Showing papers on "Monocarboxylate transporter published in 2006"

Comparison of Metabolic Pathways between Cancer Cells and Stromal Cells in Colorectal Carcinomas: a Metabolic Survival Role for Tumor-Associated Stroma

Abstract: Understanding tumor metabolism is important for the development of anticancer therapies. Immunohistochemical evaluation of colorectal adenocarcinomas showed that cancer cells share common enzyme/transporter activities suggestive of an anaerobic metabolism [high lactate dehydrogenase 5 (LDH5)/hypoxia-inducible factor alphas (HIFalphas)] with high ability for glucose absorption and lactate extrusion [high glucose transporter 1 (GLUT1)/monocarboxylate transporter (MCT1)]. The tumor-associated fibroblasts expressed proteins involved in lactate absorption (high MCT1/MCT2), lactate oxidation (high LDH1 and low HIFalphas/LDH5), and reduced glucose absorption (low GLUT1). The expression profile of the tumor-associated endothelium indicated aerobic metabolism (high LDH1 and low HIFalphas/LDH5), high glucose absorption (high GLUT1), and resistance to lactate intake (lack of MCT1). It is suggested that the newly formed stroma and vasculature express complementary metabolic pathways, buffering and recycling products of anaerobic metabolism to sustain cancer cell survival. Tumors survive and grow because they are capable of organizing the regional fibroblasts and endothelial cells into a harmoniously collaborating metabolic domain.

Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice.

Abstract: Mutations of the X-linked thyroid hormone (TH) transporter (monocarboxylate transporter, MCT8) produce in humans unusualabnormalitiesofthyroidfunctioncharacterizedbyhigh serum T3 and low T4 and rT3. The mechanism of these changes remains obscure and raises questions regarding the regulation of intracellular availability and metabolism of TH. To study the pathophysiology of MCT8 deficiency, we generated Mct8 knockout mice. Male mice deficient in Mct8 (Mct8 /y ) replicate the thyroid abnormalities observed in affected men. TH deprivation and replacement with L-T3 showed that suppression of TSH required higher serum levels T3 in Mct8 /y than wild-type (WT) littermates, indicating hypothalamus and/or thyrotroph resistance to T3. Furthermore, T4 is required to maintain the high serum T3 level because the latter was not different between the two genotypes during administration of T3. Mct8 /y mice have 2.3-fold higher T3 content in liver associated with 6.1- and 3.1-fold increase in deiodinase 1 mRNA and enzymatic activity, respectively. The relative T3 excess in liver of Mct8 /y mice produced a decrease in serum cholesterol (79 18 vs. 137 38 mg/dl in WT) and an increase in alkaline phosphatase (107 23 vs. 58 3 U/liter in WT) levels. In contrast, T3 content in cerebrum was 1.8-fold lower in Mct8 /y mice, associated with a 1.6- and 10.6-fold increase in D2 mRNA and enzymatic activity, respectively, as previously observed in TH-deprived WT mice. We conclude that cell-specificdifferencesinintracellularTHcontentduetodifferences incontributionofthevariousTHtransportersareresponsible for the unusual clinical presentation of this defect, in contrast to TH deficiency. (Endocrinology 147: 4036–4043, 2006)

Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism.

Abstract: Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability.

The H+-linked monocarboxylate transporter (MCT1/SLC16A1): a potential therapeutic target for high-risk neuroblastoma.

