Branched-chain amino acids in health and disease: metabolism, alterations in blood plasma, and as supplements

BCAA catabolism (Fig. 1)

Unlike most amino acids, the initial step of BCAA catabolism does not take place in the liver due to low hepatic activity of branched-chain-amino-acid aminotransferase (BCAT), the first enzyme in the BCAA catabolism pathway. Therefore, the BCAA increase rapidly in systemic circulation after protein intake and are readily available to extrahepatic tissues. This phenomenon gives a unique advantage to the BCAA-based nutritional formulas compared with others, especially those targeted on muscles and brain.

The initial site of most of the BCAA catabolism is skeletal muscle because of the BCAT high activity. The BCAT reaction involves the reversible transfer of the BCAA amino group to α-ketoglutarate (α-KG) to form glutamate and the corresponding branched-chain keto acids (BCKAs), α-ketoisocaproate (KIC, ketoleucine), α-keto-β-methylvalerate (KMV, ketoisoleucine), and α-ketoisovalerate (KIV, ketovaline). Glutamate then acts as an amino group source to form alanine (ALA) from pyruvate or as a substrate for ammonia detoxification to glutamine (GLN). GLN, ALA, and a significant portion of the BCKA are released from muscles to the blood.

The second enzyme of BCAA catabolism, branched-chain α-keto acid dehydrogenase (BCKD), is a multienzyme complex located on the inner surface of the inner mitochondrial membrane, which catalyzes irreversible decarboxylation of the BCKA to the corresponding branched-chain acyl-CoA esters. The BCKD is regulated by the phosphorylation-dephosphorylation mechanism. Phosphorylation mediated by a specific kinase results in inactivation, while dephosphorylation by a specific phosphatase activates the enzyme. Changes in kinase activity may play a main role.

The BCKD activity is highest in the liver, intermediate in kidneys and heart, and low in muscles, adipose tissue, and brain [3]. When the weights of individual tissues are taken into consideration, muscles, which make up 35 to 40% of total body weight, should contribute substantially to total body BCAA utilization. Thus, BCAA degradation is under joint control of a number of tissues, among which the muscle and liver play a dominant role (Fig. 2). Many influences including cytokines, hormones, nutrients, and various metabolites affect the activity state of the enzyme [3]. The remarkable rise in BCKD activity in muscles induces endotoxin or tumor necrosis factor alpha (TNF-α) administration [4, 5].

Beyond the BCKD reaction, the metabolism of the BCAA diverges into separate pathways. Catabolism of KIC leads to acetyl-CoA and acetoacetate (KIC is ketogenic), KIV is catabolized to succinyl-CoA (KIV is glucogenic), and KMV to acetyl-CoA and succinyl-CoA (KMV is both glycogenic and ketogenic). A special product of KIC catabolism is β-hydroxy-β-methylbutyrate (HMB) synthesized in the reaction catalyzed by KIC dioxygenase.

Amination of the BCKA and interorgan cycling of the BCAA and BCKA (Fig. 3)

As the BCAT reaction is reversible and near equilibrium, its direction should respond to changes in concentrations of BCAA and BCKA, and availability of the donors and acceptors of nitrogen. In most conditions the majority of the BCAA uptake and BCKA release occurs in muscles, while amination of the BCKA to the BCAA may occur in other tissues, notably in the liver, kidneys and enterocytes. Direct evidence of the BCKA amination to the BCAAs was provided by studies using labelled BCKA showing the labelled BCAA in proteins [6]. The main sources of nitrogen for amination of the BCKA are GLN, glutamate, and ALA [3].

The studies demonstrate that BCAA synthesis from the BCKA is activated in various muscle wasting disorders, e.g. sepsis, trauma, or surgery, in which the muscles release high amounts of GLN and ALA to the blood [7–11]. A marked increase in leucine release was observed by the isolated liver of endotoxin-treated animals after addition of KIC into perfusion medium [7]; higher synthesis of the BCAA from BCKA was shown in the liver perfused with medium containing 0.5 mM GLN when compared to perfusion with medium without GLN [8]. Amination of the BCKA may have a role in the unique increase of all three BCAA in blood plasma during a brief starvation characterized by accelerated release of ALA, GLN, and BCKA from muscles and augmented gluconeogenesis in the liver [9, 10].

The above-mentioned findings indicate the existence of an interorgan cycle (Fig. 3) that attenuates the loss of essential BCAA in various physiological and pathological conditions [11].

