SUPPLEMENT

Leucine Metabolism in T Cell Activation: mTOR

Signaling and Beyond1–3
Elitsa A Ananieva,4* Jonathan D Powell,5 and Susan M Hutson6
4
Department of Biochemistry and Nutrition, Des Moines University, Des Moines, IA; 5Department of Oncology, Johns Hopkins University School of
Medicine, Baltimore, MD; and 6Department of Human Nutrition, Foods, and Exercise, Virginia Tech, Blacksburg, VA

ABSTRACT

In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding
the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response
depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has
been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin
(mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in
relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by
leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells. Adv Nutr 2016;7(Suppl):798S–805S.

Keywords: immune function, leucine, BCATc, T cells, mTORC1, KIC

Introduction reprogramming, T cells enter a highly glycolytic state (4),

T cells play a central role in the cell-mediated immunity
against pathogens, autoimmune disorders, and cancer. For
the initiation of an immune response, T cells clonally ex-
pand, acquire effector functions, and reach a state of full
activation known as signal 1 plus signal 2 activation (1, 2).
These processes are high energy demanding and are accom-
panied by distinct changes in nutrient uptake and cellular me-
tabolism (metabolic reprogramming) (3). During metabolic
1
Published in a supplement to Advances in Nutrition. Some of the reviews in this supplement
were presented at the symposium "Translational and Transformational Concepts in Amino
Acid Sensing” held 29 March 2015 at the ASN Scientific Sessions and Annual Meeting at
Experimental Biology 2015 in Boston, MA. The symposium was sponsored by the American
Society for Nutrition (ASN), the ASN Energy and Macronutrient Metabolism Research
Interest Section (RIS), and the Nutrient-Gene Interaction RIS and was supported by
Ajinimoto, Co., Inc. Other articles in this supplement are selected reviews by grant-funded
researchers from the Ajinimoto Acid Research Program (3ARP). The Supplement
Coordinators for this supplement were Susan M Hutson and Tracy G Anthony. Supplement
Coordinator disclosures: Susan M Hutson received travel and registration expenses for the
ASN Scientific Sessions and Annual Meeting at Experimental Biology 2015. Tracy G Anthony
received travel and registration expenses for the ASN Scientific Sessions and Annual
Meeting at Experimental Biology 2015. Publication costs for this supplement were defrayed
in part by the payment of page charges. This publication must therefore be hereby marked
“advertisement” in accordance with 18 USC section 1734 solely to indicate this fact. The
opinions expressed in this publication are those of the author(s) and are not attributable to
the sponsors or the publisher, Editor, or Editorial Board of Advances in Nutrition.
2
Supported by NIH grants DK34738 (SMH) and R01CA098109 (JDP).
3
Author disclosures: EA Ananieva, JD Powell, and SM Hutson, no conflicts of interest.
*To whom correspondence should be addressed. E-mail: elitsa.ananieva-stoyanova@dmu.
edu.
show a marked increase in protein synthesis (4), and have
an enhanced uptake of amino acids (5, 6). Not surprisingly,
a deficiency in either dietary protein or amino acids has long
been known to impair immune function and to increase hu-
man susceptibility to infections (7). Current advances in cel-
lular metabolism have shown that amino acids are not simply
building blocks in the polypeptide chain of proteins but are
also important regulators of cellular processes including
metabolism, protein translation, and cell growth and prolif-
eration (8–11). The expansion of actively proliferating
T cells is dependent on arginine; arginine is an important
precursor of polyamines (via ornithine), creatine, agmatine,
and protein synthesis (12, 13). Myeloid suppressor cells are
able to suppress activated T cells by manipulating the metabo-
lism of arginine through enzymes such as NO synthetase and
arginase (13). Similarly, T cells require tryptophan during expan-
sion, and the lack of tryptophan blocks their proliferation. En-
zymes, such as indoleamine 2,3-dioxygenase (IDO)7, inhibit
T cell proliferation by depleting tryptophan and play roles
in autoimmunity and anti-inflammatory responses (14, 15).
