Site-specific changes in bone microarchitecture, mineralization, and stiffness during lactation and after weaning in mice

Despite the dramatic bone loss that occurs during lactation, bone mineral density rapidly recovers after offspring are weaned and milk production stops. The goal of this study is to quantify site-specific changes in bone quantity and quality during and after lactation in a mouse model. We used micro computed tomography (μCT), individual trabecula segmentation (ITS), digital topological analysis (DTA)-based tissue mineral density (TMD) analysis, and micro finite element analysis (μFEA) to quantify the effects of lactation and weaning on bone microarchitecture, mineralization, and stiffness at the spine, tibia, and femur. We found a significant decrease in trabecular plate microarchitecture, tissue mineralization of the trabecular surface, trabecular central skeleton, and intervening envelopes, and whole bone stiffness in lactating versus nulliparous mice at all three sites. In recovered mice, all these different aspects of bone quality were comparable to nulliparous mice at the spine. In contrast, trabecular plate microarchitecture and whole bone stiffness at the tibia and femur in recovered mice were lower than nulliparous mice, as were central trabecular tissue mineralization and cortical structure at the femur. These findings are consistent with clinical observations of partial recovery of femoral bone mineral density BMD after lactation in humans. The observed differences in trabecular surface tissue mineralization in nulliparous, lactating, and recovered mice are consistent with prior observations that maternal bone turnover shifts from resorption to formation at the time of pup weaning. The significant differences in trabecular central tissue mineralization during these three states suggest that osteocytes may contribute to the reversible loss of mineral during and after lactation. Future studies are necessary to determine whether differing functions of various bone cells at individual skeletal sites cause site-specific skeletal changes during and after lactation.     

Role of Retinoid Receptors in the Prevention and Treatment of Breast Cancer

Retinoids are vitamin A-related compounds thathave been found to prevent cancer in animals and humans.In this review, we discuss the role of retinoids andtheir receptors in the treatment and prevention of breast cancer. The retinoid receptors areexpressed in normal and malignant breast cells, and arecritical for normal development. In breast cells, whenbound by retinoid hormones, these proteins regulate proliferation, apoptosis, and differentiation.The mechanism by which retinoids inhibit breast cellgrowth has not been completely elucidated, however,retinoids have been shown to affect multiple signal transduction pathways, including IGF-,TGF-, and AP-1-dependent pathways. Retinoids havealso been shown to suppress the growth and prevent thedevelopment of breast cancer in animals. These agents suppress tumorigenesis in carcinogen-treatedrats and in transgenic mice, and inhibit the growth oftransplanted breast tumors. These promising preclinicalresults have provided the rationale to test retinoids in clinical trials for the treatment andprevention of breast cancer. Several retinoids,including all trans retinoic acid and9-cis retinoic acid, have been shown to havemodest activity in the treatment of breast cancer, and theseagents are now in clinical trials in combination withcytotoxic agents and anti-estrogens. Another retinoid,4-HPR, is currently being tested in a human cancer prevention trial. Preliminary results suggestthat 4-HPR may suppress breast cancer development inpremenopausal women. Future clinical trials will focuson testing new synthetic retinoids that have reduced toxicity and enhanced therapeutic andpreventive efficacy.     

Loss of the Vitamin D Receptor in Human Breast Cancer Cells Promotes Epithelial to Mesenchymal Cell Transition and Skeletal Colonization

