Karin Pierre

Monocarboxylate transporters in the central nervous system : distribution, regulation and function

Monocarboxylate transporters (MCTs) are proton‐linked membrane carriers involved in the transport of monocarboxylates such as lactate, pyruvate, as well as ketone bodies. They belong to a larger family of transporters composed of 14 members in mammals based on sequence homologies. MCTs are found in various tissues including the brain where three isoforms, MCT1, MCT2 and MCT4, have been described. Each of these isoforms exhibits a distinct regional and cellular distribution in rodent brain. At the cellular level, MCT1 is expressed by endothelial cells of microvessels, by ependymocytes as well as by astrocytes. MCT4 expression appears to be specific for astrocytes. By contrast, the predominant neuronal monocarboxylate transporter is MCT2. Interestingly, part of MCT2 immunoreactivity is located at postsynaptic sites, suggesting a particular role of monocarboxylates and their transporters in synaptic transmission. In addition to variation in expression during development and upon nutritional modifications, new data indicate that MCT expression is regulated at the translational level by neurotransmitters. Understanding how transport of monocarboxylates is regulated could be of particular importance not only for neuroenergetics but also for areas such as functional brain imaging, regulation of food intake and glucose homeostasis, or for central nervous system disorders such as ischaemia and neurodegenerative diseases.

Cellular and subcellular distribution of monocarboxylate transporters in cultured brain cells and in the adult brain

Monocarboxylate transporters (MCTs) are involved in the uptake and release of lactate, pyruvate, and ketone bodies. Studies of their distribution at both the mRNA and protein levels have highlighted the specific expression of MCT1, MCT2, and more recently MCT4 in the central nervous system. MCT1 was found strongly expressed by cortical astrocytes both in vitro and in vivo. It was also found at high levels on blood vessels, ependymocytes, and glia limitans. A subset of neurons in vitro exhibited a weak but significant MCT1 expression. In contrast, it was determined that MCT2 represents the predominant neuronal MCT on cultured neurons as well as on neurons throughout the brain parenchyma. At the subcellular level, part of MCT2 is located in postsynaptic densities. Specific populations of astrocytes in the white matter also exhibited MCT2 expression in the rat, but not in the mouse brain. MCT4 was found exclusively in astrocytes in several areas including the cortex, the hippocampus, and the cerebellum. MCT2 expression increased in cultured neurons with days in vitro commensurate with increased synapse formation. Moreover, a significant increase in MCT2 expression was observed in cultured neurons exposed to noradrenaline, an effect involving a regulation at the translational level. The description of MCTs on different cell types in the central nervous system together with clear evidence for regulation of their expression further emphasize the important role that monocarboxylates, and particularly lactate, might play in brain energy metabolism not only during development but also in the adult.

MCT2 is a major neuronal monocarboxylate transporter in the adult mouse brain

Although previous Northern blot and in situ hybridization studies suggested that neurons express the monocarboxylate transporter MCT2, subsequent immunohistochemical analyzes either failed to confirm the presence of this transporter or revealed only a low density of immunolabeled neuronal processes in vivo. The authors report that appropriate section pretreatment (brief warming episode or proteinase K exposure) leads to extensive labeling of the neuropil, which appears as tiny puncta throughout the whole mouse brain. In addition, intense MCT2 immunoreactivity was found in cerebellar Purkinje cell bodies and their processes, on mossy fibers in the cerebellum, and on sensory fibers in the brainstem. Double immunofluorescent labeling with appropriate markers and observation with epifluorescence and confocal microscopy did not show extensive colocalization of MCT2 immunoreactivity with presynaptic or postsynaptic elements, but colocalization could be observed occasionally in the cortex with the postsynaptic density protein PSD95. Observations made at the electron microscopic level in the cortex corroborated these results and showed that MCT2 immunoreactivity was associated with wide membrane segments of neuronal processes. These data provide convincing evidence that MCT2 represents a major neuronal monocarboxylate transporter in the adult mouse brain, and further suggest that mature neurons could use monocarboxylates such as lactate as additional energy substrates.

