Renan A. Orellana

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.

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

Protein synthesis and eukaryotic initiation factor (eIF) activation are increased in skeletal muscle of neonatal pigs parenterally infused with amino acids. Leucine appears to be the most effective single amino acid to trigger these effects. To examine the response to enteral leucine supplementation, overnight food-deprived 5-d-old pigs were gavage fed at 0 and 60 min a: 1) low-protein diet (LP); 2) LP supplemented with leucine (LP+L) to equal leucine in the high-protein diet (HP); or 3) HP diet. Diets were isocaloric and equal in lactose. Fractional protein synthesis rates and translation initiation control mechanisms were examined in skeletal muscles and visceral tissues 90 min after feeding. Protein synthesis rates in longissimus dorsi, gastrocnemius, and masseter muscles, heart, jejunum, kidney, and pancreas, but not liver, were greater in the LP+L group compared with the LP group and did not differ from the HP group. Feeding LP+L and HP diets compared with the LP diet increased phosphorylation of mammalian target of rapamycin (mTOR), 4E-binding protein 1, ribosomal protein S6 kinase-1, and eIF4G and formation of the active eIF4E·eIF4G complex in longissimus dorsi muscle. In all tissues except liver, activation of mTOR effectors increased in pigs fed LP+L and HP vs. LP diets. Our results suggest that leucine supplementation of a low-protein meal stimulates protein synthesis in muscle and most visceral tissues to a rate similar to that achieved by feeding a high-protein meal and this stimulation involves activation of mTOR downstream effectors.

Stimulation of Muscle Protein Synthesis by Prolonged Parenteral Infusion of Leucine Is Dependent on Amino Acid Availability in Neonatal Pigs

The postprandial rise in amino acids, particularly leucine, stimulates muscle protein synthesis in neonates. Previously, we showed that a 1-h infusion of leucine increased protein synthesis, but this response was not sustained for 2 h unless the leucine-induced decrease in amino acids was prevented. To determine whether a parenteral leucine infusion can stimulate protein synthesis for a more prolonged, clinically relevant period if baseline amino acid concentrations are maintained, overnight food-deprived neonatal pigs were infused for 24 h with saline, leucine (400 mumol.kg(-1). h(-1)), or leucine with replacement amino acids. Amino acid replacement prevented the leucine-induced decrease in amino acids. Muscle protein synthesis was increased by leucine but only when other amino acids were supplied to maintain euaminoacidemia. Leucine did not affect activators of mammalian target of rapamycin (mTOR), i.e. protein kinase B, AMP-activated protein kinase, tuberous sclerosis complex 2, or eukaryotic elongation factor 2. There was no effect of treatment on the association of mTOR with regulatory associated protein of mammalian target of rapamycin (raptor), G-protein beta subunit-like protein, or rictor or the phosphorylation of raptor or proline-rich Akt substrate of 40 kDa. Phosphorylation of mTOR and its downstream targets, eukaryotic initiation factor (eIF) 4E binding protein and ribosomal protein S6 kinase, and the eIF4E . eIF4G association were increased and eIF2alpha phosphorylation was reduced by leucine and was not further altered by correcting for the leucine-induced hypoaminoacidemia. Thus, prolonged parenteral infusion of leucine activates mTOR and its downstream targets in neonatal skeletal muscle, but the stimulation of protein synthesis also is dependent upon amino acid availability.

Enteral leucine supplementation increases protein synthesis in skeletal and cardiac muscles and visceral tissues of neonatal pigs through mTORC1-dependent pathways

Introduction:Leucine (Leu) activates mammalian target of rapamycin (mTOR) to upregulate protein synthesis (PS).Results:PS in skeletal muscles, heart, liver, pancreas, and jejunum, but not kidney, were greater in low protein supplemented with Leu (LP+L) than LP, but lower than high protein (HP). In longissimus dorsi muscle, protein kinase B phosphorylation was similar in LP and LP+L, but lower than HP. Although less than HP, p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor (eIF) 4E binding protein 1 (4EBP1) association with regulatory associated protein of mammalian target of rapamycin was greater in LP+L than LP, resulting in higher S6K1 and 4EBP1 phosphorylation. Feeding LP+L vs. LP decreased 4EBP1·eIF4E and increased eIF4E·eIF4G formation, but not to HP. Similar results were obtained for S6K1 and 4EBP1 phosphorylation in gastrocnemius, masseter, heart, liver, pancreas, and jejunum, but not kidney. eIF2α and elongation factor 2 phosphorylation was unaffected by treatment.Dicussion:Our results suggest that enteral Leu supplementation of a low protein diet enhances PS in most tissues through mTOR complex 1 pathways.Methods:To examine enteral Leu effects on PS and signaling activation, 5-d-old piglets were fed for 24 h diets containing: (i) LP, (ii) LP+L, or (iii) HP.

