MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

The role of tanycytes in hypothalamic glucosensing

Tanycytes are elongated hypothalamic glial cells that cover the basal walls of the third ventricle; their apical regions contact the cerebrospinal fluid (CSF), and their processes reach hypothalamic neuronal nuclei that control the energy status of an organism. These nuclei maintain the balance between energy expenditure and intake, integrating several peripheral signals and triggering cellular responses that modify the feeding behaviour and peripheral glucose homeostasis. One of the most important and well‐studied signals that control this process is glucose; however, the mechanism by which this molecule is sensed remains unknown. We along with others have proposed that tanycytes play a key role in this process, transducing changes in CSF glucose concentration to the neurons that control energy status. Recent studies have demonstrated the expression and function of monocarboxylate transporters and canonical pancreatic β cell glucose sensing molecules, including glucose transporter 2 and glucokinase, in tanycytes. These and other data, which will be discussed in this review, suggest that hypothalamic glucosensing is mediated through a metabolic interaction between tanycytes and neurons through lactate. This article will summarize the recent evidence that supports the importance of tanycytes in hypothalamic glucosensing, and discuss the possible mechanisms involved in this process. Finally, it is important to highlight that a detailed analysis of this mechanism could represent an opportunity to understand the evolution of associated pathologies, including diabetes and obesity, and identify new candidates for therapeutic intervention.     

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.     

Basal and stimulated lactate fluxes in primary cultures of astrocytes are differentially controlled by distinct proteins

Lactate release by astrocytes is postulated to be of importance for neuroenergetics but its regulation is poorly understood. Basigin, a chaperone protein for specific monocarboxylate transporters (MCTs), represents a putatively important regulatory element for lactate fluxes. Indeed, basigin knockdown by RNA interference in primary cultures of astrocytes partially reduced both proton-driven lactate influx and efflux. But more strikingly, enhancement of lactate efflux induced by glutamate was prevented while the effect of sodium azide was significantly reduced by treatment of cultured astrocytes with anti-basigin small interfering RNA. Enhancement of glucose utilization was unaffected under the same conditions. Basal lactate uptake and release were significantly reduced by MCT1 knockdown, even more so than with basigin knockdown, whereas glutamate-driven or sodium azide-induced enhancement of lactate release was not inhibited by either MCT1, 2, or 4 small interfering RNAs. In conclusion, MCT1 plays a pivotal role in the control of basal proton-driven lactate flux in astrocytes while basigin is only partly involved, most likely via its interaction with MCT1. In contrast, basigin appears to critically regulate the enhancement of lactate release caused by glutamate (or sodium azide) but via an effect on another unidentified transporter at least present in astrocytes in vitro.     

Purinergic signaling in tanycytes and its contribution to nutritional sensing

Tanycytes are hypothalamic radial glial-like cells with an important role in the regulation of neuroendocrine axes and energy homeostasis. These cells have been implicated in glucose, amino acids, and fatty acid sensing in the hypothalamus of rodents, where they are strategically positioned. While their cell bodies contact the cerebrospinal fluid, their extensive processes contact neurons of the arcuate and ventromedial nuclei, protagonists in the regulation of food intake. A growing body of evidence has shown that purinergic signaling plays a relevant role in this homeostatic role of tanycytes, likely regulating the release of gliotransmitters that will modify the activity of satiety-controlling hypothalamic neurons. Connexin hemichannels have proven to be particularly relevant in these mechanisms since they are responsible for the release of ATP from tanycytes in response to nutritional signals. On the other hand, either ionotropic or metabotropic ATP receptors are involved in the generation of intracellular Ca²⁺ waves in response to hypothalamic nutrients, which can spread between glial cells and towards neighboring neurons. This review will summarize recent evidence that supports a nutrient sensor role for tanycytes, highlighting the participation of purinergic signaling in this process.     

Immunogold Cytochemistry Identifies Specialized Membrane Domains for Monocarboxylate Transport in the Central Nervous System

An efficient exchange of lactate between different cell types (such as astrocytes and neurones) would require that lactate transporters are expressed in contiguous parts of the respective plasma membranes. To settle this issue we explored the subcellular expression pattern of monocarboxylate transporters (MCTs) by use of selective antibodies and high resolution immunogold cytochemistry. We investigated whether the membrane domains containing MCT1, MCT2 and MCT4 are spatially related to each other and to other membrane domains, i.e. those containing glutamate receptors. We used retina and cerebellum as a model for our investigations. We found that MCT1 was localized in the apical membrane of pigment epithelial cells and in the photoreceptor inner segment membrane in the retina. In the brain MCT1 was present in endothelial cells. MCT2 was localized in the postsynaptic membrane of parallel fiber-Purkinje cell synapses and MCT4 was situated in the membrane of glial cells in the cerebellum.     

Astrocyte-neuron lactate transport is required for long-term memory formation

We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation.     

