ESSR Published Ahead-of-Print ACCEPTED. Potential Role of Branched Chain Amino Acid (BCAA) Catabolism in

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1 ESSR Published Ahead-of-Print Potential Role of Branched Chain Amino Acid (BCAA) Catabolism in Regulating Fat Oxidation Heikki Kainulainen, Ph.D. 1 Juha J. Hulmi, Ph.D. 1 and Urho M. Kujala, M.D. 2 Departments of 1 Biology of Physical Activity and 2 Health Sciences, University of Jyväskylä, Jyväskylä, Finland Accepted: June 28, 2013 Exercise and Sport Sciences Reviews articles in the Published Ahead-of-Print section have been peer-reviewed and accepted for publication. However, during copyediting, page composition, or proof review changes may be made that could affect the content. Copyright 2013 by the American College of Sports Medicine

2 Exercise and Sport Sciences Reviews, Publish Ahead of Print DOI: /JES.0b013e3182a4e6b6 Potential Role of Branched Chain Amino Acid (BCAA) Catabolism in Regulating Fat Oxidation D Heikki Kainulainen, Ph.D.1 Juha J. Hulmi, Ph.D.1 and Urho M. Kujala, M.D.2 Jyväskylä, Finland Correspondence to: EP Heikki Kainulainen, Ph.D. TE Departments of 1Biology of Physical Activity and 2Health Sciences, University of Jyväskylä, Department of Biology of Physical Activity University of Jyväskylä PO Box 35 C FI University of Jyväskylä Jyväskylä C Finland A heikki.kainulainen@sport.jyu.fi; phone: Disclosure of funding: The authors report no conflicts of interest. The funders have no role in the preparation, review, or approval of the manuscript.

3 ABSTRACT Insulin resistant or obese individuals have increased serum 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 β-oxidation in subjects with increased inherent or acquired aerobic capacity both during exercise and at rest. Summary for the table of contents This article presents the hypothesis that skeletal muscle BCAA catabolism mediates increased constitutive use of fatty acids for β-oxidation. Key words: exercise, aerobic capacity, obesity, insulin resistance, glyceroneogenesis, TCA cycle, malateaspartate shuttle

4 INTRODUCTION Obesity and low physical activity are associated with high risk for metabolic disease and, vice versa, leanness and high physical activity are strongly associated with lower risk for metabolic disease (37). Therefore it is important to fully understand the mechanisms that regulate our energy metabolism and determine the metabolically healthy phenotype. Increasing evidence seems to associate branched-chain amino acid (BCAAs = 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 tricarboxylic acid cycle and glyceroneogenesis to increased fatty acid oxidation thus linking enhanced BCAA catabolism with more beneficial, lean metabolic phenotype. BCAAs are nonpolar, essential amino acids. Most of our daily requirement for BCAAs comes from dairy products (milk proteins), meat, fish, eggs, and also from various beans, nuts and wholegrain products. According to the FAO (Food and Agriculture Organization), BCAAs should account for 46% of all essential amino acids, viz. 39 mg leucine, 20 mg isoleucine and 26 mg valine per kg body weight every day. Unlike other amino acids, BCAAs are not directly degraded by the liver. Due to this feature of the liver, digested BCAAs end up in the bloodstream, and are thus readily available for skeletal muscle and other tissues. However, the liver can oxidize BCAAs after their conversion to oxo-ketoacids in muscle and other target tissues. BCAAs are oxidized for energy production and thus BCAA supplementation may prevent fatigue by sparing muscle glycogen stores during endurance exercise. BCAAs have been also linked to the prevention of central fatigue by decreasing the brain levels of tryptophan and

