We first performed metabolic profiling in subjects referred for exercise treadmill testing (ETT) who had normal exercise capacity and no evidence of myocardial ischemia (). We obtained peripheral blood samples from subjects at baseline, peak exercise, and 60 minutes after completion of ETT to characterize the alterations associated with acute maximum exercise. Because unintentional overfitting of data is a concern in biomarker discovery studies (25
), we studied metabolic changes in two independent groups of subjects [i.e., a derivation cohort (N=45) and a validation cohort (N=25)]. There were no significant clinical differences between subjects in the ETT derivation and validation cohorts (). Twenty-three metabolites changed significantly (nominal P<0.005) at the peak exercise time point in the derivation cohort ( and
), with an estimated false discovery rate of <5%, or approximately one metabolite out of the 23 metabolites that changed (see Methods). In the validation cohort, significant changes were noted for 21 of the 23 metabolites. For the overall group of metabolites in , the magnitude of change in individual metabolites in the two cohorts was highly correlated (r=0.99, P<0.0001) (Table S1
provides additional data on metabolite changes in the combined cohorts).
Metabolite changes in plasma at the peak exercise time point
Relative changes in metabolites in response to exercise
The observed changes in plasma metabolites immediately after cessation of exercise, which occurred approximately 10 minutes after baseline sampling, reflect rapid upregulation of several metabolic pathways responsible for skeletal muscle substrate utilization (, top panel
). The metabolite profiling platform captured previously reported increases in plasma concentrations of glycolysis products (lactate, pyruvate), lipolysis products (glycerol) and amino acids (alanine and glutamine). Plasma concentrations of the ketone body acetoacetate fell in response to acute exercise, as described (26
Fuel substrate mobilization during exercise
Our broad metabolomics approach enabled detection of coordinate changes in multiple components of various metabolic pathways. For example, we documented increases in sequential products of adenine nucleotide catabolism (e.g. AMP, inosine, hypoxanthine, xanthine), including phosphorylated metabolites that are usually confined to intracellular compartments (). We observed particularly prominent changes in span 2 TCA cycle intermediates ( and
). Notably, the changes in individual TCA cycle intermediates in plasma closely mirrored previously reported changes in skeletal muscle obtained from invasive intramuscular biopsies taken during exercise testing (9
Figure 3 (A) Enrichment of adenine nucleotide catabolites in pulmonary arterial blood during exercise. (Left) Intramuscular and extracellular metabolic reactions in the adenine nucleotide catabolism pathway. (Right) Patterns of change of individual metabolites (more ...)
We also detected plasma metabolic changes in pathways not previously associated with exercise. For example, niacinamide, which enhances insulin release and improves glycemic control (27
), increased by 79% (IQR 42 to 182%). We observed heterogeneity in niacinamide elevation in response to exercise in that leaner individuals (subjects with BMI less than the median value of 28) had a more than two-fold greater increase in niacinamide in response to exercise than did individuals with BMI above the median (102%, IQR -1 to 117% vs. 41%, IQR -7 to 80%, respectively, P=0.04). Concentrations of allantoin, a product of uric acid oxidation that has been implicated as an indicator of oxidative stress (29
), fell after acute exercise. Finally, plasma concentrations of glycogenolysis intermediates (3-phosphoglycerate, glucose-6-phosphate) increased with ETT. Hierarchical clustering of metabolites to determine the interrelatedness of metabolic changes with exercise is displayed in Fig. S2
Although prior studies have documented that heart rate and hemodynamic parameters rapidly return to baseline after acute exercise (30
), we observed metabolic changes that persisted 60 minutes after the cessation of exercise (). Metabolites that were changed only at the 60 minute time point included TCA cycle intermediates (citrate/isocitrate and aconitate), a homocysteine metabolism pathway intermediate (methionine), and two adenine nucleotide degradation products (xanthosine and uric acid). Other metabolites that were changed only at the 60 minute time point included uridine, a pyrimidine nucleoside, and the amino acid, ornithine. Adenine nucleotides concordantly increased incrementally at 60 minutes compared to the peak exercise time point (), as did span 1 TCA intermediates citrate/isocitrate and aconitic acid, whereas span 2 TCA intermediates decreased in a concerted fashion ().
Table 3 Metabolite changes in plasma at 60 minutes after completion of exercise testing. Metabolites with P<0.005 in the derivation cohort are shown. Metabolites with identical retention times and parent-daughter ion pairs (e.g. citrate and isocitrate) (more ...)
