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.

Neurodegeneration in juvenile Iberian pigs with diet-induced nonalcoholic fatty liver disease.

The objective of this study was to investigate whether juvenile Iberian pigs with diet-induced nonalcoholic fatty liver disease (NAFLD), cholestasis, and gut dysbiosis would develop histological and metabolic markers of neurodegeneration in the frontal cortex (FC) and whether supplementing probiotics would influence the response to the diet. Twenty-eight juvenile Iberian pigs were fed for 10 wk either a control (CON) or high-fructose high-fat (HFF) diet with or without a commercial probiotic mixture. Compared with CON, HFF-fed pigs had a decreased number of neurons and an increase in reactive astrocytes in FC tissue. There was also a decrease in one-carbon metabolites choline and betaine and a marked accumulation of bile acids, cholesteryl esters, and polyol pathway intermediates in FC of HFF-fed pigs, which were associated with markers of neurodegeneration and accentuated with the severity of NAFLD. Betaine depletion in FC tissue was negatively correlated with choline-derived phospholipids in colon content, whereas primary conjugated bile acids in FC were associated with cholestasis. Plasma kynurenine-to-tryptophan quotient, as a marker of indoleamine 2,3-dioxygenase activity, and intestinal dysbiosis were also correlated with neuronal loss and astrogliosis. Recognition memory test and FC levels of amyloid-ß and phosphorylated Tau did not differ between diets, whereas probiotics increased amyloid-ß and memory loss in HFF-fed pigs. In conclusion, our results show evidence of neurodegeneration in FC of juvenile Iberian pigs and establish a novel pediatric model to investigate the role of gut-liver-brain axis in diet-induced NAFLD.

Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.

New generation lipid emulsions increase brain DHA and improve body composition, but not short-term neurodevelopment in parenterally-fed preterm piglets.

New generation, multicomponent parenteral lipid emulsions provide key fatty acids for brain growth and development, such as docosahexaenoic acid (DHA) and arachidonic acid (AA), yet the content may be suboptimal for preterm infants. Our aim was to test whether DHA and AA-enriched lipid emulsions would increase activity, growth, and neurodevelopment in preterm piglets and limit brain inflammation. Cesarean-delivered preterm pigs were given three weeks of either enteral preterm infant formula (ENT) or TPN with one of three parenteral lipid emulsions: Intralipid (IL), SMOFlipid (SMOF) or an experimental emulsion (EXP). Activity was continuously monitored and weekly blood sampling and behavioral field testing performed. At termination of the study, whole body and tissue metrics were collected. Neuronal density was assessed in sections of hippocampus (HC), thalamus, and cortex. Frontal cortex (FC) and HC tissue were assayed for fatty acid profiles and expression of genes of neuronal growth and inflammation. After 3 weeks of treatment, brain DHA content in SMOF, EXP and ENT pigs was higher (P < 0.01) in FC but not HC vs. IL pigs. There were no differences in brain weight or neuron density among treatment groups. Inflammatory cytokine TNFα and IL-1ß expression in brain regions were increased in IL pigs (P < 0.05) compared to other groups. Overall growth velocity was similar among groups, but IL pigs had higher percent body fat and increased insulin resistance compared to other treatments (P < 0.05). ENT pigs spent more time in higher physical activity levels compared to all TPN groups, but there were no differences in exploratory behavior among groups. We conclude that a soybean oil emulsion increased select brain inflammatory cytokines and multicomponent lipid emulsions enriched with DHA and AA in parenteral lipids results in increased cortical DHA and improved body composition without affecting short term neurodevelopmental outcomes.

Effect of piglet separation from dam at birth on colostrum uptake.

The objective of this study was to determine whether birth order influences piglet survival because of reduced uptake of maternal antibodies by the piglets born later in large litters. Forty-five litters were serially allocated to one of 2 study groups. The crèche group consisted of 18 litters for which the 205 piglets were removed to a warm box to prevent suckling until 4 h after delivery of the first pig, and the control group of 27 litters for which 306 piglets were allowed to suckle from birth. The protein content of piglet blood and sow colostrum was determined with Brix refractometers. Parity, farrowing duration, liveborn litter size, litter size at 12 d, and piglet weight at birth and at 24 h and 12 d of age did not differ between the 2 treatment groups (P > 0.1). There were also no significant differences (P > 0.1) at any time point in weight or in the mean percent protein in plasma between the first 3 and the last 3 piglets born to an individual sow. However, the mean percent protein in plasma was significantly higher in the control group than in the crèche group at both 24 h (P ≤ 0.05) and day 12 (P ≤ 0.01) postpartum. The lack of differences in plasma protein levels between the first and last pigs born along with the lower percent plasma protein in the piglets that were prevented from suckling immediately after birth militate against the use of this technique as a way to equalize the opportunity for adequate transfer of maternal antibodies.

