Maternal folic acid supplementation does not impact skeletal muscle function and metabolism in male and female CD-1 mouse offspring.

Folic acid fortification of all white flour, enriched pasta, and cornmeal products became mandatory in Canada to reduce risk of neural tube defects at birth. Furthermore, Health Canada and the Society of Obstetricians and Gynaecologists of Canada recommend women take daily prenatal folic acid supplements in addition to folic acid fortified foods during pregnancy. However, the influence of maternal folic acid supplementation on offspring development, specifically the highly abundant and metabolically active skeletal muscle, is currently unknown. Thus, the purpose of this study was to determine the effect of supplemental folic acid (four times higher than normal dietary consumption), in utero and throughout suckling on muscle size, function, and metabolism in male and female CD-1 mouse offspring. The major findings were that maternal exposure to supplemental folic acid (i) had no impact on postpartum growth rates or muscle mass in female and male offspring, (ii) had no impact on skeletal muscle contractile kinetics in females and male offspring, and (iii) increased maximal phosphofructokinase activity in extensor digitorum longus of female and male offspring. These findings suggest that exposure to folic acid supplementation in utero and throughout suckling at levels four times higher than recommended had minimal effect on skeletal muscle size, function, and metabolism regardless of sex. Future research is needed explore the underlying biological pathways and mechanisms affected by folic acid supplementation during pregnancy and lactation on offspring skeletal muscle tissue, specifically in humans.

Impact of moderate dietary protein restriction on glucose homeostasis in a model of estrogen deficiency.

The need to consume adequate dietary protein to preserve physical function during ageing is well recognized. However, the effect of protein intakes on glucose metabolism is still intensively debated. During age-related estrogen withdrawal at the time of the menopause, it is known that glucose homeostasis may be impaired but the influence of dietary protein levels in this context is unknown. The aim of the present study is to elucidate the individual and interactive effects of estrogen deficiency and suboptimal protein intake on glucose homeostasis in a preclinical model involving ovariectomy (OVX) and a 13 week period of a moderately reduced protein intake in 7 month-old ageing rats. To investigate mechanisms of action acting via the pancreas-liver-muscle axis, fasting circulating levels of insulin, glucagon, IGF-1, FGF21 and glycemia were measured. The hepatic lipid infiltration and the protein expression of GLUT4 in the gastrocnemius were analyzed. The gene expression of some hepatokines, myokines and lipid storage/oxidation related transcription factors were quantified in the liver and the gastrocnemius. We show that, regardless of the estrogen status, moderate dietary protein restriction increases fasting glycemia without modifying insulinemia, body weight gain and composition. This fasting hyperglycemia is associated with estrogen status-specific metabolic alterations in the muscle and liver. In estrogen-replete (SHAM) rats, GLUT4 was down-regulated in skeletal muscle while in estrogen-deficient (OVX) rats, hepatic stress-associated hyperglucagonemia and high serum FGF21 were observed. These findings highlight the importance of meeting dietary protein needs to avoid disturbances in glucose homeostasis in ageing female rats with or without estrogen withdrawal.

Effect of Low Dietary Vitamin D Fed Prior to and During Pregnancy and Lactation on Maternal Bone Mineral Density, Structure, and Strength in C57BL/6 Mice.

Several studies have shown that diets containing lower vitamin D than in the AIN-93G diet do not compromise bone structure, bone mineral density (BMD), and/or bone strength in male and female mice. This study determined if a diet containing low vitamin D from prepregnancy through to the end of lactation maintained these bone outcomes to a similar extent as a high vitamin D diet. Mice were fed an AIN-93G diet with 25 (LD diet) or 5000 (HD diet) IU vitamin D/kg diet from premating through to lactation (n = 15/group). Of the major structure outcomes, only cortical area fraction of the distal femur was lower (P <0.05) with the LD diet. Lumbar vertebra BMD was lower (P <0.05) with LD whereas distal femur BMD and bone strength at 3 sites did not differ. Dams fed an LD diet premating through to the end of lactation had largely similar bone outcomes to dams fed a HD diet.

JAK2-IGF1 axis in osteoclasts regulates postnatal growth in mice.

