Skeletal Muscle Of Neonatal Pigs Research Articles

Prematurity blunts protein synthesis in skeletal muscle independently of body weight in neonatal pigs

BackgroundPostnatal growth failure in premature infants is associated with reduced lean mass accretion. Prematurity impairs the feeding-induced stimulation of translation initiation and protein synthesis in the skeletal muscle of neonatal pigs. The objective was to determine whether body weight independently contributes to the blunted postprandial protein synthesis.MethodsPreterm and term pigs that were either fasted or fed were stratified into quartiles according to birth weight to yield preterm and term groups of similar body weight; first and second quartiles of preterm pigs and third and fourth quartiles of term pigs were compared (preterm-fasted, n = 23; preterm-fed, n = 25; term-fasted, n = 21; term-fed, n = 21). Protein synthesis rates and mechanistic target of rapamycin complex 1 (mTORC1) activation in skeletal muscle were determined.ResultsRelative body weight gain was lower in preterm compared to term pigs. Prematurity attenuated the feeding-induced increase in mTORC1 activation in longissimus dorsi and gastrocnemius muscles (P < 0.05). Protein synthesis in gastrocnemius (P < 0.01), but not in longissimus dorsi muscle, was blunted by preterm birth.ConclusionA lower capacity of skeletal muscle to respond adequately to feeding may contribute to reduced body weight gain and lean mass accretion in preterm infants.ImpactThis study has shown that the feeding-induced increase in protein synthesis of skeletal and cardiac muscle is blunted in neonatal pigs born preterm compared to pigs born at term independently of birth weight.These findings support the notion that preterm birth, and not low birth weight, impairs the capacity of skeletal and cardiac muscle to upregulate mechanistic target of rapamycin-dependent anabolic signaling pathways and protein synthesis in response to the postprandial increase in insulin and amino acids.These observations suggest that a blunted anabolic response to feeding contributes to reduced lean mass accretion and altered body composition in preterm infants.

255 Leucine Supplementation During Continuous Feeding Enhances Lean Growth in a Preterm Piglet Model

Abstract Extrauterine growth restriction is a common complication of preterm birth and is associated with reduced lean growth and long-term morbidities. We previously showed that intermittent pulses of leucine during continuous orogastric feeding increases mTORC1 signaling to protein synthesis in skeletal muscle of neonatal pigs born at term. The objective of this study was to determine the extent to which leucine pulses during continuous feeding would promote lean growth by enhancing mTORC1 signaling in a preterm piglet model. Pigs delivered by cesarean section at 105 d gestation were continuously fed a protein and energy balanced milk-replacer diet [195 kcal ME and 13.5 g protein (kg body weight; BW)-1·d-1)]. Pigs (n=11-12) were randomly assigned to Leucine (LEU) or Alanine (ALA, isonitrogenous control) groups and treatments were administered as a pulse (1.6 mmol·kg BW-1·h-1) for 1 h every 4 h for 21 d. Body composition was determined by DXA and indices of amino acid signaling and mTORC1 activation were determined postprandially, 60 min after initiation of the last pulse. LEU pigs had a 10% greater average daily gain (ADG) (P &amp;lt; 0.05) than ALA pigs. Total lean mass tended to be greater (+13%; P &amp;lt; 0.06) in LEU compared with ALA, and longissimus dorsi muscle weight was 17% heavier in LEU than ALA pigs (P=0.01). Indices of mTORC1 activation, i.e., phosphorylation of S6K1 and 4EBP1 and abundance of the eIF4E-eIF4G complex, were increased in longissimus dorsi and gastrocnemius muscle of LEU compared with ALA pigs (P &amp;lt; 0.05). These results show that leucine supplementation during continuous feeding enhances mTORC1-activated translation initiation in skeletal muscle leading to an increase in muscle mass and lean growth in a preterm piglet model. Research was supported by NIH and USDA.

