Mechanisms for Altered Glucose Homeostasis During HAART

描述（由申请人提供）：该项目的长期目标是了解高活性抗逆转录病毒治疗（HAART）期间葡萄糖稳态改变的分子机制。与使用HIV蛋白酶抑制剂（Pis）相关的代谢变化具有显著增加心脏相关发病率和死亡率的潜力。本研究的直接目标是确定GLUT4抑制对胰岛素应答组织中葡萄糖摄取改变的具体贡献，阐明葡萄糖转运改变在脂细胞因子产生变化的发展中的作用，并确定介导PI与GLUT4结合的精确分子相互作用。我们将在野生型和转基因小鼠肌肉或脂肪中特异性缺失GLUT4的情况下，通过测量基础和高胰岛素正糖钳夹条件下心脏、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中的作用。瘦素、脂联素和抵抗素RNA和蛋白质水平的变化、外周葡萄糖处理和肝脏葡萄糖生成将在对照和GLUT4缺失小鼠在存在和不存在半慢性nrti治疗的情况下半急性暴露于Pis后确定。GLUT4介导HIV蛋白酶抑制剂结合和失活的结构特征将在原代和/或培养的大鼠脂肪细胞中确定，这些脂肪细胞被光激活肽标记，其中含有GLUT4抑制表位zHFF和FLAG表位偶联。修饰蛋白将使用FLAG抗体亲和柱分离，并通过质谱法鉴定。Pis影响已知与GLUT4羧基端相互作用的蛋白质结合的能力也将通过Western blot分析和使用因子特异性抗体的共免疫沉淀实验进行研究。将突变引入鉴定的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。综上所述，这些研究将为Pis促进胰岛素抵抗和糖尿病发展的机制提供重要信息。这将极大地有助于制定有效的策略，以预防和/或治疗HAART患者和具有代谢综合征特征的艾滋病毒阴性个体中发生的代谢变化。

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.