Sandra M. Sacco

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.

Flaxseed combined with low-dose estrogen therapy preserves bone tissue in ovariectomized rats.

Objective: Flaxseed is rich in lignans and &agr;-linolenic acid, compounds that may promote healthy skeletons. Many postmenopausal women consume complementary health products such as flaxseed or its components in addition to pharmacological agents such as low-dose estrogen therapy for additional support for menopausal symptoms and related conditions. However, their combined effect on bone health is unknown. The aim of this study was to determine the effects of 10% dietary flaxseed, low-dose estrogen therapy, or their combination on bone mineral density, biomechanical strength, and skeletal fatty acid composition in an ovariectomized rat model of postmenopausal osteoporosis. Methods: Ovariectomized rats received (1) basal diet (negative control), (2) 10% flaxseed, (3) low-dose estrogen implant (13 &mgr;g, 90 day release), or (4) flaxseed + low-dose estrogen implant for 12 weeks. A sham-operated group was included as a positive control. Bone mineral density, biomechanical strength, and fatty acid composition were measured at multiple skeletal sites. Results: Flaxseed + low-dose estrogen therapy resulted in the highest bone mineral density and peak load at the lumbar vertebrae, with no effect on bone mineral density or strength in the tibia and femur. Flaxseed and flaxseed + low-dose estrogen therapy resulted in significantly higher relative levels of &agr;-linolenic acid and eicosapentaenoic acid and lower levels of linoleic acid, arachidonic acid, and n-6/n-3 ratio in the lumbar vertebrae and tibia compared with all other groups. Conclusion: Flaxseed + low-dose estrogen therapy provides the greatest protection against ovariectomy-induced bone loss at the lumbar vertebrae. Moreover, this study is the first to demonstrate that flaxseed, rich in &agr;-linolenic acid, alters fatty acid composition in the ovariectomized rat skeleton.

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.

Interaction of Sesame Seed and Tamoxifen on Tumor Growth and Bone Health in Athymic Mice

Some premenopausal breast cancer patients use phytoestrogen-rich soy and flaxseed to alleviate side effects induced by drugs such as tamoxifen (TAM). Lignan-rich flaxseed protects against breast cancer and increases the effectiveness of TAM. This study determined the interactive effects of lignan-rich sesame seed (SS) and TAM on estrogen-responsive MCF-7 breast tumor growth and bone health in ovariectomized athymic mice under premenopausal-simulated conditions. Ovariectomized mice with an estrogen implant and established MCF-7 tumors were treated for 8 weeks as follows: (i) positive control fed basal diet (BD), (ii) SS group fed BD supplemented with 10% ground SS, (iii) TAM group with TAM implant fed BD, (iv) SS + TAM group with TAM implant fed BD supplemented with 10% SS, and (v) negative control fed BD with no estrogen implant. Palpable tumor data, adjusted for body weight, showed that SS does not inhibit MCF-7 tumor growth and tends to negate the tumor inhibitory effect of TAM by increasing cell proliferation and reducing apoptosis. SS alone and combined with TAM enhanced femur biomechanical strength but caused no differences in bone mineral content or bone mineral density in either the femur or lumbar vertebrae. SS is not protective and interacts adversely with TAM in MCF-7 breast tumors but induces beneficial effects on bone both alone and when combined with TAM.

Maternal vitamin D beneficially programs metabolic, gut and bone health of mouse male offspring in an obesogenic environment

Background/Objectives:Vitamin D is an anti-inflammatory nutrient and a determinant of bone health. Some prospective studies suggest that maternal vitamin D status is positively associated with offspring bone mass. We found that serum concentrations of lipopolysaccharide (LPS), an inflammatory molecule related to adiposity, insulin resistance and bone resorption, is lower in healthy mouse offspring exposed to high dietary vitamin D during pregnancy and lactation. LPS reaches the circulation via the gut. This study investigated whether maternal vitamin D programs metabolic, gut and bone health of male offspring in an obesogenic environment.Methods:C57BL/6J dams received an AIN-93G diet with high (H) or low (L) vitamin D during pregnancy and lactation. At weaning, offspring remained on their dam’s vitamin D level (LL or HH) or were switched (LH or HL) and fed a high fat (44.2%) and sucrose (19.8%) diet. Glucose response, adiposity, systemic inflammation (LPS, cytokines), intestinal permeability and mass, strength and microarchitecture of trabecular and cortical bone were assessed in 7-month-old male offsprings.Results:Higher maternal dietary vitamin D resulted in lower intestinal permeability (fecal albumin, P=0.010) and benefited trabecular but not cortical bone structure at the distal femur (higher trabecular number, P=0.022; less trabecular separation, P=0.015) and lumbar vertebra 2 (bone volume/total volume%, P=0.049). Higher maternal and offspring vitamin D resulted in lower fasting glucose (HH versus LL, P=0.039) and serum LPS concentrations (dam diet, P=0.011; pup diet, P=0.002). Higher offspring vitamin D resulted in lower epididymal fat pad relative weight (P=0.006). The serum concentrations of IL-6 and TNF-α did not differ among groups.Conclusions:Maternal dietary vitamin D beneficially programs intestinal permeability and systemic LPS concentration, which is accompanied by stronger trabecular bone in an obesogenic environment. Thus, the gut may mediate vitamin D effects. Moreover, optimizing vitamin D in early life may be critical for later health.

