Protein quality affects bone status during moderate protein restriction in growing mice

Adequate protein intake during development is critical to ensure optimal bone gain and to attain a higher peak bone mass later on. We hypothesized that the quality of the dietary protein is of prime importance for bone physiology during moderate protein restriction. The target population was growing Balb/C mice. We compared two protein restricted diets (6% of total energy as protein), one based on soy (LP-SOY) and one based on casein (LP-CAS). For comparison, a normal protein soy-based control group (NP-SOY) and a low protein group receiving an anabolic daily parathyroid hormone (PTH) 1-34 injection (LP-SOY + PTH) were included in the protocol. After 8 weeks, LP-SOY mice had reduced body weights related to a lower lean mass whereas LP-CAS mice were not different from the NP-SOY group. LP-SOY mice were characterized by lower femoral cortical thickness, bone volume, trabecular number and thickness and increased medullar adiposity when compared to both the LP-CAS and NP-SOY groups. However, the dietary intervention had no effect on the vertebral parameters. The negative effect of the LP-SOY diet was correlated to an impaired bone formation as shown by the reduced P1NP serum level as well as the reduced osteoid surfaces and bone formation rate in the femur. PTH injection in LP-SOY mice had no effect on total weight or lean mass, but improved all bone parameters at both femoral and vertebral sites, suggesting that amino acid deficiency was not the primary reason for degraded bone status in mice consuming soy protein. In conclusion, our study showed that under the same protein restriction (6% of energy), a soy diet leads to impaired bone health whereas a casein diet has little effect when compared to a normal protein control.     

Apolipoprotein E gene polymorphism and bone loss: estrogen status modifies the influence of apolipoprotein E on bone loss.

The identification of genes that contribute to bone mineral density (BMD) and bone loss has widespread implications for the understanding and prevention of osteoporosis. The objective of this study was to examine the relationship between the presence and absence of the apolipoprotein E*4 (APOE*4) allele and both BMD and annualized percentage rate of change in BMD at the lumbar spine and hip in a population of 392 healthy, pre-, peri-, and postmenopausal white women participating in the Women's Healthy Lifestyle Project. APOE genotype was analyzed by restriction enzyme analysis from genomic DNA. BMD at the lumbar spine and hip was measured at baseline and after a mean of 2.5 years using dual-energy X-ray absorptiometry (DXA). In premenopausal women, there were no significant differences in BMD or in the annualized percentage rate of change in BMD at the spine or hip when comparing women with and without the APOE*4 allele. In contrast, spine bone loss was significantly greater in peri- and postmenopausal women having an APOE*4 allele than in women without this allele (-1.75 + 1.5% per year vs. -0.98 +/- 1.4% per year, respectively, p = 0.018). Among peri- and postmenopausal women currently using hormone replacement therapy (HRT), there were no differences in the annualized percentage rate of change in spine BMD; whereas, among non-HRT users, there was a 2-fold higher rate of spine bone loss in women with an APOE*4 allele compared with women without this allele (-2.31 +/- 1.5% per year vs. -1.27 +/- 1.3% per year, respectively, p = 0.033; APOE*4 x HRT interaction, p = 0.076). In conclusion, this study shows the importance of APOE*4 allele in spine bone loss in peri- and postmenopausal women and, more importantly, it provides evidence for a genetic and lifestyle interaction in modulating spine bone loss.     

A well-balanced diet combined or not with exercise induces fat mass loss without any decrease of bone mass despite bone micro-architecture alterations in obese rat

The association of a well-balanced diet with exercise is a key strategy to treat obesity. However, weight loss is linked to an accelerated bone loss. Furthermore, exercise is known to induce beneficial effects on bone. We investigated the impact of a well-balanced isoenergetic reducing diet (WBR) and exercise on bone tissue in obese rats. Sixty male rats had previously been fed with a high fat/high sucrose diet (HF/HS) for 4 months to induce obesity. Then, 4 regimens were initiated for 2 months: HF/HS diet plus exercise (treadmill: 50 min/day, 5 days/week), WBR diet plus exercise, HF/HS diet plus inactivity and WBR diet plus inactivity. Body composition and total BMD were assessed using DXA and visceral fat mass was weighed. Tibia densitometry was assessed by Piximus. Bone histomorphometry was performed on the proximal metaphysis of tibia and on L2 vertebrae (L2). Trabecular micro-architectural parameters were measured on tibia and L2 by 3D microtomography. Plasma concentration of osteocalcin and CTX were measured. Both WBR diet and exercise had decreased global weight, global fat and visceral fat mass (p < 0.05). The WBR diet alone failed to alter total and tibia bone mass and BMD. However, Tb.Th, bone volume density and degree of anisotropy of tibia were decreased by the WBR diet (p < 0.05). Moreover, the WBR diet had involved a significant lower MS/BS and BFR/BS in L2 (p < 0.05). Exercise had significantly improved BMD of the tibia possibly by inhibiting the bone resorption, as evidenced by no change in plasma osteocalcin levels, a decrease of CTX levels (p < 0.005) and trabecular osteoclast number (p < 0.05). In the present study a diet inducing weight and fat mass losses did not affected bone mass and BMD of obese rats despite alterations of their bone micro-architecture. The moderate intensity exercise performed had improved the tibia BMD of obese rats without any trabecular and cortical adaptation.     

Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males.

To determine the contributions of estrogen receptor (ER)alpha and ERbeta in bone growth and remodeling in male and female mice, we generated and analyzed full knockouts for each receptor, and a double ER knockout. Although suppression of the ligand to the ERs (i.e., estradiol) after menopause or gonadectomy in females led to a catastrophic increase in bone turnover and concomitant bone loss, deletion of one or both ERs failed to show such an effect. Complete deletion of ERalpha led to a decrease, not an increase, in bone turnover and an increase, not a decrease, in trabecular bone volume in both male and female animals. Deletion of ERbeta led to different responses in males, where bone was unaffected, and in females, where bone resorption was decreased and trabecular bone volume increased. In contrast, deletion of both ERs led to a profound decrease in trabecular bone volume in females, which was associated with a decrease, not an increase, in bone turnover. Finally, deletion of ERalpha, but not ERbeta, led to major changes in circulating levels of estradiol and/or testosterone, indirectly affecting bone remodeling and bone mass. Thus, only ERalpha was shown to regulate bone remodeling in males, whereas in females both receptor subtypes influenced this process and could, at least under basal knockout conditions, compensate for each other.     

The presence of both an energy deficiency and estrogen deficiency exacerbate alterations of bone metabolism in exercising women

BACKGROUND: Bone loss in amenorrheic athletes has been attributed to energy deficiency-related suppression of bone formation, but not increased resorption despite hypoestrogenism. OBJECTIVE: To assess the independent and combined effects of energy deficiency and estrogen deficiency on bone turnover markers in exercising women. DESIGN: PINP, osteocalcin, U-CTX-I, TT3, leptin, and ghrelin were measured repeatedly, and bone mineral density (BMD) was measured once in 44 exercising women. Resting energy expenditure (REE) was used to determine energy status (deficient or replete) and was corroborated with measures of metabolic hormones. Daily levels of urinary estrone and pregnanediol glucuronides (E1G, PdG), were assessed to determine menstrual and estrogen status. Volunteers were then retrospectively categorized into 4 groups: 1) Energy Replete+Estrogen Replete (EnR+E2R), (n=22), 2) Energy Replete+Estrogen Deficient (EnR+E2D), (n=7), 3) Energy Deficient+Estrogen Replete (EnD+E2R), (n=7), and 4) Energy Deficient+Estrogen Deficient (EnD+E2D), (n=8). RESULTS: The groups were similar (p>0.05) with respect to age (24.05+/-1.75 yrs), weight (57.7+/-2.2 kg), and BMI (21.05+/-0.7 kg/m2). By design, REE/FFM (p=0.028) and REE:pREE (p<0.001) were lower in the EnD vs. EnR group, and the E2D group had a lower REE:pREE (p=0.005) compared to the E2R group. The EnD+E2D group had suppressed PINP (p=0.034), and elevated U-CTX-I (p=0.052) and ghrelin (p=0.028) levels compared to the other groups. These same women also had convincing evidence of energy conservation, including TT3 levels that were 29% lower (p=0.057) and ghrelin levels that were 44% higher (p=0.028) than that observed in the other groups. Energy deficiency was associated with suppressed osteocalcin, and TT3 (p<0.05), whereas estrogen deficiency was associated with decreased E1G (p<0.02), and lower L2-L4 BMD (p=0.033). Leptin was significant in predicting markers of bone formation, but not markers of bone resorption. CONCLUSIONS: When the energy status of exercising women was adequate (replete), there were no apparent perturbations of bone formation or resorption, regardless of estrogen status. Estrogen deficiency in exercising women, in the presence of an energy deficiency, was associated with bone loss and involved suppressed bone formation and increased bone resorption. These findings underscore the importance of avoiding energy deficiency, which is associated with hypoestrogenism, to avoid bone health problems.     

