Source: UNIV OF CONNECTICUT submitted to NRP
HOST-MICROBIOTA FACTORS REGULATING ZINC BIOAVAILABILITY ATTENUATED BY PHYTATE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027827
Grant No.
2022-67018-36234
Cumulative Award Amt.
$300,000.00
Proposal No.
2021-09056
Multistate No.
(N/A)
Project Start Date
May 1, 2022
Project End Date
Apr 30, 2025
Grant Year
2022
Program Code
[A1343]- Food and Human Health
Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
Nutritional Sciences
Non Technical Summary
Plant-based food products are growing popular in the U.S., as shown in a sales growth of 31.3% from 2017 to 2019. With consumption of red meat stated by the World Health Organization's International Agency for Research on Cancer as 'probably carcinogenic,' more and more populations reveal a preference for healthier diet options offered by plant-based meals. While not all plant-based foods contain high phytate contents, the soy-based texture vegetable protein--an example of the most common meat substitute--includes a ratio of phytate to zinc of 25-42.5. This ratio range far exceeds the threshold of predicted ratios--11.1 and 15.1 for men and women, respectively--that prevent people from attaining the amount of zinc satisfying the Estimated Average Requirement. Given that marginal zinc deficiency may hinder proper growth and increase risks for various diseases, in order to develop strategies that overcome zinc inadequacy resulting from phytate intake, it is crucial to build a thorough understanding of mechanisms underlying the phytate-induced reduction of zinc bioavailability.The overarching goal of this project is to understand these mechanisms by determining the effects of the genes of human and small intestine microbiota in zinc bioavailability attenuated by phytate. We recently identified a new putative zinc transporter gene that is already known to regulate myo-inositol, a degradation product of phytate. Based on this preliminary data and existing knowledge in the role of microbiota in phytate degradation, we hypothesize that the extent of phytate degradation is regulated by small intestine microbiota and that the products of degradation, including myo-inositol, impact zinc bioavailability via their interactions with the new putative zinc transporter gene. To test these hypotheses, we will adopt gene manipulation approaches in intestinal cell lines and mice and compare zinc uptake rates in these transgenic cells and animals versus controls. Also, we will analyze microbes in mice's small intestine and investigate essential microbes and their enzymes controlling phytate degradation and zinc absorption. Altogether, this project will produce critical information on the genetic factors deciding zinc bioavailability and promise further extensive research on identifying the roles of genetic variants in host and commensal microbes.
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
7023840101050%
7027010103050%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals; 7010 - Biological Cell Systems;

Field Of Science
1030 - Cellular biology; 1010 - Nutrition and metabolism;
Goals / Objectives
Our long-term goal is to identify the genetic factors in humans and microbiota that regulate micronutrient metabolisms in order to build foundational knowledge on practicing precision nutrition. In this project, we aim to determine the genetic factors of host and microbiota that regulate zinc bioavailability attenuated by phytate. Our main hypotheses are that: (1) the extent of phytate hydrolysis is regulated by small intestine microbiota and (2) the products of hydrolysis--myo-inositol (MI) and inositol phosphates (IPs)--impact zinc bioavailability via their interactions with an intestinal transporter. We will test the hypotheses with three objectives: 1) determine the role of phytate-hydrolyzed products in zinc absorption and its dependence on SMIT1; 2) determine whether MI co-treatment induces the bioavailability of zinc complexed with phytate; 3) determine the impact of phytase genes in small intestine microbiota on phytate hydrolysis and zinc bioavailability.
Project Methods
Objective 1. We will examine whether the phytate-hydrolyzed products, MI and IP1-3, facilitate zinc uptake in mouse small intestine and whether this effect depends on SMIT1 using gene knockout (KO) and knockdown (KD) approaches. Mice will be fed with diet supplemented with MI or IP1-3 and zinc, zinc concentration in blood and small intestine tissues will be measured using ICP-OES. To determine the essential role of SMIT1 in zinc uptake in the presence of MI/IPs, we will create SMIT1 KO Caco-2 enterocytes using CRISPR and test zinc uptake rate in the presence of IPs. For in vivo study, we will adopt lentivirus containing shRNAs against SMIT1, and zinc absorption rate will be compared by ICP-OES in SMIT1 KD and control shRNA-transduced mice upon zinc and IP treatment.Objective 2: We will examine intracellular zinc concentrations directly with FluoZin-3 zinc-specific fluorescent probe using live-cell imaging microscope. To test the hypothesis of the involvement of SMIT1 activity, we will utilize both SMIT1 KO and SMIT1 activity induction approaches. In the SMIT1 KO Caco-2 cells that will be created in Objective 1, we anticipate that MI does not augment the zinc bioavailability. We will clone the human SGK1 gene into pEGFP-N1 plasmid and transfect Caco-2 cells with this pEGFP-SGK1 plasmid. We anticipated that, in the cells with amplified SMIT1 activity, phytate-zinc complex treatment in culture media will increase zinc uptake compared to the cells without SMIT1 activation.Objective 3: We will test whether the microbiota facilitates phytate hydrolysis and zinc absorption in the small intestine and whether the microbial composition is altered by extended intakes of the diet with high phytate contents. Then, we will identify a dominant group of phytase genes and detailed information on phytate metabolites that are produced by specific phytase enzymes using the bacterial phytase cloning method