Leucine is an essential amino acid that also plays a role in
muscle atrophy, obesity, and metabolic disorders; liver disease;
and even cancer (16–20). Evidence suggests that leucine is
important for the adaptive immune response in which leu-
cine plays a role in T cell activation. This is associated with the

798S ã2016 American Society for Nutrition. Adv Nutr 2016;7(Suppl):798S–805S; doi:10.3945/an.115.011221.
long-known role of leucine as an activator of mammalian tar- induced obesity (31). These changes were associated with a
get of rapamycin complex 1 (mTORC). More recently, research leucine-induced increase in resting energy expenditure
on the mTORC in association with the immune response iden- (31). Consistent with this study, mice deficient in the mito-
tified the mTOR pathway as a critical regulator of immune chondrial branched-chain aminotransferase (BCATm), which
function (21–24). This review provides an overview of leucine catalyzes the first step in leucine degradation (see below), re-
in health and disease and then summarizes the most current mained lean after being fed a high-fat diet (32). The lean phe-
understanding of the effects that leucine and leucine metab- notype of global BCATm knockout (BCATm2/2) mice was
olism have on T cell activation. attributed to increased protein turnover and increased energy
expenditure. These mice showed elevated plasma leucine con-
centrations that correlated with improved insulin sensitivity
Current Status of Knowledge and glucose tolerance with high-fat diet feeding, indicating
Leucine is the most common proteinogenic amino acid that disruption in leucine metabolism may be a potential ther-
with major metabolic roles. Leucine, together with the other apeutic target for obesity (32). Thus, it is evident that leucine is
BCAAs, isoleucine and valine, comprise ;40% of the free es-
an important regulator of a variety of cellular functions with
sential amino acids in blood plasma (25). The first catabolic
important effects on metabolic health and disease.
step is limited in liver; and leucine is available to skeletal muscle
where it functions as a nutrient signal, is used for protein syn-
Leucine transport and metabolism in health and disease.
thesis, and serves as a metabolic fuel and/or a nitrogen donor
As an essential amino acid, leucine cannot be synthesized
for the synthesis of glutamine and alanine (26, 27). Leucine is
in the body but must be obtained from the diet in humans.
not limited to acting as a substrate for protein synthesis. It is
Soon after consumption of a protein-containing meal, the
actually a well-described regulator of protein turnover that
concentration of leucine increases and is transported
stimulates protein synthesis and inhibits protein degradation
across the cell membrane by a family of L-type amino acid
(11, 28, 29). Many leucine-supplementation studies linked leu-
transporters (LATs). This family consists of 4 Na+-independent
cine to body-weight control, whole-body energy expenditure,
neutral amino acid transporters: LAT1–LAT4. LAT1 and
and/or postexercise recovery of muscle protein (28–30). For
LAT2 are also known as solute carrier (Slc) 7 (Slc7a5 and
example, recovery of rat muscle protein synthesis after a
Slc7a8, respectively), whereas LAT3 and LAT4 are known
strenuous 2-h treadmill run was stimulated by oral leucine
as Slc43 (Slc43a1 and Slc43a2, respectively) (34). LAT1
supplementation in combination with glucose and sucrose.
and LAT2 require a binding partner and deliver a wider
The efficient restoration of muscle glycogen was accom-
range of neutral amino acids compared with LAT3 and
plished by the supplementation of glucose and sucrose,
LAT4; the latter are facilitated diffusers and more specific
whereas leucine had a pronounced stimulatory effect
to the leucine, isoleucine, valine, phenylalanine, and me-
on muscle protein synthesis (19). Apart from leucine’s role
thionine (34, 35). The 4 transporters have different expres-
in high-performance physical activity and postexercise mus-
sion patterns and tissue localization, although they overlap
cle recovery, leucine intake was linked to reduced adiposity
to some extent (36). LAT1 is mainly associated with spleen,
and the prevention of age- or diet-induced obesity (31–33).
activated lymphocytes, and brain, and binding of leucine
Adult male Wistar rats maintained on a food-restricted and
to LAT1 (Slc7a5) has been the most studied (6, 37–39).
