Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and 13C-tracing enrichments

OBJECTIVE: Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced METHODS: By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in RESULTS: Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of CONCLUSIONS: Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling

Cardiac myocyte KLF5 regulates body weight via alteration of cardiac FGF21

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Kriippel-like factor (KLF)-5 regulates cardiomyocyte PPAR alpha and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (alpha MEIC-KLF5(-/-)) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. alpha MHC-KLF5(-/-) mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed alpha MHC-KLF5(-/-) mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPAR gamma inactivation, alpha MHC-KLF5(-/-) mice had less SUMO-PPAR gamma in WAT. Increased diet-induced obesity found in aMHC-KLF5-/- mice was absent in alpha MHC-[KLF5(-/-);FGF21(-/-)] double knockout mice, as well as in alpha MHC-FGF21(-/-) mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT