Oral Administration Of Leucine Research Articles

Pre-heating stress associated with acute oral leucine supplementation effects in rat gastrocnemius muscle: Implications for protein synthesis signaling pathways

Skeletal muscle is a highly plastic tissue. The role of heat shock protein 72 (Hsp72) in heat stress-induced skeletal muscle hypertrophy has been well demonstrated; however, the precise mechanisms remain unclear. Essential amino acids, such as leucine, mainly mediate muscle protein synthesis. We investigated the effects of pre-heating and increased Hsp72 expression on the mechanistic target of rapamycin (mTOR) signaling and protein synthesis following leucine administration in rat gastrocnemius muscle. To ensure increased Hsp72 expression in both the red and white portions of the muscle, one leg of male Wistar rats (10-week-old, n = 23) was heat-stressed in 43 °C water for 30 min twice at a 48-h-interval (heat-stressed leg, HS leg). The contralateral leg served as a non-heated internal control (CT leg). After the recovery period (48 h), rats were divided into the pre-administration or oral leucine administration groups. We harvested the gastrocnemius muscle (red and white parts) prior to administration and 30 and 90 min after leucine treatment (n = 7–8 per group) and intramuscular signaling responses to leucine ingestion were determined using western blotting. Heat stress significantly upregulated the expression of Hsp72 and was not altered by leucine administration. Although the phosphorylation levels of mTOR/S6K1 and ERK were similar regardless of heating, 4E-BP1 was less phosphorylated in the HS legs than the CT legs after leucine administration in the red portion of the muscles (P < 0.05). Moreover, c-Myc expression differed significantly after leucine administration in both the red and white portions of the muscles. Our findings indicate that following oral leucine administration, pre-heating partially blunted the muscle protein synthesis signaling response in the rat gastrocnemius muscle.

The efficiency of oral leucine administration on the progressing of melanoma in a murine sarcopenic model

The efficiency of oral leucine administration on the progressing of melanoma in a murine sarcopenic model

Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle.

Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.

Repressors of mTORC1 act to blunt the anabolic responseto feeding in the soleus muscle of a cast-immobilized mouse hindlimb.

We recently reported results showing that cast immobilization of a rat hindlimb rapidly leads to development of anabolic resistance as demonstrated by failure of oral leucine administration to activate the mechanistic target of rapamycin complex 1 (mTORC1) and stimulate protein synthesis in the soleus muscle. The goal of this study was to assess the possible contribution of several mTORC1 regulatory proteins to the development of anabolic resistance. To accomplish this, 14‐week‐old male C57BL/6 mice (n = 21) were subjected to unilateral cast immobilization of the hindlimb for either 1 or 3 days, and the immobilized limb was compared to its contralateral control. The mass of the soleus muscle was decreased in the immobilized compared to the non‐immobilized limb within 72‐h in association with diminished protein synthesis. In agreement with our previous report, a 24‐h casting period was sufficient to induce anabolic resistance, as demonstrated by blunted re‐feeding‐induced activation of mTORC1. Moreover, resistance of mTORC1 activation was associated not only with upregulated expression of REDD1, but also with altered expression of other mTORC1 regulatory proteins, that is, Sestrin1 and DEP domain‐containing mTOR interacting protein (DEPTOR). In addition, re‐feeding‐induced phosphorylation of DEPTOR was significantly impaired in the immobilized compared to the non‐immobilized limb. This work builds upon previous discoveries by our laboratory to elucidate the blunted mTORC1 response to stimuli during disuse of skeletal muscle induced by cast immobilization while highlighting new potential therapeutic targets for future countermeasures against muscle atrophy.

The Effect of the Oral Administration of Leucine on Endothelial Function, Glucose and Insulin Concentrations in Healthy Subjects

The aim of our study was to investigate the potential differential effect of hyperglycaemia and hyperinsulinaemia induced by glucose infusion alone and in combination with leucine consumption on endothelial function in healthy individuals. Ten male volunteers were examined in random order twice. In one visit, they consumed 250 ml water (baseline) and 30 min later glucose was infused iv. In the other visit, they consumed 250 ml water with 25 g of leucine and 30 min later the same amount of glucose was infused. Serum glucose and insulin were measured at baseline and every 10 min after glucose infusion for 1 h. Endothelial function was evaluated by measurement of flow mediated vasodilatation (FMD) at baseline, 10 and 60 min after glucose infusion. In both visits, glucose levels increased to the same degree, whereas insulin response was significantly higher after leucine administration. FMD values declined significantly compared to baseline 10 min after glucose infusion in the control visit (6.9±2.7 vs. 3.2±3.5%, respectively, p=0.006), while no significant change was observed when glucose infusion was followed by leucine consumption. Acute hyperglycaemia impairs endothelial function in healthy male individuals. Leucine administration prevents hyperglycaemia-mediated endothelial dysfunction probably due to enhanced insulin secretion.

Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice.

Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis.

MTORC1 is involved in the regulation of branched-chain amino acid catabolism in mouse heart.

The branched‐chain α‐ketoacid dehydrogenase (BCKDH) complex regulates branched‐chain amino acid (BCAA) catabolism by controlling the second step of this catabolic pathway. In the present study, we examined the in vivo effects of treatment with an mTORC1 inhibitor, rapamycin, on cardiac BCKDH complex activity in mice. Oral administration of leucine in control mice significantly activated the cardiac BCKDH complex with an increase in cardiac concentrations of leucine and α‐ketoisocaproate. However, rapamycin treatment significantly suppressed the leucine‐induced activation of the complex despite similar increases in cardiac leucine and α‐ketoisocaproate levels. Rapamycin treatment fully inhibited mTORC1 activity, measured by the phosphorylation state of ribosomal protein S6 kinase 1. These results suggest that mTORC1 is involved in the regulation of cardiac BCAA catabolism.

A (14)C-leucine absorption, distribution, metabolism and excretion (ADME) study in adult Sprague-Dawley rat reveals β-hydroxy-β-methylbutyrate as a metabolite.

Leucine is an essential branched-chain amino acid that acts as a substrate for protein synthesis and as a signaling molecule. Leucine not incorporated into muscle protein is ultimately oxidized through intermediates such as β-hydroxy-β-methylbutyrate (HMB) which itself is reported to enhance muscle mass and function in rats and humans. HMB has been reported in the plasma following oral leucine administration in sheep and pigs but not in Sprague–Dawley rats, the standard preclinical model. Therefore, we conducted radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in rats using a low (3 mg/kg) or high dose (1,000 mg/kg) of 14C-leucine. Blood, tissue, and urine samples were analyzed for 14C-leucine and its metabolites by HPLC–MS. Our results show for the first time that 14C-HMB appears in plasma and urine of rats following an oral dose of 14C-leucine. 14C-leucine appears in plasma as 14C-α-ketoisocaproic acid (KIC) with a slower time course than 14C-HMB, a putative product of KIC. Further, two novel metabolites of leucine were detected in urine, N-acetyl leucine and glycyl leucine. Mass balance studies demonstrate that excretory routes accounted for no more than 0.9 % of the radiolabel and approximately 61 % of the dose was recovered in the carcass. Approximately 65 % of the dose was recovered in total, suggesting that approximately one-third of the leucine dose is oxidized to CO2. In conclusion, this study demonstrates endogenous production of HMB from leucine in adult rats, a standard preclinical model used to guide design of clinical trials in nutrition.

Differential Dose Response of mTOR Signaling to Oral Administration of Leucine in Skeletal Muscle and Liver of Rats

Phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1) in the rat liver increased in proportion to the amount of leucine administered, ranging from 0.169 to 1.35 g/kg of body weight. In the skeletal muscle, phosphorylation of these factors reached a plateau at 0.675 g/kg of body weight. The sensitivity of mammalian target of rapamycin (mTOR) signaling to leucine in the skeletal muscle appeared to be higher than that in the liver.

Oral administration of L‐leucine reduces nitric oxide synthesis by endothelial cells of rats

High concentrations of branched‐chain amino acids in plasma are commonly associated with endothelial dysfunction in obese humans and animals. This study tested the hypothesis that elevated levels of leucine in plasma can reduce nitric oxide (NO) synthesis by endothelial cells (EC). Beginning at 19 weeks of age, male Sprague‐Dawley rats were fed a semi‐purified diet containing a normal fat content and received drinking water containing either 1% L‐leucine or 0.68% L‐alanine (isonitrogenous control) (n=8/group). Twenty‐four‐hour whole‐body energy expenditure of rats was determined every 4 weeks using Oxymas (an open circuit calorimeter, Columbus Instruments, Ohio). At 31 weeks of age, rats were euthanized to obtain tissues. Oral administration of leucine increased (P &lt; 0.01) concentrations of leucine in plasma by 35%, but did not affect (P &gt; 0.05) water consumption (25 mL/kg BW per day), food intake, body weight, skeletal muscle weight, white adipose tissue weight, or whole‐body heat production. Leucine administration enhanced (P &lt; 0.05) glutamine:fructose‐6‐phosphate transaminase activity and decreased (P &lt; 0.05) NO synthesis in EC and whole‐body insulin sensitivity. These novel results indicate that elevated concentrations of leucine promote glucosamine synthesis and reduce NO production by EC, contributing to increased risk for cardiovascular disease. (Supported by AHA and NIH grants).