Prematurity Attenuates Skeletal Muscle Anabolism of Neonatal Pigs Independently of Birth Weight

ObjectivesPremature infants are at risk for postnatal growth failure that reduces lifelong lean mass. We have shown previously that preterm birth blunts the feeding-induced activation of the mechanistic target of rapamycin (mTOR) signaling pathway, translation initiation, and protein synthesis in skeletal muscle of neonatal pigs. Considering that body weight can vary widely at birth, we sought to separate the effect of prematurity from birth weight on the anabolic response to feeding. MethodsPigs were delivered by cesarean section at preterm (103 d) or term (112 d) gestation and fed parenterally for 4 d. On day 4, pigs were fasted for 4 h and were either fasted one additional hour or fed by oral gavage, yielding four groups: 1) preterm fasted (PT-FAST); 2) preterm fed (PT-FED); 3) term fasted (T-FAST); and 4) term fed (T-FED). All pigs were injected with a flooding dose of L-[4-3H]Phe to determine fractional protein synthesis rates and euthanized 60 min after feeding or the additional fast. Pigs were stratified by birth weight such that the highest birth weight preterm pigs were compared with the lowest birth weight term pigs (n = 10–13 per group). ResultsDespite similar birth weight in preterm and term pigs (1035 vs 993 ± 54 g, P > 0.10), relative weight gain of preterm was 33% lower than term pigs (22.0 vs. 32.9 ± 1.6 g·kg–1·d–1, P < 0.001). Longissimus dorsi (LD), gastrocnemius, and diaphragm protein synthesis rates were not different between PT-FAST and T-FAST groups. Gastrocnemius protein synthesis was 18% lower in PT-FED than T-FED group (P < 0.01), but protein synthesis was not different between PT-FED and T-FED groups in LD and diaphragm muscles. The abundance of the active eIF4E·eIF4G complex and phosphorylation of 4E-BP1 and S6K1, readouts of mTOR activation and translation initiation signaling, were increased after feeding in all muscles (P < 0.001). Translation initiation signaling was lower in LD and gastrocnemius muscles in PT-FED than T-FED groups (P < 0.05) but was not different in diaphragm muscle (P > 0.10). ConclusionsThese findings imply that prematurity, independent of birth weight, blunts mTOR activation and protein synthesis after feeding. The blunted anabolic response to feeding likely underpins postnatal growth failure often observed in infants born preterm. Funding SourcesNIH and USDA.

Intermittent Bolus Compared With Continuous Feeding Enhances Insulin and Amino Acid Signaling to Translation Initiation in Skeletal Muscle of Neonatal Pigs

BackgroundNutrition administered as intermittent bolus feeds rather than continuously promotes greater protein synthesis rates in skeletal muscle and enhances lean growth in a neonatal piglet model. The molecular mechanisms responsible remain unclear. ObjectivesWe aimed to identify the insulin- and/or amino acid-signaling components involved in the enhanced stimulation of skeletal muscle by intermittent bolus compared to continuous feeding in neonatal pigs born at term. MethodsTerm piglets (2–3 days old) were fed equal amounts of sow milk replacer [12.8 g protein and 155 kcal/(kg body weight · d)] by orogastric tube as intermittent bolus meals every 4 hours (INT) or by continuous infusion (CTS). After 21 days, gastrocnemius muscle samples were collected from CTS, INT-0 (before a meal), and INT-60 (60 minutes after a meal) groups (n = 6/group). Insulin- and amino acid-signaling components relevant to mechanistic target of rapamycin complex (mTORC) 1 activation and protein translation were measured. ResultsPhosphorylation of the insulin receptor, IRS-1, PDK1, mTORC2, pan-Akt, Akt1, Akt2, and TSC2 was 106% to 273% higher in the skeletal muscle of INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but phosphorylation of PTEN, PP2A, Akt3, ERK1/2, and AMPK did not differ among groups, nor did abundances of PHLPP, SHIP2, and Ubl4A. The association of GATOR2 with Sestrin1/2, but not CASTOR1, was 51% to 52% lower in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but the abundances of SLC7A5/LAT1, SLC38A2/SNAT2, SLC38A9, Lamtor1/2, and V-ATPase did not differ. Associations of mTOR with RagA, RagC, and Rheb and phosphorylation of S6K1 and 4EBP1, but not eIF2α and eEF2, were 101% to 176% higher in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05). ConclusionsThe enhanced rates of muscle protein synthesis and growth with intermittent bolus compared to continuous feeding in a neonatal piglet model can be explained by enhanced activation of both the insulin- and amino acid-signaling pathways that regulate translation initiation.