Expression of the vitamin D receptor (VDR) is thought to be associated with neoplastic progression. However, the role of the VDR in breast cancer metastasis to bone and the molecular mechanisms underlying this process are unknown. Employing a rodent model (female Balb/c nu/nu mice) of systemic metastasis, we here demonstrate that knockdown of the VDR strongly increases the metastatic potential of MDA-MB-231 human breast cancer cells to bone, resulting in significantly greater skeletal tumor burden. Ablation of VDR expression promotes cancer cell mobility (migration) and invasiveness, thereby facilitating skeletal colonization. Mechanistically, these changes in tumor cell behavior are attributable to shifts in the expression of proteins involved in cell adhesion, proliferation, and cytoskeletal organization, patterns characteristic for epithelial-to-mesenchymal cell transition (EMT). In keeping with these experimental findings, analyses of human breast cancer specimens corroborated the association between VDR expression, EMT-typical changes in protein expression patterns, and clinical prognosis. Loss of the VDR in human breast cancer cells marks a critical point in oncogenesis by inducing EMT, promoting the dissemination of cancer cells, and facilitating the formation of tumor colonies in bone. © 2019 American Society for Bone and Mineral Research.     

Inhibiting activin‐A signaling stimulates bone formation and prevents cancer‐induced bone destruction in vivo

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma‐induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer. © 2010 American Society for Bone and Mineral Research.     

Direct Crosstalk Between Cancer and Osteoblast Lineage Cells Fuels Metastatic Growth in Bone via Auto‐Amplification of IL‐6 and RANKL Signaling Pathways

The bone microenvironment and its modification by cancer and host cell interactions is a key driver of skeletal metastatic growth. Interleukin‐6 (IL‐6) stimulates receptor activator of NF‐κB ligand (RANKL) expression in bone cells, and serum IL‐6 levels are associated with poor clinical outcomes in cancer patients. We investigated the effects of RANKL on cancer cells and the role of tumor‐derived IL‐6 within the bone microenvironment. Using human breast cancer cell lines to induce tumors in the bone of immune‐deficient mice, we first determined whether RANKL released by cells of the osteoblast lineage directly promotes IL‐6 expression by cancer cells in vitro and in vivo. We then disrupted of IL‐6 signaling in vivo either via knockdown of IL‐6 in tumor cells or through treatment with specific anti‐human or anti‐mouse IL‐6 receptor antibodies to investigate the tumor effect. Finally, we tested the effect of RANK knockdown in cancer cells on cancer growth. We demonstrate that osteoblast lineage‐derived RANKL upregulates secretion of IL‐6 by breast cancers in vivo and in vitro. IL‐6, in turn, induces expression of RANK by cancer cells, which sensitizes the tumor to RANKL and significantly enhances cancer IL‐6 release. Disruption in vivo of this auto‐amplifying crosstalk by knockdown of IL‐6 or RANK in cancer cells, or via treatment with anti‐IL‐6 receptor antibodies, significantly reduces tumor growth in bone but not in soft tissues. RANKL and IL‐6 mediate direct paracrine‐autocrine signaling between cells of the osteoblast lineage and cancer cells, significantly enhancing the growth of metastatic breast cancers within bone. © 2014 American Society for Bone and Mineral Research.     

Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Absence of Calcitriol Causes Increased Lactational Bone Loss and Lower Milk Calcium but Does Not Impair Post‐lactation Bone Recovery in Cyp27b1 Null Mice

We hypothesized that adaptation to calcium supply demands of pregnancy and lactation do not require calcitriol. Adult Cyp27b1 null mice lack calcitriol and have hypocalcemia, hypophosphatemia, and rickets. We studied wild‐type (WT) and null sister pairs raised on a calcium‐, phosphorus‐, and lactose‐enriched “rescue” diet that prevents hypocalcemia and rickets. Bone mineral content (BMC) increased >30% in pregnant nulls, declined 30% during lactation, and increased 30% by 4 weeks post‐weaning. WT showed less marked changes. Micro‐CT revealed loss of trabecular bone and recovery in both genotypes. In lactating nulls, femoral cortical thickness declined >30%, whereas endocortical perimeter increased; both recovered to baseline after weaning; there were no such changes in WT. Histomorphometry revealed a profound increase in osteoid surface and thickness in lactating nulls, which recovered after weaning. By three‐point bend test, nulls had a >50% decline in ultimate load to failure that recovered after weaning. Although nulls showed bone loss during lactation, their milk calcium content was 30% lower compared with WT. Serum parathyroid hormone (PTH) was markedly elevated in nulls at baseline, reduced substantially in pregnancy, but increased again during lactation and remained high post‐weaning. In summary, pregnant Cyp27b1 nulls gained BMC with reduced secondary hyperparathyroidism, implying increased intestinal calcium delivery. Lactating nulls lost more bone mass and strength than WT, accompanied by increased osteoid, reduced milk calcium, and worsened secondary hyperparathyroidism. This implies suboptimal intestinal calcium absorption. Post‐weaning, bone mass and strength recovered to baseline, whereas BMC exceeded baseline by 40%. In conclusion, calcitriol‐independent mechanisms regulate intestinal calcium absorption and trabecular bone metabolism during pregnancy and post‐weaning but not during lactation; calcitriol may protect cortical bone during lactation. © 2017 American Society for Bone and Mineral Research.     

