Role of Slc2a2/Glut2 in Embryo and Stem Cell Metabolism, Self-Renewal, and Pathways Involved in Diabetic Embryopathy

 描述(由申请人提供)：小鼠胚胎在移植前和移植后发育早期表达SLc2a2。Slc2a2编码高Km(~16 mM)葡萄糖转运蛋白Glut2，表达于胰腺、肝脏、肾脏和肠道。移植前和早期胚胎也表达SLC2a1和SLc2a3，它们分别编码低Km(~5 mM)葡萄糖转运蛋白Glut1和GLUT3。由于胚胎通常会接触到母体循环中接近过剩1和过剩3的Km的葡萄糖浓度，因此预计在正常情况下，它们是主要的葡萄糖转运体。然而，在糖尿病妊娠期间，血糖浓度可能接近或超过Glut2的Km。我们已经证明，Slc2a2+/-和Slc2a2-/-胚胎可以防止母体高血糖导致的先天性畸形。然而，Glut2在正常发育过程中可能很重要，因为它是一种低千米(0.8 mM)的氨基葡萄糖(GlcN)转运体。我们推测，Glut2对早期胚胎存活很重要，促进了GlcN从母体循环中的运输。然而，使用i vivo系统很难研究这一点。胚胎干细胞(ESC)可以分化为特定的组织谱系，对研究胚胎发生的分子调控非常有用。我们使用可以被诱导形成神经元前体(NPs)的小鼠胚胎干细胞来研究胚胎神经上皮中葡萄糖代谢增加所激活的通路。大多数现有ESC株系的一个限制是它们不表达Glut2。我们最近发现，在低糖(100 mg/dl或5.5 mM)培养液中分离的胚胎干细胞(称为LG-ESC)表达一个功能性的Glut2转运体，并表现出与胚胎一样的对高糖培养的生化和基因表达反应。利用LG-ESC，我们发现Glut2介导的GlcN转运刺激ESC的自我更新和合成代谢，我们已经发现的受过量糖代谢调节的与先天性畸形相关的途径调节SLc2a2的表达。这一建议的中心假设是，由Glut2转运的外源GlcN通过增加底物以驱动合成代谢来促进胚胎和干细胞的增殖和多能性，并且Glut2介导的GlcN转运不仅在幼稚的未分化细胞中很重要，而且在它们开始采用命运时也是如此，例如神经上皮；进一步的糖尿病胚胎病变可能部分是由于葡萄糖竞争GlcN摄取。我们将使用LG-ESC来测试这一点，包括从与糖尿病胚胎病变相关的基因通路突变的小鼠品系建立的LG-ESC系(目标1)；通过测试在低糖环境中产生的人诱导多能干细胞(IPSC)是否表达SLC2A2，并对外源GlcN促进合成代谢、自我更新和多能性(目标2)做出响应；通过确定Glt2缺陷胚胎是否由于GlcN的吸收和代谢不足而具有生存劣势。

 DESCRIPTION (provided by applicant): Mouse embryos express Slc2a2 early during pre-and postimplantation development. Slc2a2 encodes at high KM (~16 mM) glucose transporter, Glut2, which is expressed by pancreatic ß cells, the liver, kidney, and intestine. Pre- and early postimplantation embryos also express Slc2a1 and Slc2a3, which encode the low KM (~5 mM) glucose transporters, Glut1 and Glut3, respectively. Because embryos would normally be exposed to glucose concentrations from maternal circulation that are near the KM's of Glut 1 and 3, it is expected that they are the predominant glucose transporters under normal circumstances. However, during diabetic pregnancy, blood glucose concentration could approach or surpass the KM of Glut2. We have shown that Slc2a2+/- and Slc2a2-/- embryos are protected from congenital malformations induced by maternal hyperglycemia. However, Glut2 may be important during normal development because functions as a low KM (0.8 mM) glucosamine (GlcN) transporter. We speculated that Glut2 is important for early embryo survival to facilitate GlcN transport from maternal circulation. However, this is difficult to study using i vivo systems. Embryonic stem cells (ESC) that can be differentiated into particular tissue lineages can be very useful to study molecular regulation of embryogenesis. We have used murine ESC that can be induced to form neuronal precursors (NPs) to study pathways that are activated by increased glucose metabolism in embryonic neuroepithelium. A limitation of most existing ESC lines is that they do not express Glut2. We recently showed that ESC isolated in low glucose (100 mg/dl or 5.5 mM) media (called, LG-ESC) express a functional Glut2 transporter and display the same biochemical and gene expression responses to high glucose culture as does the embryo. Using LG-ESC, we have found that Glut2-mediated GlcN transport stimulates ESC self-renewal and anabolic metabolism, and that a pathway that we had found to be regulated by excess glucose metabolism and associated with congenital malformations regulates Slc2a2 expression. The central hypothesis for this proposal is that exogenous GlcN transported by Glut2 promotes proliferation and pluripotency of embryo and stem cells by increasing substrates to drive anabolic metabolism, and that Glut2-mediated GlcN transport is important, not just in naïve, undifferentiated cells, but also as they begin to adopt a fate, suchas neuroepithelium; further diabetic embryopathy may in part be due to glucose competition for GlcN uptake. We will test this using LG-ESC, including LG-ESC lines that were established from mouse strains with mutations in gene pathways that are involved in diabetic embryopathy (Aim 1); by testing whether human induced pluripotent stem cells (iPSC) generated in a low glucose environment express SLC2A2, and are respond to exogenous GlcN to promote anabolic metabolism, self-renewal, and pluripotency (Aim 2); by determining whether Glut2-deficient embryos have a survival disadvantage to due insufficient GlcN uptake and metabolism.