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Supplement

Role of Leucine in the Regulation of mTOR by Amino Acids: Revelations from Structure–Activity Studies^{1 ,2}

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033

³To whom correspondence and reprint requests should be addressed at The Pennsylvania State University College of Medicine, Department of Cellular and Molecular Physiology, 500 University Drive, Hershey, PA 17033. E-mail: clynch{at}psu.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
In this study an overview is presented of the mTOR signaling pathway and its regulation by amino acids, particularly L-leucine. Our laboratory is studying amino acid regulation of mTOR in adipocytes. Potential roles for mTOR in adipocytes that were previously posited include hypertrophic growth, leptin secretion, protein synthesis and adipose tissue morphogenesis. A current area of interest in the field is how amino acids regulate mTOR and which amino acids are regulatory. Revelations concerning mechanism and recognition are emerging from different laboratories that examined the structural requirements for stimulation and inhibition of the mTOR signaling pathway by leucine and amino acid analogs. In adipocytes and some other cell types, leucine appears to be the main regulatory amino acid. However, this is not uniformly the case. In those cells where mTOR is regulated by several amino acids, there is evidence that the mechanism of mTOR activation may be different from cells where mainly leucine is regulatory. Furthermore, in tissues where leucine regulates mTOR, the possible existence of different tissue-specific leucine recognition sites may be indicated.

KEY WORDS: • leucine • amino acids • mTOR • 4E-BP1 • p70 S6 kinase • protein synthesis • adipose tissue

	INTRODUCTION

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
The topics covered by this review include an overview of mTOR⁴ and its potential roles in adipocytes, the effects of amino acids on mTOR targets and the role of leucine in this regard as well as recent studies on proposed mechanism(s) and structural requirements for leucine activation of the mTOR signaling pathway.

	The mTOR cell signaling pathway: overview and potential roles in adipocytes

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
The mammalian Target of Rapamycin (mTOR) is a large (2549 amino acids) proline-directed serine threonine protein kinase related to phosphatidylinositol kinases. Other PI-kinases include DNA-dependent protein kinase and ATM, which can be mutated in the disease ataxia telangiectasia (Muller et al. 1999 , Taylor 1998 , Vaziri 1997 ). In addition to lipid kinase–like domains relating it to PI kinases, mTOR also contains the "HEAT" domain found in the alpha subunit of PP2A and other proteins. These were previously posited to be involved in protein scaffolding. A possibly related observation is that mTOR is very sticky. For example, mTOR immunoprecipitates typically contain many proteins. This complicates the task of finding the physiologically relevant in vivo interacting partners of mTOR.

In freshly isolated rat adipocytes and many other cells, insulin, growth factors and amino acids activate a rapamycin-sensitive signaling cascade that involves stimulation of mTOR, leading to changes in the phosphorylation of p70^s6k and 4E-BP1 (Fox et al. 1998a , 1998b ), and probably other substrates as well. It is thought that both the intrinsic mTOR kinase activity and an mTOR-associated/regulated protein phosphatase activity contribute to phosphorylation of p70^s6k and 4E-BP1 (Hara et al. 1998 , Peterson et al. 1999 , 2000 ). Although the exact proportion of their individual contributions to substrate phosphorylation state and the mechanism(s) whereby they are regulated have not yet been determined, nonetheless it is known that these phosphorylation events ultimately affect protein synthesis, mainly at the level of initiation of mRNA translation (Dumont and Su 1996 , Jackson and Wickens 1997 , Kleijn et al. 1998 , Lawrence and Abraham 1997 , Lawrence et al. 1997 , Thomas and Hall 1997 ). The regulation of mRNA translation, as it relates to p70^s6k and 4E-BP1 phosphorylation, occurs as follows. The phosphorylation of p70^s6k is associated with increased p70^s6k activity (Weng et al. 1998 ) and phosphorylation of ribosomal protein S6. Activation of p70^s6k was previously linked to preferential translation of messenger RNAs containing a polypyrimidine stretch in their 5' untranslated region (for review see Thomas and Hall 1997 ). On the other hand, the phosphorylation of 4E-BP1 decreases its binding to eIF4E (Fadden et al. 1997 , Fox et al. 1998B ) and, in certain situations, this release of 4E-BP1 from eIF4E allows eIF4E to become part of the multiprotein translation complex eIF4F (Brunn et al. 1997 , Pause et al. 1994 ). Active eIF4E is associated with improved translation of messenger RNAs with significant secondary structure in their 5'UTR.

