GLUCOSE-DEPENDENT GLUT-1 EXPRESSION IN EMBRYONIC HEART

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DESCRIPTION (the applicant's description verbatim): Little is understood regarding the mechanisms underlying cardiac dysmorphogenesis. The embryonic heart relies on glycolytic metabolism during its early development and is thus highly dependent on glucose as a substrate for energy production and growth. Glucose uptake by embryonic cells is mediated primarily by the glucose transporter, Glut-1, which is highly expressed in the embryonic heart and is critical for delivery of glucose to embryonic heart cells for normal metabolism and growth during organogenesis. Glut-1 glucose uptake and expression are influenced in adult cells by glucose concentration and duration of exposure, but little is known regarding glucose-dependent Glut-1 expression in the embryonic heart. The central hypothesis is that the embryonic heart responds to glucose excess and deficiency by altering cardiac morphogenesis, as manifested at embryonic and fetal stages, glucose transport and disposition as an acute response, cellular localization of Glut-1 as an intermediate response, and Glut-1 mRNA expression as a chronic response. To address this hypothesis, gd 9.5 (early organogenesis) mouse embryos will be exposed in vivo and in vitro to three glucose levels (600 mg/dl, hyperglycemia; 40 mg/dl, hypoglycemia; 150 mg/dl, normoglycemia) for three durations (0.5 hr, acute; 6 hr, intermediate; 12 hr, chronic), and hearts will be evaluated according to the following specific aims: 1) Cardiac morphogenesis will be evaluated by gross and histologic examination; 2) glucose transport and phosphorylation will be calculated using [3H]2-deoxy-D-glucose, U-[14C]-glucose, and the lumped constant. Uptake will be evaluated with and without inhibition by cytochalasin B, and metabolites will be evaluated by NMR; 3) cellular Glut-1 localization will be determined using immunogold labeling with EM evaluation and adenoviral vector mediated expression of a GFP fusion tag with fluorescence microscopy; 4) Glut-1 mRNA expression will be evaluated using in situ hybridization and RT-qcPCR. Hyperglycemia is expected to decrease glucose uptake and phosphorylation acutely and cause Glut-1 translocation to intracellular membranes after intermediate exposure and decreased Glut-1 mRNA after chronic exposure. Hypoglycemia is expected to increase glucose uptake and Glut-1 expression in the same temporal pattern. This project will produce important information regarding the role of Glut-1 in glucose delivery to the embryonic heart and contribute to a long-range goal of understanding embryonic heart metabolism in response to glucose extremes, such as those occurring in the diabetic environment, and its potential role in cardiac dysmorphogenesis.