Advanced Review

Branched-chain amino acid

supplementation: impact on
signaling and relevance to critical
illness
John S. A. Mattick,1 Kubra Kamisoglu,1 Marianthi G. Ierapetritou,1
Ioannis P. Androulakis1,2 and Francois Berthiaume2
The changes that occur in mammalian systems following trauma and sepsis, termed
systemic inflammatory response syndrome, elicit major changes in carbohydrate,
protein, and energy metabolism. When these events persist for too long they
result in a severe depletion of lean body mass, multiple organ dysfunction, and
eventually death. Nutritional supplementation has been investigated to offset
the severe loss of protein, and recent evidence suggests that diets enriched in
branched-chain amino acids (BCAAs) may be especially beneficial. BCAAs are
metabolized in two major steps that are differentially expressed in muscle and
liver. In muscle, BCAAs are reversibly transaminated to the corresponding -keto
acids. For the complete degradation of BCAAs, the -keto acids must travel to the
liver to undergo oxidation. The liver, in contrast to muscle, does not significantly
express the branched-chain aminotransferase. Thus, BCAA degradation is under
the joint control of both liver and muscle. Recent evidence suggests that in
liver, BCAAs may perform signaling functions, more specifically via activation of
mTOR (mammalian target of rapamycin) signaling pathway, influencing a wide
variety of metabolic and synthetic functions, including protein translation, insulin
signaling, and oxidative stress following severe injury and infection. However,
understanding of the system-wide effects of BCAAs that integrate both metabolic
and signaling aspects is currently lacking. Further investigation in this respect will
help rationalize the design and optimization of nutritional supplements containing
BCAAs for critically ill patients. 2013 Wiley Periodicals, Inc.
How to cite this article:

WIREs Syst Biol Med 2013, 5:449460. doi: 10.1002/wsbm.1219

INTRODUCTION

evere insults, such as burns, trauma, and infection,

elicit systemic inflammatory and metabolic responses in humans as well as other mammalian
organisms. These responses have presumably evolved
Correspondence

to: fberthia@rci.rutgers.edu

Department of Chemical and Biochemical Engineering, Rutgers,

The State University of New Jersey, Piscataway, NJ, USA
2

Department of Biomedical Engineering, Rutgers, The State

University of New Jersey, Piscataway, NJ, USA
These authors contributed equally to this article.
The authors have declared no conflicts of interest for this article.

Volume 5, July/August 2013

to isolate and destroy invading pathogens, as well as to

promote rapid wound closure and subsequent repair
of the injured tissues. Although variations depending
on the type of injury and preexisting pathologies
do exist, the general pattern is strikingly similar for
many large-scale injuries. The early-phase postinjury
involves activation of the coagulation and complement
cascades to stop bleeding and provide a first line
of defense against invading microorganisms. Immune
cells, such as neutrophils and T-helper cells, are also
recruited to the injured areas, and the liver releases
acute-phase proteins into the circulation, for the
purpose of killing and clearing bacteria from the host.

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This massive immune response requires redirecting of

significant energy and metabolic resources away from
other tissues, especially skeletal muscle.
Critically ill patients are at risk of complications, often stemming from infections contracted after
admission to the hospital, which can lead to prolongation and potential intensification of their illness.
The ensuing condition is called systemic inflammatory response syndrome (SIRS) and puts further
strain on the hosts resources and, unless controlled,
leads to a significant loss of lean body mass, disseminated microvascular dysfunction, and eventually
multiple organ dysfunction syndrome (MODS) and
death.1 Nutritional supplementation in critically ill
patients is essential for replenishing endogenous nutrients and for alleviating the loss of lean body mass
because of increased proteolysis in peripheral tissues. Recent developments suggest that nutritional
supplementation should not only be viewed as adjunctive supportive care but also as an active therapeutic strategy, hence the term pharmaconutrition,
where basic supplements have the potential to modulate various detrimental effects of severe trauma at
the cellular level, such as oxidative stress, excessive proteolysis, and exacerbated proinflammatory
signaling.2
Most studies on nutritional supplementation
have focused on the amino acid composition because
the hallmarks of SIRS and associated metabolic
derangements are the accelerated proteolysis and
increased utilization of endogenous amino acids for
hepatic production of acute-phase proteins and gluconeogenesis, maintenance of gut mucosal integrity, and
mounting of the immune response.3 SIRS is associated
with a hypermetabolic state characterized by increased
energy expenditure, negative nitrogen balance, and net
efflux of amino acids from muscle.4 Reversing these
trends through the supplementation of amino acids
could prevent excessive loss of lean body mass, and
allow for an eventual recovery through the resolution
of the inflammatory response.
Glutamine and arginine are two amino acids
that received a great deal of attention in the past
decades. Glutamine, the most abundant free amino
acid in the body (60% of intracellular amino acid
pool in skeletal muscle), serves as an important
carrier of nitrogen to viscera and the immune system.
Depletion of glutamine stores within the muscle is
thought to be indicative of severe muscle wasting,5
and by increasing glutamine levels in the circulation,
one may reverse or slow down this depletion,
thus preserving skeletal muscle mass. Glutamine is
also important as a precursor in the synthesis of
glutathione, a major antioxidant molecule in the
450

