María de los Angeles García

Differential subcellular distribution of glucose transporters GLUT1–6 and GLUT9 in human cancer: Ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2–6 and 9. The classical expression of GLUT1–5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low‐to‐negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT‐PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over‐expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers. J. Cell. Physiol.

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.

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

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 β 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 α and β, 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 α and β 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 β 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.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium–vitamin C cotransporters. Recently, two different isoforms of sodium–vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high‐affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.

Vitamin C Uptake and Recycling Among Normal and Tumor Cells From the Central Nervous System

Specialized cells transport vitamin C in its reduced form using sodium‐dependent cotransporters (SVCT1 and SVCT2). Additionally, different cells transport the oxidized form of vitamin C, dehydroascorbic acid, through glucose transporters (GLUTs). We have proposed recently a model for vitamin C uptake that resolves the apparent contradiction that although only ascorbic acid is detectable in vivo, there are cells that transport only dehydroascorbic acid. We carried out a detailed kinetic analysis to compare the mechanisms of vitamin C uptake in normal human melanocytes, neurons isolated from brain cortex, hypothalamic ependymal‐glial cells, and astrocytes. Uptake of ascorbic acid was also analyzed in the human oligodendroglioma cell line TC620, in human choroid plexus papilloma cells (HCPPC‐1), and in the neuroblastoma cell line Neuro‐2a. Melanocytes were used to carry out a detailed analysis of vitamin C uptake. Analysis of the transport data by the Lineweaver‐Burk plot revealed the presence of one functional component (Km 20 μM) involved in ascorbic acid transport by melanocytes. Vitamin C sodium‐dependent saturable uptake was also observed in neurons and hypothalamic tanycytes. We confirmed SVCT2 expression in neurons by in situ hybridization; however, SVCT2 expression was not detected in astrocytes in situ. Functional data indicate that astrocytes transport mainly dehydroascorbic acid, using the glucose transporter GLUT1. Our functional uptake analyses support the hypothesis that astrocytes are involved in vitamin C recycling in the nervous system. This recycling model may work as an efficient system for the salvage of vitamin C by avoiding the hydrolysis of dehydroascorbic acid produced by antioxidative protection.

Human brain synembryn interacts with Gsα and Gqα and is translocated to the plasma membrane in response to isoproterenol and carbachol

Heterotrimeric G‐proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization‐induced neurotransmitter release for synaptic transmission is mediated by Gsα and Gqα. To identify effectors for Gα‐proteins, we performed a yeast two‐hybrid screening of a human brain cDNA library, using the human Gαs protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Gα subunits, we found that Gqα also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqα regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

The Na+‐dependent l‐ascorbic acid transporter SVCT2 expressed in brainstem cells, neurons, and neuroblastoma cells is inhibited by flavonoids

Ascorbic acid (AA) is best known for its role as an essential nutrient in humans and other species. As the brain does not synthesize AA, high levels are achieved in this organ by specific uptake mechanisms, which concentrate AA from the bloodstream to the CSF and from the CSF to the intracellular compartment. Two different isoforms of sodium–vitamin C co‐transporters (SVCT1 and SVCT2) have been cloned. Both SVCT proteins mediate high affinity Na+‐dependent l‐AA transport and are necessary for the uptake of vitamin C in many tissues. In the adult brain the expression of SVCT2 was observed in the hippocampus and cortical neurons by in situ hybridization; however, there is no data regarding the expression and distribution of this transporter in the fetal brain. The expression of SVCT2 in embryonal mesencephalic neurons has been shown by RT‐PCR suggesting an important role for vitamin C in dopaminergic neuronal differentiation. We analyze SVCT2 expression in human and rat developing brain by RT‐PCR. Additionally, we study the normal localization of SVCT2 in rat fetal brain by immunohistochemistry and in situ hybridization demonstrating that SVCT2 is highly expressed in the ventricular and subventricular area of the rat brain. SVCT2 expression and function was also confirmed in neurons isolated from brain cortex and cerebellum. The kinetic parameters associated with the transport of AA in cultured neurons and neuroblastoma cell lines were also studied. We demonstrate two different affinity transport components for AA in these cells. Finally, we show the ability of different flavonoids to inhibit AA uptake in normal or immortalized neurons. Our data demonstrates that brain cortex and cerebellar stem cells, neurons and neuroblastoma cells express SVCT2. Dose‐dependent inhibition analysis showed that quercetin inhibited AA transport in cortical neurons and Neuro2a cells.

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, α, β1 and β2. The β2 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 α and β1 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 β2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes α, β1, and β2 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 α and β1 tanycytes, but were abundant in regions containing the barrier‐forming β2 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 α and β1 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. J. Cell. Biochem. 80:491–503, 2001.

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.

Glial glucokinase expression in adult and post-natal development of the hypothalamic region

It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic β-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT–PCR [quantitative RT–PCR (reverse transcription–PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immunoultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of β1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.