N-terminal protein sequence analysis of the rabbit erythrocyte lactate transporter suggests identity with the cloned monocarboxylate transport protein MCT1.
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ABSTRACT:
SUBMITTER: Poole RC
PROVIDER: S-EPMC1137611 | biostudies-other | 1994 Nov
REPOSITORIES: biostudies-other
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Project description:BACKGROUND: Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. EXPERIMENTAL APPROACH: Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. RESULTS: In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. CONCLUSIONS: Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.
| S-EPMC3729688 | biostudies-literature
Project description:Highly malignant brain tumors harbor the aberrant propensity for aerobic glycolysis, the excessive conversion of glucose to lactic acid even in the presence of ample tissue oxygen. Lactic acid is rapidly effluxed to the tumor microenvironment via a group of plasma-membrane transporters denoted monocarboxylate transporters (MCTs) to prevent "self-poisoning." One isoform, MCT2, has the highest affinity for lactate and thus should have the ability to respond to microenvironment conditions such as hypoxia, lactate, and pH to help maintain high glycolytic flux in the tumor. Yet, MCT2 is considered to not respond to hypoxia, which is counterintuitive. Its response to tumor lactate has not been reported. In this report, we experimentally identify the transcription initiation site/s for MCT2 in astrocytes (normal) and glioma (tumor). We then use a BACmid library to isolate a 4.2-kbp MCT2 promoter-exon I region and examine promoter response to glycolysis-mediated stimuli in glioma cells. Reporter analysis of nested-promoter constructs indicated response of MCT2 to hypoxia, pH, lactate, and glucose, the major physiological "players" that facilitate a tumor's growth and proliferation. Immunoblot analysis of native MCT2 expression under altered pH and hypoxia reflected the reporter data. The pH-mediated gene-regulation studies we describe are the first to record H+-based reporter studies for any mammalian system and demonstrate the exquisite response of the MCT2 gene to minute changes in tumor pH. Identical promoter usage also provides the first evidence of astrocytes harnessing the same gene regulatory regions to facilitate astrocyte-neuron lactate shuttling, a metabolic feature of normal brain.
| S-EPMC5238458 | biostudies-literature
Project description:Inhibition of the monocarboxylate transporter MCT1 by AZD3965 results in an increase in glycolysis in human tumor cell lines and xenografts. This is indicated by changes in the levels of specific glycolytic metabolites and in changes in glycolytic enzyme kinetics. These drug-induced metabolic changes translate into an inhibition of tumor growth in vivo. Thus, we combined AZD3965 with fractionated radiation to treat small cell lung cancer (SCLC) xenografts and showed that the combination provided a significantly greater therapeutic effect than the use of either modality alone. These results strongly support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.
| S-EPMC4258406 | biostudies-literature
Project description:Metabolic imaging using hyperpolarized magnetic resonance can increase the sensitivity of MRI, though its ability to inform on relevant changes to biochemistry in humans remains unclear. In this work, we image pyruvate metabolism in patients, assessing the reproducibility of delivery and conversion in the setting of primary prostate cancer. We show that the time to max of pyruvate does not vary significantly within patients undergoing two separate injections or across patients. Furthermore, we show that lactate increases with Gleason grade. RNA sequencing data demonstrate a significant increase in the predominant pyruvate uptake transporter, monocarboxylate transporter 1. Increased protein expression was also observed in regions of high lactate signal, implicating it as the driver of lactate signal in vivo. Targeted DNA sequencing for actionable mutations revealed the highest lactate occurred in patients with PTEN loss. This work identifies a potential link between actionable genomic alterations and metabolic information derived from hyperpolarized pyruvate MRI.
| S-EPMC6949382 | biostudies-literature
Project description:1. Intact erythrocytes were incubated with 100 microM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig erythrocytes, were detected by this technique. In human erythrocytes, which have 10-fold less transport activity, no labelled polypeptide in this molecular mass range was detected. 2. Labelling of these 35-50 kDa polypeptides was decreased markedly in the presence of the specific inhibitors of lactate transport alpha-cyano-4-hydroxycinnamate and 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), which compete with DIDS for binding to the transporter. However, the weakly bound inhibitor 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) afforded little protection against labelling by DIDS. 3. The lactate transporter from rat erythrocytes was solubilized with decanoyl-N-methyl glucamide (MEGA-10) and partially purified by Mono-Q anion-exchange chromatography, with transport activity eluting at 0.1-0.15 M-NaCl. The 35-45 kDa DIDS-labelled polypeptide from rat erythrocytes was eluted in the same peak of protein as lactate transporter activity during Mono-Q chromatography. 4. These observations provide strong evidence that the lactate transporter is a polypeptide of 35-45 kDa in rat erythrocytes and of 40-50 kDa in rabbit and guinea pig erythrocytes.