5 serotonin (5). However, the existing evidence of the effects of BCAAs on aerobic performance is controversial, some studies showing positive and some non-existing effect (11, 14). In addition, leucine stimulates translation initiation and thus protein synthesis in skeletal muscle (3). Adequate dietary intake of BCAAs also has beneficial effect on body composition, e.g. 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 β-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 low serum BCAA concentration (22), high muscle BCAA degradation (24), and further with improved body composition. Furthermore, we have shown that inherited high aerobic capacity is associated with leaner phenotype as well as with improved signature of muscle BCAA degradation (21). In the following chapters we elaborate the above mentioned research and further studies related to muscle BCAA and lipid metabolism. Based on those results, we present the hypothesis that catabolism of BCAAs and energy production from fatty acids are linked by a metabolic cycle commencing with BCAA degradation. In this cycle BCAA degradation products via the tricarboxylic acid (TCA, also called citric acid or Krebs) cycle and malate-aspartate shuttle are partially directed to glyceroneogenesis, the synthesis of glyceride-glycerol from precursors other than glucose. More specifically, on this pathway a cytosolic reaction catalyzed by PEPCK

6 (phosphoenolpyruvate carboxykinase) leads to formation of glyceraldehyde-3-phosphate, which is further used for intramyocellular triglyceride synthesis into lipid droplets. This fatty acid source via β-oxidation again serves the acetyl-coa to TCA cycle and together with BCAA degradation products directs more substrates to glyceroneogenesis. The suggested metabolic cycle appears more active in subjects with 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 BCAAs A metabolomic approach in a 12-year follow-up study of over 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 correlated positively with the principle component comprised of BCAA-related metabolites when compared to their lean counterparts (28). In our studies, the metabolomics of twin pairs discordant for their leisure time physical activity for 30 years revealed lower concentrations of serum BCAAs, especially isoleucine, in the active vs. inactive co-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 up-regulated mrna expression signature of BCAA catabolism and fatty acid metabolism in active compared to inactive co-twins (24). Also, adipose tissue transcription profiles of monozygotic twin pairs revealed that BCAA catabolism was down-regulated in the adipose tissue of the obese compared to lean co-twins (32).

7 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 above-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 non-diabetic mice indicate that reduced BCAA catabolism is related to diabetic state (8). Our global transcriptomic study showed that skeletal muscles of rats selectively bred for high running capacity (HCR rats) have higher expression of BCAA degradation and fatty acid metabolism genes than rats with low running capacity (LCR rats) (21). Gene sets both for 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, β-oxidation and TCA cycle (6). Notably, the mice that were resistant for high-fat diet induced obesity expressed even higher upregulation of β-oxidation and leucine catabolism than the mice with strong predisposition to obesity (6). Since 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 higher physical activity and leanness as well as increased fat oxidation. On the other hand, decreased BCAA catabolism and thus

8 elevated levels in blood are associated with low physical activity, increased adiposity and other risk factors for metabolic diseases. DIETARY EFFECTS OF BCAAs In a recent cross-sectional, population-based study, higher dietary intake of BCAAs was associated with lower prevalence of overweight and obesity in Asian, UK and US 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-blinded cross-over 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 BCAAs in general have caused consistent improvement in body composition (4, 27, 28). Leucine may influence body composition at least in 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 weeks 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 the idea that BCAAs may increase basal metabolic rate (29). BCAAs have also rapid effects since

9 their infusion for 8 h increased mitochondrial ATP production in young (although not in elderly) adults (36). In the above-cited study by Newgard et al. (28), high-fat-fed rats with BCAA supplementation despite normalizing effects on their body composition remained insulinresistant. 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 BCAAs, may be required (28). BCAAs 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 BCAAs 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 diet induced 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 BCAAs 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 BCAAs (23). Thus, it remains to be shown if prolonged supplementation with all BCAAs would have beneficial or any effects. However, in light of all the abovementioned 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.

10 BCAAs AND METABOLISM BCAA Degradation BCAAs are catabolized mainly in muscle cells. The first step of catabolism is the removal of the aminogroup by mitochondrial branched chain aminotransferase (BCATm) (Fig.1). The resulting branched-chain α-ketoacids (α-ketoisocaproate from leucine, α-ketoisovalerate from valine and α-keto-β-methylvalerate from isoleucine) undergo oxidative decarboxylation in the reactions catalyzed by branched-chain α-ketoacid dehydrogenase (BCKD). BCKD is 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 is also inhibited by BCKD kinase that phosphorylates the E1 subunits of the BCKD complex. Accordingly, it is activated by the protein phosphatase (PP2Cm)-catalyzed dephosphorylation (41). Finally, BCAAs 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 BCAAs are transferred by BCATm to α-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-α-ketoglutarate antiporter. Since, in our model, oxaloacetate is proposed to be largely