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:The primary audience for this research includes individuals who follow plant-based diets or consume diets high in phytates, nutritionists, dietitians, healthcare providers, and researchers in the fields of nutrition, microbiology, and genetics. This research aims to elucidate the genetic factors in humans and gut microbiota that influence zinc bioavailability, which is crucial for preventing chronic diseases such as immune deficiencies and cardiovascular diseases. Individuals on Plant-Based Diets. People who consume plant-based diets are at a higher risk of zinc deficiency due to the high phytate content in plant foods. This research aims to provide insights into how these individuals can optimize their zinc intake and absorption, potentially through dietary modifications or supplementation strategies. Nutritionists and Dietitians. Healthcare professionals involved in dietary planning and nutritional counseling will benefit from understanding the genetic and microbial factors that influence zinc bioavailability. This knowledge will enable them to provide more personalized and effective dietary recommendations to their clients. Healthcare Providers. Doctors and other healthcare providers need to be aware of the potential for zinc deficiency in patients who follow plant-based diets. This research will equip them with the information necessary to diagnose and manage zinc deficiency more effectively. Researchers in Nutrition, Microbiology, and Genetics. This research contributes to the broader scientific understanding of the interactions between diet, gut microbiota, and host genetics. It will be of particular interest to researchers focusing on micronutrient metabolism, gut microbiota, and precision nutrition. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this period, two graduate students presented their research at multiple on-campus and regional conferences. The recent in vivo experiment was presented at the international conference of the American Society of Nutrition, NUTRITION 2024, in Chicago, IL. The PI presented the research about SMIT1's role in zinc uptake at the FASEB Trace Element in Biology and Medicine in Melbourne, FL. How have the results been disseminated to communities of interest?While presenting the research outcomes at regional and international conferences, we prepare manuscripts for publication in scientific journals, including the Journal of Nutritional Biochemistry. We recognize that growing numbers of young college students are interested in plant-based diets as their dietary choice. The PI continues to attend students' research events and promote the importance of mineral nutrition in plant-based diets. Our approach to examining the impacts of host and microbiota genes has received a lot of attention from students, which has led to an increasing number of students who want to learn research in the lab as volunteers. What do you plan to do during the next reporting period to accomplish the goals?We will examine IPs' impact on zinc bioavailability more since one type of IPs were in back-ordered over 10 months and now we received it. Phytase gene experiments will also be conducted to understand microbiota's impact.

Impacts
What was accomplished under these goals? During this period, we investigated MI's impact on zinc bioavailability more deeply. We found that MI decreases cellular zinc accumulation in intestinal cells by regulating gene transcription rather than directly impacting zinc transporters' activities. This data suggests that MI plays a role as a cell signaling molecule to regulate zinc accumulation, which requires further research examining the transcriptomicprofiles upon MI treatment. In this period, we also focused on optimizing in vivo zinc deficiency and supplementation experiments. Given that zinc intakes vary in the US population, examining the phytate metabolites' impact in varying dietary zinc intake conditions is critical. Using zinc-deficient diets with varying levels of zinc supplements, we have determined how these diets affect zinc levels in serum and other tissues.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Choi S, Wang Y, Choi S. Intestinal Zinc Homeostasis Upon Dietary Zinc Variations in Mice. June 30, 2024. NUTRITION 2024, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Terrell K, Choi S. PTFS12-06-23 Investigation of Cellular Zinc Influx in In Vitro Senescent Enterocyte Models. Current Developments in Nutrition. 2023; 7. Available from: https://doi.org/10.1016/j.cdnut.2023.101866 DOI: 10.1016/j.cdnut.2023.101866
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Choi S. UNVEILING THE ROLE OF SMIT1 IN ZINC ACCUMULATION. June 12, 2024. FASEB Trace Element in Biology and Medicine. Melbourne, FL.


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:The current products can help nutrition and food science researchers investigatingmineral absorption mechanisms and the impactof phytic acid in diets and clinical nutrition scientists seeking an accurate strategy to conductzinc supplementation research. Changes/Problems:We planned to test the effects of various myo-inositol phophosphate forms (IPs) on zinc absorption in a mouse model, but we found that the cost of purified IPs was too expensive to use in animal experiments. We are using only myo-inositol, which is the final form of hydrolysis of phytic acid, and still looking for alternative forms such as products of phytase-mediated hydrolysis. What opportunities for training and professional development has the project provided?Two PhD students were partially funded by this project and presented their worksin NUTRITION 2023, whichis an annual conference of the American Society of Nutrition. How have the results been disseminated to communities of interest?This project is still in an early stage, results are not available for publicly dissemination. However, project progress was presented at the annual American Society of Nutrition conference. What do you plan to do during the next reporting period to accomplish the goals?We will further analyze the 16s rRNA sequencing data and investigate genetics in certain microbes. We also want to study the genes' effects on MI's actions on zinc uptake.

Impacts
What was accomplished under these goals? During this reporting period, we found that MI and phytic acid in diets significantly alter microbiome composition in zinc-fed mice using 16s rRNA-seq analysis. Also, we found that MI indeed regulates zinc uptake in intestinal cells.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Microbiome Analysis From Small Intestines of Mice Fed With Zinc Supplement and Phytic Acid. Suyun Choi. NUTRITION 2023 Poster Presentation