low-dose leucine supplementation showed increased body
Leucine transport is dependent on glutamine and proceeds
fat loss and increased liver protein concentrations (33). How-
via a 2-step transport mechanism (38). First, glutamine is
ever, a leucine-rich (4%) diet fed to aged rats decreased body
transported inside the cell via the glutamine transporter
fat but did not have an effect on metabolic indicators of
(Slc1a5) that regulates glutamine intracellular concentra-
chronic diseases, such as total cholesterol, TGs, and glycemia
tions. Next, a complex of Slc7a5 and Slc3a2 (another glu-
(30). Nevertheless, several other studies highlighted the ther-
tamine transporter) uses intracellular glutamine as an
apeutic potential of leucine supplementation for the preven-
efflux substrate to regulate the uptake of extracellular leucine
tion or treatment of diabetes and obesity (31, 32). Leucine
into the cells (38). Once inside the cell, leucine can either
supplementation via drinking water in mice fed a high-fat diet
regulate cellular processes, be incorporated into protein,
led to significantly reduced weight gain and improved hy-
or undergo degradation starting with transfer of its amino
perglycemia and hypercholesterolemia and prevented diet-
group to a-ketoglutarate (transamination). The initia-
tion of leucine degradation occurs primarily in the mito-
7
Abbreviations used: ASS1, argininosuccinate synthase 1; BCATc, cytosolic branched-chain chondria of skeletal muscle and other tissues, because
aminotransferase; BCATm, mitochondrial branched-chain aminotransferase; BCKA,
branched-chain a-keto acid; BCKDC, branched-chain a-keto acid dehydrogenase complex; leucine as well as isoleucine and valine transamination is
BDK, branched-chain a-keto acid dehydrogenase kinase; HMB, limited in the liver (26). Leucine transamination is cata-
b-hydroxy-b-methylbutyrate; IDO, indoleamine 2,3-dioxygenase; KIC, a-ketoisocaproate; lyzed by the BCATm enzyme (40). This is a reversible trans-
LAT, L-type amino acid transporter; MSUD, maple syrup urine disease; mTOR, mammalian
target of rapamycin; mTORC, mammalian target of rapamycin complex; NALA, fer of the a-amino group of leucine to a-ketoglutarate to
N-acetyl–leucine amide; NFAT, nuclear factor of activated T cells; PBMC, peripheral blood form glutamate and a-ketoisocaproate (KIC) (Figure 1).
mononuclear cell; PPM1K, protein phosphatase, Mg2+/Mn2+ dependent 1K; Rheb, Ras Approximately 20% of leucine is converted into KIC, whereas
homolog enriched in brain; S6K1, p70 ribosomal S6 kinase 1; Slc, solute carrier; TCR, T cell
receptor; Th, T helper; Treg, regulatory T cell; TSC1, tuberous sclerosis complex 1; 4E-BP1, the rest of leucine is used for protein synthesis in skeletal
eukaryotic translation initiation factor 4E-binding protein 1. muscle (42). Glutamate, on the other hand, undergoes either

Leucine and immunity 799S

FIGURE 1 Overview of leucine metabolism.
BCATc and BCATm catalyze the first step in
leucine degradation by transferring nitrogen
from leucine to a-ketoglutarate to produce
glutamate and KIC. After leucine is metabolized
to KIC, KIC is either metabolized into isovaleryl-
CoA by BCKDC or into HMB by an enzyme
referred as to KIC dioxygenase (41). Alternatively,
one of the derivatives of isovaleryl-CoA, MC-CoA,
can be converted into HMB. Ultimately, these
metabolites yield acetoacetyl-CoA and
acetoacetate and are used for energy production.
Apart from being metabolized, leucine stimulates
protein synthesis by activating the mTORC1
signaling pathway. BCATc, cytosolic branched-
chain aminotransferase; BCATm, mitochondrial
branched-chain aminotransferase; BCKDC,
branched-chain a-keto acid dehydrogenase
complex; HMB, b-hydroxy-b-methylbutyrate; KIC,
a-ketoisocaproate; MC-CoA, b-methyl-crotonyl-CoA;
mTORC1, complex 1 of the mammalian target of
rapamycin; S6K, p70 ribosomal S6 kinase;
4E-BP1, eukaryotic translation initiation factor
4E–binding protein 1.

amidation to glutamine or transamination to a-ketoglutarate
to generate alanine in multiple tissues (42, 43).
BCATm is expressed in most human tissues except for
liver hepatocytes (26). Likewise, BCATm is constitutively ex-
pressed in T cells (44). Another enzyme that transaminates
leucine is the cytosolic branched-chain aminotransferase
(BCATc). In contrast to BCATm, BCATc is expressed in the
nervous system but has limited expression in other adult hu-
man tissues (26). However, many cancer types as well as ac-
tivated T cells express BCATc, and BCATc is implicated as
an important prognostic marker for cancer and a potential
candidate for an immunosuppressive enzyme (44–47).
