Pulsatile Leucine Administration during Continuous Enteral Feeding Enhances Skeletal Muscle Mechanistic Target of Rapamycin Complex 1 Signaling and Protein Synthesis in a Preterm Piglet Model.

BACKGROUND: Continuous feeding does not elicit an optimal anabolic response in skeletal muscle but is required for some preterm infants. We reported previously that intermittent intravenous pulses of leucine (Leu; 800 µmol Leu·kg-1·h-1 every 4 h) to continuously fed pigs born at term promoted mechanistic target of rapamycin complex 1 (mTORC1) activation and protein synthesis in skeletal muscle. OBJECTIVES: The aim was to determine the extent to which intravenous Leu pulses activate mTORC1 and enhance protein synthesis in the skeletal muscle of continuously fed pigs born preterm. METHODS: Pigs delivered 10 d preterm was advanced to full oral feeding >4 d and then assigned to 1 of the following 4 treatments for 28 h: 1) ALA (continuous feeding; pulsed with 800 µmol alanine·kg-1·h-1 every 4 h; n = 8); 2) L1× (continuous feeding; pulsed with 800 µmol Leu·kg-1·h-1 every 4 h; n = 7); 3) L2× (continuous feeding; pulsed with 1600 µmol Leu·kg-1·h-1 every 4 h; n = 8); and 4) INT (intermittent feeding every 4 h; supplied with 800 µmol alanine·kg-1 per feeding; n = 7). Muscle protein synthesis rates were determined with L-[2H5-ring]Phenylalanine. The activation of insulin, amino acid, and translation initiation signaling pathways were assessed by Western blot. RESULTS: Peak plasma Leu concentrations were 134% and 420% greater in the L2× compared to the L1× and ALA groups, respectively (P < 0.01). Protein synthesis was greater in the L2× than in the ALA and L1× groups in both the longissimus dorsi and gastrocnemius muscles (P < 0.05) but not different from the INT group (P > 0.10). Amino acid signaling upstream and translation initiation signaling downstream of mTORC1 largely corresponded to the differences in protein synthesis. CONCLUSIONS: Intravenous Leu pulses potentiate mTORC1 activity and protein synthesis in the skeletal muscles of continuously fed preterm pigs, but the amount required is greater than in pigs born at term.

Preterm birth alters the feeding-induced activation of Akt signaling in the muscle of neonatal piglets.

BACKGROUND: Postnatal lean mass accretion is commonly reduced in preterm infants. This study investigated mechanisms involved in the blunted feeding-induced activation of Akt in the skeletal muscle of preterm pigs that contributes to lower protein synthesis rates. METHODS: On day 3 following cesarean section, preterm and term piglets were fasted or fed an enteral meal. Activation of Akt signaling pathways in skeletal muscle was determined. RESULTS: Akt1 and Akt2, but not Akt3, phosphorylation were lower in the skeletal muscle of preterm than in term pigs (P < 0.05). Activation of Akt-positive regulators, PDK1 and mTORC2, but not FAK, were lower in preterm than in term (P < 0.05). The formation of Akt complexes with GAPDH and Hsp90 and the abundance of Ubl4A were lower in preterm than in term (P < 0.05). The abundance of Akt inhibitors, PHLPP and SHIP2, but not PTEN and IP6K1, were higher in preterm than in term pigs (P < 0.05). PP2A activation was inhibited by feeding in term but not in preterm pigs (P < 0.05). CONCLUSIONS: Our results suggest that preterm birth impairs regulatory components involved in Akt activation, thereby limiting the anabolic response to feeding. This anabolic resistance likely contributes to the reduced lean accretion following preterm birth. IMPACT: The Akt-mTORC1 pathway plays an important role in the regulation of skeletal muscle protein synthesis in neonates. This is the first evidence to demonstrate that, following preterm birth, the postprandial activation of positive regulators of Akt in the skeletal muscle is reduced, whereas the activation of negative regulators of Akt is enhanced. This anabolic resistance of Akt signaling in response to feeding likely contributes to the reduced accretion of lean mass in premature infants. These results may provide potential novel molecular targets for intervention to enhance lean growth in preterm neonates.

