Transporters MCT1 Research Articles

Biotin.

Biotin is a water-soluble vitamin and serves as a coenzyme for five carboxylases in humans. Biotin is also covalently attached to distinct lysine residues in histones, affecting chromatin structure and mediating gene regulation. This review describes mammalian biotin metabolism, biotin analysis, markers of biotin status, and biological functions of biotin. Proteins such as holocarboxylase synthetase, biotinidase, and the biotin transporters SMVT and MCT1 play crucial roles in biotin homeostasis, and these roles are reviewed here. Possible effects of inadequate biotin intake, drug interactions, and inborn errors of metabolism are discussed, including putative effects on birth defects.

Genetic Variations in the MCT1 (SLC16A1) Gene in the Chinese Population of Singapore

MCT1(SLC16A1) is the first member of the monocarboxylate transporter (MCT) and its family is involved in the transportation of metabolically important monocarboxylates such as lactate, pyruvate, acetate and ketone bodies. This study identifies genetic variations in SLC16A1 in the ethnic Chinese group of the Singaporean population (n=95). The promoter, coding region and exon-intron junctions of the SLC16A1 gene encoding the MCT1 transporter were screened for genetic variation in the study population by DNA sequencing. Seven genetic variations of SLC16A1, including 4 novel ones, were found: 2 in the promoter region, 2 in the coding exons (both nonsynonymous variations), 2 in the 3' untranslated region (3'UTR) and 1 in the intron. Of the two mutations detected in the promoter region, the -363-855T>C is a novel mutation. The 1282G>A (Val(428)Ile) is a novel SNP and was found as heterozygotic in 4 subjects. The 1470T>A (Asp(490)Glu) was found to be a common polymorphism in this study. Lastly, IVS3-17A>C in intron 3 and 2258 (755)A>G in 3'UTR are novel mutations found to be common polymorphisms in the local Chinese population. To our knowledge, this is the first report of a comprehensive analysis on the MCT1 gene in any population.

Increased densities of monocarboxylate transporter MCT1 after chronic hyperglycemia in rat brain

The brain is capable of taking up monocarboxylates as energy substrates. Under physiological conditions, plasma levels of monocarboxylates are very low and glucose is the primary energy substrate in brain metabolism. However, given conditions such as hyperglycemia and ketosis, levels of circulating monocarboxylates such as lactate and pyruvate are elevated. Previous studies reported an increased expression of monocarboxylate transporter MCT1 in brain following ketotic diet. The major aim of the present study was to answer the question whether chronic hyperglycemia is likewise sufficient to change local densities of MCT1 in the brain. Moreover, chronic hyperglycemia increases local cerebral glucose utilization (LCGU) in particular brain areas. Glucose hereby enters the brain parenchyma via glucose transporters and is partially metabolised by astrocytes, which then release lactate to meet the energetic demands of surrounding neurons. Streptozotocin was given intravenously to induce chronic hyperglycemia and local densities of MCT1 were measured by immunoautoradiographic methods in cryosections of rat brains. The density of monocarboxylate transporter MCT1 was significantly increased in 10 of 24 brain structures investigated (median increase 11.7 ± 3.4 %). Immunocytochemical stainings of these substructures revealed an expression of MCT1 within endothelial cells and astrocytes. A comparison of MCT1 densities with LCGU measured in a previous study under normo- and hyperglycemic conditions revealed a partial correlation between both parameters and under both conditions. Four out of 10 brain areas, which showed a significant increase in MCT1 density due to hyperglycemia, also showed a significant increase in LCGU. In summary, our data show that chronic hyperglycemia induces a moderate increase of local and global density of MCT1 in several brain structures. However, in terms of brain topologies and substructures this phenomenon did only partially match with increased LCGU. It is concluded that MCT1 transporters were up-regulated during chronic hyperglycemia at the level of brain substructures and independently of LCGU.

RNA interference screen for human genes associated with West Nile virus infection

West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.