body, and as a signaling molecule involved in the

regulation of immune functions, including neutrophil
phagocytosis and the expression of heat shock
proteins.6
Arginine has been defined as a conditionally
essential amino acid due to the fact that, during
times of stress, endogenous sources do not meet the
demand for the amino acid, and therefore external
intake is required. Like glutamine, arginine is an
important regulator of the immune system and also
precursor for the synthesis of nitric oxide (NO), which
has immunoregulatory functions including killing of
pathogens and modulating cytokine production, as
in addition to acting as scavenger of superoxide
anion.7 NO is also a well known vasodilator, which
allows increased blood flow to the sites of injury.
Various isoforms of NO synthase are responsible for
NO production. The constitutively expressed form
endothelial NO synthase is activated by various
agonists through the release of intracellular calcium,
which then triggers synthesis of relatively small
amounts of NO whose effects are primarily local
relaxation of the smooth muscle and vasodilatation.
The inducible NO synthase is transcriptionally
regulated by inflammatory mediators, and NO
synthesis is then only limited by the available
concentration of arginine. Disproportionate NO
production caused by supraphysiological arginine
supplementation may be detrimental, leading to
excessive vasodilatation and hemodynamic instability,
which are often seen in SIRS patients.8
Branched-chain amino acids (BCAAs, namely
leucine, isoleucine, and valine) have been shown to
promote muscle protein synthesis and reduce protein
catabolism; therefore, BCAA-enriched nutrition has
been suggested as potential therapeutic for critically
ill patients.9 Clinical studies using BCAA supplementation, summarized in Table 1, are however extremely
diverse with respect to the study design, pathologic
states, patient enrollment criteria, and supplementation regimens. As a result, to date, there is no
consensus whether BCAA-enriched nutritional supplementation is really effective and should be used.
BCAAs also tend to be seen as a group of amino acids
with similar properties, but it has been suggested that
leucine is the amino acid primarily responsible for the
beneficial effects of BCAAs on protein synthesis and
energy regulation, whereas isoleucine and valine may
be dispensable.9
There is evidence that BCAAs may impart their
beneficial effects not only by replenishing nutrients but
also by controlling the cellular redox state. A recent
study showed that BCAA-enriched amino acid diets
increased longevity of mice by inducing mitochondrial

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1 month
preop

6 months

8 weeks

2 years

1 year

6 months

11 days

15 days

4896 h

5 days

Duration

BCAA, branched-chain amino acids; HCV, hepatitis C virus; KIC, -ketoisocaproate.

Living donor liver

transplantation

236

Ken et al.19

2011

HCV-positive
cirrhosis

27

2008

Decompen-sated
cirrhosis

Ohno
et al.18

Fukushima
et al.17

2007

646

Advanced
cirrhosis

Critically ill

Sepsis

Cirrhosis

Muto
et al.16

2005

174

84

69

Severe burn

Severe burn

Severe burn

Condition

Marchesini
et al.15

2003

Garcia-deLorenzo
et al.13

1997

Griffiths
et al.14

King
et al.12

1990

1997

12

Yu et al.11

1988

14

22

No. of
Patients

Manelli
et al.10

Author

1984

Year

Oral supplementation with BCAAs

1 month before surgery or no
supplementation

Oral granular preparations of BCAAs

versus no supplementation

All subjects were received oral granular

preparations consisting of BCAAs
alone

Orally administered BCAAs versus diet

therapy with defined daily food
intake

BCAAs versus lactoalbumin or

maltodextrin

Glutamine-containing parenteral
nutrition versus isonitrogenous
isoenergetic control

Total parenteral nutrition, either 1.5 g

amino acids/kg/day with 23 or 45%
BCAA or 1.1 g/kg/day with 45%
BCAA content

Standard regimen (16% BCAA) versus

BCAA-enriched (31%) or a similar
regimen where 65% of the leucine is
replaced by KIC

