Tag Archives: animal

Melatonin Promotes Osteoblasts in Mouse Cells

Abstract

Melatonin promotes osteoblastic differentiation through the BMP/ERK/Wnt signaling pathways.

Although melatonin has a variety of biological actions such as antitumor, antiangiogenic, and antioxidant activities, the osteogenic mechanism of melatonin still remains unclear.Thus, in the present study, the molecular mechanism of melatonin was elucidated in the differentiation of mouse osteoblastic MC3T3-E1 cells. Melatonin enhanced osteoblastic differentiation and mineralization compared to untreated controls in preosteoblastic MC3T3-E1 cells. Also, melatonin increased wound healing and dose-dependently activated osteogenesis markers such as runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenic protein (BMP)-2 and -4 in MC3T3-E1 cells. Of note, melatonin activated Wnt 5 α/β, β-catenin and the phosphorylation of c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in a time-dependent manner while it attenuated phosphorylation of glycogen synthase kinase 3 beta (GSK-3β) in MC3T3-E1 cells. Consistently, confocal microscope observation revealed that BMP inhibitor Noggin blocked melatonin-induced nuclear localization of β-catenin. Furthermore, Western blotting showed that Noggin reversed activation of β-catenin and Wnt5 α/β and suppression of GSK-3β induced by melatonin in MC3T3-E1 cells, which was similarly induced by ERK inhibitor PD98059. Overall, these findings demonstrate that melatonin promotes osteoblastic differentiation and mineralization in MC3T3-E1 cells via the BMP/ERK/Wnt pathways.

Park KH, Kang JW, Lee EM, Kim JS…
J. Pineal Res. Sep 2011
PMID: 21470302p

Melatonin+Estrogen Increases Bone Formation in Ovariectomized Rats

Abstract

Melatonin increases oestradiol-induced bone formation in ovariectomized rats.

To assess the effect of melatonin on bone metabolism in ovariectomized rats, receiving oestradiol therapy or not, melatonin was administered in the drinking water (25 microg/mL water) and oestradiol (10 microg/kg body weight) or vehicle was given subcutaneously 5 days/week for up to 60 days after surgery. Urinary deoxypyridinoline (a marker of bone resorption) and circulating levels of bone alkaline phosphatase activity (a marker of bone formation), as well as serum calcium and phosphorus levels, were measured every 15 days. Bone area (BA), bone mineral content (BMC), bone mineral density (BMD) and total body fat (expressed as 100 g body weight) were measured by dual-energy X-ray absorptiometry at the end of the experiment. Body weight and total body fat were augmented after ovariectomy, and decreased after melatonin or oestradiol treatment. The effect of melatonin on body weight was seen in sham-operated rats only. Ovariectomy augmented, and melatonin or oestradiol lowered, urinary deoxypyridinoline excretion. This effect of melatonin and oestradiol was seen mainly in ovariectomized rats. The efficacy of oestradiol to counteract ovariectomy-induced bone resorption was increased by melatonin. Melatonin or oestradiol lowered serum bone alkaline phosphatase activity. Melatonin inhibition was seen mainly on the increase of bone alkaline phosphatase activity that followed ovariectomy. Serum phosphorus levels decreased after melatonin administration and were augmented after oestradiol injection; overall, melatonin impaired the increase of serum phosphorus caused by oestradiol. Ovariectomy decreased, and oestradiol increased, serum calcium levels while melatonin augmented serum calcium in sham-operated rats only. On day 60 after surgery, BMD and content decreased after ovariectomy and were increased after oestradiol injection. Melatonin augmented BA of spine and BMC of whole of the skeleton and tibia. The highest values observed were those of rats treated concurrently with oestradiol and melatonin. The present results indicate that: (i) melatonin treatment restrained bone remodelling after ovariectomy; (ii) the effect of melatonin required adequate concentrations of oestradiol; (iii) melatonin augmented oestradiol effects on bone in ovariectomized rats; (iv) a counter-regulation by melatonin of the increase in body fat caused by ovariectomy was uncovered. The melatonin doses employed were pharmacological in terms of circulating melatonin levels but not necessarily for some other fluids or tissues.

Ladizesky MG, Boggio V, Albornoz LE, Castrillón PO…
J. Pineal Res. Mar 2003
PMID: 12562506

Milk Basic Protein Inhibits Resorption in Ovariectomized Rats

Abstract

Milk basic protein: a novel protective function of milk against osteoporosis.

