Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.     

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

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.     

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.     

GLUT1 and CAIX expression profiles in breast cancer correlate with adverse prognostic factors and MCT1 overexpression

The goal of the present work was to evaluate the correlation of glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX) with the monocarboxylate transporters 1 (MCT1) and 4 (MCT4) and their chaperone, CD147, in breast cancer. The clinico-pathological value of GLUT1 and CAIX was also evaluated. For that, we analysed the immunohistochemical expression of GLUT1 and CAIX, in a large series of invasive breast carcinoma samples (n=124), previously characterized for MCT1, MCT4 and CD147 expression. GLUT1 expression was found in 46% of the cases (57/124), while CAIX was found in 18% of the cases (22/122). Importantly, both MCT1 and CD147, but not MCT4, were associated with GLUT1 and CAIX expression. Also, GLUT1 and CAIX correlated with each other. Concerning the clinico-pathological values, GLUT1 was associated with high grade tumours, basal-like subtype, absence of progesterone receptor, presence of vimentin and high proliferative index as measured by Ki-67. Additionally, CAIX was associated with large tumour size, high histological grade, basal-like subtype, absence of estrogen and progesterone receptors and presence of basal cytokeratins and vimentin expression. Finally, patients with CAIX positive tumours had a significantly shorter disease-free survival. The association between MCT1 and both GLUT1 and CAIX may result from hypoxia-mediated metabolic adaptations, which confer a glycolytic, acid-resistant and more aggressive phenotype to cancer cells.     

Glucose Transporter Isoform Expression in Huntington's Disease Brain

Abstract: Several reports have suggested a characteristic decrease in glucose use in the striatum of patients with Huntington's disease (HD) may contribute to the cellular atrophy of the caudate and putamen. We examined the expression of the two major glucose transporter isoforms of brain, GLUT1 and GLUT3. GLUT1 is found largely in capillary endothelial cells and to a lesser extent in the brain parenchyma, whereas GLUT3 is localized primarily in neurons. Membranes prepared from postmortem samples of HD caudate and cortex and non-HD caudate and cortex were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and probed with antisera to GLUT1 and GLUT3 by western blotting. Compared with controls, GLUT1 and GLUT3 transporter expression in caudate was decreased by three- and fourfold, respectively, in grade 3 of the disease. At earlier stages (grade 1), there was no significant difference in the expression of the two transporter isoforms compared with nondiseased controls. It is surprising that despite a substantial increase in glial fibrillary acidic protein immunoreactivity (an indicator of the extent of gliosis), glucose transporter expression was diminished significantly in HD caudate. The results suggest in the absence of a significant number of neurons, as in grade 3, glial cell GLUT1 and GLUT3 expression is down-regulated, perhaps reflecting the decreased metabolic demand of this brain region in HD.     

Adult neural stem cells express glucose transporters GLUT1 and GLUT3 and regulate GLUT3 expression

In the brain, glucose is transported by GLUT1 across the blood-brain barrier and into astrocytes, and by GLUT3 into neurons. In the present study, the expression of GLUT1 and GLUT3 mRNA and protein was determined in adult neural stem cells cultured from the subventricular zone of rats. Both mRNAs and proteins were coexpressed, GLUT1 protein being 5-fold higher than GLUT3. Stress induced by hypoxia and/or hyperglycemia increased the expression of GLUT1 and GLUT3 mRNA and of GLUT3 protein. It is concluded that adult neural stem cells can transport glucose by GLUT1 and GLUT3 and can regulate their glucose transporter densities.     

Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxia-inducible factor-1

Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1 as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels. glucose transporter 1; glucose transporter 3; glucose transport     

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.     

MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction

Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.     

MCT1 confirmed in rat striated muscle mitochondria.

