Figure 4
From: Acetylation turns leucine into a drug by membrane transporter switching
Both enantiomers of N-acetyl-leucine are transported by the monocarboxylate transporter (MCT1) but only the l-enantiomer is metabolized. Concentration–response curves for the uptake of (a) N-acetyl-l-leucine and (b) N-acetyl-d-leucine. Concentration-inhibition curves for the inhibition of uptake of the known substrate (c, d) 2-thiophene glyoxylate (500 µM). DMSO (0.5%) was the solvent control (SC) and the known inhibitor phloretin (500 µM) was the positive control (PC). (e) Chemical structure of deuterated N-acetyl-d,l-leucine incubated with cellular fraction S9 from liver to determine metabolism using liquid chromatography and mass spectrometry. (f, g) Time courses for loss of deuterated N-acetyl-d,l-leucine (1 µM initial concentration) and appearance of deuterated L-leucine for extracts derived from human and mouse livers. Data are colour-coded according to the chemical structures and names shown in e with deuterated N-acetyl-d,l-leucine in blue and deuterated l-leucine in red. (h) Concentration versus initial velocities relationship for metabolism yielded a Km values of 216 and 69 µM and Vmax values of 6.8 and 2.6 µmol/min/mg protein for human and mouse, respectively. Data were fit to the Michalis–Menten equation for transporter uptake and metabolism or the Hill equation for transporter inhibition using the solvent control to define the top and the positive control inhibitor to define the bottom. Symbols represent the mean ± SEM, n = 3. When the error bars are smaller than the symbol, they are not visible.