Effects on protein metabolism

BCAAs not only serve as substrates for protein synthesis, but also exert stimulatory effect on protein synthesis and an inhibitory effect on proteolysis. The effects are realized by the BCAAs themselves, especially by leucine, and their metabolites. Leucine stimulates protein synthesis through the mTOR signaling pathway and phosphorylation of translation initiation factors and ribosomal proteins [12]. A role in protein anabolic effect of leucine plays also its stimulatory effect on insulin secretion [13]. The inhibitory effect of the BCAA on proteolysis is mediated mainly by BCKAs and HMB. BCKAs have been shown to prevent proteolysis in muscles under in vitro conditions [14]. Infusions of KIC were more effective than leucine in maintaining nitrogen balance in fasted subjects and in patients undergoing major abdominal surgery [15, 16]. HMB decreases the activity of the ubiquitin-proteasome proteolytic pathway and exerts beneficial effects on muscle in various conditions of health and disease [17].

Effects on neurotransmission

BCAAs are transported into the brain via the same carrier that transports aromatic amino acids (AAA; phenylalanine, tyrosine, tryptophan), and competition between BCAAs and AAAs may influence synthesis of some neurotransmitters, notably dopamine, norepinephrine, and 5-hydroxytryptamine (serotonin). Therefore, elevation of the BCAA in blood plasma is able to influence neurotransmitter levels in the brain with effects on behavior and brain function. This phenomenon is the rationale for use of the BCAAs in patients with liver cirrhosis, in which a decreased ratio of BCAAs to AAAs plays a role in pathogenesis of hepatic encephalopathy [18]. It is believed that BCAA supplementation attenuates production of serotonin, which is responsible for fatigue during exercise. Furthermore, BCAA transamination in the brain plays a role in the synthesis of glutamate and gamma-aminobutyric acid, and in ammonia detoxification to GLN in astrocytes. The studies have shown that leucine decreases appetite and may decrease body adiposity [19].

Effects on glucose metabolism

There are close associations between BCAAs and plasma glucose levels. The fact that BCAAs upregulate glucose transporters and activate insulin secretion has been widely demonstrated [13, 20, 21]. However, several researchers have suggested that excessive intake of amino acids could lead to inhibition of insulin signaling [22, 23]. Recent studies have suggested differential effects of each BCAA on glucose utilization and that BCAAs may induce insulin resistance through mTOR activation [24]. Further investigation is needed to understand variable reports ranging from improving glucose utilization to inducing insulin resistance.

Effects mediated by ALA and GLN

The rate of BCAA degradation in skeletal muscle is highly responsive to their availability [25]. The consequences of this phenomenon are that the primary effects of the consumption of a BCAA-enriched diet are activated catabolism of the BCAAs and enhanced levels of the BCKAs, ALA, and GLN in peripheral circulation [26]. Therefore, a number of effects of BCAA supplementation are mediated by ALA and GLN. ALA is the main gluconeogenic amino acid, and GLN availability is essential for immune system, glutathione production, maintenance of acid-base balance by the kidneys, and expression of heat shock proteins.

Other effects

During recent years, a number of novel functions of BCAAs, including benefits for mammary health and milk quality, intestinal development, immune response, mitochondrial biogenesis and oxidative stress have been reported [21].

Starvation

Brief starvation uniquely increases BCAA concentrations in plasma. In humans, the increase is evident within a day, and reaches maximum by the second or third day [27, 28].

Both increased proteolysis and reduced protein synthesis in muscles have been reported during brief starvation and may explain the enhanced availability of BCAAs for muscles [10, 29, 30]. In this condition, BCAAs in muscles act as a source of nitrogen for synthesis of ALA and GLN, which are released into the blood and used in visceral tissues, especially as gluconeogenic substrates. Increased BCAT activity in muscles during starvation has been reported by several laboratories [31, 32].

Together with ALA and GLN, the BCKAs generated in BCAT reaction are released into circulation and their concentration in the blood increases [26]. It may be supposed that a portion of nitrogen released during catabolism of GLN and ALA in visceral tissues escapes utilization in the urea cycle and is used for amination of BCKAs. Higher rates of BCAA synthesis from the BCKAs were observed by the liver perfused with GLN-containing medium than that perfused with GLN-deficient medium [8]. A role in the increase of BCAAs may have also their decreased uptake from the blood due to the decreased levels of insulin. An unresolved possibility is the activated breakdown of proteins in the liver, which may, due to low activity of hepatic BCAT, result in the release of the BCAA into the blood.

Prolonged starvation lowers the BCAA concentration to basal levels and gradually increases the activity of the BCKD complex. Marked increase in BCKD activity in muscles and heart occurs in the terminal phase of starvation, when amino acids replace fatty acids and ketone bodies as the predominant energy substrate [33].

Effects of a low-protein diet

Feeding healthy human volunteers or animals a diet devoid of protein, but adequate in caloric content, lowered the plasma BCAA concentrations below basal levels [27, 34]. The amino acid pattern of children with severe kwashiorkor shows severe decrease of BCAAs [35].

