Maximal exercise-associated oxidative capacity is strongly correlated with health and longevity in humans. Rats selectively bred for high running capacity (HCR) have improved metabolic health and are longer-lived than their low-capacity counterparts (LCR). Using metabolomic and proteomic profiling, we show that HCR efficiently oxidize fatty acids (FAs) and branched-chain amino acids (BCAAs), sparing glycogen and reducing accumulation of short- and medium-chain acylcarnitines. HCR mitochondria have reduced acetylation of mitochondrial proteins within oxidative pathways at rest, and there is rapid protein deacetylation with exercise, which is greater in HCR than LCR. Fluxomic analysis of valine degradation with exercise demonstrates a functional role of differential protein acetylation in HCR and LCR. Our data suggest that efficient FA and BCAA utilization contribute to high intrinsic exercise capacity and the health and longevity benefits associated with enhanced fitness.

In this study, we found that the respiratory quotient (RQ) is lower at rest in HCR compared to LCR, indicative of enhanced FA oxidation, and that FA oxidation is even more markedly enhanced in HCR during exercise. Metabolomic and fluxomic profiling demonstrate that during exercise, HCR use FA and branched-chain amino acids (BCAAs) more efficiently than LCR. Assessment of the muscle mitochondrial proteome of HCR and LCR, as well as posttranslational modifications (phosphorylation and acetylation), show specific differences between HCR and LCR within oxidative pathways of FA and BCAA metabolism and provide evidence that rapid changes in protein acetylation during exercise could play a role in augmenting the fuel selection differences. These differences in fuel selection and proteome modification mirror those found in caloric restriction () and implicate fuel selection and mitochondrial oxidative efficiency as mechanisms linking enhanced exercise capacity with improved metabolic status and longevity.

Recent advances in metabolomics and proteomics allow the quantification of tens to thousands of metabolites or peptides in a single biological sample. Integrating these techniques can provide insight into the changes in nutrient utilization under different physiological conditions. In these studies, we employed a combination of metabolomics and proteomics to investigate fuel selection in rats selectively bred for high and low intrinsic running capacity (HCR and LCR). The HCR-LCR rat model was derived from a heterogeneous founder population (N:NIH) with breeder selection based solely on intrinsic (untrained) treadmill running capacity (). In this model, as in humans, exercise capacity is a heritable trait (), and like humans who differ in running capacity, HCR and LCR diverge in susceptibility to metabolic and related disease traits (). Compared to LCR, HCR animals diverge more strongly in running capacity from the founder stocks and show a 2.4-fold increased running capacity over the highest capacity observed in inbred lines (). HCR animals weigh significantly less than LCR animals throughout their lifespan, despite similar food consumption, and there is evidence of increased capacity of substrate oxidation (). A recent study () showed that HCR and LCR have similar resting energy expenditure, but HCR animals have small elevations in exercise energy expenditure and greater exercise-induced heat production from their skeletal muscle. The phenotype of HCR is coincident with a host of health benefits (), including a 28%–40% increased lifespan ().

Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle.

Spontaneous activity, economy of activity, and resistance to diet-induced obesity in rats bred for high intrinsic aerobic capacity.

Skeletal muscle mitochondrial and metabolic responses to a high-fat diet in female rats bred for high and low aerobic capacity.

Exercise capacity and cardiovascular fitness are highly predictive of metabolic health, including lower fat mass, higher insulin sensitivity, lower blood pressure, and, importantly, age-adjusted mortality (). The mechanisms underlying these associations are not fully understood. One important link between exercise capacity and overall metabolic health is the fuel selection for energy production. Higher exercise capacity is associated with increased fatty acid (FA) oxidation during exercise (), while poor metabolic health is associated with high basal use of carbohydrates and impaired fuel switching during the fast-fed transition (). The glucose-FA cycle described bystates that fat availability will drive fat oxidation and reciprocally lead to decreased glucose oxidation; however, this theory cannot explain instances when fat availability is high but carbohydrates are preferentially oxidized, as is the case during high-intensity exercise and insulin resistance ().

Effect of aging on glucose and lipid metabolism during endurance exercise.

The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.

Determinants of fat oxidation during exercise in healthy men and women: a cross-sectional study.

The relationship between aerobic fitness level and metabolic profiles in healthy adults.

Fat oxidation, fitness and skeletal muscle expression of oxidative/lipid metabolism genes in South Asians: implications for insulin resistance?.

