Normal cerebral development requires a constant supply of metabolizable substrate, predominantly glucose, to the brain during the perinatal period. Systemic metabolic stresses occurring during this period, such as hypoxia- ischemia and hypoglycemia, can severely compromise glucose delivery tot he brain and frequently produce permanent brain damage. Glucose delivery to the brain requires the passage of glucose across the endothelial cells of the blood-brain barrier and the plasma membranes of neurons and glia, mediated by the glucose transporter proteins, GLUT1 and GLUT3. GLUT1 is most concentrated in the blood-brain barrier and choroid plexus; GLUT3 is probably the major neuronal glucose transporter. A third glucose transporter protein, GLUT5, present in microglia, may contribute to cerebral glucose utilization following injury. Both GLUT1 and GLUT3 are developmentally regulated in rat brain and attain adult levels of expression by 21-30 days postnatally. However, the levels of both of these proteins are low in the 7-day postnatal rat and are likely to be limiting to glucose utilization, especially at a time of increased demand in response to stress. The goal of this proposal is to determine the relationship between the known derangements in cerebral glucose metabolism associated with perinatal hypoxia-ischemia and hypoglycemia and the developmental expression of the glucose transporter proteins. The grant will pursue three objectives; 1) the complete characterization of GLUT1, 3, & 5 in relation to cerebral glucose transport and utilization during normal development int he rat; these studies will employ tissue and membrane isolation and immunodetection techniques to assess the expression of these proteins in several brain regions, cerebral microvessels and choroid plexus; glucose transport rate constants (including Michaelis- Menten kinetics for estimation of maximal transport capacity) and cerebral glucose utilization will be determined in vivo in same aged animal with radioisotopes; 2) the determination of the temporal and regional patterns of the levels of GLUT1, 3 & 5 following hypoxia-ischemia, and acute and chronic hypoglycemia in the 7-day rat; these studies will apply the same methodology s in (1) to rats following hypoxia-ischemia or insulin-induced hypoglycemia; 3) the determination of the mechanism of regulation of transporter expression in the blood-brain barrier, neurons and glia, by mapping the associated changes of mRNA expression for each of the proteins in response to hypoxia-ischemia and hypoglycemia, in association with cell- specific markers; these studies will employ in situ hybridization histochemistry, combined with either 14C-2-deoxyglucose administration or immunocytochemical labeling. Northern blot analysis will be employed to aid in the quantitation of the observed changes. The results of this study should help elucidate the mechanisms by which the immature brain responds to injury and may provide opportunities for intervention in these, and other, perinatal diseases.
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