No major effect of estrogen receptor gene polymorphisms on bone mineral density or bone loss in postmenopausal Danish women

The polymorphisms of the estrogen receptor (ER) gene defined by the restriction enodonucleases PvuII and XbaI have recently been reported to be associated with bone mineral density (BMD) in postmenopausal women. To investigate the possible relation of the PvuII and XbaI restriction fragment-length polymorphisms of the ER gene with BMD in Danish postmenopausal women, two studies were undertaken: 1) a cross-sectional study of 499 postmenopausal women, where the ER genotypes and alleles were related to BMD of the hip, spine, and lower forearm; and 2) a longitudinal study of 101 postmenopausal women followed up for 18 years. In the latter study, late postmenopausal bone loss in the hip and spine was determined over a period of 6 years in women (mean age of 63 to 69 years), and long-term postmenopausal bone loss in the lower forearm was determined over a period of 18 years in women (mean age of 51 to 69 years). Genotyping was performed through the restriction cleavage of polymerase chain reaction-amplified genomic DNA with the two restriction enzymes, PvuII and XbaI. Restriction fragment-length polymorphisms were represented as P or p (PvuII) and X or x (XbaI), with the lower case letters signifying the presence of the restriction site. The frequencies of the ER genotypes were similar to previously published genotype frequencies in Caucasian and Asian populations. No significant effect of the ER genotypes or alleles on BMD was found at any site, nor was there a relation between ER genotypes and the rate of bone loss either in the hip and spine over 6 years, or in the lower forearm over 18 years. In conclusion, we could not demonstrate any major effect of the ER gene polymorphisms on BMD or rate of bone loss in healthy postmenopausal Danish women.     

Recovery of proximal tibia bone mineral density and strength, but not cancellous bone architecture, after long-term bisphosphonate or selective estrogen receptor modulator therapy in aged rats.

Various bisphosphonates and the selective estrogen receptor modulator (SERM) raloxifene are approved treatments of postmenopausal osteoporosis. They increase bone mineral density (BMD), decrease bone turnover, and reduce vertebral fracture incidence through different cellular mechanisms. We investigated the bone cellular activities, architecture, mineral content/density, and strength of ovariectomized (ovx) rats on a long-term bisphosphonate or SERM treatment, at doses of either agent correcting bone strength. Eleven weeks postovariectomy, 6-month-old rats were treated with the SERM MDL 103,323 or with the bisphosphonate pamidronate for 5 months. Doses of pamidronate and MDL 103,323 were selected from previous studies showing correction of the ovx-induced decrease of ultimate strength of proximal tibia. Ultimate and yield strengths, BMD, and histomorphometric parameters were all quantified at the same site, i.e., the proximal tibia metaphysis. Long-term pamidronate decreases bone turnover and bone formation activity, leading to trabecular thinning. MDL 103,323 decreases bone turnover to a lesser extent, and slightly protects trabecular architecture by uncoupling bone resorption and formation activities. The yield strength is corrected by pamidronate, but not by MDL 103,323 treatment. However, neither compound restores the ovariectomy-induced cancellous bone loss. Total tissue area and cortical thickness are unchanged with pamidronate or MDL 103,323 treatment, indicating that cortical bone mass, thickness, and cross-sectional area are not modified. The discrepancy between proximal tibia BMD and mechanical resistance to fracture modifications, on the one hand, and cancellous bone volume, on the other hand, could be due to changes in the degree of mineralization of bone matrix and/or of the intrinsic properties of the bone matrix.     

Parathyroid hormone is not required for normal milk composition or secretion or lactation-associated bone loss in normocalcemic rats

