Potential Role of Branched-Chain Amino Acid Catabolism in Regulating Fat Oxidation

ARTICLE Potential Role of Branched-Chain Amino Acid Catabolism in Regulating Fat Oxidation Heikki Kainulainen 1, Juha J. Hulmi 1, and Urho M. Kujala 2 1 Departments of Biology of Physical Activity, and 2 Health Sciences, University of Jyväskylä, Jyväskylä, Finland KAINULAINEN, H., J.J. HULMI, and U.M. KUJALA. Potential Role of Branched-Chain Amino Acid Catabolism in Regulating Fat Oxidation. Exerc. Sport Sci. Rev., Vol. 41, No. 4, pp. 194Y200, 2013. Insulin-resistant or obese individuals have increased serum branched-chain amino acid (BCAA) levels. Recent findings relate increased BCAA catabolism to increased fatty acid oxidation and better metabolic health in physically active individuals. We hypothesize that, via glyceroneogenesis, BCAA catabolism mediates increased constitutive use of fatty acids for A-oxidation in subjects with increased inherent or acquired aerobic capacity both during exercise and at rest. Key Words: exercise, aerobic capacity, obesity, insulin resistance, glyceroneogenesis, TCA cycle, malate-aspartate shuttle INTRODUCTION Obesity and low physical activity are associated with a high risk for metabolic disease and, whereas, leanness and high physical activity strongly are associated with a lower risk for metabolic disease (37). Therefore, it is important to understand the mechanisms that regulate our energy metabolism fully and determine the metabolically healthy phenotype. Increasing evidence seems to associate branched-chain amino acids ((BCAA) valine, leucine, and isoleucine) catabolism to increased fatty acid oxidation and better metabolic health. In this review, we present a hypothesis for a novel mechanism that may explain how increased BCAA degradation is connected via the tricarboxylic acid cycle (TCA) and glyceroneogenesis to increased fatty acid oxidation, thus, linking enhanced BCAA catabolism with more beneficial lean metabolic phenotype. BCAA are nonpolar essential amino acids. Most of our daily requirement for BCAA comes from dairy products (milk proteins), meat, fish, eggs, and also from various beans, nuts, and whole-grain products. According to the Food and Agriculture Organization, BCAA should account for 46% of all essential amino acids, namely 39 mg leucine, 20 mg isoleucine, and 26 mg valine per kilogram body weight every day. Unlike other amino acids, BCAA are not degraded directly by the liver. Because of Address for correspondence: Heikki Kainulainen, Ph.D., Department of Biology of Physical Activity, University of Jyväskylä, P. O. Box 35, FI-40014 University of Jyväskylä, Jyväskylä, Finland (E-mail: heikki.kainulainen@sport.jyu.fi). Accepted for publication: June 28, 2013. Associate Editor: Barry Braun, Ph.D., FACSM 0091-6331/4104/194Y200 Exercise and Sport Sciences Reviews Copyright * 2013 by the American College of Sports Medicine this feature of the liver, digested BCAA end up in the bloodstream and are, thus, readily available for skeletal muscle and other tissues. However, the liver can oxidize BCAA after their conversion to oxo-ketoacids in muscle and other target tissues. BCAA are oxidized for energy production, and, thus, BCAA supplementation may prevent fatigue by sparing muscle glycogen stores during endurance exercise. BCAA also have been linked to the prevention of central fatigue by decreasing the brain levels of tryptophan and serotonin (5). However, the existing evidence of the effects of BCAA on aerobic performance is controversial, with some studies showing positive effects and some showing nonexisting effects (11,14). In addition, leucine stimulates translation initiation and, thus, protein synthesis in skeletal muscle (3). Adequate dietary intake of BCAA also has beneficial effects on body composition, for example, by increasing the release of fatty acids from adipocytes and thus decreasing fat mass (4). Acknowledging these, BCAA-rich food/supplements such as whey protein are popular in sports (18). Hence, accumulating evidence indicates that protein-rich and especially BCAA-rich diets improve muscle protein synthesis, body composition, and perhaps also aerobic performance. Leucine, along with other amino acids, also has an insulinogenic effect in pancreatic A-cells (18). Large BCAA intake, on the other hand, can, at least in rodents with caloric excess in their diet, lead to insulin resistance (28). Recently, many findings related specifically to lipid and BCAA metabolism have accumulated, but no mechanistic explanation for their possible linkage has yet emerged. Our omics studies have shown that elevated long-term leisure-time physical activity is associated with a low serum BCAA concentration (22), a high muscle BCAA degradation (24), and further with improved body composition. Furthermore, we have shown that 194

