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Edited by Mike Shipston Monocarboxylate transporter 4 (MCT4) is an H ؉-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mM, the reported K m of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective K m values for lactate uptake were 1.7, 1.2, and 0.7 mM in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a K m for pyruvate of 4.2 mM, as opposed to >150 mM reported previously. Parallel assays with the pH-sensitive dye 2,7-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous K m estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in highlactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate. Cancer cells ferment glucose to lactate in the presence of oxygen, a phenomenon originally described by Otto Warburg and colleagues in the 1920s and later found to promote tumor growth and malignancy (1-4). In addition to fostering glycolysis by end product removal, cytosolic alkalinization, and NADH recycling, the co-extrusion of lactate and protons causes inter

Abstract-D-Glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine Key Words: humans Ⅲ endothelium Ⅲ glucose Ⅲ arginine Ⅲ nitric oxide T he cationic amino acid L-arginine is the substrate for nitric oxide (NO) synthesis via endothelial NO synthase (eNOS) 1 and is taken up primarily by the Na ϩ -independent high-affinity (K m Ϸ100 to 400 mol/L) systems y ϩ /CAT-1 and y ϩ /CAT-2B (where CAT indicates cationic amino acid transporter) in human umbilical vein endothelial cells (HUVECs). 2,3 L-Arginine transport and NO synthesis (Larginine/NO pathway) are increased in HUVECs from patients with gestational diabetes. 2 Interestingly, long-term incubation (24 hours) of HUVECs from normal pregnancies with elevated D-glucose mimics the effect of gestational diabetes on the L-arginine/NO pathway. 4 In addition, elevated D-glucose for 24 hours 4,5 or 5 days 6 increases eNOS gene expression. A recent report shows that D-glucose infusion induces vasodilatation in humans, 7 and in animal models, an elevation of plasma D-glucose results in rapid (seconds to minutes) vasodilatation. 8 -10 Therefore, rapid fluctuations in the D-glucose level are crucial in maintaining human fetal endothelial function. [2][3][4][5]11 D-Glucose activates protein kinase C (PKC), an enzyme involved with long-term stimulation of the L-arginine/NO pathway, 5,12-14 and (within 1 hour) p42 and p44 mitogen-activated protein (MAP) kinases (p42/44 mapk ). 5,14,15 p42/44 mapk activation may itself be dependent on PKC activation and NO synthesis. 5,14 However, the effect of short-term incubation with elevated D-glucose on the endothelial L-arginine/NO pathway has not been investigated. 4,11,16,17 The present study shows that a 2-minute incubation with 25 mmol/L D-glucose increases L-arginine transport and NO synthesis in HUVECs. The underlying cellular mechanisms involve phosphorylation of eNOS at Ser 1177 via phosphatidylinositol 3-kinase (PI3-k) and activation of eNOS and p42/ p44 mapk by D-glucose. 18 Materials and Methods Cell CultureHuman umbilical vein endothelium was isolated (collagenase digestion 0.25 mg/mL) and cultured (37°C, 5% CO 2 , confluent passage 2) in medium 199 containing 5 mmol/L D-glucose, 10% newborn calf serum, 10% fetal calf serum, 3.2 mmol/L L-glutamine, 100 mol/L L-arginine, and 100 U/mL penicillin-streptomycin (primary culture medium). [2][3][4] Before an experiment (24 hours), the incubation medium was changed to serum-free medium 199.

This internet appendix provides proofs of the two propositions in the paper, a description of the estimation and inference procedure employed, as well as supporting material for the empirical analysis of the wage e¤ects of Job Corps and the simulation study.