Research Project: Modification of Diurnal Patterns to Promote Health in Models for Human Metabolic Dysfunction

Location: Dietary Prevention of Obesity-related Disease Research

2020 Annual Report

Objectives
Objective 1 - Define how dietary fatty acids and exercise alter peripheral biological rhythms and metabolic dysfunction. • Subobjective 1.A. Define whether long-chain n3 polyunsaturated fatty acids correct the obesity-mediated peripheral circadian clock dysfunction. • Subobjective 1.B. Define the extent to which exercise overrides peripheral clock dysfunction and metabolic dysfunction. Objective 2 - Define the impact of diet timing on colonic bile acid pathways and inflammation. Objective 3 - Define the impact of dietary fiber composition on colonic bile acid pathways and inflammation. Objective 4 - Define the mechanisms and the influence of daily physical activity timing to improve bone health. • Subobjective 4.A. Determine the mechanisms through which the timing of exercise alters the diurnal pattern of bone turnover, bone cell physiology, calcium utilization, and bone structure. • Subobjective 4.B. Determine the efficacy of morning vs evening exercise to maximize bone anabolic effects.

Approach
Disruption of biological rhythms in peripheral organs by environmental cues leads to metabolic dysfunction and disorders, including obesity. Food and physical exercise can drive the biological rhythms in peripheral organs. This project will examine the ability of dietary components (dietary fatty acids and fiber), exercise, and the timing of food consumption and exercise to correct the disrupted biological rhythms in peripheral organs and restore metabolic homeostasis. This project will address three questions: (1) Do changes in dietary fatty acid composition and exercise override the disrupted peripheral biological rhythms and restore metabolic homeostasis? (2) Does the timing of food intake and dietary fiber composition regulate bile acid pathways and attenuate colonic inflammation? (3) Does the timing of physical exercise make differences in regulating the diurnal pattern of bone metabolism and improving bone formation? Rodent studies will be performed to address each of these questions. In addition, a human clinical trial will be performed to translate question 3 results to humans. This project takes innovative approaches to addressing these questions in the context of modifying the diurnal patterns to promote health. Results from this research will provide valuable information of how dietary fatty acids and exercise minimize metabolic dysfunction and prevent associated disorders, a greater understanding of food timing and dietary fiber in regulating bile acid pathways and informing guidance for reducing colonic inflammation, and a greater understanding of timing of exercise training in improving bone health, particularly to people with bone loss associated with advancing age.

Progress Report
Objective 1. Research continued on investigating the effects of diet on metabolic health in rodent models of obesity. All mammals exhibit biological rhythms that control daily physiological functions (for example, fast vs. feeding), and obesity disrupts these rhythms. It has been shown that fish oil can prevent obesity, is anti-inflammatory, and improves insulin sensitivity in mice. Fish oil is a rich source of long chain n3-polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA). This study is a three-group feeding study (normal control diet, an obesity-causing diet, and a DHA-supplemented obesity-causing diet) in a mouse model of obesity. Objective 3. Research continued on defining the impact of dietary fiber composition on colonic bile acid pathways and inflammation. Dietary fiber, vitamin D and calcium may protect against human colonic inflammation and cancer. However, the mechanism remains to be determined. Using murine and cellular models for human colonic inflammation and cancer, ARS scientists have initiated (1) a mouse study to determine whether an increase in dietary fiber intake reduces secondary bile acids, inflammation, and tumorigenic signaling in the colon. This is a five-group feeding study (normal control diet, an obesity-causing diet, and three elevated levels of dietary fiber-supplemented obesity-causing diets) in a mouse model of obesity; and (2) a cellular study to determine the inhibitory efficacy of butyrate (an intestinal bacterial metabolite of dietary fiber) against human colon cancer cell proliferation via cellular and molecular biology approach. As a subordinate project of Objective 3, ARS scientists have been analyzing samples collected from a mouse study which was designed to determine the interactions between colonic inflammation, tumorigenic signaling, and dietary supplementation with calcium and vitamin D. This is a three-group feeding study (normal control diet, an obesity-causing diet, and a calcium and vitamin D-supplemented obesity-causing diet) in a mouse model of obesity. Objective 4. Research continued on investigating the extent to which the timing of exercise affects bone health. ARS scientists completed a rat study investigating the mechanisms and the influence of daily physical activity timing on bone metabolism. Rats were subjected to no exercise or two hours of directed exercise at either rest phase, early active phase, or late active phase five days per week. Body weight, body composition, blood, bones, and bone marrow cells were collected to assess the effects of the timing of exercise on bone markers, bone cell functions, and bone structure. The animal study is complete. Sample analysis is underway. Furthermore, ARS scientists have a protocol that is under development for Subobjective 4B determining the timing of exercise in postmenopausal women, which will be ready for Institutional Review Board review before the end of fiscal year 2020. As a subordinate project of Objective 4, ARS scientists analyzed samples collected from a two-arm, parallel-designed, randomized controlled community-based eight-week feeding intervention trial to investigate the extent to which providing extra vegetables at the Dietary Guidelines for Americans recommended amount affects urinary calcium excretion and serum bone turnover markers.

