Human choroid plexus papilloma cells efficiently transport glucose and vitamin C.

In vitro and in vivo studies suggest that the basolateral membrane of choroid plexus cells, which is in contact with blood vessels, is involved in the uptake of the reduced form of vitamin C, ascorbic acid (AA), through the sodium-vitamin C cotransporter, (SVCT2). Moreover, very low levels of vitamin C were observed in the brains of SVCT2-null mice. The oxidized form of vitamin C, dehydroascorbic acid (DHA), is incorporated through the facilitative glucose transporters (GLUTs). In this study, the contribution of SVCT2 and GLUT1 to vitamin C uptake in human choroid plexus papilloma (HCPP) cells in culture was examined. Both the functional activity and the kinetic parameters of GLUT1 and SVCT2 in cells isolated from HCPP were observed. Finally, DHA uptake by GLUT1 in choroid plexus cells was assessed in the presence of phorbol-12-myristate-13-acetate (PMA)-activated human neutrophils. A marked increase in vitamin C uptake by choroid plexus cells was observed that was associated with superoxide generation and vitamin C oxidation (bystander effect). Thus, vitamin C can be incorporated by epithelial choroid plexus papilloma cells using the basolateral polarization of SVCT2 and GLUT1. This mechanism may be amplified with neutrophil infiltration (inflammation) of choroid plexus tumors. In choroid plexus papilloma cells, the vitamin C transporters SVCT2 and GLUT1 are polarized to the basolateral epithelial membrane, where SVCT2 is essential for AA flux from the blood vessels into the brain. However, neutrophils, attracted by inflammation or the tumor microenvironment, can oxidize extracellular AA to DHA, thereby enabling its uptake through GLUT1. For the first time, we show the in vivo and in vitro basolateral co-distribution of functional SVCT2 and GLUT1 in epithelial cells. We postulate that patients with choroid plexus papillomas may continue to transport vitamin C from the blood to CSF. However, increased transport of oxidized vitamin C could generate pro-oxidative conditions that may help control tumor growth.

Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue.

Over-expression of hexose transporters (Gluts), specifically Glut-1, is a common event in human malignancies. In prostate cancer (CaP), however, expression of Gluts has been characterized poorly. In this study, expression and distribution of Glut-1 and Glut-5 proteins were characterized using immunohistochemistry in 76 specimens of benign prostate, 10 specimens of high-grade intraepithelial neoplasia (HGPIN), and 28 specimens of CaP. In addition, mRNA expression of Glut-2, Glut-7, Glut-9, and Glut-11 was analyzed in a set of five specimens of benign prostate and CaP. In benign prostate, Glut-1 localized to the basal cells and to the basolateral membrane of secretory/luminal epithelial cells. Glut-5, however, localized to the apical membrane of secretory/luminal epithelial cells. In HGPIN, Glut-1 was immunohistochemically undetectable. Glut-5, however, localized to the apical membrane of the neoplastic epithelial cells. In CaP, Glut-1 and Glut-5, were immunohistochemically undetectable. However, over-expression of GLUT1 was observed in some specimens of highly proliferative intraductal CaP. Glut-7, Glut-9, and Glut-11 mRNAs were detected in benign prostate and CaP, however, only Glut-11 mRNA was consistently up-regulated in CaP compared to benign prostate. Low levels of expression of Glut-1 protein in the majority of CaP could explain, at least in part, the limited clinical applicability of positron emission tomography using 2-[18F]-fluoro-2-deoxy-D-glucose for imaging CaP. Moreover, expression of Glut-5 in HGPIN suggested that fructose could be utilized as potential metabolic substrate in HGPIN. Understanding the molecular mechanisms involved in regulation/dysregulation of Gluts in CaP could provide insight in the understanding of hexose metabolism in CaP.

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 beta-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 immuno-ultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of beta1-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.

Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney.

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.

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.

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.

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 (K(m) 20 microM) 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.

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.

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.