The process of neurotransmission is critically dependent upon a constant supply of glucose. This vital requirement is satisfied, in large measure, by a group of glucose transporters, perhaps the most important of which is Glut 3. Glut 3 is the cell-specific facilitative transporter that is localized to axons and is subject to developmental- and neuronal cell differentiation- linked transcriptional regulation. We hypothesize that Glut 3 is essential in normal central nervous system development and neuronal function. Our long term goal is to understand the mechanisms which regulate the fetal and postnatal expression and site-specific functional activity of this transporter. To this end, we will pursue the following specific aims: First, in the mouse we will establish the site- specific expression of Glut 3 (in comparison with Glut 1, the blood-brain barrier and glial cell glucose transporter isoform) as a function of perinatal and postnatal brain development and correlate this expression with an analysis of localized glucose transport. Next, we will quantitate Glut 3 (in comparison with Glut 1) mRNA abundance, transcriptional rate and protein levels in brain as a function of perinatal and postnatal development. This will be accomplished by a combination of in-situ hybridization, using cloned mouse Glut 3 (and Glut 1) cDNAs, and immunohistochemistry using antibodies generated against Glut 3 (and Glut 1) peptides, followed by Northern, Western and nuclear run-on analyses. Second, we will clone, size, and precisely define the 5'-regulatory regions of the Glut 3 gene. The cloning will be accomplished using a Glut 3 cDNA to screen a mouse genomic library for genomic Glut 3. This will be followed by (1) sequencing, (2) transfection into an SV-T immortalized hippocampal neuronal cell line to determine the minimal sequence necessary to regulate transcription in-vitro and (3) DNA footprinting using brain extracts taken from different stages of development, and neuronal cell extracts from different stages of differentiation, to identify sequences involved in DNA-protein interactions. Our proposed studies will characterize the developmentally-induced transcriptional regulation of neuronal cell- and region-specific Glut 3 expression. These investigations will provide the essential tools necessary for producing Glut 3 overexpressing and Glut 3 deficient mice in the future. The phenotype of these animals will fully test the hypothesis that Glut 3 is essential in normal central nervous system development and neuronal function.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD033997-03
Application #
2403584
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1995-09-01
Project End
1999-06-30
Budget Start
1997-09-01
Budget End
1999-06-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Magee-Women's Hospital of Upmc
Department
Type
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Calkins, Kara L; Thamotharan, Shanthie; Dai, Yun et al. (2018) Early dietary restriction in rats alters skeletal muscle tuberous sclerosis complex, ribosomal s6 and mitogen-activated protein kinase. Nutr Res 54:93-104
Garg, Meena; Thamotharan, Manikkavasagar; Becker, Dorothy J et al. (2014) Adolescents with clinical type 1 diabetes display reduced red blood cell glucose transporter isoform 1 (GLUT1). Pediatr Diabetes 15:511-8
Ganguly, Amit; Chen, Yongjun; Shin, Bo-Chul et al. (2014) Prenatal caloric restriction enhances DNA methylation and MeCP2 recruitment with reduced murine placental glucose transporter isoform 3 expression. J Nutr Biochem 25:259-66
Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny et al. (2014) GLUT3 gene expression is critical for embryonic growth, brain development and survival. Mol Genet Metab 111:477-83
Chen, Yongjun; Shin, Bo-Chul; Thamotharan, Shanthie et al. (2014) Differential methylation of the micro-RNA 7b gene targets postnatal maturation of murine neuronal Mecp2 gene expression. Dev Neurobiol 74:407-425
Thamotharan, Shanthie; Stout, David; Shin, Bo-Chul et al. (2013) Temporal and spatial distribution of murine placental and brain GLUT3-luciferase transgene as a readout of in vivo transcription. Am J Physiol Endocrinol Metab 304:E254-66
Londhe, Vedang A; Maisonet, Tiffany M; Lopez, Benjamin et al. (2013) Retinoic acid rescues alveolar hypoplasia in the calorie-restricted developing rat lung. Am J Respir Cell Mol Biol 48:179-87
Tomi, Masatoshi; Zhao, Yuanzi; Thamotharan, Shanthie et al. (2013) Early life nutrient restriction impairs blood-brain metabolic profile and neurobehavior predisposing to Alzheimer's disease with aging. Brain Res 1495:61-75
Thamotharan, Shanthie; Raychaudhuri, Nupur; Tomi, Masatoshi et al. (2013) Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1? in transactivating murine neuroblastic glucose transporter. Am J Physiol Endocrinol Metab 304:E583-98
Janzen, C; Lei, M Y Y; Cho, J et al. (2013) Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction. Placenta 34:1072-8

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