Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
Hepatology. 2013 Feb;57(2):740-52. doi: 10.1002/hep.26012. Epub 2013 Jan 8.
Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in Förster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na(+)-taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter αβ (OSTαβ) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis.
A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time.
胆汁酸是吸收膳食脂质和维生素的关键，也是代谢中重要的信号分子。在这里，我们描述了一种基因编码的荧光胆汁酸传感器（BAS），它可以实时监测单个活细胞中胆汁酸的转运。在多种细胞类型中，多种生理和病理生理胆汁酸的浓度变化都表现为荧光共振能量转移（FRET）的显著变化。传感器的特定亚细胞靶向显示胆汁酸迅速流入细胞质和细胞核，但在过氧化物酶体中没有观察到 FRET 变化。此外，肝脏脂肪酸结合蛋白的表达减少了核内胆汁酸的可用性。该传感器允许对通过 Na(+)-牛磺胆酸钠共转运蛋白（NTCP）介导的结合胆汁酸的摄取和积累进行单细胞可视化。此外，通过基于斑马鱼法尼醇 X 受体的传感器可视化了假定的斑马鱼顶端钠胆汁酸转运蛋白同源物介导的 cyprinol 硫酸盐摄取。传感器的可逆性质还允许在活细胞中测量胆汁酸的外排，并且有机溶质转运蛋白αβ（OSTαβ）的表达导致共轭鹅脱氧胆酸的内流和外排。最后，胆汁酸摄取的联合可视化和几种 NTCP 变体的荧光标记表明，该传感器还可用于研究影响胆汁酸动态平衡的基因中患者突变对功能的影响。
开发了一种基因编码的荧光 BAS，它可以实时在单个活细胞内对胆汁酸动态平衡进行细胞内成像。