Once BCATm converts leucine to KIC in muscle, a sub-
stantial amount of KIC is released into the bloodstream and
further metabolized in the liver by the branched-chain
a-keto acid dehydrogenase complex (BCKDC), a large mul-
tienzyme complex that contains multiple copies of 3 en-
zymes: a branched-chain a-keto acid decarboxylase (E1),
a dihydrolipoyl transacylase (E2), and a dihydrolipoyl de-
hydrogenase (E3) (26). BCKDC activity is regulated by
phosphorylation/dephosphorylation. It is inhibited by
the branched-chain a-keto acid dehydrogenase kinase
(BDK), which phosphorylates the E1a subunit of the E1 en-
zyme. This process is reversed by protein phosphatase
(PPM1K), which activates BCKDC (48, 49). Leucine trans-
amination by BCATm and oxidative decarboxylation by
BCKDC regulate the supply of leucine for tissue protein
synthesis and other leucine functions. They are also impor-
tant for the prevention of buildup of toxic metabolites or
excessive leucine concentrations. A congenital deficiency
in BCKDC leading to maple syrup urine disease (MSUD) is
associated with elevated plasma leucine concentrations and
the presence of branched-chain a-keto acids (BCKAs) in
the urine (50). One mechanism that explains leucine toxic-
ity in MSUD is leucine interference with neurotransmitter
synthesis. Leucine competes for amino acid transporters
with other amino acids such as tyrosine and phenylalanine,
which are precursors of neurotransmitters (51). However,
toxic leucine concentrations may also contribute to disrup-
tion in the energy metabolism of the brain where leucine
can inhibit pyruvate dehydrogenase and a-ketoglutarate de-
hydrogenase (52, 53). Apart from leucine, the leucine me-
tabolite KIC also accumulates in patients with MSUD and
can affect the brain bioenergetic homeostasis (54). The
mechanism includes uncoupling of oxidative phosphoryla-
tion and inhibition of a-ketoglutarate dehydrogenase activ-
ity by KIC (54). In addition, in cancer patients with
cachexia, leucine supplementation enhanced tumor pro-
gression, although it was intended to protect against cancer-
induced cachexia. This is evident from a recent study
that showed that leucine supplementation enhanced tumor
growth in both lean and overweight mice with pancreatic
cancer (18). Thus, although leucine has important functions
in stimulating protein synthesis, excess leucine and KIC con-
centrations may have a negative impact on biological processes.
Another key metabolite of leucine metabolism is b-hydroxy-
b-methylbutyrate (HMB) (Figure 1). Approximately 5%
of leucine is irreversibly converted to HMB, and HMB has
been used as a dietary substitute of leucine with no adverse
effects (42). HMB can stimulate protein synthesis (55) and
attenuate protein degradation (56) in a much smaller dos-
age than leucine and, as such, HMB has the potential to
substitute for leucine as a nutrient signal when leucine sup-
plementation is not practical or desirable (42).

800S Supplement
Leucine and leucine metabolism in immune impairment.
A number of studies from the 1970s to the present have
shown that inadequate uptake of leucine or the other BCAAs
leads to immune impairment (57–60). Jose and Good (57)
showed that dietary restriction of leucine caused a signifi-
cant decrease in the lysis of tumor cells by lymphocytes.
Likewise, decreased concentrations of BCAAs, commonly
seen in patients with advanced liver cirrhosis, were associ-
ated with impairment of the function and maturation of
dendritic cells (58). Oral administration of BCAAs had stim-
ulatory effects on peripheral blood mononuclear cells
(PBMCs) in these patients and led to increased IFN-g pro-
duction (58). Patients with advanced chronic hepatitis C
suffered from malnutrition, and plasma BCAAs were de-
creased to similar concentrations seen in patients with liver
cirrhosis (58, 59). Malnutrition impaired IFN-g signaling in
these patients; however, an increase in the plasma concen-
trations of BCAAs upregulated IFN-g signaling and was pro-
posed as a therapeutic approach for chronic hepatitis C (59).
The mechanism of BCAA function in chronic hepatitis C
was further explored in a human hemochromatotic cell
line (Huh-7.5) grown in low–amino acid media and infected
with hepatitis C virus followed by supplementation with
BCAAs. This mechanism involved the activation of the
mTORC1 signaling pathway, restoration of IFN-g signaling,
and repression of the replication of hepatitis C virus by
BCAAs (59).