Intermittent Bolus Feeding Has a Greater Stimulatory Effect on Protein Synthesis in Skeletal Muscle Than Continuous Feeding in Neonatal Pigs

Orogastric tube feeding, using either continuous or intermittent bolus delivery, is common in infants for whom normal feeding is contraindicated. To compare the impact of different feeding strategies on muscle protein synthesis, after withholding food overnight, neonatal pigs received a complete formula orally as a bolus feed every 4 h or were continuously fed. Protein synthesis rate and translational mechanisms in skeletal muscle were examined after 0, 24, and 25.5 h. Plasma amino acid and insulin concentrations increased minimally and remained constant in continuously fed compared to feed-deprived pigs; however, the pulsatile meal feeding pattern was mimicked in bolus-fed pigs. Muscle protein synthesis was stimulated by feeding and the greatest response occurred after a bolus meal. Bolus but not continuous feeds increased polysome aggregation, the phosphorylation of protein kinase B, tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kDa, eukaryotic initiation factor (eIF) 4E binding protein (4EBP1), and rp S6 kinase and enhanced dissociation of the 4EBP1 ·eIF4E complex and formation of the eIF4E ·eIF4G complex compared to feed deprivation (P < 0.05). Activation of insulin receptor substrate-1, regulatory associated protein of mammalian target of rapamycin, AMP-activated protein kinase, eukaryotic elongation factor 2, and eIF2α phosphorylation were unaffected by either feeding modality. These results suggest that in neonates, intermittent bolus feeding enhances muscle protein synthesis to a greater extent than continuous feeding by eliciting a pulsatile pattern of amino acid- and insulin-induced translation initiation.

Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs.

Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine infusion can be used to enhance protein synthesis during continuous feeding, neonatal piglets received the same amount of formula enterally by orogastric tube for 25.25 h continuously (CON) with or without LEU or intermittently by bolus every 4 h (BOL). For the CON+LEU group, leucine pulses were administered parenterally (800 μmol·kg(-1)·h(-1)) every 4 h. Insulin and glucose concentrations increased after the BOL meal and were unchanged in groups fed continuously. LEU infusion during CON feeding increased plasma leucine after the leucine pulse and decreased essential amino acids compared with CON feeding. Protein synthesis in longissimus dorsi (LD), gastrocnemius, and soleus muscles, but not liver or heart, were greater in CON+LEU and BOL than in the CON group. BOL feeding increased protein synthesis in the small intestine. Muscle S6K1 and 4E-BP1 phosphorylation and active eIF4E·eIF4G complex formation were higher in CON+LEU and BOL than in CON but AMPKα, eIF2α, and eEF2 phosphorylation were unchanged. LC3-II-to-total LC3 ratio was lower in CON+LEU and BOL than in CON, but there were no differences in atrogin-1 and MuRF-1 abundance and FoxO3 phosphorylation. In conclusion, administration of leucine pulses during continuous orogastric feeding in neonates increases muscle protein synthesis by stimulating translation initiation and may reduce protein degradation via the autophagy-lysosome, but not the ubiquitin-proteasome pathway.

Anabolic signaling and protein deposition are enhanced by intermittent compared with continuous feeding in skeletal muscle of neonates.

Orogastric tube feeding is indicated for neonates with impaired ability to ingest and can be administered by intermittent bolus or continuous schedule. Our aim was to determine whether feeding modalities affect muscle protein deposition and to identify mechanisms involved. Neonatal pigs were overnight fasted (FAS) or fed the same amount of food continuously (CON) or intermittently (INT; 7 × 4 h meals) for 29 h. For 8 h, between hours 20 and 28, pigs were infused with [(2)H(5)]phenylalanine and [(2)H(2)]tyrosine, and amino acid (AA) net balances were measured across the hindquarters. Insulin, branched-chain AA, phenylalanine, and tyrosine arterial concentrations and whole body phenylalanine and tyrosine fluxes were greater for INT after the meal than for CON or FAS. The activation of signaling proteins leading to initiation of mRNA translation, including eukaryotic initiation factor (eIF)4E·eIF4G complex formation in muscle, was enhanced by INT compared with CON feeding or FAS. Signaling proteins of protein degradation were not affected by feeding modalities except for microtubule-associated protein light chain 3-II, which was highest in the FAS. Across the hindquarters, AA net removal increased for INT but not for CON or FAS, with protein deposition greater for INT. This was because protein synthesis increased following feeding for INT but remained unchanged for CON and FAS, whereas there was no change in protein degradation across any dietary treatment. These results suggest that muscle protein accretion in neonates is enhanced with intermittent bolus to a greater extent than continuous feeding, mainly by increased protein synthesis.