Osteoclasts are specialized cells of the hematopoietic lineage that are responsible for bone resorption and play a critical role in musculoskeletal disease. JAK2 is a key mediator of cytokine and growth factor signaling; however, its role in osteoclasts in vivo has yet to be investigated. To elucidate the role of JAK2 in osteoclasts, we generated an osteoclast-specific JAK2-KO (Oc-JAK2-KO) mouse using the Cre/Lox-P system. Oc-JAK2-KO mice demonstrated marked postnatal growth restriction; however, this was not associated with significant changes in bone density, microarchitecture, or strength, indicating that the observed phenotype was not due to alterations in canonical osteoclast function. Interestingly, Oc-JAK2-KO mice had reduced osteoclast-specific expression of IGF1, suggesting a role for osteoclast-derived IGF1 in determination of body size. To directly assess the role of osteoclast-derived IGF1, we generated an osteoclast-specific IGF1-KO mouse, which showed a similar growth-restricted phenotype. Lastly, overexpression of circulating IGF1 by human transgene rescued the growth defects in Oc-JAK2-KO mice, in keeping with a causal role of IGF1 in these models. Together, our data show a potentially novel role for Oc-JAK2 and IGF1 in the determination of body size, which is independent of osteoclast resorptive function.

Bone structure is largely unchanged in growing male CD-1 mice fed lower levels of vitamin D and calcium than in the AIN-93G diet.

BACKGROUND: Calcium (Ca) and vitamin D (vit D) in the AIN-93G diet may be higher than required for healthy bone development, and mask the potential benefit of a dietary intervention. OBJECTIVE: The objective was to determine if lower levels of Ca and vit D than is present in the AIN-93G diet supports bone development in growing male CD-1 mice. METHODS: Weanling male CD-1 mice were randomized to modified AIN-93G diets containing either 100 (Trial 1) or 400 (Trial 2) IU vit D/kg diet within one of two or three Ca levels (0.35, 0.30, or 0.25% Ca diet in Trial 1 or 0.35% or 0.25% in Trial 2) or the AIN-93G diet (1000 IU/kg vit D and 0.5% Ca) from weaning to 4 months of age (n = 13-15/group). At 2 and 4 months of age, BMD and structural properties of the tibia were analyzed in vivo. Structure of lumbar vertebra 4 (L4) and mandible, and femur strength were assessed ex vivo at age 4 months. RESULTS: There were no differences in tibia, L4, and mandible structure between the AIN-93G diet and the 0.35% Ca groups at either vit D level. A few structure outcomes were compromised with the 0.25 and/or 0.3% Ca diets but there were no differences in femur biomechanical strength compared to AIN-93G group in either Trial. CONCLUSION: At 400 or 100 IU vit D/kg diet, Ca can be lowered to 0.35% without detriment to BMD or bone structure while bone strength is not altered at lower Ca (0.25%) compared to CD-1 mice fed AIN-93G diet. Because of genetic variation in CD-1 mice among different breeding facilities, results in CD-1 mice from other facilities may differ from the present study.

Bone development in growing female mice fed calcium and vitamin D at lower levels than is present in the AIN-93G reference diet.

BACKGROUND: The AIN-93G reference (REF) diet is used to allow the comparison within and between studies of different research groups but its levels of vitamin D (vit D) and calcium (Ca) may be higher than required for healthy bone structure and bone mineral density (BMD). OBJECTIVE: To determine if lower dietary levels of Ca (3.5, 3 or 2.5 g Ca/kg diet) at 1 of 2 levels of vit D (100 or 400 IU/kg diet) supports similar development of bone structure and BMD compared to AIN-93G reference (REF) diet in female CD-1 mice at 2 and 4 months of age. METHODS: Within a trial, weanling female mice (n = 12-15/group) were randomized to 1 of 4 diets until necropsy at 4 months of age: Trial 1: 100 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g Ca/kg diet (REF); and Trial 2: 400 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g/kg diet (REF). At age 2 and 4 months, in vivo bone structure and BMD were assessed using micro-computed tomography (µCT) at the proximal and midpoint tibia. At age 4 months, lumbar vertebra 4 (L4) and mandible structure were analyzed ex vivo, femur strength at midpoint and neck was assessed and serum 25(OH)D3 and PTH were quantified. RESULTS: For Trial 1 (100 IU vit D/kg), there were no differences in tibia structure at age 2 and 4 months nor L4 or mandible structure or femur strength at the midpoint or neck at 4 months of age despite lower serum 25(OH)D3 among all groups compared to REF. For Trial 2 (400 IU vit D/kg), mice fed 2.5 g Ca/kg diet had lower (p < 0.05) Ct.Ar/Tt.Ar and Ct.Th at the tibia midpoint compared to REF. Furthermore, Ct.Th. was greater in REF and 3.5 g Ca/kg diet compared to 2.5 g Ca/kg diet at age 2 but not 4 months of age. At L4, BV/TV was lower (p < 0.05) in the 3 g Ca/kg diet group compared to REF at age 4 months. There were no differences among groups for serum 25(OH)D3 or femur strength at the midpoint or neck. Serum PTH was not elevated compared to REF in either Trial. CONCLUSION: Lowering both dietary vit D (100 IU/kg) and Ca (2.5 g/kg) in AIN-93G diet did not result in differences in bone development of female CD-1 mice at early adulthood. Translational relevance of bone studies conducted using the AIN-93G diet may be affected by its high vit D and Ca content.