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

BACKGROUND: Postnatal 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. METHODS: Preterm 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. RESULTS: Relative 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. CONCLUSION: A lower capacity of skeletal muscle to respond adequately to feeding may contribute to reduced body weight gain and lean mass accretion in preterm infants. IMPACT: This 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.

Intermittent bolus feeding does not enhance protein synthesis, myonuclear accretion, or lean growth more than continuous feeding in a premature piglet model.

Optimizing enteral nutrition for premature infants may help mitigate extrauterine growth restriction and adverse chronic health outcomes. Previously, we showed in neonatal pigs born at term that lean growth is enhanced by intermittent bolus compared with continuous feeding. The objective was to determine if prematurity impacts how body composition, muscle protein synthesis, and myonuclear accretion respond to feeding modality. Following preterm delivery, pigs were fed equivalent amounts of formula delivered either as intermittent boluses (INT; n = 30) or continuously (CONT; n = 14) for 21 days. Body composition was measured by dual-energy X-ray absorptiometry (DXA) and muscle growth was assessed by morphometry, myonuclear accretion, and satellite cell abundance. Tissue anabolic signaling and fractional protein synthesis rates were determined in INT pigs in postabsorptive (INT-PA) and postprandial (INT-PP) states and in CONT pigs. Body weight gain and composition did not differ between INT and CONT pigs. Longissimus dorsi (LD) protein synthesis was 34% greater in INT-PP than INT-PA pigs (P < 0.05) but was not different between INT-PP and CONT pigs. Phosphorylation of 4EBP1 and S6K1 and eIF4E·eIF4G abundance in LD paralleled changes in LD protein synthesis. Satellite cell abundance, myonuclear accretion, and fiber cross-sectional area in LD did not differ between groups. These results suggest that, unlike pigs born at term, intermittent bolus feeding does not enhance lean growth more than continuous feeding in pigs born preterm. Premature birth attenuates the capacity of skeletal muscle to respond to cyclical surges in insulin and amino acids with intermittent feeding in early postnatal life.NEW & NOTEWORTHY Extrauterine growth restriction often occurs in premature infants but may be mitigated by optimizing enteral feeding strategies. We show that intermittent bolus feeding does not increase skeletal muscle protein synthesis, myonuclear accretion, or lean growth more than continuous feeding in preterm pigs. This attenuated anabolic response of muscle to intermittent bolus feeding, compared with previous observations in pigs born at term, may contribute to deficits in lean mass that many premature infants exhibit into adulthood.

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.

Leucine Supplementation Does Not Restore Diminished Skeletal Muscle Satellite Cell Abundance and Myonuclear Accretion When Protein Intake Is Limiting in Neonatal Pigs.

BACKGROUND: Rapid growth of skeletal muscle in the neonate requires the coordination of protein deposition and myonuclear accretion. During this developmental stage, muscle protein synthesis is highly sensitive to amino acid supply, especially Leu, but we do not know if this is true for satellite cells, the source of muscle fiber myonuclei. OBJECTIVE: We examined whether dietary protein restriction reduces myonuclear accretion in the neonatal pig, and if any reduction in myonuclear accretion is mitigated by restoring Leu intake. METHODS: Neonatal pigs (1.53 ± 0.2 kg) were fitted with jugular vein and gastric catheters and fed 1 of 3 isoenergetic milk replacers every 4 h for 21 d: high protein [HP; 22.5 g protein/(kg/d); n= 8]; restricted protein [RP; 11.2 g protein/(kg/d); n= 10]; or restricted protein with Leu [RPL; 12.0 g protein/(kg/d); n= 10]. Pigs were administered 5-bromo-2'-deoxyuridine (BrdU; 15 mg/kg) intravenously every 12 h from days 6 to 8. Blood was sampled on days 6 and 21 to measure plasma Leu concentrations. On day 21, pigs were killed and the longissimus dorsi (LD) muscle was collected to measure cell morphometry, satellite cell abundance, myonuclear accretion, and insulin-like growth factor (IGF) system expression. RESULTS: Compared with HP pigs, postprandial plasma Leu concentration in RP pigs was 37% and 47% lower on days 6 and 21, respectively (P < 0.05); Leu supplementation in RPL pigs restored postprandial Leu to HP concentrations. Dietary protein restriction reduced LD myofiber cross-sectional area by 21%, satellite cell abundance by 35%, and BrdU+ myonuclear abundance by 25% (P < 0.05); Leu did not reverse these outcomes. Dietary protein restriction reduced LD muscle IGF2 expression by 60%, but not IGF1 or IGF1R expression (P < 0.05); Leu did not rescue IGF2 expression. CONCLUSIONS: Satellite cell abundance and myonuclear accretion in neonatal pigs are compromised when dietary protein intake is restricted and are not restored with Leu supplementation.