Milk is recommended as an excellent calcium source for bone health. Moreover, milk is considered to contain other components effective for bone health. In our previous studies, using an unfractionated bone cell culture system, we found that milk whey protein, especially its basic fraction (milk basic protein [MBP]), suppressed bone resorption. In this present study, we investigated whether MBP could prevent bone loss in aged ovariectomized rats. Twenty-one 51-week-old female Sprague-Dawley rats were ovariectomized (ovx), and another seven rats received a sham operation (sham). After a 4-week recovery period, the ovx rats were separated into three groups, and they were then fed a control diet, a 0.01% MBP diet (0. 01% casein of the control diet replaced with MBP), or a 0.1% MBP diet for 17 weeks. The sham rats were fed the control diet. Bone mineral density (BMD) of the femur was measured by dual-energy X-ray absorptiometry in vivo. The BMD in the ovx-control group noticeably decreased during the experimental period in comparison with that in the sham group. However, the BMD in the OVX-0.1% MBP group was significantly higher than that in ovx-control group at weeks 12 and 16 (p < 0.05). After the 17-week feeding period, the breaking energy of the excised femur of all groups was determined by use of a three-point bending rheolometer. The breaking energy in the ovx-control group was significantly lower than that in the sham group (p < 0.05). However, the breaking energy in the ovx-0.1% MBP group was significantly higher than that of the ovx-control group (p < 0.05). Urinary deoxypyridinoline (D-Pyr) level of the ovx-control group was higher than that of the sham group, whereas the level of D-Pyr excretion in the ovx-0.01% MBP and ovx-0.1% MBP groups was significantly lower than that of the ovx-control group (p < 0.05). These results suggest that MBP suppresses the osteoclast-mediated bone resorption and prevents bone loss caused by ovariectomy. Moreover, we performed an in vitro study using isolated osteoclasts from rabbit bone to investigate the possible mechanism. MBP dose-dependently suppressed the number of pits formed by these osteoclasts. This result indicates that MBP suppresses bone resorption by its direct effects on osteoclasts. To our knowledge, this study provides the first evidence that MBP directly suppresses osteoclast-mediated bone resorption, resulting in the prevention of the bone loss that occurs in ovx rats.

Toba Y, Takada Y, Yamamura J, Tanaka M…
Bone Sep 2000
PMID: 10962352

Hypothesis: Animal Protein Associated with Hip Fracture – 1992

Abstract

Cross-cultural association between dietary animal protein and hip fracture: a hypothesis.

Age-adjusted female hip fracture incidence has been noted to be higher in industrialized countries than in nonindustrialized countries. A possible explanation that has received little attention is that elevated metabolic acid production associated with a high animal protein diet might lead to chronic bone buffering and bone dissolution. In an attempt to examine this hypothesis, cross-cultural variations in animal protein consumption and hip fracture incidence were examined. When female fracture rates derived from 34 published studies in 16 countries were regressed against estimates of dietary animal protein, a strong, positive association was found. This association could not plausibly be explained by either dietary calcium or total caloric intake. Recent studies suggest that the animal protein-hip fracture association could have a biologically tenable basis. We conclude that further study of the metabolic acid-osteoporosis hypothesis is warranted.

Abelow BJ, Holford TR, Insogna KL
Calcif. Tissue Int. Jan 1992
PMID: 1739864

This study from 1992 was one of the earlier proposals that animal protein may cause osteoporosis. The hypothesis has since been discredited.

Protein Associated with Reduced Fractures – June 2012

Abstract

Protein intake and fracture risk in elderly people: a case-control study.

We investigated whether protein intake (PI) is related to osteoporotic fractures (OP) in the elderly by analyzing vegetable protein intake (VPI), animal protein intake (API), and animal/vegetable protein intake ratio (AVR) and by calcium intake (CaI).
A 1:1 matched by age and sex case-control study with 167 cases was carried out at the Hospital of Jaen (Spain). Cases were patients aged ≥65 years with a low-energy fracture. Controls were people without previous fractures. Diet was assessed by a food frequency questionnaire. Multivariable analyses were fitted using analysis of covariance (for comparison of adjusted means) and conditional logistic regression (estimating adjusted odds ratios [ORs]).
The control-group showed a higher API (p = 0.046) even when CaI was <800 mg/day (p = 0.041). ORs for AVR were 0.68 (0.38-1.19) and 0.38 (0.15-0.98), respectively with a p for trend = 0.046. A PI<15% of the total energy intake showed an OR of 2.86 (1.10-7.43).
Patients with fracture history have lower API suggesting that high API reduce the occurrence of OP in elderly even if CaI is <800 mg/day. A PI<15% of total calories were associated with an increased risk of OP in elderly.

Martínez-Ramírez MJ, Delgado-Martínez AD, Ruiz-Bailén M, de la Fuente C…
Clin Nutr Jun 2012
PMID: 22182947

Low Protein Increases Bone Loss in Vitamin D Deficiency or Ovariectomy in Rats

Abstract

Low protein intake magnifies detrimental effects of ovariectomy and vitamin D on bone.

