Source: UNIVERSITY OF ILLINOIS submitted to NRP
EXPANDING THE BENEFITS OF BRASSICA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030148
Grant No.
2023-67017-39758
Cumulative Award Amt.
$633,000.00
Proposal No.
2022-09454
Multistate No.
(N/A)
Project Start Date
May 15, 2023
Project End Date
May 14, 2026
Grant Year
2023
Program Code
[A1343]- Food and Human Health
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
This projectresponds to USDA-NIFA AFRI Foundation Program for Food Safety, Nutrition and Health in the Program Area of Food and Human Health (A1343). We will examine interactions between brassica, gut microbiome (GM) and GLP-1 secretion. Brassica vegetables are a rich source of glucosinolates (GSLs), a group of sulfur compounds that are unique to these vegetables (broccoli, kale, and others). Upon hydrolysis, GSLs are converted to bioactive isothiocyanates (ITCs). When consumed raw, GSLs can be converted to ITCs by the plant enzyme myrosinase. Heat (cooking) inactivates myrosinase, then gut microbiota (GM) become responsible for any GSL conversion to ITCs. We hypothesize that non-ITC producing pathways present in the GM are competing for GSLs and thus explain the dramatic inter-individual differences seen in ITC production (Aim 1). Once thought to be only present in the mouth, bitter-taste receptors (T2Rs) are now found to be present in many tissues including the gut. Since gut T2Rs can trigger GLP-1 secretion and ITCs can serve as T2R agonists, we hypothesize that ITCs produced by the GM bind intestinal T2R(s) to trigger GLP-1-dependent control of obesity and glucose homeostasis (Aim 2). Optimizing ITC production in the gut, both amount and location, has the potential to reverse glucose intolerance and obesity in humans. Our findings will inform the public about the health promoting properties of brassica, explain the variation in GSL bioactivation seen in human studies, where non-ITC metabolism of GSLs by GM have been little considered, and will further increase brassica intake worldwide.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70114401010100%
Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
1440 - Cole crops;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Our long-term goal is to optimize the positive impact of brassica on mammalian health. The overall objective of this project is to determine the interaction between the GM (gut microbiota) and dietary brassica (kale and broccoli), focusing on GM metabolism of GSL (glucosinolates) and GLP-1 secretion. Our central hypothesis is that frequent brassica ingestion alters the GM resulting in increased ITC (isothiocyanate) production in situ. Concomitantly, this results in increased gut T2R activation and GLP-1 secretion, and is thus expected to improve glucose homeostasis and control weight gain. We have chosen to perform this basic research in mice, giving the greatest flexibility to dietary manipulation and access to extensive tissue evaluation which would be impossible with humans.To achieve the objective of this application, we will pursue the following specific aims:Aim 1. Characterize the impact of brassica on GM composition and GSL metabolism. The working hypothesis is that brassica diet frequency and preparation (cooked vs. raw) both significantly impact microbial GSL metabolism and thus ITC production.Aim 2. Determine the impact of dietary brassica on (1) Secretion of GLP-1, (2) Weight gain, (3) Glucose homeostasis, and (4) Obesity-linked GM dysbiosis. The working hypothesis is that ITCs promote GLP-1 secretion and thus improve glucose homeostasis and control weight gain.
Project Methods
For Aim 1, we will look at how sevendays' pre-feeding brassica impacts GSL metabolism by cecal microbiota and the cecal microbiota composition. After acclimation, 72 seven-week old, lean (C57BL/6J) mice will be fed one of ninediets for sevendays (eightmice per treatment). These diets were selected to enable valuable comparisons such as broccoli vs. kale, raw vs. cooked, with vs. without GSL, and purified GSL vs. whole plant. Feeding a 10% broccoli/kale diet over sevendays has previously resulted in significant effects, thus we will use this dietary composition for the proposed work. After seven days, the mice will be euthanized and their blood and tissues will be used for GSL metabolomics and transcriptomics analysis, respectively (Aim 1.1), their ceca will be removed anaerobically and used for ex vivo GSL metabolism product assessment (Aim 1.2), identification of microbial GSL metabolizing isolates (Aim 1.3), and microbiota community assessment (Aim 1.4). To remove the cecum, both ends of the cecum will be tied off, followed by surgical removal, and rapid transportation to the nearby anaerobic chamber, as we have done previously. Once in the anaerobic chamber, the cecum will be sliced open and the contents distributed for the various analyses described below.Aim 2.1 is a short-term animal study. Based on our in vitro data, SF (the main ITC produced from broccoli) and AITC (the main ITC produced from kale) exhibited different potency in inducing GLP-1 secretion, potentially through activating different target T2R(s). Five different dietary treatments will be fed to C57BL/6J male mice (lean mice) at sixteenweeks of age (n=10 for each) for sevendays: D12450J (10 kcal% fat diet), D12450J-based 10% raw broccoli, 10% cooked broccoli, 10% raw kale, and 10% cooked kale. Similarly, D12492 (60 kcal% fat diet), D12492-based 10% raw broccoli, 10% cooked broccoli, 10% raw kale, and 10% cooked kale will be fed to C57BL/6J DIO (diet induced obese) male mice at sixteenweeks of age (n=10 for each) for sevendays. Lean and obese fifteen-week old mice will be acclimated to D12450J and D12492 for one week, respectively, then randomized into fivegroups (n=10 for each) and fed with their assigned diet. Various host parameters will be evaluated including fasting blood glucose, insulin, total GLP-1, intestinal T2R gene expression and cecal microbiome.Aim 2.2 is a long-term animal study. To investigate the impact of long-term consumption of broccoli and kale on glucose regulation and obesity prevention mediated through GLP-1 secretion, we selected cooked broccoli and cooked kale as the treatment for Aim 2.2. Forty C57BL/6J male mice will be purchased at sixweeks of age. After oneweek of acclimation, mice will be randomized into fourtreatment groups (n=10 for each) and fed either D12450J (10 kcal% fat diet), D12492 (60 kcal% fat diet), D12492 supplemented with 10% cooked broccoli, or D12492 supplemented with 10% cooked kale. Food will be provided ad libitum for twelveweeks with occasional fasting as required for proposed measurements (OGTT, postprandial GLP-1, etc.). In addition to the host parameters measured in Aim 2.1, we will also measure body weight, food intake, fasting plasma lipid profile, fat mass measurement, and inflammatory marker measurement in Aim 2.2.