Immunomodulatory effects of leucine metabolites
(HMB, KIC) were studied in human PBMCs and in sheep
(61–63). Treatment with HMB reduced TNF-a concentra-
tions in the PBMC culture medium, modified T helper
(Th) 1/Th2 cytokine production toward a Th2 profile, and
impaired lymphocyte proliferation and progression through
the cell cycle (61). Similarly, when KIC was tested in acti-
vated PMBCs, it was found that KIC suppressed lymphocyte
DNA synthesis (62). On the contrary, KIC was shown to
stimulate lymphocyte blastogenesis and antibody responses
in sheep (63). Moreover, feeding lambs with KIC prevented
adrenocorticotropin-induced suppression of lymphocyte
function, and this effect was not achieved by feeding the
lambs with leucine (63). Considering that KIC transami-
nates with glutamate to form leucine (64), the effect of
KIC could also reflect changes in intracellular leucine or glu-
tamate and its metabolites or a direct action of high concen-
trations of intracellular KIC. Elucidating the underlying
mechanism or mechanisms may reveal therapeutic uses
for leucine metabolites.
Molecular mechanisms of leucine function. There is evi-
dence that shows a role for leucine in regulating the
mTOR signaling pathway (16, 17, 65–68). As elegantly de-
scribed by Laplante and Sabatini (69, 70), mTOR signaling
integrates extracellular and intracellular signals to regulate
protein translation, and cell metabolism, growth, prolifera-
tion, and survival. Thus, mTOR is activated during cellular
processes that use energy and nutrients, such as tumor for-
mation, angiogenesis, insulin resistance, adipogenesis, and
T cell activation (22, 70–72). The mTOR signaling path-
way contains 2 multiprotein complexes, mTORC1 and
mTORC2. The 2 complexes have different sensitivity to
the drug rapamycin, with mTORC1 being the primary target
of rapamycin. The 2 complexes also differ in their upstream
regulators, downstream outputs, and protein composition,
all of which are described in detail by Laplante and Sabatini
(69). Here, attention is given to one of the upstream proteins
of mTORC1 called Rag GTPase, because leucine is known to
activate mTORC1 in a Rag GTPase–dependent manner (73,
74). Mammals have 4 Rag GTPases (A–D), which can form
heterodimers. Leucine promotes the loading of Rag A and
Rag B with GTP, thus enabling this heterodimer to interact
with mTORC1 (via Raptor) leading to mTORC1 activation
(73). The exact mechanism of the regulatory role of leucine
is dependent on the enzyme leucyl–transfer RNA synthetase
that catalyzes the ligation of leucine to its transfer RNA. This
enzyme senses leucine cellular concentrations and activates
the Rag complex (74). Rag GTPase activates mTORC1 and
triggers the translocation of mTORC1 to the lysosomal sur-
face. There, mTORC1 interacts with another protein, Ras
homolog enriched in brain (Rheb), a small GTPase that ac-
tivates mTORC1 (75). The interaction between mTORC1,
Rag GTPases, and Rheb on the lysosomal surface is possible
only if amino acids such as leucine are available, signifying
the important role of the lysosome in amino acid sensing
by the mTORC1 signaling pathway (70, 75). The stimula-
tory effects of leucine on protein synthesis during exercise,
protein-energy malnutrition, and adipogenesis are associ-
ated with the leucine-dependent activation of mTORC1 as
well as activation of downstream targets of mTORC1 such
as the p70 ribosomal S6 kinase 1 (S6K1) and the eukaryotic
translation initiation factor 4E–binding protein 1 (4E-BP1)
(Figure 1) (16, 76, 77). On the other hand, withdrawal of
leucine was shown to be as effective in inhibiting mTORC1
signaling as was withdrawal of all amino acids (65). These
findings strongly suggest a central role for leucine in regulat-
ing the mTORC1 signaling pathway in a variety of cellular
processes.