To determine if parathyroid hormone (PTH) is essential for lactation in rats, the parathyroid glands were removed surgically during the first week of lactation and the rats were given a diet containing a high calcium-phosphorus ratio to maintain a normal serum calcium concentration. Lactating rats were placed on diet containing 1.2% calcium (Ca) and 0.8, 0.6, or 0.4% phosphorus (P) on day 2 postpartum (PP) and were parathyroidectomized (PTX) at 4-6 days PP. At 10 days PP serum Ca was 10.5 +/- 0.2 mg/dl (mean +/- SEM) for PTX rats and 10.4 +/- 0.3 mg/dl in sham-operated lactating rats when the diet contained 0.6% P. When the diet P was 0.8%, the litters gained little or no weight and serum Ca fell to 6.9 +/- 0.6 mg/dl by day 10 PP in PTX rats compared with 10.2 +/- 0.2 mg/dl in sham rats. PTX rats fed the diet containing 1.2% Ca and 0.6% P maintained a normal serum Ca level until at least day 18 PP, but their serum P levels fell gradually from approximately 5 mg/dl at 10 days to 3 mg/dl at 18 days PP. In spite of this hypophosphatemia, the litters of PTX and sham rats had gained the same amount of weight by age 16 days, indicating equal milk production in the two groups. Milk Ca, P, and total solids were not significantly different between PTX and sham rats on day 11 PP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure to antiretroviral agents during pregnancy does not alter bone status in infants

The use of combined antiretroviral agents during pregnancy is important to prevent mother-to-child transmission of human immunodeficiency virus (HIV). Antiretroviral treatment (ARV) is associated with reduced bone mass and altered bone metabolism in HIV-infected patients. There are no data regarding the effect of ARV exposure during pregnancy on newborns and infants. We therefore studied 38 subjects born from HIV-infected mothers, and we measured the speed-of-sound (SOS) at the tibia by quantitative ultrasonography (QUS) just after birth. QUS measurements at mid-tibia is easily performed in infants with the appropriate probe. Nevertheless, at this skeletal site only cortical bone is present, and therefore QUS measurements reflect the status of only one kind of bone tissue. We also measured bone alkaline phosphatase (BAP) and C-terminal telopeptide of type I collagen (CTX) in the cord blood as bone formation and resorption markers, respectively. SOS measurements were repeated at 4 and 12 months of age. As a control group we studied 94 subjects born from HIV-negative mothers. At birth the median (range) SOS of ARV-exposed neonates was 3006 (2870-3168) m/s, while that of control subjects was 3007 (2757-3311) m/s. The difference was not significant. BAP concentration of ARV-exposed was 103.6 (31.6-182.8) U/L, not different from that of control subjects (104.4 [43.2-227.2] U/L). CTX concentrations were 1.07 (0.26-2.8) ng/mL, and 1.38 (0.34-4.2) ng/mL in ARV-exposed and control subjects, respectively. SOS measurements at 4 months and 12 months of age were available for 17 ARV-exposed subjects and for 57 control subjects. SOS values changed significantly over time in both groups (F = 6.1; P < 0.0001). No differences were present between ARV-exposed and control subjects at 4 and 12 months. Our study suggests that ARV exposure during intrauterine life does not affect negatively bone metabolism and bone development, and that the changes occurring in bone QUS measurements during the first year of life in ARV-exposed subjects are similar to those occurring in healthy control infants.     

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.     

PTH level but not 25 (OH) vitamin D level predicts bone loss rates in the elderly

Summary  

We assessed the impact of calciotropic hormones on bone loss in 195 elderly subjects. After a median follow up of 4 years, parathyroid hormone (PTH) correlated negatively with changes in bone mineral density (BMD) at all skeletal sites. After adjustment for potential predictors of bone loss in the elderly, PTH level alone explained 3% of the variance in BMD changes at the hip.     

Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life

Summary  

Postmenopausal hemodialysis patients are at risk of complications related to renal mineral and bone disorder, and postmenopausal osteoporosis. In 112 postmenopausal hemodialysis patients, free estrogen index was positively correlated with bone mineral density (BMD) Z-score and the annual percent change of BMD in multiple regression analysis. Endogenous estrogen may prevent bone loss in postmenopausal hemodialysis patients throughout life.     

Exercise enhances angiogenesis during bone defect healing in mice

The aim of the present study was to investigate the effect of exercise on angiogenesis during bone defect healing in mice. We evaluated angiogenesis during cranial bone defect healing by intravital fluorescence microscopy (IVM) at days 0–21. To characterize the type of bone repair, we performed additional histomorphometric analyses at days 3–15. IVM was conducted in mice, which were housed in cages supplied with running wheels (exercise group; n = 7) and compared to IVM results of mice, which were housed in cages without running wheels (controls; n = 7). In the exercise group, we additionally performed correlation analyses between results of the IVM and the running distance. IVM showed an accelerated decrease of bone defect area in the exercise group compared to the control group. This was associated with a significantly higher blood vessel diameter in animals undergoing exercise at days 9 and 12 and a significant correlation between running distance and blood vessel density at day 9 (r = 0.74). Histomorphometry showed osseous bridging of the defect at day 9. The newly woven bone was covered by a neo‐periosteum containing those blood vessels, which were visible by IVM. We conclude that exercise accelerates bone defect healing and stimulates angiogenesis during bore repair. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1086–1092, 2011     

