Leucine Metabolism in T Cell Activation: mTOR Signaling and Beyond^,,

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.

Introduction

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 expand, 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 accompanied by distinct changes in nutrient uptake and cellular metabolism (metabolic reprogramming) (3). During metabolic reprogramming, T cells enter a highly glycolytic state (4), 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 human susceptibility to infections (7). Current advances in cellular 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 proliferation (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 metabolism of arginine through enzymes such as NO synthetase and arginase (13). Similarly, T cells require tryptophan during expansion, and the lack of tryptophan blocks their proliferation. Enzymes, 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 leucine plays a role in T cell activation. This is associated with the long-known role of leucine as an activator of mammalian target of rapamycin complex 1 (mTORC). More recently, research on the mTORC in association with the immune response identified the mTOR pathway as a critical regulator of immune function (21–24). This review provides an overview of leucine in health and disease and then summarizes the most current understanding of the effects that leucine and leucine metabolism have on T cell activation.

Current Status of Knowledge

Leucine is the most common proteinogenic amino acid with major metabolic roles.

Leucine, together with the other BCAAs, isoleucine and valine, comprise ∼40% of the free essential amino acids in blood plasma (25). The first catabolic step is limited in liver; and leucine is available to skeletal muscle where it functions as a nutrient signal, is used for protein synthesis, and serves as a metabolic fuel and/or a nitrogen donor for the synthesis of glutamine and alanine (26, 27). Leucine is not limited to acting as a substrate for protein synthesis. It is actually a well-described regulator of protein turnover that stimulates protein synthesis and inhibits protein degradation (11, 28, 29). Many leucine-supplementation studies linked leucine to body-weight control, whole-body energy expenditure, and/or postexercise recovery of muscle protein (28–30). For example, recovery of rat muscle protein synthesis after a strenuous 2-h treadmill run was stimulated by oral leucine supplementation in combination with glucose and sucrose. The efficient restoration of muscle glycogen was accomplished by the supplementation of glucose and sucrose, whereas leucine had a pronounced stimulatory effect on muscle protein synthesis (19). Apart from leucine’s role in high-performance physical activity and postexercise muscle recovery, leucine intake was linked to reduced adiposity and the prevention of age- or diet-induced obesity (31–33). Adult male Wistar rats maintained on a food-restricted and low-dose leucine supplementation showed increased body fat loss and increased liver protein concentrations (33). However, a leucine-rich (4%) diet fed to aged rats decreased body fat but did not have an effect on metabolic indicators of chronic diseases, such as total cholesterol, TGs, and glycemia (30). Nevertheless, several other studies highlighted the therapeutic potential of leucine supplementation for the prevention or treatment of diabetes and obesity (31, 32). Leucine supplementation via drinking water in mice fed a high-fat diet led to significantly reduced weight gain and improved hyperglycemia and hypercholesterolemia and prevented diet-induced obesity (31). These changes were associated with a leucine-induced increase in resting energy expenditure (31). Consistent with this study, mice deficient in the mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step in leucine degradation (see below), remained lean after being fed a high-fat diet (32). The lean phenotype of global BCATm knockout (BCATm^−/−) mice was attributed to increased protein turnover and increased energy expenditure. These mice showed elevated plasma leucine concentrations that correlated with improved insulin sensitivity and glucose tolerance with high-fat diet feeding, indicating that disruption in leucine metabolism may be a potential therapeutic target for obesity (32). Thus, it is evident that leucine is an important regulator of a variety of cellular functions with important effects on metabolic health and disease.

Leucine transport and metabolism in health and disease.

