GLUCOSE-DEPENDENT GLUT-1 EXPRESSION IN EMBRYONIC HEART

描述（申请人的描述逐字）：很少被理解 关于心脏畸形发生的机制。胚胎 心脏在其早期发育过程中依赖于糖酵解代谢， 高度依赖葡萄糖作为能量产生和生长的底物。 胚胎细胞对葡萄糖的摄取主要由葡萄糖 转运蛋白Glut-1在胚胎心脏中高度表达， 对于将葡萄糖输送到胚胎心脏细胞进行正常代谢至关重要 和器官形成过程中的生长。Glut-1葡萄糖摄取和表达是 在成体细胞中受葡萄糖浓度和暴露持续时间的影响， 但关于葡萄糖依赖性Glut-1在细胞中的表达知之甚少。 胚胎心脏核心假设是胚胎心脏对 通过改变心脏形态发生来改变葡萄糖过量和缺乏，如 在胚胎和胎儿阶段，葡萄糖转运和处置作为一种急性 反应，Glut-1的细胞定位作为中间反应，和 Glut-1 mRNA表达作为慢性反应。为了解决这个问题，GD 9.5（早期器官发生）小鼠胚胎将在体内和体外暴露于 三种葡萄糖水平（600 mg/dl，高血糖; 40 mg/dl，低血糖; 150 mg/dl，低血糖） mg/dl，正常人）三个持续时间（0.5小时，急性; 6小时，中间; 12小时，慢性），并根据以下内容评价心脏 具体目的：1）通过大体和 组织学检查; 2）葡萄糖转运和磷酸化将 使用[3 H]2-脱氧-D-葡萄糖、U-[14 C]-葡萄糖和集总 常数将在有和无细胞松弛素抑制的情况下评价摄取 B，代谢产物将通过NMR进行评价; 3）细胞Glut-1定位 将使用免疫金标记和EM评价以及腺病毒 用荧光显微镜观察载体介导的GFP融合标签的表达; 4） Glut-1 mRNA表达将使用原位杂交进行评价， RT-qcPCR。预计高血压会降低葡萄糖摄取， 急性磷酸化，导致Glut-1易位到细胞内 膜后中期曝光和减少Glut-1 mRNA后慢性 exposure.预计低血糖会增加葡萄糖摄取和Glut-1 在相同的时间模式中表达。该项目将产生重要的 关于Glut-1在葡萄糖递送至胚胎中的作用的信息 并有助于了解胚胎心脏的长期目标 葡萄糖代谢的极端反应，如那些发生在 糖尿病环境及其在心脏畸形中的潜在作用。

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.