METABOLIC AND ENDOCRINE CONTROL OF SUGAR TRANSPORT

这项建议代表了我们实验室继续努力 了解内分泌和代谢的分子基础 细胞糖运输的调控。代谢耗竭 似乎刺激肌肉和禽类红细胞(ARBC)的运输 通过增加细胞表面糖载体的内在活性 (即通过载波激活)。这与胰岛素刺激不同。 细胞表面增加对脂肪细胞和肌肉运输的影响 数字。这项提案的广泛目标是将 载体激活的生物化学及其与胰岛素的关系 对糖运输的监管。这些研究将有助于我们的长期 理解蛋白质分子基础的术语目标 介导的糖运输，并最终可能在 对糖尿病等紊乱状态的管理。 葡萄糖转运蛋白含有一个转运蛋白 三磷酸腺苷结合位点。该网站最近被特别贴上了标签 使用叠氮三磷酸腺苷。特定的目标1是通过以下方式识别该站点 对标记后释放的标记多肽进行测序 承运人。特定目标2问：由于承运人最近被 表现出极易被激活/抑制的 特异性膜双层溶血磷脂，DO载体 双层脂质改变导致代谢耗竭激活 构图？我们使用重组系统和磷脂 控制和消耗ARBC的分析和操作 回答这个问题。具体目标3问：由于刺激性LPL减少 抑制脂多糖可增加葡萄糖载体对 ATP，ATP和LPL之间的这种协同作用是否起到放大作用 它们对交通的影响是什么？我们执行配基结合和 对重组承运人进行的运输研究回答了这一问题。 特定目标4直接测试激活和招募 利用最新发展的运输调节假说 抗血清(与胞外结构域特异性反应) 载体)来定量对照中的细胞表面载体数量 和新陈代谢耗尽的ARBC。特定目标5扩展目标 2~4对胰岛素刺激的脂肪细胞糖转运。如果 这些研究的成功将为今后的工作奠定基础。 载体ATP结合位点在运输中的作用评估 规范和理解承运人之间的关系 活动和细胞代谢状态。

This proposal represents a continuing effort by our laboratory to understand the molecular basis of endocrine and metabolic regulation of cellular sugar transport. Metabolic depletion appears to stimulate muscle and avian red cell (ARBC) transport by increasing the intrinsic activity of cell surface sugar carriers (i.e. by carrier activation). This contrasts with insulin stimulation of adipocyte and muscle transport by increased cell surface numbers. The broad goal of this proposal is to characterize the biochemistry of carrier activation and its relationship to insulin regulation of sugar transport. These studies will assist in our long term goal of understanding the molecular basis of protein mediated sugar transport and could ultimately be of value in the management of disordered states such as diabetes. The glucose transport protein contains a transport modulating ATP-binding site. The site has recently been specifically labeled using azido-ATP. Specific AIM 1 is to identify this site by sequencing labelled peptides released on hydrolysis of labelled carrier. Specific Aim 2 asks: since the carrier has recently been shown to be exquisitely susceptible to activation/inhibition by specific membrane bilayer lysophospholipids (LPLs), does carrier activation by metabolic depletion result from altered bilayer lipid composition? We employ reconstituted system and phospholipid analyses and manipulation of control and depleted ARBCs to answer this. Specific Aim 3 asks: since stimulatory LPLs reduce and inhibitory LPLs increase the affinity of glucose carrier for ATP, does this synergism between ATP and LPLs act to amplify their effects on transport? We perform ligand binding and transport studies with reconstituted carrier to answer this. Specific Aim 4 directly tests activation and recruitment hypotheses for transport regulation using recently developed antisera (exclusively reactive against an extracellular domain of the carrier) to quantitate cell surface carrier numbers in control and metabolically depleted ARBCs. Specific Aim 5 extends Aims 2 to 4 to insulin stimulation of adipocyte sugar transport. If successful, these studies will provide the groundwork for future assessment of the role of the carrier ATP-binding site in transport regulation and for understanding the relationship between carrier activity and cellular metabolic status.