FOOD SPECIFIC MOLECULAR PROFILES AND BIOMARKERS OF FOOD AND NUTRIENT INTAKE, AND DIETARY EXPOSURE (R01 CLINICAL TRIAL OPTIONAL)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022139
• Grant No.: 2020-67001-30716
• Project No.: WN.W-2019-07916
• Proposal No.: 2019-07916
• Program Code: A1342
• Project Start Date: Apr 1, 2020
• Project End Date: Mar 31, 2025
• Grant Year: 2020

Project Director
den Hartigh, L.

Recipient Organization
UNIVERSITY OF WASHINGTON
4333 BROOKLYN AVE NE
SEATTLE,WA 98195

Performing Department
Medicine

Non Technical Summary
Obesity has reached epidemic proportions, and is an expanding problem world-wide. Even though most overweight people can lose some weight in the short term by changing their diet and exercise habits, maintaining weight loss through diet and exercise alone is extremely difficult, and most people will regain some of the weight lost. Extreme diets are hard to sustain, and weight loss trains the body to hold on to any excess energy, which then makes it even harder to lose weight. There are some successful interventions for obesity treatment, including bariatric surgery and pharmaceutical drugs, but these are typically reserved for the severely obese, leaving people with mild to moderate overweight with few options. Therefore, we are in desperate need developing new strategies for weight loss. We are beginning to understand that the trillions of bacteria that live in our guts, called the gut microbiota, play a very important role in maintaining our body weight. We also know that the composition of our diet is very important in shaping our gut microbiota and maintaining its health. In this proposal, we will test whether a particular type of fat that is known to cause weight loss does so by changing our gut microbiota. This type of fat, called conjugated linoleic acid (CLA), is found in dairy and beef products, and is also marketed as a weight loss supplement. In this proposal, we will use obese animal models to determine if this remarkable dietary fat triggers weight loss by changing the gut bacteria. To confirm that the gut bacteria are important for weight loss by CLA, we will also use obese mice to determine if changing the gut bacteria through the use of probiotics can lead to weight loss. It is possible that this proposal will result in the identification of new probiotic therapies that could promote weight loss, giving people with mild to moderate overweight a relatively simple and inexpensive strategy to regain health.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3899	1010	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3899 - Other animals, general;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

conjugated linoleic acid

microbiota

microbiome

gut microbiota

gut microbiome

gut bacteria

obesity

weight loss

butyrate

short chain fatty acids

fatty acids

butyrivibrio

bifidobacteria

lactobacillus

mouse models

antibiotics

germ free mice

high fat high sucrose diet

prebiotics

probiotics

inulin

arabinoxylans

gpr43

energy balance

energy expenditure

indirect calorimetry

body weight

body composition

adiposity

white adipose tissue

16s rrna sequencing

metabolomics

fecal transplant

Goals / Objectives
Obesity continues to be a major problem in the United States, despite known treatment strategies such as lifestyle modifications, pharmaceuticals, and surgical options, necessitating the development of novel weight loss approaches. It is becoming increasingly clear that there is a link between obesity and the gut microbiota, and that therapies that modulate our gut bacteria could in turn impact body weight and energy balance. Preliminary data suggest that a naturally occurring fatty acid with inherent weight loss properties, 10,12 conjugated linoleic acid (10,12 CLA), significantly alters the gut microbiota of mice by enriching the gut with species that produce the short chain fatty acid (SCFA) butyrate and produce endogenous CLA, including species of Butyrivibrio and Roseburia (known butyrate-producers) and Bifidobacteria (known CLA-producers). Supplemental butyrate and CLA exert beneficial effects on the host by promoting weight loss due to increased energy expenditure.There are two major goals in this project. The first goal is to determine if specific changes to the gut microbiota are required for 10,12 CLA-mediated weight loss. This will be achieved by completing the following objectives:A loss-of-function experiment will be performed, in which the gut microbiota will be depleted from obese mice using broad-spectrum antibiotics prior to and 10,12 CLA supplementation. This will determine whether gut bacteria are required for 10,12 CLA-mediated weight loss.A second loss-of-function experiment will be performed in mice that have been raised germ-free, meaning they are free from bacteria. This technique is the gold-standard for loss-of-function experiments involving the gut microbiota, and is expected to yield similar results as in the first objective.Next, to determine if gut bacteria are sufficient for modulating 10,12 CLA-induced weight loss, a gain-of-function experiment will be performed. Bacterial contents from the intestinal tract of mice supplemented with 10,12 CLA will be transplanted into recipient obese mice, to determine if 10,12 CLA-mediated changes to the gut microbiota can promote weight loss.The second goal is to determine if engineering the gut microbiome to increase the endogenous production of CLA and/or butyrate recapitulates such weight loss. This will be achieved by completing the following objectives:A specific pre-biotic approach will be used to enrich the gut with bacteria that produce CLA and/or butyrate. Mice will be fed a diet that contains pre-biotic compounds such as inulin and arabinoxylans, which are known to promote the growth of CLA- and butyrate-producing bacteria. This will determine if weight loss can be achieved by altering gut metabolite production through a simple dietary intervention that could easily be applied to humans in future studies.Next, a specific pro-biotic strategy will be used to directly administer bacteria that have been screened for butyrate and CLA production to obese mice. The novel approach to this objective is that potential probiotic strains will be isolated, cultured, identified, and screened directly from mice undergoing weight loss.Finally, a specific mechanism by which butyrogenic gut bacteria might modulate body weight and adiposity will be examined. Butyrate primarily signals through a specific receptor in adipose tissue, GPR43. We would expect that mice that are deficient in GPR43 will be resistant to weight loss resulting from the gut microbial modulation strategies listed above. Completing the objectives listed above will inform new strategies for weight loss in humans that target the gut microbiota largely by dietary interventions.

