Leucine Stimulates Protein Synthesis Research Articles

L-Leucine stimulates proliferation and casein secretion in porcine mammary epithelial cells through mTOR-p70S6K-4EBP1 signalling pathway

Milk production determines health status and optimal growth of suckling neonates. Recent studies revealed that the secretion of leucine into sow milk from the mammary gland is significantly lower than the amount taken up by the mammary gland from the blood, suggesting the potential and fundamental role in udder metabolism. However, little is known about the underlying mechanism. In this study, porcine mammary epithelial cells (PMECs) were employed to investigate the effects of leucine on protein synthesis and mammalian target of rapamycin (mTOR) signalling. Results showed that leucine supplementation significantly increased the proliferation of PMECs in a dose-dependent manner from 0 to 1 mmol/l. Secretions of four casein subunits were significantly increased by addition of leucine. The phosphorylation of proteins related to mTOR signalling, including mTOR, eukaryotic translation initiation factor 4E-binding protein, and p70 ribosomal protein S6 kinase, were accordingly upregulated by leucine supplementation. Casein secretion was not affected by leucine treatment when mTOR signalling was inhibited by rapamycin. This suggests that leucine stimulates protein synthesis through the mTOR signalling pathway. These findings facilitate the utilization of dietary leucine supplementation to improve milk production in lactating sows.

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 identified a family of three proteins, Sestrins 1, 2, and 3, that 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 Sestin1 expression is higher than Sestrin3 while Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine‐induced dissociation of the Sestrin•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 Sestrin3•GATOR2 complex in skeletal muscle. 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.Support or Funding InformationSupported by NIH grants DK13499 and DK15658.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

L-leucine dietary supplementation modulates muscle protein degradation and increases pro-inflammatory cytokines in tumour-bearing rats

Cancer cachexia is characterised by involuntary weight loss associated with systemic inflammation and metabolic changes. Studies aimed at maintaining lean body mass in cachectic tumour-bearing hosts have made important contributions reducing the number of deaths and improving the quality of life. In recent years, leucine has demonstrated effective action in maintaining lean body mass by decreasing muscle protein degradation. Currently, there is a growing need to understand how leucine stimulates protein synthesis and acts protectively in a cachectic organism. Thus, this study aimed to assess the effects of a leucine-rich diet on protein degradation signalling in muscle over the course of tumour growth. Animals were distributed into four experimental groups, which did or did not receive 2×106 viable Walker-tumour cells. Some were fed a leucine-rich diet, and the groups were subsequently sacrificed at three different time points of tumour evolution (7th, 14th, and 21st days). Protein degradation signals, as indicated by ubiquitin-proteasome subunits (11S, 19S, and 20S) and pro- and anti-inflammatory cytokines, were analysed in all experimental groups. In tumour-bearing animals without nutritional supplementation (W7, W14, and W21 groups), we observed that the tumour growth promoted a concurrent decrease in muscle protein, a sharp increase in pro-inflammatory cytokines (TNFα, IL-6, and IFNγ), and a progressive increase in proteasome subunits (19S and 20S). Thus, the leucine-supplemented tumour-bearing groups showed improvements in muscle mass and protein content, and in this specific situation, the leucine-rich diet led to an increase on the day in cytokine profile and proteasome subunits mainly on the 14th day, which subsequently had a modulating effect on tumour growth on the 21st day. These results indicate that the presence of leucine in the diet may modulate important aspects of the proteasomal pathway in cancer cachexia and may prevent muscle wasting due to the decrease in the cachexia index.

Enteral β-hydroxy-β-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs.

Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were studied immediately (F) or fed one of five diets for 24 h: low-protein (LP), high-protein (HP), or LP diet supplemented with 4 (HMB4), 40 (HMB40), or 80 (HMB80) μmol HMB·kg body wt(-1)·day(-1) Cell replication was assessed from nuclear incorporation of BrdU in the longissimus dorsi (LD) muscle and jejunum crypt cells. Protein synthesis rates in LD, gastrocnemius, rhomboideus, and diaphragm muscles, lung, and brain were greater in HMB80 and HP and in brain were greater in HMB40 compared with LP and F groups. Formation of the eIF4E·eIF4G complex and S6K1 and 4E-BP1 phosphorylation in LD, gastrocnemius, and rhomboideus muscles were greater in HMB80 and HP than in LP and F groups. Phosphorylation of eIF2α and eEF2 and expression of SNAT2, LAT1, MuRF1, atrogin-1, and LC3-II were unchanged. Numbers of BrdU-positive myonuclei in the LD were greater in HMB80 and HP than in the LP and F groups; there were no differences in jejunum. The results suggest that enteral supplementation with HMB increases skeletal muscle protein anabolism in neonates by stimulation of protein synthesis and satellite cell proliferation.

