EFFECTS OF NONNUTRITIVE SWEETENERS ON GUT MICROFLORA AND PROINFLAMMATORY PATHWAYS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0229808
• Project Start Date: Jul 1, 2012
• Project End Date: Jun 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Food Science & Human Nutrition

Non Technical Summary
Obesity rates among children in the United States have tripled in the past three decades and approximately one third of all children or adolescents are currently overweight or obese (CDC). Obese children are more likely to be pre-diabetics and have one or more risk factors associated with cardiovascular disease, problems that become exacerbated as they enter adulthood. To combat weight gain and obesity related disease a number of low energy foods, usually containing non-nutritive sweeteners (NNS) such as saccharine, sucralose, and aspartame have become increasingly prevalent and are recommended sugar substitutes for individuals with Type 2 diabetes. However, two recent human observational studies found positive correlations between consumption of artificial sweeteners, particularly diet sodas, with increased obesity and metabolic syndrome.Weight gain is thought to occur when energy intake surpasses energy expenditure; and foods sweetened with NNS's are designed to reduce caloric consumption while enhancing the flavor of many foods. However, new insights into the role of inteastinal flora in harvesting energy from the diet and in contributing to both increased appetite and insulin resistance suggest that the "energy in/energy out" equation may be oversimplified. In particular, several studies support that the relative proportion of the two major intestinal bacterial phyla, Firmicutes and Bacteriodetes, affect the harvest of calories from the diet. Therefore, if NNS alter the intestinal flora such that there are proportionately higher levels of Firmicutes, foods that would normally have passed undigested from the body are metabolized and absorbed through the intestines, increasing the total calories available to the host. We hypothesize that consumption of NNS may later the intestinal microflora to favor greater dietary energy harvest and/or increased inflammation associated with obesity and this study is designed to explore this hypothesis.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	4010	1010	50%
702	5010	1010	50%

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

Subject Of Investigation
4010 - Bacteria; 5010 - Food;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

obesity

non-nutritive sweeyener

inflammation

intestinal microflora

firmicutes

bacteriodetes

Goals / Objectives
Although non-nutritive sweeteners such as sucralose and saccharine do not directly contribute to dietary energy intake, there are emerging lines of evidence that they alter the community structure of the gut microflora and may negatively impact many essential gut functions, including nutrient metabolism, normal immune system functioning, and gastrointestinal mobility. We hypothesize that repeated use of NNS alter intestinal bacterial populations to favor increased energy harvest and trigger inflammation signaling,consequently altering blood sugar homeostasis resulting in insulin resistance and accumulation of visceral fat. The following objectives will be completed to test this hypothesis: 1. To determine the effects of NNS on fecal bacterial community structure and activation of pro-inflammatory pathways in vitro by exploring sweetener-related changes induced by ex vivo incubation of human fecal material and analysis of inflammatory pathway activation in human colonic epithelial cell lines. 2. To determine the effects of NNS on fecal and intestinal bacterial communities,inflammatory response, and glucose tolerance in C57BL/6 mice during a 12-week feeding study using 454 pyrosequencing analysis and qPCR, and glucose tolerance tests. Outputs include- Conducting ex vivo fecal incubations to identify sweetener-induced changes in microflora. Determining effects of fecal extracts on markers of inflammation in human colon epithelial cell lines. Conducting a sweetener feeding study in mice to explore in vivo microflora changes and inflammatory responses elicited by long-term sweetener consumption.Training and mentoring a M.S. student and undergraduate students that will conduct these studies.