Project Methods
The methods that will be employed to complete the proposed project are described below, stratified by each goal.1.Goal 1: To determine if the gut microbiota are necessary or sufficient for 10,12 CLA-mediated weight loss.Whether changes in gut microbial populations play causal roles in 10,12 CLA-mediated weight loss will be determined using both loss- and gain-of-function approaches. First, to determine if microbiota are required for these effects, germ-free or antibiotic-treated mice will be given 10,12 CLA, and body weight, energy expenditure, and adiposity will be compared with control mice. In parallel, to determine whether the microbial populations altered by 10,12 CLA exposure are sufficient to mediate weight loss, cecal contents will be transplanted from 10,12 CLA-supplemented or control donors into weight-matched recipient mice.Antibiotic loss-of-function: In this experiment, mice will be made obese by consumption of a diet high in fat and sucrose (HFHS). Their gut microbiota will then be depleted by exposure to broad-spectrum antibiotics, then they will continue to consume a HFHS diet that is supplemented with 10,12 CLA (while still consuming antibiotics). Body weight, adiposity, and energy expenditure will be assessed throughout the dietary and antibiotic interventions. Germ-free loss-of-function: This experiment will address the same question as the antibiotic study, namely whether gut microbes are required for 10,12 CLA-mediated weight loss. Mice that are reared germ-free (i.e. have never been exposed to bacteria) will be made obese by HFHS diet feeding, then will undergo 10,12 CLA supplementation to determine if the complete lack of bacteria hinders weight loss. Body weight will be monitored continuously, and adiposity assessed at the end of this experiment.Cecal microbial transfer: This experiment will determine if the changes in gut microbes induced by 10,12 CLA supplementation are sufficient to promote weight loss. Obese donor mice will be supplemented with 10,12 CLA, their cecal contents will be harvested anaerobically, and transferred orally to recipient obese mice. Body weight, adiposity, and energy expenditure will be monitored before and after cecal microbial transfer.2.Goal 2: To determine if targeted enrichment with butyrate- and/or CLA-producing microbes promotes weight loss.Preliminary data show that weight loss by 10,12 CLA is associated with increased butyrate- and CLA-producing gut bacteria. Using pro- and pre-biotic strategies, we will determine whether enrichment in these microbes plays a role in negative energy balance. In separate experiments, mice will be: (1) given oral prebiotics such as inulin or arabinoxylans that support butyrate- and CLA-producing bacteria; or (2) given oral butyrate- or CLA-producing probiotics to determine if microbiota-derived butyrate and/or CLA can impact negative energy balance. These experiments will be performed in wild type and Gpr43-/- mice to elucidate a specific role for short-chain fatty acids such as butyrate.Prebiotic supplementation: This experiment will determine if supplementing the diet with prebiotic compounds that are known to increase the abundance of particular butyrate- and CLA-producing bacteria will have an impact on body weight and adiposity. Mice that had been rendered obese by HFHS diet feeding will be switched to a diet containing either inulin or arabinoxylans, two prebiotic compounds that are abundant in particular types of food such as bananas, garlic, artichokes, and wheat, and have been shown to increase butyrogenic bacteria. Body weight, adiposity, and energy expenditure will be monitored before and after prebiotic supplementation.Probiotic administration: This experiment will determine if administering particular probiotics to obese mice can promote weight loss. Mice that had been rendered obese by HFHS diet feeding will be given three doses of a probiotic strain of bacteria that has been previously screened to be either a (1) high butyrate/low CLA producer, (2) low butyrate/high CLA producer, (3) high butyrate/high CLA producer, or (4) low butyrate/high CLA producer. Body weight, adiposity, and energy expenditure will be monitored before and after probiotic supplementation. There are several unique aspects about this experiment. Rather than using arbitrary and commonly used probiotic strains, such as strains of Lactobacillus and Bifidobacteria, probiotics will be chosen specifically for to their butyrate and/or CLA-producing capabilities. In addition, probiotic strains will be cultured and identified directly from donor mice that had consumed 10,12 CLA. Thus, this probiotic approach is highly innovative and likely to yield novel probiotic strains with clear translational appeal.GPR43 requirement: This experiment will determine if augmenting microbial butyrate production promotes weight loss. Obese mice that are deficient in GPR43, the major butyrate receptor on adipocytes, will be given pre- or pro-biotics as described above, and body weight, adiposity, and energy expenditure will be monitored.Analysis and interpretation of results: We predict that gut microbiota will be sufficient and required for the weight loss effects of 10,12 CLA. Moreover, we predict that particular modulation of the gut microbiota will phenocopy the weight loss effects of 10,12 CLA, and that this will be at least partially dependent on GPR43. In order to evaluate the efficiency of the proposed gut microbiota modulation strategies, we will collect fecal samples for bacterial sequencing and plasma samples for microbial metabolite quantification. Statistical analyses will be performed using one- and two-way ANOVA (as appropriate) to identify differences between groups in body weight, energy expenditure, and adiposity. Interpretation of results will be guide by the use of appropriate scientific and experimental controls.Efforts: Efforts to reach our target audience will be centered around the publication of the results of this proposed project and the presentation of our results to the general scientific community at local, national, and international meetings and conferences. These milestones will be accompanied by press releases, as appropriate. In addition, the results obtained through the completion of the proposed project will be incorporated into scientific review articles and lectures given by the Project Director and Co-Project Director for dissemination to the greater scientific community and general population.Evaluation: The results collected from the proposed project will be analyzed and interpreted as described above. Successful communication of these results will include publication of all findings in reputable and peer-reviewed scientific journals, presentation of results at local, national, and international conferences, and incorporation of these findings into review articles, textbooks, and lectures. The additional questions that will undoubtedly be raised by completion of the proposed studies will lead to new grant applications, for which additional funding would signify the ultimate level of success for this project.