an inherited high aerobic capacity is associated with a leaner phenotype and with improved signature of muscle BCAA degradation (21). In the following sections, we elaborate on the previously mentioned research and further studies related to muscle BCAA and lipid metabolism. Based on those results, we present the hypothesis that catabolism of BCAA and energy production from fatty acids are linked by a metabolic cycle commencing with BCAA degradation. In this cycle, BCAA degradation products via the TCA (also called citric acid or Krebs) cycle and malate-aspartate shuttle are directed partially to glyceroneogenesis, the synthesis of glyceride-glycerol from precursors other than glucose. More specifically, on this pathway, a cytosolic reaction catalyzed by phosphoenolpyruvate carboxykinase (PEPCK) leads to formation of glyceraldehyde- 3-phosphate, which is further used for intramyocellular triglyceride synthesis into lipid droplets. This fatty acid source via A-oxidation again serves the acetyl-coa to TCA cycle and, together with BCAA degradation products, directs more substrates to glyceroneogenesis. The suggested metabolic cycle seems more active in subjects with a high inherited or acquired aerobic capacity both during exercise and at rest and contributes to improved aerobic capacity and reduced body fat accumulation. GLOBAL OMICS EVIDENCE FOR THE SIGNIFICANCE OF BCAA A metabolomic approach in a 12-yr follow-up study of more than 2400 normoglycemic individuals (Framingham Offspring Study) showed an explicit association between high serum BCAA and aromatic amino acid levels and future type 2 diabetes (38). Concurrently, recent metabolomic and transcriptomic studies strongly suggest that the rate of BCAA catabolism differs in various physiological and disease states. Metabolomic profiling showed that, in obese persons, plasma BCAA concentrations were higher and that the HOMA index positively correlated with the principal component composed of BCAA-related metabolites when compared with their lean counterparts (28). In our studies, the metabolomics of twin pairs discordant for their leisure time physical activity for 30 yr revealed lower concentrations of serum BCAA, especially isoleucine, in the active versus inactive twin (22). Serum isoleucine was confirmed to be low in physically active persons in three different population-based cohorts (22). Similarly, the transcriptomics of skeletal muscle and adipose tissue yielded upregulated mrna expression signature of BCAA catabolism and fatty acid metabolism in active compared with inactive cotwins (24). In addition, adipose tissue transcription profiles of monozygotic twin pairs revealed that BCAA catabolism was downregulated in the adipose tissue of the obese compared with lean cotwins (32). Thus, especially human metabolomic and transcriptomic studies performed in twins suggest that lifestyle factors, including physical activity, can modulate BCAA catabolism and that this is accompanied by changes in fatty acid metabolism. Animal studies strongly support the previously described human data. Integrated metabolomics of urine and transcriptomics data obtained from muscle, liver, and adipose tissue of obese and diabetic db/db mice and respective nondiabetic mice indicate that reduced BCAA catabolism is related to the diabetic state (8). Our global transcriptomic study showed that skeletal muscles of rats selectively bred for high running capacity (HCR rats) have a higher expression of BCAA degradation and fatty acid metabolism genes than rats with a low running capacity (LCR rats) (21). Gene sets for both BCAA degradation and fatty acid metabolism correlated positively with aerobic performance and metabolism and negatively with glucose tolerance (21). Interestingly, a urine metabolomic approach in mice showed that increased fatty acid availability (high-fat diet) induces a rapid and consistent upregulation of BCAA catabolism, A-oxidation, and TCA cycle (6). Notably, the mice that were resistant to high-fat dietyinduced obesity expressed even higher upregulation of A-oxidation and leucine catabolism than the mice with a strong predisposition to obesity (6). Because skeletal muscle is the main organ responsible for BCAA catabolism and oxidative metabolism, it is presumable that the observed shifts in urine metabolome largely reflect changes in muscle metabolism. In sum, the existing metabolomic and transcriptomic studies suggest that constitutively (also at rest) increased BCAA catabolism is associated with a higher physical activity and leanness as well as increased fat oxidation. Decreased BCAA catabolism and, thus, elevated