13C metabolic flux analysis is the gold standard approach for quantifying rates of biochemical reactions inside living cells. It has been widely applied to debottleneck the metabolism of microbial cell factories, but it is now being increasingly used to investigate disease mechanisms in animal models and human subjects. Our lab previously developed a novel microscale method to assess hepatic fluxes in conscious, unrestrained mice through simultaneous intravenous infusions of three different 2H/13C tracers. Gluconeogenic, citric acid cycle (CAC), and anaplerotic fluxes were determined through GC-MS analysis of a 40μL plasma glucose sample, followed by model-enabled flux regression. The methodology was recently applied to investigate the effects of fatty liver disease progression in genetically obese melanocortin-4-receptor–deficient (MC4R-/-) mice. Eight weeks of Western diet (WD) feeding increased liver inflammation and accelerated liver pyruvate cycling, while 20 weeks of WD feeding extensively upregulated liver gluconeogenic and CAC metabolism. Unexpectedly, vitamin E supplementation did not improve markers of steatohepatitis, but instead increased some indicators of metabolic flux dysregulation after 8 weeks of WD. We are now adapting these technologies to assess flux in peripheral tissues using tracer data from stable and radioactive isotope infusions. Specifically, we have expanded our metabolic flux models and mass spectrometry measurement capabilities to enable simultaneous estimation of in vivo metabolic fluxes in liver, kidney, muscle, and adipose by analyzing plasma and tissue samples collected at the end of each isotope infusion. The ability to profile flux changes in several different metabolic tissues from a single mouse provides an unprecedented opportunity to dissect in vivo regulatory circuits and elucidate metabolic interactions that are not discernible using current methods that assess fluxes at the level of individual tissues. A central feature of our future research will be to apply this multi-compartment 13C flux analysis platform to study metabolic phenotypes at various stages of disease, recognizing that metabolic tissues do not function in isolation, but as an integrated network that communicates through the supply and demand of metabolic substrates in addition to hormonal cues.

Toward integrated multi-tissue assessment of metabolic flux regulation in vivo

ACS Spring 2022

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