Nutritional Programming of Bone Structure in Male Offspring by Maternal Consumption of Citrus Flavanones.

Maternal exposure to hesperidin (HSP) and naringin (NAR) during pregnancy and lactation transiently compromised bone mineral density (BMD) and bone structure at the proximal tibia in female CD-1 offspring. We examined whether maternal consumption of HSP + NAR during pregnancy and lactation compromises BMD, bone structure, and bone strength in male CD-1 offspring. Male CD-1 offspring, from mothers fed a control diet (CON, n = 10) or a 0.5% HSP + 0.25% NAR diet (HSP + NAR, n = 8) for 5 weeks before mating and throughout pregnancy and lactation, were weaned and fed CON until 6 months of age. In vivo micro-computed tomography (µCT) measured tibia BMD and structure at 2, 4, and 6 months of age. Ex vivo µCT measured femur and lumbar vertebrae (LV) structure at age 6 months. Ex vivo BMD (femur, LV) and biomechanical strength (femur and tibia midpoint, femur neck) were assessed at age 6 months by dual energy x-ray absorptiometry and strength testing, respectively. At all ages, HSP + NAR offspring had greater (p < 0.05) proximal tibia cortical structure compared to CON offspring. At age 4 months, proximal tibia trabecular structure was greater (p < 0.05) than CON offspring. At age 6 months, femur neck and LV trabecular structure were greater (p < 0.05) than CON offspring. Our results demonstrate that unlike our previous study of female offspring, maternal consumption of HSP + NAR resulted in greater bone structure at the proximal tibia in male CD-1 offspring that persisted to 6 months of age. Thus, maternal programming of offspring BMD and bone structure from consumption of HSP + NAR occurred as a sex-specific response.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography.

The use of in vivo micro-computed tomography (µCT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo µCT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo µCT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Influence of longitudinal radiation exposure from microcomputed tomography scanning on skeletal muscle function and metabolic activity in female CD-1 mice.

Microcomputed tomography (µCT) is an imaging technology to assess bone microarchitecture, a determinant of bone strength. When measured in vivo, µCT exposes the skeletal site of interest to a dose of radiation, in addition to nearby skeletal muscles as well. Therefore, the aim of this study was to determine the effects of repeated radiation exposure from in vivo µCT on muscle health - specifically, muscle morphometrics, contractile function, and enzyme activity. This study exposed the right hind limb of female mice to either a low (26 cGy) or moderate (46 cGy) dose, at 2, 4, and 6 months of age, while the left hind limb of the same animal was exposed to a single dose at 6 months to serve as a nonirradiated control. Muscle weight, cross-sectional area, isometric contractile function, and representative maximal enzyme activities of amino acid, fatty acid, glucose, and oxidative metabolism in extensor digitorum longus (EDL) and soleus were assessed. Low-dose radiation had no effect. In contrast, moderate-dose radiation resulted in a 5% increase in time-to-peak tension and 16% increase in half-relaxation time of isometric twitches in EDL, although these changes were not seen when normalized to force. Moderate-dose radiation also resulted in an ~33% decrease in citrate synthase activity in soleus but not EDL, with no changes to the other enzymes measured. Thus, three low doses of radiation over 6 months had no effect on contractile function or metabolic enzyme activity in soleus and EDL of female mice. In contrast, three moderate doses of radiation over 6 months induced some effects on metabolic enzyme activity in soleus but not EDL Future studies that wish to investigate muscle tissue that is adjacent to scanned bone should take radiation exposure dose into consideration.