Abstract: Neuroblastomas produce high amounts of lactic acid and upregulate the H(+)-linked monocarboxylate transporter isoform 1 (MCT1/SLC16A1). We found elevated MCT1 mRNA levels in fresh neuroblastoma biopsy samples that correlated positively with risk of fatal disease and amplification of the "proto-oncogenic" transcription factor MYCN. We further investigated MCT as a potential therapeutic target in vitro. The neuroblastoma cell lines evaluated were Sk-N-SH, CHP134, IMR32, and NGP. All lines exhibited decreased intracellular pH at low tumor-like extracellular pH. Lonidamine or exogenous lactate further lowered intracellular pH. Immediate early lowering of intracellular pH with lonidamine or lactate at extracellular pH 6.5 correlated positively with diminished cell viability within 48 h. These findings indicate that MCT1 is a potential therapeutic target and that neuroblastoma therapy may be enhanced by therapeutic strategies to inhibit or overwhelm MCT. Additional experiments indicated that the mechanism of cell death by lonidamine or exogenous lactate is similar to that obtained using alpha-cyano-4-OH-cinnamate, a well established MCT inhibitor. Because lactate production is also high in melanoma and many other tumor types, MCT inhibitors may have broad application in cancer treatment. Such treatment would have selectivity by virtue of the acidic milieu surrounding tumors, because MCT is increasingly active as extracellular pH decreases below 7.0 and lactic acid production increases.

Cellular expression of monocarboxylate transporters (MCT) in the digestive tract of the mouse, rat, and humans, with special reference to slc5a8

Abstract: Short-chain fatty acids (SCFA) are monocarboxylates produced by bacterial fermentation that play a crucial role in maintaining homeostasis in the large intestine. Two major transporters for SCFA, monocarboxylate transporter (MCT) and slc5a8 (or SMCT), exist in the digestive tract. The present histochemical study using in situ hybridization and immunohistochemistry revealed the distribution and subcellular localization of the MCT family in the digestive tract of mice, rats, and humans, comparing these with that of slc5a8. The expression of mucosal MCT1 in the mouse and rat was most intense in the cecum, followed by the colon, but low in the stomach and small intestine. Among other MCT subtypes, only MCT2 was detected in the parietal cell region of the gastric mucosa. Slc5a8 had predominant expression sites in the distal half of the large bowel and in the most terminal ileum. The mucosal MCT1 was localized in the basolateral membrane of enterocytes, while slc5a8 was restricted to the apical cell membrane, suggesting the involvement of slc5a8 in the uptake of luminal SCFA, and of MCT1 in the efflux of SCFA and monocarboxylate metabolites towards blood circulation. The large intestine expressed both types of the transporter, but their distribution patterns differed along the longitudinal axis of the intestine and along the perpendicular axis of the mucosa.

Identity of SMCT1 (SLC5A8) as a neuron‐specific Na+‐coupled transporter for active uptake of l‐lactate and ketone bodies in the brain

Abstract: SMCT1 is a sodium-coupled (Na+-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, β-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, α-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na+-coupled and electrogenic. The Michaelis constant is 1.4 ± 0.1 mm for β-d-hydroxybutyrate, 0.21 ± 0.04 mm for acetoacetate and 0.21 ± 0.03 mm for α-ketoisocaproate. The Na+ : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na+-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na+-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na+ gradient and hence, contribute to the maintenance of the energy status and function of neurons.

Na(+)/monocarboxylate transport (SMCT) protein expression correlates with survival in colon cancer: molecular characterization of SMCT.

Abstract: We report an extensive characterization of the Na+/monocarboxylate transporter (SMCT), a plasma membrane protein that mediates active transport of monocarboxylates such as propionate and nicotinate, and we show that SMCT may play a role in colorectal cancer diagnosis. SMCT, the product of the SLC5A8 gene, is 70% similar to the Na+/I− symporter, the protein that mediates active I− uptake in the basolateral surface of thyrocytes and other cells. SMCT was reported in the apical surface of thyrocytes and formerly proposed also to transport I− and was called the apical I− transporter. However, it is now clear that SMCT does not transport I−. Here we demonstrate a high-affinity Na+-dependent monocarboxylate transport system in thyroid cells, which is likely to be SMCT. We show that, whereas thyroidal Na+/I− symporter expression is thyroid-stimulating hormone (TSH)-dependent and basolateral, SMCT expression is TSH-independent and apical not only in the thyroid but also in kidney and colon epithelial cells and in polarized Madin–Darby canine kidney cells. We determine the kinetic parameters of SMCT activity and show its inhibition by ibuprofen (Ki = 73 ± 9 μM) in Xenopus laevis oocytes. SMCT was proposed to be a tumor suppressor in colon cancer [Li, H., et al. (2003) Proc. Natl. Acad. Sci. USA 100, 8412–8417]. Significantly, we show that higher expression of SMCT in colon samples from 113 colorectal cancer patients correlates with longer disease-free survival, suggesting that SMCT expression may be a favorable indicator of colorectal cancer prognosis.

