Mechanisms for Altered Glucose Homeostasis During HAART

HAART 期间改变血糖稳态的机制

• 批准号: 7284726
• 负责人: PAUL W HRUZ
• 金额: $ 30.44万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7284726
• 关键词: Acute; Adipocytes; Adipose tissue; Animals; Antibodies; Antibody Affinity; Atazanavir; Binding; Binding Proteins; Binding Sites; Cardiac; Chronic; Co-Immunoprecipitations; Condition; Continuous Intravenous Infusion; Control Animal; Coupled; Deoxyglucose; Development; Diabetes Mellitus; Disruption; Epitopes; Euglycemic Clamping; Fatty acid glycerol esters; Funding; GLUT 4 protein; GLUT4 gene; Glucose Clamp; Glucose Transporter; Goals; HIV Protease Inhibitors; Heart; Hepatic; Highly Active Antiretroviral Therapy; Indinavir; Individual; Insulin; Insulin Resistance; Knockout Mice; Label; Leptin; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Metabolic syndrome; Mitochondria; Molecular; Molecular Target; Morbidity - disease rate; Muscle; Mutation; Nonesterified Fatty Acids; Nucleosides; Patients; Peptides; Peripheral; Pharmaceutical Preparations; Polymerase Chain Reaction; Post-Translational Protein Processing; Production; Proteins; RNA; Radiolabeled; Rattus; Retinol Binding Proteins; Reverse Transcriptase Inhibitors; Ritonavir; Role; SLC2A1 gene; Serum; Skeletal Muscle; Stavudine; System; Time; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Week; Western Blotting; Work; Xenopus oocyte; Zidovudine; abacavir; adiponectin; antiretroviral therapy; basal insulin; blood glucose regulation; functional loss; glucose disposal; glucose production; glucose transport; glucose uptake; intraperitoneal; mortality; prevent; radiotracer; research study; resistin; uptake

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the molecular mechanisms responsible for altered glucose homeostasis during highly active antiretroviral therapy (HAART). The metabolic changes that occur in association with the use of HIV protease inhibitors (Pis) have significant potential for increasing cardiac- associated morbidity and mortality. The immediate objectives of this proposal are to determine the specific contribution of GLUT4 inhibition to altered glucose uptake into insulin responsive tissues, to elucidate the role of altered glucose transport in the development of changes in adiocytokine production, and to identify the precise molecular interactions that mediate PI binding to GLUT4. The role of GLUT4 in Pi-induced effects on peripheral glucose disposal will be studied by measuring radiolabeled 2-deoxyglucose uptake into cardiac, skeletal muscle and adipose tissue under basal and hyperinsulinemic euglycemic clamp conditions in wild-type and transgenic mice with specific deletion of GLUT4 from muscle or fat. Changes in leptin, adiponectin and resistin RNA and protein levels, peripheral glucose disposal, and hepatic glucose production will be determined following semi-acute exposure of control and GLUT4 null mice to Pis in the presence and absence of semi-chronic treatment with NRTIs. The structural features in GLUT4 that mediate HIV protease inhibitor binding and inactivation will be determined in primary and/or cultured rat adipocytes labeled with a photoactivatable peptide containing the GLUT4 inhibiting epitope zHFF coupled to the FLAG epitope. Modified proteins will be isolated using a FLAG antibody affinity column and identified by mass spectrometry. The ability of Pis to influence the binding of proteins known to interact with the GLUT4 carboxy terminus will also be investigated by Western blot analysis and co-immunoprecipitation experiments using factor specific antibodies. Mutations will be introduced into the identified GLUT4 PI binding site and the resulting functional effects on transport activity and PI sensitivity will be determined. Taken together, these studies will provide crucial information about the mechanism(s) by which Pis contribute to the development of insulin resistance and diabetes mellitus. This will greatly assist efforts to develop effective strategies for preventing and/or treating the metabolic changes that occur both in patients on HAART and in the wider context of HIV-negative individuals with features of the metabolic syndrome.

描述（由申请人提供）：该项目的长期目标是了解在高效抗逆转录病毒治疗（HAART）期间导致葡萄糖稳态改变的分子机制。与使用 HIV 蛋白酶抑制剂 (Pis) 相关的代谢变化极有可能增加与心脏相关的发病率和死亡率。该提案的直接目标是确定 GLUT4 抑制对改变胰岛素响应组织中的葡萄糖摄取的具体贡献，阐明改变的葡萄糖转运在脂肪细胞因子产生变化中的作用，并确定介导 PI 与 GLUT4 结合的精确分子相互作用。通过测量在基础和高胰岛素正常血糖钳夹条件下，在肌肉或脂肪中特异性删除 GLUT4 的野生型和转基因小鼠中心脏、骨骼肌和脂肪组织中放射性标记的 2-脱氧葡萄糖摄取，来研究 GLUT4 在 Pi 诱导的外周葡萄糖处理影响中的作用。在存在或不存在NRTIs半慢性治疗的情况下，将对照小鼠和GLUT4缺失小鼠半急性暴露于Pis后，测定瘦素、脂联素和抵抗素RNA和蛋白质水平、外周葡萄糖处理和肝葡萄糖产生的变化。介导HIV蛋白酶抑制剂结合和失活的GLUT4的结构特征将在用含有与FLAG表位偶联的GLUT4抑制表位zHFF的光激活肽标记的原代和/或培养的大鼠脂肪细胞中确定。将使用 FLAG 抗体亲和柱分离修饰的蛋白质，并通过质谱法进行鉴定。 Pis 影响已知与 GLUT4 羧基末端相互作用的蛋白质结合的能力也将通过蛋白质印迹分析和使用因子特异性抗体的免疫共沉淀实验进行研究。将突变引入已识别的 GLUT4 PI 结合位点，并确定由此产生的对转运活性和 PI 敏感性的功能影响。总而言之，这些研究将提供有关 Pis 导致胰岛素抵抗和糖尿病发展的机制的重要信息。这将极大地有助于制定有效策略来预防和/或治疗HAART患者以及具有代谢综合征特征的HIV阴性个体中发生的代谢变化。