GLUCOSE-DEPENDENT GLUT-1 EXPRESSION IN EMBRYONIC HEART

胚胎心脏中葡萄糖依赖性 GLUT-1 的表达

• 批准号: 6613725
• 负责人: IDA M. WASHINGTON
• 金额: $ 12.01万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-05 至 2005-01-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613725
• 关键词: congenital heart disorder; glucose transport; glucose transporter; heart metabolism; histogenesis; histology; hyperglycemia; hypoglycemia; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rat; mammalian embryology; morphometry; protein localization

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.

描述（申请人的逐字描述）：了解甚少 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，因此 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介导 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以进行正常代谢至关重要 和器官发生过程中的生长。 Glut-1 葡萄糖摄取和表达是 成体细胞受葡萄糖浓度和暴露时间的影响， 但对于葡萄糖依赖性 Glut-1 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来控制葡萄糖过量和缺乏，如所表明的 在胚胎和胎儿阶段，葡萄糖的转运和处置是一种急性的 反应，Glut-1 的细胞定位作为中间反应，以及 Glut-1 mRNA 表达是一种慢性反应。为了解决这个假设，gd 9.5（早期器官发生）小鼠胚胎将在体内和体外暴露于 三个葡萄糖水平（600 mg/dl，高血糖；40 mg/dl，低血糖；150 mg/dl，正常血糖）三个持续时间（0.5小时，急性；6小时，中间； 12小时，慢性），并且心脏将根据以下进行评估 具体目标： 1) 心脏形态发生将通过总体和 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]-葡萄糖和集总值计算 持续的。将在有或没有细胞松弛素抑制的情况下评估摄取 B、代谢物将通过NMR评估； 3) 细胞Glut-1定位 将使用免疫金标记结合 EM 评估和腺病毒来确定 使用荧光显微镜载体介导 GFP 融合标签的表达； 4） Glut-1 mRNA 表达将使用原位杂交进行评估 RT-qcPCR。高血糖预计会减少葡萄糖的摄取 剧烈磷酸化并导致 Glut-1 易位至细胞内 中间暴露后膜和慢性暴露后 Glut-1 mRNA 减少 接触。低血糖预计会增加葡萄糖摄取和 Glut-1 以相同的时间模式表达。该项目将产生重要 有关 Glut-1 在葡萄糖输送至胚胎中的作用的信息 心脏并为了解胚胎心脏的长期目标做出贡献 代谢对葡萄糖极端的反应，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。

项目成果

13C-NMR study of hypoglycemia-induced glycolytic changes in embryonic mouse heart.

低血糖引起的胚胎小鼠心脏糖酵解变化的 13C-NMR 研究。

• DOI: 10.1002/tera.10103
• 发表时间: 2002
• 期刊: Teratology.
• 作者: Ghatnekar,GautamS;Gracz,HannaS;Smoak,IdaW
• 通讯作者: Smoak,IdaW

Hyperglycemia-induced TGFbeta and fibronectin expression in embryonic mouse heart.

高血糖诱导胚胎小鼠心脏中 TGFbeta 和纤连蛋白的表达。

• DOI: 10.1002/dvdy.20123
• 发表时间: 2004
• 期刊: Developmental dynamics : an official publication of the American Association of Anatomists.
• 作者: Smoak,IdaWashington

Hypoglycemia induced changes in cell death and cell proliferation in the organogenesis stage embryonic mouse heart.

低血糖引起器官发生阶段胚胎小鼠心脏细胞死亡和细胞增殖的变化。

• DOI: 10.1002/bdra.20000
• 发表时间: 2004
• 期刊: Birth defects research. Part A, Clinical and molecular teratology.
• 作者: Ghatnekar,GautamS;Barnes,JillA;Dow,JanetL;Smoak,IdaW
• 通讯作者: Smoak,IdaW