The above-mentioned effects of amino acids on amino acid stimulation of p70^s6k and 4E-BP1 phosphorylation in freshly isolated rat adipocytes appear to be mediated by mTOR because they are inhibited by rapamycin, but not by a number of other kinase inhibitors.

We became interested in the mTOR cell signaling pathway during our studies of multicellular clustering of adipocytes. When adipocytes are suspended in three-dimensional cultures within a thin layer of Matrigel (a mixture of basement membrane components), they organize initially into primitive organoids and eventually into a gross multicellular structure (Fig. 1 ). Very early in this process p70^s6k is activated. Rapamycin blocks both the activation of p70^s6k as well as the multicellular clustering and increase in protein synthesis that accompanies it. Since multicellular clustering is posited to reflect events associated with adipose tissue morphogenesis (Brown et al. 1997 , Fox et al. 1998a ), these in vitro observations raise the possibility that mTOR may play a role in that process.

View larger version (75K): [in this window] [in a new window]   Figure 1. Adipocytes cultured in Matrigel organize into multicellular clusters. This behavior is stimulated by amino acids and inhibited by rapamycin.

Concentrations of amino acids above 1X are also required to observe effects on translation initiation in cardiac and skeletal muscle (e.g., Anthony et al. 2000 , Tischler et al. 1982 ). Similarly, in freshly isolated hepatocytes, effects of amino acids on protein synthesis and rapamycin-sensitive S6 phosphorylation are also within the range one would expect for a nutritional signal (Blommaart et al. 1995 ). Because amino acids concentrations increase above 1X after eating, it is tempting to infer that these in vitro effects of amino acids may reflect a form of direct nutritional regulation in vivo.

	The role of leucine

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
In freshly isolated adipocytes (Fox et al. 1998b , Lynch et al. 2000 ), as well as in a number of other cell types (e.g., Anthony et al. 2000 , Buse et al. 1979 ; Buse and Reid 1975 , Buse and Weigand 1977 , Kimball et al. 1999 , Patti et al. 1998 , Shigemitsu et al. 1999 , Xu et al. 1998 ), most of the effects of amino acids on mTOR signaling are abolished by lowering the concentration of leucine, or mimicked by adding leucine and, to a lesser extent, the other branched-chained amino acids. At 4X concentrations, leucine was the only amino acid capable of stimulating rapamycin-sensitive 4E-BP1 phosphorylation in adipocytes (Fox et al. 1998b ). At much higher concentrations, other amino acids structurally related to leucine were able to stimulate 4E-BP1 phosphorylation. This is similar to the responses seen in pancreatic beta cells (Xu et al. 1998 ). We proposed that freshly isolated rat adipocytes contain a recognition site that is directly or indirectly coupled to mTOR signaling and stimulated by leucine and, to a lesser extent, by leucine analogs in a way that is predicted by their structural relationship to leucine. In adipocytes the order of potency for stimulation of 4E-BP1 phosphorylation was: leucine > norleucine > Ile threo-L-ß-hydroxyleucine > Met Val.

