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Transcriptomics informs design of a planar human enterocyte culture system that reveals metformin enhances fatty acid export

View ORCID ProfileIsmael Gomez-Martinez, View ORCID ProfileR. Jarrett Bliton, View ORCID ProfileKeith A. Breau, View ORCID ProfileMichael J. Czerwinski, View ORCID ProfileIan A. Williamson, View ORCID ProfileJia Wen, View ORCID ProfileJohn F. Rawls, Scott T. Magness
doi: https://doi.org/10.1101/2022.01.24.477515
Ismael Gomez-Martinez
2Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599 (USA)
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  • ORCID record for Ismael Gomez-Martinez
R. Jarrett Bliton
1Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill 911 Oval Dr., Raleigh, NC, 27695 (USA)
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Keith A. Breau
2Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599 (USA)
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Michael J. Czerwinski
2Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599 (USA)
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Ian A. Williamson
5Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, 27710
6Department of Biomedical Engineering, Woo Center for Big Data and Precision Health, Duke University Pratt School of Engineering, Durham, NC 27708
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Jia Wen
5Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, 27710
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John F. Rawls
5Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, 27710
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Scott T. Magness
1Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill 911 Oval Dr., Raleigh, NC, 27695 (USA)
2Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599 (USA)
5Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, 27710
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  • For correspondence: magness@med.unc.edu
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Abstract

Background & Aims Absorption, metabolism, and export of dietary lipids occurs in the small intestinal epithelium. Caco-2 and organoids have been used to study these processes but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we develop a high-throughput planar human absorptive enterocyte (AE) monolayer system for investigating lipid-handling, then evaluate the role of fatty acid oxidation (FAO) in fatty acid (FA) export, using etomoxir, C75, and anti-diabetic drug, metformin.

Methods Single-cell RNA-sequencing (scRNAseq), transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo AE maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo AE maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation (FAO) was pharmacologically modulated and BODIPY™ (B)-labelled FAs were used to evaluate FA-handling via fluorescence and thin layer chromatography (TLC).

Results scRNAseq shows increasing expression of lipid-handling genes as AEs mature. Culture conditions promote ISC differentiation into confluent AE monolayers. FA-handling gene expression mimics in vivo maturational states. FAO inhibitor, etomoxir, decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 FAs whereas CPT1 agonist, C75, and antidiabetic drug, metformin, increased apical-to-basolateral export. Short-chain B-C5 was unaffected by FAO inhibition and diffused through AEs.

Conclusions Primary human ISCs in culture undergo programmed maturation. AE monolayers demonstrate in vivo maturational states and lipid-handling gene expression profiles. AEs create strong epithelial barriers in 96-Transwell format. FA export is proportional to FAO. Metformin enhances FAO and increases basolateral FA export, supporting an intestine-specific role.

Competing Interest Statement

Scott Magness would like to disclose a financial interest in Altis Biosystems Inc., which licenses the ISC monolayer technology used in this study.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 26, 2022.
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Transcriptomics informs design of a planar human enterocyte culture system that reveals metformin enhances fatty acid export
Ismael Gomez-Martinez, R. Jarrett Bliton, Keith A. Breau, Michael J. Czerwinski, Ian A. Williamson, Jia Wen, John F. Rawls, Scott T. Magness
bioRxiv 2022.01.24.477515; doi: https://doi.org/10.1101/2022.01.24.477515
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Transcriptomics informs design of a planar human enterocyte culture system that reveals metformin enhances fatty acid export
Ismael Gomez-Martinez, R. Jarrett Bliton, Keith A. Breau, Michael J. Czerwinski, Ian A. Williamson, Jia Wen, John F. Rawls, Scott T. Magness
bioRxiv 2022.01.24.477515; doi: https://doi.org/10.1101/2022.01.24.477515

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