Cardiorespiratory fitness as a quantitative predictor of all-cause mortality and cardiovascular events in healthy men and women: a meta-analysis.

Exercise capacity and body composition as predictors of mortality among men with diabetes.

Influences of cardiorespiratory fitness and other precursors on cardiovascular disease and all-cause mortality in men and women.

The BCKDH complex can be activated by dephosphorylation, but the role of dephosphorylation in exercise-associated increase in BCAA utilization is unclear (). Our proteomics analysis showed a nonsignificant decrease in Ser293 phosphorylation of BCKDHA subunit with exercise in HCR ( Figure 6 Table S3 B). Notably, dihydrolipoamide dehydrogenase (DLD), a subunit of the BCKDH complex, contained ten acetylation sites that were all less acetylated in HCR compared to LCR with exercise ( Figure 6 Table S3 C). As DLD is a subunit of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complex, future studies are warranted to dissect the potential modulation of these complexes secondary to changes in DLD acetylation.

The irreversible branched-chain keto-acid dehydrogenase (BCKDH) complex is the rate-limiting step of BCAA degradation. With exercise, there is greaterC-labeled C4-carnitine and β-hydroxyisobutryate (HIB) in muscle in both HCR and LCR ( Figures 6 C and 6D), indicating increased metabolism of valine. HCR showed greater flux through the BCKDH complex and downstream enzymes, as demonstrated by greater accumulation ofC-labeled C4- and C3-carnitines and succinate in muscle of HCR-Run versus LCR-Run ( Figures 6 C, 6E, and 6F). However, accumulation ofC-labeled succinate was quantitatively quite small ( Table S4 A), suggesting that valine contributes minimally to citric acid cycle anaplerosis.

To further assess the functional consequence of differential acetylation in HCR and LCR, we estimated flux through the BCAA degradation pathway by intraperitoneal injection of U-N valine. We determined the isotopic enrichment of downstream metabolites in serum and gastrocnemius muscle at rest and immediately after 10 min of exercise ( Tables S4 A and S4B; Figure 6 ). We confirmed that valine injection does not significantly alter running capacity or estimated fuel preference ( Figure S5 ) and that serum and muscle isotopic enrichment of valine was not significantly different between HCR and LCR ( Table S4 A; Figure 6 A). U-N valine (mass shift of 6 Da; M+6 isotope) is reversibly transaminated to M+5 KIV ( Figure 6 B) and can be reaminated to M+5 valine by the incorporation ofN. HCR-Run had a greater M+5:M+6 ratio of valine in serum and muscle ( Table S4 A), indicating greater valine transamination-reamination in HCR. Muscle glutamate nitrogen enrichment (M+1) was also significantly greater in HCR-Run, supporting the conclusion that HCR have greater valine transamination in skeletal muscle ( Table S4 B).

Proteins involved in BCAA metabolism (ellipses) are labeled by gene symbol with identified acetylation (circle) and phosphorylation (diamond) sites. The color scale designates log2 fold-change (HCR/LCR comparison for protein; or HCR Run/LCR Run comparison for acetylation and phosphorylation). Animals were injected with U- 13 C 15 N valine and rested for 10 min before an additional 10 min rest or 10 min run. LCR and HCR have similar amounts of labeled valine in gastrocnemius (A) but show differences in amount of labeled metabolites downstream of valine: α-ketoisovaleric acid (KIV; B), isobutyryl-carnitine (C4; C), β-hydroxyisobutyrate (HIB; D), propionyl-carnitine (C3; E) and succinate (F). Values are mean ± SEM relative to LCR rest for each group (n = 4–6). ∗ p < 0.05 between HCR and LCR; #p < 0.05 difference within strain between Rest and Run.

Deacetylation of Proteins in the BCAA Pathway Is Associated with Increased BCAA Catabolism during Exercise

To assess the role of differential acetylation of MDH2, we measured MDH activity, previously shown to be modulated by acetylation state (), in isolated mitochondria from HCR and LCR. While MDH2 protein levels were similar in HCR and LCR ( Table S3 A), there was a significant inverse relationship between average acetylation state of MDH2 and MDH activity, but less-significant inverse relationship between individual acetylation sites and MDH activity ( Figure S4 ). These data suggest a complex interplay between sites, and potentially other posttranslational modifications, in regulation of individual enzyme activity.

