THE EFFECTS OF DIETARY PREBIOTICS, MONOSACCHARIDES, AND FIBERS ON GUT MICROBIOME AND METABOLIC HEALTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1021426
• Project Start Date: Nov 20, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Nutrition

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: human nutrition; molecular biology; biotechnology.Chronic metabolic diseases including heart disease, diabetes, Alzheimer's disease, kidney disease, cancer and stroke make up the vast majority of the top 10 causes of death in the US according to the Centers for Disease Control and Prevention (https://www.cdc.gov/nchs/data-visualization/mortality-leading-causes/index.htm). It is clear that inappropriate diet is linked with inflammation as a key driver of these diseases and that the gut microbiome is a key aspect of how diets can be inflammatory. Dietary interventions to reduce inflammation and improve the gut microbiome are needed to prevent disease and improve health outcomes in a growing number of Californians and Americans.The objective of this project is to conduct two pilot dietary supplementation studies to test whether dietary fiber supplements can improve markers of metabolic health, including specifically, markers related to the gut microbiome and markers of inflammation, which have been linked with the risk of a number of chronic diseases. In the first part of the project a study will be conducted to test the effectiveness of a mixed fiber supplement in young overweight individuals who consume low-fiber diets, and in the second part of the project a study will be conducted to test the effectiveness of supplemental galactose, N-acetyl glucosamine, or spirulina in healthy individuals consuming normal diets. In both cases, the effects of the supplements will be evaluated by measuring changes in markers of inflammation and metabolic function using state of the art analytical methodology and bioinformatic tools. Statistical analysis of the data will be performed to evaluate the effectiveness of the supplements and the results will be disseminated widely to target audiences including publication in scientific journals, presentation at scientific conferences, presentation at local workshops and symposia, and as part of training and educational curricula at UC Davis. In addition, information will be disseminated through social media and news outlets. Ultimately, the successful completion of this project will provide actionable knowledge concerning the utility of fiber supplements in improving health and preventing chronic metabolic disease.

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	7310	1010	100%

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

Subject Of Investigation
7310 - Experimental design and statistical methods;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

fiber supplements

chronic metabolic disease prevention

inflammation

dietary supplementation study

Goals / Objectives
As science improves our understanding of the causes of diseases related to diet and nutrition, including obesity, type 2 diabetes, cardiovascular disease, Alzheimer's disease, and a number of other inflammatory and auto-immune diseases, recommendations and policies can be designed to help prevent and treat these diseases. There is growing evidence that the gut microbiome plays a critical role in determining metabolic health. However, we do not yet know how to modify diet to improve the functioning of the gut microbiome and improve metabolic health. Thus the overall objective of this project is to characterize the effectiveness of different dietary strategies to modify the gut microbiome and metabolic health toward a healthier profile. Specifically, the goals are to investigate the effects of different dietary prebiotics, monosaccharides, and fibers on gut microbiome composition and metabolite production, inflammation, lipoprotein composition and function.

