Submitted to: Circulation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/8/2025
Publication Date: 11/12/2025
Citation: Robbins, J.M., Benson, M., Verkerke, A.R., Tiwari, G., Deng, S., Rao, P., Tahir, U.A., Avila-Pacheco, J., Shi, X., Guan, Y., Tendoh, F.G., Barber, J.L., Miller, P.E., Perry, A.S., Hall, M.E., Wood, A.C., Taylor, K.D., Post, W.S., Rich, S.S., Nayor, M., Wilson, J.G., Lewis, G.D., Shah, R.V., Rotter, J.I., Summers, S.A., Raffield, L.M., Kajimura, S., Bouchard, C., Clish, C.B., Sarzynski, M.A., Gerszten, R.E. 2025. N-palmitoyl glutamine is a candidate mediator of cardiorespiratory fitness. Circulation. https://doi.org/10.1161/CIRCULATIONAHA.125.074187.
DOI: https://doi.org/10.1161/CIRCULATIONAHA.125.074187

Interpretive Summary: Cardiorespiratory fitness (CRF) is an important measure of health that is strongly associated with long-term outcomes (e.g. cardiovascular disease- and all-cause mortality). This study measured metabolites in the blood that were associated with CRF and identified a previously unidentified metabolite, N-palmitoyl glutamine, that was strongly, positively associated with CRF, that increases after exercise training, and is inversely associated with risk of death. The study also found that N-palmitoyl glutamine stimulates mitochondrial biogenesis and improves mitochondrial efficiency in cell-based studies. These findings suggest that N-palmitoyl glutamine may be an exercise-responsive blood metabolite that mediates CRF, and points to the discovery potential of non-targeted metabolomics profiling.

Technical Abstract: 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. 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 [VO2max]) measured by cardiopulmonary exercise testing and underwent 20 weeks of supervised endurance training. Metabolite-VO2max 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. An unknown mass spectrometry peak (mass-to-charge, 385.3056; retention time, 3.69 minutes) had the strongest, positive relationship with VO2max (mL×kg**-1min**-1) after adjustment for age, sex, race, and lean body mass (Beta =1.29; false discovery rate q=5.3×10**-6); was identified as N-palmitoyl glutamine (NPG) using tandem mass spectrometry and bioinformatics; and was confirmed with an authentic chemical standard. The biological role of NPG has not been described previously. The relationship of NPG with VO2max was validated in 408 participants from the FHS (Beta=1.2; P=3.8×10**-5), and its levels increased after exercise training (log fold change=0.22; q=5.3×10**-12). NPG 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. NPG 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 NPG, respectively [P=0.04 and P=0.02]) and improved bioenergetics (NPG at 26 nM increased the phosphate:oxygen ratio across ADP concentrations from 0 to 100 uM; ANOVA P=0.0027). We identified a novel, lipidated amino acid, NPG, that is positively associated with VO2max, increases after regular aerobic exercise, and is inversely associated with incident mortality. NPG stimulates mitochondrial biogenesis and efficiency, demonstrating its potential role as an exercise-stimulated transducer of cardiorespiratory fitness.