N‐cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage–Specific

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N‐cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N‐cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin‐insensitive Lrp5^A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2‐deleted mice, suggesting N‐cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β‐catenin accumulation after administration of an anti‐Dkk1 antibody are enhanced in N‐cadherin–deficient mice. Thus, although lack of N‐cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N‐cadherin in osteolineage cells favors bone formation. Hence, N‐cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.     

Prostate Cancer Cells Preferentially Home to Osteoblast‐rich Areas in the Early Stages of Bone Metastasis: Evidence From In Vivo Models

It has been suggested that metastasis‐initiating cells gain a foothold in bone by homing to a metastastatic microenvironment (or “niche”). Whereas the precise nature of this niche remains to be established, it is likely to contain bone cell populations including osteoblasts and osteoclasts. In the mouse tibia, the distribution of osteoblasts on endocortical bone surfaces is non‐uniform, and we hypothesize that studying co‐localization of individual tumor cells with resident cell populations will reveal the identity of critical cellular components of the niche. In this study, we have mapped the distribution of three human prostate cancer cell lines (PC3‐NW1, LN‐CaP, and C4 2B4) colonizing the tibiae of athymic mice following intracardiac injection and evaluated their interaction with potential metastatic niches. Prostate cancer cells labeled with the fluorescent cell membrane dye (Vybrant DiD) were found by two‐photon microscopy to be engrafted in the tibiae in close proximity (～40 µm) to bone surfaces and 70% more cancer cells were detected in the lateral compared to the medial endocortical bone regions. This was associated with a 5‐fold higher number of osteoblasts and 7‐fold higher bone formation rate on the lateral endocortical bone surface compared to the medial side. By disrupting cellular interactions mediated by the chemokine (C‐X‐C motif) receptor 4 (CXCR4)/chemokine ligand 12 (CXCL12) axis with the CXCR4 inhibitor AMD3100, the preferential homing pattern of prostate cancer cells to osteoblast‐rich bone surfaces was disrupted. In this study, we map the location of prostate cancer cells that home to endocortical regions in bone and our data demonstrate that homing of prostate cancer cells is associated with the presence and activity of osteoblast lineage cells, and suggest that therapies targeting osteoblast niches should be considered to prevent development of incurable prostate cancer bone metastases. © 2014 American Society for Bone and Mineral Research.     

The calcium‐sensing receptor mediates bone turnover induced by dietary calcium and parathyroid hormone in neonates

