高糖激活的wnt/β-catenin/ TCF7L2信号通路诱导糖尿病心肌能量代谢紊乱的作用及机制

The disorders of cardiac metabolism can result in diabetic cardiomyopathy, but the mechanism is still not clear. Emerging evidence has shown the canonical Wnt/β-catenin signaling pathway is associated with metabolic disorders of Diabetes Mellitus. The TCF7L2, as a member of T cell factor(TCF)/lymphoid enhancer factor(LEF) family transcriptional factors, is important in conveying Wnt signaling in prompting gene expression. Epidemiological evidence suggests that intronic SNPs in TCF7L2 is strongly associated with type 2 diabetes. The variants in the TCF7L2 and its misregulation can cause the metabolic disorders. However, how Wnt/β-catenin/ TCF7L2 pathway is regulated by diabetic conditions in the heart is also poorly understood. Our preliminary data showed that the expression of TCF7L2 and β-catenin was higher in the nuclear extracts from the heart of db mice. In HL-1 cells, high glucose increased the TCF7L2 expression at 1, 6 ,12 and 24 hours after treatment relative to control; the translocation of β-catenin was seen after high glucose treatment 1 hour. And overexpression of TCF7L2 induced by the adenovirus containing TCF7L2 full length increased the PDK4 and CPT-1 expression, which were involved in regulation of the glucose and fatty acid metabolism in heart. These data indicated that Wnt/β-catenin/TCF7L2 pathway activated by hyperglycemia may contribute to the pathogenesis of metabolic disorder in diabetic cardiomyopathy. The acetylation of β-catenin can regulate the transcriptional activity of TCF7L2. Recently, one study on cancer showed that high glucose activated-p300 triggered β-catenin acetylation, and consequently activated the promoters of target genes. Accordingly, the hypothesis of this program is TCF7L2 upregulates the expression of these genes involved in the glucose and fatty acid metabolism in cardiomyocytes; and hyperglycemia activated-Wnt/β-catenin/ TCF7L2 misregulates these gene expression, and finally results in the disorders of cardiac metabolism. The results that generate from this project will provide a novel mechanism and a therapeutic target for the metabolic disorder in diabetic cardiomyopathy.

心肌能量代谢紊乱是导致糖尿病心肌病重要原因但机制未明。TCF7L2是经典Wnt/β-catenin通路核转录因子，其基因多态性与糖尿病发病率显著相关，其基因变异或活性异常致代谢紊乱。课题组预实验结果显示Wnt/β-catenin/TCF7L2参与心肌能量代谢调节，但在糖尿病心肌代谢紊乱的作用不清楚。组蛋白修饰调节该通路转录活性，高糖激活肿瘤细胞乙酰基转移酶增强其转录作用。由此提出中心假说：高糖激活心脏Wnt/β-catenin/TCF7L2诱导糖、脂代谢基因表达异常导致能量代谢紊乱；乙酰化修饰调控其转录活性。本课题拟通过db/db小鼠、高糖处理的乳鼠心肌细胞研究TCF7L2对心肌代谢基因的转录调控，并用特异性抑制剂和分子生物学手段调节TCF7L2、β-catenin表达，明确高糖对心脏Wnt/β-catenin/TCF7L2的调控作用及机制，为糖尿病心肌能量代谢紊乱发病机制提供新线索。

Tcf7l2是经典Wnt/β-catenin通路核转录因子，其基因多态性与糖尿病发病率显著相关，其基因变异或活性异常致代谢紊乱，但经典Wnt/β-catenin/ TCF7L2通路在糖尿病心肌能量代谢紊乱中的作用尚不清楚。本项目通过体内外模型研究高糖及糖尿病环境下Wnt/β-catenin/ Tcf7l2通路与心肌能量代谢的关系。结果发现：（1）慢病毒表达载体lenti-Tcf7l2 WT、lenti-Tcf7l2 shRNA分别诱导Tcf7l2 过表达和表达降低能调控心肌糖代谢相关基因Slc2a4、Pdha1；脂质代谢相关基因Pdk4、Acot1、Acot7、Apoc1表达。高糖与Tcf7l2慢病毒共同处理心肌细胞后，对上述糖脂代谢基因表达的调控作用进一步增强。经免疫染色质沉淀验证Slc2a4、Pdha1、Pdk4是Tcf7l2调控心肌糖脂代谢的主要靶基因。（2）STZ诱导的1型糖尿病小鼠、瘦素受体缺失的db/db 2型糖尿病小鼠和高糖处理的原代乳鼠心肌细胞中，持续升高的血糖能上调心脏β-catenin、Tcf7l2表达，诱导β-catenin入核，提高Tcf7l2活性，促进β-catenin与Tcf7l2结合。（3）连续给予1型糖尿病小鼠β-catenin通路抑制剂iCRT14 8周，小鼠空腹血糖降低，心脏体重比例降低，心肌细胞核内β-catenin 、Tcf7l2表达减少，β-catenin 与Tcf7l2结合明显降低。心肌肥大标志基因ANF、β-mhc mRNA水平表达下降；Tcf7l2调控的糖脂代谢靶基因： Slc2a4、Pdha1 mRNA表达升高，Pdk4 mRNA表达降低，能量代谢紊乱能显著改善。（4）腺相关病毒AAV9-Tcf7l2 shRNA靶向抑制1型糖尿病小鼠心脏Tcf7l2表达16周，小鼠随机血糖、空腹血糖下降，心脏体重比例降低，心脏舒张功能改善，糖脂代谢基因Pdk4 mRNA表达降低，Slc2a4、Pdha1 mRNA表达增强，能量代谢紊乱表征得到明显改善。这些研究结果提示高糖能激活心脏Wnt/β-catenin/ Tcf7l2信号通路，该通路通过活化Tcf7l2诱导糖脂代谢基因表达异常参与糖尿病心肌能量代谢紊乱的发生发展。