Molecular Signaling of Rapamycin Induced Hyperlipidemia

描述(由申请人提供)： 高甘油三酯血症是心血管疾病(CVD)的主要危险因素，并与胰岛素抵抗有关。雷帕霉素(RAPA)是一种有效的抗增殖和免疫抑制药物，可减少移植急性排斥反应。然而，RAPA诱导高脂血症的机制尚不清楚。了解哺乳动物雷帕霉素靶点(MTOR)通路是如何调节胰岛素信号和引发高脂血症的，将有助于降低心血管疾病和胰岛素抵抗的风险。经过检验的假设是，RAPA通过胰岛素依赖的信号通路诱导高甘油三酯血症。雷帕霉素干扰胰岛素介导的甘油三酯在脂肪细胞中的储存，导致自由脂肪酸(FFA)释放到循环中的增加。随后，FFA的流入导致肝脏VLDL-甘油三酯分泌增加和高甘油三酯血症。我们在肾移植患者中的初步研究以及观察到RAPA通过FK506结合蛋白(FKBP)/哺乳动物靶标雷帕霉素(MTOR)/p70s6激酶途径(13-17)取消胰岛素信号提供了支持证据。研究将利用基于基因组的方法和生化方法来确定3T3-L1脂肪细胞中RAPA诱导信号的细胞路径。基因表达将通过特定途径的基因芯片进行分析，并将与蛋白质水平和磷酸化状态、磷酸酶(PP2A)活性、游离脂肪酸(FFA)、甘油三酯(TG)水平、激素敏感脂肪酶(HSL)和脂蛋白脂肪酶(LPL)活性相关。本研究将在与对照组相同的基础上，研究用反义寡核苷酸阻断RAPA处理的3T3-L1脂肪细胞mTOR翻译的功能意义。目的：1.明确RAPA诱导的豚鼠高甘油三酯血症的体内机制，以及RAPA对肝脏甘油三酯分泌、脂质和脂蛋白调节酶的影响。动物将被注射RAPA或赋形剂，并将用于两项平行研究，以确定RAPA对肝脏甘油三酯分泌、胰岛素信号和脂肪代谢的影响。这些研究的结果将确定雷帕霉素改变的中间分子，并将解决mTOR和胰岛素信号之间的串扰。

DESCRIPTION (provided by applicant): Hypertriglyceridemia is a major risk factor for cardiovascular disease (CVD), and is associated with insulin resistance. Rapamycin (RAPA) is a potent antiproliferative and immunosuppressive drug that reduces acute graft rejection. RAPA, however, induces hyperlipidemia by unknown mechanisms. Understanding how the Mammalian Target of Rapamycin (mTOR) pathway regulates insulin signaling and triggers hyperlipidemia will help reduce risk of CVD, and insulin resistance. The hypothesis tested is that RAPA induces hypertriglyceridemia via an insulin-dependent signaling pathway. Rapamycin interferes with insulin-mediated triglycerides storage in adipocytes leading to increased release of free fatty acids (FFA) to the circulation. Subsequently, influx of FFA lead to augmented hepatic secretion of VLDL-triglycerides and hypertriglyceridemia. Supportive evidence is provided by our preliminary studies in renal transplant patients, as well as observations that RAPA abolishes insulin signals via the FK506 binding protein (FKBP)/mammalian target of rapamycin (mTOR)/p70s6kinase pathway (13-17).SPECIFIC AIM 1. To characterize in vitro the molecular component(s) of the signal transduction of rapamycin in insulin-responsive 3T3-L1 adipocytes. Studies will utilize both genome-based and biochemical approaches to define the cellular pathways of RAPA-induced signals in 3T3-L1 adipocytes. Gene expression will be analyzed by pathway-specific cDNA microarray and will be correlated to protein levels and phosphorylation state; phosphatase (PP2A) activities; FFA; TG levels; hormone sensitive lipase (HSL), and lipoprotein lipase (LPL) activities.SPECIFIC AIM 2. To determine in vitro if inhibition of mTOR expression will eliminate RAPA-induced effects via an insulin-dependent pathway. Studies will investigate the functional significance of blocking mTOR translation by antisense oligonucleotides in 3T3-L1 adipocytes treated with RAPA, compared to the controls, on the same parameters determined in aim 1. SPECIFIC AIM 3. To define in vivo the mechanisms of RAPA-induced hypertriglyceridemia, and impact of RAPA on hepatic triglyceride secretion, lipid and lipoprotein regulatory enzymes in guinea pigs. Animals will be injected with either RAPA or vehicle and will be used in two parallel studies to determine the effect of RAPA on hepatic triglyceride secretion, insulin signaling and lipid metabolism. Findings from these studies will identify the intermediate molecules altered by rapamycin and will address the cross-talk between mTOR and insulin signaling.