Leucine is indispensable for mTORC1 regulation of
T cell activation. The mTOR signaling pathway is a vital
link between the development of immune response, the sur-
rounding environment, and cellular metabolism (22). In T
cells, a primary role of mTOR signaling pathway is to sense
and integrate environmental cues that dictate the fate of na-
ive T cells upon T cell receptor engagement and is essential
for Th1 and Th17 differentiation (78). Thus, it is not sur-
prising that knocking out mTORC1 in T cells affects their
lineage commitment (21, 22, 24). Delgoffe et al. (21) showed
that the absence of mTORC1 impaired the ability of T cells
to differentiate into Th1 or Th17 cells. Similarly, T cells lack-
ing the mTORC1 activator Rheb failed to differentiate to
Th1 and Th17 cells (79). On the other hand, deletion of tu-
berous sclerosis complex 1 (TSC1), an upstream inhibitor of
mTORC1, caused multiorgan inflammation in mice not ex-
pressing TSC1 as a consequence of hyperactivation of T cells
Leucine and immunity 801S
FIGURE 2 Model of Slc7a5 and
BCATc regulation of mTORC1 activity
in T cells. (A) Slc7a5 expression is
induced by TCR in T cells leading to
increased leucine uptake. Leucine
activates mTORC1, which stimulates
protein translation and glycolysis
that ultimately results in T cell
activation. BCATc is also induced by
TCR and can initiate cytosolic
transamination of leucine, providing
a negative feedback regulation of
mTORC1 activity. The BCKA product
of leucine transamination (KIC) is
transported outside the cells. (B)
When BCATc is lost, the supply of
leucine to mTORC1 is increased, which could result in T cell hyperactivation. BCATc, cytosolic branched-chain aminotransferase; BCKA,
branched-chain a-ketoacid; KIC, a-ketoisocaproate; mTORC1, complex 1 of the mammalian target of rapamycin; TCR, T cell receptor;
Slc7a5, solute carrier family 7a5 [L-type amino acid transporter (LAT1)]; a-KG, a-ketoglutarate.
by greater activity of mTORC1 and elevated Th1 and Th17 re-
sponses (80). Interestingly, a deficiency in the amino acid
transporters Slc7a5 (LAT1) and Slc1a5 (ASCT2) in mice also
impaired the differentiation of Th1 and Th17 cells in an
mTORC1-dependent manner (5, 39). As discussed above,
Slc1a5 is a glutamine transporter that controls glutamine up-
take and glutamine intracellular concentrations (38). Although
glutamine has been studied extensively in T cells and immunity
(81–86), the role of leucine in T cell activation is only now
emerging. Leucine availability was shown to be essential for
T cell activation and proliferation (87, 88). In Jurkat T cells
and activated primary mouse T cells, the leucine structural an-
tagonist N-acetyl–leucine amide (NALA) exerted similar effects
on cell cycle progression, cell proliferation, cytokine produc-
tion, and downstream targets of mTORC1 such as rapamycin,
suggesting the restriction of leucine availability (87, 88). Rapa-
mycin inhibits mTORC1 and renders T cells hyporesponsive
(anergic) even when they are given full signal 1 and signal 2 ac-
tivation (87). The stimulation of T cells in the presence of ra-
pamycin makes them tolerant, such that they fail to produce
substantial IL-2 or IFN-g upon rechallenge (even in the ab-
sence of rapamycin) (89). By promoting tolerance, rapamycin
as well as other inhibitors of mTOR have become attractive
agents for preventing transplant rejection (78, 90, 91). Other
means to inhibit mTOR signaling in T cells and reduce graft
rejection is by limiting essential amino acids, including leucine.
Skin grafts in T cell–deficient mice were shown to express tran-
scripts of 4 amino acid–consuming enzymes (BCATc being
one of them) (92). Transplanted tissues are enriched in regula-
tory T cells (Tregs), which are known to maintain peripheral
tolerance to both self- and nonself-antigens (78, 93). Cobbold
et al. (92) postulated that tolerant Tregs could establish “infec-
tious tolerance” by inducing amino acid–consuming enzymes.
This resulted in localized depletion of essential amino acids
including leucine depletion and promoted the induction of
anergy and more Tregs (92). Our preliminary analysis of
CD4+CD25+ Tregs infiltrating prostate cancer revealed that
BCATc is markedly upregulated in these cells along with
802S Supplement
Foxp3. These data support a potential role of BCATc in tu-
mor-induced Tregs (J Powell, unpublished data, 2010).
