Wendy E. Ward

Adiponectin Is a Negative Regulator of Bone Mineral and Bone Strength in Growing Mice

Obesity is associated with increased bone mineral density (BMD) but the mechanism for this is unclear. Serum levels of the adipokine adiponectin are inversely correlated with obesity, but results from studies on its relationship to bone mass are conflicting. The objective of this study was to compare bone mineral content (BMC), BMD and biomechanical strength properties of femur and lumbar vertebrae in 8- and 16-week old adiponectin transgenic mice (AdTg). These mice exhibit significantly elevated circulating adiponectin but have similar body weights compared to wild-type (WT) littermates that were used as controls. Female AdTg mice displayed significantly lower femur BMC at 8 and 16 weeks of age and femur neck peak load was significantly lower in 8-week old AdTg mice of both genders compared to controls. The peak load from compression testing of an individual lumbar vertebra was significantly lower in female AdTg mice compared to WT at 8 weeks, and this difference persisted at 16 weeks of age. In addition, lumbar vertebrae BMC was significantly lower in 16-week old male AdTg mice compared to WT although vertebra peak load was not different. Serum adiponectin levels were inversely correlated with femur BMC. In summary, elevated circulating adiponectin inhibits the acquisition of bone mass in growing mice and results in decreased biomechanical measures of functional strength that are surrogate measures of susceptibility to fractures. These results support a role for circulating adiponectin as a metabolic link that can explain, at least in part, the positive relationship between obesity and both bone mass and reduced susceptibility to fractures.

Exposure to Flaxseed or Its Purified Lignan during Suckling Inhibits Chemically Induced Rat Mammary Tumorigenesis

Previous studies have shown that feeding flaxseed (FS) or its lignan secoisolariciresinol diglucoside (SDG) to rat dams during lactation enhances the differentiation of rat mammary gland in the female offspring. This study determined whether exposure to a diet with 10% FS or SDG (equivalent to the amount in 10% FS) during suckling could protect against 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced rat mammary tumorigenesis later in life. Dams were fed the AIN-93G basal diet (BD) throughout pregnancy. After delivery, dams were randomized to continue on BD or were fed BD supplemented with 10% FS or SDG during lactation. Three-day urine of dams was analyzed for mammalian lignans. After weaning, all offspring were fed BD. At postnatal Days 49 to 51, during proestrus phase, offspring were gavaged with 5 mg of DMBA. At Week 21 post-DMBA administration, compared with the BD group, the FS and SDG groups had significantly lower (P < 0.05) tumor incidence (31.3% and 42.0% lower, respectively), total tumor load (50.8% and 62.5% lower, respectively), mean tumor size (43.9% and 67.7% lower, respectively), and tumor number (46.9% and 44.8% lower, respectively) per rat. There was a significant decreasing trend (P < 0.05) in final tumor weights in rats fed FS or SDG. The high urinary lignan excretion in dams fed with FS or SDG corresponded with the reduced tumor development. The FS and SDG groups did not differ significantly in tumor indices, indicating that the effect of FS is primarily due to its SDG. There were no significant changes in selective reproductive indices measured among dams and offspring. In conclusion, exposure to FS or SDG during suckling suppressed DMBA-induced rat mammary tumorigenesis, suggesting that exposure to lignans at this early stage of mammary gland development reduces susceptibility to mammary carcinogenesis later in life without adverse effects on selective reproductive Indices in dams or offspring.

Growth and body composition of human milk-fed premature infants provided with extra energy and nutrients early after hospital discharge: 1-year follow-up.

Objectives: Human milk (HM) is the optimal source of nutrition for premature infants; however, it is unclear whether HM alone is sufficient to meet their elevated nutritional requirements early after hospital discharge. We previously reported that premature infants (750–1800 g birth weight) fed HM containing extra nutrients for 12 weeks after discharge had dietary intakes closer to recommended levels and grew more rapidly than those fed HM alone. The objectives of the present article are to examine the impact of this intervention on bone mineralization, body composition, and HM use up to 1 year. Data are also presented on general developmental level at 18-month corrected age (CA). Patients and Methods: At discharge, predominantly HM-fed infants were randomized to receive for 12 weeks either approximately half of their feedings containing a multinutrient fortifier (intervention, n = 19) or all of their feedings as HM alone (control, n = 20). Results: Intervention infants remained longer (P < 0.001) and had greater whole-body bone mineral content (P = 0.02) until 12-month CA compared with controls. Intervention infants born less than or equal to 1250 g continued to have a larger mean head circumference throughout the first year of life (P < 0.0001). Human milk feeding (mL · kg−1 · day−1) differed between groups at 6- (P = 0.035), but not 12-month CA. No statistically significant differences were found between groups in the mental, motor, or behavior rating scale scores of the Bayley II at 18-month CA. Conclusions: Adding a multinutrient fortifier to HM provided to predominantly HM-fed premature infants early after discharge results in sustained differences in weight, length, and whole-body bone mineral content, and in smaller babies, head circumference for the first year of life.

