title: Metabolic profiling of ob/ob mouse fatty liver using HR-MAS1H-NMR combined with gene expression analysis reveals alterations in betaine metabolism and the transsulfuration pathway.
abstract: 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)1H-NMR. In addition, after demonstrating that HR-MAS1H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS1H-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-MAS1H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS1H-NMR and qRT-PCR, respectively.
authors.affiliation: Leibniz Institut für Analytische Wissenschaften - ISAS e.V., Bunsen-Kirchhoff-Strasse 11, 44139, Dortmund, Germany. mikheil.gogiashvili@isas.de.
authors.forename: Mikheil
authors.initials: M
authors.lastname: Gogiashvili
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
authors.forename: Karolina
authors.initials: K
authors.lastname: Edlund
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Gianmoena
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Marchan
authors.affiliation: Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Institute of the Ruhr-Universität Bochum, Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Brik
authors.affiliation: Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstrasse 30, 48149, Münster, Germany.
authors.forename: Jan T
authors.initials: JT
authors.lastname: Andersson
authors.affiliation: Leibniz Institut für Analytische Wissenschaften - ISAS e.V., Bunsen-Kirchhoff-Strasse 11, 44139, Dortmund, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Lambert
authors.affiliation: Faculty of Statistics, TU Dortmund University, Mathematics Building, 44221, Dortmund, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Madjar
authors.affiliation: Faculty of Statistics, TU Dortmund University, Mathematics Building, 44221, Dortmund, Germany.
authors.forename: Birte
authors.initials: B
authors.lastname: Hellwig
authors.affiliation: Faculty of Statistics, TU Dortmund University, Mathematics Building, 44221, Dortmund, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Rahnenführer
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
authors.forename: Jan G
authors.initials: JG
authors.lastname: Hengstler
authors.affiliation: Leibniz Institut für Analytische Wissenschaften - ISAS e.V., Bunsen-Kirchhoff-Strasse 11, 44139, Dortmund, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Hergenröder
authors.affiliation: Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystrasse 67, 44139, Dortmund, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cadenas
mesh_terms: D000818:Animals; D001622:Betaine / Q000378:metabolism*; D005234:Fatty Liver / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D019344:Lactic Acid / Q000378:metabolism; D020738:Leptin / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D053858:Metabolic Networks and Pathways; D055442:Metabolome; D055432:Metabolomics / Q000379:methods; D051379:Mice; D066244:Proton Magnetic Resonance Spectroscopy / Q000379:methods*; D059467:Transcriptome
chemical_list: D020738:Leptin; D019344:Lactic Acid; D001622:Betaine
keywords: Fatty liver; HR-MAS 1H-NMR spectroscopy; Lactate editing; NAFLD; One-carbon metabolism; ob/ob mice