| S-EPMC1130965 | biostudies-other
Project description:Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50-80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.
| S-EPMC4087088 | biostudies-literature
Project description:Metabolite exchange between stromal and tumor cells or among tumor cells themselves accompanies metabolic reprogramming in cancer including pancreatic adenocarcinoma (PDAC). Some tumor cells import and utilize lactate for oxidative energy production (reverse Warburg-metabolism) and the presence of these "reverse Warburg" cells associates with a more aggressive phenotype and worse prognosis, though the underlying mechanisms are poorly understood. We now show that PDAC cells (BxPc3, A818-6, T3M4) expressing the lactate-importer monocarboxylate transporter-1 (MCT1) are protected by lactate against gemcitabine-induced apoptosis in a MCT1-dependent fashion, contrary to MCT1-negative PDAC cells (Panc1, Capan2). Moreover, lactate administration under glucose starvation, resembling reverse Warburg co a phenotype of BxPc3 and T3M4 cells that confers greater potential of clonal growth upon re-exposure to glucose, along with drug resistance and elevated expression of the stemness marker Nestin and reprogramming factors (Oct4, KLF4, Nanog). These lactate dependent effects on stemness properties are abrogated by the MCT1/lactate-uptake inhibitor 7ACC2 or MCT1 knock-down. Furthermore, the clinical relevance of these observations was supported by detecting co-expression of MCT1 and reprogramming factors in human PDAC tissues. In conclusion, the MCT1-dependent import of lactate supplies "reverse Warburg "PDAC cells with an efficient driver of metabostemness. This condition may essentially contribute to malignant traits including therapy resistance.
| S-EPMC7139999 | biostudies-literature
Project description: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 K(m) value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for L-lactate K(m) values of 17+/-3 mM (pH-electrode) and 34+/-5 mM (flux measurements with L-[U-(14)C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. K(m) values of other monocarboxylates were either similar to the K(m) value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (beta-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 alpha-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.
| S-EPMC1221245 | biostudies-other
Project description:Membrane monocarboxylate transporter 1 (SLC16A1/MCT1) plays an important role in hepatocyte homeostasis, as well as drug handling. However, there is no available information about the impact of liver pathology on the transporter levels and function. The study was aimed to quantify SLC16A1 mRNA (qRT-PCR) and MCT1 protein abundance (liquid chromatography-tandem mass spectrometry (LC---MS/MS)) in the livers of patients diagnosed, according to the standard clinical criteria, with hepatitis C, primary biliary cirrhosis, primary sclerosing hepatitis, alcoholic liver disease (ALD), and autoimmune hepatitis. The stage of liver dysfunction was classified according to Child-Pugh score. Downregulation of SLC16A1/MCT1 levels was observed in all liver pathology states, significantly for ALD. The progression of liver dysfunction, from Child-Pugh class A to C, involved the gradual decline in SLC16A1 mRNA and MCT1 protein abundance, reaching a clinically significant decrease in class C livers. Reduced levels of MCT1 were associated with significant intracellular lactate accumulation. The MCT1 transcript and protein did not demonstrate significant correlations regardless of the liver pathology analyzed, as well as the disease stage, suggesting posttranscriptional regulation, and several microRNAs were found as potential regulators of MCT1 abundance. MCT1 membrane immunolocalization without cytoplasmic retention was observed in all studied liver pathologies. Overall, the study demonstrates that SLC16A1/MCT1 is involved in liver pathology, especially in ALD.
| S-EPMC7084425 | biostudies-literature
Project description:Cells are separated from the environment by a lipid bilayer membrane that is relatively impermeable to solutes. The transport of ions and small molecules across this membrane is an essential process in cell biology and metabolism. Monocarboxylate transporters (MCTs) belong to a vast family of solute carriers (SLCs) that facilitate the transport of certain hydrophylic small compounds through the bilipid cell membrane. The existence of 446 genes that code for SLCs is the best evidence of their importance. In-depth research on MCTs is quite recent and probably promoted by their role in cancer development and progression. Importantly, it has recently been realized that these transporters represent an interesting target for cancer treatment. The search for clinically useful monocarboxylate inhibitors is an even more recent field. There is limited pre-clinical and clinical experience with new inhibitors and their precise mechanism of action is still under investigation. What is common to all of them is the inhibition of lactate transport. This review discusses the structure and function of MCTs, their participation in cancer, and old and newly developed inhibitors. Some suggestions on how to improve their anticancer effects are also discussed.
| S-EPMC10918235 | biostudies-literature