levels in blood are associated with low physical activity, increased adiposity, and other risk factors for metabolic diseases. DIETARY EFFECTS OF BCAA In a recent cross-sectional population-based study, a higher dietary intake of BCAA was associated with a lower prevalence of overweight and obesity in Asian, U.K., and U.S. populations (33). In support of this finding, a growing number of rodent and human studies suggest that BCAA-rich protein supplementation has beneficial effects on several health- and fitness-related factors, such as body composition, exercise performance, muscle properties, and glucose control (1,4,10). A double-blind crossover design study found that, especially after exercise-induced glycogen depletion, BCAA supplementation may promote resistance to fatigue and increase lipid oxidation during exercise (15). In human and animal studies, protein diets aimed at increasing the plasma concentration of leucine or BCAA in general have caused consistent improvement in body composition (4,27,28). Leucine may influence body composition in at least three ways: it is a metabolic trigger of muscle protein synthesis and growth via the mtor signaling pathway (18), it has been proposed to increase diet-induced energy expenditure in skeletal muscle (34), and, centrally, hypothalamic leucine decreases food intake (9). Increased diet-induced energy expenditure may be related to futile protein turnover (34) or, more probably, to BCAA-induced increase in mitochondrial energy production and/or thermogenesis. A BCAA-enriched protein mixture increased the biogenesis of mitochondria, improved muscle function (running capacity), and increased average survival in mice (10). Isoleucine supplementation for 4 wk increased the expression of the proton uncoupler UCP3 and the molecular markers of lipid mobilization and, furthermore, reduced body and adipose tissue weight gain during a high-fat diet, supporting Volume 41 & Number 4 & October 2013 BCAA Catabolism and Fat Oxidation 195

the idea that BCAA may increase basal metabolic rate (29). BCAA also have rapid effects because their infusion for 8 h increased mitochondrial ATP production in young (although not in elderly) adults (36). In the previously cited study by Newgard et al. (28), high fatyfed rats with BCAA supplementation V despite normalizing effects on their body composition V remained insulin resistant. In fact, these results suggest that a moderate high-fat diet per se is not enough to induce insulin resistance, but a further factor, such as BCAA, may be required (28). BCAA in the presence of excess lipids may induce insulin resistance by chronically activating the mtor pathway that leads to increased serine residue phosphorylation of IRS1 and consequently to downregulation of insulin signaling (28). Another explanation is that high availability of BCAA boosts and maintains increased fatty acid oxidation, which is then observed as reciprocal decreased glucose oxidation and elevated blood glucose (insulin resistance). In a recent study with mice, increased dietary leucine improved fatty dietyinduced abnormalities, such as glucose tolerance, insulin signaling, hepatic steatosis, and inflammation in adipose tissue (25). In contrast to the aforementioned studies, long-term interventions for several months using leucine without other BCAA as a dietary supplement in humans have not shown any effects on muscle mass or glycemic control (4,23). Supplementation of leucine for long periods reduces the serum concentration of other BCAA (23). Thus, it remains to be shown if prolonged supplementation with all BCAA would have beneficial or any effects. However, in light of all the previously mentioned data on diets containing BCAA and with BCAA supplementation, an intimate mechanistically still undefined connection between BCAA metabolism and fat metabolism related to body composition seems to exist. BCAA AND METABOLISM BCAA Degradation BCAA are catabolized mainly in muscle cells. The first step of catabolism is the removal of the amino group by mitochondrial branched-chain aminotransferase (BCATm) (Fig. 1). The resulting branched-chain >-ketoacids (>-ketoisocaproate from leucine, >-ketoisovalerate from valine, and >-keto-amethylvalerate from isoleucine) undergo oxidative decarboxylation in the reactions catalyzed by branched-chain >-ketoacid dehydrogenase (BCKD). BCKD is a rate-limiting enzyme complex that structurally and functionally resembles the pyruvate dehydrogenase complex. This enzyme complex is activated by increased availability of isoleucine and especially leucine but not valine (2). NADH and CoA esters derived from BCAA catabolism are allosteric inhibitors of BCKD. BCKD also is inhibited by BCKD kinase