Dietary Leucine Supplementation Decreases Whole-Body Protein Turnover before, but Not during, Immune System Stimulation in Pigs.

BACKGROUND: Immune system stimulation (ISS) adversely affects protein metabolism and reduces pig productivity. Leu has a regulatory role in skeletal muscle and whole-body protein turnover, which may be affected by ISS. OBJECTIVE: We sought to determine the effect of supplemental Leu intake on whole-body protein turnover in pigs before and during ISS. METHODS: Pigs [mean ± SD initial body weight (BW): 10.6 ± 1.1 kg] were surgically fitted with jugular vein catheters and assigned to 1 of 3 treatments: 1.36% standardized ileal-digestible (SID) Leu (CON; n = 13); 2.04% SID Leu (LEU-M; n = 8); and 2.72% SID Leu (LEU-H; n = 7). Pigs were infused continuously with 0.66 ± 0.05 mmol 15N ⋅ kg BW-1 ⋅ d-1 to determine whole-body protein kinetics. The study consisted of a 72-h prechallenge period followed by a 36-h challenge period. At the start of the challenge period, ISS was induced in all LEU-M and LEU-H pigs and half of the CON pigs with LPS (ISS+); the remaining CON pigs were administered saline (ISS-). RESULTS: Whole-body protein synthesis (309, 273, and 260 ± 14 mmol N ⋅ kg BW-1 ⋅ d-1 for CON, LEU-M, and LEU-H pigs, respectively) and protein degradation (233, 203, and 185 ± 14 mmol N ⋅ kg BW-1 ⋅ d-1 for CON, LEU-M, and LEU-H pigs, respectively) were reduced with increasing Leu intake during the prechallenge period (P < 0.05). ISS reduced whole-body protein synthesis (203 compared with 169 ± 12 mmol N ⋅ kg BW-1 ⋅ d-1 for ISS- and ISS+ pigs fed CON, respectively; P < 0.05) and protein deposition (PD) (64.9 compared with 45.0 ± 2.9 mmol N ⋅ kg BW-1 ⋅ d-1 for ISS- and ISS+ pigs fed CON, respectively; P < 0.01), whereas ISS did not affect whole-body protein degradation. Leu intake did not affect whole-body protein synthesis or degradation in ISS+ pigs. CONCLUSIONS: Our results indicate that supplemental Leu intake improves the efficiency of PD rather than PD directly in healthy pigs but did not affect whole-body protein turnover during ISS.

An epidemiological investigation into the association between biomarkers and growth performance in nursery pigs.