Repeated irradiation from micro-computed tomography scanning at 2, 4 and 6 months of age does not induce damage to tibial bone microstructure in male and female CD-1 mice.

Long-term effects of repeated in vivo micro-computed tomography (µCT) scanning at key stages of growth and bone development (ages 2, 4 and 6 months) on trabecular and cortical bone structure, as well as developmental patterns, have not been studied. We determined the effect of repetitive µCT scanning at age 2, 4 and 6 months on tibia bone structure of male and female CD-1 mice and characterized developmental changes. At 2, 4 and 6 months of age, right tibias were scanned using in vivo µCT (Skyscan 1176) at one of three doses of radiation per scan: 222, 261 or 460 mGy. Left tibias of the same mice were scanned only at 6 months to serve as non-irradiated controls to determine whether recurrent radiation exposure alters trabecular and cortical bone structure at the proximal tibia. In males, eccentricity was lower (P<0.05) in irradiated compared with non-irradiated tibias (222 mGy group). Within each sex, all other structural outcomes were similar between irradiated and non-irradiated tibias regardless of dose. Trabecular bone loss occurred in all mice due to age while cortical development continued to age 6 months. In conclusion, repetitive µCT scans at various radiation doses did not damage trabecular or cortical bone structure of proximal tibia in male and female CD-1 mice. Moreover, scanning at 2, 4 and 6 months of age highlight the different developmental time course between trabecular and cortical bone. These scanning protocols can be used to investigate longitudinal responses of bone structures to an intervention.

Maternal Consumption of Hesperidin and Naringin Flavanones Exerts Transient Effects to Tibia Bone Structure in Female CD-1 Offspring.

Hesperidin (HSP) and naringin (NAR), flavanones rich in citrus fruits, support skeletal integrity in adult and aging rodent models. This study determined whether maternal consumption of HSP and NAR favorably programs bone development, resulting in higher bone mineral density (BMD) and greater structure and biomechanical strength (i.e., peak load) in female offspring. Female CD-1 mice were fed a control diet or a HSP + NAR diet five weeks before pregnancy and throughout pregnancy and lactation. At weaning, female offspring were fed a control diet until six months of age. The structure and BMD of the proximal tibia were measured longitudinally using in vivo microcomputed tomography at 2, 4, and 6 months of age. The trabecular bone structure at two and four months and the trabecular BMD at four months were compromised at the proximal tibia in mice exposed to HSP and NAR compared to the control diet (p < 0.001). At six months of age, these differences in trabecular structure and BMD at the proximal tibia had disappeared. At 6 months of age, the tibia midpoint peak load, BMD, structure, and the peak load of lumbar vertebrae and femurs were similar (p > 0.05) between the HSP + NAR and control groups. In conclusion, maternal consumption of HSP and NAR does not enhance bone development in female CD-1 offspring.

Maternal Dietary Vitamin D Does Not Program Systemic Inflammation and Bone Health in Adult Female Mice Fed an Obesogenic Diet.

Obesity is associated with systemic inflammation and impaired bone health. Vitamin D regulates bone metabolism, and has anti-inflammatory properties and epigenetic effects. We showed that exposure to high dietary vitamin D during pregnancy and lactation beneficially programs serum concentration of lipopolysaccharide (LPS) and bone structure in male offspring fed an obesogenic diet. Here we assessed if this effect is also apparent in females. C57BL/6J dams were fed AIN93G diet with high (5000 IU/kg diet) or low (25 IU/kg diet) vitamin D during pregnancy and lactation. Post-weaning, female offspring remained on their respective vitamin D level or were switched and fed a high fat and sucrose diet (44.2% fat, 19.8% sucrose) until age seven months when glucose response, adiposity, serum LPS, and bone mineral, trabecular and cortical structure, and biomechanical strength properties of femur and vertebra were assessed. There was no evidence for a programming effect of vitamin D for any outcomes. However, females exposed to a high vitamin D diet post-weaning had higher bone mineral content (p = 0.037) and density (p = 0.015) of lumbar vertebra. This post-weaning benefit suggests that in females, bone mineral accrual but not bone structure is compromised with low vitamin D status in utero until weaning in an obesogenic context.