During activation, the T cell receptor (TCR) engagement,
coupled with CD28 signaling, upregulates the mTOR signal-
ing pathway, which, in turn, stimulates glycolysis for energy
and metabolites necessary for the increased biosynthetic de-
mands of the proliferating T cells (94). This metabolic reprog-
ramming increases the nutrient demands of T cells and leads
to an increased expression of glucose and amino acid trans-
porters (39). Sinclair et al. (39) found that the leucine trans-
porter Slc7a5 was induced by the TCR signaling in a nuclear
factor of activated T cells (NFAT)–dependent manner. Cyclo-
sporine A, an immunosuppressive drug that inhibits this
pathway, suppressed the expression of Slc7a5 in TCR-acti-
vated T cells. Slc7a5 mediated the intracellular transport of
leucine, which was essential for the activity of mTORC1 dur-
ing T cell activation. The role of Slc7a5 in intracellular leucine
uptake during T cell activation was further confirmed in hu-
man T cells (6). Thus, leucine transport was established as an
important factor controlling mTORC1 activity in T cells.
The significance of Slc7a5 involvement in T cell activa-
tion was further shown in Slc7a5 null T cells, which failed
to properly differentiate into CD4+ and CD8+ T cells (39).
However, the severe phenotype of Slc7a5 null T cells was
not solely due to the loss of mTORC1 activity caused by
failed leucine uptake but also reflected the inability of these
cells to express c-myc (39). The transcriptional factor c-MYC
was identified as a critical regulator of T cell activation–induced
metabolic reprogramming associated with global changes in
genes important for glucose catabolism, glutaminolysis, or-
nithine and polyamine biosynthesis, and glucose and amino
acid transport (GLUT1 and SLC7A5 among them) (82). The
c-MYC–mediated induction of glycolysis and glutaminolysis
in activated T cells is reminiscent of the same processes in
cancer cells (95). Although a direct connection between
c-MYC, leucine metabolism, and T cells has not been found,
BCATc has been described as one of the c-MYC target genes
in cancer cells (47, 95, 96).
 To better understand the mechanism of c-MYC leucine
regulatory role during T cell activation, our group ex-
plored the impact of leucine metabolic enzymes in this
process. We found that BCATc, which is not normally ex-
pressed in naive resting T cells, was induced by the TCR in
a manner similar to Slc7a5 (39, 44). TCR alone was suffi-
cient to trigger BCATc expression, whereas the mitochon-
drial enzyme, BCATm, was expressed constitutively.
Furthermore, activated T cells from global BCATc 2/2
mice showed increased phosphorylation of mTORC1
downstream targets ribosomal protein S6 and 4E-BP1.
These cells had higher intracellular leucine concentrations
and also showed higher rates of glycolysis, glycolytic ca-
pacity, and glycolytic reserve when compared with acti-
vated wild-type cells (44). Our results, along with the
role of Slc7a5 in T cell activation, are consistent with a
model in which TCR triggers the expression of Slc7a5
and BCATc to regulate the uptake and the cytosolic con-
centrations of leucine, respectively (Figure 2). In this
model, BCATc is a part of a negative feedback loop con-
trolling the input of leucine toward the mTORC1 signaling
pathway (Figure 2A). Excess leucine is transaminated to
KIC, which exits the cells (44). Loss of BCATc expression
eliminates cytosolic leucine catabolism and, as a result, the
availability of leucine to activate mTORC1 is not limited,
potentially resulting in T cell hyperactivation (Figure 2B).
Future studies in animal models of human diseases will
be important to verify this paradigm in vivo. Nevertheless,
these studies show that leucine uptake and leucine metab-
olism in T cells are critical for the regulation of the
mTORC1 signaling pathway during T cell activation, and
manipulating the immune response by targeting leucine
could prove useful in treating infections, autoimmunity,
and/or cancer.
Conclusions
Substantial progress has been made in our understanding
of leucine and its role as a nutrient signal activating the
mTORC1 pathway in processes as diverse as muscle func-
tion, insulin resistance, and the activation of the immune
response. Moreover, new and exciting studies have con-
nected leucine metabolism via BCATc with cancer pro-
gression; thus, BCATc has emerged as a new prognostic
marker for cancer (45, 97). Amino acid metabolic enzymes
in tryptophan and arginine metabolism, such as IDO, ar-
gininosuccinate synthase 1 (ASS1), and arginase, have
been well established in cancer and cancer immunother-
apy and are subjects of preclinical and clinical trials (98,
99). Further studies targeting BCATc and leucine metabo-
lism in cancer and immune disorders will expand our
knowledge and opportunities for development of novel
therapeutic approaches.
Acknowledgments
We thank Adele Addington for critical evaluation of the
manuscript. All authors read and approved the final
manuscript.
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