Localization of metabolic changes through multi-site blood sampling
We performed metabolic profiling on 8 subjects who underwent comprehensive cardiopulmonary exercise testing (CPET), which included bicycle ergometry with invasive hemodynamic monitoring and multi-site blood sampling to assess the cause of shortness of breath on exertion. The CPET subjects showed normal exercise responses, although estimated peak oxygen uptake (VO2
) values were lower in the CPET cohort than in the ETT cohort (), commensurate with the smaller metabolic demand imposed by bicycle ergometry CPET than by treadmill ETT (32
). We compared samples from the superior vena cava (SVC), which contains blood from the non-exercising upper body, with samples from the pulmonary artery (PA), which reflects the venous effluent from the exercising lower extremity skeletal muscle as well as cardiac muscle. Prior studies suggest that there is a 50% contribution of inferior vena cava blood at rest while upright on a bicycle, and a 73% contribution during bicycle ergometry exercise (33
). Thus, we could assess instantaneous metabolite gradients in distinct vascular beds and so localize the site of metabolic changes. Analysis of bicycle ergometry also allowed us to test whether our findings from treadmill ETT could be generalized to other exercise modalities.
Cycle ergometry exercise yielded very similar metabolic changes to those observed with treadmill ergometry ( Heatmap and
). At peak exercise, the majority of metabolites showed significantly larger changes in PA plasma than in SVC plasma, implicating a sub-diaphragmatic skeletal muscle or cardiac source of these metabolic changes, likely from the exercising lower limbs. As expected, steep instantaneous PA-to-SVC gradients were evident for the purine degradation products (ΔPA/ΔSVC ratio range: 2.2 to 9.0, ) and span 2 TCA cycle intermediates (ΔPA/ΔSVC ratio range 1.7 to 2.3, ). By contrast, a subset of metabolites that were significantly changed in both the ETT and CPET cohorts, including several amino acids, acetoacetate, and glucose-6-phosphate, were similarly changed in the SVC and PA samples. This may be due to endocrine-like signaling effects from the exercising muscle (34
). At 60 minutes after exercise, significant instantaneous PA-to-SVC gradients were no longer present for any of the metabolite classes.
Relationship between metabolic changes and fitness in ETT
We next examined whether exercise-induced excursions of metabolites that were confirmed in both the ETT derivation and validation cohorts, either at peak exercise or 60 minutes (N=28 metabolites total), were correlated with exercise performance. For these analyses, we divided the ETT cohort of individuals with overall normal exercise capacity into two groups on the basis of median percent predicted peak oxygen uptake (%pVO2
). The more- and less-fit subgroups did not differ with regard to age (58±11 vs. 59±14 years), sex (94 vs. 86% male), weight (193±35 vs. 194±31 lbs) or the product of heart rate and systolic blood pressure achieved (27,500±4770 vs. 26750±6900 mmHg/min), respectively (P>0.05 for all comparisons). At peak exercise, glycerol increased to a greater extent in the group of individuals who achieved higher %pVO2
(98%, IQR 38 to 143%) compared to individuals who achieved lower %pVO2
(48%, IQR, 24 to 69%, P<0.005, ), suggesting that more fit individuals have a greater capacity for lipolysis in response to acute exercise. The magnitude of the changes in glycerol were most closely related to %pVO2
(r=0.54, P<0.001). We further investigated glycerol excursions in a distinct cohort of subjects with and without exercise-induced myocardial ischemia, with no differences in exercise exposure or cardiac risk factors including age, sex, BMI, or diabetes status (see Table S2
). Exercise-induced increases in plasma glycerol concentrations were signficantly smaller in subjects with inducible ischemia than in controls (18%, IQR -10 to 79%, vs. 66%, IQR 24 to 109% in controls, P=0.0015). The evidence for reduced exercise-induced lipolysis in the ischemic and less-fit cohorts, as indicated by the smaller elevations in glycerol, may be indicative of maladaptive responses to exercise in which lipid utilization is impaired.
Fitness levels and differential metabolic changes during ETT
Pantothenate also increased to a greater extent in more-fit individuals, whereas methionine excursions were greater in the less-fit subgroup (). Changes in glutamine concentrations were also greater in the less-fit group, likely reflecting greater skeletal muscle release of ammonia during exercise. The metabolic changes in each of these metabolites persisted 60 minutes after cessation of exercise (). By contrast, lactate concentrations did not differ between more and less fit individuals at either peak exercise or 60 minutes after exercise. Notably, the differential changes in glycerol, pantothenate, glutamine and methionine between more and less fit subjects persisted after we adjusted for the amount of exercise performed (Table S3
Relationship between metabolic changes and prolonged exercise
We acquired metabolic profiles of 25 subjects who ran the 26.2 mile Boston Marathon, with an average time of 247±46 minutes. As expected, extensive changes in plasma metabolite concentrations were evident at the end of the race as compared to pre-race concentrations ( and ). We documented marked elevations in glycerol (1,128%, IQR 897-1315%, P<0.0001) and β-hydroxybutyrate (401%, IQR 224-1060%, P<0.0001, ), consistent with extensive lipolysis and ketone body production, respectively, in subjects who completed the marathon. In contrast to our findings after acute exercise, we saw a reduction in gluconeogenic amino acids (alanine, threonine, serine, proline, valine, histidine, glutamine, asparagine, ) and unexpected increases in tryptophan metabolites (kynurenate, quinolinate, anthranilate).