PGC-1α increases skeletal muscle lactate uptake by increasing the expression of MCT1 but not MCT2 or MCT4

We examined the relationship between PGC-1alpha protein; the monocarboxylate transporters MCT1, 2, and 4; and CD147 1) among six metabolically heterogeneous rat muscles, 2) in chronically stimulated red (RTA) and white tibialis (WTA) muscles (7 days), and 3) in RTA and WTA muscles transfected with PGC-1alpha-pcDNA plasmid in vivo. Among rat hindlimb muscles, there was a strong positive association between PGC-1alpha and MCT1 and CD147, and between MCT1 and CD147. A negative association was found between PGC-1alpha and MCT4, and CD147 and MCT4, while there was no relationship between PGC-1alpha or CD147 and MCT2. Transfecting PGC-1alpha-pcDNA plasmid into muscle increased PGC-1alpha protein (RTA +23%; WTA +25%) and induced the expression of MCT1 (RTA +16%; WTA +28%), but not MCT2 and MCT4. As a result of the PGC-1alpha-induced upregulation of MCT1 and its chaperone CD147 (+29%), there was a concomitant increase in the rate of lactate uptake (+20%). In chronically stimulated muscles, the following proteins were upregulated, PGC-1alpha in RTA (+26%) and WTA (+86%), MCT1 in RTA (+61%) and WTA (+180%), and CD147 in WTA (+106%). In contrast, MCT4 protein expression was not altered in either RTA or WTA muscles, while MCT2 protein expression was reduced in both RTA (-14%) and WTA (-10%). In these studies, whether comparing oxidative capacities among muscles or increasing their oxidative capacities by PGC-1alpha transfection and chronic muscle stimulation, there was a strong relationship between the expression of PGC-1alpha and MCT1, and PGC-1alpha and CD147 proteins. Thus, MCT1 and CD147 belong to the family of metabolic genes whose expression is regulated by PGC-1alpha in skeletal muscle.

Negligible direct lactate oxidation in subsarcolemmal and intermyofibrillar mitochondria obtained from red and white rat skeletal muscle

We examined the controversial notion of whether lactate is directly oxidized by subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria obtained from red and white rat skeletal muscle. Respiratory control ratios were normal in SS and IMF mitochondria. At all concentrations (0.18-10 mm), and in all mitochondria, pyruvate oxidation greatly exceeded lactate oxidation, by 31- to 186-fold. Pyruvate and lactate oxidation were inhibited by alpha-cyano-4-hydroxycinnamate, while lactate oxidation was inhibited by oxamate. Excess pyruvate (10 mm) inhibited the oxidation of palmitate (1.8 mm) as well as lactate (1.8 mm). In contrast, excess lactate (10 mm) failed to inhibit the oxidation of either palmitate (1.8 mm) or pyruvate (1.8 mm). The cell-permeant adenosine analogue, AICAR, increased pyruvate oxidation; in contrast, lactate oxidation was not altered. The monocarboxylate transporters MCT1 and 4 were present on SS mitochondria, but not on IMF mitochondria, whereas, MCT2, a high-affinity pyruvate transporter, was present in both SS and IMF mitochondria. The lactate dehydrogenase (LDH) activity associated with SS and IMF mitochondria was 200- to 240-fold lower than in whole muscle. Addition of LDH increased the rate of lactate oxidation, but not pyruvate oxidation, in a dose-dependent manner, such that lactate oxidation approached the rates of pyruvate oxidation. Collectively, these studies indicate that direct mitochondrial oxidation of lactate (i.e. an intracellular lactate shuttle) does not occur within the matrix in either IMF or SS mitochondria obtained from red or white rat skeletal muscle, because of the very limited quantity of LDH within mitochondria.

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney

Recently, we cloned two Na +-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na +-coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.

H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine.

The H(+)-electrochemical gradient was originally considered as a driving force for solute transport only across cellular membranes of bacteria, plants and yeast. However, in the mammalian small intestine, a H(+)-electrochemical gradient is present at the epithelial brush-border membrane in the form of an acid microclimate. Over recent years, a large number of H(+)-coupled cotransport mechanisms have been identified at the luminal membrane of the mammalian small intestine. These transporters are responsible for the initial stage in absorption of a remarkable variety of essential and non-essential nutrients and micronutrients, including protein digestion products (di/tripeptides and amino acids), vitamins, short-chain fatty acids and divalent metal ions. Proton-coupled cotransporters expressed at the mammalian small intestinal brush-border membrane include: the di/tripeptide transporter PepT1 (SLC15A1); the proton-coupled amino-acid transporter PAT1 (SLC36A1); the divalent metal transporter DMT1 (SLC11A2); the organic anion transporting polypeptide OATP2B1 (SLC02B1); the monocarboxylate transporter MCT1 (SLC16A1); the proton-coupled folate transporter PCFT (SLC46A1); the sodium-glucose linked cotransporter SGLT1 (SLC5A1); and the excitatory amino acid carrier EAAC1 (SLC1A1). Emerging research demonstrates that the optimal intestinal absorptive capacity of certain H(+)-coupled cotransporters (PepT1 and PAT1) is dependent upon function of the brush-border Na(+)-H(+) exchanger NHE3 (SLC9A3). The high oral bioavailability of a large number of pharmaceutical compounds results, in part, from absorptive transport via the same H(+)-coupled cotransporters. Drugs undergoing H(+)-coupled cotransport across the intestinal brush-border membrane include those used to treat bacterial infections, hypercholesterolaemia, hypertension, hyperglycaemia, viral infections, allergies, epilepsy, schizophrenia, rheumatoid arthritis and cancer.