BACKGROUND: Biomarkers are useful tools in research and clinical practice where they are often used to detect and monitor differences in the physiological state of an animal. The proteins IGF-1, IGFBP-3, GHR, CRP, SAA, Hp, IFN-α, IFN-γ, TNF-α, IL-1ß, IL-6, IL-10, and IL-18 have been proposed as potential biomarkers for monitoring growth in livestock. The objective of this study was to determine whether hepatic gene expression of these proposed biomarkers is associated with growth performance in nursery pigs. Herd information and growth parameters were collected for 168 piglets from 8 commercial farms in southern Ontario. From these pigs, a subset of liver tissue samples (n = 74) was used for gene expression analysis of the proposed biomarkers. Multivariable linear regression methods were used to determine whether genetic expression of the proposed biomarkers was associated with growth performance in the nursery. RESULTS: Modelling the herd information and individual piglet traits in relation to growth performance revealed that the weight at weaning and the age at weaning are significantly associated with nursery performance. Average daily gain (ADG) was significantly associated with hepatic IGFBP-3 and GHR expression in the liver (P < 0.05), and tended to be associated with hepatic IGF-1 expression (P = 0.071). Similarly, 9-week body weight was significantly associated with hepatic expression of IGFBP-3 and GHR expression (P < 0.05), and tended to be associated with hepatic expression of IGF-1 (P = 0.055). CONCLUSION: The age and weight at which pigs are weaned is an important determinant for nursery performance. Hepatic gene expression of IGF-1, IGFBP-3, and GHR can be useful biomarkers for monitoring growth performance in nursery pigs.

Technical note: simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.

Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 mm × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± SD across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; and GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.

The synthesis of glutathione, a major intracellular antioxidant, in animal tissue accounts for a considerable fraction of the intake of the sulfur amino acids methionine and cysteine. Animal scientists accordingly need methods suitable for measuring the abundance of metabolites related to sulfur amino acid metabolism in solid tissue. However, methods currently available are either validated for measuring these metabolites in plasma, serum, or urine, do not fully describe all procedures needed to prepare tissue samples for analysis, or are validated for measuring only cysteine and glutathione in tissue. The focus of this work was to describe the sample preparation and analysis methods needed to measure these metabolites in solid tissue. Sample preparation time is less than 2 h and sample analysis time is less than 5 min. The method is robust and reproducible and is applied to identify weaning-induced differences in sulfur amino acid metabolism in liver and small intestine in pigs. The method will also help evaluate the impact of diet, stress, or inflammation on cysteine and glutathione metabolism on a tissue-by-tissue basis to help optimize levels of sulfur amino acids in swine diets.

Evaluation of protected benzoic acid on growth performance, nutrient digestibility, and gut health indices in starter pigs.

Benzoic acid is a common alternative for antibiotic and zinc oxide use in nursery diets. Free benzoic acid (BZA) is often supplied, but this form is absorbed before it can exert any effect on distal segments of the gut. The study aimed to evaluate the effects of protected benzoic acid on growth performance, nutrient digestibility, plasma metabolites, and gut health indices in starter pigs. A total of 192 pigs were weaned at 28 ± 1 d age (initial body weight, 8.72 ± 1.13 kg). Pens were assigned to one of four treatment diets (n = 8 pens per treatment): (1) no additive (NC), (2) free benzoic acid (BZA; 0.6%), (3) protected benzoic acid (BC50; 0.2%, supplied at a ratio of one to three equivalents of BZA), and (4) antibiotic growth promoter (AGP; Carbadox, 50 ppm). Diets were fed for three weeks over two periods (period 1, 7 d; period 2, 14 d). Body weight and feed intake were measured for each period. Feces were collected at the end of each period to determine apparent total tract digestibility (ATTD) of organic matter (OM), gross energy (GE), and crude protein (CP). One pig per pen was euthanized per period to determine plasma metabolites; jejunum and ileum morphology; jejunum, ileum, and colon cytokine abundance; and jejunum, ileum, and colon tight junction protein expression. The AGP group had increased average daily gain (ADG) and average daily feed intake (ADFI) compared to other groups in period 1 and overall (P < 0.05); however, ADG and ADFI of the BC50 group was intermediate between the NC and BZA groups and the AGP group in period 2. The ATTD of OM, GE, and CP were greater in the AGP group compared to the NC and BC50 groups (P < 0.05), whereas the BZA group was intermediate. Jejunum and ileum villus height and crypt depth increased from period 1 to period 2 (P < 0.01) but were similar across groups. Ileum and colon tumor necrosis factor-α (TNF-α) abundances were greater, whereas colon interleukin (IL)-1ß and colon and ileum IL-8 abundances were less, in the AGP group compared to the BZA group (P < 0.05); the NC and BC50 groups exhibited intermediate TNF-α, IL-1ß, and IL-8 abundance in the ileum and colon. Jejunum cytokine abundance did not vary among groups but declined from period 1 to period 2 (P < 0.05). Tight junction protein expression also did not vary among groups. In summary, protected BZA supported a slight increase in growth performance in starter pigs, suggesting its potential as an alternative feed additive in nursery diets.

