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; 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（由适用提供）：星形胶质细胞，基本心肌细胞，内皮细胞，红细胞和平滑肌的葡萄糖吸收是由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，并且细胞代谢需求的增加迅速增加了细胞表面glut1的内在活性或GLUT1含量。还会发生较慢的自适应反应，其中GLUT1表达增加。内皮细胞和红细胞中的糖转运速度比代谢快得多，但这些细胞也显示出适应性和/或快速转运反应。原因可能是在这些细胞中GLUT1介导的转运限制了受血液组织屏障（例如脑，周围神经，心肌，视网膜）保护的其他时间的葡萄糖利用，并且该Glut1对急性催化性调节是独特的。血结 - 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通过GLUT1介导的跨细胞转运在屏障中的葡萄糖转移。障碍障碍转运损害会损害组织功能，导致凋亡，癫痫发作，局灶性神经系统缺陷和昏迷，并且可能具有遗传性，内分泌和药物来源。长期糖浆症破坏了发展。该提案代表了我们不断努力了解GLUT1催化调节，其在有机稳态中的作用以及这带来了其他主要促进者超级家族蛋白的见解。我们的长期目标是将这些见解转化为对临床糖症的实际干预措施。 GLUT1介导的葡萄糖摄取涉及通过跨膜“通道”的快速，不敏感的葡萄糖翻译成由Glut1细胞质环6和C末端结构域形成的“ CAGE”。从笼子到细胞质的糖释放要慢得多，并且受到限制GLUT1环6，外叶环7和C末端的ATP进一步抑制。这些变化涉及特定的环路6和C末端赖氨酸保留并将笼子转换为现在比A-D-葡萄糖20倍的现在更喜欢-d-葡萄糖的笼子。 H+和AMP拮抗这些变化。该机制可能代表所有细胞中GLUT1可用的基本调节机制。特定目标1通过ESI MS-MS分析纯化的GLUT1分析，用光电反应性核苷酸类似物和已鉴定的标记氨基酸的诱变，对纯化的GLUT1进行了ESI MS-MS分析，检验了细胞质环8是ATP结合结构域的假设。具体目标2检验了以下假设：通过交换GLUT1环路6和C端域具有与ATP不敏感的GLUT3＆4的等效序列，在GLUT1调控中起主要作用的假设，并且从ATP敏感的构建体中丧失ATP响应性。特定的目标3检验了ATP将GLUT1转换为sugar-prefrring载体的假设，并询问GLUT1 C-terminus-L6相互作用和/或ATP结合是否介导了特异性变化。特定目标4检验了以下假设：通过比较急性急性降糖刺激BEND3细胞糖吸收与ATP-Deppletion-Depletion刺激的红细胞转运，通过比较了急性降糖刺激急性降糖刺激，对红细胞和血脑屏障内皮细胞糖转运的快速上调代表了单个基本GLUT1调节机制。