Location: Dairy and Functional Foods Research
Project Number: 8072-41000-108-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 31, 2021
End Date: Jun 30, 2024
Objective:
The project aims to understand how microbially derived metabolites from bioactive ingredients and functional foods impact the health of the intestinal epithelia with the goal of encouraging healthy food choices in the American people. This project will explore how the luminal fatty acid metabolites support homeostatic proliferation/differentiation in the intestine. This project will explore the role of bile derived acylcarnitines which we hypothesize may compensate for low butyrate and be protective against the development of inflammatory bowel disease (IBD). We will be utilizing both in vivo mouse models and in vitro enteroids derived from mice and humans to look for both overt phenotypic changes as well as underlying changes in biochemical signaling pathways.
Approach:
This agreement will systematically characterize the effect of carnitine conjugation on fatty acid oxidization in two dimensional (2D) enteroids based on apical vs. basolateral delivery of 1) acetate vs. acetylcarnitine; 2) butyrate vs. butyrylcarnitine; and 3) palmitate vs. palmitoylcarnitine by quantifying consumption rate and measuring transepithelial resistance (TER). In three dimensional (3D) enteroids, determine the basolateral delivery of these same compounds on colony formations, growth, and differentiation. Studies will also be performed examining the effect of diet (high fiber vs. fermentable fiber-free as well as fasting) on acylcarnitine and SCFA levels in the systemic circulation, bile, and throughout the length of the intestinal tract.
Intestinal enteroids are self-organizing three-dimensional structures that are derived from and maintained by stem cells that normally reside at the base of the intestinal crypts. In addition to stem cells, these structures contain the full complement of partially and fully differentiated cells types present in the intestinal epithelia. Typical cells lines do not have this same cellular diversity and are often immortalized thus making their similarity to the in vivo tissue questionable. Enteroids more closely model the tissue environment in vivo, which makes them ideal for studying host metabolic functions. These enteroids, or colonoids when derived from the small intestinal (i.e. duodenum, jejunum, ileum) or colonic tissue, can be maintained in vitro suspended in Matrigel bathed in specialized culture media to support continued stem cell proliferation. Utilizing established techniques we will harvest crypts from different regions of the mouse intestinal tract, expand enteroid cultures for use in experiments and freeze down stocks for later use. These enteroids can also be grown in two dimensional monolayers on plates or Transwells which can be used to assess epithelial barrier function.
3D enteroids from different sites along the gastrointestinal tract (GIT) will be treated with acetate, acetylcarnitine, butyrate, butyrylcarnitine, palmitate and palmitoylcarnitine and observed over time for changes in colony formation, enteroid growth and budding. 2D cultures treated with the same metabolites will be used to measure transepithelial resistance compared to control. Immunofluorescence can be used to probe proliferation and apoptotic status of cells in the enteroids. RNAseq will be utilized to investigate the transcriptional changes upon the addition of the different metabolites for the purposes of molecular phenotyping. We will probe protein expression using Western blot and use loss or gain of function studies to further investigate the involvement of signaling pathway suggested by the transcriptional data. We can delve into the bioenergetic mechanisms driving the observed phenotype by monitoring cellular oxidation rates using a Seahorse XF Analyzer after metabolite addition. Metabolic flux can also be measured 13C-label incorporation into central carbon metabolism over time.