Project Methods
The objective of this project is to conduct two intervention trials to investigate the effects of dietary carbohydrate sources on gut microbiome composition and function, and HDL composition and function. The objective will be achieved through two specific aims:Specific Aim 1Prebiotics are a source of fermentable substrate that induce the growth of saccharolytic bacteria, which are associated with beneficial health effects by displacing deleterious bacteria and by producing microbial metabolites that have beneficial health effects on the host. It is clear from multiple lines of evidence that increasing the relative composition of certain bacterial genera including Bifidobacterium and Lactobacillus leads to beneficial health effects. The beneficial effects of bifidobacteria and lactobacilli are associated with the production of short chain fatty acids, which have been shown to improve appetite and metabolic regulation, mucin production, epithelial integrity and immune function, along with anti-inflammatory effects in adipose tissue. Akkermansia muciniphila is a mucin-degrading bacterium that has also been found to be associated with a variety of beneficial health effects, including anti-obesity effects, reduced endotoxemia, reduced adipose tissue inflammation, reduced insulin resistance and improved gut barrier function. Thus, prebiotic approaches that can increase the relative proportions of bifidobacteria, lactobacilli, and Akkermansia muciniphila would be predicted to improve overall health and wellness by decreasing inflammation. In this aim we will determine whether a prebiotic formulation can alter the gut microbiome and markers of cardiometabolic health in young, overweight individuals consuming a low-fiber diet. Twenty individuals will be recruited and evaluated for eligibility based on exclusion and inclusion criteria. Following informed consent, eligible participants will be enrolled into the study and randomized to treatment order. Standard inclusion/exclusion criteria will apply, with the addition that overweight (BMI 25.0-29.9) will be a requirement for inclusion, while intake of >15g fiber/day will be an exclusion factor. All participants will be randomly assigned to treatment order and take 12g of the prebiotic and 12 g of the placebo supplement for a period of 4 weeks each, with a 4-week washout between intervention arms. Participants will record their diet for 3 days using dietary records twice during each 4-week segment, as well as fill out questionnaires (including tolerance for intervention, perceived energy, and visual analog scales for: satiety, hunger, gastrointestinal tolerance [bloating, stool consistency, flatulence]), have anthropometric measurements taken, including body composition assessment by scale (e.g., Tanita), have blood drawn, and provide stool samples at each study visit. Gut microbiota composition will be measured with 16S sequencing, short chain fatty acids will be measured by mass spectrometry, blood samples will be analyzed for basic metabolic profiles, lipid profiles, marker of gut permeability, and markers of inflammation. HDL isolated from plasma will be tested for anti-inflammatory and cholesterol efflux capacity and analyzed for changes in microbial RNA contents. Participants will fill out a general wellness and gut health questionnaire to assess the tolerability of the prebiotic and effects on gut symptoms. For both aims, data will be analyzed using both univariate and multivariate statistical approaches. All data will be assessed for normality and transformed as needed, or if normal distribution cannot be achieved non-parametric tests will be used.Specific Aim 2In addition to sucrose and fructose as part of sugars and starches in common carbohydrate-containing foods, we also consume an array of other monosaccharides in the diet. These include galactose, N-acetyl-glucosamine (GlcNac), and fucose, all of which are incorporated as integral components of a variety of glycosylated structures throughout the body, and are part of dietary oligosaccharides that are consumed by gut microbiota. However, it is not currently known whether supplementation with these monosaccharides affects plasma-based markers of both host metabolism and gut microbiome activity.In this aim we will determine whether consuming a galactose, GlcNac, or fucose-enriched supplement (spirulina) can change plasma glycosylation markers in healthy, young individuals. A total of 24 subjects will be enrolled and randomized to the following treatment arms: A) 6 subjects - GlcNac supplementation, 2.8 grams daily, B) 6 subjects - Galactose supplementation, 25 grams daily, C) 6 subjects - Spirulina supplementation, 3000 mg daily, and D) 6 subjects - Placebo (1 teaspoon cellulose powder). Participants will be blinded to which supplement they will be taking and will be randomized by block design. At enrollment and at each visit, vitals will be taken, as well as waist/height measurements. All subjects will have a blood draw (venipuncture) in the morning after an overnight fast at their baseline visit and at their week 2 and week 4 visits, and an optional week 8 visit (4 weeks after discontinuing the supplement to assess washout). Plasma will be analyzed for markers of inflammation, as well as changes in the glycosylation of circulating proteins (i.e. immunoglobulins). HDL particles will be isolated from plasma and analyzed for anti-inflammatory and cholesterol efflux capacity, protein glycosylation changes, and changes in microbial RNA contents.EffortsThe efforts that will be pursued to deliver the knowledge gained from these studies to the intended target audience will include: inclusion of knowledge in classroom instruction in the Clinical Nutrition course (NUT116B) taught by Dr. Zivkovic every winter quarter; opportunities for undergraduate interns and graduate students to gain laboratory skills and practicum experiences; presentation at workshops and conferences including for example the annual American Society for Nutrition and American Microbiology Society meetings as well as on-campus meetings such as the Graduate Group in Nutritional Biology Graduate Student Symposium; and extension and outreach through interviews with social media and news outlets.EvaluationThe project evaluation plan includes assessment of the successful completion of key milestones for each specific aim including: IRB approval, initiation of the study, subject recruitment, execution of the supplementation trial, collection of samples, processing of samples, sample analysis, data analysis and interpretation, and results publication and presentation. The successful completion of these milestones is linked to the expected project outcomes of providing actionable knowledge about the specific effects of prebiotic and monosaccharide supplements for improving health and preventing disease, and the assessment tools needed to measure these effects. Successful completion of the milestones will also provide knowledge about how the methods used in these studies could be adopted by others to evaluate the effects of other nutritional and health interventions. Finally, successful completion of this project will pave the way toward understanding how individual behaviors and health choices are influenced by personal health knowledge that could be gained from participating in each study.

