1a. Objectives (from AD-416)
To determine how nutritional, hormonal, and physiological factors affect bone loss/gain in obesity through modifying obesity-induced inflammatory stress. Specifically, we will determine the extent to which obesity is associated with elevated levels of pro-inflammatory cytokines known to promote bone resorption, determine how obesity affects functions of bone cells and bone metabolism, determine the extent to which existing chronic inflammatory stress (induced experimentally by lipopolysaccharide implantation), estrogen deficiency (affected by ovariectomy), and subclinical magnesium intake impair bone health in obese animal models and in obese human subjects, and determine how moderate physical activity preserves bone structure as compared to caloric restriction during weight reduction in an obese animal model.
1b. Approach (from AD-416)
Studies will utilize cell culture, animal models and human subjects. We will use diet-induced obese mice or rats to determine the mechanisms by which adiposity interacts with other dietary, hormonal and physiological factors, such as estrogen deficiency, chronic inflammation, magnesium intake, and moderate exercise, and affects bone structure and functions of osteoblasts and osteoclasts. Human studies will use the in-house Community Studies Unit and the Metabolic Research Unit to conduct supplementation and controlled feeding experiments, respectively. We will determine whether 300 mg/d Mg supplementation to obese postmenopausal women with suspected marginal magnesium deficiency, ameliorates pro-inflammatory cytokine production and improves biomarkers of bone resorption and formation balance.
3. Progress Report
An experiment was conducted to determine the effect of obesity on osteoclast activity and bone microstructure. Six-wk-old male C57BL/6mice were fed either a normal fat diet (10 kcal% energy as fat) or a high-fat diet (45 kcal% energy as fat) for 14 weeks. Serum levels of osteocalcin and tartrate-resistant acid phosphatase (TRAP) were analyzed. Bone marrow cells were cultured for osteoclast formation. Expression of osteoblast and osteoclast specific genes was measured by using quantitative real-time PCR. Bone structure of proximal and mid-shaft femur was evaluated by micro-computed tomography. An experiment was conducted to determine whether estrogen deficiency exacerbates the detrimental effect of a high-fat diet on bone structure in a mouse model. Forty-eight female C57BL/6J ovariectomized or sham operated mice (24 each) at 4-mo-old were used. Then, ovariectomized and sham-operated mice were randomly assigned to either a normal-fat diet or a high-fat diet for 3 mos. Changes in bone structure and other serum markers related to bone metabolism were measured. Data will be analyzed. An animal study is in progress to determine whether obesity exacerbates the bone loss induced by chronic inflammation. Forty-eight female C57BL/6J mice aged 6-wks were randomly assigned to four groups in a 2 x 2 factorial design (control or 1.5 µg/d LPS) and fed either a normal-fat control diet or a high-fat diet. Animals were implanted with a slow release LPS or a placebo pellet subcutaneously in the dorsal region of the neck. The experiment will last for 3 months and will be finished by the end of this year. An experiment was completed in which male rats were fed 50%, 100%, or 150% of the magnesium requirement and either normal or high-fat (butter oil) diet for 12 and 24 weeks. The objective of the experiment was to determine whether marginal magnesium deficiency exacerbates inflammatory stress associated with obesity. Some results have been published in abstract form for a presentation at the Experimental Biology 2011 meeting held in Washington, DC, and a manuscript is in progress. An experiment was started in June 2011 to determine whether magnesium deficiency enhances or magnesium supplementation alleviates chronic inflammation and bone loss in obese and estrogen-deficient female rats. Female rats aged 65-75 days have been assigned to dietary treatments of 50%, 100%, or 150% of the magnesium requirement and 10% or 45% dietary fat for 24 weeks; one-half of the rats have been ovariectomized. Increased fat is being supplied by high-oleic sunflower oil. No reportable findings have been obtained to date. A protocol has been written for a human study with the objective to determine whether magnesium supplementation decreases chronic inflammation and bone loss in obese postmenopausal women.
