Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance

Abstract

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43-79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and alpha-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Fig. 1.

Running time and distance of mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) and wild-type (WT) mice during endurance exercise on treadmill. Mice fed normal chow (NC)/branched-chain amino acid-free (−BCAA) choice diets were trained for 3 days to run and then run on treadmill to exhaustion, according to the exercise protocol described in materials and methods. Total running time was recorded (A), and running distance was calculated (B). Values are means ± SE; n = 7 for each group. *P < 0.05.

Fig. 2.

Plasma concentrations of lactate, ammonia, glucose, and BCAA before and during exercise and at exhaustion. In both BCATm KO (open bars) and WT (solid bars) mice, blood samples were collected from tail clippings immediately before and during exercise when BCATm KO mice were exhausted. Blood samples were also collected from WT mice when they were exhausted. Plasma lactate (A) and glucose (C) were measured in samples collected from mice fed NC/−BCAA choice diets. Plasma ammonia (B) and BCAA (D) were measured in sample collected from mice fed +BCAA/−BCAA choice diets. When fed these different diets, the time for BCATm KO and WT mice to reach exhaustion was not the same, but did not differ statistically. Plasma lactate when BCATm KO was exhausted was also higher in BCATm KO (14.5 ± 2.0 mM) than WT (6.5 ± 1.3 mM, P < 0.01, n = 7) mice fed +BCAA/−BCAA choice diets. Values are means ± SE; n = 7 for each group. *P < 0.05.

Fig. 3.

Muscle glycogen content during exercise and fasted-refeeding. A: gastrocnemius glycogen content in mice at rest and with a brief exercise, as described in materials and methods. Values are means ± SE; n = 10 for WT at rest and exercise, and n = 7 for BCATm KO at rest and exercise. ^aP < 0.05 compared with respective resting level. B: muscle glycogen content in mixed hindlimb muscle of mice during fasting and refeeding. Male mice were fasted for 21 h and then refed with NC/−BCAA choice diets for 3 h and killed to collect mixed hind muscle for glycogen measurement. Values are means ± SE; n = 8 for BCATm WT and KO during refeeding, and n = 3 for BCATm WT and KO after a 21-h fast. *P < 0.05.

Fig. 4.

Concentrations of lactate and pyruvate (A), TCA cycle intermediates (B), phosphocreatine (C), and ammonia (D) in quadriceps of mice at rest and with a brief exercise. All mice fed NC/−BCAA choice diets were trained for 4 days to run and rest for 1 day before death. Mice were killed either at rest or after running for 7 min at 13 m/min and 10% slope, and skeletal muscles were collected for analyses of metabolites. Values are means ± SE; n = 10 for WT at rest and exercise, and n = 7 for BCATm KO at rest and exercise. α-KG, α-ketoglutarate. *P < 0.05.

Fig. 5.

Glucose uptake (A) and lactate release (B) during hindlimb perfusion. The perfusion protocol was described in materials and methods. Overnight fasted mice were perfused with 6.1 mM glucose/insulin at 1 mU/ml for 40 min and then 19.1 mM glucose/insulin at 1 mU/ml for another 40 min. Arterial and venous samples were collected every 10 min. Glucose uptake was calculated by the following formula: (arterial glucose − venous glucose) × flow rate/weight of tissue perfused. Lactate release was calculated as (venous lactate − arterial lactate) × flow rate/weight of tissue perfused. Values are means ± SE; n = 5 for WT and 4 for BCATm KO mice. *P < 0.05.

Fig. 6.

Transmission electron image of gastrocnemius from BCATm WT (+/+) and KO (−/−) mice. Electron photomicroscope images were developed from gastrocnemius of 3 WT and 3 KO mice. A representative image is shown.

Fig. 7.

Metabolic impairments in skeletal muscle caused by disruption of the first step of BCAA metabolism. A: blockage of BCAA transamination leads to decreased alanine glutamine synthesis from pyruvate and glutamate. The scheme emphasizes the importance of translocation of glutamate from mitochondria to cytosol via aspartate aminotransferanses and glutamate-aspartate antiporter. B: model of perturbed malate/aspartate shuttle in mice lacking BCAA metabolism. The diagram emphasizes the importance of BCATm-catalyzed transamination to provide glutamate and aspartate for the malate/aspartate shuttle and the role of this shuttle to transfer electrons from cytosol to mitochondria to avoid lactate overproduction during exercise. BCATm disruption (shown as black rectangle) leads to decreases in metabolites shown in red and as “↓”, and increases in metabolites are shown in bold characters and as “↑”. The changes in branched-chain α-ketoacids, OAA (oxaloacetate), and NADH concentrations were not measured and are deduced. The enzymes or proteins involved are as follows: 1, BCATm; 2, mitochondrial aspartate aminotransferanse; 3, glutamate-aspartate antiporter; 4, cytosolic aspartate aminotransferanse; 5, cytosolic malate dehydrogenase (the “=” sign donates a diminished flux in BCATm KO mice); 6, lactate dehydrogenase; 7, malate-α-ketoglutarate antiporter; 8, mitochondrial malate dehydrogenase; 9, alanine aminotransferanse; and 10, glutamine synthetase.