Monthly Archives: May 2014

Lower NAD+ Associated with Decline in Osteogenesis

Abstract

Nampt expression increases during osteogenic differentiation of multi- and omnipotent progenitors.

Despite emerging data showing that metabolic changes occur with stem cell differentiation, the cross-talk between factors governing energy metabolism and epigenetic modification is not understood. Nicotinamide adenine dinucleotide (NAD) participates in both energy metabolism and protein modification processes. Changes of the intracellular NAD concentration have been shown to correlate with differentiation of adult and embryonic stem cells. In the present study, we investigated the expression pattern of Nampt, the rate-limiting enzyme in NAD salvaging pathway, during osteogenic differentiation of the multipotent mouse fibroblast C3H10T1/2 and the omnipotent preosteoblast MC3T3-E1 cells. We found that Nampt was increasingly expressed during differentiation in both cell models. The increase of Nampt was associated with higher NAD concentration and Sirt1 activity. Knockdown of Nampt or addition of its specific inhibitor FK866 leads to lower intracellular NAD concentration and decline in osteogenesis. These findings indicate that osteogenic differentiation correlates with intracellular NAD metabolism in which Nampt plays a regulatory role.

Li Y, He J, He X, Li Y…
Biochem. Biophys. Res. Commun. Apr 2013
PMID: 23537654

NAD+ Related to Mitochondria Function of Osteoblasts

Abstract

Involvement of PI3K/Akt/CREB and redox changes in mitochondrial defect of osteoblastic MC3T3-E1 cells.

Antimycin A (AMA) is an inhibitor of mitochondrial electron transport via its binding to complex III. In the present study, the mechanisms involved in AMA-induced cell damage were investigated. Treatment of osteoblastic MC3T3-E1 cells with AMA decreased adenosine 3′,5′-cyclic monophosphate (cAMP) level, activities of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B), and phosphorylated CREB (cAMP-response element-binding protein).
To examine whether AMA-induced cell damage involves altered metabolism of pyridine nucleotides, the levels of NAD(+), NADH, NADP(+), and NADPH were measured. Treatment with AMA significantly decreased the levels of NAD(+) and NADPH. Moreover, the activities of aconitase and thioredoxin reductase were decreased by AMA treatment. These results suggest that PI3K/Akt/CREB pathway and pyridine nucleotide (NAD(+) and NADPH) are related to mitochondria function of osteoblasts.

Choi EM, Lee YS
Toxicol In Vitro Aug 2011
PMID: 21466842

NAD+ Involved in Stimulation and Maintenance of Osteoblasts In Vitro

Abstract

Extracellular NAD+: a novel autocrine/paracrine signal in osteoblast physiology.

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.

Romanello M, Bicego M, Pirulli D, Crovella S…
Biochem. Biophys. Res. Commun. Dec 2002
PMID: 12445818

SIRT6 Boosts Bone Formation

Abstract

SIRT6 regulates osteogenic differentiation of rat bone marrow mesenchymal stem cells partially via suppressing the nuclear factor-kappa B signaling pathway.

Sirtuin 6 (SIRT6) is a NAD-dependent deacetylase involved in lifespan regulation. To evaluate the effect of SIRT6 on osteogenesis, rat bone marrow mesenchymal stem cells (rBMSCs) with enhanced or reduced SIRT6 function were developed. We observed that SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN), while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor-kappa B (NF-κB) transcriptional activity and upregulated the expression of acetyl-NF-κB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF-κB inhibitor BAY 11-7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of collagen/chitosan/HA scaffold (CHHS) could significantly boost new bone formation in rat cranial critical-sized defects, as determined by microcomputed tomography and histological examination. These data confirm that SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF-κB signaling.

Sun H, Wu Y, Fu D, Liu Y…
Stem Cells Feb 2014
PMID: 24510807

Review: Vitamin K2 (MK-4) Monotherapy Modestly Increases Bone Density and Reduces Fractures in Eight Studies

Abstract

Vitamin k2 therapy for postmenopausal osteoporosis.

Vitamin K may play an important role in the prevention of fractures in postmenopausal women with osteoporosis. Menatetrenone is the brand name of a synthetic vitamin K2 that is chemically identical to menaquinone-4. The present review study aimed to clarify the effect of menatetrenone on the skeleton in postmenopausal women with osteoporosis, by reviewing the results of randomized controlled trials (RCTs) in the literature. RCTs that investigated the effect of menatetrenone on bone mineral density (BMD), measured by dual-energy X-ray absorptiometry and fracture incidence in postmenopausal women with osteoporosis, were identified by a PubMed search for literature published in English. Eight studies met the criteria for RCTs. Small RCTs showed that menatetrenone monotherapy decreased serum undercarboxylated osteocalcin (ucOC) concentrations, modestly increased lumbar spine BMD, and reduced the incidence of fractures (mainly vertebral fracture), and that combined alendronate and menatetrenone therapy enhanced the decrease in serum ucOC concentrations and further increased femoral neck BMD. This review of the literature revealed positive evidence for the effects of menatetrenone monotherapy on fracture incidence in postmenopausal women with osteoporosis. Further studies are required to clarify the efficacy of menatetrenone in combination with bisphosphonates against fractures in postmenopausal women with osteoporosis.