Protein-induced changes in bone and calcium homeostasis could potentially be greater in the elderly and in women at risk for osteoporosis. We hypothesize that a low protein intake would magnify the negative changes in bone metabolism seen in vitamin D (vitD) insufficiency and/or estrogen deficiency. The present study was undertaken to better understand how a low protein diet along with vitD insufficiency could affect bone metabolism using a rodent ovariectomized (OVX) model. Rats (n = 60) underwent ovariectomy (OVX) or sham operation. The first 15 days after surgery, all rats were fed a standard rodent diet. Thereafter, rats (n = 10/group) were fed a low protein diet (LP; 2.5 %) or a control diet (NP; 12.5 %) with 100 IU% vitD (+D; cholecalciferol) or without vitD (-D) for 45 days. The groups were as follows: SHAM + NP + D (control); SHAM + LP + D; SHAM + LP – D; OVX + NP + D; OVX + LP + D; OVX + LP – D. Body weight (BW) of control and OVX + NP + D groups increased while those feeding the LP diet, independently of vitD feedings, decreased (p < 0.05). The OVX + LP – D group presented the lowest serum Ca, phosphorus and osteocalcin levels and the highest CTX levels (p < 0.05). At the end of the study, total skeleton bone mineral content, proximal tibia bone mineral density, bone volume and trabecular number levels decreased as follows: SHAM + NP + D (controls) > SHAM + LP + D > OVX + NP + D > SHAM + LP – D > OVX + LP + D > OVX + LP – D (p < 0.05). A low protein diet negatively affected bone mass and magnified the detrimental effects of vitD and/or estrogen deficiencies.

Marotte C, Gonzales Chaves MM, Pellegrini GG, Friedman SM…
Calcif. Tissue Int. Aug 2013
PMID: 23708885

Protein and Energy Deficiencies May Increase Bone Loss in Rats – 2008

Abstract

Influence of high and low protein intakes on age-related bone loss in rats submitted to adequate or restricted energy conditions.

Low energy and protein intake has been suggested to contribute to the increased incidence of osteoporosis in the elderly. The impact of dietary protein on bone health is still a matter of debate. Therefore, we examined the effect of the modulation of protein intake under adequate or deficient energy conditions on bone status in 16-month-old male rats. The animals were randomly allocated to six groups (n = 10/group). Control animals were fed a diet providing either a normal-protein content (13%, C-NP) or a high-protein content (26%) (C-HP). The other groups received a 40% protein/energy-restricted diet (PER-NP and PER-HP) or a normal protein/energy-restricted diet (ER-NP and ER-HP). After 5 months of the experiment, protein intake (13% or 26%) did not modulate calcium retention or bone status in those rats, although a low-grade metabolic acidosis was induced with the HP diet. Both restrictions (PER and ER) decreased femoral bone mineral density and fracture load. Plasma osteocalcin and urinary deoxypyridinoline levels were lowered, suggesting a decrease in bone turnover in the PER and ER groups. Circulating insulin-like growth factor-I levels were also lowered by dietary restrictions, together with calcium retention. Adequate protein intake in the ER condition did not elicit any bone-sparing effect compared to PER rats. In conclusion, both energy and protein deficiencies may contribute to age-related bone loss. This study highlights the importance of sustaining adequate energy and protein provision to preserve skeletal integrity in the elderly.

Mardon J, Habauzit V, Trzeciakiewicz A, Davicco MJ…
Calcif. Tissue Int. May 2008
PMID: 18437274

Onion Decreases Osteopenia in Rats

Abstract

Onion decreases the ovariectomy-induced osteopenia in young adult rats.

It has been suggested that fruit and vegetable consumption are associated with good bone health. Onion, in particular, has been verified in its efficacy in bone resorption activity. In this study, we further investigated the effects of an onion-containing diet on ovariectomy-induced bone loss using methods of serum marker assay, histomorphometric analysis and biomechanical tests. Sixty-four female Wistar rats (14-week-old) with sham operations or ovariectomy were assigned to 6 groups: CON, sham-operated control group; OVX, ovariectomized group; ALN, ovariectomized rats treated with alendronate (1 mg/kg/day, p.o.); and 3% ON, 7% ON and 14% ON, ovariectomized rats fed with diets containing 3%, 7% and 14% (wt/wt) onion powder, respectively. Animals were sacrificed after a six-week treatment course. In the serum marker assay, alendronate and all three onion-enriched diets significantly decreased serum calcium level (p<0.05). Both 14% ON group and the ALN group even showed similarly lower level of serum osteocalcin (p<0.05), suggesting a down-regulation of bone turnover. The histomorphometric analysis showed that ovariectomy markedly decrease bone trabeculae. The ALN and 14% ON rats were 80% and 46% higher, respectively, in BV/TV than the OVX rats (p<0.05), and the rats fed with onion-enriched food showed a lesser ovariectomy-induced bone loss in a dose-dependent manner. Additionally, both ALN and 14% ON groups had significantly more trabecular number, less separated trabeculae, and fewer osteoclasts (p<0.05), but the protective efficacy from the 14% onion-enriched diet was slightly inferior to that of alendronate. Ovariectomy also significantly decreased tissue weight and biomechanical strength in the OVX group (p<0.05). The ALN and 14% ON groups equivalently showed a lesser decrease in tissue weight, though the difference was not significant. On the other hand, both the ALN and 14% ON groups represented similar biomaterial properties of femurs, and both reduced the ovariectomy-induced decrease in bending load and bending energy (p<0.05). The present study further verified that an onion-enriched diet could counteract ovariectomy-induced bone loss and deterioration of biomechanical properties.

