Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism

Abstract

ObjectiveThe dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways.MethodsWe designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS).ResultsWe show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses.ConclusionsOur data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.

Extra-mitochondrial mouse frataxin and its implications for mouse models of Friedreich’s ataxia

“…However, it would have required > 95% of the mitochondria to have been disrupted in order to observe truncated mature frataxin (79-207) in the cytosolic fraction if it had actually been present in the mitochondria at the time of collection. The method for subcellular fractionation has been used in many studies to obtain mitochondria 17,31 . Furthermore, we consistently saw less mature frataxin (78-207) in the brain (7.3 ± 1.7%) than in both liver and heart as well as less mature frataxin (78-207) in the liver (11.3 ± 2.5%) than in the heart (14 ± 2.9%) ( Table 1).…”

“…Tissue sub-cellular fractionation. A piece of frozen mouse liver sample (mouse information provided above) was homogenized and separated in into cytosolic, mitochondrial, and nuclear fractions using a modification of the method of Trefely et al 31 The tissue samples were weighed (approximately 30 mg) and cut into 2-3 mm 2 pieces. All the pieces were collected in a LoBind Eppendorf tube containing 1 mL of MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tri-HCl (pH 7.5) and 1 mM EDTA (pH 7.5).…”

Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation

“…We next asked whether SILEC-SF accounts for metabolic activity during processing. We previously demonstrated that post-harvest metabolism occurs during fractionation by assessing the incorporation of isotope labeled substrates introduced in the fractionation buffer into downstream metabolic products (Trefely et al 2019). We applied post-harvest labeling to assess the extent to which SILEC internal standards account for post-harvest metabolism (Figure S3B).…”

“…We predicted that the dose-responsive change in HMG-CoA abundance occurs specifically in the cytosol, since our prior sub-cellular tracing analyses demonstrated a close kinetic relationship between cytosolic acetyl-CoA and HMG-CoA labeling from acetate (Trefely et al, 2019). We performed SILEC-SF to compare acyl-CoA levels specifically in the cytosolic and mitochondrial compartments in response to exogenous acetate (Figure 4D).…”

“…Yet, distinct challenges in measuring metabolites in subcellular compartments remain. In particular, sub-cellular fractionation is inherently disruptive and metabolic analysis after fractionation is subject to multiple confounding factors (Dietz, 2017; Lu et al, 2017; Trefely et al, 2019).…”