Protease Inhibitor Related Adipogenesis in HIV Infection

描述：(申请人提供)HIV-1蛋白酶抑制剂(PI)在高效抗逆转录病毒治疗(HART)中的临床应用已导致 HIV-1感染者预后和生活质量的显著改善 病人。然而，长期使用PIs已导致副作用，如 外周性脂肪营养不良、高脂血症、胰岛素抵抗和 成脂过程。我们的初步研究表明，PI抑制了 3T3-L1细胞成脂分化及肿瘤坏死因子α的进一步加入 抑制成脂率。相比之下，胰岛素增敏 曲格列酮甚至在致敏的肿瘤坏死因子α中也阻断了这种抑制 细胞。拟议研究的主要目标是调查 PI诱导脂肪生成调控的分子机制及机制 验证HIV-1诱导前脂肪细胞致敏的假说 炎性细胞因子TNFα和/或HIV-1 Tat蛋白对PI诱导的作用 脂肪生成受阻。这将通过以下具体措施来实现 目标：1.)测定PIs对成脂作用的体外效应 人骨髓基质祖细胞的分化。会议记录全文 早、中、晚期遗传标记，即Pref-1、脂蛋白脂酶(LPL) 和GAPDH值将分别确定。核转录水平 因子、PPAR-伽马和C/EBP-α将通过瞬变来确定 转染法和凝胶迁移率改变法。2.)要确定 HIV-1诱导的炎性细胞因子、肿瘤坏死因子α和/或致敏作用 HIV-1Tat蛋白对PI抑制小鼠成脂细胞分化的影响 人骨髓基质祖细胞。3.)为了测定体外实验中的 PIs对人基质细胞外基质降解酶活性的影响 成脂祖细胞。未分化和非分化型的纤溶活性 分化的细胞将通过发色纤溶酶进行监测 底物。不同分化阶段的ECM产品将是 SDS-PAGE电泳法测定细胞外基质降解活性 蛋白水解酶(MMPs)将通过明胶酶谱进行监测。实时 RT-PCR研究将监测tPA、PAI-1/2和MMPs/TIMPs的基因表达 它们参与了纤溶级联反应。4.)为了调查 胰岛素增敏剂对PI诱导的脂肪营养不良的改善作用。我们 将研究噻唑烷二酮类药物(罗格列酮和 吡格列酮)和双胍(二甲双胍)对大鼠脑缺血再灌注损伤的抑制作用 PI对成脂分化的抑制作用。这些研究将 阐明可能导致脂肪生成的分子机制 在存在HIV感染的情况下，PIs引起的副作用。

DESCRIPTION: (Provided by applicant) The clinical use of HIV-1 protease inhibitors (PIs) in highly active anti-retroviral therapy (HART) has led to significant improvements in the prognosis and quality of life in HIV-1 infected patients. However, long-term use of PIs has resulted in side effects such as peripheral lipodystrophy, hyperlipidemia, insulin resistance, and disruption of the adipogenic process. Our preliminary studies have shown that PIs suppress adipogenic differentiation in 3T3-L1 cells and the addition of TNFalpha further suppressed the rate of adipogenesis. In contrast, the insulin sensitizing agent, troglitazone, blocked this suppression even in TNFalpha sensitized cells. The primary goal of the proposed research is to investigate the molecular mechanisms involved in the PI-induced modulation of adipogenesis and to test the hypothesis that preadipocytes are sensitized by HIV-1 induced inflammatory cytokine TNFalpha and/or HIV-1 Tat protein, to PI-induced disruption of adipogenesis. This will be achieved by the following specific aims: 1.) To determine the in vitro effects of PIs on adipogenic differentiation in human bone marrow stromal progenitor cells. Transcripts of early, middle and late genetic markers i.e., pref-1, lipoprotein lipase (LPL) and GAPDH, respectively will be determined. Levels of nuclear transcription factors, PPAR-gamma and C/EBP-alpha will be determined by transient transfection assays and gel mobility shift assays. 2.) to determine the sensitizing effect of the HIV-1 induced inflammatory cytokine, TNFalpha and/or HIV-1 Tat protein on PI-induced inhibition of adipogenic differentiation in human bone marrow stromal progenitor cells. 3.) To determine the in vitro effects of PIs on the activity of ECM degrading proteases in human stromal adipogenic progenitor cells. Fibrinolytic activity in undifferentiated and differentiated cells will be monitored by using a chromogenic plasmin substrate. The ECM production at different stages of differentiation will be determined by SDS-PAGE electrophoresis and the activation of ECM degrading proteolytic enzymes (MMPs) will be monitored by gelatin zymography. Real time RT-PCR studies will monitor gene expression of tPA, PAI-1/2 and MMPs/TIMPs which are involved in the fibrinolytic cascade. 4.) To investigate the ameliorative effects of insulin sensitizers on PI-induced lipodystrophy. We will investigate the efficacy of thiazolidinediones (rosiglitazone and pioglitazone) and biguanides (metformin) in suppressing the effects of PI-induced inhibition of adipogenic differentiation. These studies will delineate the molecular mechanisms that may be responsible for the adipogenic side effects induced by the PIs in the presence of HIV infection.