Expression and distribution of facilitative glucose (GLUTs) and monocarboxylate/H+ (MCTs) transporters in rat olfactory epithelia

Cell-to-cell metabolic interactions are crucial for the functioning of the nervous system and depend on the differential expression of glucose transporters (GLUTs) and monocarboxylate transporters (MCTs). The olfactory receptor neurons (ORNs) and supporting cells (SCs) of the olfactory epithelium exhibit a marked polarization and a tight morphological interrelationship, suggesting an active metabolic interaction. We examined the expression and localization of MCTs and GLUTs in the olfactory mucosa and found a stereotyped pattern of expression. ORNs exhibited GLUT1 labeling in soma, dendrites, and axon. SCs displayed GLUT1 labeling throughout their cell length, whereas MCT1 and GLUT3 localize to their apical portion, possibly including the microvilli. Additionally, GLUT1 and MCT1 were detected in endothelial cells and GLUT1, GLUT3, and MCT2 in the cells of the Bowman's gland. Our observations suggest an energetic coupling between SCs and Bowman's gland cells, where glucose crossing the blood-mucosa barrier through GLUT1 is incorporated by these epithelial cells. Once in the SCs, glucose can be metabolized to lactate, which could be transported by MCTs into the Bowman's gland duct, where it can be used as metabolic fuel. Furthermore, SCs may export glucose and lactate to the mucous layer, where they may serve as possible energy supply to the cilia.     

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

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.     

The Neuroplastin Adhesion Molecules Are Accessory Proteins That Chaperone the Monocarboxylate Transporter MCT2 to the Neuronal Cell Surface

Background

The neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABA_A receptor subtypes in hippocampal neurones. The brain is dependent not only on glucose but on monocarboxylates as sources of energy. The. monocarboxylate transporters (MCTs) 1–4 are responsible for the rapid proton-linked translocation of monocarboxylates including pyruvate and lactate across the plasma membrane and require association with either embigin or basigin, proteins closely related to neuroplastin, for plasma membrane expression and activity. MCT2 plays a key role in providing lactate as an energy source to neurons.

Methodology/Findings

Here we use co-transfection of neuroplastins and monocarboxylate transporters into COS-7 cells to demonstrate that neuroplastins can act as ancillary proteins for MCT2. We also show that Xenopus laevis oocytes contain endogenous neuroplastin and its knockdown with antisense RNA reduces the surface expression of MCT2 and associated lactate transport. Immunocytochemical studies show that MCT2 and the neuroplastins are co-localised in rat cerebellum. Strikingly neuroplastin and MCT2 are enriched in the same parasagittal zebrin II-negative stripes.

Conclusions

These data strongly suggest that neuroplastins act as key ancillary proteins for MCT2 cell surface localisation and activity in some neuronal populations, thus playing an important role in facilitating the uptake of lactate for use as a respiratory fuel.     

Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.     

Cellular expression of monocarboxylate transporters in the female reproductive organ of mice: implications for the genital lactate shuttle

The present study examined the cellular localization of monocarboxylate transporters (MCTs), glucose transporters (GLUTs), and some glycolysis-related molecules in the murine female genital tract to demonstrate existence of lactate/pyruvate-dependent energy systems. MCT1, a major MCT subtype, was localized selectively in the ovarian granulosa, oviductal-ciliated cells, and vaginal epithelium; all localizations were associated with intense expressions of glycolytic enzymes. MCT1 was localized in the cell membrane of granulosa cells, including fine processes extending from cumulus cells toward oocytes. The cumulus cells and oocytes showed intense signals for lactate dehydrogenase (LDH)-A and -B, respectively. The basolateral membrane of oviductal-ciliated cells expressed MCT4 as well as MCT1, while adjacent non-ciliated cells contained an intense immunoreactivity for aldolase-C, a glycolytic enzyme. The expression of GLUTs in the ovary was generally weak with an intense expression of GLUT1 only in some vascular endothelia. The oviductal epithelium expressed GLUT1 and GLUT3, respectively, in the basolateral and apical membrane of non-ciliated cells. In the vagina, the basal layers of epithelium were immunolabeled for MCT1 with the entire length of cell membrane, and expressed abundantly both GLUT1 and LDH-A. The findings correspond well with the rich existence of lactate in the genital fluids and strongly suggest the active transport of lactate/pyruvate in the female reproductive tract, which provides favorable conditions for oocytes, sperms, and zygotes.     