Project Methods
A number of methods will be used to identify the effects of non-nutritive sweeteners (NNS)on intestinal microflora and inflammation including ex vivo fecal incubations from human samples, 454 pyrosequencing, ARISA, and qPCR to evaluate bacterial community changes, targeted qPCR, PCR arrays, and ELISA to explore gene expression changes in inflammatory pathways, and exploring weight gain, glucose tolerance, and visceral fat accumulation in animals after prolonged feeding of NNS. Microbial bioinformatics data will be analyzed for quality, aligned, and clustered using freeware (Mothur) and community comparisons will be determined with UNIFRAC. Taxonomic assignments will be made by alignment with Silva bacteria and RDP databases. Gene expression from PCR data will be normalized to housekeeping genes and identified as fold change over control animals and significance will be determined by t-test. Animal weights and other parameters will be analyzed using standard statistical analyses. Number of animals used in the study will be determined by power calculation. Efforts to disseminate the information gained from these experiments will include presenting talks and posters at scientific meetings, peer-reviewed publications, and working with extension personnel within the department to develop material that are suitable for the general public.The project will be evaluated to ensure that project milestones are met in a timely manner. Biweekly lab meetings and data presentation will be required between project personnel to ensure timely completion of objectives. In addition, the project graduate student will be required to prepare a poster or short talk for presentation at a scientific meeting after completion of the first objective.

Progress 07/01/12 to 06/30/14

Outputs
Target Audience: THis research has been disseminated through various efforts, including formal and informal classroom settings and a graduate student research exhibition. Specifically, the primary graduate student on the project, Tirrel Miller presented a poster displaying her research findings at the Cell and Molecular Biology Graduate Research symposium at Colorado State University. This event is widely attended by faculty and students across campus as well as being a research showcase for potential new student recruits. She also presented her research at her thesis seminar presntation and at a nutrition science group meeting, both of which primarily target faculty and students in Food Science and Human Nutrition. ASpects of this research have also been included in presentations given by Dr. Weir for her FTEC 350 Fermentation Microbiology class which consists of CSU undergraduates in Fermentation Science, Microbiology, and ahandful of other majors. Finally, Dr. Weir has been interviewed by a number of different news outlets over the past year and has disseminated findings of this research through these venues, which would reach a broad national target audience. Some examples include the Nutrition Action Health Letter and Health and Wellness Magazine. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate student Tirrel Miller had the opportunity to learn cell culturing techniques from a post-doctoral student in another department as well as to learn measurement of trans-epithelial electical resistance in fixed cell monolayers. How have the results been disseminated to communities of interest? Research results are of interest to the general public. The findings of the animal study have been prepared for publication and research results have been disseminated in talks, interviews, and poster presentations. These venues have targeted both general audiences as well as the scientific community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We determined that addition of non-nutritive sweeteners did not dramatically alter the bacterial phyla composition when incubated with fresh human stool samples. Likewise, usingextracts of these incubations to treat immortalized human colonic epithelial cells (CACO-2 cell line) grown in culture had no effect on TNF-a expression or cytokins release. 2. We identified the following effects of NNS consumption in mice: - effects on metabolism and gut bacteria were observed in animals treated with the sugar alcohol, sorbitol, but not in animals given aspartame -sorbital altered glucose metabolism, the ratio of Bacteroidetes:Firmicutes, increased the inflammatory marker IL-1B, and decreased production of short chain fatty acids (as measured in the cecal contents). There was also an overall increase in cecal weight, which indicates lower microbial activity in the sorbitol group. - To isolate the effects of the sweetener consumption we terminated the experiment after 4 weeks before differences in overall body weight and food intake differed, which would have confounded results of the measured parameters.

Publications

• Type: Journal Articles Status: Other Year Published: 2015 Citation: Miller,T. Foster, M.T., Weir T.L. Effects of Artificial Sweeteners on Intestinal Microbiota. In preparation for Metabolism.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The target audience for this proposal are consumers ofnon-nutritivesweetners. This comprises a large portion of the general public as these are predominantly found in diet sodas, but is specifically relevant to overweight/obese individuals and diabetics or others that must limit their intake of natural sugars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One Master's student is being trained on this project. She has currently learned several new laboratory techniques including cell culture, qPCR, and TEER. She recently completed a proposed animal study and will be analyzing results for the next reporting period. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We have recebntly completed an animal feeding study and are analyzing results. For the next reporting period we hope to identify the impact of aspartame and the sugar alcohol, xylitol, on gut bacteria, intestinal cytokines, glucose toleranceand adipose tissue deposition in vivo.

