Protease Inhibitor Related Adipogenesis in HIV Infection

说明：（申请人提供）HIV-1蛋白酶的临床用途 高活性抗逆转录病毒治疗（哈特）中的抑制剂（PI）导致 显著改善HIV-1感染者的预后和生活质量 患者然而，长期使用PI会导致副作用，例如 外周脂肪营养不良、高脂血症、胰岛素抵抗和 脂肪形成过程我们的初步研究表明，PI抑制 3 T3-L1细胞中的脂肪形成分化和TNF α的加入进一步 抑制脂肪生成的速率。相反，胰岛素增敏 药物曲格列酮甚至在TNF α致敏的 细胞这项研究的主要目的是调查 PI诱导的脂肪形成调节的分子机制， 为了验证前脂肪细胞被HIV-1诱导的 炎性细胞因子TNF α和/或HIV-1达特蛋白，对PI诱导的 破坏脂肪生成。这将通过以下具体措施来实现 目的：1.）确定PI对成脂细胞的体外作用， 人骨髓基质祖细胞的分化。成绩单 早期、中期和晚期遗传标记，即，脂蛋白脂酶（LPL） 和GAPDH，将分别确定。核转录水平 因子，PPAR-gamma和C/EBP-α将通过瞬时 转染测定和凝胶迁移率变动测定。2.）的情况。确定 HIV-1诱导的炎性细胞因子TNF α和/或 HIV-1达特蛋白对PI诱导的成脂细胞分化抑制的影响 人骨髓基质祖细胞3.）第三章为了确定体外 PI对人基质细胞ECM降解蛋白酶活性的影响 成脂祖细胞在未分化和未分化的 将通过使用显色纤溶酶监测分化的细胞 衬底在不同分化阶段的ECM产生将是 通过SDS-PAGE电泳和ECM降解活性测定 蛋白水解酶（MMP）将通过明胶酶谱法监测。真实的时间 RT-PCR研究将监测tPA、派-1/2和MMPs/TIMPs的基因表达 参与纤维蛋白溶解级联反应。4.）探讨 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）和双胍类（二甲双胍）抑制 PI诱导的成脂分化抑制。这些研究将 描绘可能负责脂肪形成的分子机制， 在存在HIV感染的情况下PI诱导的副作用。

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.