Regulation of skeletal muscle protein synthesis in the preterm pig by intermittent leucine pulses during continuous parenteral feeding.

BACKGROUND: Extrauterine growth restriction is a common complication of preterm birth. Leucine (Leu) is an agonist for the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway that regulates translation initiation and protein synthesis in skeletal muscle. Previously, we showed that intermittent intravenous pulses of Leu to neonatal pigs born at term receiving continuous enteral nutrition increases muscle protein synthesis and lean mass accretion. Our objective was to determine the impact of intermittent intravenous pulses of Leu on muscle protein anabolism in preterm neonatal pigs administered continuous parenteral nutrition. METHODS: Following preterm delivery (on day 105 of 115 gestation), pigs were fitted with umbilical artery and jugular vein catheters and provided continuous parenteral nutrition. Four days after birth, pigs were assigned to receive intermittent Leu (1600 µmol kg-1 h-1 ; n = 8) or alanine (1600 µmol kg-1 h-1 ; n = 8) parenteral pulses every 4 h for 28 h. Anabolic signaling and fractional protein synthesis were determined in skeletal muscle. RESULTS: Leu concentration in the longissimus dorsi and gastrocnemius muscles increased in the leucine (LEU) group compared with the alanine (ALA) group (P < 0.0001). Despite the Leu-induced disruption of the Sestrin2·GATOR2 complex, which inhibits mTORC1 activation, in these muscles (P < 0.01), the abundance of mTOR·RagA and mTOR·RagC was not different. Accordingly, mTORC1-dependent activation of 4EBP1, S6K1, eIF4E·eIF4G, and protein synthesis were not different in any muscle between the LEU and ALA groups. CONCLUSION: Intermittent pulses of Leu do not enhance muscle protein anabolism in preterm pigs supplied continuous parenteral nutrition.

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

Leucine (Leu) and its metabolite ß-hydroxy-ß-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 ß-hydroxy-ß-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 Muscle Growth in Early Postnatal Life in a Swine Model.

Skeletal muscle growth during the early postnatal period is rapid in the pig and dependent on the capacity of muscle to respond to anabolic and catabolic stimuli. Muscle mass is driven by the balance between protein synthesis and degradation. Among these processes, muscle protein synthesis in the piglet is exceptionally sensitive to the feeding-induced postprandial changes in insulin and amino acids, whereas muscle protein degradation is affected only during specific catabolic states. The developmental decline in the response of muscle to feeding is associated with changes in the signaling pathways located upstream and downstream of the mechanistic target of rapamycin protein complex. Additionally, muscle growth is supported by an accretion of nuclei derived from satellite cells. Activated satellite cells undergo proliferation, differentiation, and fusion with adjacent growing muscle fibers. Enhancing early muscle growth through modifying protein synthesis, degradation, and satellite cell activity is key to maximizing performance, productivity, and lifelong pig health.

Effects of dietary leucine supplementation and immune system stimulation on plasma AA concentrations and tissue protein synthesis in starter pigs.

