The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1-4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1-4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.
butyrate; gamma-hydroxybutyrate; lactate; monocarboxylate transporters; SLC16A
Overdoses of γ-hydroxybutyrate (GHB), a drug of abuse, result in coma, respiratory arrest, and death. The objective of this study was to evaluate a potential GHB detoxification strategy by inhibiting the monocarboxylate transporter (MCT)-mediated renal reabsorption of GHB in rats, using the MCT substrate L-Lactate. The use of the osmotic diuretic D-mannitol alone or combined with L-Lactate was also explored. GHB (208 mg/h/kg) was infused i.v. for 3 h in the absence or presence of L-Lactate (60.5, 121, and 302.5 mg h−1 kg−1), D-mannitol (0.5 g/kg), or L-Lactate (60.5 mg h−1 kg−1) combined with D-mannitol (0.5 g/kg). GHB in plasma and urine samples was determined along with blood pH, electrolytes, glucose, and L-Lactate. Administration of L-Lactate, or the combination of L-Lactate and D-mannitol, but not D-mannitol alone, significantly increased the renal and total clearances of GHB in rats. Blood pH and electrolyte concentrations exhibited small changes with GHB, GHB/lactate, and GHB/mannitol treatments, although most values remained within their normal range. The concomitant administration of lactated Ringer's solution (28 mM L-Lactate) at 300 µl/min with mannitol (0.5 g/kg) resulted in a significant increase in GHB clearance and a decrease in sleep time after an i.v. dose of 1 g/kg. Overall, our results indicated the following: 1) the use of the MCT inhibitor L-Lactate can increase the renal and total clearances of GHB, and 2) the combination of lactated Ringer's solution and D-mannitol significantly alters GHB toxicokinetics and toxicodynamics and represents a potential clinical detoxification strategy for the treatment of GHB overdoses.
Monocarboxylate transporter 1 (MCT1) has been previously reported as an important determinant of the renal reabsorption of the drug of abuse, γ-hydroxybutyrate (GHB). Luteolin is a potent MCT1 inhibitor, inhibiting the uptake of GHB with an IC50 of 0.41 μM in MCT1-transfected MDA-MB231 cells. The objectives of this study were to characterize the effects of luteolin on GHB pharmacokinetics and pharmacodynamics in rats, and to investigate the mechanism of the interaction using model-fitting methods. GHB (400 and 1,000 mg/kg) and luteolin (0, 4 and 10 mg/kg) were administered to rats via iv bolus doses. The plasma or urine concentrations of luteolin and GHB were determined by HPLC and LC/MS/MS, respectively. The pharmacodynamic parameter sleep time in rats after GHB administration was recorded. A pharmacokinetic model containing capacity-limited renal reabsorption and metabolic clearance was constructed to characterize the in vivo interaction. Luteolin significantly decreased the plasma concentration and AUC, and increased the total and renal clearances of GHB. Moreover, luteolin significantly shortened the duration of GHB (1,000 mg/kg)-induced sleep in rats (161 ± 16, 131 ± 14 and 121 ± 5 min for control, luteolin 4 and 10 mg/kg groups, respectively, p < 0.01). An uncompetitive inhibition model, with an inhibition constant of 1.1 μM, best described the in vivo pharmacokinetic interaction. The results of this study indicated that luteolin significantly altered the pharmacokinetics of GHB by inhibiting its MCT1-mediated transport. The interaction between luteolin and GHB may offer a potential clinical detoxification strategy to treat GHB overdoses.
