Ling H-Y, Hu B, Hu X-B, Zhong J, Feng S-D, Qin L, Liu G, Wen G-B, Liao D-F
Department of Physiology, School of Medicine, University of South China, Hengyang, China.
Exp Clin Endocrinol Diabetes. 2012 Oct;120(9):553-9. doi: 10.1055/s-0032-1311644. Epub 2012 Sep 6.
AIMS/HYPOTHESIS: Our previous study showed there was a change of microRNA (miRNA) expression profile, and miR-21 was significantly down regulated in insulin-resistant adipocytes (IR-adipocytes). Phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, was identified to be a target gene of miR-21, which suggested miR-21 might be associated with insulin resistance (IR) or diabetes. However, it is not known whether miR-21 play any role in the development of IR in 3T3-L1 adipocytes.
Normal adipocytes and adipocytes transfected with pre-miR-21(pmiR-21) or negative control (pNeg) were treated with high glucose and high insulin for 24 h, insulin-stimulated glucose uptake was determined by 2-Deoxyglucose transport assay, miR-21 expression level was measured by using quantitative real-time RT-PCR (qRT-PCR). The protein expression levels of PTEN, Akt, phospho-Akt (Ser473), IRβ, GSK3β, phospho-GSK3β (Ser9) and GLUT4 were detected by western blotting assay.
We further confirmed that miR-21 was down regulated in IR-adipocytes by qRT-PCR. Over-expression of miR-21 significantly increased insulin-induced glucose uptake and decreased PTEN protein expression, while it had no significant effect on PTEN mRNA expression in IR-adipocytes. Moreover, over-expressing miR-21 significantly increased insulin-induced phosphorylation of AKT (Ser473), GSK3β (Ser9) and the translocation of glucose transporter 4 (GLUT4) in IR-adipocytes.
In this study, our data demonstrate that miR-21 reverses high glucose and high insulin induced IR in 3T3-L1 adipocytes, possibly through modulating the PTEN-AKT pathway, and miR-21 may be a new therapeutic target for metabolic diseases such as T2DM and obesity.
目的/假设：我们之前的研究表明，微小RNA（miRNA）表达谱存在变化，且在胰岛素抵抗脂肪细胞（IR-脂肪细胞）中miR-21显著下调。10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）是磷脂酰肌醇3激酶（PI3K）/AKT通路的负调节因子，被确定为miR-21的靶基因，这表明miR-21可能与胰岛素抵抗（IR）或糖尿病有关。然而，尚不清楚miR-21在3T3-L1脂肪细胞IR的发生发展中是否发挥作用。
将正常脂肪细胞以及转染了pre-miR-21（pmiR-21）或阴性对照（pNeg）的脂肪细胞用高糖和高胰岛素处理24小时，通过2-脱氧葡萄糖转运试验测定胰岛素刺激的葡萄糖摄取，使用定量实时RT-PCR（qRT-PCR）测量miR-21表达水平。通过蛋白质印迹法检测PTEN、Akt、磷酸化Akt（Ser473）、IRβ、GSK3β、磷酸化GSK3β（Ser9）和GLUT4的蛋白表达水平。
我们通过qRT-PCR进一步证实了IR-脂肪细胞中miR-21下调。miR-21的过表达显著增加了胰岛素诱导的葡萄糖摄取并降低了PTEN蛋白表达，而对IR-脂肪细胞中PTEN mRNA表达无显著影响。此外，过表达miR-21显著增加了胰岛素诱导的IR-脂肪细胞中AKT（Ser473）、GSK3β（Ser9）的磷酸化以及葡萄糖转运蛋白4（GLUT4）的转位。
在本研究中，我们的数据表明miR-21可逆转高糖和高胰岛素诱导的3T3-L1脂肪细胞中的IR，可能是通过调节PTEN-AKT通路实现的，并且miR-21可能是2型糖尿病和肥胖症等代谢性疾病的新治疗靶点。
