range of concentrations with mean correlation coefficients R2 of 0.980 for K1, 0.994 for MK-4 and 0.978 for MK-7. The first point of calibration curves (0.03 ng/mL) corresponds to the lower limit of quantification (LLOQ). Limits of detection (LoD) were determined by a further dilution of the first calibration point (LLOQ). Concentrations of 0.0019 ng/mL for K1 and MK-7 and 0.00375 ng/mL for MK-4 were obtained.

Results

We measured the vitamin K levels in a healthy Central European population. The group of patients consisted of 191 volunteers, 22% of them male, ages 51 ± 16 years (mean ± SD). Serum levels of K1, MK-4 and MK-7 from the healthy Central European population were: 0.03–2.19 (0.20) ng/mL, 0.05–2.79 (0.19) ng/mL, and 0.05–1.39 (0.09) ng/mL, respectively (min—max (median)).

Abstract

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, β-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic β-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

2.1. MK-4 Enhanced GSIS in Isolated Mouse Pancreatic Islets and Rat INS-1 Cells

To confirm the effect of MK-4 on GSIS, isolated islets and INS-1 cells were used in the present study. To determine the dosage of MK-4 for INS-1 cells, we analyzed the cell survival rate under 0–100 µM MK-4 treatment for 24 h by using the WST-1 assay (), which suggested that the concentration of 3 µM MK-4 was harmless to the cells. Accordingly, MK-4 at 1 and 3 µM was used in INS-1 cells for the subsequent experiments. We then evaluated the effect of MK-4 on GSIS in INS-1 cells, as shown in . MK-4 enhanced insulin levels in both basal (2.8 mM) and high (16.8 mM) glucose-stimulated INS-1 cells in a dose-dependent manner. We used mouse pancreatic islets isolated from genetically diabetic KK-Ay mice subsequently. The isolated pancreatic islets were stimulated with 2.8 mM Glc and MK-4 for 1 h, and 20 µM MK-4 was found to enhance GSIS about two fold compared with the 0 µM MK-4 group; thus, MK-4 amplified GSIS in the isolated mouse pancreatic islets of KK-Ay mice (). Moreover, we also found that MK-4 enhanced GSIS in C57BL/6J mice (). Hence, MK-4 enhanced insulin secretion in both isolated islets and INS-1 cells under glucose stimulation, and pancreatic β-cells were the target of MK-4.

2.2. MK-4 Increased cAMP Levels in INS-1 Cells

To investigate the mechanism by which MK-4 regulates GSIS in β-cells, INS-1 cells were used in the following experiments. Initially, intracellular cAMP was measured in INS-1 cells because increased cAMP levels promote nutrient-induced insulin secretion in enteroendocrine cells. As shown in , the level of cAMP tended to increase with the lower dose (1 µM MK-4), and was significantly increased in the group treated with 3 µM MK-4 for 1 h. These results revealed that MK-4 might enhance insulin secretion by regulating the cAMP-dependent pathway in INS-1 cells.

2.3. MK-4 Amplified GSIS by Regulating the cAMP/Epac-Dependent Pathway but not the cAMP/PKA-Dependent Pathway in INS-1 Cells

To verify the effect of MK-4 on cAMP-dependent pathways, the cAMP/PKA pathway was investigated first (). However, through the cAMP response element (CRE)-reporter gene assay, it was found that luciferase activity did not change with either 1 or 3 µM of MK-4 treatment (A). Not unexpectedly, treatment with the protein kinase A (PKA) inhibitor H89 also did not affect the influence of MK-4 on GSIS (B), which showed that MK-4 might not regulate PKA activity in INS-1 cells. Therefore, we analyzed another cAMP-dependent pathway, the cAMP/Epac2 pathway of GSIS, in INS-1 cells. We blocked the cAMP/Epac2 pathway using the exchange protein directly activated by the cAMP 2 (Epac2) inhibitor ESI-05. As shown in , the insulinotropic effect of MK-4 might have been abolished by the Epac2 inhibitor, suggesting that MK-4 amplified GSIS through the regulation of the cAMP/Epac-dependent pathway but not the cAMP/PKA-dependent pathway in INS-1 cells.

