The Roles of Dietary Nitrate in Bone and the Gut in an Ovarian Hormone-deficient Rodent Model Public Deposited


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  • Osteoporosis is a common metabolic bone disease, affecting a third of women and a fifth of men over age 65. In the US, annual health care costs associated with osteoporosis are estimated to be over $20 billion. Osteoporosis is associated with increased fracture risk, which has been demonstrated to predict mortality rates and nursing home admittance. Altered bone remodeling, defined by excessive bone resorption and/or impaired bone formation, is a major risk factor for osteoporotic fracture. In menopause-induced ovarian hormone deficiency, bone resorption exceeds formation. Menopause-induced bone resorption is associated with a reduced bone mineral density (BMD), and there is a relationship between low BMD and increased fracture risk. Given the importance of bone remodeling and fracture risk, there have been significant efforts to understand the ability of dietary components to slow bone loss. Some observational studies report that fruit and vegetable consumption is associated with an increase in bone mineral content (BMC) and BMD. Dietary nitrates, present in high concentrations in leafy green vegetables, may serve as a dietary component that supports bone health. Vegetable intake accounts for ~80% of dietary nitrate consumption in the typical human diet. Dietary nitrates can be reduced to nitric oxide (NO) via non-enzymatic reduction by lingual bacteria and a variety of mammalian reductases through the nitrate-nitrite-NO pathway. There is evidence suggesting that strategies to increase NO bioavailability using organic nitrates (i.e., nitroglycerin) as an NO donor may decrease bone turnover and loss in ovariectomized (OVX) animals, which are animals that have had their ovaries surgically removed to induce a postmenopausal-like state, defined by diminished ovarian production of female hormones, particularly estrogen and progesterone. Some follow up trials in postmenopausal women have demonstrated similar results. Since non-enzymatic reduction of dietary nitrate can account for up to 50% of NO production, this provides a biologically plausible link between dietary nitrate and bone health and suggests that dietary nitrate, as an alternate NO donor, may offer efficacious means of decreasing bone loss in postmenopausal women. Here, using an in vivo approach, we examined the dose-response relationship between dietary nitrates and bone density, microarchitecture, and turnover in OVX rats. We show that dietary nitrates do not improve bone density, microarchitecture, or turnover. Further, dietary nitrate had no beneficial effect on mineral apposition rate, bone formation rate, or bone volume/tissue volume in OVX rats. Our novel findings demonstrate dietary nitrate does not slow ovx-induced bone loss. This knowledge clarified the relationship between a dietary compound found in large quantities in leafy greens and bone loss in an OVX model. As an exploratory component of this study, we used 16S rRNA gene sequencing to profile the composition of fecal microbiota associated with sham surgical controls, OVX, and nitrate-treated groups to evaluate the ability of OVX or dietary nitrate to modulate the gut microbiome in OVX mammals. While dietary nitrate did not alter the gut microbiome in the context of OVX, we did observe gut microbiome changes in structure and composition associated with OVX status and identified specific phylotypes whose abundance stratify sham and OVX rats, including Proteobacteria, Firmicutes, and Bacteroidetes. These results add to the growing body of evidence that there is an association between the gut microbiome and sex steroid deficiency. Our use of the OVX model also provided an important added opportunity to further our understanding of the tissue effects of sex steroid deficiency observed in menopause. While both progesterone and estrogen influence metabolism, most attention has been given to the effects of ovarian hormone deficiency. Complex interactions between estrogen and host metabolism suggest that tissues in addition to serum and urine must be examined to clarify the mechanistic underpinnings of ovarian hormone deficiency and associated disease risk. Insufficient data are available from tissue-based studies using NMR-based platforms in estrogen-deficient models. Metabolomic studies in OVX rats, a model for osteoporosis, and postmenopausal women have demonstrated major metabolic shifts in serum, plasma, and urine as a result of ovarian hormone deficiency. To our knowledge, metabolomics has not been used for investigating the metabolic phenotype observed in the postmenopausal colon. Metabolomic studies of ovarian hormone deficiency is limited to bone, adipose, skeletal muscle, serum, plasma, and urine. Further, while it is established that ovarian hormone deficiency causes metabolic dysregulation, and the gut is associated with metabolic diseases and bone metabolism, there is also limited information available on the effects of estrogen loss on both gut tissue and gut microbiota. In light of this and our observed gut microbiome differences in OVX rats, we evaluated the effects of OVX on the colon using a metabolomics approach. We found the OVX colon is characterized by elevated levels of antioxidants, such as taurine and hypotaurine, and osmolytes, such as glycerophospholine, glycine, and glutamate. These novel findings suggest alterations to the colonic transsulfuration and methylation pathways, and they are suggestive of hyperosmotic and oxidative stress in the OVX colon. Previous work has associated hyperosmotic and oxidative stress with inflammation and intestinal permeability. These processes may also be present in the OVX colon; however, we did not quantify either inflammation or intestinal permeability in the current body of work. This dissertation takes advantage of a postmenopausal osteoporosis animal model and integrative techniques, such as osteologic, immunological, metabonomic, microbiomic and bioinformatic methods, to evaluate different metrics of osteoporosis, including physiological determinants influencing the rate of bone loss and gut-associated changes in ovarian hormone deficiency. The central findings of this dissertation show that dietary nitrate does not slow bone loss in ovarian hormone deficiency-induced rat model of bone loss. Further, dietary nitrate does not appear to influence the gut microbiome in the context of OVX. This is important in furthering our understanding of the relationship between the consumption of this ubiquitous component of fruit and vegetables and bone health. We also demonstrate that OVX is associated with shifts in gut microbiome structure and composition. Finally, we show OVX influences the colon metabolic phenotype, inducing the upregulation of metabolites involved with hyperosmotic and oxidative stress. Since estrogen protects against oxidative stress in many other tissues, we hypothesize that ovarian hormone deficiency promotes oxidative and hyperosmotic stress in the colon, promoting intestinal permeability and elevating the inflammatory response. In response, the colonic transsulfuration and methylation pathways may adapt by upregulating osmolytes and antioxidants. However, the specific causative agents of oxidative stress cannot be derived from our current work. Further investigation of the role these metabolites and processes play in the development of inflammation and metabolic dysregulation will yield insights into the physiological effects of ovarian hormone deficiency in postmenopausal women, allowing for more informed diagnosis and treatment of susceptible individuals.
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