Metabolic Control of Sugar Transport

描述（由申请方提供）：星形胶质细胞、基底心肌细胞、内皮细胞、红细胞和平滑肌中的葡萄糖摄取由GLUT 1介导。在大多数组织中，葡萄糖利用受到葡萄糖摄取的限制，细胞代谢需求的增加迅速增加细胞表面GLUT 1内在活性或GLUT 1含量。GLUT 1表达增加时也会发生较慢的适应性反应。内皮细胞和红细胞中的糖转运比代谢快得多，但这些细胞也显示出适应性和/或快速转运反应。原因可能是这些细胞中GLUT 1介导的转运限制了受血液组织屏障保护的其他组织（例如脑、外周神经、心肌、视网膜）中的葡萄糖利用，并且GLUT 1独特地适合于急性催化调节。血液组织屏障包括通过紧密连接连接的内皮细胞。受保护组织中的葡萄糖代谢需要葡萄糖通过GLUT 1介导的跨细胞转运跨越屏障。屏障转运受损损害组织功能，引起细胞凋亡、癫痫发作、局灶性神经功能缺损和昏迷，可能具有遗传、内分泌和药理学起源。长期低血糖会破坏发育。该提案代表了我们不断努力了解GLUT 1催化调节，其在生物体内平衡中的作用以及这给其他主要促进剂超家族转运蛋白带来的见解。我们的长期目标是将这些见解转化为临床低血糖症的实际干预。GLUT 1介导的葡萄糖摄取涉及快速、ATP不敏感的葡萄糖转运，通过跨膜“通道”进入由GLUT 1胞质环6和C末端结构域形成的“笼”。糖从笼释放到细胞质中要慢得多，并且被ATP进一步抑制，ATP重建GLUT 1环6、外表面环7和C末端。这些变化涉及特定的环6和C-末端赖氨酸残基，并将笼转化为现在比α-D-葡萄糖更喜欢Δ-D-葡萄糖20倍的笼。H+和AMP可拮抗这种变化。这种机制可能代表了所有细胞中GLUT 1的基本调节机制。具体目的1通过对经光反应性核苷酸类似物共价修饰的纯化GLUT 1进行ESI MS-MS分析，并通过对已鉴别标记氨基酸进行诱变，检验细胞质环8为ATP结合结构域的假设。特定目的2通过将GLUT 1环6和C末端结构域与来自ATP不敏感GLUT 3和4的等效序列交换并测试构建体的ATP响应性丧失，检验了C末端和细胞质环6在GLUT 1调节中发挥主要作用的假设。特异性目的3检验了ATP将GLUT 1转化为戊糖偏好载体的假设，并询问GLUT 1 C-末端-L 6相互作用和/或ATP结合是否介导特异性变化。具体目的4通过比较bEnd 3细胞糖摄取的急性低血糖刺激与ATP耗竭刺激的红细胞转运，检验红细胞和血脑屏障内皮细胞糖转运的快速上调代表单一基本GLUT 1调节机制的假设。

DESCRIPTION (provided by applicant): Glucose uptake in astrocytes, basal cardiomyocytes, endothelial cells, erythrocytes and smooth muscle is mediated by GLUT1. In most tissues, glucose utilization is limited by glucose uptake and increases in cellular metabolic demand rapidly increase cell surface GLUT1 intrinsic activity or GLUT1 content. A slower, adaptive response also occurs in which GLUT1 expression increases. Sugar transport in endothelial cells and erythrocytes is much faster than metabolism yet these cells also show adaptive and/or rapid transport responses. The reason may be that GLUT1-mediated transport in these cells limits glucose utilization in other tissues protected by blood-tissue barriers (e.g. brain, peripheral nerve, myocardium, retina) and that GLUT1 is uniquely amenable to acute catalytic regulation. Blood-tissue barriers comprise endothelial cells connected by tight junctions. Glucose metabolism in protected tissues requires glucose transfer across the barrier by GLUT1-mediated, trans-cellular transport. Impaired barrier transport compromises tissue function causing apoptosis, seizures, focal neurologic deficits and coma and may have genetic, endocrine and pharmacologic origins. Long-term glycopenia disrupts development. This proposal represents our continuing efforts to understand GLUT1 catalytic regulation, its role in organismal homeostasis and the insights this brings to other Major Facilitator Superfamily transport proteins. Our long-term goal is to translate these insights into practical intervention in clinical glycopenia. GLUT1-mediated glucose uptake involves rapid, ATP-insensitive, glucose translocation through a membrane-spanning "channel" into a "cage" formed by GLUT1 cytoplasmic loop 6 and C-terminal domains. Sugar release from the cage into cytoplasm is much slower and is further inhibited by ATP which restructures GLUT1 loop 6, exofacial loop 7 and the C-terminus. These changes involve specific loop 6 and C-terminal lysine residues and convert the cage to one which now prefers ¿-D-glucose 20-fold over a-D-glucose. H+ and AMP antagonize these changes. This mechanism may represent a fundamental regulatory mechanism available to GLUT1 in all cells. Specific Aim 1 tests the hypothesis that cytoplasmic loop 8 is the ATP binding domain by ESI MS-MS analysis of purified GLUT1 covalently modified with photoreactive nucleotide analogs and by mutagenesis of identified, labeled amino acids. Specific Aim 2 tests the hypothesis that the C-terminus and cytoplasmic loop 6 play a primary role in GLUT1 regulation by swapping GLUT1 loop 6 and C-terminal domains with equivalent sequence from ATP-insensitive GLUT3 & 4 and testing constructs for loss of ATP-responsiveness. Specific Aim 3 tests the hypothesis that ATP converts GLUT1 to a ¿-sugar-preferring carrier and asks whether GLUT1 C-terminus-L6 interactions and/or ATP binding mediate specificity changes. Specific Aim 4 tests the hypothesis that rapid up-regulation of erythrocyte and blood brain barrier endothelial cell sugar transport represent a single fundamental GLUT1 regulatory mechanism by comparison of acute hypoglycemic stimulation of bEnd3 cell sugar uptake with ATP-depletion-stimulated red cell transport.