Metabolic profiling of ob/ob mouse fatty liver using HR-MAS 1H-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) H-NMR. In addition, after demonstrating that HR-MASH-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-MASH-NMR and qRT-PCR, respectively.

Adenosine accumulation causes metabolic disorders in testes and associates with lower testosterone level in obese mice

“…Sertoli cells export lactate for the metabolism of germ cells including pachytene spermatocyte and spermatid, and the level of lactate in testis may an effective indicator of sperm cell viability (Jutte et al, 1982). Lactate in plasma and liver showed a downward trend in obese mice models, similar to our observation in testis (Gogiashvili et al, 2017; Shearer et al, 2008). Besides, elevated alanine level indicated a lower redox/higher oxidative state for the cells (Oliveira et al, 2011).…”

“…Sertoli cells export lactate for the metabolism of germ cells including pachytene spermatocyte and (Jutte et al, 1982). Lactate in plasma and liver showed a downward trend in obese mice models, similar to our observation in testis (Gogiashvili et al, 2017;Shearer et al, 2008).…”

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high‐resolution magic angle spinning 1H NMR spectroscopy

“…Overall, the use of HR MAS NMR to assess metabolite concentrations in tumor tissue may be advantageous, particularly when tissue availability is a limiting factor, as the method is non‐destructive and allows the same specimen to be re‐analyzed using a complementary technique, such as RNA‐based analyses. This is supported by reports of high‐quality RNA being extracted after HR MAS NMR . However, a main disadvantage of the technique is its poor sensitivity, which restricts the analysis to metabolites present in relatively high concentrations.…”

Metabolomic and Lipidomic Biomarkers for Premalignant Liver Disease Diagnosis and Therapy

“…1 H signals from lipids were highly statistically significantly elevated in ob/ob livers, as expected. Several other molecules involved in betaine (N,N,N-trimethylglycine) metabolism were altered (Table 4) [232].…”