Intermittent Bolus Compared with Continuous Feeding Promotes Higher Activation of Insulin and Amino Acid Signaling to Protein Synthesis in Skeletal Muscle of Neonatal Pigs

Intermittent Bolus Compared with Continuous Feeding Promotes Higher Activation of Insulin and Amino Acid Signaling to Protein Synthesis in Skeletal Muscle of Neonatal Pigs

Prematurity blunts the insulin- and amino acid-induced stimulation of translation initiation and protein synthesis in skeletal muscle of neonatal pigs.

Extrauterine growth restriction in premature infants is largely attributed to reduced lean mass accretion and is associated with long-term morbidities. Previously, we demonstrated that prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in skeletal muscle of neonatal pigs. The objective of the current study was to determine whether the blunted feeding response is mediated by reduced responsiveness to insulin, amino acids, or both. Pigs delivered by cesarean section preterm (PT; 103 days, n = 25) or at term (T; 112 days, n = 26) were subject to euinsulinemic-euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps four days after delivery. Indices of mechanistic target of rapamycin complex 1 (mTORC1) signaling and fractional protein synthesis rates were measured after 2 h. Although longissimus dorsi (LD) muscle protein synthesis increased in response to both INS and AA, the increase was 28% lower in PT than in T. Upstream of mTORC1, Akt phosphorylation, an index of insulin signaling, was increased with INS but was 40% less in PT than in T. The abundances of mTOR·RagA and mTOR·RagC, indices of amino acid signaling, increased with AA but were 25% less in PT than in T. Downstream of mTORC1, eIF4E·eIF4G abundance was increased by both INS and AA but attenuated by prematurity. These results suggest that preterm birth blunts both insulin- and amino acid-induced activation of mTORC1 and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the high prevalence of extrauterine growth restriction in prematurity.NEW & NOTEWORTHY Extrauterine growth faltering is a major complication of premature birth, but the underlying cause is poorly understood. Our results demonstrate that preterm birth blunts both the insulin-and amino acid-induced activation of mTORC1-dependent translation initiation and protein synthesis in skeletal muscle, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced accretion of lean mass and extrauterine growth restriction of premature infants.

Global DNA methylation pattern involved in the modulation of differentiation potential of adipogenic and myogenic precursors in skeletal muscle of pigs

BackgroundSkeletal muscle is a complex and heterogeneous tissue accounting for approximately 40% of body weight. Excessive ectopic lipid accumulation in the muscle fascicle would undermine the integrity of skeletal muscle in humans but endow muscle with marbling-related characteristics in farm animals. Therefore, the balance of myogenesis and adipogenesis is of great significance for skeletal muscle homeostasis. Significant DNA methylation occurs during myogenesis and adipogenesis; however, DNA methylation pattern of myogenic and adipogenic precursors derived from skeletal muscle remains unknown yet.MethodsIn this study, reduced representation bisulfite sequencing was performed to analyze genome-wide DNA methylation of adipogenic and myogenic precursors derived from the skeletal muscle of neonatal pigs. Integrated analysis of DNA methylation and transcription profiles was further conducted. Based on the results of pathway enrichment analysis, myogenic precursors were transfected with CACNA2D2-overexpression plasmids to explore the function of CACNA2D2 in myogenic differentiation.ResultsAs a result, 11,361 differentially methylated regions mainly located in intergenic region and introns were identified. Furthermore, 153 genes with different DNA methylation and gene expression level between adipogenic and myogenic precursors were characterized. Subsequently, pathway enrichment analysis revealed that DNA methylation programing was involved in the regulation of adipogenic and myogenic differentiation potential through mediating the crosstalk among pathways including focal adhesion, regulation of actin cytoskeleton, MAPK signaling pathway, and calcium signaling pathway. In particular, we characterized a new role of CACNA2D2 in inhibiting myogenic differentiation by suppressing JNK/MAPK signaling pathway.ConclusionsThis study depicted a comprehensive landmark of DNA methylome of skeletal muscle-derived myogenic and adipogenic precursors, highlighted the critical role of CACNA2D2 in regulating myogenic differentiation, and illustrated the possible regulatory ways of DNA methylation on cell fate commitment and skeletal muscle homeostasis.