As an essential amino acid, leucine cannot be synthesized in the body but must be obtained from the diet in humans. Soon after consumption of a protein-containing meal, the concentration of leucine increases and is transported across the cell membrane by a family of l-type amino acid transporters (LATs). This family consists of 4 Na⁺-independent neutral amino acid transporters: LAT1–LAT4. LAT1 and LAT2 are also known as solute carrier (Slc) 7 (Slc7a5 and Slc7a8, respectively), whereas LAT3 and LAT4 are known as Slc43 (Slc43a1 and Slc43a2, respectively) (34). LAT1 and LAT2 require a binding partner and deliver a wider range of neutral amino acids compared with LAT3 and LAT4; the latter are facilitated diffusers and more specific to the leucine, isoleucine, valine, phenylalanine, and methionine (34, 35). The 4 transporters have different expression patterns and tissue localization, although they overlap to some extent (36). LAT1 is mainly associated with spleen, activated lymphocytes, and brain, and binding of leucine to LAT1 (Slc7a5) has been the most studied (6, 37–39). Leucine transport is dependent on glutamine and proceeds via a 2-step transport mechanism (38). First, glutamine is transported inside the cell via the glutamine transporter (Slc1a5) that regulates glutamine intracellular concentrations. Next, a complex of Slc7a5 and Slc3a2 (another glutamine transporter) uses intracellular glutamine as an efflux substrate to regulate the uptake of extracellular leucine into the cells (38). Once inside the cell, leucine can either regulate cellular processes, be incorporated into protein, or undergo degradation starting with transfer of its amino group to α-ketoglutarate (transamination). The initiation of leucine degradation occurs primarily in the mitochondria of skeletal muscle and other tissues, because leucine as well as isoleucine and valine transamination is limited in the liver (26). Leucine transamination is catalyzed by the BCATm enzyme (40). This is a reversible transfer of the α-amino group of leucine to α-ketoglutarate to form glutamate and α-ketoisocaproate (KIC) (Figure 1). Approximately 20% of leucine is converted into KIC, whereas the rest of leucine is used for protein synthesis in skeletal muscle (42). Glutamate, on the other hand, undergoes either amidation to glutamine or transamination to α-ketoglutarate to generate alanine in multiple tissues (42, 43).

FIGURE 1.

Overview of leucine metabolism. BCATc and BCATm catalyze the first step in leucine degradation by transferring nitrogen from leucine to α-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 α-keto acid dehydrogenase complex; HMB, β-hydroxy-β-methylbutyrate; KIC, α-ketoisocaproate; MC-CoA, β-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.

BCATm is expressed in most human tissues except for liver hepatocytes (26). Likewise, BCATm is constitutively expressed 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 human tissues (26). However, many cancer types as well as activated 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 substantial amount of KIC is released into the bloodstream and further metabolized in the liver by the branched-chain α-keto acid dehydrogenase complex (BCKDC), a large multienzyme complex that contains multiple copies of 3 enzymes: a branched-chain α-keto acid decarboxylase (E1), a dihydrolipoyl transacylase (E2), and a dihydrolipoyl dehydrogenase (E3) (26). BCKDC activity is regulated by phosphorylation/dephosphorylation. It is inhibited by the branched-chain α-keto acid dehydrogenase kinase (BDK), which phosphorylates the E1α subunit of the E1 enzyme. This process is reversed by protein phosphatase (PPM1K), which activates BCKDC (48, 49). Leucine transamination by BCATm and oxidative decarboxylation by BCKDC regulate the supply of leucine for tissue protein synthesis and other leucine functions. They are also important 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 α-keto acids (BCKAs) in the urine (50). One mechanism that explains leucine toxicity 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 disruption in the energy metabolism of the brain where leucine can inhibit pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (52, 53). Apart from leucine, the leucine metabolite KIC also accumulates in patients with MSUD and can affect the brain bioenergetic homeostasis (54). The mechanism includes uncoupling of oxidative phosphorylation and inhibition of α-ketoglutarate dehydrogenase activity by KIC (54). In addition, in cancer patients with cachexia, leucine supplementation enhanced tumor progression, 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 concentrations may have a negative impact on biological processes. Another key metabolite of leucine metabolism is β-hydroxy-β-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 dosage than leucine and, as such, HMB has the potential to substitute for leucine as a nutrient signal when leucine supplementation is not practical or desirable (42).

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 significant decrease in the lysis of tumor cells by lymphocytes. Likewise, decreased concentrations of BCAAs, commonly seen in patients with advanced liver cirrhosis, were associated with impairment of the function and maturation of dendritic cells (58). Oral administration of BCAAs had stimulatory effects on peripheral blood mononuclear cells (PBMCs) in these patients and led to increased IFN-γ production (58). Patients with advanced chronic hepatitis C suffered from malnutrition, and plasma BCAAs were decreased to similar concentrations seen in patients with liver cirrhosis (58, 59). Malnutrition impaired IFN-γ signaling in these patients; however, an increase in the plasma concentrations of BCAAs upregulated IFN-γ signaling and was proposed 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-γ 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-α concentrations 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 activated 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 transaminates with glutamate to form leucine (64), the effect of KIC could also reflect changes in intracellular leucine or glutamate and its metabolites or a direct action of high concentrations of intracellular KIC. Elucidating the underlying mechanism or mechanisms may reveal therapeutic uses for leucine metabolites.