We sought to test the hypothesis that monocarboxylate transporter isoform 1 (MCT1) is the inner mitochondrial membrane lactate/pyruvate transporter, and, as such, contributes to functioning of the intracellular lactate shuttle. However, presence of a mammalian mitochondrially localized MCT1 (mMCT1) has been contested. We sought to confirm by Western blotting the mitochondrial localization of MCT1 in rat cardiac, soleus, and extensor digitorum longus muscles utilizing three different cell fractionation methods and three different antibodies. We performed Western blotting using antibodies to cell membrane glucose transporter isoform GLUT1, inner mitochondrial constituent cytochrome oxidase, the monocarboxylate transporter protein chaperone CD147, as well as custom and commercially available MCT1 antibodies. Western blots demonstrated similar results with each MCT1 antibody and two of three methods of fractionation. MCT1 was found in the mitochondria, as well as in the sarcolemmal membrane and whole muscle homogenates. Probing with GLUT1 and CD147 demonstrated that mitochondrial fractions were not contaminated with sarcolemmal remnants. Probing with cytochrome oxidase showed mitochondrial localization of MCT1. Comparison of these results to the findings of others indicates that the most likely source of discrepancy is the cell fractionation procedure utilized.     

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.     

Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.     

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.     

Glucose deprivation and hypoxia increase the expression of the GLUT1 glucose transporter via a specific mRNA cis-acting regulatory element

The expression of the glucose transporter type-1 (GLUT1) gene is up-regulated in hypoxia and glucose deprivation. A 10 nucleotide (nt) cis-acting regulatory element (CAE), which is located within nt 2181-2190 of the GLUT1 3'-untranslated region (CAE2181-2190), increases the expression of a GLUT1-luciferase reporter gene and decreases its mRNA decay. The present study investigated the role of the GLUT1 CAE2181-2190 in glucose deprivation and hypoxia using stable transfectants. Glucose and O2 deprivation produced a marked increase in the expression of the GLUT1 reporter gene carrying the CAE2181-2190, and this effect was additive. Glucose deprivation and/or hypoxia induced no significant changes in the expression of the reporter gene wherein the GLUT1 CAE2181-2190 was site-directed deleted. Data presented here suggest that the GLUT1 CAE2181-2190 participates in the increase of GLUT1 gene expression in glucose deprivation and hypoxia.     

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential.Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1.These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.     

Ependymal Cell Differentiation and GLUT1 Expression is a Synchronous Process in the Ventricular Wall

Ependymal cells appear to be totally differentiated during the first 3 weeks in the mouse brain. Early during postnatal development ependymal cells differentiate and undergo metabolic activation, which is accompanied by increased glucose uptake. We propose that ependymal cells induce an overexpression of the glucose transporter, GLUT1, during the first 2 weeks after delivery in order to maintain the early metabolic activation. During the first postnatal day, GLUT1 is strongly induced in the upper region of the third ventricle and in the ventral area of the rostral cerebral aqueduct. During the next 4 days, GLUT1 is expressed in all differentiated ependymal cells of the third ventricle and in hypothalamic tanycytes. At the end of the first week, ependymal cell differentiation and GLUT1 overexpression is concentrated in the latero-ventral area of the aqueduct. We propose that ependymal cell differentiation and GLUT1 overexpression is a synchronous process in the ventricular wall.     

Glucose transporter expression in English sparrows (Passer domesticus)

Patterns of glucose transporter expression have been well-characterized in mammals. However, data for birds is currently restricted to isolated cells, domestic chickens and chicks, and ducklings. Therefore, in the present study, protein and gene expression of various glucose transporters (GLUTs) in English sparrow extensor digitorum communis, gastrocnemius and pectoralis muscles as well as heart, kidney, and brain tissues were examined. The hypothesis is that the expression pattern of avian GLUTs differs from mammals to maintain the high plasma glucose levels of birds and insulin insensitivity. Our studies failed to identify a GLUT4-like insulin responsive transporter in sparrows. GLUT1 gene expression was identified in all tissues examined and shares 88% homology with chicken and 84% homology with human GLUT1. Compared to the rat control, GLUT1 immunostaining of sparrow extensor digitorum communis muscle was weak and appeared to be localized to blood vessels whereas immunostaining of gastrocnemius muscles was comparable to rat muscle controls. Gene expression of GLUT3 was identified in all tissues examined and shares 90% gene sequence homology with chicken embryonic fibroblast and 75% homology with human GLUT3. Protein expression of GLUT3 was not determined as an avian antibody is not available. Moreover, the C-terminus of the mammalian GLUT3 transporter, against which antibodies are typically designed, differs significantly among species. The predominant difference of chicken and sparrow GLUT expression patterns from that of mammals is the lack of an avian GLUT4. The absence of this insulin responsive GLUT in birds may be a contributing factor to the observed high blood glucose levels and insulin insensitivity.