Developmental switch in brain nutrient transporter expression in the rat

Normal development of both human and rat brain is associated with a switch in metabolic fuel from a combination of glucose and ketone bodies in the immature brain to a nearly total reliance on glucose in the adult. The delivery of glucose, lactate, and ketone bodies from the blood to the brain requires specific transporter proteins, glucose and monocarboxylic acid transporter proteins (GLUTs and MCTs), respectively. Developmental expression of the GLUTs in rat brain, i.e., 55-kDa GLUT1 in the blood-brain barrier (BBB), 45-kDa GLUT1 and GLUT3 in vascular-free brain, corresponds to maturational increases in cerebral glucose uptake and utilization. It has been suggested that MCT expression peaks during suckling and sharply declines thereafter, although a comparable detailed study has not been done. This study investigated the temporal and regional expression of MCT1 and MCT2 mRNA and protein in the BBB and the nonvascular brain during postnatal development in the rat. The results confirmed maximal MCT1 mRNA and protein expression in the BBB during suckling and a decline with maturation, coincident with the switch to glucose as the predominant cerebral fuel. However, nonvascular MCT1 and MCT2 levels do not reflect changes in cerebral energy metabolism, suggesting a more complex regulation. Although MCT1 assumes a predominantly glial expression in postweanling brain, the concentration remains fairly constant, as does that of MCT2 in neurons. The maintenance of nonvascular MCT levels in the adult brain implies a major role for these proteins, in concert with the GLUTs in both neurons and astrocytes, to transfer glycolytic intermediates during cerebral energy metabolism.     

脑部乳酸供能的方式

脑部存在星形胶质细胞-神经元的乳酸穿梭方式供能。单羧酸转运蛋白2(monocarboxylate transporter 2,MCT2)和MCT4的细胞特异性分布与乳酸穿梭的方向存在密切联系。MCT2和MCT4分别集中于神经元突触后膜致密区与星形胶质细胞终足膜,可能有利于N-甲基-D-天冬氨酸(N-methyl-D-asparticacid,NMDA)受体抑制的解除及循环中乳酸穿越血脑屏障。神经元和星形胶质细胞均能利用乳酸或血糖作为氧化底物。神经元中,乳酸是比血糖更为优势的氧化底物,其原因有待研究。脑部乳酸不仅来源于血糖酵解,还来源于血液循环中的乳酸及星形胶质细胞的糖原酵解。     

Androgen-responsive and nonresponsive prostate cancer cells present a distinct glycolytic metabolism profile

Prostate cancer (PCa) progresses from an early stage, confined to prostate, to a more aggressive metastasized cancer related with loss of androgen responsiveness. Although, it has been recognized that PCa cells have unique metabolic features, their glycolytic profile in androgen-dependent and androgen-independent stages of disease is much less known. Hence, the main purpose of this study was to compare glucose metabolism in androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) PCa cells. Cell culture medium was collected and differences in glucose consumption and, lactate and alanine production were measured using Proton Nuclear Magnetic Resonance ((1)H NMR) spectra analysis. The mRNA and protein expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) were determined by real-time PCR and Western Blot, respectively. The obtained results demonstrate that androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) cells consumed similar amounts of glucose, whereas PC3 cells present higher lactate production. This increase in lactate production was concomitant with higher levels of MCT4 protein, increased LDH activity and higher lactate/alanine ratio, also suggesting increased levels of oxidative stress in PC3 cells. However, protein levels of LDH, associated with lactate metabolism, and GLUT3, involved in glucose uptake, were decreased in PC3 comparatively with LNCaP. Androgen-responsive and nonresponsive PCa cells present distinct glycolytic metabolism profiles, which suggest that targeting LDH and MCT4 metabolic pathways may be an important step for the development of new diagnostic and therapeutic strategies in the different stages of PCa.     

Facilitated Lactate Transport by MCT1 when Coexpressed with the Sodium Bicarbonate Cotransporter (NBC) in Xenopus Oocytes

Monocarboxylate transporters (MCT) and sodium-bicarbonate cotransporters (NBC) transport acid/base equivalents and coexist in many epithelial and glial cells. In nervous systems, the electroneutral MCT1 isoform cotransports lactate and other monocarboxylates with H⁺, and is believed to be involved in the shuttling of energy-rich substrates between astrocytes and neurons. The NBC cotransports bicarbonate with sodium and generates a membrane current. We have expressed these transporter proteins, cloned from rat brain (MCT1) and human kidney (NBC), alone and together, by injecting the cRNA into oocytes of the frog Xenopus laevis, and measured intracellular pH changes and membrane currents under voltage-clamp with intracellular microelectrodes, and radiolabeled lactate uptake into the oocytes. We determined the cytosolic buffer capacity, the H⁺ and lactate fluxes as induced by 3 and 10 mM lactate in oocytes expressing MCT1 and/or NBC, and in water-injected oocytes, in salines buffered with 5 mM HEPES alone or with 5% CO₂/10 mM HCO₃⁻ (pH 7.0). In MCT1 + NBC- but not in MCT1- or NBC-expressing oocytes, lactate activated a Na⁺- and HCO₃⁻-dependent membrane current, indicating that lactate/H⁺ cotransport via MCT1, due to the induced pH change, stimulates NBC activity. Lactate/H⁺ cotransport by MCT1 was increased about twofold when MCT1 was expressed together with NBC. Our results suggest that the facilitation of MCT1 transport activity is mainly due to the increase in apparent buffer capacity contributed by the NBC, and thereby suppresses the build-up of intracellular H⁺ during the influx of lactate/H⁺, which would reduce MCT1 activity. Hence these membrane transporters functionally cooperate and are able to increase ion/metabolite transport activity.