that phosphorylates the E1 subunits of the BCKD complex. Accordingly, it is activated by the protein phosphatase (PP2Cm)Ycatalyzed dephosphorylation (41). Finally, BCAA are catabolized further by a cascade of enzyme reactions to end-products (leucine to acetyl-coa, isoleucine to acetyl-coa and succinyl-coa, and valine to succinyl-coa) that can enter the TCA cycle (Fig. 1). In addition to rendering their carbon skeletons available for decarboxylation and oxidation, the amino groups of BCAA are Figure 1. Schematic presentation of BCAA catabolism. BCAA are catabolized, especially during exercise, to acetyl-coa and/or succinate-coa, which supply the TCA cycle. The main steps of catabolic reactions (transamination by BCATm and the decarboxylation by BCKD) are shown. BCKD catalyzed decarboxylation is the main regulatory step of BCAA catabolism. BCKD is inhibited by BCKD kinaseymediated phosphorylation and increased concentrations of BCAA ketoacids and activated by dephosphorylation catalyzed by protein phosphatase PP2Cm. Note that there is no comprehensive evidence from human studies on the functionality of all parts of the cycles presented in the figures of this article (for details, see text), thus, representing important foci for future research. >-KG, >-ketoglutarate; >-KIC, >-ketoisocaproate; >-KIV, >-ketoisovalerate; >-KMV, >-keto-amethylvalerate; BCAA, branched-chain amino acid; BCATm, mitochondrial branched-chain aminotransferase; BCKD, branched-chain >-ketoacid dehydrogenase; BCKD kinase, branched-chain >-ketoacid dehydrogenase kinase; Glu, glutamate; Ile, isoleucine; Leu, leucine; OAA, oxaloacetate; PP2Cm, BCKD phosphatase; TCA cycle, tricarboxylic acid cycle; Val, valine. transferred by BCATm to >-ketoglutarate to form mitochondrial glutamate (Fig. 2). Thereafter, via the malate-aspartate shuttle (Fig. 2), in addition to other intermediary metabolites, there is increased formation of cytosolic oxaloacetate. Oxaloacetate is a substrate for the synthesis of phosphoenolpyruvate by cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C). Normally, in the malate-aspartate shuttle, oxaloacetate is catalyzed by cytosolic malate dehydrogenase to malate that is then shuttled back to mitochondria via the malate->-ketoglutarate antiporter. Because, in our model, oxaloacetate is proposed to be catalyzed largely to phosphoenolpyruvate by PEPCK-C, we propose that the cytosolic malate, required for the export of >-ketoglutarate via the malatey>-ketoglutarate antiporter, is supplied from mitochondria via another malate transporter, the dicarboxylate carrier (Fig. 2, 3). This carrier has been shown to act analogously in supplying malate for citrate transport required in fatty acid synthesis (26). In addition to oxaloacetate, the malate-aspartate shuttle produces cytosolic glutamate that participates further in the formation of glutamine and alanine. Both of these amino acids are known to be synthesized in and released from skeletal muscle via BCAA catabolism. 196 Exercise and Sport Sciences Reviews www.acsm-essr.org

Figure 2. Schematic presentation of modified malate-aspartate shuttle. In BCATm catalyzed transamination, the amino groups of BCAA enter the malateaspartate shuttle. >-ketoglutarate receives the amino group and forms glutamate, which reacts with oxaloacetate to form aspartate. Aspartate is transferred to cytosol by the glutamate-aspartate antiporter and reacts with cytosolic >-ketoglutarate, producing cytosolic oxaloacetate. We propose that, in situations where increased BCAA catabolism is required (e.g., during aerobic exercise), cytosolic oxaloacetate is catalyzed by PEPCK-C to phosphoenolpyruvate and further to glyceroneogenesis. Normally, in the malate-aspartate shuttle, cytosolic oxaloacetate forms malate, which is transported back to mitochondria via the malate->ketoglutarate antiporter. We propose that, during increased BCAA catabolism, the cytoplasmic malate concentration essential for the proper function of the malate-aspartate shuttle is secured by an alternative malate transporter (dicarboxylate carrier). >-KG, >-ketoglutarate; Asp, aspartate; BCAA, branched-chain amino acid; Gln, glutamine; Glu, glutamate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK-C, cytosolic phosphoenolpyruvate carboxykinase; TCA cycle, tricarboxylic acid cycle; Val, valine. a, glutamate-aspartate antiporter; b, malate->-ketoglutarate antiporter; c, dicarboxylate carrier. BCAA, TCA Cycle, and Energy Metabolism In addition to acetyl-coa, anaplerotic reactions produce four- and five-carbon intermediates that are incorporated into the TCA cycle. Because the TCA cycle cannot oxidase these four- and five-carbon products completely, it is necessary to remove such intermediates