持续性红细胞生成素受体激动剂（CERA）改善db/db小鼠骨骼肌胰岛素抵抗的实验研究

Insulin resistance is a main feature of the metabolic syndrome and chronic kidney disease (CKD), particularly in patients with end-stage renal disease (ESRD).Erythropoietin stimulating agents(ESAs)reduces serum insulin levels, increases insulin sensitivity, and improves insulin resistance. However, the mechanisms behind this are unclear. Our previous studies indicated rHuEPO treatment improved glucose uptake, increased adiponectin levels, and reduced TNF-α and leptin levels in dexamethasone-induced insulin resistance 3T3L1 adipocytes. EPOR protein and gene expression was present in mature 3T3L1 adipocytes. However, the expression of EPOR in insulin resistant 3T3L1 adipocytes was significantly decreased. rHuEPO increased the expression of EPOR, and upregulated the expression of pAKT/AKT and pSTAT5/STAT5 in 3T3L1 adipocytes, which was blocked by the phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002, STAT5 inhibitor and siRNA, respectively. We suggest that the EPO-EPOR system may play a role in glucose metabolism within adipocytes. However, there is few study to investigate the effect of CREA on improving insulin resistant of adipocyte or skeletal muscle. As a new long-term ESA, CERA has large molecular weight, is not easy to be dialysised clearly and long half-life. Based on the above study, the objective of this study is to investigate the effects of continuous erythropoietin receptor activator（CERA） on insulin resistance in skeletal muscle of obese db/db mice, and its potential mechanisms.

红细胞生成刺激剂(ESAs)长期应用于慢性肾脏疾病（CKD）纠正肾性贫血。持续性红细胞生成素受体激动剂(CERA)是新型长效ESA，因其分子量大不易被透析清除以及超长的体内半衰期，具有临床优势。CKD相关的胰岛素抵抗（IR）长期以来倍受关注。我课题组首先报道了成熟3T3-L1脂肪细胞上存在EPO受体（EPOR），在地塞米松诱导的IR模型中EPOR表达下降，给予不同浓度rHuEPO刺激，呈剂量依赖地促进脂肪细胞的脂联素分泌，抑制瘦素的表达，该作用可能与STAT5和Akt信号通路激活有关，提示EASs具有改善胰岛素抵抗的作用并独立于贫血的纠正。此次课题申请拟在前期研究的基础上观察CERA对2型糖尿病模型小鼠（db/db）外周组织骨骼肌上EPOR的表达，胰岛素受体敏感性及下游PI3K/AKT信号通路以及葡萄糖转运蛋白4（GLUT4）的表达转位的影响，旨在阐明CERA改善胰岛素抵抗的作用机制。

胰岛素抵抗（IR）是导致2型糖尿病的主要原因之一，脂肪组织、骨骼肌等外周组织是IR主要累及的器官。我们过去的研究已经证实促红细胞生成素（EPO）可以改善脂肪组织的IR，但是能否改善骨骼肌IR尚不明确。本研究主要观察了EPO对改善2型糖尿病模型小鼠（db/db）骨骼肌胰岛素抵抗的作用及相关机制。通过体内研究证实EPO具有改善db/db骨骼肌胰岛素抵抗的作用，表现为血清血糖的下降，IGTT葡萄糖的曲线下面积的下降，以及ITT胰岛素曲线下面积的下降，而小鼠体重变化不明显。血液中EPO浓度与血糖浓度无关，但是骨骼肌中的EPOR受体表达升高，骨骼肌中NO浓度的升高，结果提示EPO改善db /db胰岛素抵抗可能通过其受体EPOR下游通路以及增加骨骼肌中的NO浓度有关。进一步检测EPO对小鼠骨骼肌IRS/PI3K/AKT信号通路检测发现，EPO可以增加骨骼肌局部的IRS/PI3K/AKT的磷酸化水平，同时增加eNOS的含量，并通过原代分离小鼠骨骼肌细胞，给予培养和鉴定，并构建了慢病毒包裹shRNA EPOR 载体敲低内源性EPOR，同时给予EPO刺激。证实了EPO可以增加骨骼肌局部的IRS/PI3K/AKT的磷酸化水平，同时增加eNOS的含量，该过程可以被shRNA EPOR阻断。明确了EPO增加了骨骼肌细胞GLUT4的转位，同时抑制骨骼肌细胞的凋亡，以及促进骨骼肌细胞的自噬。本研究为改善2型糖尿病IR提供了新的治疗靶点。