Lactate as a Metabolic Signal in Gene Expression

PURPOSE: Lactate plays an important role in adaptive response of muscle to exercise. The possible contribution gene transcription in causing lactate-induced adaptive responses remains to be discovered. The goal of this study was to assess genome-wide responses of L6 myocytes to lactate. METHODS: L6 myocytes were cultured in Dulbecco's modified Eagle's Medium (high glucose). Eight days following seeding of the cells they were incubated with media containing 0 mM or 20 mM of sodium lactate. The cells were then harvested 1 hr or 6 hr after incubation with lactate. GeneChip analysis (Rat genome 230A2.0, Affymetrix, Santa Clara, CA) was performed as described by manufacturer. Quantitative real-time RT-PCR was performed to validate the data of GeneChip analysis. RESULTS: Twenty mM lactate significantly increased the expression of 33 genes and decreased the expression of 76 genes at 1 hr, and increased the expression of 673 genes and decreased the expression of only 3 genes at 6 hr. Interestingly, 79 genes involved in metabolism and mitochondria were up-regulated at 6 hr and among them, large numbers of genes are related to mitochondria biogenesis. Furthermore, large numbers of genes which are related to trans crip tional activation, transporter, oxidative stress, apoptosis, cell growth, or Ca2+ and muscle were up-regulated at 6 hr. The inductions of monocarboxylate transporter MCT1 and cytochrome c oxidase genes detected by the GeneChip assay were confirmed by real-time RT-PCR. CONCLUSIONS: The GeneChip analysis suggests that lactate accumulation increases mitochondrial biogenesis in L6 myocytes.

Monocarboxylate transporter expression is associated with the absorption of benzoic acid in human intestinal epithelial cells

AbstractA diet rich in fruit and vegetables is associated with a decreased risk of developing a number of chronic diseases including colon cancer. The active components of the diet which exert these protective effects are unknown but may include small aromatic and phenolic acids. To elicit a physiological response, these acids must first be absorbed by the intestinal epithelium and the present study has focussed on one potential absorptive pathway—the monocarboxylate transporter MCT1. Using Caco‐2 cells (a human intestinal cell line) we have established that benzoic acid uptake is pH‐dependent (resulting in intracellular acidification) and is enhanced in cells expressing higher levels of MCT1 protein. A number of monohydroxybenzoic acids (including salicylic acid) also induced a decrease in intracellular pH which was of a similar magnitude to that elicited by benzoic acid itself, suggesting that they may all be substrates for the same transport pathway. The role of these small organic acids in maintaining normal cell physiology remains to be fully explored. Copyright © 2006 Society of Chemical Industry

Expression of CD147 and monocarboxylate transporters MCT1, MCT2 and MCT4 in porcine small intestine and colon

Lactate, formed mainly in the stomach and small intestines, and short-chain fatty acids (SCFAs) formed in the colon, are ionised and require transporter proteins such as monocarboxylate transporters (MCTs) for absorption. The amounts of MCT1, MCT2, MCT4 and CD147, an ancillary protein for MCT1 and MCT4, were measured by immunoblotting the small intestine and colon of 40 pigs (Landrace, Yorkshire and Landrace × Yorkshire). MCT1 and MCT4 were found in both small intestine and colon, but MCT2 only in the small intestine. In both small intestine and colon, Yorkshire pigs had more CD147 than Landrace pigs, while no interbreed differences were found in MCT isoforms. Since CD147 is essential for the activity of MCT1 and MCT4, the breed difference suggests that MCT activity is higher in Yorkshire than in Landrace pigs. The absence of MCT2 in the colon suggests that it is mainly a lactate transporter, while MCT1 and MCT4 facilitate the transport of both lactate and SCFA.