Accomplishments
1. Consumption of fruits and vegetables benefits bone health in overweight and obese adults. Fruits and vegetables are rich in nutrients and bioactive compounds that are beneficial to health and an essential part of a healthy diet. Results from controlled intervention trials are lacking to support the positive association between fruit and vegetable intake and bone health. ARS scientists in Grand Forks, North Dakota, analyzed blood and urine bone-related markers in response to changes of fruit and vegetable intake in overweight and obese people. They demonstrated that incorporating extra fruits and vegetables at the amount recommended by the Dietary Guidelines for Americans improves indicators of bone health. These results will have impact for health professionals, policy makers, and the general public. These findings support eating fruits and vegetables at or above those recommended by the Dietary Guidelines for Americans.

2. Obesity alters the biological rhythms in mammary glands of pubertal female mice. Childhood obesity in girls is associated with early breast growth, which is a risk factor for breast cancer in adulthood. Using a mouse model of breast development, ARS scientists in Grand Forks, North Dakota, found that an obesity-causing diet alters the expression of genes that control the biological rhythms in the mammary glands of young mice. Furthermore, the obesity-causing diet alters concentrations of the female sex hormone estrogen and its receptors in mammary glands. These findings indicate that disruption of the biological clock in pubertal mammary glands by obesity may cause aberrant breast development and growth and contribute to adulthood breast diseases including cancer. This research will have impact for scientists in government, academia, and industry studying the roles of diet in breast health.

3. Advanced liver disease is concomitant with increased inflammatory bacterial metabolites in the colon. Obesity is linked to nonalcoholic fatty liver disease (NAFLD) which is the most common chronic liver disease in Western countries. The gut-liver connection is implicated in liver disease development; however, the molecular mechanism remains to be determined. ARS scientists in Grand Forks, North Dakota, along with collaborators, (1) established a diet-induced NAFLD mouse model to study the molecular basis of NAFLD and (2) demonstrated that advanced liver disease is accompanied with increases in liver inflammation and biochemical changes in the colon. These findings provide direct molecular evidence on gut-liver connection during NAFLD progression. This research provides mechanistic, nutritional insight into the roles of diet and colonic health in preventing liver disease. This work is of interest to health professionals, basic and clinical scientists, and the general public.

4. Time-restricted feeding alters biological metabolites in the plasma of mice bearing lung metastases. Obesity is a risk factor for metastatic cancer. Time-restricted feeding (also known as intermittent fasting) reduces body fat mass in laboratory mice. ARS scientists in Grand Forks, North Dakota, found that, using a mouse model of lung cancer, time-restricted feeding reduces the obesity-enhanced tumor growth in the lungs and alleviates the obesity-mediated metabolic disruption. These findings indicate that time-restricted feeding restores the metabolic homeostasis disrupted by obesity. This improvement may contribute to the inhibition of metastatic growth. Findings from this study indicate that maintaining a healthy eating habit may be beneficial in reducing the risk of obesity and associated diseases including cancer. Scientists, and university collaborators and investigators are interested in this work to understand the roles of timing of food intake in cancer prevention. This work will build the foundation for clinical trials evaluating the role of diet in disease prevention and health promotion in humans.

5. Metabolism of mammary tumors differs from normal mammary glands but is not altered by time-restricted feeding. Obesity is a risk factor for breast cancer. Time-restricted feeding (known as intermittent fasting) is effective in restoring metabolic control and improving insulin sensitivity in mice. Using a mouse model of breast cancer, ARS scientists in Grand Forks, North Dakota, found that time-restricted feeding to the night mitigates mammary tumor growth and reduces body fat mass in mice. However, restricted feeding does not alter the metabolism of mammary tumors. These findings indicate that the protection by the time-restricted feeding against breast cancer is likely due to changes in host-tumor interactions rather than a direct regulation of nutrients to the tumor. This research provides insight into the role of dietary patterns in the prevention of breast cancer and is of interest to scientists in government, academia, and industry. Understanding the roles of timing of food intake in breast cancer prevention will build the foundation for clinical trials on diet in disease prevention and health promotion in humans.

6. Selenium inhibits breast tumor growth but does not prevent breast cancer-induced bone loss. Wasting is a risk factor for cancer-related death. Selenium is an essential nutrient to humans with demonstrated capability of breast cancer prevention. Using a mouse model of breast cancer, ARS scientists in Grand Forks, North Dakota, found that dietary supplementation with selenium inhibits the growth of breast tumors, but it does not prevent the bone loss in this model of breast cancer. The lack of effect in preventing bone loss by selenium may be due to its inhibition of estrogen, which is needed in maintaining bone health. Findings from this study indicate the need for additional strategies in combination with selenium supplementation to achieve the goal of preventing breast cancer and its associated bone wasting. This research is of interest to scientists in government, academia, and industry and to the general public.

7. Butyrate protects against colon cancer cell growth. Butyrate, an intestinal bacterial metabolite of dietary fiber, exhibits colon cancer preventive effects. In contrast, intake of an obesity-causing diet increases fecal secondary bile acids, such as deoxycholic acid (DCA, a potential cancer promoter). However, the molecular mechanism remains to be determined. ARS scientists in Grand Forks, North Dakota, along with collaborators, established an experimental cell model to study the biological interaction between butyrate and DCA and demonstrated that butyrate inhibits DCA-induced cellular mutation. These findings provide a proof-of-concept that butyrate can protect against colon carcinogenesis through a specific targeting of DCA-resistant colonic cells. This research will have impact for basic and translational research by scientists in government, academia, and industry.

8. Genetic deletion of inflammatory proteins blocks the colonic tumorigenic signaling in diet-induced obese mice. Obesity is a risk factor for colon cancer. However, the mechanisms responsible for this relationship are not adequately delineated. ARS scientists in Grand Forks, North Dakota, along with collaborators, demonstrated that (1) the genetic deletion of tumor necrosis factor alpha attenuated the tumor signaling and inflammation, that were elevated by diet-induced obesity in laboratory mice and (2) a causal role of tumor necrosis factor alpha in mediating obesity-associated tumor signaling and inflammation. These findings provide a potential mechanism of inflammation-driven tumor signaling for obesity-associated colon cancer and may lead to identifying new molecular targets for cancer prevention. This research will have impact for basic and translational research by scientists in government, academia, and industry.

9. Increasing fish oil intake reduces adiposity and improves bone health in growing obese mice. Long-chain n–3 polyunsaturated fatty acids supplied as fish oil to diets are beneficial to skeletal health. ARS scientists in Grand Forks, North Dakota demonstrated that the intake of fish oil, a source of long chain omega 3 polyunsaturated fatty acids decreases inflammation and adiposity while improving bone related outcome measures in growing, obese mice. The findings support the concept that increasing intake of long-chain polyunsaturated fatty acids is beneficial to bone health and reduces the risk of obesity. Scientists in government, academia, and industry and nutrition professionals are interested in this work to understand the roles of dietary modification in maintenance of bone health. This research builds the foundation for clinical trials on diet in disease prevention and health promotion in humans.

Review Publications
Cao, J.J., Gregoire, B.R., Michelsen, K.G., Picklo, M.J. 2020. Decreasing the dietary ratio of n-6:n-3 polyunsaturated fatty acids by reducing intake of linoleic acid does not prevent adiposity or bone deterioration in obese mice. Journal of Nutrition. https://doi.org/10.1093/jn/nxaa044.
Guo, C., Kim, S.J., Frederick, A., Li, J., Jin, Y., Zeng, H., Mason, J.B., Liu, Z. 2019. Genetic ablation of tumor necrosis factor-alpha attenuates colonic Wnt-signaling associated with obesity. Journal of Nutritional Biochemistry. https://doi.org/10.1016/j.jnutbio.2019.108302.
Cao, J.J., Gregoire, B.R., Michelsen, K.G., Picklo, M.J. 2019. Increasing dietary fish oil reduces adiposity and mitigates bone deterioration in growing C57BL/6 mice fed a high-fat diet. Journal of Nutrition. https://doi.org/10.1093/jn/nxz215.
Yan, L., Nielsen, F., Sundaram, S., Cao, J.J. 2019. Dietary selenium supplementation does not attenuate mammary tumorigenesis-mediated bone loss in male MMTV-PyMT mice. Biological Trace Element Research. https://doi.org/10.1007/s12011-019-01767-7.
Sundaram, S., Johnson, L., Yan, L. 2020. High-fat diet disrupts diurnal expression of circadian genes in mammary glands of prepubertal mice. Frontiers in Endocrinology. https://doi.org/10.3389/fendo.2020.00349.
Yan, L., Rust, B.M., Picklo, M.J. 2020. Plasma metabolomic changes in mice with time-restricted feeding-attenuated spontaneous metastasis of Lewis lung carcinoma. Anticancer Research. https://doi.org/10.21873/anticanres.14137.
Yan, L., Sundaram, S., Rust, B.M., Picklo, M.J., Bukowski, M.R. 2020. The metabolome of mammary tumors differs from normal mammary glands but is not altered by time-restricted feeding under obesogenic conditions. Anticancer Research. https://doi.org/10.21873/anticanres.14358.
Zeng, H., Larson, K.J., Cheng, W., Bukowski, M.R., Safratowich, B.D., Liu, Z., Hakkak, R. 2020. Advanced liver steatosis accompanies an increase in hepatic inflammation, colonic secondary bile acids and lactobacillaceae/lachnospiraceae bacteria in C57BL/6 mice fed a high-fat diet. Journal of Nutritional Biochemistry. https://doi.org/10.1016/j.jnutbio.2019.108336.
Zeng, H., Safratowich, B.D., Wang, T.T., Hamlin, S., Johnson, L. 2020. Butyrate inhibits deoxycholic-acid-resistant colonic cell proliferation via cell cycle arrest and apoptosis. Molecular Nutrition and Food Research. https://doi.org/10.1002/mnfr.201901014.
Cao, J.J., Ding, K., Rosario, R., Su, Y., Lawrence, M., Hamrick, M., Isales, C., Shi, X. 2020. Deletion of PPARy in mesenchymal lineage cells protects against aging-induced cortical but not trabecular bone loss in mice. Journal of Gerontology Biological Science. https://doi.org/10.1093/gerona/glaa049.