Overall, mitochondrial acetylation was lower in HCR at rest ( Figure 5 A), and the difference was amplified following exercise ( Figure 5 B). KEGG pathway analysis revealed that acetylation sites were enriched within the oxidative phosphorylation, citric acid cycle, BCAA degradation, propionate metabolism, and FA metabolism pathways. By estimating the relative level of acetylation of all peptides within each of these pathways, we found that there was significantly lower mitochondrial acetylation in each pathway in HCR compared to LCR, both at rest and following exercise ( Figure 5 C). In addition, HCR had significant deacetylation in four of the five acetyl-rich pathways with 10 min of exercise, while LCR had no significant pathway changes in acetylation. These pathways are targets of the mitochondrial lysine deacetylase SIRT3 (). In particular, MDH2 K239 is a major SIRT3 target () and is significantly differentially acetylated between HCR and LCR at 10 min of exercise (p = 0.02) and becomes less acetylated in HCR with exercise (p = 0.057; see Table S3 C), suggesting a possible role of SIRT3 in mediating the differences in acetylation. However, we found no difference in the SIRT3 levels between HCR and LCR in mitochondrial extracts by proteomic analysis ( Table S3 A) or western blotting ( Figure S3 ). If SIRT3 is mediating these differences, then it is likely due to differential activation of SIRT3.

Log2 fold-change (HCR/LCR) values for the 141 acetyl-lysine sites were plotted against p values at rest (A) and at 10 min run (B; n = 5). Acetylation sites were enriched within oxidative pathways; for each enriched pathway, HCR proteins are less acetylated than LCR proteins (C). Average protein acetylation (small circles) is dynamic with exercise, and average pathway acetylation (large circles) is lower in HCR than LCR at 10 min of exercise. The fat oxidation pathway (D) is less acetylated in HCR with exercise. Proteins (ellipses) are labeled by gene symbol with identified acetylation (circle) and phosphorylation (diamond) sites. The color scale designates log2 fold-change (HCR/LCR comparison for protein, or HCR Run/LCR Run comparison for acetylation and phosphorylation).

Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways.

Changes in fuel use, though dependent on fuel availability, are largely controlled by enzyme availability and activity. Previous studies have demonstrated increased mRNA and protein expression in pathways related to oxidative metabolism in HCR versus LCR (). To investigate potential changes in the proteome and posttranslation modifications in skeletal muscle mitochondria with exercise, we performed quantitative proteomic analysis. Mitochondria were isolated from HCR and LCR extensor digitorum longus muscle at rest and after 10 min of exercise, and isobaric tags were used for multiplexed quantification (). We identified 428 mitochondrial proteins ( Table S3 A), of which 73 were phosphorylated and 85 were acetylated ( Figures 4 A and 4B ; Tables S3 A–S3C). In prior studies, HCR were reported to have no change () or a fiber type-specific upregulation of mitochondrial mass () when compared to LCR rats; thus we normalized our proteomic data to mitochondrial protein. In the mitochondrial proteome, 174 proteins were significantly different (p < 0.05, permutation t test) between HCR and LCR. Pathway analysis is a common tool to understand global changes in phenotypes in cells and tissues. Due to our focus on metabolism, we chose to use KEGG Pathway Ontology for analysis, which has well-annotated metabolic pathways. KEGG pathway analysis by Enrichr () showed that the differentially expressed proteins were enriched in oxidative phosphorylation, FA metabolism, BCAA degradation, and tRNA biosynthesis pathways ( Figure 4 C). Of the 174 proteins, only 22 were significantly higher in HCR and included multiple enzymes involved in FA oxidation ( Figure 4 C; Table S3 A). When we examined changes with exercise, we found few alterations in the proteome or phosphoproteome in either strain ( Figures 4 D and 4E). However, we found evidence for decreased acetylation in both HCR and LCR mitochondria following exercise ( Figure 4 F).

We identified 428 mitochondrial proteins, of which 73 were phosphorylated and 85 were acetylated (A and B). For pathway enrichment, log2 fold-change values of differentially expressed proteins between HCR and LCR were input into Enrichr; the numbers of identified proteins out of total number of proteins in the ontology are listed to the right of the bars (C). Log2 fold-change (Run/Rest) values were used to plot changes with exercise in the proteome (D), phosphoproteome (E), and acetylome (F).

Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle.

Skeletal muscle mitochondrial and metabolic responses to a high-fat diet in female rats bred for high and low aerobic capacity.

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity.

