IMPROVING ANIMAL MODELS FOR NUTRITION AND CHRONIC DISEASE STUDIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006536
• Project Start Date: Jul 1, 2015
• Project End Date: Jun 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322

Performing Department
Nutrition, Dietetics and Food Sciences

Non Technical Summary
The link between our food system, nutrition and chronic human diseases is increasingly being realized. In order to understand the mechanistic links between nutrition and disease, animal models are used. However, current animal models of chronic disease poorly emulate nutritional patterns of Americans and thus decrease the applicability of the disease model to the human disease. Moreover, the gut microbiome is now known to play a large role in the interplay between diet and chronic disease. Therefore, the primary goal of this project is to increase the fidelity of translation between animal models of chronic disease and human populations through more appropriate animal diets and manipulation of the gut microbiome.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	5010	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
5010 - Food;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

western diet

colon cancer

obesity

gut microbiome

Goals / Objectives
Goals and Objectives1) Formulation of a food based, Total Western Diet. To better understand the correlations between the Western diet and chronic disease, a different dietary model is needed to better replicate the actual human digestive experience when using rodents as human models. For that reason, a whole-food based rodent diet that emulates American dietary pattern will be created, called the Total Western Diet 2 (TWD2). The TWD2 will have the same micro and macronutrient profile as the TWD but instead of using purified ingredients, whole foods will be used to formulate the diet.2) Determine the effects of a Western diet on the gut microbiome of pigs. In a previous study, using the same approach as the TWD for mice, a similar Western diet was formulated for pigs. Pigs were fed either the Western diet or a healthy control diet for 12 weeks to determine effects on reproductive fitness. Fecal samples were collected at baseline, week 6 and week 12. Gut microbiome taxonomy will be compared between all diets and timepoints.3) Determine the effects of TWD2 on colorectal cancer (CRC). Previous work has demonstrated that the TWD increases colorectal cancer in mouse models (Fig. X). However, although the TWD emulates American nutrition, it is a purified diet with minimal ingredients and is not representative of the complex food matrix consumed by humans. Therefore, the TWD2 will be compared to the TWD and a control diet in the inflammation driven DSS+AOM CRC model.4) Determine the effects of TWD2 on obesity/metabolism. Similar to Objective 2, the effects of the TWD2 on feeding behavior, weight gain, body composition, glucose tolerance and systemic inflammation will be compared to the TWD and a control diet.5) Determine the effects of TWD2 on the gut microbiome. Due to the complex food matrix of the TWD2 compared to the TWD and other purified diets, it is likely that consumption of the TWD2 will result in a unique gut microbiome compared to mice fed the TWD or control diet. Therefore, gut microbiome taxonomy will be compared between mice fed the TWD2, TWD and a control diet.6) Determine if the broad-scope antibiotic, human to mouse fecal transfer method conveys human phenotypes to recipient mice. To test this objective mice will be depleted of inherent microbiota through our broad scope antibiotic method. Mice will be repleted with microbiota from either obese or lean human donors and fed either a high-fat, obesity inducing diet or low-fat control diet in a 2X2 factorial design. Feeding behavior, weight gain, body composition, glucose tolerance and systemic inflammation will be compared between treatments.

Project Methods
Objective 1. Development of the Total Western Diet 2 (TWD2). Using the latest version of the USDA Food Intakes Converted to Retail Commodities Database, we will modify the TWD by replacing purified ingredients with 30 food ingredients. The TWD2 will improve the translational relevance of the planned studies, as the food matrix, secondary compounds and cooking byproducts have all been shown to influence the gut microbiota.Objective 2. Determine the effects of a Western diet on the gut microbiome of pigs. The feeding portion of the swine study has been completed. Sows were fed either a low-fat control diet, optimized in micronutrients or a high energy, high fat Western diet with a suboptimal micronutrients (n=5/treatment). Fecal samples were taken at baseline, 6 weeks and, 12 weeks. Using these collected fecal samples, the gut microbiome will be characterized for each diet group at each timepoint and compared using Roche 454 GS FLX Titanium System, QIIME pyrosequencing data analysis pipeline and UniFrac analysis.Objective 3. Determine the effects of TWD2 on colorectal cancer (CRC). In previous work, mice fed the TWD had a higher incidence of AOM+DSS induced CRC compared to mice fed the control AIN93-G diet. To determine effects of the food matrix on CRC, we have developed a new Western diet (TWD2) that is formulated entirely from food products as opposed to purified ingredients. To determine if the food matrix plays a role in the etiology of CRC, the following experimental dietary treatments are planned: 1) Total western diet (TWD), 2). Total Western Diet, 5% fiber (TWD-F), 3) Food based Total Western Diet (TWD2), 4). AIN93-G - Control diet. To determine the influence of diets on colon cancer, the AOM+DSS chemically-induced colorectal cancer model will be used.Objective 4 Determine Effects of the TWD2 on Metabolism. To determine diet effects on metabolism, the following endpoints will be measured from sham-injected mice from Objective 3: food intake, weight gain, body composition and oral glucose tolerance. Body composition will be determined by magnetic resonance image (MRI) scan every 4 weeks. Fasting glucose will be assessed every 4 weeks for the duration of the study. Oral glucose tolerance tests will be performed at week 7 and 15 of the study.Objective 5. Determine the effects of TWD2 on the gut microbiome. Fresh fecal samples will be collected weekly from sham-injected mice from the experiment described in Objective 3. Microbiome analysis will be performed as described in Objective 2.Objective 6. Determine if the broad-scope antibiotic, human to mouse fecal transfer method conveys human phenotypes to recipient mice. We will humanize mice with microbiota from obese or lean donors to determine if phenotype is transferable using our model. Mice will be fed either an obesegenic, 60% fat diet (DIO60) or a low fat control diet (AIN93G) resulting in the following treatments: 1) Lean microbiome, control diet; 2) Lean microbiome, obesegenic diet; 3) Obese microbiome, control diet; 4) Obese microbiome, obesegenic diet. To determine treatment effects on obesity and metabolism, the same endpoints in Objective 4 will be measured.

