Robbins JM; Beth Israel Deaconess Medical Center, Boston, MA USA
Benson M; Verkerke ARP; Tiwari G; et al

Circulation. 153(1):47-58, 2026 Jan 06.

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 [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.

RESULTS: An unknown mass spectrometry peak (mass-to-charge, 385.3056;
retention time, 3.69 minutes) had the strongest, positive relationship
with VO2max (mLxkg-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 (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 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). 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 mcM; ANOVA P=0.0027).

CONCLUSIONS: We identified a novel, lipidated amino acid, N-pal-gln, that
is positively associated with VO2max, 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.