Acute Acidosis Attenuates Leucine Stimulated Signal Transduction and Protein Synthesis in Rat Skeletal Muscle

Background: Critical illnesses are often complicated by acute metabolic acidosis, which if persistent, adversely affects outcome. Among the harmful effects that it might cause are impaired utilization of nutrients, increased proteolysis and depressed protein synthesis, leading to muscle wasting. As the amino acid leucine stimulates protein synthesis by activating mTOR signaling, we explored whether in acidosis, impaired leucine-stimulated signaling might be a contributor to the depressed protein synthesis. Methods: Male pair-fed rats were gavaged with NH₄Cl (acidosis) or NaCl (control) for 2 days and then gavaged once with leucine and sacrificed 45 min later. Extensor digitorum longus muscles were isolated, incubated with or without leucine and protein synthesis measured. The anterior tibial muscle signaling was analysed by Western immunobloting. Results: Despite pair-feeding, acidotic rats lost body and muscle weight vs. controls. Moreover, leucine-induced protein synthesis in isolated muscle from acidotic rats was impaired. In-vivo, 45 min after an oral leucine load, anterior tibial muscle mTOR and 4E-BP1 phosphorylation increased significantly and comparably in control and acidotic rats. In contrast, leucine-stimulated phosphorylation of S6K1, a regulator of translation initiation and protein synthesis, was attenuated to approximately 56% of the control value (p < 0.05). Conclusion: This study reveals that an acute metabolic acidosis impairs leucine-stimulated protein synthesis and activation of signaling downstream of mTOR at the level of S6K1. We propose that this S6K1abnormality may account in part, for the resistance to leucine-stimulated muscle protein synthesis, and may thereby contribute to the impaired nutrient utilization and ultimately the muscle wasting that develops in acidosis.

Protein synthesis in skeletal muscle of neonatal pigs is enhanced by administration of β-hydroxy-β-methylbutyrate

Many low-birth-weight infants experience failure to thrive. The amino acid leucine stimulates protein synthesis in skeletal muscle of the neonate, but less is known about the effects of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB). To determine the effects of HMB on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were infused with HMB at 0, 20, 100, or 400 μmol·kg body wt(-1)·h(-1) for 1 h (HMB 0, HMB 20, HMB 100, or HMB 400). Plasma HMB concentrations increased with infusion and were 10, 98, 316, and 1,400 nmol/ml in the HMB 0, HMB 20, HMB 100, and HMB 400 pigs. Protein synthesis rates in the longissimus dorsi (LD), gastrocnemius, soleus, and diaphragm muscles, lung, and spleen were greater in HMB 20 than in HMB 0, and in the LD were greater in HMB 100 than in HMB 0. HMB 400 had no effect on protein synthesis. Eukaryotic initiation factor (eIF)4E·eIF4G complex formation and ribosomal protein S6 kinase-1 and 4E-binding protein-1 phosphorylation increased in LD, gastrocnemius, and soleus muscles with HMB 20 and HMB 100 and in diaphragm with HMB 20. Phosphorylation of eIF2α and elongation factor 2 and expression of system A transporter (SNAT2), system L transporter (LAT1), muscle RING finger 1 protein (MuRF1), muscle atrophy F-box (atrogin-1), and microtubule-associated protein light chain 3 (LC3-II) were unchanged. Results suggest that supplemental HMB enhances protein synthesis in skeletal muscle of neonates by stimulating translation initiation.

Leucine Supplementation of a Low Protein Meal Increases Skeletal Muscle and Visceral Tissue Protein Synthesis in Neonatal Pigs by Stimulating mTOR‐Dependent Translation Initiation