Progress 07/01/15 to 06/30/20

Outputs
(N/A)

Impacts
What was accomplished under these goals? All of the proposed Objectives were completed. The results of these studies resulted in the major findings: 1) The whole food, TWD2 induces a greater degree of obesity and metabolic syndrome in mice compared to an identical diet made from purified ingredients. 2) Pigs fed a Western diet, based on American intakes, became obese, diabetic, and have a microbiome that is similar to obese humans. This has resulted in a change of knowledge in terms of the physiological relevance of animal models. 3) Mice fed a diet formulated with a complex food matrix have a microbiome that is unique from mice fed purified diets. However, the food matrix did not alter response to inflammation driven colorectal cancer. This has resulted in a change of knowledge in terms of the physiological relevance of animal models. 4) Mice inoculated with bacteria from obese human donors consumed more calories compared to mice inoculated from lean human donors but only when fed the TWD. 5) We demonstrated that both vancomycin treatment and the TWD had significant main effects for increasing tumor burden and aberrant colon pathology. These results have changed our knowledge about interactions between diet, the microbiome, feeding behavior, and colon cancer. 6) We also demonstrated that the mouse microbiome is most heavily influenced by diet compared to human donor. In a separate study, we demonstrated that certain bacterial taxa, notably Bifidobacteria, are associated with lower tumor burden and that antibiotic use coupled with a poor diet increases colon cancer risk. These findings have resulted in a change of knowledge regarding diet, the microbiome, feeding behavior, and colon cancer. We have disseminated that knowledge in the form of conference presentations and peer-reviewed publications.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Researchers who use animal models, nutrition scientists, dietitians, physicians and health professionals. Local, national, international. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Nikklas Aardema, a graduate student working on these projects successfully completed and defended his MS degree. Data from this project was presented at the American Society for Nutrition Meeting by the project director. How have the results been disseminated to communities of interest?We published 1 peer reviewed paper based on work from this project: Basal Diet Determined Long-Term Composition of the Gut Microbiome and Mouse Phenotype to a Greater Extent than Fecal Microbiome Transfer from Lean or Obese Human Donors. Rodriguez DM, Benninghoff AD, Aardema NDJ, Phatak S, Hintze KJ. Nutrients. 2019 Jul 17;11(7). pii: E1630. doi: 10.3390/nu11071630. Data from this project was also presented at the American Society for Nutrition Annual Meeting, held in Baltimore, MD. There is one remaining manuscript resulting from this project that is in the final stages of preparation. We anticipate submission in early 2020. What do you plan to do during the next reporting period to accomplish the goals?In the final 6 months of this project we plan to publish one additional paper from this project.