Enteral feeding with BCAA-enriched

(44%) versus conventional egg
protein formulation

BCAA-enriched (41%) versus

conventional (22% BCAA) parenteral
nutrition

Administered Supplements

Outcomes

Incidence of bacteremia after

transplantation was lower in BCAA
group

BCAA supplementation reduced production

of oxidative stress and
microinflammation, which lead to a
decreased occurrence of hepatocellular
carcinoma (HCC)

Basal levels observed initially were low

total albumin and high oxidized/reduced
albumin ratio. BCAA supplementation
improved oxidized/reduced albumin ratio

The incidence of events decreased in the

BCAA group. Serum albumin
concentration increased significantly

BCAA significantly reduced combined event

rates compared with lactoalbumin and
nonsignificantly compared with
maltodextrin

Survival at 6 months was significantly

improved in patient group receiving
glutamine. Total ICU and hospital cost
per survivor was reduced by half

Lower mortality rate observed in patient

groups with high BCAA loads. Higher
plasma concentrations of BCAAs and
rapid protein turnover are shown

Leucine enrichment reduced muscle protein

breakdown compared with a standard
feed, whereas KIC enrichment did not

BCAA-enriched feeding failed to

demonstrate significant benefits in terms
of protein synthesis or degradation

BCAA supplementation demonstrated

improvement in protein catabolism

TABLE 1 Clinical Studies Investigating the Effects of Amino Acid-Enriched Nutritional Supplementation on Survival and Recovery

No controls. BCAA supplementation

increases the turnover leading to
reduction in its circulation half-life
and therefore reduced level of
oxidation

Liver function tests of the patients were

stable or improved with BCAA
treatment

Nitrogen balance or serum albumin

levels were not significantly affected

Cross-over design study, no parallel

controls

No beneficial effect upon nitrogen loss

or nitrogen balance was shown

Notes

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biogenesis and reducing oxidative stress through

signaling mechanisms that increase defenses against
oxidative stress.20 Moreover, in patients with liver
disease, BCAA-enriched supplements have been
shown to induce a marked reduction of oxidative
stress accompanied by a positive effect on protein
synthesis and glucose metabolism.17,18
In sum, amino acid supplements can significantly
impact on intracellular signaling networks, redox
state, and protein synthesis rate, all of which can
have far-reaching therapeutic consequences. A better
understanding of the comprehensive metabolic and
signaling effects of amino acid supplementation
in normal and critical states will be important
to rationally design nutritional regimens containing
optimal combinations of amino acids. In this review,
we summarize current knowledge of the cellular
mechanisms whereby BCAAs impact metabolism and
cell signaling under stress.

METABOLISM OF BCAAs
BCAAs go across the cell membrane via a single
transport mechanism, and as a result compete with
each other for transport into the cell.9,21 Transport
into the mitochondria is governed by yet another
mechanism,22 which maintains different BCAA levels in the cytosol, where they putatively function as
signaling molecules, and in the mitochondria, where
they are ultimately degraded to produce energy. These
transport mechanisms are also differentially regulated
among tissues. For example, inflammatory signals
tend to inhibit BCAA transport into muscle, while
promoting the same into liver.23 Studies also indicate
a decrease in BCAA absorption through the gut in
systemic inflammatory states,24 which increases the
importance of intertissue transport of BCAAs in these
pathological states.
Metabolic degradation of all three naturally
occuring BCAAs proceeds first by deamination followed by breakdown of branched carbon structures
into simpler ones that are further oxidized via major
lipid oxidation pathways in mitochondria. BCAA
deamination is catalyzed by mitochondrial branchedchain aminotransferase (BCATm), which removes the
amino group from the BCAA carbon backbone and
transfers it to -ketoglutarate to form glutamate
and the corresponding branched-chain -keto acid
(BCKA). This reaction is reversible and therefore provides a mechanism to redistribute nitrogen among the
various BCAAs with glutamate as the intermediate.25
BCATm is either not expressed or barely expressed in
liver; therefore, the initial step in BCAA metabolism
primarily occurs in other tissues, with skeletal muscle
452

