组蛋白乙酰化调控Wnt∕β-catenin通路在猪肝脏氨代谢中的作用及机理研究

Nitrogen emissions have become a grave social concern, which seriously restricts the sustainable development of China's pig industry. In contrast to conversion of ammonia into glutamine and other amino acids in perivenous hepatocytes, ammonia in periportal hepatocytes is responsible for urea synthesis. With regard to the difference of hepatic ammonia metabolism, it has been demonstrated that the distinct metabolic patterns are determined (influenced) by Wnt∕β-catenin pathway. According to our further endeavor, hepatic Wnt∕β-catenin pathway was modified by histone acetylation. Given this research background, we are wondering what is the key histone acetylase regulating the different ammonia metabolism in periportal and perivenous hepatocytes. Moreover, what is the sophisticated mechanism involved, discrepancy in gene expression as a result of binding between Wnt/β-catenin pathway to different region of DNA due to different histone acetylation, or different acetylated sites of β-catenin by histone acetylase? This program is based on the periportal and perivenous hepatocytes isolation and culture system, both of which were successfully established to explore the differences of ammonia metabolism and histone acetylation characteristics. TAP/MS techniques are used to investigate the acetylation characteristics of β-catenin by histone acetylase. On the other hand, the histone acetylation regular pattern of key protein DNA binding domain in the Wnt/β-catenin pathway is analyzed by ChIP techniques. By the combination of epigenetic and molecular nutrition, we finally attempt to reveal the mechanism of hepatic ammonia metabolism in pigs.

氮排放问题制约我国养猪业可持续发展。氨在猪肝脏门静脉周肝细胞代谢为尿素，而在肝静脉周肝细胞合成谷氨酰胺等氨基酸。氨代谢差异受Wnt∕β-catenin通路调控，课题组发现猪肝脏Wnt∕β-catenin通路受组蛋白乙酰化酶修饰。那么调控猪肝细胞氨代谢差异的乙酰化酶是什么？是由于组蛋白乙酰化酶对β-catenin的乙酰化位点不同，还是组蛋白乙酰化差异引起了Wnt∕β-catenin通路被招募到不同的DNA区域调控基因表达，导致了门静脉周和肝静脉周肝细胞的氨代谢差异？本研究拟在已建立的门静脉周、肝静脉周肝细胞分离与培养基础上，研究肝细胞氨代谢差异及组蛋白乙酰化特点；采用TAP/MS等技术研究组蛋白乙酰化酶对β-catenin乙酰化特点；采用ChIP技术，分析Wnt∕β-catenin通路关键蛋白DNA结合区域组蛋白乙酰化差异规律。通过表观遗传学和分子营养学结合，旨在揭示猪肝脏氨代谢的差异机理。

氮排放问题制约我国养猪业可持续发展。氨在猪肝脏门静脉周肝细胞代谢为尿素，而在肝静脉周肝细胞合成谷氨酰胺等氨基酸。氨代谢差异受Wnt∕β-catenin通路调控，课题组发现猪肝脏Wnt∕β-catenin通路受组蛋白乙酰化酶修饰。本项目成功建立了猪门静脉周、肝静脉周肝细胞分离与培养技术，发现两类肝细胞在氨代谢上存在差异；发现了氨在猪门静脉代谢生成尿素过程中，会加速氨基酸分解代谢；Sirt3和Sirt5分别通过去乙酰化调控尿素循环中的关键基因OTC和CPS1，促进了尿素的生成。其次，本项目还发现了现β-catenin在门静脉周肝细胞DNA结合区域中负调控尿素生成关键基因CPS1、Ag1、AST、ASS的表达，而在肝静脉周肝细胞DNA结合区域中正调控谷氨酰胺生成关键基因Gs和Rhbg的表达；之后我们通过在两类肝细胞中转染乙酰转移酶PCAF，发现PCAF显著提高门静脉周和肝静脉周β-catenin乙酰化水平，促进了尿素和谷氨酰胺生成。进一步地，本项目还利用TMT标记定量蛋白组学技术集中对乙酰化的氨基酸代谢与尿素循环关键酶进行鉴定及筛选，发现门静脉周肝细胞中氨基酸代谢生成尿素途径的关键酶AST和ASS也受到了大量的乙酰化修饰。另外，本课题组成功建立了猪尿氮排放监控技术平台—养殖环境控制箱，为进一步在猪机体上研究氨代谢奠定了实践基础。我们还发现β-catenin在门静脉周肝细胞DNA结合区域中负调控的尿素生成关键基因ALT和AST受到乙酰化修饰，且通过免疫共沉淀术的技术（CoIP）发现ALT和AST均被乙酰转移酶PCAF乙酰化，并且也筛选得到控制ALT和AST酶活性的乙酰化位点。本项目还利用猪门静脉灌注技术，探究了亮氨酸对不同来源氨在猪肝脏代谢的调控作用及机理利用。总体来看，项目的研究结果揭示了PCAF通过乙酰化β-catenin，调控两类细胞氨代谢基因的表达以及尿素和谷氨酰胺的生成，阐明了猪肝脏氨代谢的差异机理，为减少氨排放、促进我国养殖业可持续发展提供了理论基础和试验依据。