from the cycle by cataplerosis. Obviously, anaplerotic and cataplerotic reactions are harmonized and operate in equilibrium. During exercise, the speed of the TCA cycle can intensify up to 70- to 100-fold (13). Interestingly, during exercise, the intramuscular concentration of >-ketoglutarate decreases whereas the concentrations of the other TCA intermediates (especially malate) are elevated; the total concentration increasing up to three- to fourfold (13). This suggests that exercise induces efficient removal of >-ketoglutarate from the TCA cycle. Because the malate-aspartate shuttle removes >-ketoglutarate from the TCA cycle, it is important to replenish the TCA intermediates for cataplerosis. BCAA are oxidized and used for energy production during exercise. Interestingly, prevention of amino acid catabolism by deleting the BCATm gene renders mice exercise intolerant, affirming that BCAA catabolism is necessary for exercise performance (35). Acute exercise activates the BCKD complex (the main regulator of BCAA oxidation) by decreasing BCKD kinase activity (20,40). As a result, during exercise, BCAA are to an increasing extent metabolized to acetyl-coa and succinyl-coa, thereby replenishing the TCA cycle for energy production and cataplerosis during exercise. It is worth noting that, in addition to isoleucine and valine, A-oxidation of odd-numbered fatty acids also produces succinyl-coa. Long-term exercise has been reported to decrease BCKD kinase content in rat skeletal muscle (12) but to increase it in human muscle (17). It remains to be shown if this discrepancy is caused by species differences or perhaps other reasons, such as different muscle tissues or type, length or intensity of training, or nutritional status. As mentioned earlier, BCKD activity also is regulated by BCKD phosphatase (PP2Cm) (41); however, the effects of physical activity on PP2Cm remain unknown. Muscle Glyceroneogenesis Glyceroneogenesis is defined as de novo synthesis of glycerol- 3-phosphate from pyruvate, lactate, or amino acids, the key enzyme of synthesis being PEPCK-C (7). PEPCK-C is a major cataplerotic, gluconeogenic/glyceroneogenic enzyme that catalyzes the synthesis of phosphoenolpyruvate from oxaloacetate. In adipose tissue, glyceroneogenesis is important for the maintenance of lipid homeostasis. Indeed, the gene for PEPCK-C is a candidate gene for diabetes and obesity, suggesting that dysregulation of the glyceroneogenic pathway may have pathophysiological effects (7). It has been shown that thiazolidinediones (TZD) that are used to treat type 2 diabetes increase PEPCK-C expression in adipocytes. This suggests that one of the antidiabetic effects of TZD is to induce adipocyte glyceroneogenesis, thus, decreasing fatty acid output and increasing insulin sensitivity (7). It is feasible that PEPCK-C is important also for lipid homeostasis of skeletal muscle. Constitutive overexpression of the cytosolic form of PEPCK in mouse skeletal Volume 41 & Number 4 & October 2013 BCAA Catabolism and Fat Oxidation 197

muscle produced an extraordinary phenotype (16). The aerobic (running) capacity of these mice is multifold compared with that of wild-type mice; they are hyperactive, live longer, and have more mitochondria in their muscles. In addition, transgenic PEPCK-C mice are leaner, having strikingly smaller visceral and subcutaneous fat depots compared with those in wild-type mice, although their intramuscular lipid stores are elevated highly. One likely possibility for the phenotype difference is the metabolic change resulting in increased removal of the TCA intermediates and subsequent increased muscle glyceroneogenesis in PEPCK-C mice (16). In another animal model, high- and low-capacity runner rats (HCR and LCR rats, respectively), phenotype differences strikingly similar to those in PEPCK-C and wild-type mice have been observed. These rats were derived from a common founder population by selective breeding according to their inherent running capacity (39). Indeed, HCR rats are superior runners compared with LCR, with more than 500% higher capacity when running to exhaustion (21). HCR are more active, have higher V O 2max and ample muscle mitochondria, live longer, and are leaner, with smaller fat depots than those in LCR (21). On the other hand, LCR rats have elevated risk factors (e.g., high blood glucose and serum insulin levels) for metabolic diseases (39). Importantly, HCR have higher PEPCK levels in skeletal muscle (30). As previously mentioned, HCR rats have high expression levels in skeletal muscle of the genes involved in BCAA degradation and fatty acid metabolism compared with LCRs (21). These data from PEPCK-C mouse and HCR/LCR rat models imply that high aerobic