having the major contribution. Furthermore, glutamate produced as a byproduct of BCKA generation
may be further transaminated to glutamine,26 which
can be exported. Thus, muscle-derived products of
BCAA metabolism include BCKAs and indirectly
glutamine, both of which can be taken up by the
liver.
Unlike BCAAs, BCKA transport is not governed
by a single transporter, but is instead mediated by
monocarboxylate transporters (MCTs), which also
control the transport of lactate, pyruvate, and ketone
bodies through the plasma membrane.27 There are
many different MCTs that are active at multiple
tissues, indicative of organ-specific metabolic regulation through transport activity. Notably, muscle has
been shown to coexpress multiple MCTs, including
MCTs 1 through 7 indicating a need in muscle cells
for high-throughput transport mechanisms capable
of moving multiple metabolites, including BCKAs.
Studies have been conducted to assess the Km values for some of these transporters, and although
many remain unknown, it is worth noting that BCKA
transport is significantly higher in MCT4 than in
MCTs 1 or 2.28 Utilizing these transport mechanisms,
newly metabolized BCKAs can travel from muscle
to other peripheral tissues, notably liver, for further
metabolism.
BCKAs undergo oxidative decarboxylation,
which is catalyzed by the multienzyme complex BCKA
dehydrogenase (BCKDH) within the mitochondria,
with the end products being branched-chain acylcoenzyme A esters of the BCKA precursors. An
important fact to note here is that the enzyme activity
ratio of BCAT:BCKDH is high in muscle, which tends
to favor the release of BCKAs into the circulation
instead of their oxidation. In liver, the ratio is reversed
(because BCATm is virtually absent and BCKDH
activity is high), and oxidation of BCKAs is favored.29
As a result, there is a continuous exchange of BCKAs
from skeletal muscle to liver.
Unlike BCATm, BCKDH catalyzes an irreversible reaction and therefore controls the flux
of BCKAs toward complete oxidation. BCKDH is
regulated by a posttranslational mechanism involving BCKDH kinase, which inactivates the BCKHD
enzyme complex by phosphorylation. BCKAs, which
are the substrates of BCKDH, also serve as inhibitors
of BCKDH kinase. Therefore, accumulation of BCKAs
causes dephosphorylation and activation of BCKHD.
The leucine-derived BCKA, -ketoisocaproate (KIC),
is a potent inhibitor of the regulatory kinase.
BCKAs derived from isoleucine and valine, -keto-methylbutyrate and -ketoisovalerate, respectively,
have similar effects, albeit with lower inhibitory

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potency on the kinase.30 Therefore, it is thought that

leucine-mediated regulation of this pathway is dominant. This regulatory scheme conserves BCAAs, putatively for protein synthesis, when their concentration
is low, while favoring BKCA oxidation when BCAA
concentration is high.31
The foregoing has important implications for
livermuscle communication during systemic inflammation, as the major source of BCKAs reaching the
liver are muscle-derived. Furthermore, BCAAs stored
in the liver must either be exported to the muscle
for oxidation or locally used for protein synthesis.
As detailed further below, BCAAs also have important signaling effects in addition to their metabolic
fates, which creates a dual role for these molecules,
namely signaling elements in the liver, and metabolic
substrates in the muscle. The distributed nature of
the BCAA degradation pathways between muscle
and liver may suggest competing functions (in this
case, the signaling properties of BCAAs potentially
compete with their use as energy sources through
BCKA dehydrogenase activity), which can be resolved
through the division of BCAAs and BCKAs that are
achieved through interorgan compartmentalization.
Figure 1 summarizes the pathways involved in BCAA
metabolism in liver and muscle.

SIGNALING FUNCTIONS OF BCAAs

Levels of amino acids, and in particular BCAAs,
can affect relevant metabolic fluxes through mass
action effects (i.e., via a direct effect of concentration
on reaction rate), but in most cases these levels
are monitored through specific sensing mechanisms
that initiate or terminate processes that utilize amino
acids, including protein synthesis.32 Thus, BCAAs,
besides their ability to serve as substrates for protein
synthesis or energy production, also act as signaling
molecules. For example, provision of BCAAs mimics
the effect of a more complete mixture of amino acids
and stimulates the initiation of mRNA translation in
skeletal muscle. Among BCAAs, leucine appears to be
the most potent effector of protein synthesis,33 which
is not surprising given its role as a primary regulator
of BCAA degradation.

Leucine-Based Regulation of Translational

Initiation in Eukaryotes
Leucine promotes translation through a stimulatory
effect on eukaryotic initiation factor (eIF) 4F complex (eIF4F). Assembly of eIF4F complex is regulated
by the repressor protein eIF-binding protein-1 (4EBP1).33 4E-BP1 prevents formation of active eIF4F

FIGURE 1 | Branched-chain amino acid (BCAA) metabolism in liver and muscle. BCAAs absorbed from the gut reach the liver where they are
taken up and play various signaling and protein synthetic roles. The BCAA catabolic pathway takes place within mitochondria, and consists of two
major steps: reversible transamination with -ketoglutarate to form branched-chain -keto acids (BCKAs), followed by irreversible decarboxylation to
form coenzyme A (CoA) compounds that enter the tricarboxylic acid (TCA) cycle. The transamination step catalyzed by branched-chain
aminotransferase (BCAT) mostly occurs in muscle because this is where BCAT expression is highest. On the other hand, BCKA decarboxylation,
catalyzed by branched-chain ketoacid dehydrogenase (BCKDH), as well as later steps leading to complete oxidation occur mainly in liver.27