γ-hydroxybutyrate; luteolin; MCT; pharmacokinetic interactions
Monocarboxylate transporters (MCTs) are known to mediate the transport of short chain monocarboxylates such as lactate, pyruvate and butyrate. Currently, fourteen members of this transporter family have been identified by sequence homology, of which only the first four members (MCT1- MCT4) have been shown to mediate the proton-linked transport of monocarboxylates. Another transporter family involved in the transport of endogenous monocarboxylates is the sodium coupled MCTs (SMCTs). These act as a symporter and are dependent on a sodium gradient for their functional activity. MCT1 is the predominant transporter among the MCT isoforms and is present in almost all tissues including kidney, intestine, liver, heart, skeletal muscle and brain. The various isoforms differ in terms of their substrate specificity and tissue localization. Due to the expression of these transporters in the kidney, intestine, and brain, they may play an important role in influencing drug disposition. Apart from endogenous short chain monocarboxylates, they also mediate the transport of exogenous drugs such as salicylic acid, valproic acid, and simvastatin acid. The influence of MCTs on drug pharmacokinetics has been extensively studied for γ-hydroxybutyrate (GHB) including distribution of this drug of abuse into the brain and the results will be summarized in this review. The physiological role of these transporters in the brain and their specific cellular localization within the brain will also be discussed. This review will also focus on utilization of MCTs as potential targets for drug delivery into the brain including their role in the treatment of malignant brain tumors.
Monocarboxylate transporters; γ-hydroxybutyrate; brain; lactate
γ-Hydroxybutyric acid (GHB), a drug of abuse, exhibits saturable renal clearance and capacity-limited metabolism. The objectives of this study were to construct a mechanistic toxicokinetic (TK) model describing saturable renal reabsorption and capacity-limited metabolism of GHB and to predict the effects of inhibition of renal reabsorption on GHB TK in the plasma and urine. GHB was administered by iv bolus (200–1,000 mg/kg) to male Sprague-Dawley rats and plasma and urine samples were collected for up to 6 h post-dose. GHB concentrations were determined by LC/MS/MS. GHB plasma concentration and urinary excretion were well-described by a TK model incorporating plasma and kidney compartments, along with two tissue and two ultrafiltrate compartments. The estimate of the Michaelis-Menten constant for renal reabsorption (Km,R) was 0.46 mg/ml which is consistent with in vitro estimates of monocarboxylate transporter (MCT)-mediated uptake of GHB (0.48 mg/ml). Simulation studies assessing inhibition of renal reabsorption of GHB demonstrated increased time-averaged renal clearance and GHB plasma AUC, independent of the inhibition mechanism assessed. Co-administration of GHB (600 mg/kg iv) and l-lactate (330 mg/kg iv bolus plus 121 mg/kg/h iv infusion), a known inhibitor of MCTs, resulted in a significant decrease in GHB plasma AUC and an increase in time-averaged renal clearance, consistent with the model simulations. These results suggest that inhibition of renal reabsorption of GHB is a viable therapeutic strategy for the treatment of GHB overdoses. Furthermore, the mechanistic TK model provides a useful in silico tool for the evaluation of potential therapeutic strategies.
gamma-hydroxybutyrate; kidney reabsorption; pharmacokinetic model; renal clearance; toxicokinetics
Background and objective
The purpose of the current study was to demonstrate proof-of-concept that monocarboxylate transporter (MCT) inhibition with L-lactate combined with osmotic diuresis increases renal clearance of γ-hydroxybutyrate (GHB) in human subjects. GHB is a substrate for human and rodent MCTs, which are responsible for GHB renal reabsorption, and this therapy increases GHB renal clearance in rats.
Ten healthy volunteers were administered GHB orally as sodium oxybate 50 mg/kg (4.5 gm maximum dose) on two different study days. On study day 1, GHB was administered alone. On study day 2, treatment of L-lactate 0.125 mmol/kg and mannitol 200 mg/kg followed by L-lactate 0.75 mmol/kg/hr was administered intravenously 30 minutes after GHB ingestion. Blood and urine were collected for 6 hours, analyzed for GHB, and pharmacokinetic and statistical analyses performed.
L-lactate/mannitol administration significantly increased GHB renal clearance compared to GHB alone, 439 vs. 615 mL/hr (P=0.001), and increased the percentage of GHB dose excreted in the urine, 2.2 vs. 3.3% (P=0.021). Total clearance was unchanged.
MCT inhibition with L-lactate combined with osmotic diuresis increases GHB renal elimination in humans. No effect on total clearance was observed in this study due to the negligible contribution of renal clearance to total clearance at this low GHB dose. Considering the nonlinear renal elimination of GHB, further research in overdose cases is warranted to assess the efficacy of this treatment strategy for increasing renal and total clearance at high GHB doses.