Mechanisms of disease: psychomotor retardation and high T3 levels caused by mutations in monocarboxylate transporter 8.

Abstract: Thyroid hormone transport across the plasma membrane is essential for hormone functions. As detailed here, novel mutations in monocarboxylate transporter 8 reveal important roles in thyroid hormone access to the brain and might explain the pathogenesis of Allan–Herndon–Dudley syndrome, which is now known to feature thyroid hormone resistance. The actions and the metabolism of thyroid hormone are intracellular events that require the transport of iodothyronines across the plasma membrane. It is increasingly clear that this process does not occur by simple diffusion, but is facilitated by transport proteins. Only recently have iodothyronine transporters been identified at the molecular level, of which organic anion transporting polypeptide 1C1 and monocarboxylate transporter 8 (MCT8) deserve special mention, because of their high activity and specificity for iodothyronines. Organic anion transporting polypeptide 1C1 is almost exclusively expressed in brain capillaries, and may be crucial for the transport of the prohormone T4 across the blood–brain barrier. MCT8 is also expressed in the brain—in particular, in neurons—but also in other tissues. MCT8 seems to be especially important for the uptake of active hormone T3 into neurons, which is essential for optimal brain development. T3 is produced from T4 by type 2 deiodinase in neighboring astrocytes. Neurons express type 3 deiodinase, the enzyme that terminates T3 activity. The SLC16A2 (formerly MCT8) gene is located on chromosome Xq13.2 and has recently been associated with a syndrome combining severe, X-linked, psychomotor retardation and high serum T3 levels. In over 20 families, where affected males have developed this syndrome, several mutations in MCT8 have been identified. The disease mechanism is thought to involve a defect in the neuronal entry of T3 and, therefore, in the action and metabolism of T3 in these cells. This defect results in impaired neurological development and a decrease in T3 clearance.

Testosterone increases lactate transport, monocarboxylate transporter (MCT) 1 and MCT4 in rat skeletal muscle

Abstract: We have examined the effects of administration of testosterone for 7 days on monocarboxylate transporter (MCT) 1 and MCT4 mRNAs and proteins in seven metabolically heterogeneous rat hindlimb muscles and in the heart. In addition, we also examined the effects of testosterone treatment on plasmalemmal MCT1 and MCT4, and lactate transport into giant sarcolemmal vesicles prepared from red and white hindlimb muscles and the heart. Testosterone did not alter MCT1 or MCT4 mRNA, except in the plantaris muscle. Testosterone increased MCT1 (20%-77%, P 0.05), and MCT 4 mRNA and protein were not detected. There was no correlation between the testosterone-induced increments in MCT1 and MCT4 proteins. Muscle fibre composition was not associated with testosterone-induced increments in MCT1 protein. In contrast, there was a strong positive relationship between the testosterone-induced increments in MCT4 protein and the fast-twitch fibre composition of rat muscles. Lactate transport into giant sarcolemmal vesicles was increased in red (23%, P< 0.05) and white muscles (21%, P< 0.05), and in the heart (58%, P< 0.05) of testosterone-treated animals (P< 0.05). However, plasmalemmal MCT1 protein (red, +40%, P< 0.05; white, +39%, P< 0.05) and plasmalemmal MCT4 protein (red, +25%, P< 0.05; white, +48%, P< 0.05) were increased only in skeletal muscle. In the heart, plasmalemmal MCT1 protein was reduced (-20%, P< 0.05). In conclusion, these studies have shown that testosterone induces an increase in both MCT1 and MCT4 proteins and their plasmalemmal content in skeletal muscle. However, the testosterone-induced effect was tissue-specific, as MCT1 protein expression was not altered in the heart. In the heart, the testosterone-induced increase in lactate transport cannot be explained by changes in plasmalemmal MCT1 content, but in skeletal muscle the increase in the rate of lactate transport was associated with increases in plasmalemmal MCT1 and MCT4.