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complex by sequestering mRNA cap-binding protein eIF4E and rendering it inactive. Leucine induces
hyperphosphorylation of 4E-BP1 leading to its dissociation from eIF4E, allowing eIF4F active complex
formation by association of eIF4E with a large scaffold protein eIF4G.34,35 Leucine administration also
enhances the phosphorylation of ribosomal p70S6
kinase.33
Recent studies suggest that all of these interactions are in fact downstream events controlled by
the mammalian target of rapamycin (mTOR), which
is a highly conserved serine/threonine kinase that
regulates cell growth in response to nutrient status.
mTOR appears to act as a critical signaling element
sensing the intracellular availability of amino acids
and whose activation triggers a downstream cascade
leading to activation of eIFs.36,37 However, the exact
mechanism by which leucine activates mTOR remains
elusive.
Data obtained from manipulating the activity
of another eIF, EIF2, suggest differences in leucinemediated stimulation of protein synthesis between
liver and muscle. GCN2 is a kinase tasked with
the regulation of EIF2 through inhibition, and
furthermore, it has been shown to mediate leucinebased regulation of protein synthesis by being
negatively regulated itself by leucine. Anthony et al.
used GCN2 knockout mice to demonstrate some of
the interplay between leucine and GCN2. In wildtype animals that had functional GCN2 activity,
leucine deprivation caused loss of both liver and
lean body mass in the animals, but no mortality
was observed.38 Because GCN2 mediates leucines
interaction with EIF2, among other initiation factors,
knockout mice were unable to respond appropriately
to leucine restriction with the repression of EIF2.
Only skeletal muscle loss was observed in these
animal, while other tissues were largely spared, despite
significantly increased mortality. Decoupling leucine
sensitivity from EIF2 regulation in these animals
appears to alter the host response to the lack of
BCAAs from a systemic reduction of protein synthesis
toward an unhealthy depletion of peripheral tissue
mass. Furthermore, leucine has been implicated in the
stimulation of protein synthesis through EIF2,39 which
strongly suggests that there is a regulatory element
toward BCAA activity in protein synthesis beyond
their use as substrates. In summary, these findings
suggest that leucine is an important metabolite that
is capable of regulating protein synthesis in an
mTOR-dependent pathway that is likely mediated
through EIF proteins. When leucine is scarce, the
appropriate response appears to involve a reduction
in overall protein synthesis and a distributed minor
454

loss of lean body mass with minimal impact on

the host. The loss of GCN2 function, leading to
an unregulated activation of EIF2 in the knockout
animals in spite of the absence of leucine, causes
an abnormal loss of skeletal muscle protein and
metabolic complications that are strikingly similar
to the observed hypermetabolism after burns and
trauma. The addition of leucine has been shown to
stimulate EIF4E activity in skeletal muscle following
burn injury in rats, albeit there was little evidence of
EIF2 activity,34 and has been implicated in increased
protein synthesis following burn. With regard to burn,
trauma, and sepsis-related stress, it is worth noting
that the GCN2/EIF2 regulatory pathway described
above is similar in function to other EIF families,
which have been shown to be regulated by a
much wider array of cellular functions than leucine
deprivation, including apoptosis-related factors.40
Furthermore, the regulatory structure that governs
the mediation of protein synthesis through amino
acid sensing is not completely elucidated.41 While
evidence suggests that BCAAs, and in particular,
leucine, play an important role in linking metabolic
functions to protein synthesis, one also needs to take
into consideration the fact that these amino acids
may affect protein degradation rates as well. The
same pathways discussed above are in fact involved
in apoptosis and protein catabolism. The net effect
of BCAAs on protein turnover appears to vary
depending on the tissue and the specific protein in
question.
For example, although BCAAs stimulate protein
translation and increase protein synthesis in skeletal
muscle, they do not increase total protein production
in liver.35 Administration of leucine to fasted rats
increases the translation of ribosomal proteins L26,
S4, and S8 by enhancing polysome size, where their
translation occurs. This contrasts with -actin and
albumin, for which there is no change in polysomal
association of their mRNAs.32 These three ribosomal mRNAs have a terminal oligopyrimidine (TOP)
sequence at the 5 end, which is common for proteins
involved in the translational apparatus.35 Association of TOP mRNAs with the polysomes requires
phosphorylation of p70S6K, and rapamycin selectively suppresses their translation through inhibition
of mTOR activity induced by leucine. These results
suggest that BCAAs, particularly leucine, have a regulatory role in liver, which is distinct from skeletal
muscle. This is also reflected by the fact that liver
does not oxidize BCAAs, in contrast to skeletal muscle. In cases of severe injury and/or infection, hepatic
BCAA uptake increases in order to meet the increased
demand of amino acids for protein synthesis imposed

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by the acute-phase response.42 This increased BCAA

uptake may also affect fundamental processes of translation and transcription, which also play a key role in
the acute-phase response, but have not yet been fully
explored.

