The Kinetics, Substrate, and Inhibitor Specificity of the Monocarboxylate (Lactate) Transporter of Rat Liver Cells Determined Using the Fluorescent Intracellular pH Indicator, 2′,7′-Bis(carboxyethyl)-5(6)-carboxyfluorescein

Abstract

The kinetics of transport of L-lactate, pyruvate, ketone bodies, and other monocarboxylates into isolated hepatocytes from starved rats were measured at 25 degrees C using the intracellular pH-sensitive dye, 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein, to detect the associated proton influx. Transport kinetics were similar, but not identical, to those determined using the same technique for the monocarboxylate transporter (MCT) of Ehrlich Lettré tumor cells (MCT1) (Carpenter, L., and Halestrap, A. P. (1994) Biochem. J. 304, 751-760). Km values for L-lactate (4.7 mM), D-lactate (27 mM), D,L-2-hydroxybutyrate (3.3 mM), L-3-hydroxybutyrate (12.7 mM), and acetoacetate (6.1 mM) were very similar in both cell types, whereas in hepatocytes the Km values were higher than MCT1 for pyruvate (1.3 mM, cf. 0.72 mM), D-3-hydroxybutyrate (24.7 mM, cf. 10.1 mM), D-2-chloropropionate (1.3 mM, cf. 0.8 mM), 4-hydroxybutyrate (18.1 mM, cf. 7.7 mM), and acetate (5.4 mM, cf. 3.7 mM). In contrast, the hepatocyte carrier had lower Km values than MCT1 for glycolate, chloroacetate, dichloroacetate, and 2-hydroxy-2-methylpropionate. Differences in stereoselectivity were also detected; both carriers showed a lower Km for L-lactate than D-lactate, while hepatocyte MCT exhibited a lower Km for D- than L-2-chloropropionate and for L- than D-3-hydroxybutyrate; this is not the case for MCT1. A range of inhibitors of MCT1, including alpha-cyanocinnamate derivatives, phloretin, and niflumic acid, inhibited hepatocyte MCT with K0.5 values significantly higher than for tumor cell MCT1, while stilbene disulfonate derivatives and p-chloromercuribenzene sulfonate had similar K0.5 values in both cell types. The branched chain ketoacids alpha-ketoisocaproate and alpha-ketoisovalerate were also potent inhibitors of hepatocyte MCT with K0.5 values of 270 and 340 microM, respectively. The activation energy of L-lactate transport into hepatocytes was 58 kJ mol-1, and measured rates of transport at 37 degrees C were considerably greater than those required for maximal rates of gluconeogenesis. The properties of the hepatocyte monocarboxylate transporter are consistent with the presence of a distinct isoform of MCT in liver cells as suggested by the cloning and sequencing of MCT2 from hamster liver (Garcia, C. K., Brown, M. S., Pathak, R. K., and Goldstein, J. L. (1995) J. Biol. Chem. 270, 1843-1849).