Iwamoto J
Nutrients 2014
PMID: 24841104 | Free Full Text


One interesting passage from the full text talks about the unpublished dose range study from Japan:

Orimo, H., et al. “Clinical evaluation of soft capsule menatetrenone (Ea-0167) in the treatment of osteoporosis: late phase II dose study.” J New Remedies Clinics 41 (1992): 1249-79.

A dose-finding study of menatetrenone in Japan [7] 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) [8]. No toxic effects of menatetrenone (45 mg/day) have been reported [7]. High-dose vitamin K is needed to prevent fractures in postmenopausal women with osteoporosis [9]. However, the effect of menatetrenone on the skeleton remains a matter of controversy [10–17], and the role of menatetrenone in the treatment of osteoporosis therefore needs to be clarified.

Skipping Breakfast Associated with Lower Bone Density in Young Women

Abstract

Relationship between skipping breakfast and bone mineral density in young Japanese women.

It is well known that insufficient nutrient intake leads to poor bone status. To find a simple evaluation method for prevention of nutrition intake disorder, a cross-sectional study with 275 healthy Japanese female students aged 19-25 was conducted.
Anthropometric parameters, bone mineral density (BMD) at lumbar and total hip, bone metabolic markers and physical activity were measured in study participants and the frequency of skipping meals (breakfast, lunch, supper), and absolute values for nutrient intakes were assessed using a Diet History Questionnaire.
The frequency of skipping breakfast significantly correlate to total energy intake (ρ= -0.276, p<0.001). BMI, total intake of energy, intake of protein, intake of phosphate, and energy expenditure positively correlated significantly to BMD at lumbar and total hip (p<0.05) using simple linear regression. BMI (regression coefficient (b))=0.088, p<0.001), bone alkaline phosphatase (b= -0.050, p=0.012), total energy expenditure (b=0.019, p<0.001), and frequency of skipping breakfast (b= -0.018, p=0.048) were independent risk factors for lower total hip BMD by multiple regression analysis. The total hip BMD in participants who skipped breakfast three or more times was significantly lower than in those who did not skip breakfast (p=0.007).
In conclusion, managing the frequency of skipping breakfast and reducing it to <3 times per week may be beneficial for the maintenance of bone health in younger women.

Kuroda T, Onoe Y, Yoshikata R, Ohta H
Asia Pac J Clin Nutr 2013
PMID: 24231019 | Free Full Text

Breakfast and Exercise in High School Increases Bone Density in Men

Abstract

Consuming breakfast and exercising longer during high school increases bone mineral density in young adult men.

We examined the bone mineral densities (BMDs) of young adult men and analyzed the factors associated with BMD differences. Between 1993 and 2002, all male freshmen in the Wakayama Medical University, Japan were recruited into the present study, which included a self-administrated questionnaire survey, anthropometric measurements, and BMD measurements of the spine and hip. Of a total of 387 freshmen, 382 (98.7 %; mean age, 20.3 years; age range, 18-29 years) completed the study. The mean BMDs of the spine (L2-4) and femoral neck (FN) were 1.21 (standard deviation, 0.13) g/cm(2) and 1.12 (0.14) g/cm(2), respectively. The L2-4 BMDs were not associated with age, while FN BMDs were significantly inversely associated with age. The BMDs at L2-4 and FN were significantly associated with body mass index (BMI). After adjustment for age and BMI, multivariate regression analysis indicated that BMDs at L2-4 and FN were associated with current longer exercise duration (L2-4, p = 0.024; FN, p = 0.001), those at L2-4 with milk intake (p = 0.024), and those at FN with consuming breakfast (p = 0.004). Similarly, habits of consuming breakfast and exercising longer (on a weekly basis) during high school were linked with significantly higher L2-4 and FN BMDs. High-impact activities during high school significantly influenced the later BMDs. In conclusion, to maximize peak bone mass, consuming breakfast and completing a longer duration of stronger exercise in the late high school years for at least 10 h per week is recommended.

Ishimoto Y, Yoshida M, Nagata K, Yamada H…
J. Bone Miner. Metab. May 2013
PMID: 23263782

Breakfast and Exercise Important for Bone Mass in Young Adults

Abstract

Skipping breakfast and less exercise are risk factors for bone loss in young Japanese adults: a 3-year follow-up study.