	Studies on the mechanism(s) of mTOR activation by amino acids

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
There is much interest in how amino acids activate mTOR. As a first step, the possibility was considered that amino acids may be activating some of the cell signaling elements upstream from mTOR in the insulin/growth factor signaling pathway (Patti et al. 1998 ) such as PI 3-kinase and serine/threonine protein kinase, protein kinase B (AKT). It is thought that insulin-mediated increases in the membrane concentration of the product of PI 3-kinase recruits AKT to the plasma membrane (Klippel et al. 1997 ). A change in the conformation state of AKT is thought to uncover phosphorylation sites that are subsequently phosphorylated by one or more constitutively active protein kinases, e.g., PDK1 (Cohen 1999 ). Phosphoryation of AKT on its activation loop at Ser 473 is required for activation (for review see Downward 1998 ). Studies with dominant-negative mutants of AKT and other experiments indicate that the activation of AKT by insulin is required for the rapamycin-sensitive phosphorylation of 4E-BP1 that is stimulated by insulin (Gingras et al. 1998 , Kohn et al. 1998 , Mendez et al. 1996 , Scott et al. 1998 , Takata et al. 1999 , Von Manteuffel et al. 1997 ). Thus it appears that in the insulin signaling pathway, AKT activation is upstream from mTOR activation (Fig. 2 ). Whether AKT can directly phosphorylate mTOR is a matter of controversy, although phosphorylation of mTOR is associated with insulin stimulation of the kinase (Scott et al. 1998 , Nave 1999). However, in contrast to the situation with insulin and growth factors, amino acids do not stimulate PI 3-kinase or AKT activity in any tissue where this has been examined (e.g., Hara et al. 1998 , Iiboshi et al. 1999 , Kimball et al. 1999 , Patti et al. 1998 , Wang et al. 1998). Thus amino acids activate mTOR signaling by an AKT-independent mechanism (Fig. 2) .

View larger version (55K): [in this window] [in a new window]   Figure 2. Amino acids and insulin/growth factors stimulate mTOR by different mechanisms.

A potential mechanism for amino acid regulation of mTOR came from the exciting work of Terada’s group (Iiboshi et al. 1999 ). Using lymphoblastoid cell lines, they showed that manipulations affecting the t-RNA charging state (e.g., incubation with amino acid alcohols or use of a temperature-sensitive mutant of t-RNA synthetase) blocked the effects of amino acids on mTOR. Thus in these cells there seems to be some mechanism coupled to mTOR signaling that recognizes uncharged t-RNA or is capable of sensing the activity of t-RNA synthetases. The former mechanism is attractive and reminiscent of the eIF-2 kinase in yeast, GCN2, that recognizes levels of uncharged t-RNA (Hinnebusch 1997 ).

We proposed that freshly isolated adipocytes may activate mTOR by an entirely different mechanism that does not rely on detection of the t-RNA charging state or t-RNA synthetase activity (Lynch et al. 2000 ). The evidence for this is as follows. First, in contrast to lymphoblastoid cells, essential amino acids other than leucine were not regulatory in adipocytes. This observation is inconsistent with a mechanism that recognizes uncharged t-RNAs. Second, in freshly isolated adipocytes leucine stimulation of mTOR was not inhibited by the amino acid alcohol L-leucinol (Lynch et al. 2000 ), as was the case in lymphoblastoid cell lines (Iiboshi et al. 1999 ). Third, mTOR signaling was activated in rat adipocytes by the leucine analog norleucine (Lynch et al. 2000 ), an amino acid that is not incorporated into mammalian proteins (ul Hassan and Greenberg 1952 ). Next, the stereospecificity of the response to leucine in rat adipocytes does not match the stereospecificity expected for leucyl-tRNA or leucyl-tRNA synthetase. For example, there was a six- to 10-fold difference in the potency of L- and D-leucine stimulating 4E-BP1 phosphorylation in rat adipocytes (Fox et al. 1998b ). D-Leucine was an agonist, but higher concentrations were required compared to those of L-leucine (Fox et al. 1998b ). D-Amino acids are not substrates for t-RNA synthetase activity, but typically inhibit RNA synthetase-catalyzed ATP–PP_i exchange reaction as do amino acid alcohols (Santi and Webster Jr. 1976 ). Thus, if t-RNA charging was the signal for L-leucine stimulation of 4E-BP1 phosphorylation in adipocytes, D-leucine should not have possessed any agonist activity (Fox et al. 1998b ). Finally, there is a large difference between the K_m for leucyl t-RNA synthetase, 10 µM (Tischler et al. 1982 ) and the EC₅₀ for leucine stimulation of mTOR signaling in rat adipocytes (250 µM) in different experiments (Fox et al. 1998b , Lynch et al. 2000 ). Thus freshly isolated rat adipocytes may use a different mechanism to regulate mTOR, one that does not involve recognition of the t-RNA charging state or t-RNA synthetase activity, as may be the case in the above-mentioned cell lines (Fig. 2) . We referred to this second mechanism as the amino acid alcohol-insensitive mechanism of mTOR activation, although little else is known about it. Although the details of this putative second mechanism remain to be described, it is noteworthy that amino acid regulation of protein synthesis initiation in skeletal muscle, cardiac muscle and liver also may be independent of t-RNA charging state (Ojamaa et al. 1993 , Tischler et al. 1982 ).