Ovariectomy-Induced Hyperphagia Does Not Modulate Bone Mineral Density or Bone Strength in Rats

The ovariectomized (OVX) rat is a widely used animal model for the development of prevention and treatment strategies for postmenopausal osteoporosis. However, ovariectomy-induced hyperphagia results in weight gain and adiposity. To prevent potential protective effects of increased body weight on bone from confounding outcomes of preclinical studies, pair-feeding is used in some but not all studies to control food intake, but its importance is not well elucidated. We investigated if the type of feeding, pair-feeding vs. consumption of diet ad libitum, modulates bone mineral and bone strength in OVX rats. Three-month-old female Sprague-Dawley rats (n = 12/group) were randomized to 1) sham-operated control (SHAM); 2) OVX pair-fed (OVX-PF); and 3) OVX ad libitum (OVX-AL). For 14 wk, OVX-PF rats were pair-fed with the SHAM group and daily food intakes and weekly body weights were obtained. At necropsy, regional body composition was measured by dual energy X-ray absorptiometry. Bone mineral density (BMD) and biomechanical bone strength of femurs and lumbar vertebrae (LV) were also measured. OVX-AL rats had higher overall food intake (P < 0.01), final body weight (P < 0.01), weight gain (P < 0.01), and fat mass (P < 0.05) than either SHAM and OVX-PF rats. Conversely, SHAM rats had higher femur (P < 0.001) and LV1-3 BMD (P < 0.001) as well as LV4 peak load (P < 0.01) than both the OVX groups, whereas bone outcomes did not differ between the OVX-PF and OVX-AL groups. In summary, ovariectomy-induced hyperphagia and weight gain do not modulate BMD or biomechanical strength at 14 wk postovariectomy, suggesting that pair-feeding is not essential.

Bone abnormalities in adolescent leptin-deficient mice

Ob/ob and db/db mice have different aberrations in leptin signaling that both lead to abnormalities in bone mineral density (BMD), and bone histological and histomorphometric outcomes. A few studies have directly compared bone metabolism in ob/ob and db/db mice, and biomechanical strength properties that are surrogate measures of fracture risk, have not been extensively studied. This study compared bone mineral content (BMC), BMD and biomechanical strength properties of femurs and lumbar vertebrae among 10 week old male ob/ob, db/db and C57Bl/6 wildtype (WT) mice. Femurs and lumbar vertebrae were specifically studied to determine if trabecular and cortical bone are regulated by leptin in a similar manner in ob/ob and db/db mice. Femurs of ob/ob and db/db mice had lower BMC, BMD and biomechanical strength properties, including peak load, compared to WT mice. In contrast, lumbar vertebrae BMC and BMD did not differ among genotypes, nor did the peak load from compression testing of an individual lumbar vertebra differ among groups. These findings suggest that leptin deficiency in adolescent male mice first results in changes in femurs, a representative long bone, and alterations in lumbar vertebrae may occur later in life.

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

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

Exposure to purified lignan from flaxseed (Linum usitatissimum) alters bone development in female rats.

Due to the potential oestrogenic effects of secoisolariciresinol diglycoside (SDG), the mammalian lignan precursor in flaxseed (Linum usitatissimum), we hypothesized that exposure to purified SDG during early life would have a positive effect on developing bone. This present study determined whether exposure to SDG purified from flaxseed during suckling via mothers milk or continuously to adolescence (postnatal day (PND) 50) or adulthood (PND 132) increased bone mineral content (BMC) or bone strength in female rat offspring. Offspring were exposed to basal diet (BD) or one of two doses of SDG (50S, 100S) equivalent to that in a 50 or 100 g flaxseed/kg diet during lactation only or through to PND 50 or 132. At PND 50 and 132, femurs were analysed for BMC by dual energy X-ray absorptiometry and biomechanical strength by a 3-point bending test. Compared with BD group, rats exposed to continuous 50S or 100S diet had stronger femurs at PND 50 without changes in BMC. At PND 132 there were no differences in femur strength despite the fact that continuous exposure to BD resulted in a higher BMC than rats exposed to 100S during lactation only or to 50S or 100S during lactation through to adulthood. In conclusion, female rat bone is more sensitive to the oestrogen-like action of lignans during early life when endogenous levels of sex hormones are low, but by adulthood the improved bone strength does not persist. Importantly, exposure to purified lignan does not have negative effects on bone strength.

