Monthly Archives: April 2014

Review: Protein and Bone Studies

Abstract

Health effects of protein intake in healthy adults: a systematic literature review.

The purpose of this systematic review is to assess the evidence behind the dietary requirement of protein and to assess the health effects of varying protein intake in healthy adults. The literature search covered the years 2000-2011. Prospective cohort, case-control, and intervention studies were included. Out of a total of 5,718 abstracts, 412 full papers were identified as potentially relevant, and after careful scrutiny, 64 papers were quality graded as A (highest), B, or C. The grade of evidence was classified as convincing, probable, suggestive or inconclusive. The evidence is assessed as: probable for an estimated average requirement of 0.66 g good-quality protein/kg body weight (BW)/day based on nitrogen balance studies, suggestive for a relationship between increased all-cause mortality risk and long-term low-carbohydrate-high-protein (LCHP) diets; but inconclusive for a relationship between all-cause mortality risk and protein intake per se; suggestive for an inverse relationship between cardiovascular mortality and vegetable protein intake; inconclusive for relationships between cancer mortality and cancer diseases, respectively, and protein intake; inconclusive for a relationship between cardiovascular diseases and total protein intake; suggestive for an inverse relationship between blood pressure (BP) and vegetable protein; probable to convincing for an inverse relationship between soya protein intake and LDL cholesterol; inconclusive for a relationship between protein intake and bone health, energy intake, BW control, body composition, renal function, and risk of kidney stones, respectively; suggestive for a relationship between increased risk of type 2 diabetes (T2D) and long-term LCHP-high-fat diets; inconclusive for impact of physical training on protein requirement; and suggestive for effect of physical training on whole-body protein retention. In conclusion, the evidence is assessed as probable regarding the estimated requirement based on nitrogen balance studies, and suggestive to inconclusive for protein intake and mortality and morbidity. Vegetable protein intake was associated with decreased risk in many studies. Potentially adverse effects of a protein intake exceeding 20-23 E% remain to be investigated.

Pedersen AN, Kondrup J, Børsheim E
Food Nutr Res 2013
PMID: 23908602 | Free Full Text

Based on a validated FFQ, a French study of postmenopausal women with a habitual HP intake (45), there was no overall association between fracture risk and total protein intake. In the presence of low calcium intake (<400 mg/1,000 kcal), there was an increased risk of fractures related to energy-adjusted total and animal protein as well as gram per kg BW, while energy-adjusted vegetable protein was associated with a decreased fracture risk. In the Framingham Offspring Study of men and women (46), there was no overall association between fracture risk and total protein intake based on FFQ. Animal protein intake was associated with an increased fracture risk provided a low (<800 mg) calcium intake and a decreased risk of fractures provided a high (>800 mg) calcium intake. The Study of Osteoporotic Fractures in postmenopausal women (42) found increased risk of hip fractures related to high animal protein intake and high A/V ratio estimated from FFQ. When the model was adjusted for BMD, the relation of A/V ratio to fracture risk became non-significant. The systematic review and meta-analysis (44) found no relationship between protein intake and risk of fractures, neither in the cohort studies nor in the supplemental studies.

[…]

A systematic review and meta-analysis assessed the relation of dietary acid load to bone health (47), quality graded as C because of the lack of information about dietary intake methods or intervention (see Appendix C, Table C10). The analysis did not support the hypothesis that ‘acid’ from the diet causes osteoporosis or that an ‘alkaline’ diet prevents osteoporosis. The systematic review also indicated that higher protein intake and animal protein were not detrimental to calcium retention. The ideal protein intake for bone health could not be determined.

[…]

Two intervention trials including postmenopausal women (48, 49), quality graded as B and A, respectively, were identified for the association between protein and calcium and bone metabolism (see Appendix C, Table C10). Harrington et al. (48) used a high-sodium–high-protein diet versus a low-sodium–UP diet in a randomized cross-over trial. Thus, it was difficult to separate the effect of protein per se. Nevertheless, they found that a high-sodium HP diet led to increased urinary calcium loss and increased bone resorption. In a high-quality feeding trial by Hunt et al. (49), high- (20 E%) or low- (10 E%) protein intake was combined with high- (1,510 mg) and low-calcium (675 mg) intake in a randomized four interventions’ cross-over design. They found that the combination of HP and low-calcium diet increased calcium retention, and it also resulted in an increase in IGF-1, an anabolic peptide hormone stimulating bone formation.