1. Obesity induced by a high-fat diet increases bone loss and fracture risk. Obesity is detrimental to bone health through an undefined mechanism. To investigate the mechanisms through which obesity affects bone health, ARS researchers in Grand Forks, ND, evaluated bone microarchitecture of the upper hind leg using a sophisticated imaging technique, measured blood substances or chemicals related to bone turnover, and measured the formation of bone resorbing cells in bone marrow cells in mice made obese by a high-fat diet. They demonstrated that obesity induced by a high-fat diet alters the balance between bone loss and gain such that it results in reduced bone mass which can increase fracture risk.
2. Obesity induced by high dietary butter fat does not result in inflammatory stress that increases the risk for chronic disease. A nutritional model of obesity in rats that induces chronic inflammatory stress that increases the risk for osteoporosis and heart disease is needed to determine whether other nutritional factors contribute to such stress and chronic disease risk. ARS researchers in Grand Forks, ND, made rats obese by increasing dietary energy from 10% to 45% with butter fat. Increasing energy intake by feeding high dietary butter fat decreased instead of increased a biomarker of inflammatory stress called C-reactive protein, in rats fed a diet conducive to inflammatory stresses. A factor in butter fat, which is found in all fat-containing dairy products apparently prevented inflammatory stress. The findings show that obesity caused by consuming high amounts of butter fat does not result in chronic low-grade inflammation that could contribute to diseases often is associated with obesity; instead butter fat contains a factor that may alleviate inflammatory stress.
3. Marginal or subclinical magnesium deficiency increases oxidative and inflammatory stress, which increases the risks for chronic disease such as osteoporosis and heart disease. Magnesium deficiency in a severity similar to that commonly occurring in humans has not been established as an inducer of chronic inflammatory and oxidative stress, which increases the risk for chronic diseases such as osteoporosis and heart disease. ARS researchers in Grand Forks, ND, fed rats 50% (a deficient level that reportedly occurs in humans), 100%, or 150% of the magnesium requirement in a diet conducive to inflammatory and oxidative stress (high in fat and simple sugars) for 24 weeks. Marginal magnesium deficiency increased urinary excretion of indicators of oxidative and inflammatory stress. These findings indicate that marginal magnesium enhances or induces inflammatory and oxidative stress that can result in an increased risk for chronic diseases when conditions are conducive to such stresses.
4. Obesity alters the response to marginal or subclinical magnesium deficiency. Whether obesity, in which low magnesium status often occurs, affects the changes in calcium metabolism that impairs bone health induced by marginal magnesium deficiency has not been established. ARS researchers in Grand Forks, ND, fed diet-induced obese rats magnesium at 50% of the requirement (a deficient level that reportedly occurs in humans) for 24 weeks. The low magnesium intake resulted in changes in blood amounts of hormones that regulate calcium use in the body, and these changes, which indicated impaired calcium metabolism, were most evident in obese rats. These findings suggest that marginal magnesium deficiency with obesity can alter calcium metabolism to increase the risk for bone loss and osteoporosis.
5. Selenium from pinto beans is bioavailable to support normal bone development. Selenium may have beneficial effects on bone health through preventing the formation of forms of oxygen that result in bone breakdown (oxidative stress). Researchers in Grand Forks, ND, conducted an experiment to determine whether selenium deficiency affects bone microarchitecture and whether selenium from pinto beans is bioavailable as selenium in the form commonly found in over-the-counter supplements (selenomethionine) in supporting normal bone development in a mouse model. Mice fed the selenometionine supplement or the pinto bean selenium diet had a higher amount of bone in the thigh bone and the bone had structural changes that indicated a stronger bone than that in mice fed a selenium-deficient diet. The selenium from pinto beans was as effective as selenomethionine in preventing selenium-deficient bone changes. The findings show that selenium deficiency is bad to bone health and pinto beans are a good source of selenium to support normal bone development.
Yan, C., Cao, J.J., Wu, M., Jiang, T., Yoshimura, A., Gao, H., Zhang, W. 2010. Suppressor of cytokine signaling 3 inhibits LPS-induced IL-6 expression in osteoblasts by suppressing CCAAT/enhancer-binding protein ß activity. Journal of Biological Chemistry. 285(48):37227-37239.