	Case for different leucine recognition sites

TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 
Because, in adipocytes, leucine does not appear to activate mTOR by a mechanism that involves t-RNA charging or t-RNA synthetase, it must interact with another biological recognition site to bring about the rapamycin-sensitive effects on 4E-BP1 and p70^s6k. This second site presumably couples to the mTOR signaling pathway by an unknown amino acid alcohol-insensitive mechanism. Thus in mammalian tissues there may be at least be two leucine recognition sites that can couple to mTOR signaling, that is, an amino acid alcohol-sensitive one (e.g., Jurkat cells) and an amino acid alcohol-insensitive one (e.g., rat adipocytes).

There may be a third recognition site. Table 1 summarizes evidence for another recognition site coupling leucine to its effects of mTOR. This is suggested by comparing the structural requirements of leucine in H4IIE cells (a transformed cell line) and freshly isolated rat adipocytes (Lynch et al. 2000 , Shigemitsu et al. 1999 ). Of the naturally occurring amino acids, only leucine and its structural relative methionine were capable of regulating p70^s6k in H4IIE cells at a "4X" concentration. Ile and Val were not active. In adipocytes, only Leu could regulate mTOR signaling at 4X concentration, but at higher concentrations effects of other amino acids could be seen. Concentration–response analysis indicated that Ile is more potent than Met in adipocytes. This represents the first difference noted between rat adipocytes and H4IIE cells. Shigemitsu et al. (1999 ) also reported that in H4IIE cells that H--methyl-D/L-Leu is an efficacious agonist of the mTOR signaling pathway. In contrast, even at very high concentrations, this compound was only a weak agonist of rapamycin-sensitive 4E-BP1 phosphorylation in rat adipocytes (Lynch et al. 2000 ). Shigemitsu et al. (1999 ) also reported the discovery of two antagonists, N-acetyl-leucine amide or N-acetyl-leucine, that blocked leucine stimulation of p70^s6k in H4IIE cells. However, these compounds had no effect on leucine stimulation of 4E-BP1 phosphorylation in freshly isolated rat adipocytes.

	FOOTNOTES

 
¹ Presented as part of the symposium "Leucine as a Nutritional Signal" given at the Experimental Biology 2000 meeting held in San Diego, CA on April 18, 2000. This symposium was sponsored by the American Society for Nutritional Sciences and was supported by the National Institutes of Health Division of Nutritional Research Corporation and Division of Digestive Diseases and Nutrition. The proceedings of the symposium are published as a supplement to The Journal of Nutrition. Editors for the symposium publication were Susan M. Hutson, Wake Forest University School of Medicine and Robert A. Harris, Indiana University School of Medicine.

² Supported by grant DK53843 from the National Institutes of Health.

