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

Background & Aims: Absorption, metabolism, and secretion of 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 dynamic and flexible planar absorptive enterocyte (AE) monolayer system, investigate how fatty acid oxidation (FAO) regulates FA mobilization mechanisms and probe a role for metformin in FA mobilization. Methods: Single-cell RNA-sequencing (scRNAseq) was performed on primary human jejunum. Transcriptomic signatures and trajectory analysis defined in vivo AE maturational signatures, which informed culture methods to differentiate ISCs and mimic in vivo AEs. The system was scaled for high-throughput drug screening and FAO was pharmacologically modulated. BODIPY (B)-labelled fatty acids (FAs) were used to easily and sensitively evaluate FA handling via fluorescence, and thin layer chromatography (TLC). Results: scRNAseq shows increasing expression of lipid-handling genes as AEs mature. In vitro, ISCs differentiate into AEs that mimic the in vivo maturational program, exhibit strong barrier function and express FA-handling genes. FA synthase inhibitor, C75, increased apical to basolateral mobilization of B-C16. Short-chain FA (B-C5) was unaffected and diffused freely through AEs. FAO inhibitor, etomoxir, decreased apical to basolateral mobilization of medium and long chain FAs (B-C12, B-C16) whereas anti-diabetic drug, metformin, augmented this mobilization. Conclusions: Primary human ISCs in culture undergo programmed maturation in vitro to generate a sensitive culture system of absorptive epithelium to investigate FA-handling. Modulating FAO impacts mobilization of FAs across absorptive epithelium and FAO enhanced by metformin increases basolateral mobilization pointing to an intestine-specific role. Overall design: One entire human intestine, hashtagged and called for the six regions. Only Jej and Ile are analyzed in this dataset Also see jejunum and ileum data from GSE185224.