Uptake and Efflux of Carnitine, Acylcarnitines, Valine, or Known Substrates

Transport experiments were performed with primary human hepatocytes (Thermo Fisher Scientific, Darmstadt, Germany) or HEK293 cells stably transfected to overexpress hOCT1 or mOCT1. As a control, cells transfected with the empty vector pcDNA5 were used. The generation and validation of the cell lines was described previously (Saadatmand et al., 2012; Meyer et al., 2020). Uptake or efflux of carnitine, acylcarnitines, or valine were performed with radiolabeled or deuterated substrates and quantified by scintillation counting or LC-MS/MS. For the latter, specific mass transitions and voltages were used (Supplementary Table S1). The cell number of each experiment was normalized by total protein measurement in representative wells by using the bicinchoninic acid assay (Smith et al., 1985). Detailed descriptions of the methods are provided in the supplementary files.