特异性脂肪组织LXRs表达抑制在白色脂肪向棕色脂肪转化及改善胰岛素抵抗中的作用和机制研究

Obesity arises from chronic energy surplus and excess lipid storage in white adipose tissue (WAT), while brown adipose tissue (BAT) burns lipid to generate heat. Recent studies reavealed the magic effect of BAT activation on the insulin sensitivity and the reversal of impaired glucose tolerance in rodent animals. A finding that adult humans also have functional BAT has fueled speculation that pharmacologic enhancement of BAT development and activity might be a useful strategy to counteract obesity. Results from mice of LXRα-/- and/or LXRβ-/- showd that LXRα or LXRβ gene knockout promotes the activity of BAT and the transformation from WAT to BAT. However, the overall LXRs geneknockout in the body will lead to cholesterol metabolism disorders and severe arterial atherosclerosis..In our previous studies, we found that activation of LXRs inhibited the transcriptional activity of PPARγ, which is an important transcriptional fator in the activation of BAT and in the recruitment of BAT in WAT in mature adipocytes. More importantly, we found that the expression of cyclooxygenase-2 (COX-2), a molecule recognized recently as the most important downstream effector of β-adrenergic signaling in WAT and also required for the recruiment of brown-like adipocyte in WAT depots was inhibited by LXRs agonist T0901317, While, LXRα knockdown by RNA interference increased the experssion of COX-2 in 3T3-L1 preadipocyte cells. Based on these results from mature adipocyte, we assumed that specific LXRs knockdown in adipose tissue would promote the BAT activity and the recruinment of WAT in BAT..In the present study, we will separate the stromal-vascular fraction （SVF）from inter-scapular brown fat tissue(iBAT), inguinal white adipose tissue(iWAT) and epididymal white fat tisse (eWAT) and induce them for differentiation with special brown adipocyte defferentiation medium. We will also adopt a high-fat diet to induce the obese and insulin-resistant mouse model. With such cell and animal models, we will investigate whether specific knockdown of LXRs genes in SVF or in white fat tissue (epididymal) would inhibit the SVF transdifferentiation from white adipocyte to brown-like adipocyte and the brown-like adipocyte recruiment of WAT in BAT. Based on the important role of BAT in insulin sensitivity, we would also like to observe the improvement of insulin function by BAT activation with adipose specific LXRs inhibition in high-fat diet induced obese mice .Manipulation of LXRs activity in adipose tissue probably represents an alternative strategy for enhancing BAT activity that could help protect against obesity and insulin resistance.

棕色脂肪组织（BAT）在成人体内存在且促进能量消耗，激活BAT可能是拮抗肥胖及胰岛素抵抗的新方向。小鼠中发现肝X受体(LXRs)基因敲有利于BAT激活以及白色脂肪（WAT）向BAT转化，但可能导致脂代谢紊乱。我们前期研究在脂肪细胞中发现LXRs激动抑制PPARγ转录活性以及负向调节环加氧酶-2（COX-2）活性，而后两者是体内WAT向BAT转化的必须分子。由此提出脂肪组织LXRs抑制可能促进PPARγ转录活性和COX-2通路活性，诱导BAT激活及WAT向BAT转化，并改善肥胖小鼠胰岛素敏感性。本研究拟分离小鼠BAT和WAT的血管基质成分（SVF）并诱导分化，建立高脂饮食肥胖小鼠模型，借助启动子报告基因、染色质免疫沉淀、RNA干扰及腺病毒脂肪转染、能量代谢和胰岛素敏感性评价试验观察特异脂肪LXRs抑制在促进BAT激活及改善胰岛素敏感性中的作用并探讨机制，旨在为干预肥胖及其代谢紊乱找到有效靶

背景：既往研究发现脂肪组织选择性的肝X受体（LXRs）敲低导致小鼠肥胖、脂肪组织肥大，而非组织特异性的LXRs基因敲除小鼠则能抵抗高糖饮食诱导的肥胖。而激活棕色脂肪被证实是非组织选择性LXRs基因敲除抵抗肥胖的重要机制。本研究拟观察肝X受体抑制剂22-S-Hydroxycholesterol（22-S-HC）及脂肪细胞特异性敲低LXRs对白色脂肪向棕色脂肪转化的作用,以及对棕色化相关信号通路的影响，探讨可能的分子机制。方法：建立MEF细胞向棕色脂肪细胞诱导分化的模型，观察22-S-HC及敲低LXRs对MEF细胞棕色化的作用。建立高脂饮食肥胖小鼠模型（HFD），以22-S-HC（30mg/kg/d，15d）腹腔注射，观察LXRs抑制剂对HFD小鼠体重、糖耐量、胰岛素敏感性的作用，并通过组织切片染色、免疫荧光、Western Blot及qRT-PCR等实验方法检测脂肪组织的棕色化改变。构建UCP1、PPARγ、PGC1α、COX2 的启动子报告基因质粒,利用双荧光素酶报告检测系统来检测 22-S-HC 和 LXRs 敲低对于上述启动子的作用。结果：1、应用棕色脂肪细胞诱导方案， MEF细胞在诱导第14天时棕色脂肪细胞的特异性蛋白-解偶联蛋白1（UCP1）表达最明显；而qRT-PCR，Western Blot及免疫荧光染色检测均显示，使用22-S-HC拮抗LXRs，或采用shRNA技术敲低LXRs均可增加UCP1的表达，同时过氧化物酶体增殖物激活受体γ（PPARγ）及其转录辅因子PGC1α转录及蛋白表达亦显著升高，细胞对葡萄糖的摄取能力也显著提高。2、22-S-HC腹腔注射组小鼠较对照组的体重下降，腹腔糖耐量试验（IPGTT）和腹腔胰岛素耐量试验（ITT）显示治疗组IPGTT血糖曲线下面积（AUCglu）水平下降（1718.4±243.8 vs 2078.2±195.4, P=0.033）, 而ITT的AUCglu更低（833.34±85.4 vs 1027.9±53.1，P=0.005）。HE染色可见22-S-HC腹腔注射组小鼠腹股沟脂肪组织脂肪细胞体积变小，脂滴沉积减少，提示棕色化特征较对照组明显。腹股沟脂肪组织的UCP1表达亦明显升高。3、22-S-HC和LXRs的敲低对UCP1、PPARγ及PGC1α的启动子均有一定程度的激活。结论：肝X受体抑制剂22-S-HC和