Trans-activators Regulating Neuronal Glucose Transporter Isoform-3 Gene Expression in Mammalian Neurons

Abstract

The murine facilitative glucose transporter isoform 3 is developmentally regulated and is predominantly expressed in neurons. By employing the primer extension assay, the transcription start site of the murine Glut 3 gene in the brain was localized to ؊305 bp 5 to the ATG translation start codon. Transient transfection assays in N2A neuroblasts using murine GLUT3-luciferase reporter constructs mapped enhancer activities to two regions located at ؊203 to ؊177 and ؊104 to ؊29 bp flanking a previously described repressor element (؊137 to ؊130 bp). Dephosphorylated Sp1 and Sp3 proteins from the 1-and 21-day-old mouse brain nuclear extracts bound the repressor elements, whereas both dephosphorylated and phosphorylated cAMP-response element-binding protein (CREB) in N2A, 1-and 21-dayold mouse brain nuclear extracts bound the 5-enhancer cis-elements (؊187 to ؊180 bp) of the Glut 3 gene, and the Y box protein MSY-1 bound the sense strand of the ؊83-to ؊69-bp region. Sp3, CREB, and MSY-1 binding to the GLUT 3 DNA was confirmed by the chromatin immunoprecipitation assay, whereas CREB and MSY-1 interaction was detected by the co-immunoprecipitation assay. Furthermore, small interference RNA targeted at CREB in N2A cells decreased endogenous CREB concentrations, and CREB mediated GLUT 3 transcription. Thus, in the murine brain similar to the N2A cells, phosphorylated CREB and MSY-1 bound the Glut 3 gene trans-activating the expression in neurons, whereas Sp1/Sp3 bound the repressor elements. We speculate that phosphorylated CREB and Sp3 also interacted to bring about GLUT 3 expression in response to development/cell differentiation and neurotransmission.Glucose, an essential substrate for brain oxidative metabolism, is transported across the blood-brain barrier and into neurons and glia by a family of structurally related membranespanning glycoproteins termed the facilitative glucose transporters (1, 2). Of the 14 major isoforms cloned to date (1-9), GLUT 1 and GLUT 3 are the isoforms predominantly expressed in the brain (10, 11). Whereas GLUT 1 is expressed by endothelial cells lining the microvasculature and glial cells, which are components of the blood-brain barrier (10), GLUT 3 is the predominant neuronal isoform (11). We and others have reported previously that although the spatial distribution of GLUT 3 in brain is not age-dependent (12), a temporal distribution exists with low amounts noted during the embryonic/ fetal and early postnatal stages and peak amounts at day 14 -21 (13), which coincides with the timing of synaptogenesis (14 -16). In addition, GLUT 3 localization to the synaptic region and its vesicular trafficking, which involves SNAP-25 and syntaxin-1, proteins of the SNARE complex present in synaptic vesicles, supports a role for GLUT 3 in neurotransmission (17). Brain 2-deoxyglucose uptake serves as a surrogate marker for neuronal activity (18); thus GLUT 3, which mediates this glucose uptake, must play a major role in fueling neurotransmission (19). Depolarization of neurons in vitro by the presence of...

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