Biochemical markers of bone turnover predict bone loss in perimenopausal women but not in postmenopausal women-the Japanese Population-based Osteoporosis (JPOS) Cohort Study

Introduction The predictive value of biochemical markers of bone turnover for subsequent change in bone density in a population sample of healthy women with a wide range of ages has not been fully established. Methods We followed 1,283 women aged 15–79 years at baseline selected randomly from the inhabitants of three areas in Japan for 6 years, and examined 1,130 subjects with no disease or administration of drugs affecting bone metabolism. The annual change in bone density at the spine, total hip, and distal one third of the radius was determined during the follow-up period by dual x-ray absorptiometry and was compared among the groups using different levels of biochemical markers at baseline, including serum osteocalcin (OC) and bone-specific alkaline phosphatase (bone ALP), free and total (tDPD) forms of immunoreactive deoxypyridinoline, and type I collagen crosslinked C-terminal telopeptide (CTX) in urine. Results Premenopausal women aged 45 years or older with elevated levels of OC, bone ALP, CTX, or tDPD showed significantly greater bone loss at most skeletal sites during the follow-up period than those with lower levels, after adjustment for the effects of age, height, weight, dietary calcium intake, regular exercise, and current smoking. The greatest coefficient of determination of the model was observed in the association between CTX and bone loss at the hip during the first 3 years of follow-up (42.8%). These subjects were pooled with perimenopausal women at baseline, and those who still menstruated at follow-up in this pooled group showed significant but more modest associations, whereas those who entered menopause during the follow-up period showed clear associations. However, early postmenopausal women with less than 5 or 10 years since menopause showed an association that was limited mostly to the distal radius, and other postmenopausal groups had virtually no association. Conclusion Biochemical markers of bone turnover may predict bone loss in women undergoing menopausal transition but may not predict bone loss in postmenopausal women.     

Bone turnover markers in peripheral blood and marrow plasma reflect trabecular bone loss but not endocortical expansion in aging mice

We examined age-related changes in biochemical markers and regulators of osteoblast and osteoclast activity in C57BL/6 mice to assess their utility in explaining age-related changes in bone. Several recently discovered regulators of osteoclasts and osteoblasts were also measured to assess concordance between their systemic levels versus their levels in marrow plasma, to which bone cells are directly exposed. MicroCT of 6-, 12-, and 24-month-old mice indicated an early age-related loss of trabecular bone volume and surface, followed by endocortical bone loss and periosteal expansion. Trabecular bone loss temporally correlated with reductions in biomarkers of bone formation and resorption in both peripheral blood and bone marrow. Endocortical bone loss and periosteal bone gain were not reflected in these protein biomarkers, but were well correlated with increased expression of osteocalcin, rank, tracp5b, and cathepsinK in RNA extracted from cortical bone. While age-related changes in bone turnover markers remained concordant in blood versus marrow, aging led to divergent changes in blood versus marrow for the bone cell regulators RANKL, OPG, sclerostin, DKK1, and serotonin. Bone expression of runx2 and osterix increased progressively with aging and was associated with an increase in the number of osteoprogenitors and osteoclast precursors. In summary, levels of biochemical markers of bone turnover in blood and bone marrow plasma were predictive of an age-related loss of trabecular surfaces in adult C57BL/6 mice, but did not predict gains in cortical surfaces resulting from cortical expansion. Unlike these turnover markers, a panel of bone cell regulatory proteins exhibited divergent age-related changes in marrow versus peripheral blood, suggesting that their circulating levels may not reflect local levels to which osteoclasts and osteoblasts are directly exposed.     

Hemodynamic and metabolic alterations in peripheral tissue during hemorrhagic shock.

In dogs subjected to hypovolemic shock (modified Wiggers model) severe enough to decrease the arterial flow in an isolated hind limb by two-thirds, a marked hyperglycemia (three times control) and an increase in blood glucose AV difference (ten times control) occur. Despite the decreased arterial flow, glucose uptake by peripheral tissues increased by a factor of three within one-half hour of hemorrhage and remained elevated for several hours. Presumably, the increased glucose uptake reflects the need for more energy substrate during the hypoxic conditions of the decreased peripheral blood flow.