It is believed that the principal factors in the decrease of BCAAs during protein deprivation are the absence of exogenous amino acids as well as curtailed muscle protein breakdown. Lowered BCKD activities in muscles and liver of protein-depleted rats indicate the effort of the body to conserve BCAAs [36].

BCAA or BCKA supplementation should be recommended when a low-protein diet is prescribed to patients with chronic renal failure or urea cycle disorders.

Effects of a high-protein diet

Increased intake of protein may increase protein synthesis, decrease protein breakdown, reduce fat accumulation, and increase fat-free mass. Therefore protein supplementation or a high-protein diet is recommended to build the muscles in athletes, to prevent muscle wasting in severe illness, and to lose fat in the treatment of obesity.

High concentrations of BCAAs and urea are found in the postprandial state in the peripheral blood and muscles after intake of a protein meal and in subjects consuming a high-protein diet. In contrast to increased BCAA levels, the increments in arterial concentrations of most remaining amino acids of the ingested protein are small or insignificant [37, 38].

The main cause of the specific BCAA increase is the unique distribution of the enzymes, which control BCAA catabolism. While complete oxidation of most individual amino acids occurs in the liver, the initial site of BCAA catabolism is skeletal muscle. Therefore, a significant portion of ingested BCAA escapes hepatic uptake and appears in peripheral circulation. The effects of protein ingestion on BCAA levels are not observed in a postabsorptive state [38].

Liver cirrhosis

The decrease in BCAAs and an increase in AAAs are characteristic alterations in the blood of subjects with liver cirrhosis, which play a role in pathogenesis of hepatic encephalopathy and muscle wasting [18, 42]. Several studies have shown an inverse relationship between plasma ammonia and BCAA concentrations in patients with cirrhosis and that ammonia infusion decreases BCAA levels [43, 44]. BCAAs decrease because they are rapidly consumed to form glutamate from α-KG as a pivotal step in ammonia detoxification to GLN in muscles and in the brain [45]. Accelerated consumption of α-KG (cataplerosis) may disturb the function of the tricarboxylic acid (TCA) cycle (Fig. 5). The AAA increase is due to the decreased ability of the diseased liver to metabolize these amino acids. The BCAA levels do not decrease in acute liver injury due the leaking of amino acids from dying hepatocytes into the circulatory system [46].

Urea cycle disorders (UCD) and phenylbutyrate

UCD result from inherited enzymatic defects in the ammonia detoxification pathway in the liver, leading to low levels of urea and high levels of ammonia in the blood. The disorders are characterized by seizures, lethargy, coma, and death in the neonatal period or severe long-term neurological impairment.

In addition to altered levels of ammonia and urea, common finding in patients with UCD is an increase in GLN and a decline in BCAA levels, notably during acute metabolic decompensation [53]. These alterations support the theory that BCAAs play a unique role in ammonia detoxification to GLN and that hyperammonemia is the cause of decreased BCAA levels in subject with liver cirrhosis [45, 54].

At present, the management of UCD is achieved by dietary protein restriction and the use of compounds that remove nitrogen, notably benzoate and phenylbutyrate. Benzoate conjugates glycine to promote the synthesis of hippuric acid that is eliminated in urine and thus attenuates catabolism of glycine to ammonia. Phenylbutyrate is converted by β-oxidation into phenylacetate that is conjugated with GLN to form phenacetylglutamine, which is excreted in the urine (Fig. 4). Unfortunately, it has been shown that phenylbutyrate activates the BCKD, resulting in decreases in BCAA and BCKA levels in blood plasma [55, 56]. Marked decrease of BCAAs in UCD after phenylbutyrate treatment has been reported by Scaglia et al. [57].

Chronic renal failure (CRF)

Most studies of amino acid patterns in CRF reported decreased BCAA and BCKA levels in the blood plasma [59–61] and reduced concentrations of valine in muscles [61, 62]. The derangements are caused by the action of multiple factors, notably acidosis and glucocorticoids. Decreased intake of proteins and hemodialysis, resulting in low concentrations of most essential and nonessential amino acids, is also a factor. In contrast to CRF, inconsistent alterations have been reported in acute renal failure.

Several articles have suggested that metabolic acidosis is responsible for accelerated proteolysis and enhanced activity of the BCKD in muscles and liver [63, 64]. More significant increases in proteolysis and leucine oxidation were reported in rats with chronic uremia and acidosis when compared with uremic rats without acidosis. A significant decrease in valine concentration in the gastrocnemius muscle was found only in rats with acidosis [61].