capacity and leanness are related to muscle glyceroneogenesis (i.e., to the synthesis of glyceraldehyde-3-phosphate from excess pyruvate and TCA intermediates via increased PEPCK activity) and hence to accelerated esterification of free fatty acids (FFA) to triglycerides. In muscle cells, FFA are stored as triglycerides in lipid droplets from which they are used for oxidative energy production, membrane biosynthesis, and other vital functions of the cell. A recent report showed that, at least in the resting state, FFA transported to muscle cells are first reesterified to triglycerides (19), showing the importance of triglyceride synthesis for the later oxidation of fatty acids. Glyceroneogenesis is, in contrast to glyceraldehyde-3-phosphate formation from glucose via the first steps of glycolysis, quantitatively the predominant source of triglyceride glycerol in skeletal muscle, adipose tissue, and liver of the rat during fasting and high feeding (31). Linking BCAA Catabolism and Fatty Acid Oxidation It is accepted widely that the lean metabolic phenotype with increased lipid metabolism and fatty acid oxidation is associated strongly with a lower risk for metabolic diseases. As described in previous chapters, increased BCAA supply and catabolism seem to be important components of this phenotype. But how is BCAA metabolism connected to enhanced lipid oxidation? A few possibilities are conceivable. One might be that BCAA degradation via TCA cycle would increase lipid synthesis concurrently with increased lipid oxidation, the net balance being on the side of increased energy consumption. Another option could be that increased oxidation of BCAA leads to increased acetyl CoA pool and consequent inhibition of pyruvate dehydrogenase that would shift substrate oxidation toward increased lipid oxidation and away from the use of carbohydrates. Both options probably play a part in the scheme of muscle metabolism. However, these metabolic features would not explain greatly increased malate and heavily decreased >-ketoglutarate concentration during prolonged submaximal exercise (13) and the presence of cytosolic PEPCK, the key glyceroneogenic enzyme, in skeletal muscle. In our hypothesis, the best candidate connecting increased BCAA supply and utilization to lean metabolic phenotype with high fat oxidation is a metabolic pathway that involves BCAA oxidation, TCA intermediate anaplerosis and cataplerosis, glyceroneogenesis originating from BCAA, and intramyocellular triglyceride synthesis (Fig. 3). Based on the previously cited research, we hypothesize that BCAA catabolism is a key factor in muscle oxidative metabolism and obesity through linking amino acid catabolism to fat metabolism. In brief, the results and facts that generated our hypothesis are: 1. Omics studies have shown that lean and physically active persons have lower blood BCAA concentrations than obese and less active persons. Higher physical activity and innate high aerobic capacity are associated with expression signatures of increased muscle BCAA catabolism and fatty acid metabolism. Furthermore, a high availability of fat in diet induces rapid and consistent upregulation of BCAA catabolism, A-oxidation, and TCA cycle. 2. BCAA-rich diets and BCAA supplementation often are associated with leanness, suggesting augmented fatty acid oxidation. 3. Gene manipulation studies have shown that BCATm deficiency, inhibiting the oxidation of BCAA, renders mice exercise intolerant. 4. Isoleucine and valine are catabolized to succinyl-coa, which is then incorporated into the TCA cycle. Addition of four- or five-carbon intermediates needs to be followed by removal of other intermediates from the cycle. Exercise leads to decreased concentration of >-ketoglutarate. In muscle, the main cataplerotic reaction is the removal of >-ketoglutarate, which is converted to glutamate and, thereafter, via the malate-aspartate shuttle to cytosolic oxaloacetate. 5. Muscle-specific overexpression of the main muscle cataplerotic enzyme PEPCK-C results in leanness and increased muscle fat stores, as well as a dramatic increase in running capacity. Also, selective breeding of rats for high aerobic capacity leads to enhanced muscle PEPCK-C expression, along with increased running capacity. 6. Glyceroneogenesis is quantitatively the predominant source of triglyceride glycerol in skeletal muscle. In the context of our hypothesis, increased synthesis of intramyocellular triglycerides provides energy during extended aerobic performance and also constitutively during rest. Thus, the high activity of this cycle leads to leaner body composition and better exercise performance. It is noteworthy that, in addition to increased physical activity, this proposed cycle seems to be activated by a higher availability of dietary BCAA as well as a higher activity of PEPCK-C. The presented hypothesis has a strong translational component. Human studies supporting our hypothesis have provided 198 Exercise and Sport Sciences Reviews www.acsm-essr.org