Skeletal site-specific effects of endurance running on structure and strength of tibia, lumbar vertebrae, and mandible in male Sprague-Dawley rats.

Bone microarchitecture, bone mineral density (BMD), and bone strength are affected positively by impact activities such as running; however, there are discrepancies in the magnitude of these effects. These inconsistencies are mainly a result of varying training protocols, analysis techniques, and whether or not the skeletal sites measured are weight bearing. This study's purpose was to determine the effects of endurance running on sites that experience different weight bearing and load. Eight-week-old male Sprague-Dawley rats (n = 20) were randomly assigned to either a group with a progressive treadmill running protocol (25 m/min for 1 h, incline of 10%) or a nontrained control group for 8 weeks. The trabecular structure of the tibia, lumbar vertebra (L3), and mandible and the cortical structure at the tibia midpoint were measured using microcomputed tomography to quantify bone volume fraction (i.e., bone volume divided by total volume (BV/TV)), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness. BMD at the proximal tibia, lumbar vertebrae (L1-L3), and mandible was measured using dual energy X-ray absorptiometry. The tibia midpoint strength was measured by 3-point bending using a materials testing system. Endurance running resulted in superior bone structure at the proximal tibia (12% greater BV/TV (p = 0.03), 14% greater Tb.N (p = 0.01), and 19% lower Tb.Sp (p = 0.05)) but not at other sites. Contrary to our hypothesis, mandible bone structure was altered after endurance training (8% lower BV/TV (p < 0.01) and 15% lower Tb.Th (p < 0.01)), which may be explained by a lower food intake, resulting in less mechanical loading from chewing. These results highlight the site-specific effects of loading on the skeleton.

Combined high-fat-resveratrol diet and RIP140 knockout mice reveal a novel relationship between elevated bone mitochondrial content and compromised bone microarchitecture, bone mineral mass, and bone strength in the tibia.

SCOPE: While resveratrol (RSV) is associated with the prevention of high-fat (HF) diet-induced insulin resistance, the effects on bone health combined with an HF-diet is unknown. Therefore, we determined the effect of RSV on bone microarchitecture in the presence of an HF-diet, while also elucidating molecular adaptations within bone that could contribute to bone health status. METHODS AND RESULTS: Male C57BL6 mice were provided control (10% fat) or HF-diet (60% fat) in the presence or absence of RSV for 12 weeks. While RSV prevented HF diet-induced glucose intolerance, HF-RSV compromised tibial microarchitecture, mineral mass, and strength. The compromised outcomes following HF-RSV corresponded with higher markers of osteoclast-activation and bone-resorption (decreased OPG/RANKL ratio; increased cathepsin K), as well as higher markers of tibial mitochondrial content. A molecular model of elevated mitochondrial content (RIP140 knock out (KO) mice) was utilized to determine proof-of-principle that increasing mitochondrial content coincides with decrements in bone health. RIP140 KO mice displayed higher markers of mitochondrial content, and similar to HF-RSV, had compromised bone microarchitecture, lower BMD/strength, and higher markers of osteoclast-activation/bone-resorption. CONCLUSION: These data show that in the presence of an HF-diet, RSV negatively alters bone health, a process associated with increased mitochondrial content and markers of bone resorption.

Longitudinal Use of Micro-computed Tomography Does Not Alter Microarchitecture of the Proximal Tibia in Sham or Ovariectomized Sprague-Dawley Rats.