We have investigated, in neonates, whether the calcium‐sensing receptor (CaR) mediates the effects of dietary calcium on bone turnover and/or modulates parathyroid hormone (PTH)–induced bone turnover. Wild‐type (WT) pups and pups with targeted deletion of the Pth (Pth^–/–) gene or of both Pth and CaR (Pth^–/–CaR^–/–) genes were nursed by dams on a normal or high‐calcium diet. Pups nursed by dams on a normal diet received daily injections of vehicle or of PTH(1–34) (80 µg/kg) for 2 weeks starting from 1 week of age. In pups receiving vehicle and fed by dams on a normal diet, trabecular bone volume, osteoblast number, type 1 collagen–positive area, and mineral apposition rate, as well as the expression of bone‐formation‐related genes, all were reduced significantly in Pth^–/– pups compared with WT pups and were decreased even more dramatically in Pth^–/–CaR^–/– pups. These parameters were increased in WT and Pth^–/– pups but not in Pth^–/–CaR^–/– pups fed by dams on a high‐calcium diet compared with pups fed by dams on a normal diet. These parameters also were increased in WT, Pth^–/–, and Pth^–/–CaR^–/– pups following exogenous PTH treatment; however, the percentage increase was less in Pth^–/–CaR^–/– pups than in WT and Pth^–/– pups. In vehicle‐treated pups fed by dams on either the normal or high‐calcium diet and in PTH‐treated pups fed by dams on a normal diet, the number and surfaces of osteoclasts and the ratio of RANKL/OPG were reduced significantly in Pth^–/– pups and less significantly in Pth^–/–CaR^–/– pups compared with WT pups. These parameters were further reduced significantly in WT and Pth^–/– pups from dams fed a high‐calcium diet but did not decrease significantly in similarly treated Pth^–/–CaR^–/– pups, and they increased significantly in PTH‐treated pups compared with vehicle‐treated, genotype‐matched pups fed by dams on the normal diet. These results indicate that in neonates, the CaR mediates alterations in bone turnover in response to changes in dietary calcium and modulates PTH‐stimulated bone turnover. © 2011 American Society for Bone and Mineral Research.     

The G60S connexin 43 mutation activates the osteoblast lineage and results in a resorption‐stimulating bone matrix and abrogation of old‐age–related bone loss

We previously isolated a low bone mass mouse, Gja1^Jrt/ + , with a mutation in the gap junction protein, alpha 1 gene (Gja1), encoding for a dominant negative G60S Connexin 43 (Cx43) mutant protein. Similar to other Cx43 mutant mouse models described, including a global Cx43 deletion, four skeletal cell conditional‐deletion mutants, and a Cx43 missense mutant (G138R/ +), a reduction in Cx43 gap junction formation and/or function resulted in mice with early onset osteopenia. In contrast to other Cx43 mutants, however, we found that Gja1^Jrt/+ mice have both higher bone marrow stromal osteoprogenitor numbers and increased appendicular skeleton osteoblast activity, leading to cell autonomous upregulation of both matrix bone sialoprotein (BSP) and membrane‐bound receptor activator of nuclear factor‐κB ligand (mbRANKL). In younger Gja1^Jrt/+ mice, these contributed to increased osteoclast number and activity resulting in early onset osteopenia. In older animals, however, this effect was abrogated by increased osteoprotegerin (OPG) levels and serum alkaline phosphatase (ALP) so that differences in mutant and wild‐type (WT) bone parameters and mechanical properties lessened or disappeared with age. Our study is the first to describe a Cx43 mutation in which osteopenia is caused by increased rather than decreased osteoblast function and where activation of osteoclasts occurs not only through increased mbRANKL but an increase in a matrix protein that affects bone resorption, which together abrogate age‐related bone loss in older animals. © 2013 American Society for Bone and Mineral Research.     

The Lrp4R1170Q Homozygous Knock‐In Mouse Recapitulates the Bone Phenotype of Sclerosteosis in Humans