Repeated post-exercise administration with a mixture of leucine and glucose alters the plasma amino acid profile in Standardbred trotters.

BackgroundThe branched chain amino acid leucine is a potent stimulator of insulin secretion. Used in combination with glucose it can increase the insulin response and the post exercise re-synthesis of glycogen in man. Decreased plasma amino acid concentrations have been reported after intravenous or per oral administration of leucine in man as well as after a single per oral dose in horses. In man, a negative correlation between the insulin response and the concentrations of isoleucine, valine and methionine have been shown but results from horses are lacking. This study aims to determine the effect of repeated per oral administration with a mixture of glucose and leucine on the free amino acid profile and the insulin response in horses after glycogen-depleting exercise.MethodsIn a crossover design, after a glycogen depleting exercise, twelve Standardbred trotters received either repeated oral boluses of glucose, 1 g/kg body weight (BW) at 0, 2 and 4 h with addition of leucine 0.1 g/kg BW at 0 and 4 h (GLU+LEU), or repeated boluses of water at 0, 2 and 4 h (CON). Blood samples for analysis of glucose, insulin and amino acid concentrations were collected prior to exercise and over a 6 h post-exercise period. A mixed model approach was used for the statistical analyses.ResultsPlasma leucine, isoleucine, valine, tyrosine and phenylalanine concentrations increased after exercise. Post-exercise serum glucose and plasma insulin response were significantly higher in the GLU+LEU treatment compared to the CON treatment. Plasma leucine concentrations increased after supplementation. During the post-exercise period isoleucine, valine and methionine concentrations decreased in both treatments but were significantly lower in the GLU+LEU treatment. There was no correlation between the insulin response and the response in plasma leucine, isoleucine, valine and methionine.ConclusionsRepeated post-exercise administration with a mixture of leucine and glucose caused a marked insulin response and altered the plasma amino acid profile in horses in a similar manner as described in man. However, the decreases seen in plasma amino acids in horses seem to be related more to an effect of leucine and not to the insulin response as seen in man.