Cell-specific expression pattern of monocarboxylate transporters in astrocytes and neurons observed in different mouse brain cortical cell cultures

Evidence suggests that lactate could be a preferential energy substrate transferred from astrocytes to neurons. Such a process implies the presence of specific monocarboxylate transporters on both cell types. Expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was studied in enriched cultures of mouse cortical astrocytes or neurons. It was observed that, at both the mRNA and the protein levels, astrocytes strongly expressed MCT1 but had very little if any MCT2. By contrast, neurons had high amounts of MCT2 mRNA, although MCT1 mRNA was also detected. Double immunofluorescent labelings with appropriate markers confirmed the cell‐specific preference in the expression of MCT1 and MCT2, but they revealed that a subset of neurons expresses low to moderate levels of MCT1. Parallel immunocytochemical stainings of cultured neurons with the presynaptic marker synaptophysin showed that MCT2 expression is correlated with synaptic development. Although MCT2 and synaptophysin were not colocalized, their distribution was similar, and they were often closely apposed, suggesting that MCT2 could be associated with postsynaptic terminals. Interaction between astrocytes and neurons, as occurring in layered cultures, did not modify the levels of MCT1 and MCT2 expression or their distribution and cell‐specific preference under the conditions used. However, a close apposition between neurites and MCT1‐expressing astrocytic processes was apparent and developed as cultures evolved. In addition to providing an extensive description of MCT distribution in cultured cells, our data underscore the potential of such preparations for future studies on the regulation of MCT expression.

Activation of Astrocytes by CNTF Induces Metabolic Plasticity and Increases Resistance to Metabolic Insults

High energy demands of neurons make them vulnerable to adverse effects of energy impairment. Recently, astrocytes were shown to regulate the flux of energy substrates to neurons. In pathological situations, astrocytes are activated but the consequences on brain energy metabolism are still poorly characterized. We found that local lentiviral-mediated gene transfer of ciliary neurotrophic factor (CNTF), a cytokine known to activate astrocytes, induced a stable decrease in the glycolytic flux in the rat striatum in vivo as measured by 2-[18F]-2-deoxy-d-glucose autoradiography and micro-positron emission tomography imaging. The activity of the mitochondrial complex IV enzyme cytochrome oxidase was not modified, suggesting maintenance of downstream oxidative steps of energy production. CNTF significantly increased the phosphorylation level of the intracellular energy sensor AMP-activated protein kinase (AMPK), supporting a specific reorganization of brain energy pathways. Indeed, we found that different key enzymes/transporters of fatty acids β-oxidation and ketolysis were overexpressed by CNTF-activated astrocytes within the striatum. In primary striatal neuron/astrocyte mixed cultures exposed to CNTF, the AMPK pathway was also activated, and the rate of oxidation of fatty acids and ketone bodies was significantly enhanced. This metabolic plasticity conferred partial glial and neuronal protection against prolonged palmitate exposure and glycolysis inhibition. We conclude that CNTF-activated astrocytes may have a strong protective potential to face severe metabolic insults.

Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice.

Monocarboxylate transporters (MCTs) are membrane carriers for lactate and ketone bodies. Three isoforms, MCT1, MCT2 and MCT4, have been described in the central nervous system but little information is available about the regulation of their expression in relation to altered metabolic and/or nutritional conditions. We show here that brains of mice fed on a high fat diet (HFD) up to 12 weeks as well as brains of genetically obese (ob/ob) or diabetic (db/db) mice exhibit an increase of MCT1, MCT2 and MCT4 expression as compared to brains of control mice fed a standard diet. Enhanced expression of each transporter was visible throughout the brain but most prominently in the cortex and in the hippocampus. Using immunohistochemistry, we observed that neurons (expressing mainly MCT2 but also sometimes low levels of MCT1 under normal conditions) were immunolabelled for all three transporters in HFD mice as well as in ob/ob and db/db mice. At the subcellular level, changes were most remarkable in neuronal cell bodies. Western blotting performed on brain structure extracts allowed us to confirm quantitatively the enhancement of MCT1 and MCT2 expression. Our data demonstrate that the expression of cerebral MCT isoforms can be modulated by alterations of peripheral metabolism, suggesting that the adult brain is sensitive and adapts to new metabolic states. This observation could be relevant in the context of obesity development and its consequences for brain function.