L'objectif de la présente étude était de déterminer si l'ordre de naissance influence la survie des porcelets à cause de la réduction d'ingestion d'anticorps maternels par les porcelets nés plus tard dans les portées nombreuses. Quarante-cinq portées ont été réparties de manière successive à l'un des deux groupes d'étude. Le groupe crèche était constitué de 18 portées pour lesquelles les 205 porcelets ont été maintenus dans une boîte chauffée pour empêcher la tétée jusqu'à 4 h après la naissance du premier porcelet, et le groupe témoin de 27 portées pour lesquelles les 306 porcelets ont pu téter dès la naissance. Le contenu en protéine du sang des porcelets et du colostrum de la truie a été déterminé avec un réfractomètre Brix. La parité, la durée de la mise-bas, le nombre de porcelets nés vivants, la taille de la portée à 12 j, et le poids des porcelets à la naissance et à 24 h et 12 j d'âge n'étaient pas différents entre les deux groupes de traitement (P > 0,1). Il n'y avait également pas de différence significative (P > 0,1) à aucun des temps mesurés pour le poids ou le pourcentage moyen de protéines dans le plasma entre les trois premiers et les trois derniers porcelets nés à une truie individuelle. Toutefois, le pourcentage moyen en protéine dans le plasma était significativement plus élevé dans le groupe témoin que dans le groupe crèche à 24 h (P ≤ 0,05) et au jour 12 (P ≤ 0,01) post-partum. Le manque de différence dans les quantités de protéines plasmatiques entre le premier et le dernier né avec également le pourcentage de protéine plasmatique plus faible chez les porcelets qui ont été empêché de boire immédiatement après la naissance milite contre l'utilisation de cette technique comme moyen d'égaliser l'opportunité pour un transfert adéquat d'anticorps maternels.(Traduit par Docteur Serge Messier).

Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets.

Necrotizing enterocolitis (NEC) is associated with low plasma arginine and vascular dysfunction. It is not clear whether low intestinal citrulline production, the precursor for arginine synthesis, occurs before and thus predisposes to NEC or if it results from tissue damage. This study was designed to test the hypothesis that whole body rates of citrulline, arginine, and nitric oxide synthesis are low in premature pigs and that they precede NEC. Piglets delivered by cesarean section at 103 days [preterm (PT)], 110 days [near-term (NT)], or 114 days [full-term (FT)] of gestation were given total parenteral nutrition and after 2 days orogastrically fed infant formula for 42 h to induce NEC. Citrulline and arginine fluxes were determined before and during the feeding protocol. Gross macroscopic and histological NEC scores and plasma fatty acid binding protein (iFABP) concentration were determined as indicators of NEC. Intestinal gene expression for enzymes of the arginine pathway were quantitated. A lower ( P < 0.05) survival rate was observed for PT (8/27) than for NT (9/9) and FT pigs (11/11). PT pigs had higher macroscopic gross ( P < 0.05) and histological NEC ( P < 0.05) scores and iFABP concentration ( P < 0.05) than pigs of more advanced gestational age. PT pigs had lower citrulline production and arginine fluxes ( P < 0.05) throughout and a reduced gene expression in genes of the citrulline-arginine pathway. In summary, intestinal enzyme expression and whole body citrulline and arginine fluxes were reduced in PT pigs compared with animals of more advance gestational age and preceded the development of NEC. NEW & NOTEWORTHY Arginine supplementation prevents necrotizing enterocolitis (NEC), the most common gastrointestinal emergency of prematurity. Citrulline (precursor for arginine) production is reduced during NEC, and this is believed to be a consequence of intestinal damage. In a swine model of NEC, we show that intestinal gene expression of the enzymes for citrulline production and whole body citrulline and arginine fluxes are reduced and precede the onset of NEC in premature pigs. Reduced citrulline production during prematurity may be a predisposition to NEC.

Amino acids, independent of insulin, attenuate skeletal muscle autophagy in neonatal pigs during endotoxemia.

BACKGROUND: Sepsis induces loss of skeletal muscle mass by activating the ubiquitin proteasome (UPS) and autophagy systems. Although muscle protein synthesis in healthy neonatal piglets is responsive to amino acids (AA) stimulation, it is not known if AA can prevent the activation of muscle protein degradation induced by sepsis. We hypothesize that AA attenuate the sepsis-induced activation of UPS and autophagy in neonates. METHODS: Newborn pigs were infused for 8 h with liposaccharide (LPS) (0 and 10 µg·kg(-1)·h(-1)), while circulating glucose and insulin were maintained at fasting levels; circulating AA were clamped at fasting or fed levels. Markers of protein degradation and AA transporters in longissimus dorsi (LD) were examined. RESULTS: Fasting AA increased muscle microtubule-associated protein light 1 chain 3 II (LC3-II) abundance in LPS compared to control, while fed AA levels decreased LC3-II abundance in both LPS and controls. There was no effect of AA supplementation on activated protein kinase (AMP), forkhead box O1 and O4 phosphorylation, nor on sodium-coupled neutral AA transporter 2 and light chain AA transporter 1, muscle RING-finger protein-1 and muscle Atrophy F-Box/Atrogin-1 abundance. CONCLUSION: These findings suggest that supplementation of AA antagonize autophagy signal activation in skeletal muscle of neonates during endotoxemia.

Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs.

Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight(-1) · day(-1)) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E · eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E · eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.