REGULATION OF GLUCOSE TRANSPORT BY CTAP-III ISOFORMS

CTAP-III 同工酶对葡萄糖转运的调节

• 批准号: 3161207
• 负责人: CHRISTIN CARTER-SU
• 金额: $ 16.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161207
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; biological signal transduction; cell growth regulation; connective tissue cells; cyclic AMP; densitometry; enzyme linked immunosorbent assay; fibroblasts; gene induction /repression; genetic promoter element; glucose metabolism; glucose transport; growth factor; growth factor receptors; high performance liquid chromatography; hormone regulation /control mechanism; human tissue; intracellular transport; leukotrienes; neutrophil; northern blottings; nuclear runoff assay; prostaglandin E; protein kinase C; protein sequence; tissue /cell culture; transfection; transport proteins; western blottings

Accumulating evidence suggests that many naturally occurring peptides activate connective tissue cells in vitro and may play important roles in promoting increased secretion of extracellular matrix and increased replication of connective tissue cells in rheumatic diseases. One such peptide, CTAP-III (connective tissue activating peptide-III), is a member of a family of homologous proteins (PF-4, NAP-1, MGSA, GRO, gamma IP-10, 9E3) thought to be important in inflammation, wound healing and neoplasia. We propose to study a vital, and one of the earliest events following addition of CTAP-III to cultured connective tissue cells - namely stimulation of glucose transport. Incubation with CTAP-III for 30 min increases glucose transport by 30-110% ("immediate" effect); a more substantial stimulation (400-800%) ("delayed effect") is evident after 17- 41 h in human fibroblast cell types that are relatively insensitive to insulin. Only the delayed effect appears to require mRNA synthesis, indicating that the effects involve different pathways. Since recent studies indicate that the active form of CTAP-III may be a product of proteolytic processing, designated CTAP-III (des1-15)/NAP-2, the effects of CTAP-III and the des 1-15 form on glucose transport in connective tissue cells will be compared. Both forms will be tested for their ability to stimulate glucose transport in neutrophils, a cell type stimulated to chemotaxis by CTAP-III (des1-15)/NAP-2. The delayed effect on transport is accompanied by increased numbers of GLUT 1 glucose transporters in murine 3T3-F442A fibroblasts. Specific polyclonal antibodies to the glucose transporter combined with immunoblotting and immunoprecipitation of [35S]methionyl-labeled transporters will be used to test whether CTAP-III increases the rate of synthesis or decreases the degradation rate of glucose transporters. Since preliminary data indicate CTAP-III increases GLUT 1 mRNA levels, the ability of CTAP-III to increase the rate of transcription and/or message stability will be tested. If CTAP-III increases the rate of transcription, CAT constructs will be used to start to determine the part of the GLUT 1 gene conferring CTAP-III sensitivity. Whether the immediate effects of CTAP-III isoforms on glucose transport are mediated via an increase in transporter number, increased intrinsic activity, or redistribution of glucose transporters will be tested as will the hypothesis that the effects of CTAP-III on glucose transport are mediated via PGE2 and subsequent production of cAMP. Whether CTAP-III uses a receptor specific for CTAP-III isoforms or ones shared by structural homologues such as NAP-1 and PF-4 will also be determined. In providing insight into the mechanism by which members of the CTAP-III functional and structural families regulate glucose transport, the molecular mechanisms by which locally released growth factors enable specific, localized relatively insulin-insensitive areas of the body to meet increased demands for glucose will be clarified. Understanding the signal transduction mechanisms used by growth factors other than insulin to regulate glucose transport will provide valuable insight into the earliest events involved in rheumatic disease, inflammation and wound healing, and possibly suggest tactics for therapeutic intervention.

越来越多的证据表明，许多天然存在的多肽 在体外激活结缔组织细胞，并可能在 促进细胞外基质分泌增加 风湿性疾病中结缔组织细胞的复制。这样的一个 多肽，CTAP-III(结缔组织激活肽-III)，是成员 同源蛋白家族(Pf-4、NAP-1、MGSA、Gro、Gamma IP-10、 9E3)被认为在炎症、伤口愈合和肿瘤形成中起重要作用。 我们打算研究一个重要的，也是最早的事件之一 在培养的结缔组织细胞中添加CTAP-III-即 刺激葡萄糖的运输。CTAP-III孵育30分钟 葡萄糖转运增加30-110%(“立竿见影”的效果)； 实质刺激(400-800%)(“延迟效应”)在17- 相对不敏感的人成纤维细胞41h 胰岛素。只有延迟效应似乎需要mRNA的合成， 这表明这些效应涉及不同的途径。自最近以来 研究表明，CTAP-III的活性形式可能是 蛋白质降解处理，命名为CTAP-III(des1-15)/NAP-2，其影响 CTAP-III和DES 1-15形式对结缔组织中葡萄糖转运的影响 我们将对细胞进行比较。这两种形式都将接受测试，以确定它们是否有能力 刺激中性粒细胞中的葡萄糖运输，这是一种被刺激到 CTAP-III(des1-15)/NAP-2的趋化作用。对运输的延迟影响是 伴有Glut 1葡萄糖转运体数量增加的小鼠 3T3-F442a成纤维细胞。抗葡萄糖的特异性多克隆抗体 转运蛋白结合免疫印迹和免疫沉淀 [35S]甲硫酰标记的转运体将用于测试CTAP-III 增加合成速度或降低降解率 葡萄糖转运蛋白。由于初步数据显示CTAP-III增加 Glut 1mRNA水平，CTAP-III的能力增加的速度 将测试转录和/或消息稳定性。如果CTAP-III 提高转录速度，将使用CAT构造启动 目的：确定GLUT 1基因中与CTAP-III敏感的部分。 CTAP-III亚型对葡萄糖转运的直接影响是否 通过增加传输器数量进行调解，增加内在的 葡萄糖转运体的活性或重新分布将进行测试 假设CTAP-III对葡萄糖转运的影响是 通过PGE2介导，随后产生cAMP。CTAP-III是否使用 CTAP-III异构体的特异性受体或由结构蛋白共享的受体 还将确定NAP-1和PF-4等同系物。在提供 洞察CTAP第三次会议成员行使职能和 结构家族调节葡萄糖转运的分子机制是通过 哪些本地发布的增长因素使特定的、相对本地化的 身体中对胰岛素不敏感的区域，以满足增加的葡萄糖需求 将会得到澄清。了解所使用的信号转导机制 通过胰岛素以外的生长因子调节葡萄糖转运 提供有关风湿性疾病最早事件的宝贵见解 疾病、炎症和伤口愈合，并可能建议策略 治疗性干预。