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:With poor long-term success rates of weight loss by lifestyle modifications and excessive health care costs due to bariatric surgery and obesity pharmaceuticals, a major goal of this project is to widen the net of potential treatment strategies for those struggling with mild to moderate levels of obesity. Pending the results of this project, it is our hope that novel therapeutic strategies for the treatment of mild to moderate overweight will include particular combinations of prebiotics and probiotics (also known as synbiotics). The target audience for this project is anyone in the general public that is concerned with general overweight and obesity. This includes the overweight/obese community itself, medical providers, the insurance industry, government and global health agencies, and pharmaceutical industries. Efforts to reach our target audience will be and have been centered around the publication of the results of this proposed project and the presentation of our results to the general scientific community at local, national, and international meetings and conferences. These milestones will be accompanied by press releases, as appropriate. In addition, the results obtained through the completion of the proposed project will be incorporated into scientific review articles and lectures given by the Project Director and Co-Project Director for dissemination to the greater scientific community and general population. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training opportunities in several ways. First, several undergraduate and graduate students, including a visiting medical student, (involved in Major Goals 1, 2, and 3) from the Director's laboratory were given valuable training in scientific design, execution/interpretation of results, and data presentation and communication. One undergraduate student began a PhD program in September 2022, but remains involved with the preparation of a manuscript for publication (Major Goal 3). A In addition, one postdoctoral scholar and two graduate students are involved with several aspects of Major Goals 2 and 3, two from the lab of the Director and one in the Co-Director's lab. This has enabled the Project Director to actively engage in mentoring activities for these trainees in study design, data acquisition and analyses, data presentation, and manuscript and grant preparation. These trainees have been provided with many opportunities for professional development, including regular presentation opportunities at various seminar series and lab meetings at the UW and Rutgers, as well as undertaking the preparation and publication of several review manuscripts related to Major Goals 2 and 3 (two recently published) and many conference abstracts. The Project Director and Co-Director engage in regular virtual meetings to share ideas, results, and future projects, including a new NIH R01 proposal related to Goal 3 that was submitted during this funding period. Opportunities for professional development include the participation of the Project Director with several national symposia, including an invitation to present at the University of Illinois Chicago's weekly seminar series, and regular presentation opportunities through the University of Washington's Diabetes Research Center annual symposia, weekly research training conference, and presentations of project results to several local groups, including collaborator's group meetings and Seattle's Informal Microbiome Research Seminars (SIMR). How have the results been disseminated to communities of interest?The current project results have been presented regularly to the local scientific community by way of group lab meetings, local symposia, weekly research training conferences, and Seattle's Informal Microbiome Research bi-weekly virtual meetings. We also continue to publish research related to the Major Goals of this project in high-impact journals that are largely available to the public. What do you plan to do during the next reporting period to accomplish the goals?During the next funding period, we expect to publish a manuscript that will complete the experiments in Major Goal 1. We also expect to publish a manuscript related to Major Goal 3. We will continue to focus the majority of our work on completing Major Goal 2. This will include the completion of experiments examining the metabolic effects of SCFA on cultured adipocytes, and the initiation of experiments to study this in mice. The experiments described in new Major Goal 3.2 will also continue. The Project Director will continue to provide mentorship to two new postdoctoral fellows on their projects related to these Major Goals, including an assessment of sex steroids on mouse metabolism that involves gut microbiota, and whether administering antibiotics to pregnant mice results in similar antibiotic-mediated phenotypes related to body weight in their offspring. In addition, the Project Director expects a visiting scholar to join her laboratory during the final project year, which will provide additional mentoring experience for both the Project Director and the visiting faculty member.