In vivo micro-computed tomography (µCT) provides the ability to measure longitudinal changes to tibia microarchitecture, but the effect of this radiation is not well understood. The right proximal tibia of Sprague-Dawley rats (n = 12/group) randomized to Sham-control (Sham) or ovariectomy (OVX) surgery at 12 weeks of age was scanned using µCT at 13, 17, 21, and 25 weeks of age, at a resolution of 18 µm and a radiation dose of 603 mGy. The left proximal tibia was scanned only at 25 weeks of age to serve as an internal non-irradiated control. Repeated irradiation did not affect tibia microarchitecture in Sham or OVX groups, although there was an increase in cortical eccentricity (P < 0.05). All trabecular outcomes and cortical BMD were different (P < 0.05) between groups after only 1 week post-surgery and differences persisted to study endpoint. Characteristic changes to trabecular bone were observed in OVX rats over time. Interactions of time and hormone status were found for cortical BMD (P < 0.001), Ps. Pm., and Ec. Pm. (P < 0.05). Repeated irradiation of the tibia at 13, 17, 21, and 25 weeks does not cause adverse effects to microarchitecture, regardless of hormone status. This radiation dose can be applied over a typical 3-month study period to comprehensively understand how an intervention alters tibia microarchitecture without confounding effects of radiation.

Flaxseed enhances the beneficial effect of low-dose estrogen therapy at reducing bone turnover and preserving bone microarchitecture in ovariectomized rats.

Our previous research showed greatest protection to vertebral bone mineral density and strength in ovariectomized (OVX) rats when lignan- and α-linolenic acid-rich flaxseed (FS) is combined with low-dose estrogen therapy (LD) compared with either treatment alone. This study determined the effects of combined FS+LD on serum and tissue markers of bone turnover and microarchitecture to explain our previous findings. Three-month-old OVX rats were randomized to negative control (NEG), FS, LD or FS+LD for 2 or 12 weeks, meaningful time points for determining effects on markers of bone metabolism and bone structure, respectively. Ground FS was added to the AIN-93M diet (100 g/kg diet) and LD (0.42 µg 17ß-estradiol/(kg body weight·day)) was delivered by subcutaneous implant. Sham rats were included as positive control. Bone formation (e.g., osteocalcin), bone resorption (e.g., tartrate-resistant acid phosphatase-5ß (TRAP-5ß)), as well as osteoprotegerin (OPG) and receptor activator of nuclear factor κ-B ligand (RANKL) were analyzed from the 2-week study by commercial assays (serum) and (or) histology (vertebra). Vertebral bone microarchitecture was measured from the 12-week study using microcomputed tomography. In serum, FS+LD and LD induced lower TRAP-5ß and osteocalcin, and higher OPG and OPG/RANKL ratio versus NEG and FS (p < 0.05). In vertebrae, FS+LD induced higher OPG and lower osteocalcin versus NEG (p < 0.01) and did not differ from LD and FS. FS+LD improved bone microarchitecture versus NEG, FS, and LD (p < 0.05). In conclusion, FS+LD protects bone tissue because of a reduction in bone turnover. However, elucidating the distinctive action of FS+LD on bone turnover compared with LD requires further investigation.

Flaxseed does not enhance the estrogenic effect of low-dose estrogen therapy on markers of uterine health in ovariectomized rats.

Flaxseed (FS) is an oilseed rich in phytoestrogens and n-3 polyunsaturated fatty acids, compounds that may attenuate bone loss during aging. We previously demonstrated using the ovariectomized (OVX) rat model of postmenopausal osteoporosis that 10% dietary FS combined with low-dose estrogen therapy (LD) preserves vertebral bone mass and strength more so than either treatment alone. However, it was prudent to also consider the effect of this intervention on uterine tissue as LD, and possibly FS, may have estrogenic, and thus negative, effects on uterine tissue. The present study investigated if FS enhances the estrogenic effect of LD on markers of uterine health in OVX rats. Three-month-old rats were randomized to groups: (1) SHAM, (2) OVX, (3) OVX+FS, (4) OVX+LD, or (5) OVX+FS+LD. Ground FS was added to the AIN-93M diet (100 g/kg of diet), and LD was delivered by subcutaneous implant (0.42 µg of 17ß-estradiol/kg of body weight/day) to mimic LD in postmenopausal women. After 12 weeks, histological analyses of uterine tissue demonstrated flattened or cuboidal luminal epithelia organized in a single layer in the OVX group, while FS, LD, and FS+LD induced a single layer of elongated luminal epithelia, columnar in shape. The SHAM group had the greatest epithelial mass. Cell proliferation was similar among all OVX groups. Therefore FS and FS+LD similarly induce estrogen-like effects on the morphology of luminal epithelia that are weaker than in the SHAM group without inducing cell proliferation in OVX rats. Thus, FS does not enhance the estrogenic effect of LD on markers of uterine health in OVX rats.