Sclerosteosis is a rare autosomal recessive bone disorder marked by hyperostosis of the skull and tubular bones. Initially, we and others reported that sclerosteosis was caused by loss‐of‐function mutations in SOST, encoding sclerostin. More recently, we identified disease‐causing mutations in LRP4, a binding partner of sclerostin, in three sclerosteosis patients. Upon binding to sclerostin, LRP4 can inhibit the canonical WNT signaling that is known to be an important pathway in the regulation of bone formation. To further investigate the role of LRP4 in the bone formation process, we generated an Lrp4 mutated sclerosteosis mouse model by introducing the p.Arg1170Gln mutation in the mouse genome. Extensive analysis of the bone phenotype of the Lrp4R1170Q/R1170Q knock‐in (KI) mouse showed the presence of increased trabecular and cortical bone mass as a consequence of increased bone formation by the osteoblasts. In addition, three‐point bending analysis also showed that the increased bone mass results in increased bone strength. In contrast to the human sclerosteosis phenotype, we could not observe syndactyly in the forelimbs or hindlimbs of the Lrp4 KI animals. Finally, we could not detect any significant changes in the bone formation and resorption markers in the serum of the mutant mice. However, the serum sclerostin levels were strongly increased and the level of sclerostin in the tibia was decreased in Lrp4R1170Q/R1170Q mice, confirming the role of LRP4 as an anchor for sclerostin in bone. In conclusion, the Lrp4R1170Q/R1170Q mouse is a good model for the human sclerosteosis phenotype caused by mutations in LRP4 and can be used in the future for further investigation of the mechanism whereby LRP4 regulates bone formation. © 2017 American Society for Bone and Mineral Research.     

N‐acetylcysteine prevents nitric oxide‐induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis

We investigated whether N‐acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)‐induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0–2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase‐3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO‐induced apoptosis, ROS overproduction, p53 up‐regulation, and caspase‐3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis‐preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO‐induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:156–163, 2010     

Levels of serotonin,sclerostin, bone turnover markers as well as bone density and microarchitecture in patients with high‐bone‐mass phenotype due to a mutation in Lrp5

Patients with an activation mutation of the Lrp5 gene exhibit high bone mass (HBM). Limited information is available regarding compartment‐specific changes in bone. The relationship between the phenotype and serum serotonin is not well documented. To evaluate bone, serotonin, and bone turnover markers (BTM) in Lrp5‐HBM patients, we studied 19 Lrp5‐HBM patients (T253I) and 19 age‐ and sex‐matched controls. DXA and HR‐pQCT were used to assess BMD and bone structure. Serum serotonin, sclerostin, dickkopf‐related protein 1 (DKK1), and BTM were evaluated. Z‐scores for the forearm, total hip, lumbar spine, forearm, and whole body were significantly increased (mean ± SD) between 4.94 ± 1.45 and 7.52 ± 1.99 in cases versus ?0.19 ± 1.19 to 0.58 ± 0.84 in controls. Tibial and radial cortical areas, thicknesses, and BMD were significantly higher in cases. In cases, BMD at the lumbar spine and forearm and cortical thickness were positively associated and trabecular area negatively associated with age (r = 0.49, 0.57, 0.74, and ?0.61, respectively, p < .05). Serotonin was lowest in cases (69.5 [29.9–110.4] ng/mL versus 119.4 [62.3–231.0] ng/mL, p < .001) and inversely associated with tibial cortical density (r = ?0.49, p < .05) and directly with osteocalcin (OC), bone‐specific alkaline phosphatase (B‐ALP), and procollagen type 1 amino‐terminal propeptide (PINP) (r = 0.52–0.65, p < .05) in controls only. OC and S‐CTX were lower and sclerostin higher in cases, whereas B‐ALP, PINP, tartrate‐resistant acid phosphatase (TRAP), and dickkopf‐related protein 1 (DKK1) were similar in cases and controls. In conclusion, increased bone mass in Lrp5‐HBM patients seems to be caused primarily by changes in trabecular and cortical bone mass and structure. The phenotype appeared to progress with age, but BTM did not suggest increased bone formation. © 2011 American Society for Bone and Mineral Research     

The effects of a two‐year randomized,controlled trial of whey protein supplementation on bone structure,IGF‐1, and urinary calcium excretion in older postmenopausal women