Branched-Chain Amino Acid Modulation

of Insulin Signaling and Glucose Uptake
The effects of amino acids on glucose homeostasis
that have been reported in the literature are extremely
variable, ranging from increasing glucose sensitization
to inducing insulin resistance. The effects are in
fact highly dependent on several considerations,
including (but not limited to) the constituents of the
regimen administered, the presence of other factors
such as insulin or lipids, the organ or cell type in
which the results are obtained, and the presence of
underlying disease, such as hepatitis, cirrhosis, or
diabetes.
First, a series of in vitro studies in which amino
acid mixtures were supplemented to muscle and liver
cells indicated that amino acids reduce insulin-induced
glucose uptake.37 In neonatal rat thigh skeletal muscle
cells, in vitro, a balanced mixture of amino acids
reduced insulin-stimulated glucose transport and this
reduction was related to increased mTOR activity.
Incubation of the cells with amino acids potentiated
the activation of mTOR/p70S6 kinase pathway by
insulin causing deactivation of phosphatidylinositol
3-kinase (PI3-kinase). This deactivation effect was
associated with Ser/Thr phosphorylation of insulin
receptor substrate 1 (IRS-1) resulting in impaired
recruitment of PI3-kinase to IRS-1 and subsequent
reduction in cellular glucose uptake.43 This study
also confirmed a prior in vitro study in which amino
acids modulated the action of insulin in hepatoma
and muscle cells bidirectionally, acting as positive
signals for protein anabolism while limiting glucose
transport.36 Activation of mTOR/p70S6 pathway was
also associated with increases in glycogen synthesis.
In human muscle cells, amino acids were shown
to stimulate mTOR/p70S6 kinase pathway and
transiently inhibit glycogen synthase kinase-3, thereby
stimulating glycogen synthesis.44
In insulin-free conditions, contrary to previous observations, amino acids, particularly BCAAs,
enhanced glucose uptake and utilization. In isolated rat skeletal muscle cells, leucine specifically increased glucose transport through a PI3kinase- and protein kinase C-dependent but mTORindependent mechanism.45 This was also confirmed
in skeletal muscle of cirrhotic rats where glucose
metabolism was impaired. Leucine, and more potently
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isoleucine, improved glucose tolerance independently

from insulin as a result of increased GLUT4 and
GLUT1 translocation to plasma membrane. Additionally, glycogen synthase activity was also augmented
by leucine but not isoleucine through an mTORdependent mechanism. Taken together, these data
suggested that leucine and isoleucine supplementation in cirrhotic patients may partially substitute
for insulin and therefore alleviate the impaired glucose homeostasis.46 Insulin resistance is frequently
observed in patients with chronic liver disease, such
as cirrhosis and prolonged hepatitis C. In the case of
cirrhosis, insulin sensitivity is impaired independent
of the etiology of the disease or clinical and nutritional state of the patients.47 In the case of hepatitis
C virus, virus-driven core protein is shown to cause
degradation of IRS-1 and IRS-2 as well as suppression of insulin signaling cascade through reduction
of PI3k and Akt phosphorylation.48 If not properly
managed, development of insulin resistance leads to
various complications of hepatic or extrahepatic origin throughout the course of these diseases.49 In both
of these conditions, the effectiveness of BCAA supplementation for improvement of glucose tolerance was
demonstrated clinically.5052 However, the molecular details of how BCAAs can help maintain glucose
homeostasis in such diverse pathologies as well as
whether there are any conditions where BCAAs aggravate the pathology instead of ameliorating remain to
be investigated. Another aspect that has been the subject of many studies was the individual effects of three
BCAAs and which one is most beneficial therapeutically.
Differential effects of each BCAA on wholebody glucose clearance and utilization mechanisms
were reported in rats. Oral administration of BCAAs
showed that isoleucine significantly increases glucose
clearance, whereas valine decreases it and leucine
has no effect. The plasma glucose-lowering effect
of isoleucine was due to insulin-independent glucose uptake in the skeletal muscle. Isoleucine did
not induce glycogen synthesis; however, leucine and
valine stimulated glycogen synthesis.53 The hypoglycemic effect of isoleucine was investigated further,
where isoleucine significantly inhibited glucose production in hepatocytes associated with a decline in
mRNA levels for two key gluconeogenic enzymes,
phosphoenolpyruvate carboxykinase and glucose-6phosphatase (G6Pase). Decreased activity of G6Pase
was also reported. Therefore, it is established that
isoleucine exerts an overall hypoglycemic effect involving a reduction in gluconeogenesis in liver in addition
to an increase in skeletal muscle glucose uptake.54
This effect was also confirmed in db/db mice which