Protodioscin Inhibits Bone Loss in Ovariectomized Rats

Abstract

In vivo antiosteoporotic activity of a fraction of Dioscorea spongiosa and its constituent, 22-O-methylprotodioscin.

The antiosteoporotic activity of the 90 % EtOH fraction of the water extract of rhizomes of Dioscorea spongiosa and methylprotodioscin, its major constituent, were examined in the model of postmenopausal bone loss using ovariectomized (OVX) rats or mice. After 6 weeks treatment, the proximal tibia of rats or mice and the distal femora of mice were scanned by peripheral quantitative computed tomography (pQCT). Both the 90 % EtOH fraction (100 mg/kg/d) and methylprotodioscin (50 mg/kg/d) significantly inhibited bone loss in bone mineral content (BMC) and bone mineral density (BMD) in total, cancellous and cortical bones, and the decrease in bone strength indexes induced by OVX, without side effect on the uterus.

Yin J, Tezuka Y, Kouda K, Le Tran Q…
Planta Med. Mar 2004
PMID: 15114498

Silibinin Promotes Osteoblasts In Vitro

Abstract

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Ying X, Sun L, Chen X, Xu H…
Eur. J. Pharmacol. Dec 2013
PMID: 24076187

Calcium Threonate in Ester-C Enhances Vitamin C’s Bone Mineralization In Vitro

Abstract

Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate).
Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence.
In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate.
In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.

Rowe DJ, Ko S, Tom XM, Silverstein SJ…
J. Periodontol. Sep 1999
PMID: 10505801

This study is on Ester-C. Ester-C, PureWay-C, and AlphaSorb-C are Vitamin C products that contain Calcium-L-Threonate. Biocalth is Calcium product which is all Calcium-L-Threonate without Vitamin C.

 

L-Threonate Inhibits Resorption In Vitro

Abstract

[Effects of L-threonate on bone resorption by osteoclasts in vitro].

To clarify if calcium L-threonate and sodium L-threonate have inhibitory effects on the bone resorption of rabbit’s osteoclasts in vitro.
This study contained a total of 16 culture groups, including one group as control and 5 groups treated by 5 drugs (calcium D-threonate, sodium L-threonate, alendronate, 17beta-estradiol and calcium gluconate) each at the final concentrations of 10(-9) mol/L, 10(-7) mol/L, 10(-5) mol/L respectively. After 7 days, eight bone slices of every group were stained with toluidine blue and the areas of resorptive pits were analyzed under light microscope; the concentrations of C-telopeptide of type I collagen (CTx or Crosslaps) in culture supernatants were measured by ELISA.
(1) The resorption area and the CTx concentration of the Calcium L-threonate groups were reduced significantly as compared with those of control and of Calcium gluconate groups respectively. The resorption area and CTx level of the Sodium L-threonate groups were significantly reduced when compared with those of the control, but the effects of Calcium gluconate groups were not so. (2) The reduction in the resorption area and CTx concentration of Calcium L-threonate group was more than that of Sodium L-threonate group. (3) The reductive effect of the high concentration (10(-5)) group of Calcium L-threonate on the area and CTx level was corresponding to that of 17beta-estradiol at a concentration between 10(-7) and 10(-9). (4) The resorption area was related to the CTx concentration (r=0.876). (5) The CTX level was much more sensitive, precise and stable than the concentration.
L-threonate, especially calcium L-threonate could inhibit the bone resorption of osteoclasts in vitro, and its effect might be related to the radical of L-threonic acid. The CTx concentration in culture supernatants might be an effective marker quantitatively reflecting the bone resorption by osteoclasts in vitro.

