Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North Carolina.
Cell Mol Gastroenterol Hepatol. 2022;14(2):409-434. doi: 10.1016/j.jcmgh.2022.04.009. Epub 2022 Apr 28.
BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.
Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.
Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.
Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.
吸收性肠细胞的脂肪酸氧化与 2 型糖尿病、肥胖症和血脂异常的病理生理学有关。Caco-2 和类器官已被用于研究包括脂肪酸氧化在内的膳食脂质处理过程，但在生理相关性方面存在局限性或排除了顶端和基底的同时进入。在这里，我们开发了一种高通量的平面人吸收性肠细胞单层系统来研究脂质处理，然后使用 etomoxir、C75 和抗糖尿病药物二甲双胍评估脂肪酸氧化在脂肪酸输出中的作用。
对原代人空肠进行单细胞 RNA 测序、转录组学和谱系轨迹分析。体内吸收性肠细胞成熟状态的信息用于分化模拟体内吸收性肠细胞成熟的人肠干细胞 (ISC)。该系统经过放大可用于高通量药物筛选。通过药理学调节脂肪酸氧化，并使用 BODIPY（Thermo Fisher Scientific，Waltham，MA）（B）标记的脂肪酸通过荧光和薄层层析来评估脂肪酸处理。
单细胞 RNA 测序显示，随着吸收性肠细胞的成熟，脂质处理基因的表达逐渐增加。培养条件促进 ISC 分化为融合的吸收性肠细胞单层。脂肪酸处理基因的表达模拟了体内成熟状态。脂肪酸氧化抑制剂 etomoxir 减少了中链 B-C12 和长链 B-C16 脂肪酸的顶端到基底外侧的输出，而 CPT1 激动剂 C75 和抗糖尿病药物二甲双胍增加了顶端到基底外侧的输出。短链 B-C5 不受脂肪酸氧化抑制的影响，并通过吸收性肠细胞扩散。
原代人 ISC 在培养中经历程序化成熟。吸收性肠细胞单层显示出体内成熟状态和脂质处理基因表达谱。吸收性肠细胞在 96 孔 Transwell 格式中形成强大的上皮屏障。脂肪酸输出与脂肪酸氧化成正比。二甲双胍增强脂肪酸氧化并增加基底外侧脂肪酸输出，支持肠道特异性作用。
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