Metabolic Control of Sugar Transport

糖运输的代谢控制

• 批准号: 8000134
• 负责人: ANTHONY CARRUTHERS
• 金额: $ 16.24万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000134
• 关键词: Acute; Aerobic; Amino Acids; Apoptosis; Astrocytes; Autonomic nervous system; Binding; Blood; Blood - brain barrier anatomy; Brain; C-terminal; Capillary Endothelial Cell; Cardiac Myocytes; Carrier Proteins; Cations; Cell physiology; Cell surface; Cells; Chimera organism; Clinical; Coma; Cytoplasm; Development; Dominant-Negative Mutation; Endocrine; Endothelial Cells; Erythrocytes; Focal Neurologic Deficits; Focal Seizure; GLUT-3 protein; Genetic; Glucose; Glucose Transporter; Goals; Heart; Homeostasis; Hour; Hypoglycemia; Immune system; Immunoglobulin G; Intervention; Label; Labyrinth; Lysine; Measures; Mediating; Membrane; Metabolic; Metabolic Control; Metabolism; Molecular; Mus; Mutagenesis; Myocardium; Neurologic; Olfactory Epithelium; Peripheral Nerves; Play; Process; Regulation; Reporting; Retina; Role; SLC2A1 gene; Seizures; Smooth Muscle; Specificity; System; Testing; Tight Junctions; Tissues; Translating; Up-Regulation; anomer; brain tissue; glucose metabolism; glucose transport; glucose uptake; in vitro Model; insight; macrophage; mutant; nucleotide analog; preference; reproductive; research study; response; skeletal; sugar; uptake

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.

描述（由申请人提供）：星形细胞、基底心肌细胞、内皮细胞、红细胞和平滑肌中的葡萄糖摄取是由GLUT1介导的。在大多数组织中，葡萄糖的利用受到葡萄糖摄取的限制，细胞代谢需求的增加会迅速增加细胞表面GLUT1的内在活性或GLUT1含量。一种较慢的适应性反应也发生在GLUT1表达增加的情况下。内皮细胞和红细胞中的糖转运比代谢快得多，但这些细胞也表现出适应性和/或快速转运反应。原因可能是这些细胞中GLUT1介导的转运限制了其他受血组织屏障保护的组织（如脑、周围神经、心肌、视网膜）对葡萄糖的利用，而且GLUT1特别适合急性催化调节。血液组织屏障由紧密连接的内皮细胞组成。受保护组织中的葡萄糖代谢需要葡萄糖通过glut1介导的跨细胞运输跨越屏障。屏障运输受损损害组织功能，导致细胞凋亡、癫痫发作、局灶性神经功能缺损和昏迷，可能有遗传、内分泌和药理学原因。长期的血糖降低会扰乱发育。这一提议代表了我们继续努力了解GLUT1的催化调节，其在生物体稳态中的作用，以及由此带来的其他主要促进物超家族运输蛋白的见解。我们的长期目标是将这些见解转化为临床血糖降低的实际干预措施。GLUT1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GLUT1细胞质环6和c端结构域形成的“笼子”。糖从笼中释放到细胞质的速度要慢得多，并且被ATP进一步抑制，ATP重组GLUT1环6、外表面环7和c端。这些变化涉及特定的环6和c端赖氨酸残基，并将笼转化为现在比a- d -葡萄糖更倾向于- d -葡萄糖的20倍。H+和AMP对抗这些变化。这一机制可能代表了所有细胞中GLUT1可用的基本调节机制。特异性Aim 1通过ESI - MS-MS分析纯化的GLUT1，用光反应性核苷酸类似物共价修饰，并通过诱变已识别的标记氨基酸，验证了细胞质环8是ATP结合域的假设。Specific Aim 2通过将GLUT1环6和c端结构域与来自atp不敏感的GLUT3和4的等效序列交换，并测试atp反应性丧失的构建体，验证了c端和细胞质环6在GLUT1调控中起主要作用的假设。特异性目的3验证ATP将GLUT1转化为糖偏好载体的假设，并询问GLUT1 c -末端- l6相互作用和/或ATP结合是否介导特异性变化。特异性目的4通过比较急性低血糖刺激bEnd3细胞糖摄取与atp消耗刺激红细胞转运，验证了红细胞和血脑屏障内皮细胞糖转运的快速上调代表GLUT1单一基本调节机制的假设。