Leucine appears to be the most effective single amino acid to trigger the activation of translation initiation factors that regulate protein synthesis in muscle. The effects of Leu in visceral tissues are not well understood. We hypothesized that enteral Leu supplementation of a low protein diet (LP) in neonatal pigs will acutely increase skeletal muscle and visceral tissue protein synthesis to a rate similar to that achieved by feeding a high protein diet (HP). Fasted 5‐d‐old piglets (n=21) were gavage fed at 0 and 60 min either: 1) LP diet, 2) low protein diet supplemented with Leu (LP+L) to equal HP diet, or 3) HP diet. Diets were isocaloric and lactose was equal. Fractional protein synthesis rates and translational control mechanisms were examined 90 min after feeding. Protein synthesis increased in longissimus dorsi, gastrocnemius, masseter, heart, jejunum, kidney and pancreas, but not liver, of the LP+L group compared to LP group alone and did not differ from HP group. LP+L and HP increased translational efficiency and phosphorylation of S6K1 and 4E‐BP1 in all tissues except liver. Our results suggest that low protein diets supplemented with leucine stimulates protein synthesis in muscle and most visceral tissues to a rate similar to that achieved by high protein diets and this stimulation involves activation of mTOR downstream effectors. (Supported by Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001).Grant Funding Source: Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001

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.

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 α-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.

Leucine stimulation of skeletal muscle protein synthesis during prolonged leucine infusion is dependent on amino acid availability

Leucine stimulates protein synthesis in cultured cells, mature rats and neonatal pigs. We have reported that leucine infusion increases protein synthesis in skeletal muscle of neonatal pigs during a 60-min infusion. When leucine infusion was prolonged for 120 min, however, protein synthesis was not increased. We hypothesized that this lack of responsiveness was due to a decrease (−50%) in plasma essential amino acids (AA). To test this hypothesis, fasted neonatal pigs were infused with leucine (0 or 400 μmol·kg−1·h−1) for 120 min to raise plasma leucine within the physiological postprandial range in the presence or absence of the infusion of an AA mixture (without leucine) to maintain plasma AA levels. Parenteral AA administration prevented the leucine induced reduction (P < 0.05) in plasma essential AA. Leucine infusion, regardless of AA treatment, increased (P < 0.05) the phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1), decreased the inactive 4E BP1·eIF4E complex, and increased the active eIF4G·eIF4E complex in longissimus dorsi muscle. Protein synthesis was increased (P < 0.02, +54%) in muscle with infusion of both leucine and AA but not with leucine alone. In liver, neither protein synthesis nor activation of translation initiation factors was affected by leucine infusion with or without AA. Thus, the stimulation of protein synthesis during prolonged leucine infusion is tissue specific and dependent on AA availability (NIH AR 44474 and USDA 58-6250-6-001)

Physiological rise in plasma leucine stimulates muscle protein synthesis in neonatal pigs by enhancing translation initiation factor activation

Protein synthesis in skeletal muscle of adult rats increases in response to oral gavage of supraphysiological doses of leucine. However, the effect on protein synthesis of a physiological rise in plasma leucine has not been investigated in neonates, an anabolic population highly sensitive to amino acids and insulin. Therefore, in the current study, fasted pigs were infused intra-arterially with leucine (0, 200, or 400 micromol.kg(-1).h(-1)), and protein synthesis was measured after 60 or 120 min. Protein synthesis was increased in muscle, but not in liver, at 60 min. At 120 min, however, protein synthesis returned to baseline levels in muscle but was reduced below baseline values in liver. The increase in protein synthesis in muscle was associated with increased plasma leucine of 1.5- to 3-fold and no change in plasma insulin. Leucine infusion for 120 min reduced plasma essential amino acid levels. Phosphorylation of eukaryotic initiation factor (eIF)-4E-binding protein-1 (4E-BP1), ribosomal protein (rp) S6 kinase, and rpS6 was increased, and the amount of eIF4E associated with its repressor 4E-BP1 was reduced after 60 and 120 min of leucine infusion. No change in these biomarkers of mRNA translation was observed in liver. Thus a physiological increase in plasma leucine stimulates protein synthesis in skeletal muscle of neonatal pigs in association with increased eIF4E availability for eIF4F assembly. This response appears to be insulin independent, substrate dependent, and tissue specific. The results suggest that the branched-chain amino acid leucine can act as a nutrient signal to stimulate protein synthesis in skeletal muscle of neonates.

Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle.