He JH, Tong NW, Li HQ, Wu J
Sichuan Da Xue Xue Bao Yi Xue Ban Mar 2005
PMID: 15807273

Silibinin Increases Osteoblasts and Inhibits Osteoclasts in Mouse Cells

Abstract

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts.

Bone-remodeling imbalance induced by decreased osteoblastogenesis and increased bone resorption is known to cause skeletal diseases such as osteoporosis. Silibinin is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum). Numerous studies suggest that silibinin is a powerful antioxidant and has anti-hepatotoxic properties and anti-cancer effects against carcinoma cells. This study investigated that silibinin had bone-forming and osteoprotective effects in in vitro cell systems of murine osteoblastic MC3T3-E1 cells and RAW 264.7 murine macrophages. MC3T3-E1 cells were incubated in osteogenic media in the presence of 1-20 µM silibinin up to 15 days. Silibinin accelerated cell proliferation and promoted matrix mineralization by enhancing bone nodule formation by calcium deposits. In addition, silibinin furthered the induction of osteoblastogenic biomarkers of alkaline phosphatase, collagen type 1, connective tissue growth factor, and bone morphogenetic protein-2. Differentiated MC3T3-E1 cells enhanced secretion of receptor activator of nuclear factor-κB ligand (RANKL) essential for osteoclastogenesis, which was reversed by silibinin. On the other hand, RAW 264.7 cells were pre-incubated with 1-20 µM silibinin for 5 days in the presence of RANKL. Non-toxic silibinin markedly attenuated RANK transcription and intracellular adhesion molecule-1 expression elevated by RANKL, thereby suppressing the differentiation of macrophages to multi-nucleated osteoclasts. It was also found that silibinin retarded tartrate-resistant acid phosphatase and cathepsin K induction and matrix metalloproteinase-9 activity elevated by RANKL through disturbing TRAF6-c-Src signaling pathways. These results demonstrate that silibinin was a potential therapeutic agent promoting bone-forming osteoblastogenesis and encumbering osteoclastic bone resorption.

Kim JL, Kang SW, Kang MK, Gong JH…
J. Cell. Biochem. Jan 2012
PMID: 21898547

Diosgenin and Lovastatin Prevent Bone Loss in Ovariectomized Rat

Abstract

Osteoprotective effect of Monascus-fermented dioscorea in ovariectomized rat model of postmenopausal osteoporosis.

This experiment established the ovariectomized (OVX) rat model of postmenopausal osteoporosis and examined the effect of the oral administration of different dosages of dioscorea, red mold dioscorea (RMD), and soy isoflavones on bone mineral density (BMD). Three months after osteoporosis had been induced and 4 weeks after feeding had begun, the tibia and femur BMD of OVX rats administered RMD showed significant increases compared with that of all other groups of OVX rats. Closer examination using microcomputed tomography also revealed that the RMD-administered rats had denser trabecular bone volume and a higher trabecular number compared to all other rat groups. Reconstructed 3D imaging indicated increases in cancellous bone mineral content, cancellous bone mineral density, and cortical bone mineral content of the proximal tibia in OVX rats. These findings indicate that administration of monacolin K and phytoestrogen diosgenin could prevent bone loss induced by estrogen deficiency.

Chiang SS, Chang SP, Pan TM
J. Agric. Food Chem. Sep 2011
PMID: 21800902

Diosgenin Stimulates Bone Formation In Mouse Osteoblasts

Abstract

Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells.