Induction of monocarboxylate transporter 2 expression and ketone transport following traumatic brain injury in juvenile and adult rats.

Abstract: Based on recent work demonstrating age-dependent ketogenic neuroprotection after traumatic brain injury (TBI), it was hypothesized that the neuroprotection among early post-weaned animals was related to induced cerebral transport of ketones after injury. Regional changes in monocarboxylate transporter 2 (MCT2) were acutely examined with immunohistochemistry after sham surgery or controlled cortical impact injury among postnatal day 35 and adult rats. Both ages showed elevated MCT2 expression in the ipsilateral cerebral vasculature after TBI. Using Western blotting, MCT2 expression was 80–88% greater in microvessels isolated from postnatal day 35 rats at all time points relative to adults. The increased MCT2 expression was temporally correlated with an age-related increase in cerebral uptake of ketones, when ketones were made available after injury.

Metabolic remodeling of malignant gliomas for enhanced sensitization during radiotherapy: An in vitro study

Abstract: Glioblastoma multiforme (GBM) is the most common of all primary brain tumors. It is also the most aggressive, with patient survival being dismal with conventional therapies. Despite numerous clinical approaches, little progress in prolonging patient survival has been achieved in the past 20 years. Similar to most highly malignant tumors, these aggressive gliomas are highly glycolytic, producing large amounts of lactic acid as a metabolic by-product. This aberrant metabolic phenotype is known as aerobic glycolysis because it occurs even in the presence of oxygen (2, 27, 30, 39). In contrast, normal cells produce lactate mainly under anaerobic conditions (anaerobic glycolysis). Thus, such malignant tumors require an effective mechanism for the rapid disposal of accumulating lactic acid to the tumor microen-vironment. Tumors, as well as normal tissue, use a family of transmembrane transporters (monocarboxylate transporter isoforms 1 to 9) (14, 15) for this purpose, in which isoforms with different substrate affinities are expressed in a tissue-specific manner throughout the organism. Previous studies from our laboratory (26) indicated differential expression of these isoforms in normal brain versus malignant glioma, in which the Type 3 isoform was predominantly expressed in normal tissue, whereas Type 1 and 2 isoforms were highly expressed in GBMs. Our in vitro studies using small interfering ribonucleic acids indicated that selective blocking of lactate efflux (by targeting glioma-expressed mono-carboxylate transporter isoforms) induced critical metabolite changes in the tumors, resulting in cell death (26). Intracellular pH changes were observed (a fourfold increase in H+ concentrations, resulting in a pH change from 7.4 to 6.8) as well as indications of altered cofactor profiles (oxidized nicotinamide adenine dinucleotide [NAD+]/reduced nicotinamide adenine dinucleotide [NADH] and oxidized nicotinamide adenine din-ucleotide phosphate [NADP+]/reduced nicotinamide adenine dinucleotide phosphate [NADPH]). Thus, tumor-specific blocking of these transporters presented an attractive anticancer therapeutic target that may debilitate such malignant tumors, either via disruption of energy flux through the tumor cells or via metabolite remodeling within the tumors. α-cyano-4-hydroxycinnamic acid (ACCA; CHC; mw 189.2) is a known competitive inhibitor of mammalian lactate transporters (13, 36) that also inhibits pyruvate entry into mitochon-dria (13). We hypothesized that application of ACCA to malignant gliomas might disrupt energy-generating metabolic cascades (both aerobic glycolysis and oxidative phosphorylation), promoting both induction of apoptosis and disruption of pyruvate-derived antioxidant biosynthesis via mitochondria-derived NADH. Disruption of glycolysis would also affect the pentose phosphate pathway-derived NADPH/glutathione-generating mechanisms and NADH synthesis at the glycer-aldehydes-3-P-dehydrogenase step of the glycolytic pathway, thus depleting the overall capacity of the tumor to defend against free radical-induced cell damage. We reasoned that a combined experimental strategy of inhibiting lactate efflux with concomitant treatment with radiation would result in the targeted gliomas being highly radiosensitized towards free radical-induced damage and also energetically debilitated because of the disruption of key metabolic pathways. Thus, a combined protocol of chemotherapy and radiotherapy should provide a more effective GBM-targeting strategy. Radiotherapy has been one of the more effective modalities for therapy of malignant glioma, and dosages of 60 Gy are routinely used (10, 21). However, these can result in toxicity to the surrounding normal brain tissue, including leukoencephalopathy and necrosis (40). Therefore, strategies to enhance radiosensitivity of targeted glioma would permit lower radiation doses to be used, thereby vastly minimizing the frequently observed normal tissue damage while maintaining the efficacy of radiotherapy treatment. Low-dose radiotherapy using doses between 0.5 and 1.0 Gy has been suggested as a potential strategy to develop improved radiotherapeutic regimens while minimizing excessive radiation-induced injury (3, 17, 22, 25). In the present investigation, we first established that ACCA acts only as a cell-surface inhibitor of lactate transporters in the model U-87MG glioma cells and that it does not internalize. Thus, we reasoned that a combined strategy of first using ACCA as an inhibitor to disrupt glioma metabolism would reduce cellular radioprotectant generation and that subjecting the targeted cells to low-dose irradiation would then provide an avenue for an improved chemoradiotherapy regimen against malignant glioma while significantly minimizing damage to normal tissues. Using in vitro studies, we first used magic angle spinning (MAS) 1H magnetic resonance spectroscopy (MRS) to demonstrate that radioprotective metabolites are significantly reduced when lactate efflux is blocked in glioma cells and that the treated cells are highly susceptible to low-dose radiation-induced cellular damage, resulting in cell death.