⁴ Abbreviations used: 1X, one times the concentration found in the plasma of a rat in the postabsorptive state; 4E-BP1, initiation factor 4E binding protein 1; ATM, protein mutated in the disease ataxia telangiectasia; eIF4E, initiation factor 4E; eIF4F, initiation factor 4F; HEAT, a tandemly repeating module containing 37–47 amino acids and occurring in a number of cytoplasmic proteins, including Huntington, Elongation factor 3, Alpha subunit of protein phosphatase 2A and TOR1 (also in mTOR); mTOR, mammalian Target of Rapamycin; p70^S6k, p70 S6 kinase; PI, phosphatidylinositol; PP2A, protein phosphatase 2A.

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TOP ABSTRACT INTRODUCTION The mTOR cell signaling... The role of leucine Studies on the mechanism(s)... Case for different leucine... REFERENCES

 

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				P. G. Cammisotto, Y. Gelinas, Y. Deshaies, and L. J. Bukowiecki Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids Am J Physiol Endocrinol Metab, July 1, 2005; 289(1): E166 - E171. [Abstract] [Full Text] [PDF]

				C. H. Lang and R. A. Frost Differential effect of sepsis on ability of leucine and IGF-I to stimulate muscle translation initiation Am J Physiol Endocrinol Metab, October 1, 2004; 287(4): E721 - E730. [Abstract] [Full Text] [PDF]

				T. Kanazawa, I. Taneike, R. Akaishi, F. Yoshizawa, N. Furuya, S. Fujimura, and M. Kadowaki Amino Acids and Insulin Control Autophagic Proteolysis through Different Signaling Pathways in Relation to mTOR in Isolated Rat Hepatocytes J. Biol. Chem., February 27, 2004; 279(9): 8452 - 8459. [Abstract] [Full Text] [PDF]

				C. H. Lang, R. A. Frost, N. Deshpande, V. Kumar, T. C. Vary, L. S. Jefferson, and S. R. Kimball Alcohol impairs leucine-mediated phosphorylation of 4E-BP1, S6K1, eIF4G, and mTOR in skeletal muscle Am J Physiol Endocrinol Metab, December 1, 2003; 285(6): E1205 - E1215. [Abstract] [Full Text] [PDF]

				A. Blais, J.-F. Huneau, L. J. Magrum, T. J. Koehnle, J. W. Sharp, D. Tome, and D. W. Gietzen Threonine Deprivation Rapidly Activates the System A Amino Acid Transporter in Primary Cultures of Rat Neurons from the Essential Amino Acid Sensor in the Anterior Piriform Cortex J. Nutr., July 1, 2003; 133(7): 2156 - 2164. [Abstract] [Full Text] [PDF]

				C. Roh, J. Han, A. Tzatsos, and K. V. Kandror Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes Am J Physiol Endocrinol Metab, February 1, 2003; 284(2): E322 - E330. [Abstract] [Full Text] [PDF]

				M. L. McDaniel, C. A. Marshall, K. L. Pappan, and G. Kwon Metabolic and Autocrine Regulation of the Mammalian Target of Rapamycin by Pancreatic {beta}-Cells Diabetes, October 1, 2002; 51(10): 2877 - 2885. [Abstract] [Full Text] [PDF]

				C. J. Lynch, S. M. Hutson, B. J. Patson, A. Vaval, and T. C. Vary Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E824 - E835. [Abstract] [Full Text] [PDF]

				C. J. Lynch, B. J. Patson, J. Anthony, A. Vaval, L. S. Jefferson, and T. C. Vary Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E503 - E513. [Abstract] [Full Text] [PDF]

				T. Peng, T. R. Golub, and D. M. Sabatini The Immunosuppressant Rapamycin Mimics a Starvation-Like Signal Distinct from Amino Acid and Glucose Deprivation Mol. Cell. Biol., August 1, 2002; 22(15): 5575 - 5584. [Abstract] [Full Text] [PDF]

				C. J. Lynch, B. J. Patson, S. A. Goodman, D. Trapolsi, and S. R. Kimball Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR Am J Physiol Endocrinol Metab, July 1, 2001; 281(1): E25 - E34. [Abstract] [Full Text] [PDF]

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