Impacts
What was accomplished under these goals? Major Goal 1 has been completed within this budget period. First, we have determined that gut bacteria are required for 10,12 CLA-mediated weight loss (Major Goal 1.1). Mice that were given oral 10,12 CLA in addition to a broad-spectrum antibiotic cocktail did not lose as much body weight as mice only given 10,12 CLA. This loss-of-function experiment suggests that an intact gut microbiome is important for the weight loss effects of 10,12 CLA. Second, we have determined that gut microbiota from 10,12 CLA-treated mice are not sufficient to promote weight loss (Major Goal 1.2). In this gain-of-function experiment, previously harvested fecal microbial contents from mice consuming 10,12 CLA or control diets were transplanted into recipient obese mice, and whether this could promote weight loss was examined. This experiment yielded unexpected results, in that transplanting microbiota from mice undergoing weight loss had NO effect on body weights in the recipient mice. However, we interestingly observed that the glucose metabolic phenotype of the donor mice was observed in the recipient mice. This unexpected finding could lead to new research questions for future proposals. Based on the intriguing results of Major Goals 1.1 and 1.2, we next completed Goal 1.3. In this experiment, the same cecal contents that were used in the gain-of-function experiment from Goal 1.2 were transplanted into germ-free mice. This design enabled a more pure gain-of-function experiment. Similar to Major Goal 1.2, this experiment determined that gut microbes were not sufficient to recapitulate the body weight phenotype of the donor mice, but did show a strong recapitulation of the glucose metabolic phenotype. Collectively, the experiments conducted in Major Goal 1 have been submitted for publication to the prestigious journal Molecular Metabolism. Work has progressed quickly on Major Goals 2.1-2.2. We have treated various cohorts of diet-induced obese mice with pre- and pro-biotics, and post-biotics, including an inulin (fiber)-containing diet, VSL3 probiotics, and acetate-containing water. After determining the best treatment delivery strategies and the most appropriate controls, we have nearly completed a large cohort of treatment mice (n=~200). These include both males and females. We have determined that particular strategies that are known to increase the endogenous production acetate and butyrate have profound effects on energy balance. Notably, the addition of fiber to an obesogenic diet effectively blunts further weight gain and rapidly improves glucose metabolism. The addition of VSL3 probiotics leads to further improvements. However, surprisingly, oral delivery of acetate (a post-biotic) had no major effects on energy metabolism. During the last budget period for this award, whether these SCFA-increasing strategies impact insulin signaling, energy intake or expenditure, or hepatic phenotypes will be explored. During the past budget period we also have nearly completed work on Major Goal 2.3, in which we sought to determine if adipocyte-GPR43 modulates the beneficial effects of inulin and/or VSL3 probiotics. We have revealed the striking finding that the robust effects of these pre- and pro-biotic treatments on body weight and composition are NOT observed in mice deficient in adipocyte-GPR43. This is a very exciting finding that will be further studied in the final year of this award. As this award has progressed, we have developed a new Major Goal 3, which is an exciting new goal because it is a natural progression of previous work from both the Director's and Co-Director's labs, and will forge a new joint line of investigation. Major Goal 3 will determine whether serum amyloid A (SAA), an inflammatory mediator that is associated with chronic metabolic diseases such as obesity, impacts gut microbiota in obesity. The following objectives will be assessed: Determine whether SAA contributes to microbiota composition with high fat diet feeding in mice. Mice that are sufficient or deficient in SAA1 and SAA2 will be fed a high fat diet with either single- or co-housing to determine if an intersection between SAA and gut microbiota contribute to body weight gain and adiposity. Because there is an emerging literature on sexual dimorphic effects of SAA in metabolic disease states, effects in male and female mice will be closely examined and compared. This will include direct comparisons in the mouse model above, as well as the effect of antibiotics on adipocyte function and metabolism in male and female mice that are sufficient or deficient in SAA1/2. In this budget period we made considerable progress on new Major Goal 3. We have completed most experiments for Major Goal 3.1, having shown that: (1) SAA expression in the gut is dependent on the presence of intact microbiota, with much higher SAA1 and SAA2 expression in the ileum than the colon; (2) gut microbial composition changes dramatically with deletion of SAA1 and SAA2; (3) SAA1/2 KO mice have a more severe reaction to acute inflammatory stimulus (LPS) and a more chronic inflammatory stimulus (DSS-induced colitis), reflected by reduced body temperature and enhanced inflammatory gene expression, suggesting that SAA is required to mount an appropriate inflammatory response to an acute insult, but may limit the inflammatory response to a more chronic insult; and (4) there is a sexually dimorphic response to the loss of SAA1/2 in obese mice. We recently completed some proteomics analyses to finalize this work, which is actively being prepared to submit for publication in the next funding period. This work was done by a former student in the laboratory of the Co-Director, who is now attending graduate school to obtain her PhD.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: den Hartigh LJ, May KS, Zhang XS, Chair A, Blaser MJ. Serum amyloid A and metabolic disease: evidence for a critical role in chronic inflammatory conditions. Front. Cardiovasc. Med. 2023, Jun 15; 10:1197432.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Kothari V, Savard C, Tang J, Lee SP, Subramanian S, Wang S, den Hartigh LJ, Bornfeldt KE, Ioannou GN. sTREM2 is a plasma biomarker for human NASH and promotes hepatocyte lipid accumulation. Hepatology Communications 2023, Oct. 12;7(11):e0265.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Zhang M, Wang SA, Koh H, den Hartigh LJ, Blaser MJ. Early life sub-therapeutic antibiotics impact adipose tissue to modulate obesity. Submitted to the Rutgers University Microbiome Program Annual Retreat (March 2023). **Recipient of best poster award
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Zaki M, Wang S, May KS, den Hartigh LJ. Sex-specific impact of serum amyloid A3 (SAA3) on adipocyte metabolism. Presented at NIDDK Medical Student Research Program symposium in Nashville, TN (July 2023).
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2024 Citation: Zhang M, Wang SA, Koh H, den Hartigh LJ, Blaser MJ. Early life sub-therapeutic antibiotics impact adipose tissue to modulate obesity. Submitted to American Society for Microbiology (ASM) Microbe 2024, to be presented June 13-17 in Atlanta, GA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: May KS, Wang SA, Bryan C, Blaser MJ, Morton GJ, den Hartigh LJ. Gut microbiota manipulation by dietary interventions impacts energy and glucose homeostasis in diet-induced obese male mice. Submitted to American Society for Nutrition (ASN) Nutrition 2024, to be presented June 29-July 2 in Chicago, IL.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Zhang M, Yin YS, May KS, Zhang X, Wang S, Blaser M, den Hartigh LJ. Gut microbiota contribute to the glucoregulatory and body compositional changes that accompany 10,12 CLA-mediated weight loss in mice. Submitted to Mol Metab.

