Gogiashvili Mikheil, Edlund Karolina, Gianmoena Kathrin, Marchan Rosemarie, Brik Alexander, Andersson Jan T, Lambert Jörg, Madjar Katrin, Hellwig Birte, Rahnenführer Jörg, Hengstler Jan G, Hergenröder Roland, Cadenas Cristina
Leibniz Institut für Analytische Wissenschaften - ISAS e.V., Bunsen-Kirchhoff-Strasse 11, 44139, Dortmund, Germany.
Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
Anal Bioanal Chem. 2017 Feb;409(6):1591-1606. doi: 10.1007/s00216-016-0100-1. Epub 2016 Nov 28.
Metabolic perturbations resulting from excessive hepatic fat accumulation are poorly understood. Thus, in this study, leptin-deficient ob/ob mice, a mouse model of fatty liver disease, were used to investigate metabolic alterations in more detail. Metabolites were quantified in intact liver tissues of ob/ob (n = 8) and control (n = 8) mice using high-resolution magic angle spinning (HR-MAS) H-NMR. In addition, after demonstrating that HR-MAS H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS H-NMR measurements. Importantly, the gene expression changes obtained agreed with those observed by Affymetrix microarray analysis performed on RNA isolated directly from fresh-frozen tissue. In total, 40 metabolites could be assigned in the spectra and subsequently quantified. Quantification of lactate was also possible after applying a lactate-editing pulse sequence that suppresses the lipid signal, which superimposes the lactate methyl resonance at 1.3 ppm. Significant differences were detected for creatinine, glutamate, glycine, glycolate, trimethylamine-N-oxide, dimethylglycine, ADP, AMP, betaine, phenylalanine, and uridine. Furthermore, alterations in one-carbon metabolism, supported by both metabolic and transcriptional changes, were observed. These included reduced demethylation of betaine to dimethylglycine and the reduced expression of genes coding for transsulfuration pathway enzymes, which appears to preserve methionine levels, but may limit glutathione synthesis. Overall, the combined approach is advantageous as it identifies changes not only at the single gene or metabolite level but also deregulated pathways, thus providing critical insight into changes accompanying fatty liver disease. Graphical abstract A Evaluation of RNA integrity before and after HR-MAS H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS H-NMR and qRT-PCR, respectively.
肝脏脂肪过度积累所导致的代谢紊乱目前仍知之甚少。因此，在本研究中，我们使用瘦素缺乏的ob/ob小鼠（一种脂肪肝疾病的小鼠模型）来更详细地研究代谢变化。使用高分辨率魔角旋转（HR-MAS）氢核磁共振（H-NMR）对ob/ob小鼠（n = 8）和对照小鼠（n = 8）的完整肝脏组织中的代谢物进行定量分析。此外，在证明HR-MAS H-NMR不影响RNA完整性之后，对HR-MAS H-NMR测量后的相同样本所提取的RNA进行定量实时PCR，以检测转录变化。重要的是，所获得的基因表达变化与对直接从新鲜冷冻组织中分离的RNA进行的Affymetrix微阵列分析所观察到结果一致。总共可以在光谱中鉴定出40种代谢物并随后进行定量分析。在应用抑制脂质信号的乳酸编辑脉冲序列后，也可以对乳酸进行定量分析，该脂质信号会在1.3 ppm处叠加乳酸甲基共振峰。检测到肌酐、谷氨酸、甘氨酸、乙醇酸、氧化三甲胺、二甲基甘氨酸、ADP、AMP、甜菜碱、苯丙氨酸和尿苷存在显著差异。此外，观察到一碳代谢的变化，这在代谢和转录变化方面均得到了支持。这些变化包括甜菜碱向二甲基甘氨酸的去甲基化减少以及转硫途径酶编码基因的表达降低，这似乎能维持蛋氨酸水平，但可能会限制谷胱甘肽的合成。总体而言，这种联合方法具有优势，因为它不仅能在单个基因或代谢物水平上识别变化，还能识别失调的途径，从而为伴随脂肪肝疾病的变化提供关键见解。图摘要A完整小鼠肝脏组织进行HR-MAS H-NMR前后的RNA完整性评估。B分别使用HR-MAS H-NMR和qRT-PCR对ob/ob（脂肪变性）和ob/+（对照）小鼠中的代谢物浓度和基因表达水平进行评估。