Immune system stimulation (ISS) adversely affects protein and AA metabolism and reduces productivity in pigs. Leucine (Leu) has a regulatory role in skeletal muscle protein turnover, which may be affected by ISS. The objective of this study was to evaluate the effects of ISS and dietary Leu supplementation on the protein fractional synthesis rate (FSR) of various tissues in pigs. Yorkshire barrows were surgically fitted with jugular vein catheters and assigned to one of three dietary treatments: (i) CON, 1.36% standardized ileal digestible (SID) Leu; (ii) LEU-M, 2.04% SID Leu; and (iii) LEU-H, 2.72% SID Leu. The diets were formulated to contain all essential AA 10% above estimated requirements for maximum whole-body protein deposition for this BW range. At the start of the 36-h challenge period (initial BW = 14.5 ± 0.8 kg), ISS was induced in pigs with lipopolysaccharide (ISS+; n = 7, 8, and 7 for CON, LEU-M, and LEU-H pigs, respectively); a subset of CON pigs was injected with sterile saline (ISS-; n = 6). During challenge period, pigs were fed every 4 h and feed intake of ISS- pigs was kept equal to ISS+ pigs. At the end of the challenge period, FSR of liver, plasma, gastrocnemius, and LD proteins were determined with a flooding dose of l-[ring-2H5]phenylalanine (40 mol%). All essential AA, most nonessential AA, and plasma urea-N peaked at 12 h and declined to baseline levels at 36 h after ISS was induced in ISS+ pigs (P < 0.05), whereas plasma AA and urea-N concentrations were constant in ISS- pigs. At 36 h, dietary Leu supplementation resulted in a linear decline in plasma isoleucine, valine, glutamine, and urea nitrogen concentrations (P < 0.05), whereas plasma Leu concentration was unaffected. Liver protein FSR was increased in ISS+ pigs (P < 0.05), whereas plasma and skeletal muscle protein FSR was not affected by ISS. Dietary Leu supplementation tended to diminish liver protein FSR (linear reduction; P = 0.052) and increase gastrocnemius protein FSR (linear increase; P = 0.085) in ISS+ pigs. Leucine supplementation above estimated requirements may support repartitioning of AA from visceral to peripheral protein deposition during ISS.

Whole-body nitrogen utilization and tissue protein and casein synthesis in lactating primiparous sows fed low- and high-protein diets.

Twenty-eight lactating Yorkshire and Yorkshire × Landrace primiparous sows were used to test the hypothesis that feeding a diet with reduced CP concentration and supplemented with crystalline AA (CAA) does not decrease milk protein yield and litter growth but improves apparent N utilization for milk protein production. Sows were assigned to 1 of 2 dietary treatments: 1) control (CON; 16.2% CP; analyzed content) or 2) low CP with CAA to meet estimated requirements of limiting AA (LCP; 12.7% CP) over a 17-d lactation period. A N balance was conducted for each sow between days 13 and 17 of lactation. On day 17, a 12-h primed continuous infusion of l-[ring-2H5]-Phe was conducted on 12 sows (n = 6) with serial blood and milk sampling to determine plasma AA concentrations and Phe enrichment, and milk casein synthesis, respectively. Thereafter, sows were sacrificed and tissues were collected to determine tissue protein fractional synthesis rates (FSR). Litter growth rate and milk composition did not differ. Sows fed the LCP diet had reduced N intake (122.7 vs. 153.2 g/d; P < 0.001) and maternal N retention (13.5 vs. 24.6 g/d; P < 0.05) and greater apparent efficiency of using dietary N intake for milk production (85.1% vs. 67.5%; P < 0.001). On day 17 of lactation, all plasma essential AA concentrations exhibited a quartic relationship over time relative to consumption of a meal, where peaks occurred at approximately 1- and 4-h postprandial (P < 0.05). Protein FSR in liver, LM, gastrocnemius muscle, mammary gland, and in milk caseins did not differ between treatments. Feeding primiparous sows with a diet containing 12.7% CP and supplemented with CAA to meet the limiting AA requirements did not reduce milk protein yield or piglet growth rate and increased the apparent utilization of dietary N, Arg, Leu, Phe+Tyr, and Trp for milk protein production. The improved apparent utilization of N and AA appears to be related exclusively to a reduction in N and AA intake.