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

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 BODIPYTM (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. Keywords: Intestinal stem cells, absorptive enterocyte monolayers, fatty acid oxidation, drug screening

A Proximal-to-Distal Survey of Healthy Adult Human Small Intestine and Colon Epithelium by Single-Cell Transcriptomics

“…AEs and ACCs perform nearly all intestinal absorption. 108 Three AE Leiden clusters and 2 ACC clusters were consistent with increasing maturity, reflecting other reports, 4 , 9 , 33 and 1 cluster largely from donor 3 ileum (AE2) separated from other AEs ( Figures 1 C and 13 B ). A DEG signature was defined by comparing DEGs from all AEs and all ACCs.…”

“… 28 , 29 , 30 , 31 We used partition-based graph abstraction (PAGA), which analyzes transcriptomic similarity between individual cells in different clusters, to define total connection strength (connectivity) between progenitor and differentiated populations and to infer temporal lineage trajectories. 32 As expected, absorptive enterocytes (AEs) and absorptive colonocyte (ACCs) are strongly and almost exclusively connected to ISCs and TA cells, 8 , 33 while PCs, GCs, and enteroendocrine cells (EECs) connect strongly to the secretory progenitor population ( Figure 4 G and H ). Consistent with murine findings, tuft cells connect weakly but exclusively to secretory progenitors in colon but not in SI.…”