Metabolite changes following completion of a 26.2 mile marathon. Metabolites with P<0.005 are shown. Metabolites with identical retention times and parent-daughter ion pairs (e.g. citrate/isocitrate) cannot be distinguished by the platform.
To investigate whether the metabolic changes that correlated with fitness after acute exercise also correlated with fitness after prolonged exercise, we stratified marathon runners into two groups on the basis of their finish time above and below the median (240 minutes). The groups did not differ in age (faster group, 41±11 vs. 42±7 years), sex (76% vs. 69% male), or BMI (25±4 vs. 24±2, all P>0.05). Fumarate changes, which correlated with fitness during acute exercise performance, were also related to marathon speed (faster group: +200%, IQR=110 to 281% vs. +108%, IQR=16 to 137%, P<0.05). Additional span 2 TCA intermediary metabolites with greater excursions in the faster marathoners included succinate (+227%, 194 to 411%, vs. +66%, 30 to 94%) and malate (+227%, 133 to 269% vs. 87%, 49 to 109%, all P<0.05). An equally weighted sum of TCA cycle intermediate concentrations after marathon completion differentiated the faster from the slower runners (). There were non-significant trends toward greater increases in both pantothenate (+40%, 6 to 88% vs. +22%, -15 to 40%,) and glycerol (+1172%, 993 to 1469% vs. 986%, 827 to 1295%,) in the faster marathon group, in the same direction as was observed in acute exercise.
TCA intermediate changes with marathon running
In the marathon runners, other metabolites that changed during acute exercise differed in the faster and slower runners. Faster runners had more modest increases in ketone bodies (β-hydroxybutyrate, +362% vs. +855%) and in allantoin (+9% vs. +28%), a marker of oxidative stress, than did slower runners. Citrulline, which modulates arginine bioavailability for nitric oxide synthesis, showed attenuated reduction in faster runners (-14% vs. -41%), while arginosuccinate concentrations (+79% vs. +25%) and niacinamide (+253% vs. +53%) were higher in faster marathon runners (P<0.05 for all).
Metabolic predictors of fitness in a large, prospective cohort
We next determined whether baseline plasma concentrations of metabolites that were altered in response to exercise () were associated with cardiovascular fitness in an independent cohort of subjects from the Framingham Heart Study (N=302, see Table S3
). Heart rate is a known independent predictor of exercise capacity (35
) that has been directly related to cardiovascular outcomes in the Framingham Heart Study (36
) and was measured at the time of blood sampling in this cohort.
We specifically evaluated whether the metabolites that were modulated by acute exercise were correlated with resting heart rate in this cohort. Of the acute exercise metabolites, 12 were also measured in a subset of subjects in the Framingham Heart Study cohort (Table S5
). Glycerol concentrations were significantly correlated with resting heart rate (R=0.22, P=0.0002) whereas glutamine concentrations were inversely related to heart rate (R=-0.21, P=0.0002). Both of these differences remained significant after adjustment for age, BMI, and gender (Table S5
). Subjects in the fourth heart rate quartile had 1.44-fold higher glycerol concentrations than those in the first quartile (P=0.001). Glutamate concentrations in the fourth heart rate quartile tended to be higher (1.21-fold, P=0.08), while glutamine concentrations were lower (0.87-fold, P=0.03). These data are notable given the greater increase in glycerol and attenuated decrease in glutamine in response to exercise in more fit subjects in the ETT cohort.
Activation of the nur77 pathway by exercise-induced metabolites
We further hypothesized that a subset of metabolites that increased in plasma after exercise could also modulate pathways relevant to cellular respiration and substrate utilization. We performed experiments with 6 exercise-induced metabolites of biological interest, specifically assaying their effects on 11 transcriptional regulators of metabolism in cultured myotubes. We found that a mixture of physiologically relevant concentrations of glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate rapidly upregulates the expression of nr4a1c (or nur77) (), a recently described transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle (37
). No individual metabolite triggered a similar response. Consistent with these findings, nur77 expression was induced 5-fold in mouse quadriceps by 30 minutes of exercise ().
Figure 6 (A) Modulation of gene expression by metabolites. (Left) mRNA expression of indicated genes (36B4: Rplp0 ribosomal protein, large, P0, HPRT: hypoxanthine guanine phosphoribosyl transferase, CYCS: cytochrome c, somatic, COX5B: cytochrome c oxidase subunit (more ...)