γ-hydroxybutyrate; Pharmacokinetics; Renal clearance; Monocarboxylate transporter
Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs). Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4), thyroid hormones (MCT8, -10) and aromatic amino acids (MCT10). Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR)-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT) and PPARα knockout (KO) mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1–4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms.
Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.
CD147; MCT1; MCT4; lactate; multiple myeloma
Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra). However, mutation of these transporters does not prevent this organism from utilizing alanine for growth. An R. leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source. Characterization of MctP confirmed that it transports alanine (Km = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (Km = 4.4 and 3.8 μM, respectively). Uptake inhibition studies indicate that propionate, butyrate, α-hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates. MctP has significant sequence similarity to members of the sodium/solute symporter family. However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters. While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium. In contrast, glutamate uptake in R. leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled. Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the β-subdivision, gram-positive bacteria, and archaea. A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression.
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.
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.
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.
Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.
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.
hypoxia; monocarboxylate transporter-4; stem cells; glioma
The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8−/−) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8−/− larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8−/− larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8−/− larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8−/− larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients.
In a wide range of brain disorders, mutations in specific genes cause alterations in the development and function of neural circuits that ultimately affect behavior. A major challenge is to uncover the mechanism and provide treatment which is capable of preventing brain damage. Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by intellectual disabilities, neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter MCT8 are associated with AHDS. Mice that lack the MCT8 protein exhibited impaired TH levels, as is the case in human patients; however, they lack neurological defects. Here, we generated an mct8 mutant (mct8−/−) zebrafish, which exhibited neurological and behavioral deficiencies and mimics pathological conditions of AHDS patients. The zebrafish is a simple transparent vertebrate and its nervous system is conserved with mammals. Time-lapse live imaging of single axons and synapses, and video-tracking of behavior revealed deficiencies in neural circuit assembly, which are associated with disturbed sleep and altered locomotor activity. In addition, since the mct8−/− larvae provides a highthroughput platform for testing therapeutic drugs, we showed that TH analogs can recover neurological deficiencies in an animal model for psychomotor retardation.
MCT3, a specific marker of differentiated RPE, is downregulated after wounding. This report demonstrates for the first time a role for cell-cell contacts in restoring MCT3 expression after injury.
MCT3 is a proton-coupled monocarboxylate transporter preferentially expressed in the basolateral membrane of the retinal pigment epithelium (RPE) and has been shown to play an important role in regulating pH and lactate concentrations in the outer retina. Decreased expression of MCT3 in response to trauma or disease could contribute to pathologic changes in the retina. The present study followed the expression of MCT3 after wounding and re-epithelialization of chick RPE explant and human fetal (hf) RPE cultures.
Immunofluorescence microscopy and immunoblotting were performed to determine changes in MCT expression after scratch wounding and re-epithelialization of chick RPE/choroid explant cultures and hfRPE cell monolayers.
MCT3 expression and basolateral polarity were maintained in chick RPE/choroid explant cultures and hfRPE monolayers. Wounding resulted in loss of MCT3 and the upregulation of MCT4 expression in migrating cells at the edge of the wound. On re-epithelialization, MCT3 was detected in chick and hfRPE cells when cells became hexagonally packed and pigmented. However, in hfRPE cells, MCT4 was consistently expressed throughout the epithelial monolayer. RPE cells at the edges of chick explants and hfRPE cultures with a free edge expressed MCT4 but not MCT3.
Wounding of RPE monolayers resulted in dedifferentiation of the cells at the edge of the wound, as evidenced by a loss of MCT3 and increased MCT4 expression. Collectively, these findings suggest that both cell-cell and cell-substrate interactions are essential in directing and maintaining differentiation of the RPE and expression of MCT3.
Tumour cells are characterized by aerobic glycolysis, which provides biomass for tumour proliferation and leads to extracellular acidification through efflux of lactate via monocarboxylate transporters (MCTs). Deficient and spasm-prone tumour vasculature causes variable hypoxia, which favours tumour cell survival and metastases. Brain metastases frequently occur in patients with advanced breast cancer.Effective treatment strategies are therefore needed against brain metastasis from breast carcinoma.
