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.

Adverse Metabolic Phenotypes in Parenterally Fed Neonatal Pigs Do Not Persist into Adolescence.

BACKGROUND: Nutrition during fetal and neonatal life is an important determinant for the risk of adult-onset diseases, especially type 2 diabetes and obesity. OBJECTIVES: We aimed to determine whether total parenteral nutrition (TPN) compared with enteral formula feeding [enteral nutrition (EN)] in term piglets during the first 2 wk after birth would increase the long-term (5-mo) development of metabolic syndrome phenotypes with adverse glucose homeostasis, fatty liver disease, and obesity. METHODS: Neonatal female pigs were administered TPN (n = 12) or fed enterally with a liquid enteral milk-replacer formula (EN, n = 12) for 14 d. After transitioning TPN pigs to enteral feeding of liquid formula (days 15-26), both groups were adapted to a solid high-fat diet (30% of the total diet) and sucrose (20% of the total diet) diet (days 27-33), which was fed until the end of the study (140 d). Body composition was measured by dual-energy X-ray absorptiometry at 14, 45, and 140 d. Serum biochemistry and glucose-insulin values (after a fasting intravenous glucose tolerance test) were obtained at 140 d. Liver and muscle were analyzed for insulin receptor signaling and triglycerides. RESULTS: Body weight was similar, but percent fat was higher, whereas percent lean and bone mineral density were lower in TPN than in EN pigs (P < 0.01) at 45 d of age but not at 140 d. At 140 d, there were no differences in serum markers of liver injury or lipidemia. Intravenous glucose tolerance test at 140 d showed a lower (P < 0.05) AUC for both glucose and insulin in TPN than in EN pigs, but the ratio of AUCs of insulin and glucose was not different between groups. CONCLUSIONS: Administration of TPN during the neonatal period increased adipose deposition that transiently persisted in early adolescence when challenged with a high-fat diet but was not sustained or manifested as glucose intolerance.

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.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

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.

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.

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

BACKGROUND: Nutrition 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. OBJECTIVES: We 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. METHODS: Term 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. RESULTS: Phosphorylation 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). CONCLUSIONS: The 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.

Development of Food Pattern Recommendations for Infants and Toddlers 6-24 Months of Age to Support the Dietary Guidelines for Americans, 2020-2025.

BACKGROUND: Developing food-based dietary guidelines (FBDGs) for infants and toddlers is a complex task that few countries have attempted. OBJECTIVES: Our objectives are to describe the process of food pattern modeling (FPM) conducted to develop FBDGs for the Dietary Guidelines for Americans, 2020-2025 for infants 6 to <12 mo and toddlers 12 to <24 mo of age, as well as the implications of the results and areas needing further work. METHODS: The US 2020 Dietary Guidelines Advisory Committee, with the support of federal staff, conducted FPM analyses using 5 steps: 1) identified energy intake targets; 2) established nutritional goals; 3) identified food groupings and expected amounts, using 3 options for the amount of energy from human milk in each age interval; 4) estimated expected nutrient intakes for each scenario, based on nutrient-dense representative foods; and 5) evaluated expected nutrient intakes against nutritional goals. RESULTS: For human milk-fed infants (and toddlers), example combinations of complementary foods and beverages were developed that come close to meeting almost all nutrient recommendations if iron-fortified infant cereals are included at 6 to <12 mo of age. These combinations would also be suitable for formula-fed infants. For toddlers not fed human milk, 2 patterns were developed: the Healthy US-Style Pattern and the Healthy Vegetarian Pattern (a lacto-ovo vegetarian pattern). Achieving nutrient recommendations left virtually no remaining energy for added sugars. CONCLUSIONS: It is challenging to meet all nutrient needs during these age intervals. Added sugars should be avoided for infants and toddlers <2 y of age. Further work is needed to 1) establish a reference human milk composition profile, 2) update and strengthen the DRI values for these age groups, and 3) use optimization modeling, in combination with FPM, to identify combinations of foods that meet all nutritional goals.

A Proposed Framework for Identifying Nutrients and Food Components of Public Health Relevance in the Dietary Guidelines for Americans.