Specific Upregulation and Differential Acetylation of Mitochondrial Proteins in HCR and LCR

Carbohydrate use increases and FA use decreases with approach to exhaustion in both HCR and LCR. To identify metabolite changes that are coincident with fuel selection and exhaustion, we determined correlation coefficients (Pearson r) of metabolites with estimated FA use, carbohydrate use, and percent exhaustion (time at collection/previous time to exhaustion). We found 19 metabolites to be correlated with both fat and carbohydrate oxidation and coincident with exhaustion; most of these increased with longer exercise duration ( Table S2 B). Six of these metabolites were confirmed to change significantly with exhaustion in a second independent metabolomic data set ( Table S2 B) in which HCR and LCR were run to quarter, half, and full exhaustion ( Table S2 C). Muscle malate consistently increased with approach to exhaustion, as did blood lactate and acetylcarnitine ( Figures 3 A–3C). In parallel, muscle glycogen levels declined with approach to exhaustion ( Figure 1 E). Muscle acetylcarnitine levels were positively correlated with carbohydrate oxidation and inversely correlated with fat oxidation ( Figures 3 D and 3E). A correlation between acetylcarnitine levels and fuel selection (RQ) has been reported previously (), but unlike previous reports, we found no association between depletion of muscle L-carnitine and the decline in fat oxidation ( Figure 3 F), suggesting that in this model, carnitine availability is not limiting fat oxidation. Rather, accumulation of acetylcarnitine indicates overproduction of acetyl units relative to downstream utilization through the citric acid cycle at exhaustion in both HCR and LCR. The similar pattern of change in metabolite levels relative to exhaustion in HCR and LCR suggests that the mechanism of exhaustion is similar in both strains but is simply delayed in HCR.

Muscle malate (A), blood lactate (B), and muscle acetylcarnitine (C) values are shown at 0, 10, and 45 min of exercise as mean ± SEM for each group (n = 4–6). Muscle acetylcarnitine (C2) is also strongly correlated with previous estimates of carbohydrate oxidation (D) and fat oxidation (E) at 0, 10, and 45 min of exercise. Muscle L-carnitine (F) was not correlated with fat oxidation. ∗ p < 0.05 between HCR and LCR at a specific time point; #p < 0.05 difference from baseline.

Although FA and carbohydrates account for 85%–95% of the fuel used during exercise, amino acids also contribute to overall energy production (). At rest, HCR have higher muscle lysine and lower plasma leucine levels than LCR ( Figures 2 D and 2E). With exercise, there are greater changes in amino acids in HCR than in LCR. Muscle BCAA (leucine, isoleucine, and valine) and ornithine were lower in HCR than LCR at 10 min; these changes were paralleled by a fall in plasma BCAA and ornithine in HCR but not LCR ( Figures 2 D and 2E), suggesting increased utilization in HCR during exercise. Of all amino acids, BCAA contribute the most toward energy production, and BCAA metabolism increases with exercise (). The first step in BCAA degradation is loss of nitrogen through transamination to yield branched-chain keto-acids (BCKA). We found that plasma BCKA, α-ketoisocaproic acid (KIC), α-ketomethlyvaleric acid (KMV), and α-ketoisovaleric acid (KIV) were significantly increased in LCR at 10 min, but not in HCR ( Figure 2 F). Further metabolism of BCKA results in the formation of CoA derivatives that can be transferred to carnitine, primarily as C3-, C4-, and C5-carnitine. These short-chain acylcarnitines were also elevated in LCR muscle and plasma near exhaustion (10 min), but only C4-carnitine increased in HCR at 10 min, likely reflecting increased n-butyryl-carnitine derived from fat oxidation. Muscle and plasma C3-, C4-, C5-carnitine similarly increased near exhaustion (45 min) in HCR ( Figures 2 B and 2C). As with FA oxidation, these results are consistent with greater utilization of BCAA in HCR during exercise.

Fuel metabolism in men and women during and after long-duration exercise.