Differential metabolic adaptation to acute and long-term hypoxia in rat primary cortical astrocytes.

Abstract: Brain astrocytes provide structural and metabolic support to surrounding cells during ischemia. Glucose and oxygen are critical to brain function, and glucose uptake and metabolism by astrocytes are essential to their metabolic coupling to neurons. To examine astrocyte metabolic response to hypoxia, cell survival and metabolic parameters were assessed in rat primary cortical astrocytes cultured for 3 weeks in either normoxia or in either 1 day or 3 weeks sustained hypoxia (5% O2). Although cell survival and proliferation were not affected by the mildly hypoxic environment, substantial differences in glucose consumption and lactate release after either acute or prolonged hypoxia suggest that astrocyte metabolism may contribute to their adaptation. Hypoxia over a period of 1 day increased glucose uptake, lactate release, and glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1) expression, whereas hypoxia over a period of 3 weeks resulted in a decrease of all parameters. Furthermore, increased glucose uptake at 1 day of hypoxia was not inhibited by cytochalasin B suggesting the involvement of additional glucose transporters. We uncovered hypoxia-regulated expression of sodium-dependent glucose transporters (SGLT1) in astrocytes indicating a novel adaptive strategy involving both SGLT1 and GLUT1 to regulate glucose intake in response to hypoxia. Overall, these findings suggest that although increased metabolic response is required for the onset of astrocyte adaptation to hypoxia, prolonged hypoxia requires a shift to an energy conservation mode. These findings may contribute to the understanding of the relative tolerance of astrocytes to hypoxia compared with neurons and provide novel therapeutic strategies aimed at maintaining brain function in cerebral pathologies involving hypoxia.

Interaction of ibuprofen and other structurally related NSAIDs with the sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8).

