基于超高效液相色谱-飞行时间质谱法测定LAMTOR1在肝脏炎症恶性转化中调控的代谢物

Identification of LAMTOR1-regulated metabolites using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry in malignant transformation of liver inflammation

LAMTOR1(晚期胞内体/溶酶体接头蛋白,MAPK以及mTOR激活蛋白1)是机体应对营养压力时重要的调控蛋白之一。公共开放基因表达数据库分析显示LAMTOR1在非酒精性脂肪肝炎(NASH)和肝癌中均异常高表达,显示LAMTOR1在NASH和肝癌发生发展中发挥重要作用,探索LAMTOR1在肝脏炎症恶性转化过程中调控的代谢机制具有重要意义。该研究中小鼠给予蛋氨酸胆碱缺乏(MCD)饮食饲养,肝脏组织的苏木精伊红(HE)染色结果显示小鼠肝脏炎症性损伤的成功构建。接下来利用蛋白免疫印迹实验验证了肝脏组织中LAMTOR1基因的特异性敲除以及一些LAMTOR1调控的蛋白变化。紧接着开展了基于超高效液相色谱-飞行时间质谱联用的肝脏组织代谢组学分析,以鉴定LAMTOR1肝脏特异性调控的重要代谢物。对检测到的134个代谢物进行多变量分析,主成分分析模型显示特异性敲除LAMTOR1对小鼠肝脏的代谢过程有明显的扰动。其中45个代谢物发生了显著性变化,表明敲除LAMTOR1可引起肝脏多条代谢通路紊乱。进一步分析显示,UDP-乙酰葡萄糖胺(UDP-GlcNAc)、S-腺苷蛋氨酸、S-腺苷高丝氨酸和三甲基赖氨酸等代谢物在LAMTOR1敲除(LAMTOR1LKO)小鼠中明显上调,说明LAMTOR1与己糖胺合成通路和生物分子甲基化过程可能存在调控关系。另外,9-氧代十八碳二烯酸、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)等不饱和脂肪酸等代谢物水平在LAMTOR1LKO小鼠中明显下降。接下来基于公共开放转录组数据库开展了基因表达相关性的预测分析,得到的相关系数R表征基因间的调控关系,R的绝对值接近或高于0.5属于中强相关,结果提示LAMTOR1可能负调控MAT1A(R=-0.47)基因,同时预测得到LAMTOR1与MGAT1(R=0.47)和ADSL(R=0.59)等基因存在正调控关系。该研究将代谢组学方法应用于疾病机制研究,通过鉴定小鼠NASH模型中LAMTOR1特异性调控的代谢物,并结合基因表达相关性分析,揭示出LAMTOR1基因在非酒精性脂肪肝炎及恶性转化过程中可能调控的重要代谢通路,为后续NASH及NASH转化的肝癌发病机制和治疗研究提供理论基础。

The late endosomal/lysosomal adaptor MAPK and mTOR activator 1(LAMTOR1)is an important regulator protein in the response to energy stress.Public gene expression data shows that the expression of LAMTOR1 is abnormally high in nonalcoholic steatohepatitis(NASH)and hepatocellular carcinoma(HCC);hence,LAMTOR1 may play an important role in the development of NASH and HCC.Therefore,exploring the LAMTOR1 regulatory mechanism in the progression of NASH and malignant transformation of liver inflammation may be crucial for translational medicine.First,a NASH mouse model was established by feeding a methionine choline-deficient(MCD)diet.Hematoxylin-eosin staining of liver tissues showed successful modeling of inflammatory injury in the mouse liver.Immunoblot analysis confirmed LAMTOR1-and LAMTOR1-mediated protein expression in LAMTOR1 specifically depleted mouse livers.Subsequently,metabolic profiling of liver tissues was performed using an ultra-performance liquid chromatography-time-of-flight mass spectrometry strategy.Based on the retention time,m/z value,and tandem mass spectra,134 metabolites were identified.Among these,the levels of 45 metabolite were significantly influenced by hepatic LAMTOR1 depletion.According to the metabolomics results,uridine diphosphate-N-acetylglucosamine(UDP-GlcNAc)was significantly upregulated in LAMTOR1-depleted(LAMTOR1LKO)hepatocyte tissues.As the final product of the hexosamine biosynthetic pathway(HBP),alteration in UDP-GlcNAc levels may regulate LAMTOR1 and metabolic regulatory genes downstream of HBP.Moreover,there was an obvious increase in the levels of several methylation-related metabolites.Thus,upregulated S-adenosylmethionine,S-adenosylhomocysteine,and N6,N6,N6-trimethyl-L-lysine indicated that LAMTOR1 may regulate the process of DNA or protein methylation.In addition,downregulation of 9-oxo-octadecadienoate,eicosapentaenoic acid(EPA),and docosahexaenoic acid(DHA)was also observed in LAMTOR1LKO mice liver tissues.Alterations in polyunsaturated fatty acids,such as EPA and DHA,link LAMTOR1 to inflammatory and immune processes,which are known to play important roles in NASH pathogenesis.These metabolic disorders demonstrated that LAMTOR1 significantly contributed to the metabolic mechanism of NASH.Furthermore,gene expression correlations were analyzed to interpret the regulatory role of LAMTOR1 from the perspective of genetic networks.Owing to a paucity of liver whole-transcriptome studies in NASH,correlation analysis was performed based on HCC transcriptome data from public databases.First,a negatively regulated relationship was observed between LAMTOR1 and MAT1A(R=-0.47).MAT1A encodes methionine adenosyltransferase 1A,an essential enzyme that catalyzes the formation of S-adenosylmethionine.Based on the upregulation of UDP-GlcNAc under hepatocyte LAMTOR1 depletion,it was predicted that LAMTOR1 positively influenced MGAT1(R=0.47),a gene encoding alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase.Together with changes in succinyladenosine caused by hepatocyte LAMTOR1 deletion,predicted correlation results showed that LAMTOR1 may also participate in the pathogenesis through the positive regulatory relationship with ADSL(R=0.59).The ADSL gene provides instructions for making an enzyme called adenylosuccinate lyase,which can dephosphorylate the substrate succinyladenosine.In this study,LAMTOR1 was identified to specifically regulate multiple key metabolic pathways based on both NASH mouse models and gene expression correlations.These results illustrate the important role of LAMTOR1 in the progression of NASH and malignant transformation of liver inflammation,which provides a theoretical basis for the diagnosis and treatment of NASH or possible NASH-driven HCC.