Noradrenaline enhances monocarboxylate transporter 2 expression in cultured mouse cortical neurons via a translational regulation

Regulation of the expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was investigated in primary cultures of mouse cortical neurons. Under basal conditions, both MCT immunoreactivities (IR) were found in the cell soma and dendrites, although IR for MCT1 appeared less bright than for MCT2. Treatment of cultured cortical neurons with 100 μm noradrenaline (NA) led, after a few hours, to a striking enhancement in fluorescence intensity associated with MCT2 IR in the cell soma as well as in dendrites. In contrast, MCT1 IR was not altered by NA treatment. Western blot experiments performed on cultured neurons treated with NA confirmed that MCT2 protein expression was increased. Forskolin and dBcAMP also enhanced MCT2 expression, suggesting the implication of a cAMP‐mediated pathway in the effect of NA. Surprisingly, neither NA, dBcAMP nor forskolin affected MCT2 mRNA expression. Application of cycloheximide, a protein synthesis inhibitor, prevented the enhancement of MCT2 IR, while the mRNA synthesis inhibitor actinomycin D also blocked the effect of NA on MCT2 IR levels. These results suggest that regulation of MCT2 expression in neurons by NA occurs at the translational level despite the requirement for an as yet unknown transcriptional step.

Enhanced cerebral expression of MCT1 and MCT2 in a rat ischemia model occurs in activated microglial cells

Monocarboxylate transporters (MCTs) are essential for the use of lactate, an energy substrate known to be overproduced in brain during an ischemic episode. The expression of MCT1 and MCT2 was investigated at 48 h of reperfusion from focal ischemia induced by unilateral extradural compression in Wistar rats. Increased MCT1 mRNA expression was detected in the injured cortex and hippocampus of compressed animals compared to sham controls. In the contralateral, uncompressed hemisphere, increases in MCT1 mRNA level in the cortex and MCT2 mRNA level in the hippocampus were noted. Interestingly, strong MCT1 and MCT2 protein expression was found in peri-lesional macrophages/microglia and in an isolectin B4+/S100β+ cell population in the corpus callosum. In vitro, MCT1 and MCT2 protein expression was observed in the N11 microglial cell line, whereas an enhancement of MCT1 expression by tumor necrosis factor-α (TNF-α) was shown in these cells. Modulation of MCT expression in microglia suggests that these transporters may help sustain microglial functions during recovery from focal brain ischemia. Overall, our study indicates that changes in MCT expression around and also away from the ischemic area, both at the mRNA and protein levels, are a part of the metabolic adaptations taking place in the brain after ischemia.

Insulin and IGF-1 enhance the expression of the neuronal monocarboxylate transporter MCT2 by translational activation via stimulation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin pathway.

MCT2 is the main neuronal monocarboxylate transporter essential for facilitating lactate and ketone body utilization as energy substrates. Our study reveals that treatment of cultured cortical neurons with insulin and IGF‐1 led to a striking enhancement of MCT2 immunoreactivity in a time‐ and concentration‐dependent manner. Surprisingly, neither insulin nor IGF‐1 affected MCT2 mRNA expression, suggesting that regulation of MCT2 protein expression occurs at the translational rather than the transcriptional level. Investigation of the putative signalling pathways leading to translation activation revealed that insulin and IGF‐1 induced p44‐ and p42 MAPK, Akt and mTOR phosphorylation. S6 ribosomal protein, a component of the translational machinery, was also strongly activated by insulin and IGF‐1. Phosphorylation of p44‐ and p42 MAPK was blocked by the MEK inhibitor PD98058, while Akt phosphorylation was abolished by the PI3K inhibitor LY294002. Phosphorylation of mTOR and S6 was blocked by the mTOR inhibitor rapamycin. In parallel, it was observed that LY294002 and rapamycin almost completely blocked the effects of insulin and IGF‐1 on MCT2 protein expression, whereas PD98059 and SB202190 (a p38K inhibitor) had no effect on insulin‐induced MCT2 expression and only a slight effect on IGF‐1‐induced MCT2 expression. At the subcellular level, a significant increase in MCT2 protein expression within an intracellular pool was observed while no change at the cell surface was apparent. As insulin and IGF‐1 are involved in synaptic plasticity, their effect on MCT2 protein expression via an activation of the PI3K–Akt–mTOR–S6K pathway might contribute to the preparation of neurons for enhanced use of nonglucose energy substrates following altered synaptic efficacy.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3‐interacting proteins, such as C‐kinase‐interacting protein 1, glutamate receptor‐interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C‐kinase‐interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three‐dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor α and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm‐enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.