Abstract: Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na+-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [14C]-nicotinate and [3H]-ibuprofen for direct measurements of uptake in oocytes. In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na+ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 ± 16 μM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na+ dependent, and competitive. However, there was no uptake of [3H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [14C]-nicotinate was demonstrable under identical conditions. Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.

Modulation and compensation of the mRNA expression of energy related transporters in the brain of glucose transporter 1-deficient mice.

Abstract: Facilitative glucose transporter 1 (GLUT1) is the molecule responsible for the entry of glucose into the brain, and its mutation is known as GLUT1 deficiency syndrome (GLUT1DS) in humans. To clarify the effect of GLUT1 gene deficiency, we have produced GLUT1-deficient mice, and investigated the developmental expression of GLUT1, monocarboxylate transporter 1 (MCT1) and MCT2 in the brains of these mice. Since the homozygotes were found to be embryonically lethal and the heterozygotes exhibited no abnormalities, GLUT1deficiency was examined using heterozygote mice. GLUT1 deficiency did not significantly affect the mRNA levels of GLUT1 at P0, P7 and in adults, or the levels of MCTs at P7, P14 and in adults. The GLUT1 level at P14 was reduced by 46.9%, although this was not statistically significant. The MCTs levels at P0 were increased about 2.0-fold in the deficient mice compared with the wild type. Furthermore, at P0, GLUT1 mRNA levels in wild type females were 1.91-fold higher than in wild type males. These results suggest that GLUT1 deficiency affects GLUT1 mRNA expression in the infant brain, and that of MCT1 and MCT2 in the neonatal brain. Furthermore, a compensatory effect of GLUT1 expression was observed in the brain of adult deficient mice. These effects of GLUT1 deficiency in the brain provide a molecular basis to assist in our understanding of the symptoms of GLUT1DS.

Placental lactate transporter activity and expression in intrauterine growth restriction.

Abstract: To compare lactate uptake in the microvillous plasma membrane (maternal facing [MVM]) in term and preterm placentas in intrauterine growth restriction (IUGR) and appropriate weight for gestational age (AGA) controls, and in the basal plasma membrane fetal facing [BM]) at term. In addition, we examine the expression of monocarboxylate transporters (MCT1 and MCT4). We measured [14C] L-lactate uptakes into vesicles prepared from MVM and BM, stimulated by an inwardly directed H gradient. MCT expression was examined by Western blotting. In term placentas, mean (± SE) [14C] L-lactate uptake into MVM vesicles of the IUGR (n = 6) and AGA (n = 11)groups at initial rate was similar (15.4 ±2.3 versus 15. 0 ± 1.1 pmol/mg protein/20 s). In preterm placentas, in IUGR (n = 3) and AGA (h = 3) groups, [14G] L-lactate uptake into MVM was also not significantly different. In BM vesicles from term placentas, [14C] L-lactate uptake was significantly lower in IUGR (n = 5) than in AGA (n = 6) controls (3.6 ± 0.4 versus 5.6 ± 0.6 pmol/mg protein/20 s, P <. 05). MCT1 and MCT4 were expressed in BM vesicles, but there was no difference in expression between the IUGR and AGA groups. These findings suggest that in IUGR placental lactate transport capacity in the BM is reduced, which may adversely affect placental lactate clearance.

Faster lactate transport across red blood cell membrane in sickle cell trait carriers