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is an established model of severe type 2 diabetes.55

However, when BCAAs were delivered in a balanced
mixture, the specific hypoglycemic effect of isoleucine
subsided. Glucose levels in plasma following a glucose
tolerance test did not change much in response to
BCAA administration, although a boost was still
observed for the glucose transport system and primary
enzyme (liver-type glucokinase) for glucose utilization
in liver.56 The fact that the BCAA mixture is not
as effective as isoleucine alone for improving glucose
uptake may be due to confounding effects of the other
two BCAAs, especially leucine, on glucose transport
as well as insulin secretion. While supplementation
of isoleucine improved glucose clearance, a similar
effect was observed for the deprivation of leucine. A
diet deficient in leucine was shown to improve wholebody insulin sensitivity as well as increased insulin
signaling in liver under both normal and insulinresistant conditions, in vivo.57 An increase in the
relative amounts of isoleucine and valine accompanies leucine deprivation; therefore, whole-body insulin
sensitivity may be the result of the shifted balance
of these metabolites. As exemplified by these cases,
combinations of BCAAs may have various effects on
insulin signaling and anabolic responses, and generating a comprehensive understanding of the way BCAAs
may crosstalk, which is currently lacking, would
be important to optimize BCAA supplementation
regimens.
Another confounding factor that needs a thorough assessment for the use of BCAA supplementation
regimens is the consumption of dietary lipids along
with the amino acids. First, isoleucine was shown to
exert its hypoglycemic and insulin-sensitizing effects
even under the metabolic burden of a high-fat diet
(HFD).55 On the contrary, leucine also improves glucose metabolism in animals on HFD, but is ineffective
in animals on a normal diet. Zhang et al.58 conducted
a study where they doubled the amount of dietary
leucine by giving leucine-containing drinking water
to mice. Leucine spiked water, in addition to normal chow diet, had no effect. However, for animals
on HFD, leucine improved glucose metabolism by
increasing insulin sensitivity and decreasing glucagon
secretion as well as cholesterol levels. Macotela et al.59
confirmed these findings and further showed that
doubling leucine reduces adipose tissue inflammation and hepatic steatosis induced by HFD, while
improving insulin signaling in muscle, liver, and fat. In
addition to individual effects of leucine and isoleucine,
combination of other amino acids was also investigated. Fortification of all ketogenic amino acids
(KAAs) in HFD (KAAs: leucine, isoleucine, valine,
lysine, and threonine) improved diet-induced hepatic
456

steatosis and glucose tolerance in mice.60 All of

these studies favor the supplementation of BCAAs
to prevent the detrimental effects of HFD on glucose
homeostasis; however, a recent metabolomics study
challenges this view. Principal component analysis
of more than 100 analytes measured in the plasma
from lean versus obese subjects revealed that, rather
than lipid-related markers, BCAAs were more strongly
associated with insulin resistance.61 BCAAs and aromatic amino acids form an independent cluster
including byproducts of BCAA catabolism such as
glutamine, alanine, and C3 and C5 acylcarnites, suggesting that not BCAAs themselves but an altered flux
through BCAA catabolism pathway is responsible for
its association with insulin resistance. Furthermore,
gastric bypass surgery, which is an established treatment for excessive obesity, is shown to improve insulin
sensitivity and glucose homeostasis in parallel with a
dramatic decline in circulating BCAAs and BCAArelated metabolites.62,63
Overall, there is no consensus on under what
conditions BCAAs are useful for the regulation of
glucose homeostasis. As the further molecular details
unfold with future studies, it may be possible to better
predict these responses and consequently formulate
better regimens for the use of BCAA supplementation
in critical illness. A systems approach to investigate
molecular events as a whole, rather than observing
perturbations in a limited number of representative
markers would help for building a solid understanding
of the mechanisms by which BCAAs impact metabolic
signaling in these conditions. Only then, it would be
possible to form standardized regimens for different
scenarios applicable in clinic.

