scholarly journals Basigin drives intracellular accumulation of l-lactate by harvesting protons and substrate anions

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0249110
Author(s):  
Anna-Lena Köpnick ◽  
Annika Jansen ◽  
Katharina Geistlinger ◽  
Nathan Hugo Epalle ◽  
Eric Beitz
Keyword(s):  
Disulfide Bridge ◽  
Monocarboxylate Transporter ◽  
Intracellular Accumulation ◽  
Biophysical Properties ◽  
Entry Site ◽  
Cytosolic Ph ◽  
Domain Identification ◽  
Surface Patches ◽  
Transporter Family ◽  
Lactate Uptake

Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant.

scholarly journals Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

2000 ◽  
Vol 279 (4) ◽  
pp. G775-G780 ◽  
Author(s):  
Christos Hadjiagapiou ◽  
Larry Schmidt ◽  
Pradeep K. Dudeja ◽  
Thomas J. Layden ◽  
Krishnamurthy Ramaswamy
Keyword(s):  
Short Chain Fatty Acid ◽  
Rnase Protection Assay ◽  
Significant Inhibition ◽  
Monocarboxylate Transporter ◽  
Rnase Protection ◽  
Monocarboxylate Transporter 1 ◽  
Antisense Orientation ◽  
Transporter Family ◽  
Saturation Kinetics

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na+independent. Butyrate uptake was unaffected by DIDS; however, α-cyano-4-hydroxycinnamate and the butyrate analogs propionate and l-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

scholarly journals 669 Lactate uptake through MCT11, a novel monocarboxylate transporter, enforces dysfunction in terminally exhausted T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A697-A697
Author(s):  
Ronal Peralta ◽  
Greg Delgoffe
Keyword(s):  
T Cells ◽  
Lactic Acid ◽  
Tumor Burden ◽  
B16 Melanoma ◽  
T Cell Subsets ◽  
Monocarboxylate Transporter ◽  
Acid Uptake ◽  
Ribonucleoprotein Complexes ◽  
Lactate Uptake ◽  
Marker Expression

BackgroundUpon infiltration into tumors, T cells experiencing persistent antigen stimulation progressively differentiate into a state of dysfunction, known as exhaustion. Exhausted T cells are characterized by the sustained upregulation of co-inhibitory molecules and reduced effector cytokine production. Additionally, exhausted T cells exist in a state of metabolic dysfunction in the tumor microenvironment (TME), due to disrupted mitochondrial biogenesis, hypoxia and lack of metabolites. Highly glycolytic tumor and stromal cells outcompete T cells for glucose, and secrete lactate into the TME, acidifying the extracellular space. Recent studies have shown lactate can be metabolized by tumor infiltrating Tregs and macrophages. We hypothesized that CD8+ tumor-infiltrating lymphocytes (TIL) may also take up lactate as an alternative carbon source to meet their metabolic demands.MethodsFor lactate uptake experiments, B16 melanoma single cell suspensions from B6 mice were loaded with the pH sensitive dye pHrodo, then pulsed with 5µM lactic acid. MCT11 KO OT-I T cells were generated via transfection of Slc16a11 sgRNA-Cas9 ribonucleoprotein complexes, and adoptively transferred into B16-OVA bearing mice.ResultsRNA sequencing and flow cytometry data from CD8+ T cell subsets in the TME revealed MCT11 (encoded by Slc16a11), a monocarboxylate transporter (MCT) only recently discovered, to be highly and uniquely expressed in terminally exhausted T cells (Tex). As lactate is an abundant monocarboxylate in tumors, we asked whether MCT11 supports lactate uptake into Tex cells. Antibody blockade of MCT11 resulted in reduced lactic acid uptake, but whether lactic acid promoted or inhibited effector function. Intriguingly, overexpression of MCT11 in OT-I T cells adoptively transferred into B16-OVA bearing mice resulted in accelerated exhaustion: increased co-inhibitory marker expression and decreased TNFa and IFN production. Conversely, knockdown of MCT11 in the same model resulted in decreased co-inhibitory marker expression and increased TNFa and IFN production. Further, MCT11 KO OT-I T cells used therapeutically had decreased tumor burden over mice treated with control OT-I T cells. As MCT11's uptake function was blocked with an antibody, we also used the antibody therapeutically, revealing that single-agent MCT11 antibody treatment reduced tumor burden and increased survival in B16 melanoma bearing mice.ConclusionsOur data support a model where exhausted CD8+ T cells upregulate MCT11, which renders them sensitive to toxic lactic acid in the TME. Our data suggest MCT11 could be deleted on therapeutic T cells or blocked using an antibody on endogenous T cells to render exhausted T cells impervious to lactic acid such and promote tumor eradication.

Impaired sarcolemmal vesicle lactate uptake and skeletal muscle MCT1 and MCT4 expression in obese Zucker rats

2001 ◽  
Vol 281 (6) ◽  
pp. E1308-E1315 ◽  
Author(s):  
Guillaume Py ◽  
Karen Lambert ◽  
Antonia Perez-Martin ◽  
Eric Raynaud ◽  
Christian Préfaut ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Tibialis Anterior ◽  
Skeletal Muscles ◽  
Monocarboxylate Transporter ◽  
Zucker Rats ◽  
Hindlimb Muscle ◽  
Obese Zucker Rats ◽  
Lactate Uptake ◽  
Sarcolemmal Vesicles ◽  
Sarcolemmal Vesicle

The present experiments were undertaken to characterize 1) the hindlimb muscle mass lactate uptake and 2) the expression of monocarboxylate transporter isoforms MCT1 and MCT4, as well as lactate dehydrogenase (LDH) isozyme distribution, in various skeletal muscles of Zucker fa/fa rats taken as a model of insulin resistance-related obesity. Initial lactate uptake at six different concentrations was measured in sarcolemmal vesicles (SV) by use ofl-[U-14C]lactate. Compared with controls, the maximal rate of lactate uptake and affinity were decreased in SV of Zucker rats (∼30%) in which MCT4 content was significantly decreased ( P < 0.05). MCT4 expression was decreased in soleus, extensor digitorum longus, and red tibialis anterior (RTA; P < 0.05), but not in white tibialis anterior, whereas MCT1 expression was decreased only in RTA of Zucker rats ( P < 0.05). Obesity led to a shift toward type M-LDH isozyme in mixed muscles. We conclude that obesity leads to changes in muscular MCT1 and MCT4 expression, which, when associated with LDH isozyme redistribution, may contribute to the hyperlactatemia noted in insulin resistance.

Significance of Short Chain Fatty Acid Transport by Members of the Monocarboxylate Transporter Family (MCT)

2012 ◽  
Vol 37 (11) ◽  
pp. 2562-2568 ◽  
Author(s):  
Ivano Moschen ◽  
Angelika Bröer ◽  
Sandra Galić ◽  
Florian Lang ◽  
Stefan Bröer
Keyword(s):  
Fatty Acid ◽  
Short Chain Fatty Acid ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Monocarboxylate Transporter ◽  
Fatty Acid Transport ◽  
Acid Transport ◽  
Transporter Family

An endogenous monocarboxylate transport inXenopus laevisoocytes

2000 ◽  
Vol 278 (5) ◽  
pp. R1190-R1195 ◽  
Author(s):  
M. Tosco ◽  
M. N. Orsenigo ◽  
G. Gastaldi ◽  
A. Faelli
Keyword(s):  
Xenopus Laevis ◽  
Monocarboxylate Transporter ◽  
Monocarboxylate Transporters ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
Ph Gradients ◽  
Monocarboxylate Transport ◽  
Functional Features ◽  
Lactate Uptake ◽  
Uptake Studies