Abstract: The physical and physiological behavior of sickle cell trait carriers (AS) is somewhat equivocal under strenuous conditions, although this genetic abnormality is generally considered to be a benign disorder. The occurrence of incidents and severe injuries in AS during exercise might be explained, in part, by the lactic acidosis due to a greater lactate influx into AS red blood cells (RBCs). In the present study, the RBC lactate transport activity via the different pathways was compared between AS and individuals with normal hemoglobin (AA). Sixteen Caribbean students, nine AS and seven AA, performed a progressive and maximal exercise test to determine maximal oxygen consumption. Blood samples were obtained at rest to assess haematological parameters and RBC lactate transport activity. Lactate influxes [total lactate influx and monocarboxylate transporter (MCT-1)-mediated lactate influx] into erythrocytes were measured at four external [14C]-labeled lactate concentrations (1.6, 8.1, 41, and 81.1 mM). The two groups had similar maximal oxygen consumption. Total lactate influx and lactate influx via the MCT-1 pathway were significantly higher in AS compared with AA at 1.6, 41, and 81.1 mM. The maximal lactate transport capacity for MCT-1 was higher in AS than in AA. Although AS and AA had the same maximal aerobic physical fitness, the RBCs from the sickle cell trait carriers took up more lactate at low and high concentrations than the RBCs from AA individuals. The higher MCT-1 maximal lactate transport capacity found in AS suggests greater content or greater activity of MCT-1 in AS RBC membranes.

MCT-4, A511/basigin and EF5 expression patterns during early chick cardiomyogenesis indicate cardiac cell differentiation occurs in a hypoxic environment

Abstract: We have identified the presence of the hypoxia marker EF5 in the stage 4/5 chick heart fields. This suggests that cardiac cell differentiation occurs in a relatively anaerobic environment. Monocarboxylate transporter (MCT) studies in adult cardiac myocytes have demonstrated that MCTs catalyze proton-linked pyruvate and lactate transport activity. 5A11/Basigin is an ancillary protein that targets MCTs to the plasma membrane for their function. MCT-4 expression is most evident in cells with a high glycolytic rate associated with hypoxic energy production. Subsequent to the immunohistochemical localization of EF5 in the early heart field, we continued in our analysis during stages 5 to 12 for the expression of indicators of cellular glycolytic metabolism in the developing heart, such as MCT-4, MCT-1, and 5A11 (Basigin/CD147). Our observations indicate that MCT-4 and 5A11/Basigin are expressed early, in a differential left-right pattern, in the bi-lateral plate mesoderm, as the cardiac compartment is forming. At stage 11, MCT-4/5A11 continues to be highly expressed in the myocardial wall of the looping heart, but not in the dorsal mesocardium. RT-PCR analyses for MCT-1, -4, and 5A11 indicate that MCT-4 and 5A11 are expressed throughout precardiac, embryonic, and fetal stages in the heart. MCT-1 is first detected in the heart on embryonic day 3 and then remains expressed throughout development to hatching. These results indicate that cardiac precursor cells are equipped for differentiating in a hypoxic environment using anaerobic metabolism for energy production. Developmental Dynamics 235:124–131, 2006. © 2005 Wiley-Liss, Inc.

Skeletal muscle expression of LDH and monocarboxylate transporters in growing rats submitted to protein malnutrition.

Abstract: In different circumstances such as infant malnutrition, old age or chronic disease, decline of muscular strength, particularly anaerobic power, is shown. In this context, our laboratory, has demonstrated a decrease in anaerobic glycolytic power in pre-pubertal Bolivian children living at low and high altitude and suffering from marginal protein malnutrition. To bring molecular support to the relationship between protein malnutrition and anaerobic glycolytic metabolism, we studied the impact of prolonged protein malnutrition on lactate metabolism in different muscles of growing rats. Lactate dehydrogenase (LDH), monocarboxylate transporters (MCT1, MCT4) and membrane protein CD147 were chosen as specific markers of anaerobic glycolytic metabolism. Two groups of 10 weaning male rats were fed for 10 weeks either ad libitum with a well-balanced diet containing 18% protein or an isocaloric-diet containing 8% protein. LDH activity and mRNA amounts of LDH isoforms, MCT, CD147 were measured. Protein deprivation during rat growth induced a decrease of LDH specific activity in skeletal muscles (mean value of −41%), accompanied by isoform distribution modifications in soleus, but not in glycolytic muscles (extensor digitorum longus (EDL) or plantaris). A reduction in mRNA amounts encoding the LDH A and B subunits was observed in EDL. A decrease in LDH B mRNA amounts was monitored in plantaris, whereas no modification in both LDH isoform mRNA quantities was observed in soleus. MCT1 mRNA quantities were decreased in EDL but MCT4 mRNA quantities remained stable. CD147 mRNA amounts were unchanged except for EDL with a 42% increase. The global decreases of LDH activity, LDH and MCT gene expressions in growing rat skeletal muscles support the observed alterations of lactate metabolism associated with lowered muscular anaerobic performances in protein malnutrition.