Figure 3. Diagram of the hypothesized connection between skeletal muscle BCAA catabolism and fatty acid metabolism. We propose that during increased demand for BCAA catabolism, for example during exercise, transamination of BCAA is essential for cytosolic oxaloacetate formation (via the malate-aspartate shuttle). In our model, oxaloacetate is metabolized further to phosphoenolpyruvate for glyceroneogenesis, which is required in skeletal muscles for the storage of fatty acids in lipid droplets. Another essential feature is BCAA decarboxylation, which supplies acetyl-coa and succinyl-coa for the TCA cycle. Similarly, A-oxidation of even-numbered fatty acids provides acetyl-coa. A-oxidation of odd-numbered fatty acids provides acetyl-coa and, in addition, also succinyl-coa. It is worth noting that, during exercise and increased oxidation of fatty acids, the acetyl-coa supply from pyruvate is inhibited by PDK4-catalyzed phosphorylation of the pyruvate dehydrogenase complex. During exercise, the muscle concentration of >-ketoglutarate decreases and the concentrations of the other TCA metabolites increase; the concentration of malate being the highest. In the presented model, malate, which is required for the functioning of the malate-aspartate cycle, is transported from the mitochondria to cytosol via the dicarboxylate carrier. BCAA, branched-chain amino acid; FFA, free fatty acid; GA3P, glyceraldehyde-3-phosphate; LD, lipid droplet; PDK4, pyruvate dehydrogenase kinase 4. Other abbreviations are as presented in Figures 1 and 2. omics-based and biochemical information of the effects of physical activity, body composition, and diet on the relation between BCAA and fat metabolism. Experimental animal research naturally has provided overlapping and confirmatory information but also a substantial amount of complementary data that are difficult or even impossible to obtain from human studies. Especially, selective breeding of HCR and LCR rat strains and gene manipulation in mice, for example, musclespecific overexpression of PEPCK-C and deletion of BCATm, have provided essential knowledge that helped us create the presented hypothesis. It remains to be shown if the presented metabolic cycle is functional in human skeletal muscle or perhaps only in rodent muscles. Proving the functional relevance of the proposed metabolic cycle requires a multifaceted approach. The metabolic fate of the different substrates could be traced by using advanced tracer techniques in vitro and in vivo. Further inhibition or activation of the function of the cycle at putative key points, for example, by inhibiting the activity of PEPCK-C or the dicarboxylate carrier, would, together with the metabolite measurements, provide some of the required information. CONCLUSIONS In summary, we hypothesize that, via glyceroneogenesis, BCAA catabolism mediates increased constitutive use of fatty acids for A-oxidation in subjects with increased inherent or acquired aerobic capacity both during exercise and at rest. These are important links in the complex human metabolism and particularly important for aerobic performance and the prevention of fat accumulation. BCAA are not catabolized directly by the liver; consequently, their appearance in the bloodstream directly is related to dietary intake. Thus, BCAA, in particular with increased physical activity, represent a good candidate for a clinically relevant modifier of the proposed regulatory function. As a consequence, serum BCAA or their metabolite levels may be a putative clinical marker predicting the development of metabolic diseases including type 2 diabetes. Existence of the proposed metabolic pathway may provide further understanding of aerobic performance and basis for the development of new therapeutic strategies against obesity and other lipid metabolismyrelated disorders. Acknowledgments The authors report no conflicts of interest. The funders have no role in the preparation, review, or approval of the article. The authors apologize that because of constraints on space, it was not possible to cite all the outstanding work in this area. The authors work related to this review and hypothesis was supported by the Academy of Finland, the Finnish Ministry of Education and Culture, and TEKES, the Finnish Funding Agency for Technology and Innovation. Volume 41 & Number 4 & October 2013 BCAA Catabolism and Fat Oxidation 199

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