11 catalyzed to phosphoenolpyruvate by PEPCK-C, we propose that the cytosolic malate, required for the export of α-ketoglutarate via the malate-α-ketoglutarate antiporter, is supplied from mitochondria via another malate transporter, the dicarboxylate carrier (Fig. 2, Fig. 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. BCAAs, TCA Cycle and Energy Metabolism In addition to acetyl-coa, anaplerotic reactions produce 4- and 5-carbon intermediates that are incorporated into the TCA cycle. Since the TCA cycle cannot completely oxidase these 4- and 5-carbon products 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 fold (13). Interestingly, during exercise the intramuscular concentration of α-ketoglutarate decreases while the concentrations of the other TCA intermediates (especially malate) are elevated, the total concentration increasing up to 3-4 fold (13). This suggests that exercise induces efficient removal of α-ketoglutarate from the TCA cycle. Since the malate-aspartate shuttle removes α-ketoglutarate from the TCA cycle, it is important to replenish the TCA intermediates for cataplerosis. BCAAs are oxidized and used for energy production during exercise. Interestingly, prevention of amino acid catabolism by deleting the BCATm gene renders mice exerciseintolerant, affirming that BCAA catabolism is necessary for exercise performance (35). Acute exercise activates the BCKD complex (the main regulator of BCAA oxidation) by decreasing

12 BCKD kinase activity (20, 40). As a result, during exercise BCAAs 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, β-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 due to species difference or perhaps other reasons such as different muscle tissues or type, length or intensity of training, or nutritional status. As mentioned earlier, BCKD activity is also 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 glyceroneogenic pathway may have pathophysiological effects (7). It has been shown that thiatzolidinediones (TZDs) that are used to treat type 2 diabetes, increase PEPCK-C expression in adipocytes. This suggests that one of the antidiabetic effects of TZDs 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 muscle produced an extraordinary phenotype (16). The aerobic (running) capacity of these mice is multifold compared to wild-type mice, they are hyperactive, live longer, and have

13 more mitochondria in their muscles. In addition, transgenic PEPCK-C mice are leaner, having strikingly smaller visceral and subcutaneous fat depots compared with wild-type mice, although their intramuscular lipid stores are highly elevated. 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 to LCRs with over 500% higher capacity when running to exhaustion (21). HCRs are more active, have higher VO 2max and ample muscle mitochondria, live longer and are leaner with smaller fat depots than LCRs (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, HCRs have higher PEPCK levels in skeletal muscle (30). As mentioned earlier, HCR rats have high expression levels in skeletal muscle of the genes involved in BCAA degradation and fatty acid metabolism compared to 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 FFAs to triglycerides. In muscle cells FFAs 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 FFAs 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,

14 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 widely accepted that the lean metabolic phenotype with increased lipid metabolism and fatty acid oxidation is strongly associated with lower risk for metabolic diseases. As described in previous chapters, increased BCAA supply and catabolism seem to be an important component 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 BCAAs leads to increased acetyl CoA pool and consequent inhibition of pyruvate dehydrogenase that would shift substrate oxidation towards 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 α-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 BCAAs, and intramyocellular triglyceride (IMTG) synthesis (Fig. 3). Based on the above-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:

15 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, high availability of fat in diet induces rapid and consistent upregulation of BCAA catabolism, β-oxidation and TCA cycle. 2. BCAA-rich diets and BCAA supplementation are often associated with leanness suggesting augmented fatty acid oxidation. 3. Gene manipulation studies have shown that BCATm deficiency, inhibiting the oxidation of BCAAs, renders mice exercise-intolerant. 4. Isoleucine and valine are catabolized to succinyl-coa which is then incorporated into the TCA cycle. Addition of 4- or 5-carbon intermediates needs to be followed by removal of other intermediates from the cycle. Exercise leads to decreased concentration of α- ketoglutarate. In muscle, the main cataplerotic reaction is the removal of α-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.