Mammary Gland Morphogenesis is Enhanced by Exposure to Flaxseed or Its Major Lignan During Suckling in Rats

The exposure of rats to 10% flaxseed (FS) or an equivalent level of its major lignan, secoisolariciresinol diglucoside (SDG), during suckling enhances mammary gland differentiation, which protects against mammary carcinogenesis at adulthood. We determined whether this diet-induced mammary gland differentiation is mediated through the estrogenic pathway via epidermal growth factor receptor (EGFR) and estrogen receptor (ER) signaling. Rats were fed the AIN-93G basal diet (BD) from day 7 of pregnancy until delivery and then randomized to consume BID, FS, or SDG during lactation. After weaning, female offspring were fed BD throughout the experiment. At postnatal day (PND) 21 and the proestrus phase on PND 49-51, mammary glands of offspring were analyzed for morphology, cell proliferation, and expression of EGFR, epidermal growth factor (EGF), transforming growth factor-α, ER-α, and ER-ß. At PND 21, compared with the BD control, the number of terminal end buds (TEBs) and terminal ducts were increased by FS, whereas mammary epithelial cell proliferation was increased by both FS and SDG, suggesting that mammary morphogenesis was enhanced. Epithelial EGFR and stromal fibroblast EGF were increased by SDG, whereas epithelial ER-ß was decreased by FS. Conversely, at PND 49-51, a lower number of TEBs but a higher ratio of lobules to TEBs with decreased expression of EGFR or EGF was observed in both treatment groups. EGFR expression was positively associated with EGF expression and cell proliferation in TEB epithelium at PND 21. Urinary lignans of lactating dams were related to their offsprings indices of mammary gland development. In conclusion, exposure to FS or SDG during suckling enhanced mammary gland morphogenesis by modulation of EGFR and ER signaling, which led to more differentiated mammary glands at PND 49-51. The physiological outcomes of FS and SDG were similar, which suggests that SDG is partly responsible for the mammary gland differentiation effect.

Exposure to flaxseed or purified lignan during lactation influences rat mammary gland structures

Previous investigation demonstrated that feeding a 10% flaxseed (10F) diet during pregnancy and lactation enhanced the differentiation of highly proliferative terminal end bud (TEB) structures of rat mammary gland into less proliferative alveolar buds and lobules. From this study, it was hypothesized that the lignan component in flaxseed mediated the observed effects. Because mammary glands with more TEBs are more susceptible to carcinogens, exposure to flaxseed during early postnatal life may reduce the risk of developing mammary cancer. Our objectives were to elucidate whether exposure to flaxseed during lactation only and during pregnancy and lactation can similarly influence the differentiation of mammary gland structures and also to identify whether the lignan component of flaxseed is the biologically active agent. Offspring were exposed to a 10F diet or a dose of purified lignan equivalent to that in a 10F diet (10S) during lactation only or from lactation to postnatal Day 50. Compared with controls, exposure to 10F or 10S during lactation only or from lactation to postnatal Day 50 reduced the number of TEBs and resulted in a rise in the number of alveolar buds. In conclusion, exposure to flaxseed or its purified lignan during lactation is a critical period in which mammary gland development may be promoted by enhancing the differentiation of the mammary gland structures. However, continuous exposure, particularly to purified lignans, resulted in the most differentiation of the mammary gland. The next step is to determine whether the changes in mammary gland structures are chemopreventive in rats challenged with a carcinogen.

Flavonoid Intake and Bone Health

Flavonoids, found in a wide diversity of plant foods from fruits and vegetables, herbs and spices, essential oils, and beverages, have the most potential of dietary components for promotion of bone health beyond calcium and vitamin D. Recent epidemiological studies show flavonoid consumption to have a stronger association with bone than general fruit and vegetable consumption. Bioactive flavonoids are being assessed for properties beyond their chemical antioxidant capacity, including anti-inflammatory actions. Some have been reported to enhance bone formation and to inhibit bone resorption through their action on cell signaling pathways that influence osteoblast and osteoclast differentiation. Future research is needed to determine which of the flavonoids and their metabolites are most effective and at what dose, as well as the mechanism of modulating cellular events, in order to set priorities for clinical trials.