An elevated sarcolemmal lactate (and proton) transport capacity is an advantage during muscle activity in healthy humans.

The etiology of muscle fatigue remains incompletely solved especially in vivo (3). The causes of muscle fatigue vary according to the type, duration and intensity of exercise, and to the physical fitness and health status of the subjects. Nevertheless, an elevated sarcolemmal lactate (and proton) transport capacity constitutes an advantage during muscle activity. During moderate-intensity exercise, efficient lactate exchanges contribute to the cell-cell lactate shuttle i.e., the delivery of lactate for its utilization as fuel by neighboring or distant active and oxidative muscles fibers or as substrate by other tissues (e.g. for neoglucogenesis by the liver) (1). During high-intensity exercise, the lactate exchange ability was positively correlated with the capacity to prolong exercise (4). Whether this correlation between the lactate exchange ability and performance was (directly or indirectly) causal or coincidental, the question is still under debate. Nevertheless, it remains that an elevated lactate exchange ability seems to constitute a protective mechanism against muscle fatigue. Since the transport of lactate across the sarcolemma is mediated mainly by the lactate-H+ cotransport via the monocarboxylate transporters MCT1 and MCT4, an elevated lactate transport capacity delays both muscle lactate accumulation and intracellular pH decrease and seems to favor muscle activity. In accordance, previous experiments have shown that metabolic alkalosis enhances the net lactate release rate from the active muscles and work capacity (2, 5) contrary to acidosis that reduces lactate efflux from muscle and exercise tolerance (5). All this makes an elevated sarcolemmal lactate (and proton) transport capacity an advantage during muscle activity.

Potent Blockers of the Monocarboxylate Transporter MCT1: Novel Immunomodulatory Compounds.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The following letters are in response to Point:Counterpoint series “Lactic acid accumulation is an advantage/disadvantage during muscle activity”

The following letters are in response to Point:Counterpoint series “Lactic acid accumulation is an advantage/disadvantage during muscle activity” that appeared in the April issue ( J Appl Physiol 100: 1410–1414, 2006; ). To the Editor :

Influence of Ageing on Monocarboxylate Transporter in Human Skeletal Muscle

Isoforms of the lactate transporter known as monocarboxylate transporter (MCT) are increased in skeletal muscle following vigorous exercise. To date, no studies have investigated how age influences the content of MCT1 and MCT4 in human skeletal muscle. PURPOSE: To test the hypothesis that, compared with the vastus lateralis (VL) of untrained subjects, the VL of endurance trained athletes would exhibit an increase in the content of MCT1 and MCT4. METHODS: Healthy male subjects (n=33) were recruited and assigned to one of six groups based on age and training status (young trained (YT), n=6, 23.2 ± 0.8 yr, young untrained (YUT), n=6, 22.3 ± 2.9 yr, middle-aged trained (MAT), n=6, 46.3 ± 4.3 yr, middle-aged untrained (MAUT), n=5, 44.6 ± 3.9 yr, senior trained (ST) n=6, 58.7 ± 3.4 yr, and senior untrained (SUT), n=4, 58.8 ± 1.9 yr. The inclusion criteria required that all trained subjects spend at least 8 hours per week training above 60% of their VO2max, and that all untrained subjects have a self-reported average of less than 3 hours per week of moderate physical activity. Assignment to training status was supported by the completion of a VO2max test, by the wearing of an Actiheart™ downloadable heart rate monitor to record daily heart rates, and by completing a physical activity questionnaire. Muscle biopsies of the VL were analyzed Western Blot for MCT1 and MCT4 content. RESULTS: There were no significant age-related differences in MCT1 or MCT4 content (Y vs. MA vs. S, P > 0.05), and no significant differences in MCT1 or MCT4 content related to training status were observed (T vs. UT, P > 0.05), although a trend toward decreased content of both isoforms was found in the untrained group when compared with the trained group. CONCLUSION: The results of this first study investigating an effect of age and training status on moncarboxylate transporters in human skeletal muscle suggest that age does not influence the level of lactate transporter proteins MCT1 and MCT4. Supported by a grant from The Gatorade Sports Science Institute.