Impacts
What was accomplished under these goals? Characterization of non-nutritive sweetner on human gut health While non-nutritive sweetners are not metabolized by the body, they may be metabolized by intestinal bacteria. This could lead to changes in the composition of the intestinal biota that alter dietary energy harvesting capability, immune function, and gut barrier integrity. In other words, although these sweetners do not contribute directly to weight gain, they may have indirect effects on weight and fat accumulation by by mechanism regulated by intestinal bacteria. We examined the effects of different non-nutritive sweetners on human gut biota by incubating donated fecal samples with sweetners and measuring changes in total bacteria as well as in the ratio of the two major phyla, Bacteriodetes and Firmicutes, using qPCR. We also used human intestinal cell-based assays to determine if these sweeteners altered the intestinal immune response. Most artificial sweeteners tested had no consistent effect on total bacteria or Bacteriodetes/Firmicutes ratios in human stool samples after a 24 hour ex vivo incubation period. Consequently, neitherdirect application of thesweeteners or stool extracts that had been incubated with the sweeteners resulted in increased expression of TNF-a in human colon epithelial cells (Caco-2). These treatments also did not compromise epithelial cell barrier integrity when measured by transepithelial electrical resistance (TEER) using Caco-2 monolayers. Therefore,preliminaryexperiments inex vivo and in vitro models suggest that non-nutritive sweeteners do not negatively affectgut bacteria and inflammation.

Publications

Progress 06/01/12 to 12/31/12

Outputs
OUTPUTS: Non-nutritive sweeteners, such as those found in diet foods and beverages, are generally considered to have little or no impact on energy balance as the compounds in these sweeteners are not absorbed by the digestive system. However, emerging studies suggest that the composition of the intestinal microflora can alter dietary energy harvest, and the impact of these artificial sweetners on host microflora have not been determined. Host microflora not only alter dietary energy harvest, but also regulate inflammatory pathways in the gut which can lead to "leaky gut", contributing to system inflammation implicated in development of obesity and metabolic disorders. This project is targeted at identifying impacts of non-nutritive sweeteners on intestinal microflora and inflammation. To date, the following activities have been conducted towards completion of this project: 1. IRB approval for the collection of human fecal samples from healthy volunters was obtained. 2. An MS student in Food Science and Nutrition was hired to conduct the study. 3. Five individuals have been recruited to the study and completed the requirements. 4. Students involved in the project were trained in the culture of human colon cancer cell line (HT-29) 5. Specific protocols for quantifying bacterial phyla in stool sample slurries were developed. State problem - Who Cares and Why b. Describe study conducted in general terms - What has been done c. State the results or accomplishments d. Evaluate the impact of the results 1) Economic 2) Social 3) Environmental 4) Scientific PARTICIPANTS: Tiffany Weir is the PI on ths project and has overseen the hiring of project personnel and assignment of duties. She has also interacted with the Colorado State University Institutional Review Board (IRB) to obtain approval for the human subjects portion of this research. Charles Condon (Soil and Crop Science) is an undergraduate student hourly who developed the initial protocols for incubating stool samples with non-nutritive sweeteners. Allie Reava (Food Science and Human Nutrition-Dietetics) is an undergraduate student hourly that helped develop the application materials for IRB submission. Tirrel Miller (Food Science and Human Nutrition) is a MS student that is conducting fecal incubations, quantifying bacterial populations, culturing human colon cancer cells, and will conduct animal studies. This project will be the basis of her MS thesis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project is still in it's early stages and has not yet yielded any concrete outcomes or impacts. However, completion of aim 1 is expected to result in identification of which non-nutritive sweeteners impact intestinal microflora populations. This information would be important for the management of intestinal health and obesity prevention.

Publications

• No publications reported this period