Triennial Growth Symposium: leucine acts as a nutrient signal to stimulate protein synthesis in neonatal pigs.

The postprandial increases in AA and insulin independently stimulate protein synthesis in skeletal muscle of piglets. Leucine is an important mediator of the response to AA. We have shown that the postprandial increase in leucine, but not isoleucine or valine, acutely stimulates muscle protein synthesis in piglets. Leucine increases muscle protein synthesis by modulating the activation of mammalian target of rapamycin (mTOR) complex 1 and signaling components of translation initiation. Leucine increases the phosphorylation of mTOR, 70-kDa ribosomal protein S6 kinase-1, eukaryotic initiation factor (eIF) 4E-binding protein-1, and eIF4G; decreases eIF2α phosphorylation; and increases the association of eIF4E with eIF4G. However, leucine does not affect the upstream activators of mTOR, that is, protein kinase B, adenosine monophosphate-activated protein kinase, and tuberous sclerosis complex 1/2, or the activation of translation elongation regulator, eukaryotic elongation factor 2. The action of leucine can be replicated by α-ketoisocaproate but not by norleucine. Interference by rapamycin with the raptor-mTOR interaction blocks leucine-induced muscle protein synthesis. The acute leucine-induced stimulation of muscle protein synthesis is not maintained for prolonged periods, despite continued activation of mTOR signaling, because circulating AA fall as they are utilized for protein synthesis. However, when circulating AA concentrations are maintained, the leucine-induced stimulation of muscle protein synthesis is maintained for prolonged periods. Thus, leucine acts as a nutrient signal to stimulate translation initiation, but whether this translates into a prolonged increase in protein synthesis depends on the sustained availability of all AA.

Feeding Rapidly Stimulates Protein Synthesis in Skeletal Muscle of Neonatal Pigs by Enhancing Translation Initiation

Food consumption increases protein synthesis in most tissues by promoting translation initiation, and in the neonate, this increase is greatest in skeletal muscle. In this study, we aimed to identify the currently unknown time course of changes in the rate of protein synthesis and the activation of factors involved in translation in neonatal muscle after a meal. After overnight food deprivation, 36 5- to 7-d-old piglets were administered a nutritionally complete bolus i.g. meal and were killed immediately before or 30, 60, 90, 120, or 240 min later. The increase in skeletal muscle protein synthesis peaked 30 min after the meal and this was sustained through 120 min, returning to baseline thereafter. The relative proportion of polysomes to nonpolysomes was higher only after 30 min. Protein kinase B phosphorylation peaked 30 min after feeding and returned to baseline by 90 min. The phosphorylation of mammalian target of rapamycin, eukaryotic initiation factor (eIF) 4E binding protein (4E-BP1), ribosomal protein S6, and eIF4G was increased within 30 min of feeding and persisted through 120 min, but all had returned to baseline by 240 min. The association of 4E-BP1.eIF4E was reduced and eIF4E.eIF4G increased 30 min after receiving a meal, remaining so for 120 min, before returning to baseline at 240 min. Thus, in neonates, food consumption rapidly increased skeletal muscle protein synthesis by enhancing translation initiation and this increase was sustained for at least 120 min after the meal but returned to baseline by 240 min after the feeding.

Regulation of muscle protein synthesis in neonatal pigs during prolonged endotoxemia

In adults, protein synthesis in skeletal muscle is reduced by as much as 50% after a septic challenge, and is associated with repression of translation initiation. Neonates are highly anabolic and their muscle protein synthesis rates are elevated and uniquely sensitive to amino acid and insulin stimulation. In the present study, neonatal piglets were infused with Endotoxin (lipopolysaccharide, LPS) for 20 h at 0 (n = 6) and 13 μg/kg•h (n = 8). During the last 2 h, dextrose and an amino acid mixture were infused to attain fed plasma concentrations of amino acids, glucose, and insulin. Fractional protein synthesis rates and translational control mechanisms were examined. LPS reduced protein synthesis in glycolytic muscles by only 13% and had no significant effect in oxidative muscles. This depression was associated with reductions in the phosphorylation of 4E-BP1 (−31%) and S6 K1 (−78%), and a decrease in eIF4G binding to eIF4E (−62%), an event required for formation of the active mRNA binding complex. By comparison, LPS increased protein synthesis in the liver (+29%), spleen (+32%), and kidney (+27%), and in the liver, this increase was associated with augmented eIF4G to eIF4E binding (+88%). In muscle and liver, LPS did not alter eIF2B activity, an event that regulates initiator met-tRNAi binding to the 40S ribosomal complex. These findings suggest that during sustained endotoxemia, the high rate of neonatal muscle protein synthesis is largely maintained in the presence of substrate supply, despite profound changes in translation initiation factors that modulate the mRNA binding step in translation initiation.