Impacts
What was accomplished under these goals? All of the experiments are now complete, the most notable accomplishment this year is that we have completed all analysis and have summarized that information for all of the experiments. We have disseminated that knowledge in the form of conference presentations and peer-reviewed publications. These findings have resulted in a change of knowledge regarding diet, the microbiome, feeding behavior, and colon cancer.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Researchers who use animal models, nutrition scientists, dietitians, physicians and health professionals. Local, national, international. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed for professional development for myself and my graduate students as I had corresponding authorship on 2 presentations given by my graduate students at the 2018 Nutrition Meeting held in Boston, MA. One of my students, Niklas Aardema, won 2nd prize in the Emerging leaders in Nutrition competition based on his work on this project. My students have undertaken most of the labor for this project. Daphne Rodriguez and Nikklas Aardema, were both MS students, and coordinated the experiments described in Objectives 5-7. Three undergraduate researchers have also been involved in these studies, Hope Tinsley and Barbara Freeman from USU and Tomohiro Shiina from Tsukuba University, Japan. How have the results been disseminated to communities of interest?Based on the success of Objectives 6 and 7, two abstracts were submitted and presented at the 2018 Nutrition Meeting. Daphne Rodriguez successfully defended her thesis and was awarded an MS degree. Moreover, one peer-reviewed publication directly related to the current project was published in 2018, while another has been submitted and is currently under review. What do you plan to do during the next reporting period to accomplish the goals?All of the animal studies have been completed. The bulk of the work for 2019 will be further interpretation and publishing results.

Impacts
What was accomplished under these goals? All of the animal experiments are now complete. The most notable discoveries for this year include: Mice inoculated with bacteria from obese human donors did not gain more weight compared to mice inoculated from lean human. We also demonstrated that the mouse microbiome is most heavily influenced by diet compared to human donor. In a separate study, we demonstrated that certain bacterial taxa, notably Bifidobacteria, are associated with lower tumor burden and that antibiotic use coupled with a poor diet increases colon cancer risk. These findings have resulted in a change of knowledge regarding diet, the microbiome, feeding behavior, and colon cancer.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Researchers who use animal models, nutrition scientists, dietitians, physicians and health professionals. Local, national, international. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed for professional development for myself as I had 2 presentations (1 of which was invited) chosen as oral presentations at the 2017 Experimental Biology Meeting. I also gave invited oral presentations at the Functional Foods and Bioactive Compounds in Health and Disease meeting (March 25th, San Diego), NIH Office of Dietary Supplements (September 12th, Rockville, MD), USDA/ARS Beltsville Research Center (September 14th, Beltsville, MD), Brigham Young University (October 27th, Provo, UT), and the Utah One Health Symposium (November 17th, Salt Lake City, UT). Much of the labor for this project has been undertaken by students. Daphne Rodriguez and Nikklas Aardema, both MS students, are coordinating the experiments described in Objectives 5-7. Three undergraduate researchers have also been involved in these studies, Hope Tinsley and Barbara Freeman from USU and Tomohiro Shiina from Tsukuba University, Japan. How have the results been disseminated to communities of interest?Based on the success of Objectives 2-5, one abstract was submitted and chosen as an oral presentations at the 2017 Experimental Biology Meeting. I also gave an invited oral presentation in a special Animal Modeling symposium. Moreover, two peer-reviewed publications directly related to the current project were published in 2017. What do you plan to do during the next reporting period to accomplish the goals?The animal studies for Objectives 6 and 7 have been completed. The bulk of the work for 2018 will be completing the biochemical analyses generated from these studies.

Impacts
What was accomplished under these goals? Objectives 1 through 5 are now complete. The animal experiments for Objectives 6 and 7 have been completed and the biochemical analyses are underway. Most notably for Objective 6, mice inoculated with bacteria from obese human donors consumed more calories compared to mice inoculated from lean human donors but only when fed the TWD. For Objective 7, we demonstrated that both vancomycin treatment and the TWD had significant main effects for increasing tumor burden and aberrant colon pathology. These results have changed our knowledge about interactions between diet, the microbiome, feeding behavior, and colon cancer.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hintze, K. J., Benninghoff, A., Ward, R. E. (2017). Improving animal diets to increase relevance to human populations. Functional Foods in Health and Disease, 7(5), 329-337. www.ffhdj.com/index.php/ffhd/article/view/340/612
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Hintze, K. J., "Improving Laboratory Animal Diets to Increase Relevance to Human Populations," NIH Office of Dietary Supplements, Rockville, MD. (September 12, 2017)
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Hintze, K. J. (Presenter & Author), Hisatome, T. (Author Only), Kellen, S. (Author Only), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2017, "Effects of the Food Matrix and Western Diet on the Mouse Microbiome," American Society of Nutrition, Chicago, IL. (April 22, 2017 - April 26, 2017)
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Hintze, K. J. (Presenter & Author), Ward, R. E. (Author Only), Benninghoff, A. (Author Only), Lefevre, M. (Author Only), Experimental Biology 2017, "The Total Western Diet: Effects on Metabolism, Colon Cancer, and the Microbiome.," American Society for Nutrition, Chicago, IL. (April 23, 2017)

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target Audience Researchers who use animal models, nutrition scientists, dietitians, physicians and health professionals. Local, national, international. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunities This project has allowed for professional development for myself as I had two presentations chosen as oral presentations at the 2016 Experimental Biology Meeting. Much of the labor for this project has been undertaken by students. Daphne Rodriguez and Nikklas Aardema, both MS students, are coordinating the experiments described in Objectives 5 and 6. Three undergraduate researchers have also been involved in these studies (Ashli Hunter, Hope Tinsley and Canyon Ness). How have the results been disseminated to communities of interest?Dissemination Based on the success of Objectives 2-5, two abstract were submitted and chosen as oral presentations at the 2016 Experimental Biology Meeting. Moreover, two peer-reviewed publications directly related to the current project were published in 2016. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work The animal study for Objective 6 is underway and will be completed early in the new year. The bulk of the work for 2017 will be completing this animal study and performing the biochemical analyses.