Progress 05/15/24 to 05/14/25

Outputs
Target Audience:The primary target audience reached during the reporting period is the scientific community. We have submitted manusripts and presented posters at scientific meetings. The PI has had several presentations where he presented the ongoing work of the project - both in the US and abroad. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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 will continue our push to publish the second manuscript from Aim 1. A major goal is to become successful with our animal model so we can demonstrate the possiblity of cecal produced ITC to stimulate GLP-1 and demonstrate an outcome. We expect this to result in a manuscript during the next reporting period.

Impacts
What was accomplished under these goals? Aim 1 We have completed most of this aim and will result in two manuscripts. We anticipate both to be published by the next submitting period. We have demonstrated that ITCs, and not GSLs, can stimulate GLP-1 production from enteroendocrine cells via cell culture assays (paper 2). We have also fed mice cooked broccoli for one week and demonstrated changes in the gut microbiota composition and function (paper 1; accepted). Cooked broccoli consumption increases the production by the cecal microbiota of total ITCs, sulforaphane and sulforaphane-cyteine conjugate when compared to animals fed control diet (no broccoli). There was no significant change in sulforaphane-nitrile production. Aim 2 No new data to report yet but I have had lab personnel trained by another USDA funded investigator (Dr. Duca at Univ. of Arizona) to perform rodent surgeries where we can cannulate the terminal ileum and mesenteric vein. With these animals, we will confirm that ITC production in the cecum, and not GSLs, can result in increased GLP-1 as measured in the mesenteric vein blood samples AND that this results in decreased food consumption. We have been successful with the surgeries and have preliminary data supportive of our hypothesis. We expect to have more conclusive data by the next reporting period.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Zhao, A., Li, J., Peterson, M., Black, M., Gaulke, C. A., Jeffery, E. H. & Miller, M. J. (2025). Cooked broccoli alters cecal microbiota and impacts microbial metabolism of glucoraphanin in lean and obese mice. Mol Nutr Food Res (accepted)


Progress 05/15/23 to 05/14/24

Outputs
Target Audience:The primary target audience reached during the reporting period is the scientific community. We have submitted manusripts and presented posters at scientific meetings. The PI has had several presentations where he presented the ongoing work of the project - both in the US and abroad. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My lab technician was able to visit Dr. Duca's lab in Tucson to be trained on the surgery needed to address our overall project goals. The two grad students have also participated in several workshops during the recording period including: computational genomics course (1-week, on-campus), next generation sequencing library preparation workshop (1 day, on-campus), and Carver metabolomics core summer workshop (2-day, on-campus). 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 will continue our push to publish our first two manuscripts. A major goal is to become successful with our animal model so we can demonstrate the possiblity of cecal produced ITC to stimulate GLP-1 and demonstrate an outcome.

Impacts
What was accomplished under these goals? Aim 1 We have completed most of this aim and have two manuscripts in review. We anticipate both to be published by the next submitting period. We have demonstrated that ITCs, and not GSLs, can stimulate GLP-1 production from enteroendocrine cells via cell culture assays (paper 1). We have also fed mice cooked broccolifor one week and demonstrated changes in the gut microbiota composition and function (paper 2). Cooked broccoli consumption increases the production by the cecal microbiotaof total ITCs, sulforaphane and sulforaphane-cyteine conjugate when compared to animals fed control diet (no broccoli). There was no significant change in sulforaphane-nitrile production. Aim 2 No new data to report yet but I have had lab personnel trained by another USDA funded investigator (Dr. Duca at Univ. of Arizona) to perform rodent surgeries where we can cannulate the terminal ileum and mesenteric vein. With these animals, we will confirm that ITC production in the cecum, and not GSLs, can result in increased GLP-1 as measured in the mesenteric vein blood samples AND that this results in decreased food consumption. I expect new data on this front with the next reporting period.

Publications