Although HCR have significantly greater fat oxidation throughout exercise, plasma NEFA declined similarly in HCR and LCR at 10 min of exercise ( Figure 1 F). However, plasma lipids (primarily triglycerides) declined more in HCR than LCR at 10 min of exercise ( Figure 2 A). Alterations in fat metabolism can also be observed through changes in acylcarnitines, which are derived from fatty acyl-chains exchanging CoA with L-carnitine in the mitochondria. At rest, muscle medium- and long-chain acylcarnitine species were lower in HCR than LCR ( Figure 2 B), while the same acylcarnitine species were higher in plasma of HCR than LCR ( Figure 2 C). With exercise, LCR muscle long-chain acylcarnitines changed minimally, and we observed only small rises in plasma long-chain acylcarnitines at 10 min, consistent with reduced FA use. In contrast, HCR muscle long-chain acylcarnitines increased at 10 min of exercise, indicative of increased FA import. In addition, minimal changes in muscle medium-chain acylcarnitines (C6-C12) were found, suggesting efficient oxidation of the FA. Only near exhaustion do HCR muscle and plasma medium- and long-chain acylcarnitines increase above resting levels ( Figures 2 B, 2C, and S2 ). The increases in plasma acylcarnitines in HCR and LCR near exhaustion likely reflect the limit of mitochondrial FA oxidation, and the delayed rise in plasma acylcarnitines in HCR reflects their greater capacity to oxidize FA during exercise.

Metabolite values at 0, 10, and 45 min of exercise (LCR and HCR) are expressed relative to LCR rest (0 min) for each group (n = 4–6). Plasma fatty acids (primarily triglycerides) (A) and muscle and plasma acylcarnitines (B and C) are listed by carbon chain length. Muscle and plasma amino acids (D and E) are less dynamic with exercise, but branched-chain keto acids (F) α-ketoisocaproic acid (KIC), α-ketoisovaleric acid (KIV), and α-ketomethylvaleric acid (KMV) show similar trends as short-chain acylcarnitines. ∗ p < 0.05 between HCR and LCR at a specific time point; #p < 0.05 difference from baseline.

Muscle and Plasma Long-Chain Acylcarnitines Increase More with Exercise in HCR

Figure 2 Muscle and Plasma Long-Chain Acylcarnitines Increase More with Exercise in HCR

We evaluated metabolites in plasma and gastrocnemius muscle from these same animals following a separate exercise bout ( Table S2 A). Subsets of rats (n = 4–6) were sampled at rest (0 min), at 10 min (mean time to exhaustion for LCR), and at 45 min (mean time to exhaustion for HCR) ( Figure 1 C). At rest, LCR and HCR show no difference in levels of primary fuel sources blood glucose ( Figure 1 D), muscle glycogen ( Figure 1 E), or plasma nonesterified fatty acid (NEFA) ( Figure 1 F). Blood glucose increased in both LCR and HCR at 10 min of exercise but was reduced in HCR at 45 min ( Figure 1 D). Muscle glycogen decreased with exhaustion in LCR (10 min) and HCR (45 min), but glycogen levels were not significantly changed in HCR from 0 to 10 min of exercise ( Figure 1 E). These data suggest that muscle glycogen contributes significantly to the higher whole-body carbohydrate oxidation in LCR during the first 10 min of exercise ( Figure 1 A), while HCR have delayed mobilization of glycogen.

Using a protocol identical to that used for selecting breeders, HCR have 1.4-fold higher VOmax and run 4.3-fold longer distance than LCR (see Table S1 available online). To estimate fuel use, we determined VOand VCO(normalized to lean mass) at rest and during exercise in HCR and LCR. Carbohydrate utilization rose rapidly in both HCR and LCR with onset of exercise ( Figure 1 A). In LCR, carbohydrate utilization continued to increase until exhaustion, with a concomitant decrease in fat oxidation. In contrast, HCR maintained comparatively high levels of fat oxidation throughout exercise. As HCR approached exhaustion, carbohydrate oxidation increased and fat oxidation decreased ( Figures 1 A and 1B). Similarly, at maintained submaximal exercise (75% VOmax), the rate of fat oxidation was significantly higher in HCR than LCR ( Figure S1 ), providing further evidence that HCR, compared to LCR, have enhanced capacity to oxidize FA.

Carbohydrate (A) and fat oxidation (B) were estimated from VO 2 and VCO 2 during an exhaustive exercise test for LCR (○) (n = 16) and HCR (●) (n = 23). Animals were separated into groups (n = 4–6) and run for 0, 10, and 45 min (HCR only) (C). Blood glucose concentration (D), muscle glycogen (E), and plasma nonesterified fatty acid (NEFA) (F) are presented as mean ± SEM for each group. ∗ p < 0.05 between HCR and LCR at a specific time point; #p < 0.05 difference from rest (0 min).

Discussion