BACKGROUND: Identification of nutrients of public health concern has been a hallmark of the Dietary Guidelines for Americans (DGA); however, a formal systematic process for identifying them has not been published. OBJECTIVES: We aimed to propose a framework for identifying "nutrients or food components" (NFCs) of public health relevance to inform the DGA. METHODS: The proposed framework consists of 1) defining terminology; 2) establishing quantitative thresholds to identify NFCs; and 3) examining national data. The proposed framework utilizes available data from 3 key data sources or "prongs": 1) dietary intakes; 2) biological endpoints; and 3) clinical health consequences such as prevalence of health conditions, directly or indirectly through validated surrogate markers. RESULTS: In identifying potential NFCs of public health concern, the 2020 DGA Committee developed a decision-tree framework with suggestions for combining the 3 prongs. The identified NFCs of public health concern for Americans ≥1 y old included fiber, calcium (≥2 y old), vitamin D, and potassium for low intakes and sodium, added sugars, and saturated fats (≥2 y old) for high intakes that were associated with adverse health consequences. Iron was identified among infants ages 6-12 mo fed human milk. For reproductive-aged and pregnant females, iron (all trimesters) and folate (first trimester) were identified for low intake, based on dietary and biomarker data (iron) or the severity of the consequence (folic acid and neural tube defects). Among pregnant women, low iodine was of potential public health concern based on biomarker data. Other NFCs that were underconsumed, overconsumed, and pose special challenges were identified across the life course. CONCLUSIONS: The proposed decision-tree framework was intended to streamline and add transparency to the work of this and future Dietary Guidelines Advisory Committees to identify NFCs that need to be encouraged or discouraged in order to help reduce risk of chronic disease and promote health and energy balance in the population.

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.

Intermittent leucine pulses during continuous feeding alters novel components involved in skeletal muscle growth of neonatal pigs.

When neonatal pigs continuously fed formula are supplemented with leucine pulses, muscle protein synthesis and body weight gain are enhanced. To identify the responsible mechanisms, we combined plasma metabolomic analysis with transcriptome expression of the transcriptome and protein catabolic pathways in skeletal muscle. Piglets (n = 23, 7-day-old) were fed continuously a milk replacement formula via orogastric tube for 21 days with an additional parenteral infusion (800 µmol kg-1 h-1) of either leucine (LEU) or alanine (CON) for 1 h every 4 h. Plasma metabolites were measured by liquid chromatography-mass spectrometry. Gene and protein expression analyses of longissimus dorsi muscle were performed by RNA-seq and Western blot, respectively. Compared with CON, LEU pigs had increased plasma levels of leucine-derived metabolites, including 4-methyl-2-oxopentanoate, beta-hydroxyisovalerate, ß-hydroxyisovalerylcarnitine, and 3-methylglutaconate (P ≤ 0.05). Leucine pulses downregulated transcripts enriched in the Kyoto Encyclopedia of Genes and Genomes terms "spliceosome," "GAP junction," "endocytosis," "ECM-receptor interaction," and "DNA replication". Significant correlations were identified between metabolites derived from leucine catabolism and muscle genes involved in protein degradation, transcription and translation, and muscle maintenance and development (P ≤ 0.05). Further, leucine pulses decreased protein expression of autophagic markers and serine/threonine kinase 4, involved in muscle atrophy (P ≤ 0.01). In conclusion, results from our studies support the notion that leucine pulses during continuous enteral feeding enhance muscle mass gain in neonatal pigs by increasing protein synthetic activity and downregulating protein catabolic pathways through concerted responses in the transcriptome and metabolome.

Prematurity blunts the feeding-induced stimulation of translation initiation signaling and protein synthesis in muscle of neonatal piglets.

Postnatal growth of lean mass is commonly blunted in preterm infants and may contribute to short- and long-term morbidities. To determine whether preterm birth alters the protein anabolic response to feeding, piglets were delivered at term or preterm, and fractional protein synthesis rates (Ks) were measured at 3 days of age while fasted or after an enteral meal. Activation of signaling pathways that regulate protein synthesis and degradation were determined. Relative body weight gain was lower in preterm than in term. Gestational age at birth (GAB) did not alter fasting plasma glucose or insulin, but when fed, plasma insulin and glucose rose more slowly, and reached peak value later, in preterm than in term. Feeding increased Ks in longissimus dorsi (LD) and gastrocnemius muscles, heart, pancreas, and kidney in both GAB groups, but the response was blunted in preterm. In diaphragm, lung, jejunum, and brain, feeding increased Ks regardless of GAB. Liver Ks was greater in preterm than term and increased with feeding regardless of GAB. In all tissues, changes in 4EBP1, S6K1, and PKB phosphorylation paralleled changes in Ks. In LD, eIF4E·eIF4G complex formation, phosphorylation of TSC2, mTOR, and rpS6, and association of mammalian target of rapamycin (mTOR1) complex with RagA, RagC, and Rheb were increased by feeding and blunted by prematurity. There were no differences among groups in LD protein degradation markers. Our results demonstrate that preterm birth reduces weight gain and the protein synthetic response to feeding in muscle, pancreas, and kidney, and this is associated with blunted insulin- and/or amino acid-induced translation initiation signaling.

