Saturable leucine, MeAIB and metformin transport
COLD
Saturable leucine uptake was measured in hepatocytes and Caco-2 cells, using a tracer-quench assay, with two solutions, following an earlier method [Caco-2 cells were maintained according to Natoli et al., To model intestinal absorption of amino acids, Caco-2 cells were seeded on permeable Transwell membrane supports (Corning) to allow cell polarization. Monolayer integrity was routinely investigated using visual analysis under a light microscope and deemed sufficient if TEER values remained above 500 mV. For experiments, media was replaced on both sides of the membrane. Drug and amino acid free EBSS was added to the basolateral pool and EBSS with/without drug added to the apical pool for 2 h. Next, the apical media was carefully removed and replaced with tracer or quench leucine +/− drug in EBSS plus cold leucine. Basolateral efflux (appearance in the media) was measured by scintillation counting.