Progress 04/01/22 to 03/31/23

Outputs
Target Audience:With poor long-term success rates of weight loss by lifestyle modifications and excessive health care costs due to bariatric surgery and obesity pharmaceuticals, a major goal of this project is to widen the net of potential treatment strategies for those struggling with mild to moderate levels of obesity. Pending the results of this project, it is our hope that novel therapeutic strategies for the treatment of mild to moderate overweight will include particular combinations of prebiotics and probiotics (also known as synbiotics). The target audience for this project is anyone in the general public that is concerned with general overweight and obesity. This includes the overweight/obese community itself, medical providers, the insurance industry, government and global health agencies, and pharmaceutical industries. Efforts to reach our target audience will be and have been centered around the publication of the results of this proposed project and the presentation of our results to the general scientific community at local, national, and international meetings and conferences. These milestones will be accompanied by press releases, as appropriate. In addition, the results obtained through the completion of the proposed project will be incorporated into scientific review articles and lectures given by the Project Director and Co-Project Director for dissemination to the greater scientific community and general population. Changes/Problems:Several challenges related to the global COVID-19 pandemic were experienced during this funding period. This stemmed largely from public health policies in place that occasionally limited the amount of time the Project Director was able to spend on-site in her laboratory. The Project Director is a parent to 3 young children that attend public elementary school, which lead to major disruptions in school attendance (and her subsequent ability to work on-site) due to heightened attendance rules that mandate that any symptomatic children remain at home. This presented an occasional hardship for the Director and her laboratory. In addition, there were some issues with the Co-Director's gnotobiotic mouse facility that has delayed the completion of Major Goal 1. The gnotobiotic vivarium at Rutgers experienced some mouse contamination, which rendered it closed and unavailable to the Co-Director for approximately 8 months. That facility is, thankfully, up and running again. In addition, the loss of a key collaborator, Dr. DePaolo, has hindered the completion of Major Goal 2. We have had to rely on the help of a different collaborator, Dr. Maria Marco (UC Davis), in his stead. What opportunities for training and professional development has the project provided?The project has provided training opportunities in several ways. First, an undergraduate student (involved in Major Goals 1 and 3) from the Co-Director's laboratory was given valuable training in scientific design, execution/interpretation of results, and data presentation and communication. This undergraduate student began a PhD program in September 2022, but remains involved with the preparation of a manuscript for publication. In addition, one postdoctoral scholar and one graduate student are involved with several aspects of Major Goals 2 and 3, one from the lab of the Director and one in the Co-Director's lab. This has enabled the Project Director to actively engage in mentoring activities for these trainees in study design, data acquisition and analyses, data presentation, and manuscript and grant preparation. These trainees have been provided with many opportunities for professional development, including regular presentation opportunities at various seminar series and lab meetings at the UW and Rutgers, as well as undertaking the preparation and publication of several review manuscripts related to Major Goals 2 and 3 (two published and one recently submitted). The Project Director and Co-Director engage in regular virtual meetings to share ideas, results, and future projects, including a new NIH R01 proposal related to Goal 3 to be submitted during this next funding period. Opportunities for professional development include the participation of the Project Director with several national symposia, including an invitation to present at the University of Illinois Chicago's weekly seminar series, and regular presentation opportunities through the University of Washington's Diabetes Research Center annual symposia, weekly research training conference, and presentations of project results to several local groups, including collaborator's group meetings and Seattle's Informal Microbiome Research Seminars (SIMR). The Project Director also had the opportunity to present research related to these projects at the Keystone Symposium on Adipose Tissue in January 2023. How have the results been disseminated to communities of interest?The current project results have been presented regularly to the local scientific community by way of group lab meetings, local symposia, weekly research training conferences, and Seattle's Informal Microbiome Research bi-weekly virtual meetings. We also continue to publish research related to the Major Goals of this project in high-impact journals that are largely available to the public. What do you plan to do during the next reporting period to accomplish the goals?During the next funding period, we expect to publish a manuscript that will complete the experiments in Major Goal 1. We also expect to publish a manuscript related to Major Goal 3. We will continue to focus the majority of our work on completing Major Goal 2. This will include the completion of experiments examining the metabolic effects of SCFA on cultured adipocytes, and the initiation of experiments to study this in mice. The experiments described in Major Goal 3.2 will also continue. The Project Director will continue to provide mentorship to two new postdoctoral fellows on their projects related to these Major Goals, including an assessment of sex steroids on mouse metabolism that involves gut microbiota, and whether administering antibiotics to pregnant mice results in similar antibiotic-mediated phenotypes related to body weight in their offspring. In addition, the Project Director expects a medical student to join her laboratory during the summer months, which will provide additional mentoring experience for both the Project Director and postdoctoral fellow. This medical student will help with the in vitro aspects of Major Goal 3.2 for approximately 2-3 months.