Amino acids and insulin have anabolic effects in skeletal muscle, but the mechanisms are poorly understood. To test the hypothesis that leucine and insulin stimulate translation initiation in human skeletal muscle by phosphorylating 70-kDa ribosomal protein S6 kinase (p70(S6k)), we infused healthy adults with leucine alone (n = 6), insulin alone (n = 6), or both leucine and insulin (n = 6) for 2 h. p70(S6k) and protein kinase B (PKB) serine(473) phosphorylation were measured in vastus lateralis muscles. Plasma leucine increased from approximately 116 to 343 micromol/l during the leucine-alone and leucine + insulin infusions. Plasma insulin increased to approximately 400 pmol/l during the insulin-alone and leucine + insulin infusions and was unchanged during the leucine-alone infusion. Phosphorylation of p70(S6k) increased 4-fold in response to leucine alone, 8-fold in response to insulin alone, and 18-fold after the leucine + insulin infusion. Insulin-alone and leucine + insulin infusions increased PKB phosphorylation, but leucine alone had no effect. These results show that physiological concentrations of leucine and insulin activate a key mediator of protein synthesis in human skeletal muscle. They suggest that leucine stimulates protein synthesis through a nutrient signaling mechanism independent of insulin, raising the possibility that administration of branched-chain amino acids may improve protein synthesis in insulin-resistant states.

Signaling Pathways Involved in Translational Control of Protein Synthesis in Skeletal Muscle by Leucine

Numerous reports established that in skeletal muscle the indispensable branched-chain amino acid leucine is unique in its ability to initiate signal transduction pathways that modulate translation initiation. Oral administration of leucine stimulates protein synthesis in association with hyperphosphorylation of the translational repressor, eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1), resulting in enhanced availability of the mRNA cap-binding protein eIF4E, for binding eIF4G and forming the active eIF4F complex. In addition, leucine enhances phosphorylation of the 70-kDa ribosomal protein S6 kinase (S6K1). These results suggest that leucine upregulates protein synthesis in skeletal muscle by enhancing both the activity and synthesis of proteins involved in mRNA translation. The stimulatory effects of leucine on translation initiation are mediated in part through the protein kinase mammalian target of rapamycin (mTOR), where both insulin signaling and leucine signaling converge to promote a maximal response.

Orally Administered Leucine Stimulates Protein Synthesis in Skeletal Muscle of Postabsorptive Rats in Association with Increased eIF4F Formation

We investigated the protein synthetic response of skeletal muscle to an orally administered dose of leucine given alone or in combination with carbohydrate. Male rats were freely fed (F) or food deprived for 18 h; food-deprived rats were then administered saline (S), carbohydrate (CHO), leucine (L) or a combination of carbohydrate plus leucine (CL). CHO and CL meals were isocaloric and provided 15% of daily energy requirements. L and CL meals each delivered 270 mg leucine. Muscle protein synthesis in S was 65% of F (P<0.01) 1 h after meal administration. Concomitant with lower rates of protein synthesis, phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), was less in S, leading to greater association of 4E-BP1.eIF4E, and reduced formation of the active eIF4G.eIF4E complex compared with F (P<0.01). Oral administration of leucine (L or CL), but not CHO, restored protein synthesis equal to that in F and resulted in 4E-BP1 phosphorylation that was threefold greater than that of S (P<0.01). Consequently, formation of 4E-BP1.eIF4E was inhibited and eIF4G.eIF4E was not different from F. The amount of eIF4E in the phosphorylated form was greater in S and CHO (P<0.01) than in all other groups. In contrast, no differences in the phosphorylation state of eIF2alpha or the activity of eIF2B were noted among treatment groups. Serum insulin was elevated 2.6- and 3.7-fold in CHO and CL, respectively, but was not different in L, compared with S (P<0.05). These results suggest that leucine stimulates protein synthesis in skeletal muscle by enhancing eIF4F formation independently of increases in serum insulin.

Effects of Leucine on in Vitro Protein Synthesis and Degradation in Rat Skeletal Muscles

Weanling rats were used to examine the role of leucine in in vitro protein turnover in skeletal muscles. In three experiments, rats were subjected to 24 or 72 hours of food deprivation or 5 days of consuming a protein-free diet or injection of streptozotocin. The soleus and extensor digitorum longus muscles were removed and utilized for measures of protein synthesis or degradation by using the isolated, incubated muscle technique of Li et al. These experiments demonstrate that supplementation of the incubation media with 0.5 mM leucine stimulates protein synthesis in these catabolic muscles and that during total starvation the stimulation decreases as the severity of the condition increases. Leucine supplementation failed to affect protein degradation in these skeletal muscles. This study demonstrates that the branched-chain amino acid leucine has the potential to stimulate protein synthesis in skeletal muscles, at least under specific catabolic conditions, but does not affect protein degradation in skeletal muscles under the conditions studied.