100 President Oral Presentation Pick: Prematurity Blunts Protein Synthesis in Skeletal Muscle of Neonatal Pigs Independently of Birth Weight

Abstract Postnatal growth failure reduces lean mass accretion and increases adiposity. Previously we showed that preterm birth impairs the feeding-induced stimulation of mechanistic target of rapamycin (mTOR), translation initiation, and protein synthesis in skeletal muscle of neonatal pigs. Considering that pig body weight can vary widely at birth, we wished to differentiate the effect of prematurity versus BW on these responses. Pigs delivered by caesarean section at preterm (103 d) or term (112 d) gestation were fed parenterally for 4 d. On day 4, all pigs were fasted for 4 h and were either fasted one additional hour or fed an elemental meal by oral gavage, yielding four treatment groups: preterm fasted, preterm fed, term fasted, and term fed. A flooding dose of L-[4-3H]Phe was injected into pigs 30 min after feeding (30 min before euthanasia). Preterm and term pigs were stratified according to birth weight such that preterm pigs with the highest birth weight were compared to term pigs with the lowest birth weight (n = 10–13 per group). Despite similar birth weight of pigs in these two subgroups, relative body weight gain of preterm pigs was 33% lower than term pigs (22.0 vs. 32.9 ± 1.6 g·kg-1·d-1, P &amp;lt; 0.001). Gastrocnemius protein synthesis increased after feeding in both preterm and term pigs, but the increase was 18% lower in preterm than in term pigs pigs (Table 1). Feeding-induced AKT phosphorylation and mTOR-dependent 4EBP1 and S6K1 phosphorylation mirrored the pattern in protein synthesis. These results suggest that prematurity (rather than low birth weight, which is frequently associated with preterm birth) perturbs mTOR activation and protein synthesis after feeding. However, it is unclear whether an inadequate response to feeding also underpins poor growth in intrauterine growth restricted piglets. Research was supported by NIH and USDA.

146 Prematurity Blunts the Protein Synthetic Response of Skeletal Muscle to Insulin and Amino Acids

Abstract Livestock animals are important dual-purpose models that benefit both agricultural and biomedical research. The neonatal pig is an appropriate model for the human infant to assess long-term effects of early life nutrition on growth and metabolic outcomes. Previously we have demonstrated that prematurity blunts the feeding-induced stimulation of translation initiation and protein synthesis in skeletal muscle of neonatal pigs. The objective of this study was to determine whether reduced sensitivity to insulin and/or amino acids drives this blunted response. Pigs were delivered by caesarean section at preterm (PT, 103 d gestation) or at term (T, 112 d gestation) and fed parenterally for 4 d. On day 4, pigs were subject to euinsulinemic-euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps for 120 min, yielding six treatments: PT-FAST (n = 7), PT-INS (n = 9), PT-AA (n = 9), T-FAST (n = 8), T-INS (n = 9), and T-AA (n = 9). A flooding dose of L-[4-3H]Phe was injected into pigs 30 min before euthanasia. Birth weight and relative body weight gain were lower in PT than T pigs (P &amp;lt; 0.001). Plasma insulin concentration was increased from ~3 to ~100 µU/mL in INS compared to FAST and AA pigs (P &amp;lt; 0.001); plasma BCAA concentration was increased from ~250 to ~1,000 µmol/L in AA compared to FAST and INS pigs (P &amp;lt; 0.001). Despite achieving similar insulin and amino acid levels, longissimus dorsi AKT phosphorylation, mechanistic target of rapamycin (mTOR)·Rheb abundance, mTOR activation, and protein synthesis were lower in PT-INS than T-INS pigs (Table 1). Although amino-acid induced dissociation of Sestrin2 from GATOR2 was not affected by prematurity, mTOR·RagA abundance, mTOR·RagC abundance, mTOR activation, and protein synthesis were lower in PT-AA than T-AA pigs. The impaired capacity of premature skeletal muscle to respond to insulin or amino acids and promote protein synthesis likely contributes to reduced lean mass accretion. Research was supported by NIH and USDA.

Differential regulation of mTORC1 activation by leucine and β-hydroxy-β-methylbutyrate in skeletal muscle of neonatal pigs.