Material and methods
In order to identify differences in the capacity for lactate exchange, human T-47D breast cancer cells and human glioblastoma T98G cells were grown under 4 % or 20 % oxygen conditions and examined for MCT1, MCT2 and MCT4 expression on plasma membranes by quantitative post embedding immunogold electron microscopy. Whereas previous studies on MCT expression in tumours have recorded mRNA and protein levels in cell extracts, we examined concentrations of the proteins in the microvillous plasma membrane protrusions specialized for transmembrane transport.
In normoxia, both tumour cell types highly expressed the low affinity transporter MCT4, which is thought to mainly mediate monocarboxylate efflux, while for high affinity transport the breast tumour cells preferentially expressed MCT1 and the brain tumour cells resembled brain neurons in expressing MCT2, rather than MCT1. The expressions of MCT1 and MCT4 were upregulated in hypoxic conditions in both breast and brain tumour cells. The expression of MCT2 also increased in hypoxic breast cancer cells, but decreased in hypoxic brain tumour cells. Quantitative immunoblots showed similar hypoxia induced changes in the protein levels.
The differential expression and regulation of MCTs in the surface membranes of hypoxic and normoxic tumour cells of different types provide a foundation for innovation in tumour therapy through the selective targeting of MCTs. Selective inhibition of various MCTs could be an efficient way to quench an important energy source in both original breast tumour and metastatic cancer tissue in the brain.
Monocarboxylate transporter; Hypoxia; Glioblastoma; Breast cancer; Tumour cell
Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas.
MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models.
MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed.
This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.
CD147; CHC; glioblastomas; gliomas; glycolytic metabolism; lactate; monocarboxylate transporters
In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3±1.4 nM, 1.29±0.09 nmol per ml of packed cells and 12.2±1.1 s−1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7–10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011–20021].
chimaeric transporter; erythrocyte; lactate transport; monocarboxylate transporter (MCT); Xenopus oocytes; BCECF, 2′-7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; DIDS, di-isothiocyanostilbene disulfonate; MCT, monocarboxylate transporter; TM, transmembrane
L-lactate represents a potential treatment for GHB overdose by inhibiting GHB renal reabsorption mediated by monocarboxylate transporters. Our objective was to assess the dose-dependence of L-lactate treatment, with and without D-mannitol, on GHB toxicokinetics/toxicodynamics (TK/TD).
Rats were administered GHB 600 mg/kg i.v. with L-lactate (low and high doses), D-mannitol, or L-lactate (low dose) with D-mannitol. GHB-induced sleep time and GHB plasma, urine and brain extracellular fluid (ECF) concentrations (by LC/MS/MS) were determined. The effect of L-lactate and D-mannitol on the uptake and efflux of GHB was assessed in rat brain endothelial RBE4 cells.
L-lactate treatment increased GHB renal clearance from 1.4 ± 0.1 ml/min/kg (control) to 2.4 ± 0.2 and 4.7 ± 0.5 ml/min/kg after low and high doses, respectively, and reduced brain ECF AUC values to 65 and 25% of control. Sleep time was decreased from 137 ± 12 minutes (control) to 91 ± 16 and 55 ± 5 minutes (low and high L-lactate, respectively). D-mannitol did not alter GHB TK/TD and did not alter L-lactate’s effects on GHB TK/TD. L-lactate, but not D-mannitol, inhibited GHB uptake, and increased GHB efflux from RBE4 cells.
L-lactate decreases plasma and brain ECF concentrations of GHB, decreasing sedative/hypnotic effects.
γ-hydroxybutyrate; monocarboxylate transporter; microdialysis; lactate; overdose
Cancer cell adopts peculiar metabolic strategies aimed to sustain the continuous proliferation in an environment characterized by relevant fluctuations in oxygen and nutrient levels. Monocarboxylate transporters MCT1 and MCT4 can drive such adaptation permitting the transport across plasma membrane of different monocarboxylic acids involved in energy metabolism.