Impacts
What was accomplished under these goals? We have continued to work on the experiments outlined in the first major goal of this project. First, we have completed experiments aimed at determining whether gut bacteria are required for 10,12 CLA-mediated weight loss (Major Goal 1.1). Mice that were given oral 10,12 CLA in addition to a broad spectrum antibiotic cocktail did not lose as much body weight as mice only given 10,12 CLA. This loss-of-function experiment suggests that an intact gut microbiome is important for the weight loss effects of 10,12 CLA. Second, we have also completed an experiment to determine whether the gut microbiota from 10,12 CLA-treated mice can itself promote weight loss (Major Goal 1.2). In this gain-of-function experiment, previously harvested fecal microbial contents from mice consuming 10,12 CLA or control diets were transplanted into recipient obese mice, and whether this could promote weight loss was examined. This experiment yielded unexpected results, in that transplanting microbiota from mice undergoing weight loss had NO effect on body weights in the recipient mice. However, we interestingly observed that the glucose metabolic phenotype of the donor mice was observed in the recipient mice. This unexpected finding could lead to new research questions for future proposals. Based on the intriguing results of Major Goals 1.1 and 1.2, we embarked on tackling Goal 1.3, which is nearly complete. In this experiment, the same cecal contents that were used in the gain-of-function experiment from Goal 1.2 were transplanted into germ-free mice. This design enables a more pure gain-of-function experiment, which is nearly complete. Similar to Major Goal 1.2, this experiment showed a trend towards a recapitulation of the body weight phenotype of the donor mice, but showed a strong recapitulation of the glucose metabolic phenotype. This experiment was unexpectedly delayed by a contamination issue in the germ-free facility at Rutgers, but is back on track and is expected to be completed within the next budget period. During this funding period we have made considerable progress in Major Goal 2.1. A postdoctoral scholar has performed many experiments using cultured adipocytes to determine if the SCFA acetate and butyrate impact adipocyte metabolism in vitro. She has found that acetate restricts adipocyte cell size, potentially by limiting lipid and glucose uptake and increasing fatty acid oxidation. Acetate also appears to reduce inflammation and improve insulin sensitivity. This work is ongoing, but is expected to be completed within the next funding period. This work has also resulted in the postdoctoral scholar, who is currently supported by a T32 training grant on nutrition, submitting an application for an American Heart Association postdoctoral fellowship. Based on results thus far from Major Goal 2.1, we are ready to begin work on Major Goals 2.2-2.4. We have recently obtained mice with adipocyte-specific deficiency of GPR43, which are currently breeding. During this time we are beginning some pilot studies using WT mice to determine the optimal dosing strategies for administering pre- and pro-biotics to mice. We will begin work on Major Goals 2.2-2.4 during the next funding period, which we anticipate will be completed in Year 5. Finally, we have also made considerable progress on our new Major Goal 3. This new third goal is to determine whether serum amyloid A (SAA), an inflammatory mediator that is associated with chronic metabolic diseases such as obesity, impacts gut microbiota in obesity. This is an exciting new goal because it is a natural progression of previous work from both the Director's and Co-Director's labs, and will forge a new joint line of investigation. The following objectives will be assessed: Determine whether SAA contributes to microbiota composition with high fat diet feeding in mice. Mice that are sufficient or deficient in SAA1 and SAA2 will be fed a high fat diet with either single- or co-housing to determine if an intersection between SAA and gut microbiota contribute to body weight gain and adiposity. Because there is an emerging literature on sexual dimorphic effects of SAA in metabolic disease states, effects in male and female mice will be closely examined and compared. This will include direct comparisons in the mouse model above, as well as the effect of antibiotics on adipocyte function and metabolism in male and female mice that are sufficient or deficient in SAA1/2. We have completed most experiments for Major Goal 3.1, having shown that: (1) SAA expression in the gut is dependent on the presence of intact microbiota, with much higher SAA1 and SAA2 expression in the ileum than the colon; (2) gut microbial composition changes dramatically with deletion of SAA1 and SAA2; (3) SAA1/2 KO mice have a more severe reaction to acute inflammatory stimulus (LPS) and a more chronic inflammatory stimulus (DSS-induced colitis), reflected by reduced body temperature and enhanced inflammatory gene expression, suggesting that SAA is required to mount an appropriate inflammatory response to an acute insult, but may limit the inflammatory response to a more chronic insult; and (4) there is a sexually dimorphic response to the loss of SAA1/2 in obese mice. Pending some proteomics analyses, these experiments have been prepared to submit for publication in the next funding period. This work was done by a former student in the laboratory of the Co-Director, who is now attending graduate school to obtain her PhD. We have also initiated a new experiment related to Major Goal 3, involving a Postdoctoral Fellow from the Director's laboratory, and one Graduate Student from the Co-Directors laboratory. In this project, plasma from male and female mice given antibiotics early in life was utilized to treat cultured adipocytes to determine if microbiota-related factors could influence adipocyte metabolism in a sex-dependent manner. Preliminary results suggest that serum factors reduce adipogenesis in fat cells, an effect that is reversed by antibiotic treatment in mice, and an effect which is stronger in male mice.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Vaisar T, Wang S, Omer M, Irwin AD, Storey C, Tang C, den Hartigh LJ. 10,12 conjugated linoleic acid supplementation improves HDL composition and function in male mice. J Lipid Res 2022 Jun 15;63(8):100241. [PMID: 35714730]
• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Valencia A, Whitson J, Wang S, Nguyen L, den Hartigh LJ, Rabinovitch P, Marcinek D. The aging heart is prone to aggravated hypertrophy by short-term high sugar consumption. Nutrients. 2022 Nov 3;14(21):4645. doi: 10.3390/nu14214645. [PMID 36364920]
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: May KS, den Hartigh LJ. Gut microbial-derived short chain fatty acids: impact on adipose tissue physiology. Nutrients. 2023 Jan 5;15(2):272. doi: 10.3390/nu15020272. [PMID 36678142]
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Kothari V, Savard C, Tang J, Lee SP, Subramanian S, Wang S, den Hartigh LJ, Bornfeldt KE, Ioannou GN. Soluble triggering receptor expressed in myeloid cells 2 (TREM2) is associated with non-alcoholic fatty liver disease progression. Submitted to Hepatology.
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Chait A, May KS, Zhang XS, Blaser MJ, den Hartigh LJ. Serum amyloid A and metabolic disease: evidence for divergent mechanisms in acute vs. chronic inflammatory conditions. Submitted to Front. Cardiovasc. Med.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Zhang M, Lee A, Patal P, Shamsi F, Shinoda K, den Hartigh LJ, Blaser MJ. Mechanisms underlying antibiotic-induced obesity. Presented as a poster at the Graduate Student Research Symposium at Rutgers University (May 2022).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Zhang M, Wang SA, Patel P, Lee A, den Hartigh LJ, Blaser MJ. Mechanisms underlying early-life antibiotic-induced obesity. Presented as a poster at the Cold Spring Harbor Lab Microbiome Meeting (October 2022).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: May K, Wang S, Goodspeed L, Ogimoto K, Morton GJ, den Hartigh LJ. Adipocyte-derived fibroblast growth factor 21 (FGF21) contributes to diet-induced obesity in mice. Presented at the Keystone Symposium on Adipose Tissue: Energizing Good Fat (January 2023).