Vagal complex monocarboxylate transporter-2 expression during hypoglycemia.

Abstract: Astrocytic provision of lactate provision to neurons may be a critical indicator of substrate fuel availability in metabolic sensing sites in the brain, including the hindbrain dorsal vagal complex. We examined the hypothesis that vagal complex monocarboxylate transporter protein levels are gender dependent and estrogen dependent, and that estrogen influences adaptation of these protein responses during repeated insulin-induced hypoglycemia. Western blot analyses showed that male and estrogen-treated ovariectomized female rats exhibit opposite changes in monocarboxylate transporter-2 levels after one insulin injection, as well as divergent patterns of adaptation to this metabolic challenge. The data suggest that sex differences in hypoglycemic patterns in vagal complex lactate transport may underlie disparate signaling of cellular energy imbalance.

Functional Purification of the Monocarboxylate Transporter of the Yeast Candida utilis

Abstract: Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-β-d-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.

Characterization of monocarboxylate permeases in yeast : functional and structural analysis

Abstract: Short chain carboxylic acids are important compounds that result from normal cell metabolism and that can be used as sole carbon and energy source by different organisms. In this context the study of monocarboxylate permeases is of great significance since the uptake of these nutrients across cellular membranes is essential for the metabolism of most cells. With the work presented in this thesis we seek to increase the current knowledge on yeast monocarboxylate permeases by studying the Saccharomyces cerevisiae lactate/pyruvate proton symporter, encoded by the JEN1gene. The first step of this work was the heterologous expression of the JEN1 gene in Pichia pastoris. JEN1 was cloned in an integrative vector (pPICZB) and a replicative vector (pZPARS), and expressed in two different strains, Mut+ and Muts. JEN1 expression was confirmed in 24 hour methanol-induced cells both at mRNA and protein level. Maximum lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol s-1 mg-1 dry weight, representing a 5 fold increase in the permease activity when compared to the wild-type lactate-grown cells. In the second part of this work the lactate permease activity was reconstituted in hybrid vesicles, obtained by fusion of plasma membranes from P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. The reconstituted lactate uptake activity presented similar properties with those of the permease evaluated in S. cerevisiae in what concerns the proton symporter mechanism and the inhibitors tested. This work demonstrated that S. cerevisiae Jen1p is a fully functional lactate transporter. The molecular identification of other genes encoding monocarboxylate permeases led us to study the uptake of monocarboxylates in the yeast Candida albicans. A lactate permease was biochemically identified in C. albicans RM1000 lactate-grown cells. Inhibition assays demonstrated that lactate uptake was competitively inhibited by pyruvic and propionic acids but not by acetic acid, that behaved as a non-competitive substrate. A ScJEN1 homologue search within the C. albicans genome revealed the existence of an ORF possessing 61% similarity, that was named CaJEN1. Deletions of both CaJEN1 alleles resulted in absence of mRNA detection and in the lack of measurable lactate uptake. In the presence of glucose no CaJEN1 expression and lactate permease activity were detected. Heterologous expression of CaJEN1 was performed in S. cerevisiae jen1Δ strain, but no activity for the permease was achieved with the native CaJEN1 gene. Due to a difference in the C. albicans genetic code, site directed mutagenesis was performed to re-establish the CaJen1p codon 217 as a serine when expressed in S. cerevisiae, and permease activity was recovered. This was the confirmation that the CaJEN1 gene codes for a monocarboxylate transporter. Additionally, studies with a CaCAT8 deletion strain demonstrated that the CaJEN1 transcription level is influenced by the expression…