AMPing Down Leucine Action in Skeletal Muscle

In recent years, several investigative teams have tested leucine as a therapeutic agent to support muscle growth. Although some studies have shown that BCAA or leucine support muscle protein synthesis (MPS) (1), others have found that leucine is ineffective at stimulating muscle protein anabolism under certain conditions, e.g. sepsis, alcohol intoxication, and glucocorticoid excess in aged rats. During these catabolic states, MPS is either precluded or blunted in response to leucine, leading the authors of the current highlighted paper, Pruznak et al. (2), which was published in the October, 2008 issue of The Journal of Nutrition, to describe the muscle as being in a state of leucine resistance. Understanding the molecular basis for why muscle is unresponsive to supplemental leucine can be used to identify novel therapeutic targets and/or optimize current nutritional support strategies. The BCAA are known for their unique ability to stimulate MPS independently of other amino acids. When administered orally, leucine alone stimulates MPS in exercised or fooddeprived rats (3). The mechanism for the anabolic effect involves stimulation of mRNA translation initiation by increasing eukaryotic initiation factor (EIF) 4F complex assembly and signaling via mammalian target of rapamycin (mTOR) (3). The mTOR kinase facilitates cell growth and proliferation by nutrients and insulin through its association with the regulatory proteins Raptor (regulatory associated protein of mTOR), mLST8/GbL, and PRAS40 (proline-rich Akt substrate of 40 kDa) in a complex referred to as mTOR complex 1 (mTORC1) (4,5). Over the past decade, many laboratories have sought to define how leucine stimulates mTORC1 activity. Although it is clear that leucine does not activate the insulin/insulin-like growth factor/phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway, identification of the relevant molecules upstream of mTORC1 remains elusive. To understand why leucine is ineffective at inducing MPS under certain stress conditions in vivo, Pruznak et al. (2) examined the relationship between leucine and AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that serves as a major energy sensor in the cell. Activated when ATP becomes limiting, AMPK stimulates catabolic pathways and inhibits anabolic pathways in an effort to supply ATP for cell survival. As such, AMPK is a negative upstream regulator of mTORC1, reducing MPS under conditions of energetic stress such as hypoxia, intense exercise, or glucose starvation (6). AMPK inhibits mTORC1 signaling in 2 ways: via phosphorylation of Raptor, which leads to decreased mTOR kinase activity, and via phosphorylation of tuberin (also known as tuberous sclerosis complex 2) at Ser1345 (7), converting the mTOR activator, Ras homolog enhanced in brain, to its inactive form. Activationof AMPK canbemimickedusing5-aminoimidazole4-carboxamide-1–4-ribofuranoside (AICAR), a cell-permeable compound whose phosphorylated metabolite activates AMPK without perturbing the cellular concentrations of ATP, ADP, or AMP in skeletal muscle (8). In the October issue, Pruznak et al. (2) utilizes AICAR treatment in rats to very effectively block the stimulation of skeletal MPS, EIF4F complex assembly, and mTORC1 signaling by oral administration of leucine (Fig. 1). Importantly, the overall block in leucine action is more complete than that achieved by other inhibitors tested previously in rats (e.g. rapamycin), demonstrating that cellular energy state plays a dominant role in the regulation of MPS by amino acids in vivo. These findings are supported by recently published work showing that leucine stimulation of mTOR and MPS is inhibited by AICAR treatment of C2C12 cells (9). Pruznak et al. (2) further advance our understanding of how leucine transmits mTORC1 signaling in vivo by identifying PRAS40 as a mediator of leucine’s effects. PRAS40 is a Raptorbinding protein that is phosphorylated directly by mTORC1 at several sites, including Ser183, and by protein kinase B at Thr246. Whereas studies using cells in culture show that phosphorylation of PRAS40 at Ser183 is modulated by amino acid-activated mTORC1 (4), the study of Pruznak et al. (2) finds that leucine enhances PRAS40 phosphorylation at the insulin-activated Thr246 site. Interpretation of this result should be viewed cautiously, because it is possible that the transient rise in insulin following leucine administration may have contributed to 1 Author disclosures: T. Anthony and J. Anthony, no conflicts of interest.

Acute Oral Leucine Administration Stimulates Protein Synthesis during Chronic Sepsis through Enhanced Association of Eukaryotic Initiation Factor 4G with Eukaryotic Initiation Factor 4E in Rats ,

Sepsis induces the loss of muscle proteins by impairing skeletal muscle protein synthesis through an inhibition of messenger RNA (mRNA) translation initiation. Amino acids and Leu (Leu) in particular stimulate mRNA translation initiation. The experiments were designed to test the effects of Leu on potential signal transduction pathways that may be important in accelerating mRNA translation initiation in skeletal muscle of rats with chronic (5–6 d) septic intra-abdominal abscess. Gastrocnemius from male Sprague Dawley rats gavaged with Leu or water were sampled 5–6 d following development of an intra-abdominal sterile or septic abscess. Gavage with Leu stimulated protein synthesis and enhanced the assembly of the active eukaryotic initiation factor (eIF)4G-eIF4E complex. Increased assembly of the active eIF4G-eIF4E complex was associated with a robust rise in phosphorylation of eIF4G(Ser1108) and a decreased assembly of inactive eIF4E binding protein-1 (4E-BP1)-eIF4E complex in both sterile inflammatory and septic rats. The reduced assembly of 4E-BP1-eIF4E complex was associated with an increase in phosphorylation of 4E-BP1 in the γ-form following Leu gavage. Phosphorylation of 70-kDa ribosomal protein S6 kinase on Thr389 was also increased following Leu gavage, as well as the phosphorylation of mammalian target of rapamycin on Ser2448 or Ser2481. In contrast, phosphorylation of protein kinase B (PKB) on Thr308 or Ser473 was not augmented following Leu gavage in septic rats. We conclude that Leu stimulates a PKB-independent signal pathway elevating the eIF4G-eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E in skeletal muscle during sepsis.

Rapamycin blunts nutrient stimulation of eIF4G, but not PKCε phosphorylation, in skeletal muscle

Phosphorylation of eukaryotic initiation factor 4G (eIF4G) is hypothesized to be an important contributor to the stimulation of protein synthesis in skeletal muscle following meal feeding. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in mediating the meal feeding-induced elevations in phosphorylation of eIF4G. Gastrocnemius from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled prior to and following 3 h of having the meal provided in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1 (TORC1). Pretreatment with rapamycin prevented the feeding-induced phosphorylation of mTOR, eIF4G, and S6K1 but only partially attenuated the shift in 4E-BP1 into the gamma-form. In contrast, the feeding-induced increase in phosphorylation of PKCepsilon was not reduced by rapamycin. Rapamycin also prevented the augmented association of eIF4G with eIF4E and the decreased association of eIF4E with 4E-BP1. Similar findings were observed in gastrocnemius from animals after oral administration of leucine. Perfusion of gastrocnemius with medium containing rapamycin partially prevented the leucine-induced increase in phosphorylation of eIF4G. Thus, rapamycin attenuated a feeding- or leucine-induced phosphorylation of eIF4G in skeletal muscle both in vivo and in situ. The latter observation implies that the effects observed with rapamycin were the result of modulation of skeletal muscle signaling mechanisms responsible for eIF4G phosphorylation.

Leucine suppresses myofibrillar proteolysis by down-regulating ubiquitin–proteasome pathway in chick skeletal muscles

In skeletal muscle, amino acids, together with hormones, are key regulators of protein metabolism. Leucine, in particular, has inhibitory effects of protein degradation in skeletal muscles, but the mechanisms are poorly understood. The present study addressed the role of leucine as a regulator of myofibrillar proteolysis in cultured chick myotubes and chick skeletal muscles, and aimed to determine which cellular responses regulate the process. In chick myotubes, leucine suppressed myofibrillar proteolysis (as measured by N τ-methylhistidine release), while also decreasing ubiquitin and proteasome C2 subunit mRNA. Oral administration of leucine also suppressed myofibrillar proteolysis (as measured by plasma N τ-methylhistidine concentration), while also decreasing proteasome C2 subunit mRNA in chick skeletal muscle. Leucine activated the phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) (but not the mammalian target of rapamycin) inhibition of these pathways and increased myofibrillar proteolysis, ubiquitin and proteasome C2 subunit mRNA. Thus, an important component of muscle proteolysis inhibition by leucine, through the PI3K and PKC, is its ability to suppress transcription of the ubiquitin and proteasome C2 subunit, and degradation of myofibrillar protein.

Effect of Orally Administered Branched-chain Amino Acids on Protein Synthesis and Degradation in Rat Skeletal Muscle

Although amino acids are substrates for the synthesis of proteins and nitrogen-containing compounds, it has become more and more clear over the years that these nutrients are also extremely important as regulators of body protein turnover. The branched-chain amino acids (BCAAs) together or simply leucine alone stimulate protein synthesis and inhibit protein breakdown in skeletal muscle. However, it was only recently that the mechanism(s) involved in the regulation of protein turnover by BCAAs has begun to be defined. The acceleration of protein synthesis by these amino acids seems to occur at the level of peptide chain initiation. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Despite our knowledge of the induction of protein synthesis by BCAAs, there are few studies on the suppression of protein degradation. The recent findings that oral administration of leucine rapidly reduced N τ -methylhistidine (3- methylhistidine; MeHis) release from isolated muscle, an index of myofibrillar protein degradation, indicate that leucine suppresses myofiblilar protein degradation. The details of the molecular mechanism by which leucine inhibits proteolysis is just beginning to be elucidated. The purpose of this report was to review the current understanding of how BCAAs act as regulators of protein turnover. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 1 : 133-140)

Oral Leucine Administration Stimulates Protein Synthesis in Rat Skeletal Muscle

Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at concentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), the association of eIF4G with the mRNA cap binding protein eIF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of eIF4G, as well as the association of 4E-BP1 with eIF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans.

TNF? mediates sepsis-induced impairment of basal and leucine-stimulated signaling via S6K1 and eIF4E in cardiac muscle

Decreased translation initiation adversely impacts protein synthesis and contributes to the myocardial dysfunction produced by sepsis. Therefore, the purpose of the present study was to identify sepsis-induced changes in signal transduction pathways known to regulate translation initiation in cardiac muscle and to determine whether the stimulatory effects of leucine can reverse the observed defects. To address this aim, sepsis was produced by cecal ligation and puncture (CLP) in anesthetized rats and the animals studied in the fasted condition 24 h later. Separate groups of septic and time-matched control rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating cap-dependent mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined. Under basal conditions, hearts from septic rats demonstrated a redistribution of the rate-limiting factor eIF4E due to increased binding of the translational repressor 4E-BP1 with eIF4E. However, this change was independent of an alteration in the phosphorylation state of 4E-BP1. The phosphorylation of mTOR, S6K1, the ribosomal protein (rp) S6, and eIF4G was not altered in hearts from septic rats under basal conditions. In control rats, leucine failed to alter eIF4E distribution but increased the phosphorylation of S6K1 and S6. In contrast, in hearts from septic rats leucine acutely reversed the alterations in eIF4E distribution. However, the ability of leucine to increase S6K1 and rpS6 phosphorylation in septic hearts was blunted. Sepsis increased the content of tumor necrosis factor (TNF)-alpha in heart and pre-treatment of rats with a TNF antagonist prevented the above-mentioned sepsis-induced changes. These data indicate that oral administration of leucine acutely reverses sepsis-induced alterations eIF4E distribution observed under basal conditions but the anabolic actions of this amino acid on S6K1 and rpS6 phosphorylation remain blunted, providing evidence for a leucine resistance. Finally, TNFalpha, either directly or indirectly, appears to mediate the sepsis-induced defects in myocardial translation initiation.

Branched-chain amino acids improve glucose metabolism in rats with liver cirrhosis

It is well established that impaired glucose metabolism is a frequent complication in patients with hepatic cirrhosis. We previously showed that leucine, one of the branched-chain amino acids (BCAA), promotes glucose uptake under insulin-free conditions in isolated skeletal muscle from normal rats. The aim of the present study was to evaluate the effects of BCAA on glucose metabolism in a rat model of CCl(4)-induced cirrhosis (CCl(4) rats). Oral glucose tolerance tests were performed on BCAA-treated CCl(4) rats. In the CCl(4) rats, treatment with leucine or isoleucine, but not valine, improved glucose tolerance significantly, with the effect of isoleucine being greater than the effect of leucine. Glucose uptake experiments using isolated soleus muscle from the CCl(4) rats revealed that leucine and isoleucine, but not valine, promoted glucose uptake under insulin-free conditions. To clarify the mechanism of the blood glucose-lowering effects of BCAA, we collected soleus muscles from BCAA-treated CCl(4) rats with or without a glucose load. These samples were used to determine the subcellular location of glucose transporter proteins and glycogen synthase (GS) activity. Oral administration of leucine or isoleucine without a glucose load induced GLUT4 and GLUT1 translocation to the plasma membrane. GS activity was augmented only in leucine-treated rats and was completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. In summary, we found that leucine and isoleucine improved glucose metabolism in CCl(4) rats by promoting glucose uptake in skeletal muscle. This effect occurred as a result of upregulation of GLUT4 and GLUT1 and also by mammalian target of rapamycin-dependent activation of GS in skeletal muscle. From these results, we consider that BCAA treatment may have beneficial effects on glucose metabolism in cirrhotic patients.

Leucine acutely reverses burn-induced alterations in translation initiation in heart.

Myocardial dysfunction is a common manifestation of thermal injury, the etiology of which appears to be multifactorial. We have previously demonstrated that burn injury impairs cardiac protein synthesis at the level of translation initiation. The purpose of the present study was to determine whether oral administration of leucine, which is known to stimulate translation initiation in skeletal muscle, can ameliorate burn-induced changes in signal transduction pathways known to regulate protein synthesis in cardiac muscle. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied in the fasted condition 24 h later; appropriate time-matched nonburned control rats were also included. Separate groups of control and burn rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined using immunoprecipitation and immunoblotting techniques. Hearts from burned rats demonstrated a redistribution of eIF4E as evidenced by the increased binding of the translational repressor 4E-BP1 with eIF4E, a decreased amount of eIF4E bound with eIF4G, and a decreased amount of the hyperphosphorylated gamma-isoform of 4E-BP1. Furthermore, constitutive phosphorylation of mTOR, the ribosomal protein S6, and eIF4G was also decreased in hearts from burned rats. In control rats, leucine failed to alter eIF4E distribution but did increase the phosphorylation of S6K1 and S6. However, in hearts from burn rats, leucine acutely reversed the alterations in eIF4E distribution as well as the changes in S6, eIF4G, and mTOR phosphorylation. These data suggest that oral administration of leucine can acutely reverse multiple defects in cardiac translation initiation produced by thermal injury.