Amino Acid- and Insulin-Induced Activation of mTORC1 in Neonatal Piglet Skeletal Muscle Involves Sestin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR Complex Formation.

Background: Feeding stimulates protein synthesis in skeletal muscle of neonates and this response is regulated through activation of mechanistic target of rapamycin complex 1 (mTORC1). The identity of signaling components that regulate mTORC1 activation in neonatal muscle has not been fully elucidated. Objective: We investigated the independent effects of the rise in amino acids (AAs) and insulin after a meal on the abundance and activation of potential regulators of mTORC1 in muscle and whether the responses are modified by development. Methods: Overnight-fasted 6- and 26-d-old pigs were infused for 2 h with saline (control group) or with a balanced AA mixture (AA group) or insulin (INS group) to achieve fed levels while insulin or AAs, respectively, and glucose were maintained at fasting levels. Muscles were analyzed for potential mTORC1 regulatory mechanisms and results were analyzed by 2-factor ANOVA followed by Tukey's post hoc test. Results: The abundances of DEP domain-containing mTOR-interacting protein (DEPTOR), growth factor receptor bound protein 10 (GRB10), and regulated in development and DNA damage response 2 (REDD2) were lower (65%, 73%, and 53%, respectively; P < 0.05) and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1/2 (LAMTOR1/2), vacuolar H+-ATPase (V-ATPase), and Sestrin2 were higher (94%, 141%, 145%, and 127%, respectively; P < 0.05) in 6- than in 26-d-old pigs. Both AA and INS groups increased phosphorylation of GRB10 (P < 0.05) compared with control in 26- but not in 6-d-old pigs. Formation of Ras-related GTP-binding protein A (RagA)-mTOR, RagC-mTOR, and Ras homolog enriched in brain (RHEB)-mTOR complexes was increased (P < 0.05) and Sestrin2-GTPase activating protein activity towards Rags 2 (GATOR2) complex was decreased (P < 0.05) by both AA and INS groups and these responses were greater (P < 0.05) in 6- than in 26-d-old pigs. Conclusion: The results suggest that formation of RagA-mTOR, RagC-mTOR, RHEB-mTOR, and Sestrin2-GATOR2 complexes may be involved in the AA- and INS-induced activation of mTORC1 in skeletal muscle of neonates after a meal and that enhanced activation of the mTORC1 signaling pathway in neonatal muscle is in part due to regulation by DEPTOR, GRB10, REDD2, LAMTOR1/2, V-ATPase, and Sestrin2.

Short- and long-term effects of leucine and branched-chain amino acid supplementation of a protein- and energy-reduced diet on muscle protein metabolism in neonatal pigs.

The objective of this study was to determine if enteral leucine or branched-chain amino acid (BCAA) supplementation increases muscle protein synthesis in neonates who consume less than their protein and energy requirements, and whether this increase is mediated via the upregulation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway or the decrease in muscle protein degradation signaling. Neonatal pigs were fed milk replacement diets containing reduced energy and protein (R), R supplemented with BCAA (RBCAA), R supplemented with leucine (RL), or complete protein and energy (CON) at 4-h intervals for 9 (n = 24) or 21 days (n = 22). On days 9 and 21, post-prandial plasma amino acids and insulin were measured at intervals for 4 h; muscle protein synthesis rate and activation of mTOR-related proteins were determined at 120 min post-feeding in muscle. For all parameters measured, the effects of diet were not different between day 9 or day 21. Compared to CON and R, plasma leucine and BCAA were higher (P ≤ 0.01) in RL- and RBCAA-fed pigs, respectively. Body weight gain, protein synthesis, and activation of S6 kinase (S6K1), 4E-binding protein (4EBP1), and eukaryotic initiation factor 4 complex (eIF4E·eIF4G) were decreased in RBCAA, RL, and R relative to CON (P < 0.01). RBCAA and RL upregulated (P ≤ 0.01) S6K1, 4EBP1, and eIF4E·eIF4G compared to R. In conclusion, when protein and energy are restricted, both leucine and BCAA supplementation increase mTOR activation, but do not enhance skeletal muscle protein synthesis and muscle growth in neonatal pigs.

Enteral ß-hydroxy-ß-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 ß-hydroxy-ß-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.