The effects of dietary protein on bone structure and metabolism have been controversial, with evidence for and against beneficial effects. Because no long‐term randomized, controlled studies have been performed, a two‐year study of protein supplementation in 219 healthy ambulant women aged 70 to 80 years was undertaken. Participants were randomized to either a high‐protein drink containing 30 g of whey protein (n = 109) or a placebo drink identical in energy content, appearance, and taste containing 2.1 g of protein (n = 110). Both drinks provided 600 mg of calcium. Dual‐energy X‐ray absorptiometric (DXA) hip areal bone mineral density (aBMD), 24‐hour urinary calcium excretion, and serum insulin‐like growth factor 1 (IGF‐1) were measured at baseline and at 1 and 2 years. Quantitative computed tomographic (QCT) hip volumetric bone mineral density (vBMD) and a femoral neck engineering strength analysis were undertaken at baseline and at 2 years. Baseline average protein intake was 1.1 g/kg of body weight per day. There was a significant decrease in hip DXA aBMD and QCT vBMD over 2 years with no between‐group differences. Femoral neck strength was unchanged in either group over time. The 24‐hour urinary calcium excretion increased significantly from baseline in both groups at 1 year but returned to baseline in the placebo group at 2 years, at which time the protein group had a marginally higher value. Compared with the placebo group, the protein group had significantly higher serum IGF‐1 level at 1 and 2 years (7.3% to 8.0%, p < .05). Our study showed that in protein‐replete healthy ambulant women, 30 g of extra protein increased IGF‐1 but did not have beneficial or deleterious effects on bone mass or strength. The effect of protein supplementation in populations with low dietary protein intake requires urgent attention. © 2011 American Society for Bone and Mineral Research     

The association of red blood cell n‐3 and n‐6 fatty acids with bone mineral density and hip fracture risk in the women's health initiative

Omega‐3 (n‐3) and omega‐6 (n‐6) polyunsaturated fatty acids (PUFA) in red blood cells (RBCs) are an objective indicator of PUFA status and may be related to hip fracture risk. The primary objective of this study was to examine RBC PUFAs as predictors of hip fracture risk in postmenopausal women. A nested case‐control study (n = 400 pairs) was completed within the Women's Health Initiative (WHI) using 201 incident hip fracture cases from the Bone Mineral Density (BMD) cohort, along with 199 additional incident hip fracture cases randomly selected from the WHI Observational Study. Cases were 1:1 matched on age, race, and hormone use with non–hip fracture controls. Stored baseline RBCs were analyzed for fatty acids using gas chromatography. After removing degraded samples, 324 matched pairs were included in statistical analyses. Stratified Cox proportional hazard models were constructed according to case‐control pair status; risk of fracture was estimated for tertiles of RBC PUFA. In adjusted hazard models, lower hip fracture risk was associated with higher RBC α‐linolenic acid (tertile 3 [T3] hazard ratio [HR]: 0.44; 95% confidence interval [CI], 0.23–0.85; p for linear trend 0.0154), eicosapentaenoic acid (T3 HR: 0.46; 95% CI, 0.24–0.87; p for linear trend 0.0181), and total n‐3 PUFAs (T3 HR: 0.55; 95% CI, 0.30–1.01; p for linear trend 0.0492). Conversely, hip fracture nearly doubled with the highest RBC n‐6/n‐3 ratio (T3 HR: 1.96; 95% CI, 1.03–3.70; p for linear trend 0.0399). RBC PUFAs were not associated with BMD. RBC PUFAs were indicative of dietary intake of marine n‐3 PUFAs (Spearman's rho = 0.45, p < 0.0001), total n‐6 PUFAs (rho = 0.17, p < 0.0001) and linoleic acid (rho = 0.09, p < 0.05). These results suggest that higher RBC α‐linolenic acid, as well as eicosapentaenoic acid and total n‐3 PUFAs, may predict lower hip fracture risk. Contrastingly, a higher RBC n‐6/n‐3 ratio may predict higher hip fracture risk in postmenopausal women. © 2013 American Society for Bone and Mineral Research.