16 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 higher availability of dietary BCAAs as well as higher activity of PEPCK-C. The presented hypothesis has a strong translational component. Human studies supporting our hypothesis have provided 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 has naturally provided overlapping and confirmatory information but also a substantial amount complementary data that is difficult or even impossible to obtain from human studies. Especially selective breeding of HCR and LCR rat strains and gene manipulation in mice, e.g. muscle specific over-expression of PEPCK-C and deletion of BCATm, have provided essential knowledge that helped us to 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, e.g. by inhibiting the activity of PEPCK-C or the dicarboxylate carrier, would, together with the metabolite measurements, provide some of the required information.

17 CONCLUSION In summary, we hypothesize that, via glyceroneogenesis BCAA catabolism mediates increased constitutive use of fatty acids for β-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. BCAAs are not directly catabolized by the liver, consequently their appearance in the blood stream is directly related to dietary intake. Thus, BCAAs, 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 metabolism-related disorders. Acknowledgements The authors apologize that owing to 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. The authors declare no conflict of interest.

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22 Figure legends: Figure 1. Schematic presentation of BCAA catabolism. BCAAs 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 kinase mediated phosphorylation and increased concentrations of BCAA ketoacids and activated by dephosphorylation catalyzed by protein phosphatase PP2Cm. Note that there is not comprehensive evidence from human studies on the functionality of all parts of the cycles presented in the figures of this paper (for details see text) thus representing important foci for future research. BCATm = mitochondrial branched chain aminotransferase, BCKD = branched-chain α-ketoacid dehydrogenase, BCKD kinase = branched-chain α-ketoacid dehydrogenase kinase, Glu = glutamate, Ile = isoleucine, α-kg = α-ketoglutarate, α-kic = α-ketoisocaproate, α-kiv = α- ketoisovalerate, α-kmv = α-keto-β-methylvalerate. Leu = leucine, OAA = oxaloacetate, PP2Cm = BCKD phosphatase, TCA cycle = tricarboxylic acid cycle, Val = valine. Figure 2. Schematic presentation of modified malate-aspartate shuttle. In BCATm catalyzed transamination, the amino groups of BCAAs enter the malate-aspartate shuttle. α-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 α-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,

23 which is transported back to mitochondria via the malate-α-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). Asp = aspartate, BCAA = branched-chain amino acid, Gln = glutamine, Glu = glutamate, α-kg = α-ketoglutarate, OAA = oxaloacetate, PEP = phosphoenolpyruvate, PEPCK-C = cytosolic phosphoenolpyruvate carboxykinase, TCA cycle = tricarboxylic acid cycle, Val = valine, a = glutamate-aspartate antiporter, b = malate-α-ketoglutarate antiporter, c = dicarboxylate carrier. 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 e.g. during exercise, transamination of BCAAs is essential for cytosolic oxaloacetate formation (via the malate-aspartate shuttle). In our model oxaloacetate is further metabolized 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 β-oxidation of even-numbered fatty acids provides acetyl-coa. β- 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 α-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 malateaspartate cycle is transported from the mitochondria to cytosol via the dicarboxylate carrier.

24 FFA = free fatty acid, GA3P = glyceraldehyde-3-phosphate, LD = lipid droplet, PDK4 = pyruvate dehydrogenase kinase 4. Other abbreviations as in Figs.1 and 2.

25 Figure 1 OAA TCA cycle α-kg Citrate Glu α-kg Succinyl-CoA Leu Ile Val α-kic α-kmv α-kiv Acetyl- CoA Succinyl- CoA Θ 1 = BCATm 2 = BCKD 3 = BCKD kinase 4 = PP2Cm 3 4

26 Figure 2 carrier Gln PEP PEPCK-C Malate OAA Glu Malate Phosphate Glu Asp α-kg Cytosol c a b Mitochondria BCAA Malate Glu Asp α-kg 1 α-kg Malate OAA Glu α-ketoacid NAD+ NADH 1 = BCATm a = glutamate-aspartate antiporter b = malate-α-ketoglutarate antiporter c = dicarboxylate

27 Figure 3 FFA Malate Cytosol Ala c Mitochondria PDK4 Malate GA3P PEP Pyruvate OAA PEPCK-C OAA Acetyl-CoA Malateaspartate cycle TCA cycle D Glu α-kg α-kg Succinyl-CoA BCAA LDs β-oxidation