Abstract
BACKGROUND: Cardiorespiratory fitness is an integrative measure of cardiometabolic health and predictor of survival, yet little is known about its molecular underpinnings. Small molecule metabolites and lipids are increasingly recognized as exercise-stimulated signaling molecules and candidate molecular transducers of cardiorespiratory fitness. METHODS: We performed nontargeted liquid chromatography mass spectrometry-based plasma metabolomics in 654 participants (mean age, 35 years; 55% women) from the HERITAGE Family Study (Health, Risk Factors, Exercise Training, and Genetics) who had cardiorespiratory fitness (maximal oxygen uptake [VO(2)max]) measured by cardiopulmonary exercise testing and underwent 20 weeks of supervised endurance training. Metabolite-VO(2)max relationships were assessed using linear regression and tested for replication in FHS (Framingham Heart Study) participants who also underwent cardiopulmonary exercise testing. Metabolite relationships with incident all-cause mortality ascertained in JHS (Jackson Heart Study) and MESA (Multi-Ethnic Study of Atherosclerosis) were tested using Cox regression. Experimental studies of cellular respiration and mitochondrial function were performed in C2C12 myotubes. RESULTS: An unknown mass spectrometry peak (mass-to-charge, 385.3056; retention time, 3.69 minutes) had the strongest, positive relationship with VO(2)max (mL×kg(-1)min(-1)) after adjustment for age, sex, race, and lean body mass (β=1.29; false discovery rate q=5.3×10(-6)); was identified as N-palmitoyl glutamine (N-pal-gln) using tandem mass spectrometry and bioinformatics; and was confirmed with an authentic chemical standard. The biological role of N-pal-gln has not been described previously. The relationship of N-pal-gln with VO(2)max was validated in 408 participants from the FHS (β=1.2; P=3.8×10(-5)), and its levels increased after exercise training (log fold change=0.22; q=5.3×10(-12)). N-pal-gln levels were inversely associated with all-cause mortality in JHS and MESA (hazard ratio, 0.91 and 0.65 [P=0.029 and P=0.028], respectively). Previous studies have shown that structurally related biochemicals modulate energy homeostasis; thus, we performed mitochondrial experiments. N-pal-gln administration led to a dose-dependent increase in mitochondrial:nuclear DNA ratio compared with control treated cells (15% and 20% increases at 6.5 nM and 26 nM N-pal-gln, respectively [P=0.04 and P=0.02]) and improved bioenergetics (N-pal-gln at 26 nM increased the phosphate:oxygen ratio across ADP concentrations from 0 to 100 μM; ANOVA P=0.0027). CONCLUSIONS: We identified a novel, lipidated amino acid, N-pal-gln, that is positively associated with VO(2)max, increases after regular aerobic exercise, and is inversely associated with incident mortality. N-pal-gln stimulates mitochondrial biogenesis and efficiency, demonstrating its potential role as an exercise-stimulated transducer of cardiorespiratory fitness.