Progress 11/20/19 to 09/30/20

Outputs
Target Audience:During this reporting period I gave talks and interacted with industry partners, published scientific articles, gave talks at scientific conferences, presented posters at scientific conferences, provided content for social media, and was interviewed by media. Changes/Problems:As described above, due to Covid, there has been an unavoidable delay in the study outlined under objective 2 of this project. Currently, study activities are on hold. As soon as the Covid related restrictions on study activities are lifted, recruitment activities will resume. What opportunities for training and professional development has the project provided?Many opportunities for training and professional development were provided by this project. Three graduate students and 6 undergraduate interns were trained on subject recruitment, screening, enrollment, and consent procedures. The students also received training on how to perform subject study days, from taking in dietary records, to assisting subjects through the study center and taking anthropometric measurements, to assisting with or performing plasma aliquoting, and sample organization and storage activities. Students also received training in and completed a number of data entry and data analysis tasks, including the use of diet analysis software, the generation of tables and figures, and statistical analysis using the programming language R. The project also offered opportunities for professional development. Three students and 3 undergraduate interns presented posters at scientific conferences, and/or used the experience/data from the study to present work in classes or to obtain internship units and credits. How have the results been disseminated to communities of interest?Results have been disseminated through presentations at scientific conferences, publication of abstracts, as well as through social media announcements. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the project will proceed toward the completion of both objectives. Specifically, for objective 1 sample analysis and then data analysis will proceed as outlined in the proposal and in the accomplishments section, and for objective 2, as soon as the Covid restrictions are lifted, study activities will resume.

Impacts
What was accomplished under these goals? During the first year of this project, steps toward the completion of objective 1 were achieved, but progress toward objective 2 has been delayed due to Covid. Objective 1: Determine whether a prebiotic formulation can alter the gut microbiome and markers of cardiometabolic health in young, overweight individuals consuming a low-fiber diet. Twenty individuals were successfully recruited, evaluated for eligibility based on exclusion and inclusion criteria, and following informed consent, enrolled into the study, randomized to treatment order, and completed the study protocol. No subjects dropped out, and all 20 out of 20 enrolled subjects completed the study protocol as specified with no adverse events. All participants were randomly assigned to treatment order and took 12g of the prebiotic and 12 g of the placebo supplement for a period of 4 weeks each, with a 4-week washout between intervention arms. Participants recorded their diet for 3 days using dietary records twice during each 4-week segment, filled out questionnaires (including tolerance for intervention, perceived energy, and visual analog scales for: satiety, hunger, gastrointestinal tolerance [bloating, stool consistency, flatulence]), had anthropometric measurements taken, had blood drawn, and provided stool samples at each study visit. The following study outcome measurement assays have already been completed: stool short chain fatty acid content was measured by mass spectrometry, and blood samples were analyzed for basic metabolic profiles and lipid profiles. The dietary intake data have been entered and processed through diet analysis software. The questionnaire data have been entered. The stool samples are currently being processed for gut microbiota compositional analysis by metagenomic sequencing, Plasma samples are currently being analyzed for inflammatory cytokines and plasma metabolomic analyses are being completed using mass spectrometry. Sample analysis and data analysis are currently under way. A poster was presented at the Experimental Biology conference in the spring of 2020 detailing the effects of the supplement on cardiometabolic markers. Additional posters are being prepared for presentation at upcoming scientific meetings. Objective 2: Determine whether consuming a galactose, GlcNac, or fucose-enriched supplement (spirulina) can change plasma glycosylation markers in healthy, young individuals A total of 24 subjects are currently being enrolled and randomized to the following treatment arms: A) 6 subjects - GlcNac supplementation, 2.8 grams daily, B) 6 subjects - Galactose supplementation, 25 grams daily, C) 6 subjects - Spirulina supplementation, 3000 mg daily, and D) 6 subjects - Placebo (1 teaspoon cellulose powder). Participants will be blinded to which supplement they were taking and were randomized by block design. At enrollment and at each visit, vitals will be taken, as well as waist/height measurements. All subjects will have a blood draw (venipuncture) in the morning after an overnight fast at their baseline visit and at their week 2 and week 4 visits, and an optional week 8 visit (4 weeks after discontinuing the supplement to assess washout). Due to Covid restrictions study recruitment has been delayed and we will resume recruitment once allowed to proceed again under county guidelines. Once all subjects are recruited and go through the study protocol analysis of plasma for markers of inflammation, changes in the glycosylation of circulating proteins (i.e. immunoglobulins), HDL isolation followed by analysis of anti-inflammatory and cholesterol efflux capacity, protein glycosylation changes, and changes in particle morphology will be completed. Data analysis will proceed when sample analysis has been completed.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhu C, Wong M, Li Q, Sawrey-Kubicek L, Beals E, Rhodes CH, Sacchi R, Lebrilla CB, Zivkovic AM. Site-Specific Glycoprofiles of HDL-Associated ApoE are Correlated with HDL Functional Capacity and Unaffected by Short-Term Diet. J Proteome Res. 2019 Nov 1;18(11):3977-3984. doi: 10.1021/acs.jproteome.9b00450. Epub 2019 Oct 1. PubMed PMID: 31545048; PubMed Central PMCID: PMC7480961.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhu C, Sawrey-Kubicek L, Bardagjy AS, Houts H, Tang X, Sacchi R, Randolph JM, Steinberg FM, Zivkovic AM. Whole egg consumption increases plasma choline and betaine without affecting TMAO levels or gut microbiome in overweight postmenopausal women. Nutr Res. 2020 Jun;78:36-41. doi: 10.1016/j.nutres.2020.04.002. Epub 2020 Apr 22. PubMed PMID: 32464420.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Johnson AJ, Zheng JJ, Kang JW, Saboe A, Knights D, Zivkovic AM. A Guide to Diet-Microbiome Study Design. Front Nutr. 2020;7:79. doi: 10.3389/fnut.2020.00079. eCollection 2020. Review. PubMed PMID: 32596250; PubMed Central PMCID: PMC7303276.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: International Society for Extracellular Vesicles Annual Meeting, Philadelphia, PA, May 2020: An extracellular small RNA-seq data processing pipeline optimized for high-performance computing. Chenghao Zhu, Angela M. Zivkovic
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: International Society for Extracellular Vesicles Annual Meeting, Philadelphia, PA, May 2020: Method to Simultaneously Capture Multiple Classes of Intact Extracellular RNA Carriers Including Extracellular Vesicles and Lipoprotein Particles. Brian Hong, Hannah Houts, Emily R. Mallick, Chenghao Zhu, Joanne Agus, Bonita H. Powell, Yiyao Huang, Kenneth Witwer, Angela M. Zivkovic
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: American Society for Nutrition Annual Meeting, Seattle, WA, April 2020: Novel Fiber Formulation Does not Alter Cardiometabolic Profiles in Overweight Participants Consuming Low-Fiber Diets. Jea Woo Kang, Chenghao Zhu, Christopher H. Rhodes, Hannah E. Houts, Jingyuan Zheng, Brian V. Hong, Xinyu Tang, Oscar M. Munoz, Joanne K. Agus, Angela M. Zivkovic
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Rodriguez A, Trigatti BL, Mineo C, Knaack D, Wilkins JT, Sahoo D, Asztalos BF, Mora S, Cuchel M, Pownall HJ, Rosales C, Bernatchez P, Ribeiro Martins da Silva A, Getz GS, Barber JL, Shearer GC, Zivkovic AM, Tietge UJF, Sacks FM, Connelly MA, Oda MN, Davidson WS, Sorci-Thomas MG, Vaisar T, Ruotolo G, Vickers KC, Martel C. Proceedings of the Ninth HDL (High-Density Lipoprotein) Workshop: Focus on Cardiovascular Disease. Arterioscler Thromb Vasc Biol. 2019 Dec;39(12):2457-2467. doi: 10.1161/ATVBAHA.119.313340. Epub 2019 Oct 10. Review. PubMed PMID: 31597448; PubMed Central PMCID: PMC6937204.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhu C, Sawrey-Kubicek L, Beals E, Rhodes CH, Houts HE, Sacchi R, Zivkovic AM. Human gut microbiome composition and tryptophan metabolites were changed differently by fast food and Mediterranean diet in 4 days: a pilot study. Nutr Res. 2020 May;77:62-72. doi: 10.1016/j.nutres.2020.03.005. Epub 2020 Mar 26. PubMed PMID: 32330749.