We investigated the existence of an endogenous system for lactate transport in Xenopus laevis oocytes.36Cl-uptake studies excluded the involvement of a DIDS-sensitive anion antiporter as a possible pathway for lactate movement.l-[14C]lactate uptake was unaffected by superimposed pH gradients, stimulated by the presence of Na+in the incubating solution, and severely reduced by the monocarboxylate transporter inhibitor p-chloromercuribenzenesulphonate (pCMBS). Transport exhibited a broad cation specificity and was cis inhibited by other monocarboxylates, mostly by pyruvate. These results suggest that lactate uptake is mediated mainly by a transporter and that the preferred anion is pyruvate. [14C]pyruvate uptake exhibited the same pattern of functional properties evidenced for l-lactate. Kinetic parameters were calculated for both monocarboxylates, and a higher affinity for pyruvate was revealed. Various inhibitors of monocarboxylate transporters reduced significantly pyruvate uptake. These studies demonstrate that Xenopus laevis oocytes possess a monocarboxylate transport system that shares some functional features with the members of the mammalian monocarboxylate cotransporters family, but, in the meanwhile, exhibits some particular properties, mainly concerning cation specificity.

scholarly journals The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells

2000 ◽  
Vol 350 (1) ◽  
pp. 219-227 ◽  
Author(s):  
Kai-Stefan DIMMER ◽  
Björn FRIEDRICH ◽  
Florian LANG ◽  
Joachim W. DEITMER ◽  
Stefan BRÖER
Keyword(s):  
Mammalian Cells ◽  
Short Chain Fatty Acids ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Cytosolic Ph ◽  
Monocarboxylate Transport ◽  
Thiol Reagent ◽  
Km Value ◽  
Flux Measurements ◽  
Disulphonic Acid

Transport of lactate and other monocarboxylates in mammalian cells is mediated by a family of transporters, designated monocarboxylate transporters (MCTs). The MCT4 member of this family has recently been identified as the major isoform of white muscle cells, mediating lactate efflux out of glycolytically active myocytes [Wilson, Jackson, Heddle, Price, Pilegaard, Juel, Bonen, Montgomery, Hutter and Halestrap (1998) J. Biol. Chem. 273, 15920–15926]. To analyse the functional properties of this transporter, rat MCT4 was expressed in Xenopus laevis oocytes and transport activity was monitored by flux measurements with radioactive tracers and by changes of the cytosolic pH using pH-sensitive microelectrodes. Similar to other members of this family, monocarboxylate transport via MCT4 is accompanied by the transport of H+ across the plasma membrane. Uptake of lactate strongly increased with decreasing extracellular pH, which resulted from a concomitant drop in the Km value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for l-lactate Km values of 17±3mM (pH-electrode) and 34±5mM (flux measurements with l-[U-14C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. Km values of other monocarboxylates were either similar to the Km value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (β-hydroxybutyrate and short-chain fatty acids). Under physiological conditions, rat MCT will therefore preferentially transport lactate. Monocarboxylate transport via MCT4 could be competitively inhibited by α-cyano-4-hydroxycinnamate, phloretin and partly by 4,4´-di-isothiocyanostilbene-2,2´-disulphonic acid. Similar to MCT1, monocarboxylate transport via MCT4 was sensitive to inhibition by the thiol reagent p-chloromercuribenzoesulphonic acid.

scholarly journals Transport of lactate and pyruvate in the intraerythrocytic malaria parasite, Plasmodium falciparum

2001 ◽  
Vol 355 (3) ◽  
pp. 733-739 ◽  
Author(s):  
Jemma L. ELLIOTT ◽  
Kevin J. SALIBA ◽  
Kiaran KIRK
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Direct Evidence ◽  
Monocarboxylate Transporter ◽  
Human Malaria Parasite ◽  
Transporter Family ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Lactate And Pyruvate ◽  
Higher Eukaryotes

The mature, intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, is reliant on glycolysis for its energetic requirements. It produces large quantities of lactic acid, which have to be removed from the parasite's cytosol to maintain the cell's integrity and metabolic viability. Here we show that the monocarboxylates lactate and pyruvate are both transported across the parasite's plasma membrane via a H+/monocarboxylate symport process that is saturable and inhibited by the bioflavonoid phloretin. The results provide direct evidence for the presence at the parasite surface of a H+-coupled monocarboxylate transporter with features in common with members of the MCT (monocarboxylate transporter) family of higher eukaryotes.