Molecular mechanisms of leucine function.

There is evidence that shows a role for leucine in regulating the mTOR signaling pathway (16, 17, 65–68). As elegantly described by Laplante and Sabatini (69, 70), mTOR signaling integrates extracellular and intracellular signals to regulate protein translation, and cell metabolism, growth, proliferation, and survival. Thus, mTOR is activated during cellular processes that use energy and nutrients, such as tumor formation, angiogenesis, insulin resistance, adipogenesis, and T cell activation (22, 70–72). The mTOR signaling pathway 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 surface. There, mTORC1 interacts with another protein, Ras homolog enriched in brain (Rheb), a small GTPase that activates 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 stimulatory effects of leucine on protein synthesis during exercise, protein-energy malnutrition, and adipogenesis are associated 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 regulating 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 surrounding 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 naive T cells upon T cell receptor engagement and is essential for Th1 and Th17 differentiation (78). Thus, it is not surprising 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 lacking the mTORC1 activator Rheb failed to differentiate to Th1 and Th17 cells (79). On the other hand, deletion of tuberous sclerosis complex 1 (TSC1), an upstream inhibitor of mTORC1, caused multiorgan inflammation in mice not expressing TSC1 as a consequence of hyperactivation of T cells by greater activity of mTORC1 and elevated Th1 and Th17 responses (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 uptake 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 antagonist N-acetyl–leucine amide (NALA) exerted similar effects on cell cycle progression, cell proliferation, cytokine production, and downstream targets of mTORC1 such as rapamycin, suggesting the restriction of leucine availability (87, 88). Rapamycin inhibits mTORC1 and renders T cells hyporesponsive (anergic) even when they are given full signal 1 and signal 2 activation (87). The stimulation of T cells in the presence of rapamycin makes them tolerant, such that they fail to produce substantial IL-2 or IFN-γ upon rechallenge (even in the absence 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 transcripts of 4 amino acid–consuming enzymes (BCATc being one of them) (92). Transplanted tissues are enriched in regulatory 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 “infectious 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 Foxp3. These data support a potential role of BCATc in tumor-induced Tregs (J Powell, unpublished data, 2010).

During activation, the T cell receptor (TCR) engagement, coupled with CD28 signaling, upregulates the mTOR signaling pathway, which, in turn, stimulates glycolysis for energy and metabolites necessary for the increased biosynthetic demands of the proliferating T cells (94). This metabolic reprogramming increases the nutrient demands of T cells and leads to an increased expression of glucose and amino acid transporters (39). Sinclair et al. (39) found that the leucine transporter Slc7a5 was induced by the TCR signaling in a nuclear factor of activated T cells (NFAT)–dependent manner. Cyclosporine A, an immunosuppressive drug that inhibits this pathway, suppressed the expression of Slc7a5 in TCR-activated T cells. Slc7a5 mediated the intracellular transport of leucine, which was essential for the activity of mTORC1 during T cell activation. The role of Slc7a5 in intracellular leucine uptake during T cell activation was further confirmed in human 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 activation 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, ornithine 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 explored the impact of leucine metabolic enzymes in this process. We found that BCATc, which is not normally expressed in naive resting T cells, was induced by the TCR in a manner similar to Slc7a5 (39, 44). TCR alone was sufficient to trigger BCATc expression, whereas the mitochondrial enzyme, BCATm, was expressed constitutively. Furthermore, activated T cells from global BCATc^−/− 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 capacity, and glycolytic reserve when compared with activated 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 concentrations of leucine, respectively (Figure 2). In this model, BCATc is a part of a negative feedback loop controlling 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 metabolism 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.

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 α-ketoacid; KIC, α-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)]; α-KG, α-ketoglutarate.

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 function, insulin resistance, and the activation of the immune response. Moreover, new and exciting studies have connected leucine metabolism via BCATc with cancer progression; 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, argininosuccinate synthase 1 (ASS1), and arginase, have been well established in cancer and cancer immunotherapy and are subjects of preclinical and clinical trials (98, 99). Further studies targeting BCATc and leucine metabolism in cancer and immune disorders will expand our knowledge and opportunities for development of novel therapeutic approaches.