Leucine (Leu) and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent protein synthesis in the skeletal muscle of neonatal pigs. This study aimed to determine whether HMB and Leu utilize common nutrient-sensing mechanisms to activate mTORC1. In study 1, neonatal pigs were fed one of five diets for 24 h: low protein (LP), high protein (HP), or LP supplemented with 4 (LP+HMB4), 40 (LP+HMB40), or 80 (LP+HMB80) μmol HMB·kg body wt-1·day-1. In study 2, neonatal pigs were fed for 24 h: LP, LP supplemented with Leu (LP+Leu), or HP diets delivering 9, 18, and 18 mmol Leu·kg body wt-1·day-1, respectively. The upstream signaling molecules that regulate mTORC1 activity were analyzed. mTOR phosphorylation on Ser2448 and Ser2481 was greater in LP+HMB40, LP+HMB80, and LP+Leu than in LP and greater in HP than in HMB-supplemented groups (P < 0.05), whereas HP and LP+Leu were similar. Rheb-mTOR complex formation was lower in LP than in HP (P < 0.05), with no enhancement by HMB or Leu supplementation. The Sestrin2-GATOR2 complex was more abundant in LP than in HP and was reduced by Leu (P < 0.05) but not HMB supplementation. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than in LP and HMB groups (P < 0.05). There were no treatment differences in RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex abundances. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was lower in LP than HP (P < 0.05) and unaffected by HMB or Leu supplementation. Our results demonstrate that HMB stimulates mTORC1 activation in neonatal muscle independent of the leucine-sensing pathway mediated by Sestrin2 and the Rag proteins.NEW & NOTEWORTHY Dietary supplementation with either leucine or its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulates protein synthesis in skeletal muscle of the neonatal pig. Our results demonstrate that both leucine and HMB stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) phosphorylation in neonatal muscle. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased binding of Rag A/C to mTOR. However, HMB's activation of mTORC1 is independent of this leucine-sensing pathway.

Regulation of Leucine‐induced mTORC1 Activation in Skeletal Muscle of Neonatal Pigs

BackgroundLeucine acts as anabolic agent that stimulates protein synthesis in skeletal muscle via activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Studies using cell cultures have proposed several models of the leucine‐induced activation of mTORC1 that involve Sestrin2, leucyl‐tRNA synthetase (LRS), and SH3 domain binding protein 4 (SH3BP4). In the first model, Sestrin2 (a GATOR2‐interacting protein) is a leucine sensor upstream of mTORC1. Lack of leucine causes Sestrin2 to interact with GATOR2, leading to inhibition of mTORC1. Under leucine‐rich condition, the binding of leucine to Sestrin2 disturbs the Sestrin2‐GATOR2 complex resulting in the activation of mTORC1. In a second proposed model, LRS acts as a leucine sensor that positively regulates mTORC1. In this model, there are two proposed pathways involving RagD and Vps34. In the first pathway, leucine binding to LRS stimulates LRS‐RagD interaction, which then promotes activation of mTORC1. In the second pathway, Vps34 mediates amino acid signaling to mTORC1. Leucine‐induced LRS‐Vps34 interaction is necessary for Vps34 activation towards Phospholipase D 1 (PLD1). PLD1 is essential for mTORC1 activation. In the third model, the binding of SH3BP4 to RagB acts as a potent inhibitor of amino acid‐induced activation of mTORC1. Leucine obstructs the interaction of SH3BP4 with RagB, allowing RagB to become fully active and participate in mTORC1 activation.ObjectiveWe aimed to determine whether these components participate in the leucine‐induced stimulation of mTORC1 in skeletal muscle in vivo.MethodsFasted 5‐d‐old piglets were gavage fed every 4 hours either: 1) low protein diet (LP), 2) LP supplemented with Leu (LP+Leu), or 3) high protein diet (HP). Diets were isocaloric and lactose was equal. At 25 h, piglets were injected with L[4‐3H] phenylalanine to measure fractional protein synthesis rates and killed 30 min later. Leucine action toward mTORC1 signaling was determined by measuring mTORC1 activation (mTORC1‐associated mTOR Ser‐2481 autophosphorylation) and the protein‐protein interactions among purported regulatory components of mTORC1 (Sestrin2‐Mios [GATOR2], RagA‐mTOR, RagC‐mTOR, RagB‐SH3BP4, RagD‐LRS, and Vps34‐LRS).ResultsMuscle protein synthesis and mTORC1 activation were higher in the LP+Leu and the HP groups than the LP group. The abundance of the Sestrin2‐GATOR2 complex was lower, and RagA‐mTOR and RagC‐mTOR complexes were greater in the LP+Leu and the HP groups than the LP group. RagB‐SH3BP4, RagD‐LRS, and Vps34‐LRS abundances did not differ among groups.ConclusionSestrin2 and the Rag proteins, but not LRS and SH3BP4, are crucial for the leucine‐induced activation of mTORC1 in skeletal muscle of neonatal pigs.Support or Funding InformationNIH AR444474, NIH HD085573, NIH HD072891, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055.