Impacts
What was accomplished under these goals? Accomplishments Objectives 1 and 2 are now complete. The animal experiments for Objectives 3-5 have been completed and a majority of the biochemical analyses have been completed and have resulted in several novel findings. Most notably, mice fed a diet formulated with a complex food matrix have a microbiome that is unique from mice fed purified diets. However, the food matrix did not alter response to inflammation driven colorectal cancer. This has resulted in a change of knowledge in terms of the physiological relevance of animal models.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Hintze, K. J. (Presenter & Author), Benninghoff, A. (Author Only), Lefevre, M. (Author Only), Ward, R. E. (Author Only), Hisatome, T. (Author Only), Kellen, S. (Author Only), Experimental Biology, "Effects of Food Matrix and Western Diet on Colorectal Cancer and Metabolism in C57BL/6 Mice," American Society for Nutrition, San Diego, CA. (April 2, 2016 - April 6, 2016)
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Presentations Hintze, K. J. (Presenter & Author), Lefevre, M. (Author Only), Isom, S. C. (Author Only), Cox, L. (Author Only), Experimental Biology, "Pigs Fed a Western Diet Develop Elevated Fasting Glucose and a Microbiome Analogous to Human Obesity," American Society for Nutrition, San Diego, CA. (April 2, 2016)

Progress 07/01/15 to 09/30/15

Outputs
Target Audience:Target Audience Researchers who use animal models, nutrition scientists, dietitians, physicians and health professionals. Local, national, international. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunities Based on the success of Objective 2, an abstract was submitted, accepted and presented at the World Congress on Targeting Microbiota in Paris, France on October 23rd, 2015. How have the results been disseminated to communities of interest?Dissemination As stated above, results from Objective 2 were presented at an international meeting. Results from both Objective 1 and 2 were submitted in abstract form to the Experimental Biology meeting which will be held in April, 2016. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work In the coming year work will continue to completely finish Objectives 1 and 2. However, the bulk of the work will involve Objectives 3 and 4.

Impacts
What was accomplished under these goals? Accomplishments Objectives 1 and 2 are nearing completion. The Total Western Diet 2 (TWD2) has been formulated (Objective 1) and fed to mice. Analysis is ongoing to determine biochemical effects of treatment detailed in Objectives 3 and 4. Objective 2 is completed, pigs were fed a Western or control diet and fecal samples were collected for gut microbiome analysis. Microbiome sequencing and analysis has been completed. The results of these studies resulted in the major findings: 1) The whole food, TWD2 induces a greater degree of obesity and metabolic syndrome in mice compared to an identical diet made from purified ingredients. 2) Pigs fed a Western diet, based on American intakes, became obese, diabetic, and have a microbiome that is similar to obese humans. This has resulted in a change of knowledge in terms of the physiological relevance of animal models.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Hintze, K. J. (Presenter & Author), Kellen, S. (Author Only), Larson, D. (Author Only), Ward, R. (Author Only), Lefevre, M. (Author Only), Benninghoff, A. (Author Only), Experimental Biology, "The Effect of Dietary Polyunsaturated Acid (PUFA) Concentration and n6: n3 Ratio on Azoxymethane+ Dextran Sodium Sulfate (AOM+ DSS) Inflammation-Associated Colorectal Cancer (CRC)," American Society for Nutrition, Boston, MA. (April 2015 - Present)
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Hintze, K. J. (Presenter & Author), Lefevre, M. (Author Only), Isom, S. C. (Author Only), Cox, L. (Author Only), 3rd World Congress on Targeting Microbiota, "Pigs Fed a Western Diet Develop Features of Metabolic Syndrome and a Microbiome Analogous to Human Obesity," International society of microbiota, Paris, France. (October 21, 2015 - Present)
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Presentations Hintze, K. J. (Presenter & Author), Isom, S. C. (Author Only), Cox, L. (Author Only), Experimental Biology, "Formulation of the Total Western Diet (TWD) for Pigs and Phenotypic Differences After a 12-week Feeding Trial," American Society for Nutrition, Boston, MA. (April 2015 - Present)