Role of MCTs in tumor-stroma metabolic relationship was investigated in vitro and in vivo using transformed prostate epithelial cells, carcinoma cell lines and normal fibroblasts. Moreover prostate tissues from carcinoma and benign hypertrophy cases were analyzed for individuating clinical-pathological implications of MCT1 and MCT4 expression.
Transformed prostate epithelial (TPE) and prostate cancer (PCa) cells express both MCT1 and MCT4 and demonstrated variable dependence on aerobic glycolysis for maintaining their proliferative rate. In glucose-restriction the presence of L-lactate determined, after 24 h of treatment, in PCa cells the up-regulation of MCT1 and of cytochrome c oxidase subunit I (COX1), and reduced the activation of AMP-activated protein kinase respect to untreated cells. The blockade of MCT1 function, performed by si RNA silencing, determined an appreciable antiproliferative effect when L-lactate was utilized as energetic fuel. Accordingly L-lactate released by high glycolytic human diploid fibroblasts WI-38 sustained survival and growth of TPE and PCa cells in low glucose culture medium. In parallel, the treatment with conditioned medium from PCa cells was sufficient to induce glycolytic metabolism in WI-38 cells, with upregulation of HIF-1a and MCT4. Co-injection of PCa cells with high glycolytic WI-38 fibroblasts determined an impressive increase in tumor growth rate in a xenograft model that was abrogated by MCT1 silencing in PCa cells. The possible interplay based on L-lactate shuttle between tumor and stroma was confirmed also in human PCa tissue where we observed a positive correlation between stromal MCT4 and tumor MCT1 expression.
Our data demonstrated that PCa progression may benefit of MCT1 expression in tumor cells and of MCT4 in tumor-associated stromal cells. Therefore, MCTs may result promising therapeutic targets in different phases of neoplastic transformation according to a strategy aimed to contrast the energy metabolic adaptation of PCa cells to stressful environments.
Aerobic glycolysis; Monocarboxylate transporters; Cancer associated fibroblasts; Warburg effect; Tumor stroma
Butyrate, a short chain fatty acid (SCFA) produced by bacterial fermentation of undigested carbohydrates in the colon, constitutes the major fuel for colonocytes. We have earlier shown the role of apically localized monocarboxylate transporter isoform 1 (MCT1) in transport of butyrate into human colonic Caco-2 cells. In an effort to study the regulation of MCT1 gene, we and others have cloned the promoter region of the MCT1 gene and identified cis elements for key transcription factors. A previous study has shown up-regulation of MCT1 expression, and activity by butyrate in AA/C1 human colonic epithelial cells, however, the detailed mechanisms of this up-regulation are not known. In this study, we demonstrate that butyrate, a substrate for MCT1, stimulates MCT1 promoter activity in Caco-2 cells. This effect was dose dependent and specific to butyrate as other predominant SCFAs, acetate, and propionate, were ineffective. Utilizing progressive deletion constructs of the MCT1 promoter, we showed that the putative butyrate responsive elements are in the −229/+91 region of the promoter. Butyrate stimulation of the MCT1 promoter was found to be independent of PKC, PKA, and tyrosine kinases. However, specific inhibitors of the NF-κB pathway, lactacystein (LC), and caffeic acid phenyl ester (CAPE) significantly reduced the MCT1 promoter stimulation by butyrate. Also, butyrate directly stimulated NF-κB-dependent luciferase reporter activity. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also stimulated MCT1 promoter activity, however, unlike butyrate, this stimulation was unaltered by the NF-κB inhibitors. Further, the combined effect of butyrate, and TSA on MCT1 promoter activity was additive, indicating that their mechanisms of action were independent. Our results demonstrate the involvement of NF-κB pathway in the regulation of MCT1 promoter activity by butyrate.
short chain fattyacids; Caco-2; HDAC; TSA
Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys38, Lys45, Lys282, and Lys413. K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO3− groups interact with positively charged Lys282 together with Lys45 and/or Lys413. This positions one DIDS isothiocyanate group close to Lys38, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys38 or Lys290 of MCT1 and Lys160 or Lys164 of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys38 in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys38 accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp302− and Asp306+, an ion pair already identified as important for transport and located adjacent to Phe360, which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.