Diosgenin, a steroid saponin extracted from the root of wild yam (Dioscorea villossa) is claimed to have osteogenic property. However, detailed studies providing evidence to this claim have not been fully undertaken. In this study, we investigated the effect of diosgenin on the osteogenesis of murine MC3T3-E1 osteoblastic cells. Cells were cultured with varying levels of diosgenin (0-10 μM) within 25 days of bone formation period. Diosgenin was found to stimulate proliferation within the range of 0.01-5 μM using MTT assay. The medium and cellular levels of Type 1 collagen and alkaline phosphatase (ALP), both of which are major bone matrix proteins, increased within the low range of diosgenin concentration (>0-3 μM), and this pattern was further confirmed by collagen and ALP staining of the extracellular matrix (ECM). The cellular protein expression of ALP and collagen Type 1 was also increased at 0.1-1 μM diosgenin treatment as analyzed by Western blot. Calcium deposition within the ECM also showed the same pattern as assessed by Alizarin Red S and Von Kossa staining. Bone-specific transcription factor runt-related transcription factor 2 (Runx2) and Runx2-regulated osteopontin protein expressions were induced at low concentration (0.1-1 μM) and again decreased with high diosgenin concentrations. Based on our findings, our study suggests that diosgenin can enhance bone formation by stimulating the synthesis and secretion of Type 1 collagen and ALP and bone marker proteins Runx2 and osteopontin expression. The increased levels of these marker proteins, in turn, can increase the formation of calcium deposits within the ECM thereby increasing bone formation.

Alcantara EH, Shin MY, Sohn HY, Park YM…
J. Nutr. Biochem. Nov 2011
PMID: 21292464

Diosgenin Promotes Angiogenesis in Preosteoblast-Like Mouse Cells

Abstract

Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts.

Diosgenin, extracted from the root of wild yam (Dioscorea villosa), has been reported to demonstrate an opportunity for medical application. Vascular endothelial growth factor-A (VEGF-A) plays an important role in bone-related angiogenesis, a critical process occurring during bone formation and fracture healing. In this study, we examine whether diosgenin is able to induce VEGF-A expression and to promote angiogenesis in osteoblasts. For murine MC3T3-E1 preosteoblast-like cells, VEGF-A mRNA and protein expression seemed to be significantly elevated in response to diosgenin in a concentration-dependent fashion. Conditioned media prepared from cells treated with diosgenin induced strong angiogenic activity in either in vitro or ex vivo angiogenesis assay. Furthermore, diosgenin treatment increased the stability and activity of HIF-1alpha protein. Inhibition of HIF-1alpha activity by transfection with DN-HIF-1alpha significantly diminished diosgenin-mediated VEGF-A up-regulation. The use of pharmacological inhibitors or genetic inhibition revealed that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways were potentially required for diosgenin-induced HIF-1 activation and subsequent VEGF-A up-regulation. It is noteworthy that an estrogen receptor binding assay revealed that diosgenin has the strong ability to replace [(3)H]estradiol bound to estrogen receptor (IC(50), 10 nM). In addition, the specific estrogen receptor antagonists ICI 182,780 (faslodex) and tamoxifen were noted to be able to strongly inhibit diosgenin-induced, src kinase-dependent Akt and p38 MAPK activation. Taken together, such results provide evidence that diosgenin up-regulates VEGF-A and promotes angiogenesis in preosteoblast-like cells by a hypoxia-inducible factor-1alpha-dependent mechanism involving the activation of src kinase, p38 MAPK, and Akt signaling pathways via estrogen receptor.

Yen ML, Su JL, Chien CL, Tseng KW…
Mol. Pharmacol. Oct 2005
PMID: 15998873 | Free Full Text

Diosgenin Inhibits Osteoclastogenesis

Abstract

Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of Akt, I kappa B kinase activation and NF-kappa B-regulated gene expression.

Diosgenin, a steroidal saponin present in fenugreek (Trigonella foenum graecum) and other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood. In the present study, we report that diosgenin inhibits receptor-activated nuclear factor-kappaB ligand-induced osteoclastogenesis, suppresses tumor necrosis factor (TNF)-induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by NF-kappaB. Diosgenin suppressed TNF-induced NF-kappaB activation as determined by DNA binding, activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of Akt activation. NF-kappaB-dependent reporter gene expression was also abrogated by diosgenin. TNF-induced expression of NF-kappaB-regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc), antiapoptosis (IAP1, Bcl-2, Bcl-X(L), Bfl-1/A1, TRAF1 and cFLIP), and invasion (MMP-9) were also downregulated by the saponin. Diosgenin also potentiated the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents.

Shishodia S, Aggarwal BB
Oncogene Mar 2006
PMID: 16331273