Lignan-rich sesame seed negates the tumor-inhibitory effect of tamoxifen but maintains bone health in a postmenopausal athymic mouse model with estrogen-responsive breast tumors

Objective: Flaxseed, the richest source of mammalian lignan precursors, enhances the tumor growth-inhibitory effect of tamoxifen while exerting no adverse effects on other estrogen-responsive tissues such as bone. Ingestion of sesame seed produces mammalian lignans comparable with flaxseed, but its anticancer potential is unknown. This study determined the interactive effects of sesame seed and tamoxifen on established MCF-7 tumor growth and bone health in ovariectomized athymic mice simulating a postmenopausal condition. Design: Mice with established MCF-7 tumors were treated for 8 weeks with (1) basal diet (negative control), (2) 10% sesame seed, (3) basal diet + tamoxifen implant, (4) 10% sesame seed + tamoxifen implant, or (5) basal diet + estrogen implant (positive control). Weekly palpable tumor size, final tumor weight, cell proliferation, and apoptosis were measured. Bone mineral content, bone mineral density, and biomechanical strength testing were performed on the femur and lumbar vertebrae. Results: Sesame seed induced regression of established tumor size similar to the negative control but tended to negate the tumor-inhibitory effect of tamoxifen, in part by reducing apoptosis. Sesame seed combined with tamoxifen induced higher bone mineral content, bone mineral density, and biomechanical strength in the femur and lumbar vertebrae than either treatment alone. A significant positive relationship was found between final tumor weight and bone strength parameters. Conclusions: Sesame seed is not protective and negatively interferes with tamoxifen in inducing regression of established MCF-7 tumor size but beneficially interacts with tamoxifen on bone in ovariectomized athymic mice.

Flaxseed does not antagonize the effect of ultra-low-dose estrogen therapy on bone mineral density and biomechanical bone strength in ovariectomized rats.

A previous study showed that flaxseed (FS) combined with low-dose (LD) estrogen therapy, resembling LD transdermal estrogen therapy in postmenopaual women, inhibited loss of bone mineral density (BMD), bone mineral content (BMC), and strength in lumbar vertebrae in ovariectomized rats. Whether FS combined with an even lower dose of estrogen is effective at preserving bone or whether FS interferes with the effect of this lower dose of estrogen is unknown. Thus, this study determined whether an ultra-low-dose (ULD) estrogen therapy, half the dose previously studied, in combination with FS preserved bone mass and strength in the lumbar vertebrae in ovariectomized rats. Rats were treated for 12 wk with (1) basal diet (BD) (ovariectomized control), (2) BD + ULD estrogen implant, or (3) BD containing 10% FS + ULD estrogen implant. A sham-operated control group was fed BD. Unlike ULD, FS + ULD attenuated loss of BMD and strength at the lumbar vertebrae and BMD in femurs and tibias. FS + ULD resulted in higher percentages of n-3 fatty acids including alpha-linolenic acid and eicosapentaenoic acid and lower percentages of n-6 fatty acids including linoleic acid compared to all other groups. Differences in fatty acid composition at the lumbar vertebrae and tibia were significantly related to BMD, BMC, and strength. No treatment-induced effects on uterus weight were observed, but histological analyses are needed to confirm safety. In conclusion, FS did not antagonize the activity of ULD, and their combination attenuated the loss of BMD and strength at the lumbar vertebrae, which was associated with differences in bone fatty acid composition.

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.

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.

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 17β-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-5β (TRAP-5β)), 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-5β 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.