Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1alpha-subunit at Ser293 (S293), which inactivates the complex. Because the alpha-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.     

Leucine promotes leptin receptor expression in mouse C2C12 myotubes through the mTOR pathway

Leptin plays a critical role in regulating muscle protein metabolism by binding with leptin receptors in a 1:1 stoichiometry. However, the role for leucine in the regulation of leptin receptor expression in muscle has not been investigated. The present study was conducted to test the hypothesis that leucine regulates leptin receptor levels in C2C12 myotubes. Cells were cultured in the presence of DMEM/F12 medium containing supplemental 0 or 5 mM l-leucine. Leptin receptor expression by C2C12 myotubes peaked at 2 h post-supplementation. Additionally, leucine stimulated leptin receptor expression at both mRNA and protein levels in a dose-dependent manner. Furthermore, leucine enhanced the phosphorylation of mammalian target of rapamycin (mTOR). Addition of rapamycin (an inhibitor of mTOR) to culture medium completely suppressed leucine-induced activation of mTOR and inhibited leucine-stimulated leptin receptor production. These results indicate that leucine affects leptin receptor expression in muscle cells via the mTOR signaling pathway.     

Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue

In freshly isolated rat adipocytes, leucine or its analog norleucine activates the mammalian target of rapamycin (mTOR)-signaling pathway. This results in phosphorylation of the ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), two proteins involved in the initiation phase of protein synthesis. The purpose of the studies reported herein was to address the question of whether or not these in vitro effects of leucine and norleucine on adipocytes could be extended to the intact animal and to other tissues. To accomplish this, food-deprived (18 h) male Sprague-Dawley rats were orally administered solutions (2.5 ml/100 g body wt) containing normal saline (0.9% NaCl), a carbohydrate mixture (26.2% D-glucose and 26.2% sucrose), leucine (5.4%), or norleucine (5.4%). The protein synthetic responses of adipose tissue were measured and compared with those of other tissues. In addition, S6K1 and 4E-BP1 phosphorylation was measured, as was the plasma concentration of insulin and tissue ATP concentrations. Leucine administration stimulated protein synthesis in adipose tissue, gastrocnemius, and kidney but not in liver and heart. Norleucine stimulated protein synthesis in all of the tissues tested but, in contrast to leucine, without affecting plasma insulin concentrations. The carbohydrate meal had no effect on protein synthesis in any tissue tested but elicited a robust increase in plasma insulin. These findings provide support for a role of leucine as a direct-acting nutrient signal for stimulation of protein synthesis in adipose tissue as well as other select tissues. In adipose tissue, the effects of the different treatment conditions on the acute regulation of protein synthesis closely correlated with changes in phosphorylation of S6K1 and 4E-BP1; however, this correlation did not exist in all tissues examined. This result implies that leucine or norleucine may acutely stimulate protein synthesis, at least in some tissues, by a mechanism that is independent of both S6K1 and 4E-BP1 phosphorylation.     

Leucine stimulates protein synthesis in skeletal muscle of neonatal pigs by enhancing mTORC1 activation

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E.eIF4G complex and increased eIF4E.4E-BP1 complex abundance. Rapamycin had no effect on the association of mTOR with rictor, a crucial component for mTORC2 activation, or G protein beta-subunit-like protein (GbetaL), a component of mTORC1 and mTORC2. Neither leucine nor rapamycin affected the phosphorylation of AMP-activated protein kinase (AMPK), PKB, or tuberous sclerosis complex (TSC)2, signaling components that reside upstream of mTOR. Eukaryotic elongation factor (eEF)2 phosphorylation was not affected by leucine or rapamycin, although current dogma indicates that eEF2 phosphorylation is mTOR dependent. Together, these in vivo data suggest that leucine stimulates muscle protein synthesis in neonates by enhancing mTORC1 activation and its downstream effectors.     

The mammalian target of rapamycin complex 1 regulates leptin biosynthesis in adipocytes at the level of translation: the role of the 5'-untranslated region in the expression of leptin messenger ribonucleic acid

Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.     

Leucine in food mediates some of the postprandial rise in plasma leptin concentrations

In vitro, leptin secretion is regulated at the level of mRNA translation by the rapamycin-sensitive mammalian target of rapamycin (mTOR) and its agonist leucine (Leu). Studies were conducted on meal-trained rats to evaluate the potential physiological relevance of these in vitro findings and the role of Leu in affecting rises in plasma leptin observed after a meal. In the first study, we correlated changes in plasma insulin and Leu to mTOR-signaling pathway activation and plasma leptin at different times during meal feeding. Rapid rises in plasma insulin and Leu, along with mTOR signaling (phosphorylation of eIF4G, S6K1, rpS6, and 4E-BP1) in adipose tissue were observed during the 3-h meal and declined thereafter. Plasma leptin rose more slowly, peaking at 3 h, and was inhibited by rapamycin (0.75 mg/kg) pretreatment. In another experiment, oral Leu or norleucine was provided instead of a meal. Leu and norleucine stimulated a rise in plasma leptin; however, the magnitude was less than the response to a complete meal. In a third study, rats were provided a meal that lacked Leu, branched-chain amino acids, or all amino acids. Stimulation of leptin secretion was reduced approximately 40% in animals provided the Leu-deficient meal. Further reductions were not observed by removing the other amino acids. Thus Leu appears to regulate most of the effects of dietary amino acids on the postprandial rise in plasma leptin but is responsible only for part of the leptin response to meal feeding.     

Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57

The mammalian target of rapamycin (mTOR) regulates cell growth and survival via two different multiprotein complexes, mTORC1 and mTORC2. The assembly of these serine-threonine kinase multiprotein complexes occurs via poorly understood molecular mechanisms. Here, we demonstrate that GRp58/ERp57 regulates the existence and activity of mTORC1. Endogenous mTOR interacts with GRp58/ERp57 in different mammalian cells. In vitro, recombinant GRp58/ERp57 preferentially interacts with mTORC1. GRp58/ERp57 knockdown reduces mTORC1 levels and phosphorylation of 4E-BP1 and p70(S6K) in response to insulin. In contrast, GRp58/ERp57 overexpression increases mTORC1 levels and activity. A redox-sensitive mechanism that depends on GRp58/ERp57 expression activates mTORC1. Although GRp58/ERp57 is known as an endoplasmic reticulum (ER) resident, we demonstrate its presence at the cytosol, together with mTOR, Raptor, and Rictor as well as a pool of these proteins associated to the ER. In addition, the presence of GRp58/ERp57 at the ER decreases in response to insulin or leucine. Interestingly, a fraction of p70(S6K), but not 4E-BP1, is associated to the ER and phosphorylated in response to serum, insulin, or leucine. Altogether, our results suggest that GRp58/ERp57 is involved in the assembly of mTORC1 and positively regulates mTORC1 signaling at the cytosol and the cytosolic side of the ER.     

Antagonistic effects of leucine and glutamine on the mTOR pathway in myogenic C<Subscript>2</Subscript>C<Subscript>12</Subscript> cells

This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C(2)C(12) cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70(S6k), S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70(S6k) and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70(S6k) pathway.     

Intracellular mediators in regulation of leptin secretion from adipocytes

Leptin is a hormone primarily secreted by adipocytes and participating in the regulation of food intake and energy expenditure. Its blood levels usually correlate with adiposity. The secretion of this hormone is affected, among others, by food consumption, insulin, fasting and cold exposure. Regulation of leptin secretion depends on many intracellular events. It is known that the activation of mTOR (the mammalian target of rapamycin) as well as increase in ATP and malonyl-CoA content in adipocytes enhance secretion of leptin. The rise in intracellular cAMP and fatty acids is thought to evoke the opposite effect. Moreover, the undisturbed action of endogenous adenosine in adipocytes and the proper intracellular Ca(2+) concentration in these cells were also found to have an important function in leptin release. The role of mTOR, ATP, cAMP, fatty acids, malonyl-CoA, adenosine and Ca(2+) in the regulation of leptin secretion from adipocytes is discussed.     

The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver

Previous studies have shown that oral administration of leucine to fasted rats results in a preferential increase in liver in the translation of mRNAs containing an oligopyrimidine sequence at the 5'-end of the message (i.e. a TOP sequence). TOP mRNAs include those encoding the ribosomal proteins (rp) and translation elongation factors. In cells in culture, the preponderance of evidence suggests that translation of TOP mRNAs is regulated by the mammalian target of rapamycin (mTOR), a protein kinase that signals through ribosomal protein S6 kinase (S6K1) to rpS6. However, the results of previous studies were recently challenged by several reports suggesting that translation of TOP mRNAs is independent of mTOR, S6K1, and S6 phosphorylation. The purpose of the present study was to evaluate the role of mTOR in the stimulation of TOP mRNA translation by leucine in vivo. Fasted rats were treated with the mTOR inhibitor, rapamycin, prior to oral administration of leucine. It was found that rapamycin severely attenuated leucine-induced signaling through mTOR in liver. In addition, rapamycin prevented the enhanced translation of TOP mRNAs in rats administered leucine, as assessed by a decrease in the proportion of TOP mRNAs associated with polysomes (i.e. those mRNAs being actively translated). Instead, in rapamycin-treated rats, ribosomal protein mRNAs accumulated in the fraction containing monosomes (mRNA bound to one ribosome). The results suggest that in liver in vivo, mTOR-dependent signaling is critical for maximal stimulation of TOP mRNA translation.     

Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase

Signaling mediated by the cellular kinase mammalian target of rapamycin (mTOR) activates cap-dependent translation under normal (nonstressed) conditions. However, translation is inhibited by cellular stress responses or rapamycin treatment, which inhibit mTOR kinase activity. We show that during human cytomegalovirus (HCMV) infection, viral protein synthesis and virus production proceed relatively normally when mTOR kinase activity is inhibited due to hypoxic stress or rapamycin treatment. Using rapamycin inhibition of mTOR, we show that HCMV infection induces phosphorylation of two mTOR effectors, eucaryotic initiation factor 4E (eIF4E) binding protein (4E-BP) and eIF4G. The virally induced phosphorylation of eIF4G is both mTOR and phosphatidylinositol 3-kinase (PI3K) independent, whereas the phosphorylation of 4E-BP is mTOR independent, but PI3K dependent. HCMV infection does not induce mTOR-independent phosphorylation of a third mTOR effector, p70S6 kinase (p70S6K). We show that the HCMV-induced phosphorylation of eIF4G and 4E-BP correlates with the association of eIF4E, the cap binding protein, with eIF4G in the eIF4F translation initiation complex. Thus, HCMV induces mechanisms to maintain the integrity of the eIF4F complex even when mTOR signaling is inhibited.     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

Assessment of cell-signaling pathways in the regulation of mammalian target of rapamycin (mTOR) by amino acids in rat adipocytes

Enhanced phosphorylation of the ribosomal protein s6 kinase, p70(s6k), and the translational repressor, 4E-BP1, are associated with either insulin-induced or amino acid-induced protein synthesis. Hyperphosphorylation of p70(s6k) and 4E-BP1 in response to insulin or amino acids is mediated through the mammalian target of rapamycin (mTOR). In several cell lines, mTOR or its downstream targets can be regulated by phosphatidylinositol (PI) 3-kinase; protein kinases A, B, and C; heterotrimeric G-proteins; a PD98059-sensitive kinase or calcium; as well as by amino acids. Regulation by amino acids appears to involve detection of levels of charged t-RNA or t-RNA synthetase activity and is sensitive to inhibition by amino acid alcohols. In the present article, however, we show that the rapamycin-sensitive regulation of 4E-BP1 and p70(s6k) in freshly isolated rat adipocytes is not inhibited by either L-leucinol or L-histidinol. This finding is in agreement with other recent studies from our laboratory suggesting that the mechanism by which amino acids regulate mTOR in freshly isolated adipocytes may be different than the mechanism found in a number of cell lines. Therefore we investigated the possible role of growth factor-regulated and G-protein-regulated signaling pathways in the rapamycin-sensitive, amino acid alcohol-insensitive actions of amino acids on 4E-BP1 phosphorylation. We found, in contrast to previously published results using 3T3-L1 adipocytes or other cell lines, that the increase in 4E-BP1 phosphorylation promoted by amino acids was insensitive to agents that regulate protein kinase A, mobilize calcium, or inhibit protein kinase C. Furthermore, amino acid-induced 4E-BP1 phosphorylation was not blocked by pertussis toxin nor was it mimicked by the G-protein agonists fluoroaluminate or MAS-7. However, amino acids failed to activate either PI 3-kinase, protein kinase B, or mitogen-activated protein kinase and failed to promote tyrosine phosphorylation of cellular proteins, similar to observations made using cell lines. In summary, amino acids appear to use an amino acid alcohol-insensitive mechanism to regulate mTOR in freshly isolated adipocytes. This mechanism is independent of cell-signaling pathways implicated in the regulation of mTOR or its downstream targets in other cells. Overall, our study emphasizes the need for caution when extending results obtained using established cell lines to the differentiated nondividing cells found in most tissues.     

Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.     

Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins

Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.     

Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.     

Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.     

Nutrient regulation of PKCepsilon is mediated by leucine, not insulin, in skeletal muscle

Nutrients enhance signaling pathways involved in skeletal muscle growth through an increased rate of protein synthesis. These studies have led to an understanding of the potential role of the mammalian target of rapamycin (mTOR) in this process. However, activation of mTOR cannot account for all the stimulatory effects of nutrients. The purpose of these experiments was to examine the effect of nutrients on the cellular distribution and activation state of novel PKC isoforms (PKCepsilon and PKCdelta) in the gastrocnemius of rats by use of modification state-dependent phosphopeptide-specific antibodies. The phosphorylation of PKCepsilon on the catalytic domain autophosphorylation site (Ser(729)) was elevated during feeding and then returned to basal levels when the feeding period ended. Meal feeding augmented the phosphorylation of the downstream effectors of mTOR, namely S6K1 and 4E-BP1. In contrast, the phosphorylation of PKCdelta on either the catalytic domain autophosphorylation site (Ser(643)) or activation loop site (Thr(505)) was unaffected. Similar results were obtained when animals were given leucine either acutely via gavage or chronically by dietary supplementations. The effect of leucine was not mimicked by injecting animals with insulin but could be induced by gavage with norleucine, a structural analog of leucine that does not increase plasma insulin concentration. Thus rises in insulin secondary to meal intake or leucine gavage are probably not responsible for increased phosphorylation of PKCepsilon in response to meal feeding. Elevating the leucine concentration stimulated the phosphorylation of PKCepsilon in gastrocnemius from perfused hindlimb and caused a shift in the distribution of PKCepsilon from the membrane fraction to the cytosolic fraction. The results indicate that leucine leads to an activation (autophosphorylation) and subcellular redistribution of PKCepsilon, but not PKCdelta, in gastrocnemius both in vivo and in vitro. Furthermore, activation of the mTOR signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKCepsilon, suggesting that multiple signaling pathways become activated with leucine.