Insulin modulates energy and substrate sensing and protein catabolism induced by chronic peritonitis in skeletal muscle of neonatal pigs.

Acute infection promotes skeletal muscle wasting and insulin resistance, but the effect of insulin on energy and substrate sensing in skeletal muscle of chronically infected neonates has not been studied. Eighteen 2-d-old pigs underwent cecal ligation and puncture (CLP) or sham surgery (CON) to induce a chronic infection for 5 d. On d 5, pancreatic-substrate clamps were performed to attain fasting or fed insulin levels but to maintain glucose and amino acids in the fasting range. Total fractional protein synthesis rates (Ks), translational control mechanisms, and energy sensing and degradation signal activation were measured in longissimus dorsi muscle. In fasting conditions, CLP reduced Ks and sirtuin 1 (SIRT1) and increased AMP-activated protein kinase α (AMPKα) activation and muscle RING-finger protein-1 (MuRF1). Insulin treatment increased Ks and mitochondrial protein synthesis, enhanced translation activation, and reduced SIRT1 in CON. In contrast, in CLP, insulin treatment increased Ks, protein kinase B (PKB) and Forkhead box O1 phosphorylation, antagonized AMPK activation, and decreased peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), MuRF1, and SIRT1. Energy and substrate sensing in skeletal muscle by the PKB-AMPK-SIRT1-PGC-1α axis is impacted by chronic infection in neonatal pigs and can be modulated by insulin.

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.

Long‐term Leucine and BCAA Inclusion in a 30% Protein and Energy Restricted Diet Increases mTORC1 Signaling in Skeletal Muscle of Neonatal Pigs

Suboptimal nutrient intake during the shift from parenteral to oral feeding represents a limiting factor for growth of low‐birth weight infants. Leucine has been shown to have anabolic properties in skeletal muscle and can promote lean gain when supplemented to neonatal pigs fed a milk replacement formula. The objective of this study was to test if enteral leucine supplementation alone, or in combination with isoleucine and valine, can increase muscle protein synthesis in neonate pigs who consume 30% less crude protein (CP) and metabolizable energy than their requirements, and whether this effect is mediated through changes in muscle protein synthesis and degradation pathways, and amino acid (AA) transporter expression. Piglets (4‐d‐old) were fed by gastric tube 1 of 4 diets providing (kg BW/d) 15 g CP and 215 kcal (CON), 10.5 g CP and 156 kcal (R), 10.5 g CP + 1.19 g leucine and 156 kcal (RL), or 10.5 g CP + 1.67 g branched‐chain amino acids and 156 kcal (RBCAA) at 4‐h intervals for 9 d (n = 24). Body weights (BW) were recorded on d 0, 3, 6, and 9. On d 9, plasma AA and insulin concentrations and muscle protein synthesis rate, abundance of Na+‐coupled neutral AA transporter (SNAT2) and L‐type AA transporter (LAT1), and phosphorylation and/or abundance of insulin receptor substrate 1 (IRS‐1, Ser1101), protein kinase B (PKB), S6 kinase (S6K1), 4E‐binding protein (4EBP1), eukaryotic initiation factor 4 complex (eIF4E·eIF4G), forkhead box O (FOXO1, Ser256), atrophy F‐Box/Atrogin‐1 and muscle RING‐finger protein (MuRF1) were determined post‐feeding. Plasma leucine levels were greater in RL and RBCAA compared to CON (P ≤ 0.01). BW gain and muscle protein synthesis rate, phosphorylation of S6K1 and 4EBP1, and formation of eIF4E·eIF4G were lower in R, RL, and RBCAA than CON (P &lt; 0.01). RBCAA and RL upregulated (P ≤ 0.01) S6K1 and 4EBP1 phosphorylation and eIF4E·eIF4G formation compared to R. Phosphorylation of FOXO1 was lower in R compared to CON (P ≤ 0.05). There were no differences in phosphorylation and/or abundance of IRS‐1, PKB, SNAT2, LAT1, atrogin‐1 and MURF1 among diets. In conclusion, when protein and energy intakes are restricted, leucine or BCAA supplementation for 9 d increases the activation of the mammalian target of rapamycin pathway, but does not enhance skeletal muscle protein synthesis or promote body weight gain in neonatal pigs. Furthermore, protein degradation signaling, AA transporter abundance, and insulin signaling are unaffected by leucine or BCAA supplementation of a protein and energy restricted diet suggesting that when both dietary protein and/or energy supplies are deficient, the anabolic effect of leucine or BCAA supplementation on muscle protein accretion is limited.Support or Funding InformationNIH AR444474, NIH HD072891, NIH AR46308, USDA NIFA 2013‐67015‐20438, and USDA/ARS 6250‐51000‐055