Activation of mTOR and ROS Scavenging

by Branched-Chain Amino Acids
Besides modulating protein translation in the cell, activation of mTOR by BCAAs critically impact oxygen
consumption, choice of substrates for energy production, and defense capacity against reactive oxidants.64
A BCAA supplementation study in mice showed
increased average lifespan, increased mitochondrial
biogenesis, and upregulation of the genes involved in
scavenging of reactive oxygen species (ROS). Furthermore, these changes appeared to be largely mediated
by increased mTOR activity.20 By acting as the indicators of available nutrient supply, BCAAs regulate
the critical cellular decisions, such as selecting the
correct substrate for energy production based on available resources and determining whether initiating new
protein synthesis action is feasible or not. As BCAA
availability in the liver is effectively controlled through

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Branched-chain amino acid supplementation

the skeletal muscle, this mechanism appears to serve

as a way for the liver to take measure of the robustness
of its supply lines during crisis.
Under conditions of stress, preserving mitochondrial function is critical for maintaining cell and tissue
viability. For example, excessive ROS production by
dysfunctional mitochondria has been implicated in
organ failure induced by inflammation and hypoxia
following trauma.65 It has been suggested that BCAAs
behave as evolutionarily conserved modulators of
lifespan through exerting control on mitochondrial
biogenesis, cellular energy metabolism, and ROS scavenging systems.66 Two studies in patients with liver
disease indicated that BCAAs markedly reduce oxidative stress and positively affect protein synthesis.17,18
As we have discussed earlier, BCAAs are implicated
in the mTOR signaling pathway, while impacting on
other anabolic regulators such as insulin, and on the
cellular translational machinery. Studies also suggest
that BCAAs are being utilized as markers of amino
acid availability, and have different effects in liver and
muscle. From this, it is clear that BCAAs have a much
greater role in regulating whole-body metabolism than
their role as protein synthesis substrates would suggest. Their ability to interact with anabolic pathways
in order to stimulate glucose uptake, protein synthesis,
and signaling pathways indicates that they may be a
critical line of communication between peripheral tissues and central metabolism in the liver, and therefore
may be worthwhile as therapeutic agents in addition
to their role as nutritional support agents.

CONCLUSIONS
SIRS and related injury-induced systemic inflammatory responses have not seen much improvement in
terms of clinical treatment options, primarily because
of the complexity and redundancy in the regulatory
pathways and in the interorgan relationships involved.
Thus, traditional approaches that attempt to target a
single pathway or molecule have largely failed. It is
also noteworthy that mediators and signaling aspects
of the inflammatory response have received the bulk
of the attention in SIRS research. On the other hand,
many of the deleterious effects of SIRS are metabolic
in nature: loss of lean body mass, a negative nitrogen
balance, and energetic failure.

Some of the most promising nutritional supplements that are currently being explored are BCAAs.
Despite being essential amino acids (therefore they
cannot be endogenously made on demand), they play
a major role in the communication between muscle
and liver. BCAAs, and their reversible degradation
products, BCKAs, are differentially regulated in the
muscle and liver, with the production of BCKAs from
BCAAs occurring almost exclusively in the muscle,
and the final irreversible catabolism of BCKAs occurring in the liver. The liver is responsible for the final
breakdown, suggesting that energy derived from these
amino acids is intended for the liver, and yet the fact
that hepatic cells have extremely limited capacities for
the breakdown of BCAAs directly indicates that the
role of BCAAs within hepatic cells is not solely as
substrates for protein synthesis. Indeed, studies have
shown that within muscle, where BCAA breakdown
to BCKA is encouraged, the addition of BCAAs will
increase total protein production. In contrast, hepatic
exposure to BCAAs causes a significantly different
response, whereby they selectively promote the synthesis of translational proteins, increasing the livers
capacity for overall protein production. Furthermore,
leucine has been shown to promote mitochondrial biogenesis and reactive oxygen defenses, both of which
enhance the livers capacity for energy production.
As the liver is incapable of directly influencing BCAA
concentration, BCAAs may serve as signals that are
controlled by the muscle. These signals may reflect
the nutritional state of the host, and therefore influence the metabolic output of the liver during periods
of stress. The interplay of BCAA supplementation
between liver and muscle highlights the importance
of a systems approach toward the study of both the
efficacy of nutritional interventions and the structure
of the regulatory architecture they seek to alter during critical illness. Too much BCAA supplementation
may induce anabolic activity in the liver at the expense
of peripheral tissues, whereas too little may create a
metabolic response appropriate for starvation, both
of which could potentially be detrimental to patient
recovery. Investigation of the mechanisms of action
of BCAAs thus represents an opportunity for systems
biology to tackle challenges inherent to the multiorgan
response that governs the mammalian inflammatory
response to severe injury and infection.

ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support from NIH grant GM082974.
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