Transient Elevations in Lactate Concentration Increase the Expression of Lactate Transporter MCT1

Transient Elevations in Lactate Concentration Increase the Expression of Lactate Transporter MCT1

The Plasma Membrane Lactate Transporter MCT4, but Not MCT1, Is Up-regulated by Hypoxia through a HIF-1α-dependent Mechanism

The monocarboxylate transporter MCT4 mediates lactic acid efflux from most tissues that are dependent on glycolysis for their ATP production. Here we demonstrate that expression of MCT4 mRNA and protein was increased >3-fold by a 48-h exposure to 1% O(2), whereas MCT1 expression was not increased. The effect was mimicked by CoCl(2) (50 microm), suggesting transcriptional regulation by hypoxia-inducible factor 1alpha (HIF-1alpha). The predicted promoters for human MCT1, MCT2, and MCT4 were cloned into the pGL3 vector and shown to be active (luciferase luminescence) under basal conditions. Only the MCT4 promoter was activated (>2-fold) by hypoxia. No response was found in cells lacking HIF-1alpha. Four potential hypoxia-response elements were identified, but deletion analysis implicated only two in the hypoxia response. These were just upstream from the transcription start site and also found in the mouse MCT4 promoter. Mutation of site 2 totally abolished the hypoxic response, whereas mutation of site 1 only reduced the response. Gel-shift analysis demonstrated that nuclear extracts of hypoxic but not normoxic HeLa cells contained two transcription factors that bound to DNA probes containing these hypoxia-response elements. The major shifted band was abolished by mutation of site 2, and supershift analysis confirmed that HIF-1alpha bound to this site. Binding of the second factor was abolished by mutation of site 1. We conclude that MCT4, like other glycolytic enzymes, is up-regulated by hypoxia through a HIF-1alpha-mediated mechanism. This adaptive response allows the increased lactic acid produced during hypoxia to be rapidly lost from the cell.

Evidence of a mitochondrial lactate oxidation complex at mitochondrial inner membrane in mammalian skeletal muscle cells

Results of previous studies suggested a role of mitochondria (MI) in an Intracellular Lactate Shuttle. Therefore, using confocal laser-scanning microscopy (CLSM) we examined the cellular locations of mitochondria, lactate dehydrogenase (LDH), the lactate/pyruvate transporter MCT1, and CD147, a purported chaperone protein for MCT1 in rat-derived L6 skeletal muscle cell line and rat plantaris muscle. As well, the interaction of MCT1 and related proteins was confirmed by Western blotting of mitochondrial (MI) cell fractions and by immunoprecipitation from L6 lysates followed by immunoblotting. CLSM showed that LDH, MCT1 and CD147 are co-localized with the mitochondrial reticulum in L6 cells and rat plantaris muscle. Western blots showed that cytochrome oxidase (COX), NADH-dehydrogenase (NADH-dh), LDH, MCT1 and CD147 are abundant in MI fractions of L6 cells. Interactions among cytochrome oxidase (COX), MCT1 and CD147 in mitochondria was confirmed by immunoblotting after immunoprecipitation in L6 cells. However, we did not find association between NADH-dh and MCT1 and LDH in L6 cells. These findings further our understanding of the mitochondrial role in cellular lactate oxidation and may be interpreted to indicate presence of a terminal mitochondrial electron transport chain component, the lactate oxidation complex, in mammalian skeletal muscle. Supported by NIH grant AR050459

Potent blockers of the monocarboxylate transporter MCT1: Novel immunomodulatory compounds

A novel series of potent blockers of the monocarboxylate transporter, MCT1, is disclosed. From very potent but lipophilic lead compounds, systematic changes to all parts of the molecule, targeting reduction in log D, afforded compounds with significantly improved overall properties. These compounds show potent immunomodulatory activity.

Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex

Results of previous studies suggested a role of mitochondria in intracellular and cell-cell lactate shuttles. Therefore, by using a rat-derived L6 skeletal muscle cell line and confocal laser-scanning microscopy (CLSM), we examined the cellular locations of mitochondria, lactate dehydrogenase (LDH), the lactate-pyruvate transporter MCT1, and CD147, a purported chaperone protein for MCT1. CLSM showed that LDH, MCT1, and CD147 are colocalized with the mitochondrial reticulum. Western blots showed that cytochrome oxidase (COX), NADH dehydrogenase, LDH, MCT1, and CD147 are abundant in mitochondrial fractions of L6 cells. Interactions among COX, MCT1, and CD147 in mitochondria were confirmed by immunoblotting after immunoprecipitation. These findings support the presence of a mitochondrial lactate oxidation complex associated with the COX end of the electron transport chain that might explain the oxidative catabolism of lactate and, hence, mechanism of the intracellular lactate shuttle.