Type 1 diabetes

High BCAA levels in subjects with defective insulin secretion were first described in dogs with experimental diabetes [67]. Further studies have shown that in addition to the increase of BCAAs, there is a decrease in levels of gluconeogenic amino acids, especially ALA [68–70]. Most data on pathogenesis of high levels of the BCAA in diabetes type 1 originate from studies using animals with diabetes induced by streptozotocin or alloxan.

There are some similarities in the pathogenesis of the increased BCAAs in diabetes and short-term starvation, which is also an insulin deficient state. As in starvation, a role play activated amination of the BCKAs in the liver and impaired uptake of the BCAA by muscles. The BCKA levels increase in blood plasma and muscles of rats with chemically-induced diabetes, but decline in the liver [71]. The role of the liver as a source of BCAAs is supported by observations of reduced activity of hepatic BCKD in rats with severe ketotic diabetes [72].

However, unlike brief starvation, the changes in diabetes are associated with marked increase in proteolysis and BCKD activity in muscles, resulting in severe cachexia [73]. While muscle nitrogen repletion occurs and BCAA levels are normalized after feeding of previously starving subjects, the BCAAs accumulate and diminished nitrogen repletion remains after feeding in subjects with type 1 diabetes [74].

Obesity and type 2 diabetes

Plasma concentrations of BCAAs are frequently elevated in obesity and type 2 diabetes [75–77]. The mechanism responsible for the increased BCAAs in these insulin-resistant states is not completely clear. A major cause might be reduced activity of the BCKA dehydrogenase, which was reported in the liver and adipose tissue in genetically obese ob/ob mice, Zucker rats and spontaneous type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats [76, 77].

The studies have shown that the BCAA levels in obesity correlate with insulin resistance and are a sensitive predictor of diabetes in the future [78, 79]. Recent studies have suggested that high levels of the BCAA interfere with oxidation of fatty acids in muscles, leading to accumulation of various acylcarnitines and insulin resistance [24].

Maple syrup urine disease (MSUD)

MSUD is recessive disorder caused by a severe deficiency of BCKD activity. All three BCAAs, as well as the corresponding BCKAs, are elevated in blood, tissues, and urine. High BCAA and BCKA levels are related to excitotoxicity, energy deficit, and oxidative stress in the brain, resulting in severe neurological symptoms.

Exercise

Physical exercise is associated with enhanced BCAA oxidation and GLN release from muscles [84, 85]. Evidence suggests that BCKD is activated by dephosphorylation mediated by falling ATP levels within the muscles during exercise. Training appears to increase mRNA expression of this enzyme [86]. The plasma BCAA levels during or after exercise have been reported to be unchanged [87], to decrease [88], or to increase [89]. The cause of inconsistent response can be explained by different work load and duration of exercise.

Hypermetabolic states accompanied by systemic inflammatory response syndrome

There are several hypermetabolic states (e.g. sepsis, burn injury, trauma, and cancer) in which alterations in BCAA levels are not consistent, with increased, unchanged, and decreased levels being reported. Present in all of these conditions is systemic inflammatory response syndrome (SIRS) characterized by a wide range of neuro-humoral abnormalities, including enhanced production of cytokines, sympathetic nervous system activation, and cortisol production. These events cause several alterations in metabolism, including insulin resistance and enhanced myofibrillar protein degradation, resulting in severe depletion of lean body mass. If the hypermetabolic state persists, multisystem organ failure and eventually death may occur (Fig. 6).

In this situation, BCAAs act as a significant energy substrate for muscles [4, 5, 94]. Increased BCAA oxidation is coupled with increased synthesis of GLN, which is released from muscles and utilized, preferably by the immune system. Utilization of GLN often exceeds its synthesis, leading to a lack of GLN in blood and tissues [95, 96]. Decreased GLN availability can become rate-limiting for key functions of immune cells, such as phagocytosis and antibody production. Decreased GLN levels have been shown to act as a driving force for BCAA utilization in muscles [97]. Studies have also indicated that inflammatory signals decrease BCAA absorption from the gut and inhibit BCAA transport from the blood to muscles, while promoting transport into the liver [98, 99]. The BCAA synthesis from the BCKA in visceral tissues is probably activated. A marked increase in leucine release was observed by the isolated liver of endotoxin-treated animals after the addition of KIC into perfusion medium [7].

The cause of inconsistent alterations in BCAA levels although their oxidation is remarkably activated are different influences of individual metabolic changes occurring in the SIRS. Increased protein breakdown or decreased protein synthesis in muscles and insulin resistance may enhance the BCAA levels. Activation of BCAA catabolism associated with enhanced ALA and GLN production in muscles and protein synthesis in visceral tissues decrease the BCAA levels. Therefore, alterations in BCAA levels are inconsistent.