叶酸调控Pdlim5甲基化改善肝脏胰岛素抵抗的作用机制及其传代遗传效应

Hepatic insulin resistance in patients with non-alcoholic fatty liver disease (NAFLD) could increase risk of developing both type 2 diabetes and cardiovascular disease, and promote the progression of NASH, liver cirrhosis and hepatocarcinoma. Recent studies have shown that metabolic risk factors, such as obesity, high fat diet, et al., can lead to increasing susceptibility to hepatic insulin resistance and NAFLD by transgenerationally altering the DNA methylation in liver, however, there is still a lack of effective measures to intervene with the epigenetic trends and control the current increasing NAFLD prevalence. Folic acid, also called vitamin B9, is an endogenous source of methyl groups, and it plays an important role in regulating hepatic DNA methylation. Our previous studies found that serum folic acid concentration is inversely associated with liver histological steatosis grades and insulin resistance level measured by HOMA-IR.Further mechanism study indicated that folic acid might down-regulate the expression of a cellular adaptor protein PDLIM5 through hypermethylation of Pdlim5 gene promoter, inhibiting PKCε cellular membrane translocation and activating insulin signaling pathway in hepatocytes. However，it is still unclear whether the effect of folic acid on Pdlim5 promoter methylation is a key link in improving hepatic insulin resistance, and whether this effect could pass on to the next generation. Therefore, we plan to detect the expression of PDLIM5 and its promoter methylation in liver tissue samples from patients with different grades of NAFLD,and construct Pdlim5 knock-down animal and cellular model to explore its crucial influence to the effects of folic acid in improving hepatic insulin resistance. Offsprings of the experimental mice will also be obtained by natural conception or IVF-ET technology, and the transgenerational effect of folic acid will be further investigated. Thus, this study will reveal the molecular mechanism and transgenetic effects of folic acid in improving hepatic insulin resistance and preventing NAFLD and type 2 diabetes.

NAFLD患者的肝脏胰岛素抵抗增加心血管疾病、2型糖尿病风险，并推动肝脏病变进展。新近发现肥胖、高脂饮食等可改变DNA甲基化推动人群肝脏胰岛素抵抗和NAFLD风险逐代累积增加，且目前对此并无有效应对措施。叶酸是机体甲基化的主要供体。我们前期研究发现人体血清叶酸水平与肝脏脂肪样变、胰岛素抵抗负相关，且动物和细胞实验初步发现叶酸可能通过调控细胞内衔接蛋白PDLIM5的DNA甲基化，下调PKCε表达，活化肝脏胰岛素信号通路。但是，尚不清楚这一机制改善肝脏胰岛素抵抗的重要性和可遗传性。因此，本项目拟进一步1)测定人体不同程度脂肪肝组织Pdlim5表达及其甲基化；2)构建Pdlim5敲减动物和细胞模型，探索叶酸调控Pdlim5启动子甲基化介导肝脏胰岛素抵抗改善的重要性；3)自然受孕和IVF-ET方法获得小鼠子代，探索叶酸调控基因表观遗传对子代影响。从而揭示叶酸改善肝脏胰岛素抵抗的机制和传代遗传效应。

非酒精性脂肪性肝病已成为全球化重要健康问题，累及25%的世界人口，并仍呈现患病率逐渐增长的趋势。已知表观遗传因素受到环境因素调控，可促进脂肪肝的发生发展，并可能通过传代遗传方式导致逐代累积的脂肪肝遗传易感性增加。DNA甲基化是目前研究最深入的普遍表观遗传机制，而叶酸作为机体甲基化的内源性供体，在DNA甲基化修饰中发挥关键作用。本项目前期研究发现人体血清叶酸水平与肝脏脂肪样变、炎症和纤维化水平显著负相关，因此，本项目拟进一步探索叶酸是否通过调控脂质代谢关键基因甲基化改善脂肪肝并发挥传代遗传获益。研究发现：（1）人体血清叶酸水平降低与肝脏组织脂质合成基因Srebp1及其下游基因的表达升高显著相关。（2）叶酸灌胃16周可显著改善高脂饮食诱导的脂肪肝小鼠的肝脏脂肪样变和糖代谢异常，其作用机制与叶酸直接呈浓度依赖地下调脂质合成基因和蛋白表达有关。（3）进一步用高脂饮食喂养叶酸灌胃干预后亲代小鼠的子一代雄性小鼠，发现亲代叶酸干预后除了改善亲代代谢表型外，还对预防子代发生脂肪肝和糖代谢异常，存在传代保护作用。进一步借助组织甲基化芯片联合亚硫酸氢盐处理后测序方法，发现叶酸干预可促进Srebf1的CpG岛的甲基化，从而下调肝脏Srebf1的表达和肝脏脂质从头合成，并且这一甲基化修饰可通过传代遗传方式直接传给子代。本研究（1）借助动物传代遗传模型提示无论是男性或是女性补充叶酸，可对后代预防脂肪肝、糖尿病等代谢性疾病发挥保护作用，为孕期男性以及女性补充适量叶酸提供新的证据，在推动优生优育方面可能有重要的意义。（2）此外，本研究发现通过调控改变表观遗传修饰可能对子代的代谢发挥调控作用，提示表观遗传修饰，特别是本研究涉及的DNA甲基化修饰，可能是生命周期最早期甚至前期干预代谢异常疾病的关键靶点。为通过干预调控甲基化减少种族代谢性疾病易感性，减少代谢性疾病负担具有长远的现实意义。