Effects of Dietary Supplementation of β-hydroxy-β-methylbutyrate on Sow Performance and mRNA Expression of Myogenic Markers in Skeletal Muscle of Neonatal Piglets.

The effects of dietary β-hydroxy-β-methylbutyrate (HMB) supplementation during gestation on reproductive performance of sows and the mRNA expression of myogenic markers in skeletal muscle of neonatal pigs were determined. At day 35 of gestation, a total of 20 sows (Landrace × Yorkshire, at third parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 4 g/day β-hydroxy-β-methylbutyrate calcium (HMB-Ca) until parturition. At parturition, the total and live litter size were not markedly different between treatments, however, the sows fed HMB diet had a decreased rate of stillborn piglets compared with the sows fed the control (CON) diets (p < 0.05). In addition, piglets from the sows fed HMB diet tended to have an increased birth weight (p = 0.08), and a reduced rate of low birth weight piglets (p = 0.05) compared with piglets from the CON sows. Nevertheless, lower feed intake during lactation was observed in the sows fed the HMB diet compared with those on the CON diet (p < 0.01). The relative weights of the longissimus dorsi (LD) and semitendinosus (ST) muscle were higher (p < 0.05) in neonatal pigs from the HMB than the CON sows. Furthermore, maternal HMB treatment increased the mRNA levels of the myogenic genes, including muscle regulatory factor-4 (MRF4, p < 0.05), myogenic differentiation factor (MyoD) and insulin-like growth factor-1 (IGF-1, p < 0.01). In conclusion, dietary HMB supplementation to sows at 4 g/day from day 35 of gestation to term significantly improves pregnancy outcomes and increases the expression of myogenic genes in skeletal muscle of neonatal piglets, but reduces feed intake of sows during lactation.

Leucine Antagonizes Protein Degradation Induced by Endotoxin in Skeletal Muscle of Neonatal Pigs

In neonatal animals, protein synthesis and degradation rates are high. Administration of the branched-chain amino acid, leucine (Leu), stimulates muscle protein synthesis in neonatal pigs but less ...

Regulation of the protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs (137.1)

The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin (Ins) and amino acids (AA), especially leucine (leu), modulate protein synthesis in skeletal muscle and how this changes with development. These studies aimed to determine whether Ins, AA or Leu alter the abundance and activation of ubiquitin‐proteasome, i.e., atrogin‐1 (MAFbx) and muscle RING‐finger protein‐1 (MuRF1), and autophagy‐lysosome, i.e., microtubule‐associated protein light chain 3 (LC3) and lysosomal‐associated membrane protein 2 (Lamp‐2), protein degradation systems and whether the responses change with age. In study 1, overnight fasted 6‐ and 26‐d‐old pigs were infused with saline or with insulin or AA to achieve fed levels. In study 2, overnight fasted 6‐d‐old pigs were infused with Leu while Ins and AA were kept at fasting levels. Abundance of atrogin‐1, but not MuRF1, was greater in 26‐ than in 6‐day‐old pigs (P&lt;0.05) and was not affected by Ins, AA, or Leu. Abundance of LC3 was higher in younger pigs and not affected by treatment (P&lt;0.05). LC3‐II/LC3‐I ratio was reduced by Ins, AA, and Leu (P&lt;0.05). This response was more profound in younger pigs. Abundance of Lamp‐2 was not affected by treatment or development. In conclusion, Ins, AA, and, in particular, Leu act as signals to modulate muscle protein degradation in neonates.Grant Funding Source: Supported by NIH AR444474 and USDA/ARS 6250‐51000‐055

Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

BackgroundThe rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the current study, we identified mechanisms involved in protein degradation regulation. In experiment 1, 6- and 26-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic, 2) euinsulinemic-euglycemic-hyperaminoacidemic, and 3) hyperinsulinemic-euglycemic-euaminoacidemic clamps for 2 h. In experiment 2, 5-d-old pigs were studied during 1) euinsulinemic-euglycemic-euaminoacidemic-euleucinemic, 2) euinsulinemic-euglycemic-hypoaminoacidemic-hyperleucinemic, and 3) euinsulinemic-euglycemic-euaminoacidemic-hyperleucinemic clamps for 24 h. We determined in muscle indices of ubiquitin-proteasome, i.e., atrogin-1 (MAFbx) and muscle RING-finger protein-1 (MuRF1) and autophagy-lysosome systems, i.e., unc51-like kinase 1 (UKL1), microtubule-associated protein light chain 3 (LC3), and lysosomal-associated membrane protein 2 (Lamp-2). For comparison, we measured ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (eIF4E) activation, components of translation initiation.ResultsAbundance of atrogin-1, but not MuRF1, was greater in 26- than 6-d-old pigs and was not affected by insulin, amino acids, or leucine. Abundance of ULK1 and LC3 was higher in younger pigs and not affected by treatment. The LC3-II/LC3-I ratio was reduced and ULK1 phosphorylation increased by insulin, amino acids, and leucine. These responses were more profound in younger pigs. Abundance of Lamp-2 was not affected by treatment or development. Abundance of eIF4E, but not rpS6, was higher in 6- than 26-d-old-pigs but unaffected by treatment. Phosphorylation of eIF4E was not affected by treatment, however, insulin, amino acids, and leucine stimulated rpS6 phosphorylation, and the responses decreased with development.ConclusionsThe rapid growth of neonatal muscle is in part due to the positive balance between the activation of protein synthesis and degradation signaling. Insulin, amino acids, and, particularly, leucine, act as signals to modulate muscle protein synthesis and degradation in neonates.

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.

Insulin ameliorates skeletal muscle degradation during sepsis in neonatal pigs

Insulin decreases whole body protein breakdown during sepsis in adolescents, but not in adults. Neonates have higher rates of muscle deposition, protein turnover, and response to insulin than adults. To determine the effects of insulin on skeletal muscle protein degradation during sepsis, neonatal pigs (n=16) underwent cecal ligation and puncture (CLP) or sham surgery and were pair‐gavage fed for 5 days. Pancreatic‐substrate clamps were performed for 2 h providing insulin to attain fasting or fed (+Ins) insulin levels and to maintain glucose and amino acids (AA) in the fasting range. Protein synthesis (PS) and protein degradation signals were examined in longissimus dorsi. Circulating insulin and total AA did not differ between CLP and Shams, but CLP had reduced plasma BCAA and Leu than Shams. CLP+Ins had lower AA deposition rates and higher circulating 3‐methylhistidine than Shams+Ins. Compared to Shams, CLP had decreased PS and Fox03 phosphorylation and increased AMPK phosphorylation and MuRF abundance in muscle. Compared to CLP, CLP+Ins had higher PS, PKB and Fox01 phosphorylation and caspase‐3 abundance, and lower PCG‐1α mRNA expression, AMPK phosphorylation and MuRF abundance in muscle. The results suggest that insulin anatagonizes the CLP‐induced decrease in PS and suppresses proteosomal degradation signal activation in skeletal muscle of neonatal pigs. NIH AR44474, NIH AR51563, and USDA 58–6250‐6–001