Huang TH, Mühlbauer RC, Tang CH, Chen HI…
Bone Jun 2008
PMID: 18387868

Genistein Increases Bone Density in Rats, Cooked Soybeans and Stachyose Don’t

Abstract

Influence of a low dose of dietary soybean on bone properties and mineral status in young rats.

The aim of this study was to evaluate effects of dietary supplementation with genistein, daidzein stachyose, and raw or cooked soybean on mineral content, optical density, and mechanical properties of bones in growing rats. The experiment was performed on 70 male young Wistar rats (4 weeks old at the start of the experiment) divided into seven groups. Genistein, daidzein, or stachyose were administered by gavage. Raw or cooked soybean was added directly to the diet (1%) The experiment lasted 28 days. Femurs were removed postmortem and kept until analysis at -20°C. Mineral content in bones was determined by atomic absorption flame spectrometry, and inductively coupled plasma atomic emission spectrometry. Optical density was analyzed with a KODAK 1D 3.5 system. Mechanical properties were tested using INSTRON 4301 equipment. Genistein increased mineral content in bones of growing rats. Biological action of genistein and daidzein on the mineralization of bone tissues in growing rats was different. Addition of stachyose (1.9 mg/day/rat) did not affect bone tissues, nor did the addition of raw or cooked soybean. None of the studied biologically active substances: genistein (0.26 mg/day/rat), daidzein (0.104 mg/day/rat), stachyose (1.9 mg/day/rat), or soybean had an effect on bone optical density.

Piastowska-Ciesielska AW, Gralak MA
Biofactors
PMID: 20806285

Isoflavones + Calcium Better Than Isoflavones or Calcium in Ovariectomized Rats

Abstract

Isoflavones with supplemental calcium provide greater protection against the loss of bone mass and strength after ovariectomy compared to isoflavones alone.

Although hormone replacement therapy (HRT) and calcium (Ca) supplementation preserve bone mass more when combined, there is a growing concern over the safety of HRT that necessitates thorough investigation of effective, alternative treatments for bone loss. While plant-derived estrogen-like compounds such as isoflavones preserve bone, it is not known whether isoflavones and Ca supplementation attenuate losses in bone mass and strength to a greater extent when combined. This study compared the effects of an isoflavone extract + high Ca to isoflavone extract or high Ca alone on preservation of bone mineral density (BMD) and biomechanical strength in ovariectomized (ovx) rats. Rats were sham-operated (n = 10) or ovx (n = 40). Shams were fed a 0.2% Ca diet. Ovx rats were randomized to a 0.2% Ca diet alone (OVX) or with isoflavone extract (IE; 1.6 g/kg diet) or to a high Ca diet (Ca; 2.5%) alone or a high Ca diet with the isoflavone extract (IE + Ca) for 8 weeks. BMD of femur and lumbar spine were measured by dual-energy X-ray absorptiometry. The biomechanical strength of femurs and individual vertebra was measured by three-point bending and compression testing, respectively. The average food intake was lowest (P < 0.05) among sham and IE groups and greatest (P < 0.05) among the OVX group. Final body weight was lowest (P < 0.05) among shams and highest (P < 0.05) among the OVX group while IE + Ca were lighter (P < 0.05) than all ovx groups. Femur and vertebra BMD was greater (P < 0.05) among IE + Ca and sham rats compared to IE, Ca, or OVX rats. Although there were differences in femur BMD among groups, biomechanical properties at the femur midpoint did not differ among groups, possibly due to the lack of cortical bone loss at this site. Conversely, vertebra biomechanical strength was greater (P < 0.05) among IE + Ca and Ca alone groups compared to IE alone. Uterine weight was higher (P < 0.05) among shams than OVX and IE with no difference among shams, Ca, or IE + Ca rats, suggesting that the isoflavones did not have an uterotrophic effect. In conclusion, isoflavones combined with high Ca are more protective against the loss of femur and vertebra BMD than isoflavones or high Ca diet alone.

Breitman PL, Fonseca D, Cheung AM, Ward WE
Bone Oct 2003
PMID: 14555264

Also, it’s interesting that Calcium alone was superior to Isoflavones alone.