Progress 04/01/21 to 03/31/22

Outputs
Target Audience:With poor long-term success rates of weight loss by lifestyle modifications and excessive health care costs due to bariatric surgery and obesity pharmaceuticals, a major goal of this project is to widen the net of potential treatment strategies for those struggling with mild to moderate levels of obesity. Pending the results of this project, it is our hope that novel therapeutic strategies for the treatment of mild to moderate overweight will include particular combinations of prebiotics and probiotics (also known as synbiotics). The target audience for this project is anyone in the general public that is concerned with general overweight and obesity. This includes the overweight/obese community itself, medical providers, the insurance industry, government and global health agencies, and pharmaceutical industries. Efforts to reach our target audience will be and have been centered around the publication of the results of this proposed project and the presentation of our results to the general scientific community at local, national, and international meetings and conferences. These milestones will be accompanied by press releases, as appropriate. In addition, the results obtained through the completion of the proposed project will be incorporated into scientific review articles and lectures given by the Project Director and Co-Project Director for dissemination to the greater scientific community and general population. Changes/Problems:The ongoingproblem that was encountered during this reporting period was the continuation of the global pandemic. Progress on this project was hindered by the need to socially distance while in the laboratory, and the frequent need to isolate when COVID-19 symptoms or exposures occur. This has been particularly problematic for the Project Director, who is a parent to 3 young children that experience major disruptions to school attendance due to the pandemic. Another problem encountered during this funding period is the abrupt retirement of our collaborator, Dr. Will dePaolo. Dr. dePaolo planned to provide us with bacterial isolates to screen for CLA and butyrate production. Without his assistance, we are currently brainstorming alternative approaches to obtain bacterial strains to screen. What opportunities for training and professional development has the project provided?The project has provided training opportunities in several ways. First, an undergraduate student from the Co-Director's laboratory was given valuable training in scientific design, execution, evaluation/interpretation of results, and data presentation and communication. This undergraduate student has now begun the graduate school application process. In addition, a new postdoctoral fellow has joined the laboratory of the Project Director, and is actively engaged in work related to Major Goal 2 of this project. This has enabled the Project Director to directly engage in mentoring activities, including training the postdoctoral fellow on study design, data acquisition and analysis, data presentation, and manuscript and grant preparation. The postdoctoral fellow has been provided with many opportunities for professional development, including regular presentation opportunities at various seminar series and lab meetings at the UW, as well as undertaking the preparation and publication of a review manuscript in her area of interest related to Project 2. The Project Director has facilitated regular virtual meetings between their lab and the Co-Director's lab to share ideas, results, and future projects. Opportunities for professional development include the participation of the Project Director with several local symposia, including chairing the annual University of Washington Diabetes Research Center Symposium, which showcased prominent researchers in obesity from across the United States; participation in the local University of Washington weekly Research Training Conference; and presentation of the results of this project to several local groups, including collaborator's group meetings and the University of Washington Center for Microbiome Science and Technology (CMiST) Microbiome Club bi-weekly meetings. How have the results been disseminated to communities of interest?The current project results have been presented regularly to the local scientific community by way of group lab meetings, local symposia, weekly research training conferences, and the University of Washington Center for Microbiome Science and Technology (CMiST) Microbiome Club bi-weekly meetings. The Project Director has recently published a comprehensive review article related to the new postdoctoral fellow's research project on the impact of short-chain fatty acids on adipocyte metabolism. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the projects pertaining to Major Goal 1.1, 1.2, and 1.3 will be prepared for publication. This will include the completion of the experimental procedures, the presentation of results, and the composition of a manuscript. We will continue to make progress on new Major Goal 3, which should result in 1-2 publications in the next funding period. The Project Director will continue to provide mentorship to a new postdoctoral fellow, which will provide not only experiences in technical and grantsmanship training to the postdoc, but also valuable mentoring experience for the Project Director. Results from this postdoctoral project will inform the pro-biotic strains to be used in Major Goal 2, which we expect to begin in the next funding period. In addition, the Project Director expects to mentor an undergraduate summer intern, which will provide additional mentoring experience for both the Project Director and postdoctoral fellow. This undergraduate intern will help with the in vitro aspects of Major Goal 2 for approximately 3 months. In addition, the Project Co-Director anticipates the arrival of a new graduate student to contribute to Major Goal 3.

Impacts
What was accomplished under these goals? We have continued to work on two major experiments outlined in the first major goal of this project. First, we have completed experiments aimed at determining whether gut bacteria are required for 10,12 CLA-mediated weight loss (major goal 1.1). Mice that were given oral 10,12 CLA in addition to a broad spectrum antibiotic cocktail did not lose as much body weight as mice only given 10,12 CLA. This loss-of-function experiment suggests that an intact gut microbiome is important for the weight loss effects of 10,12 CLA. Second, we have also completed an experiment to determine whether the gut microbiota from 10,12 CLA-treated mice can promote itself promote weight loss (major goal 1.3). In this gain-of-function experiment, previously harvested fecal microbial contents from mice consuming 10,12 CLA or control diets were transplanted into recipient obese mice, and whether they can promote body weight loss was examined. This experiment yielded unexpected results, in that transplanting microbiota from mice undergoing weight loss had NO effect on the body weights of the recipient mice. However, we interestingly observed that the glucose metabolic phenotype of the donor mice was observed in the recipient mice. This unexpected finding could lead to new research questions for future proposals. Based on the intriguing results of Goals 1.1 and 1.3, we have now just embarked on tackling Goal 1.2. In this experiment, the same cecal contents that were used in the gain-of-function experiment from Goal 1.3 will be transplanted into germ-free mice. This design enables a more true gain-of-function experiment, and is currently underway. A third accomplishment from this reporting period, which will henceforth be termed Major Goal 3, stems from a new collaborative direction which the Project Director and Co-Director have initiated together. This third experiment was to determine what effect serum amyloid A, an inflammatory mediator that is associated with chronic disease states, has on gut microbial communities in obesity. This new experiment is well underway, and we expect to publish a joint paper in the coming funding period. Some of these experiments were performed by a graduate student. As such, some mentoring of trainees was an activity related to this project. A fourth accomplishment that was initiated during the second funding period is related to Major Goal 2. A new postdoctoral fellow in the den Hartigh laboratory has initiated a project to test whether short-chain fatty acids, major metabolites produced by certain bacterial species to be used as pro-biotics for Major Goal 2, can modulate adipocyte metabolism and inflammation. The results of these experiments will inform which pro-biotic strains to use for the mouse studies in Major Goal 2.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: May K, den Hartigh LJ (2021). Modulation of adipocyte metabolism by microbial short-chain fatty acids. Nutrients Oct 19;13(10):3666.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Spann RA, Morrison CD, den Hartigh LJ. The nuanced metabolic functions of endogenous FGF21 depend on the nature of the stimulus, tissue source, and experimental model. Frontiers Endocrinology (Lausanne) 2022 Jan 3;12:802541.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Gomes D, Wang S, Goodspeed L, Turk KE, Wietecha T, Liu Y, Bornfeldt KE, OBrien KD, Chait A, den Hartigh LJ. Comparison between genetic and pharmaceutical disruption of LDLR expression for the development of atherosclerosis. J Lipid Res. 2022 Jan 28:100174.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: 1. Vaisar T, Wang S, Omer M, Irwin AD, Storey C, Tang C, den Hartigh LJ. Anti-atherosclerotic effect of 10,12 conjugated linoleic acid is associated with improved HDL anti-inflammatory potential and increased cholesterol capacity in mice. Submitted to Journal of Lipid Research.