Accessibility of ³H-secoisolariciresinol diglycoside lignan metabolites in skeletal tissue of ovariectomized rats.

Flaxseed, rich in the phytoestrogen lignan secoisolariciresinol diglycoside (SDG), provides protection against bone loss at the lumbar vertebrae primarily when combined with low-dose estrogen therapy in the ovariectomized rat model of postmenopausal osteoporosis. Whether SDG metabolites are accessible to skeletal tissue, and thus have the potential to interact with low-dose estrogen therapy to exert direct local action on bone metabolism, is unknown. The objective of this study was to determine whether metabolites of SDG are accessible to the skeleton of ovariectomized rats and to compare the distribution of SDG metabolites in skeletal tissue with that in other tissues. Rats were fed a 10% flaxseed diet and gavaged daily with tritium-labeled SDG (7.4 kBq/g of body weight) in deionized water (500 µL) (n=3) or deionized water alone (n=3) for 7 days, after which tissues were collected for liquid scintillation counting. Radioactivity was detected in similar concentrations in the lumbar vertebrae, femurs, and tibias. Compared with non-skeletal tissues, total radioactivity in the skeleton was significantly lower than in the liver, heart, kidney, thymus, and brain (P < .001). There were no significant differences in levels of radioactivity between skeletal tissue versus the spleen, lung, bladder, uterus, vagina, and mammary gland. In conclusion, SDG metabolites are accessible to skeletal tissue of ovariectomized rats. Thus, it is biologically plausible that SDG metabolites may play a direct role in the protective effects of flaxseed combined with low-dose estrogen therapy against the loss of bone mass and bone strength in the ovariectomized rat model of postmenopausal osteoporosis.

Revisiting estrogen: efficacy and safety for postmenopausal bone health.

The rapid decline in endogenous estrogen production that occurs during menopause is associated with significant bone loss and increased risk for fragility fracture. While hormone therapy (HT) is an effective means to re-establish endogenous estrogen levels and reduce the risk of future fracture, its use can be accompanied by undesirable side effects such as stroke and breast cancer. In this paper, we revisit the issue of whether HT can be both safe and effective for the prevention of postmenopausal bone loss by examining standard and alternative doses and formulations of HT. The aim of this paper is to continue the dialogue regarding the benefits and controversies of HT with the goal of encouraging the dissemination of-up-to date evidence that may influence how HT is viewed and prescribed.

Influence of high-fat diet from differential dietary sources on bone mineral density, bone strength, and bone fatty acid composition in rats.

Previous studies have suggested that high-fat diets adversely affect bone development. However, these studies included other dietary manipulations, including low calcium, folic acid, and fibre, and (or) high sucrose or cholesterol, and did not directly compare several common sources of dietary fat. Thus, the overall objective of this study was to investigate the effect of high-fat diets that differ in fat quality, representing diets high in saturated fatty acids (SFA), n-3 polyunsaturated fatty acids (PUFA), or n-6 PUFA, on femur bone mineral density (BMD), strength, and fatty acid composition. Forty-day-old male Sprague-Dawley rats were maintained for 65 days on high-fat diets (20% by weight), containing coconut oil (SFA; n = 10), flaxseed oil (n-3 PUFA; n = 10), or safflower oil (n-6 PUFA; n = 11). Chow-fed rats (n = 10), at 105 days of age, were included to represent animals on a control diet. Rats fed high-fat diets had higher body weights than the chow-fed rats (p < 0.001). Among all high-fat groups, there were no differences in femur BMD (p > 0.05) or biomechanical strength properties (p > 0.05). Femurs of groups fed either the high n-3 or high n-6 PUFA diets were stronger (as measured by peak load) than those of the chow-fed group, after adjustment for significant differences in body weight (p = 0.001). As expected, the femur fatty acid profile reflected the fatty acid composition of the diet consumed. These results suggest that high-fat diets, containing high levels of PUFA in the form of flaxseed or safflower oil, have a positive effect on bone strength when fed to male rats 6 to 15 weeks of age.