GLUCOSE-DEPENDENT GLUT-1 EXPRESSION IN EMBRYONIC HEART

描述(申请人的描述逐字逐句)：不太懂 关于心脏畸形发生的机制。萌芽状态 心脏在早期发育过程中依赖糖酵解代谢，因此 高度依赖葡萄糖作为能量生产和生长的底物。 胚胎细胞对葡萄糖的摄取主要由葡萄糖介导。 转运蛋白GLUT-1，在胚胎心脏中高表达，是 葡萄糖对胚胎心脏细胞正常代谢的关键作用 以及器官发生过程中的生长。GLUT-1葡萄糖摄取和表达是 在成年细胞中受葡萄糖浓度和暴露时间的影响， 但对葡萄糖依赖的GLUT-1在血管内皮细胞中的表达知之甚少 胚胎心脏。中心假设是胚胎心脏对 通过改变心脏形态发生而导致的葡萄糖过多和不足 在胚胎和胎儿阶段，葡萄糖的转运和处置是急性的 应答，GLUT-1的细胞定位作为中间应答，以及 GLUT-1mRNA表达作为一种慢性反应。为了解决这一假设，gd 9.5(早期器官发生)小鼠胚胎将在体内和体外暴露于 三种血糖水平(600 mg/dl，高血糖；40 mg/dl，低血糖；150 Mg/dl，血糖正常)，共3个疗程(急性0.5小时；中度6小时； 12小时，慢性)，心脏将根据以下内容进行评估 具体目标：1)心脏形态发生将通过粗略和 组织学检查；2)葡萄糖转运和磷酸化 使用[~3H]2-脱氧-D-葡萄糖、U-[14C]-葡萄糖和集总 常量。摄取将在有和没有细胞松弛素抑制的情况下进行评估 3)细胞GLUT-1的定位 将使用免疫金标记法结合EM评估和腺病毒来确定 荧光显微镜下载体介导的绿色荧光蛋白融合标签的表达 GLUT-1mRNA的表达将通过原位杂交和 RT-qcPCR。高血糖预计会减少葡萄糖的摄取和 急性磷酸化并导致GLUT-1转位到细胞内 中度暴露后膜和慢性暴露后GLUT-1mRNA的降低 曝光。低血糖预计会增加葡萄糖摄取和过剩-1 在相同的时间模式中表达。这个项目将产生重要的 GLUT-1在葡萄糖转运到胚胎中的作用 心脏，并为理解胚胎心脏的长期目标做出贡献 新陈代谢对葡萄糖的极端反应，例如发生在 糖尿病环境及其在心脏畸形发生中的潜在作用。

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.