Progress 04/01/20 to 03/31/21

Outputs
Target Audience:With poor long-term success rates of weight loss by lifestyle modifications and excessive health care costs due to bariatric surgery and obesity pharmaceuticals, a major goal of this project is to widen the net of potential treatment strategies for those struggling with mild to moderate levels of obesity. Pending the results of this project, it is our hope that novel therapeutic strategies for the treatment of mild to moderate overweight will include particular combinations of prebiotics and probiotics (also known as synbiotics). The target audience for this project is anyone in the general public that is concerned with general overweight and obesity. This includes the overweight/obese community itself, medical providers, the insurance industry, government and global health agencies, and pharmaceutical industries. Efforts to reach our target audience will be and have been centered around the publication of the results of this proposed project and the presentation of our results to the general scientific community at local, national, and international meetings and conferences. These milestones will be accompanied by press releases, as appropriate. In addition, the results obtained through the completion of the proposed project will be incorporated into scientific review articles and lectures given by the Project Director and Co-Project Director for dissemination to the greater scientific community and general population. Changes/Problems:The obvious problem that was encountered during this reporting period was the global pandemic. As a result of "shelter-in-place" mandates in the Spring and Summer of 2020, progress on this project was hindered, and continues to be somewhat hindered as the pandemic wears on. New workplace safety practices demand a slower pace for some of the major goals, including Major Goal 1.2. What opportunities for training and professional development has the project provided?The project has provided training opportunities in several ways. First, a graduate student from the Co-Director's laboratory was given valuable training in scientific design, execution, evaluation/interpretation of results, and data presentation and communication. The Project Director has facilitated regular virtual meetings between their lab and the Co-Project Director's lab to share ideas, results, and future projects. Opportunities for professional development include the participation of the Project Director with several local symposia, including chairing the annual University of Washington Diabetes Research Center Symposium, which showcased prominent researchers in obesity from across the United States; participation in the local University of Washington weekly Research Training Conference; and presentation of the results of this project to several local groups, including collaborators group meetings and the University of Washington Center for Microbiome Science and Technology (CMiST) Microbiome Club bi-weekly meetings. As a result of this project, the Project Director has been able to attend several virtual international meetings, including the American Society for Nutrition Annual Meeting (2020), the American Diabetes Association Scientific Sessions (2020), and the Keystone Conference on Obesity (2021). How have the results been disseminated to communities of interest?The current project results have been presented regularly to the local scientific community by way of group lab meetings, local symposia, weekly research training conferences, and the University of Washington Center for Microbiome Science and Technology (CMiST) Microbiome Club bi-weekly meetings.In addition, some results related to the new SAA project are in the process of being published in a publically accessibly journal (Arteriosclerosis, Thrombosis, and Vascular Biology), which has a wide readership. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the projects pertaining to Major Goal 1.1 and 1.3 will be completed. This will include the completion of the experimental procedures, the presentation of results, and the composition of a manuscript. We will continue to make progress on new Major Goal 3. When it is safe to do so (re: COVID-19), we will initiate Major Goal 1.2. The Project Director will welcome a new postdoctoral fellow to her team, which will provide not only experiences in technical and grantsmanship training to the postdoc, but also valuable mentoring experience for the Project Director.

Impacts
What was accomplished under these goals? We have begun to work on two major experiments outlined in the first major goal of this project. First, we have begun to address whether gut bacteria are required for 10,12 CLA-mediated weight loss (major goal 1.1). Mice that were given oral 10,12 CLA in addition to a broad spectrum antibiotic cocktail exhibited did not lose as much body weight as mice only given 10,12 CLA. This loss-of-function experiment suggests that an intact gut microbiome is important for the weight loss effects of 10,12 CLA. Second, we have begun an experiment to determine whether the gut microbiota from 10,12 CLA-treated mice can promote itself promote weight loss (major goal 1.3). In this gain-of-function experiment, previously harvested fecal microbial contents from mice consuming 10,12 CLA or control diets were transplanted into recipient obese mice, and whether they can promote body weight loss was examined. This study is currently underway, and there are not yet any results to report. A third accomplishment from this reporting period, which will henceforth be termed Major Goal 3, stems from a new collaborative direction which the Project Director and Co-Director have initiated together. This third experiment was to determine what effect serum amyloid A, an inflammatory mediator that is associated with chronic disease states, has on gut microbial communities in obesity. This new experiment is still in the initiation stage. Some of these experiments were performed by a graduate student. As such, some mentoring of trainees was an activity related to this project.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Chait A, Wang S, Goodspeed L, Gomes D, Turk KE, Wietecha T, Tang J, Storey C, OBrien KD, Rubinow KB, Tang C, Vaisar T, Gharib SA, Lusis AJ, den Hartigh LJ. Sexually dimorphic relationships among serum amyloid A3, inflammation, and cholesterol metabolism modulate atherosclerosis in mice. Under review for Arteriosclerosis Thrombosis and Vascular Biology.