Although bone loss contributes to osteoporosis (OP) in the elderly, little is known about changes in bone mineral density (BMD) in young adults that lead to bone loss. Here, we evaluated the rate of bone change and risk factors for bone loss in young men and women using data from a 3-year prospective study of Japanese medical students. The study included a self-administrated questionnaire survey, anthropometric measurements, and BMD measurements of the spine (L2-L4) and femoral neck (FN). After 3 years, the BMD of the participants was again measured at the same sites. In all, 458 students (95.4 %; 298 men and 160 women; age range, 18-29 years; mean age, 20.2 years) completed both the baseline and follow-up surveys. The mean L2-L4 BMD value at baseline increased significantly within 3 years. This tendency was also observed for the FN in men but not in women. The annual changes at L2-L4 were 1.78 % in men and 0.97 % in women per year; those for FN were 1.08 % in men and 0.08 % in women per year. However, 20.3 % and 38.5 % of the total freshmen lost BMD in the lumbar spine and FN, respectively. After adjustment for age and body mass index, logistic regression analysis revealed that bone loss in men at L2-L4 at the baseline was affected by skipping breakfast. In contrast, exercise (>2 h/week) increased lumbar spine BMD in both genders. These findings indicate that breakfast and exercise are important for maintaining BMD in young men and women.

Nagata K, Yoshida M, Ishimoto Y, Hashizume H…
J. Bone Miner. Metab. Sep 2013
PMID: 24052206

Vitamin K1 and K2 (MK-4, MK-7) Don’t Prevent Bone Loss in Rats Fed Adequate Nutrients

Abstract

Vitamin K supplementation does not prevent bone loss in ovariectomized Norway rats.

Despite plausible biological mechanisms, the differential abilities of phylloquinone (PK) and menaquinones (MKn) to prevent bone loss remain controversial. The objective of the current study was to compare the effects of PK, menaquinone-4 (MK-4) and menaquinone-7 (MK-7) on the rate of bone loss in ovariectomized (OVX) Norway rats. A secondary aim was to compare the effects of vitamin K with those of bisphosphonates (BP) on bone loss.
Rats (n = 96) were randomized to 6 dosing groups [n = 16/group; Sham; OVX; OVX + BP (100 μg/kg/100 μg/mL saline sc); OVX + PK; OVX + MK-4; and OVX + MK-7] for 6 wk. Equimolar daily doses of 107 mg PK/kg, 147 mg MK-4/kg, and 201 mg MK-7/kg diet were provided.
BP significantly increased bone strength and bone mineral density (BMD) vs. OVX (P < 0.05). However, PK, MK-4 or MK-7 did not change bone strength or BMD compared to the OVX group. Whereas supplementation of PK, MK-4 and MK-7 increased serum and tibia concentrations of each respective form, PK concentrations were consistently higher despite equimolar intakes.
PK, MK-4, and MK-7 do not appear to prevent bone loss in OVX rats when administered concurrent with adequate intake of other nutrients.

Fu X, Moreines J, Booth SL
Nutr Metab (Lond) 2012
PMID: 22348311 | Free Full Text


In conclusion, supplementation of PK, MK-4 or MK-7 did not confer a beneficial effect on bone loss in ovariectomized Norway rats fed a diet that meets nutritional requirements for other nutrients, including calcium and vitamin D. This would suggest that equivocal findings in the literature regarding the effect of various forms of vitamin K on bone cannot be attributed to differences among the forms studied. These data are also consistent with a growing number of clinical studies that report no beneficial effect of vitamin K supplementation on bone loss in the elderly who are otherwise calcium and vitamin D-replete [1,18,19].

Ginkgo Biloba Stimulates Osteoblasts and Decreases Resorption with SERM-Like Effect in Rats

Abstract

Effects of ginkgo biloba on in vitro osteoblast cells and ovariectomized rat osteoclast cells.

Ginkgo biloba extract (GBE) has a selective estrogen receptor modulator (SERM)-like biphasic effect on estrogen, and could be a potential alternative to hormone replacement therapy (HRT). Here, we investigated whether GBE can ameliorate estrogen-depleted osteoporosis in in vitro osteoblast cells and in estrogen-deprived ovariectomized (OVX) rats, a classical animal model for postmenopausal osteoporosis. GBE (50-150 microg/mL) significantly increased ALP (Alkaline phosphatase) activity of osteoblast cells, indicating that GBE promotes osteoblast mineralization. OVX rats exposed to GBE (100 and 200 mg/kg/day, oral treatment), raloxifene (3 mg/kg/day, oral treatment) or estradiol (E2, 10 microg/kg/day, subcutaneous injection) decreased osteoclast resorptive activity compared with OVX rats. GBE and raloxifene did not increase uterine weight compared with OVX rats, while E2 and Sham control did, suggesting that GBE has no uterotrophic activity, which is a disadvantage of estrogen therapy. In OVX rats, GBE did not restore severe bone density loss induced by OVX, indicating that GBE may be insufficient as therapeutic material for severe osteoporosis. However, despite its no effects on bone density loss in OVX rats, GBE did stimulate osteoblast differentiation and antiosteoclastic activity in vitro. Therefore, GBE may have preventive potential on osteoporosis as do other phytoestrogens.

Oh SM, Kim HR, Chung KH
Arch. Pharm. Res. Feb 2008
PMID: 18365693