Abstract

Cerebral ischemia/reperfusion (I/R) injury causes cognitive deficits, excitotoxicity, neuroinflammation, oxidative stress and brain edema. Vitamin K2 (Menaquinone 4, MK-4) as a potent antioxidant can be a good candidate to ameliorate I/R consequences. This study focused on the neuroprotective effects of MK-4 for cerebral I/R insult in rat’s hippocampus. The rat model of cerebral I/R was generated by transient bilateral common carotid artery occlusion for 20 min. Rats were divided into control, I/R, I/R+DMSO (solvent (1% v/v)) and I/R+MK-4 treated (400 mg/kg, i.p.) groups. Twenty-four hours after I/R injury induction, total brain water content, superoxide dismutase (SOD) activity, nitrate/nitrite concentration and neuronal density were evaluated. In addition to quantify the apoptosis processes, TUNEL staining, as well as expression level of Bax and Bcl2, were assessed. To evaluate astrogliosis and induced neurotoxicity by I/R GFAP and GLT-1 mRNA expression level were quantified. Furthermore, pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α were measured. Seven days post I/R, behavioral analysis to quantify cognitive function, as well as Nissl staining for surviving neuronal evaluation, were conducted. The findings indicated that administration of MK-4 following I/R injury improved anxiety-like behavior, short term and spatial learning and memory impairment induced by I/R. Also, MK-4 was able to diminish the increased total brain water content, apoptotic cell density, Bax/ Bcl2 ratio and GFAP mRNA expression following I/R. In addition, the high level of nitrate/nitrite, IL-6, IL-1β and TNF-α induced by I/R was reduced after MK-4 administration. However, MK-4 promotes the level of SOD activity and GLT-1 mRNA expression in I/R rat model. The findings demonstrated that MK-4 can rescue transient global cerebral I/R consequences via its anti-inflammatory and anti-oxidative stress features. MK-4 administration ameliorates neuroinflammation, neurotoxicity and neuronal cell death processes and leads to neuroprotection.

Animals

172 Adult male Wistar rats with weighing 250–300 gr selected at random from the animal facility of Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran. Animals were housed in standard cages under controlled room temperature (22–24°C) and humidity (45–50%) and exposed to 12:12 h light–dark cycle conditions (lights on at 08:00 am) (). The experimental procedures were approved by the National Food Chain Safety and conducted according to Institutional Animal Ethics Committee guidelines for the care and use of laboratory animals [, ].

Surgical procedure for induction global cerebral I/R model

Rats were anesthetized by an intraperitoneal (i.p.) injection of Xylazine 20 mg/kg and Ketamine 100 mg/kg. The body temperature was monitored and maintained between 37.1°C and 37.3°C under free-regulating conditions with using a thermometer and heating pad during and after the surgery up to the animals recovered from anesthesia. After separation of the vagus nerve, bilateral common carotid arteries occlusion (two-vessel occlusion; 2-VO) was conducted by 20 min clamping [, , ]. After 20 min, clamps were removed for reperfusion onset either for 24 hours or for 7 days depends on different experimental sets. After surgery, animals were placed in home cages with free access to food and water. Some of the rats were removed from experiments because of: 1) death induced by ischemia, 2) movement impairment, 3) visual deficiency, 4) difficulties in reperfusion (not operated in both common carotids) and 5) seizure.

Experimental design

216 animals were divided in 4 groups as follows: Age-matched control group, I/R injury group (sham control group), I/R injury + 2 times DMSO i.p. injection (solvent of MK-4, 1% v/v) [], I/R injury + 400 mg/kg MK-4 (2 times 200 mg/kg i.p. injections, immediately and 2h after reperfusion). This dosage was selected empirically. At first, the dose of 100 mg/kg MK-4 i.p. was selected due to the previous investigations [–], however, the mortality rate of ischemic animals was high (equal I/R group). To this reason, we had to enhance the treatment dose to 400 mg/kg in two injections (200 mg/kg i.p. immediately and 200 mg/kg i.p. 2h after reperfusion). The high number of survived rats was observed subsequently. Besides, earlier studies showed that a high dose of vitamin K2 has very low toxicity [, ]. Behavioral tests were conducted 7 days post reperfusion in healthy animals. TUNEL and Nissl histological staining were performed 24h and 7 days after reperfusion respectively. The brain volume changes for total brain water content assessment was measured 24 hours after reperfusion. In addition, 24h after I/R injury ELISA was used for detection of pro-inflammatory cytokines including TNF-α, IL-1β, IL-6 and real-time PCR was utilized for evaluation of mRNA level of GFAP, Bax, Bcl-2 and GLT-1 (EAAT2). Then, the activity of SOD enzyme and NO concentration were assessed ().