Muscle contraction increases lactate transport while reducing sarcolemmal MCT4, but not MCT1

2002 ◽  
Vol 282 (5) ◽  
pp. E1062-E1069 ◽  
Author(s):  
Mio Tonouchi ◽  
Hideo Hatta ◽  
Arend Bonen
Keyword(s):  
Plasma Membrane ◽  
Muscle Contraction ◽  
Soleus Muscle ◽  
Muscle Glycogen ◽  
Lactate Concentration ◽  
Monocarboxylate Transporter ◽  
Intrinsic Activity ◽  
Lactate Transport ◽  
Lactate Uptake ◽  
Sarcolemmal Vesicles

Rates of lactate uptake into giant sarcolemmal vesicles were determined in vesicles collected from rat muscles at rest and immediately after 10 min of intense muscle contraction. This contraction period reduced muscle glycogen rapidly by 37–82% in all muscles examined ( P < 0.05) except the soleus muscle (no change P > 0.05). At an external lactate concentration of 1 mM lactate, uptake into giant sarcolemmal vesicles was not altered ( P > 0.05), whereas at an external lactate concentration of 20 mM, the rate of lactate uptake was increased by 64% ( P < 0.05). Concomitantly, the plasma membrane content of monocarboxylate transporter (MCT)1 was reduced slightly (−10%, P < 0.05), and the plasma membrane content of MCT4 was reduced further (−25%, P < 0.05). In additional studies, the 10-min contraction period increased the plasma membrane GLUT4 ( P < 0.05) while again reducing MCT4 (−20%, P < 0.05) but not MCT1 ( P > 0.05). These studies have shown that intense muscle contraction can increase the initial rates of lactate uptake, but only when the external lactate concentrations are high (20 mM). We speculate that muscle contraction increases the intrinsic activity of the plasma membrane MCTs, because the increase in lactate uptake occurred while plasma membrane MCT4 was decreased and plasma membrane MCT1 was reduced only minimally, or not at all.

scholarly journals Localization of members of MCT monocarboxylate transporter family Slc16 in the kidney and regulation during metabolic acidosis

2010 ◽  
Vol 299 (1) ◽  
pp. F141-F154 ◽  
Author(s):  
Helen M. Becker ◽  
Nilufar Mohebbi ◽  
Angelica Perna ◽  
Vadivel Ganapathy ◽  
Giovambattista Capasso ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Mrna Level ◽  
Distal Tubule ◽  
Transient Increase ◽  
Monocarboxylate Transporter ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Transporter Family ◽  
And Function ◽  
Lactate Excretion

The monocarboxylate transporter family (MCT) comprises 14 members with distinct transport properties and tissue distribution. The kidney expresses several members of the MCT family, but only little is known about their exact distribution and function. Here, we investigated selected members of the MCT family in the mouse kidney. MCT1, MCT2, MCT7, and MCT8 localized to basolateral membranes of the epithelial cells lining the nephron. MCT1 and MCT8 were detected in proximal tubule cells whereas MCT7 and MCT2 were located in the thick ascending limb and the distal tubule. CD147, a β-subunit of MCT1 and MCT4, showed partially overlapping expression with MCT1 and MCT2. However, CD147 was also found in intercalated cells. We also detected SMCT1 and SMCT2, two Na+-dependent monocarboxylate cotransporters, on the luminal membrane of type A intercalated cells. Moreover, mice were given an acid load for 2 and 7 days. Acidotic animals showed a marked but transient increase in urinary lactate excretion. During acidosis, a downregulation of MCT1, MCT8, and SMCT2 was observed at the mRNA level, whereas MCT7 and SMCT1 showed increased mRNA abundance. Only MCT7 showed lower protein abundance whereas all other transporters remained unchanged. In summary, we describe for the first time the localization of various MCT transporters in mammalian kidney and demonstrate that metabolic acidosis induces a transient increase in urinary lactate excretion paralleled by lower MCT7 protein expression.

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