Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we perform in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Isotope tracing allowed qualitative assessment of a wide range of metabolic pathways within a single experiment, confirming well-known features of liver metabolism but also revealing unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Glucose production ex vivo correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes, and could be suppressed by postprandial levels of nutrients and insulin, and also by pharmacological inhibition of glycogen utilization. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.

Background

Both RNA-Seq and sample freeze-thaw are ubiquitous. However, knowledge about the impact of freeze-thaw on downstream analyses is limited. The lack of common quality metrics that are sufficiently sensitive to freeze-thaw and RNA degradation, e.g. the RNA Integrity Score, makes such assessments challenging.

Results

Here we quantify the impact of repeated freeze-thaw cycles on the reliability of RNA-Seq by examining poly(A)-enriched and ribosomal RNA depleted RNA-seq from frozen leukocytes drawn from a toddler Autism cohort. To do so, we estimate the relative noise, or percentage of random counts, separating technical replicates. Using this approach we measured noise associated with RIN and freeze-thaw cycles. As expected, RIN does not fully capture sample degradation due to freeze-thaw. We further examined differential expression results and found that three freeze-thaws should extinguish the differential expression reproducibility of similar experiments. Freeze-thaw also resulted in a 3' shift in the read coverage distribution along the gene body of poly(A)-enriched samples compared to ribosomal RNA depleted samples, suggesting that library preparation may exacerbate freeze-thaw-induced sample degradation.

Conclusion

The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.

Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor S-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.