In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.
basigin; embigin; erythrocyte; lactate transport; monocarboxylate transporter 1 (MCT1); monocarboxylate transporter 2 (MCT2); BCECF, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein; CFP, cyan fluorescent protein; EST, expressed sequance tag; FRET, fluorescence resonance energy transfer; HA, haemagglutinin; MCT, monocarboxylate transporter; MCT1trn, MCT1 without C-terminus; MCT1/2c, MCT1 with MCT2 C-terminus; MCT2trn, MCT2 without C-terminus; MCT2/1c, MCT2 with MCT1 C-terminus; pCMBS, p-chloromercuribenzene sulfonate; RT, reverse transcription; TM, transmembrane; WT, wild-type; YFP, yellow fluorescent protein
Lactate-proton cotransport via MCT1, -2, and -4 is present in corneal endothelium. Lactate-dependent proton flux can activate Na+/H+ exchange and is facilitated by maximizing intracellular buffering capacity through the presence of HCO3−, HCO3− transport, NHE, and CA activity.
To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO3−, sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux.
MCT1–4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pHi were measured in BCEC in HCO3−-free or HCO3−-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na+/H+ exchange blocker), disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells.
MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pHi measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpHi/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO3− but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO3−, was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA.
MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H+ transport. Lactate dependent proton flux can activate Na+/H+ exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO3−, HCO3− transport, NHE and CA activity.
Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.
Gamma-hydroxybutyric acid (GHB) is used as an illicit drug and is implicated in drug-facilitated sexual assault, but it also has some therapeutic uses. Detection of GHB in urine is important for forensic testing and could be of clinical benefit in overdose management. Urine GHB concentration-time profiles have not been well-characterized or correlated with doses used therapeutically. GHB levels were measured by gas chromatography–mass spectrometry in urine collected over 24 h from 16 adults administered single doses of 50 mg/kg GHB (Xyrem®) alone and combined with 0.6 g/kg ethanol. Peak GHB urine concentrations averaged 150–200 mg/L and occurred in the 0–3 h urine collection. Significant variability in GHB urine levels between individuals was observed. Caucasians had lower urine concentrations than other races/ethnicities (p = 0.03). Men had lower GHB levels than women in the first 3 h after dosing (p = 0.038). Coingestion of ethanol did not significantly affect renal clearance of GHB, but urine GHB concentrations were lower in the first 3 h when ethanol and GHB were coingested (p = 0.039). At a proposed cut-off of 10 mg/L to distinguish endogenous versus exogenous GHB levels, 12.5% of the samples collected from 3 to 6 h, 81.3% of samples collected from 6 to 12 h, and 100% of urine specimens collected from 12 to 24 h were below this level. We conclude that the detection time for GHB in urine may be shorter than the previously reported 12-h window in some people taking therapeutic doses of GHB.
Cancer cells generally have a high rate of glycolysis and produce larger quantities of lactate as compared to the surrounding normal cells. Monocarboxylate transporter 4 (MCT4) is one of the proton pumps exchanging the lactate through the plasma membrane. The prognostic significance of MCT4 expression has not been evaluated in patients with colorectal cancer (CRC). Surgical specimens from 105 CRC patients were immunohistochemically stained using a polyclonal anti-MCT4 antibody. The relationships among the MCT4 expression, clinicopathological factors and prognosis were evaluated. A total of 53 (50.5%) of the 105 patients with CRC were determined to have tumors positive for MCT4 expression. The expression of MCT4 significantly correlated with the tumor size, depth of invasion, lymph node metastasis, distant metastasis and TNM staging. The survival rate of the patients who were positive for MCT4 expression was significantly lower than that of patients with negative MCT4 expression. Positive MCT4 expression was a significantly poor prognostic factor, as determined by both univariate and multivariate analyses. Therefore, positive MCT4 expression appears to be a useful marker for tumor progression and prognosis in patients with CRC.
monocarboxylate transporter; monocarboxylate transporter 4; colorectal cancer