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.

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.

Gut development following insulin-like growth factor-1 supplementation to preterm pigs.

BACKGROUND: Reduced insulin-like growth factor-1 (IGF-1) levels may contribute to impaired organ development in preterm infants. Using preterm pigs as a model, we hypothesized that IGF-1 supplementation improves health and gut development during the first three weeks of life. METHODS: First, clinical and organ endpoints were compared between artificially-reared, cesarean-delivered preterm pigs and vaginally-delivered, sow-reared term pigs at 5, 9 and 19 days. Next, preterm pigs were treated with recombinant human IGF-1 for 19 days (2.25 mg/kg/day, systemically). RESULTS: Relative to term pigs, preterm pigs had lower body weight, fat, bone contents, relative weights of liver and spleen and a longer and thinner intestine at 19 days. Preterm birth reduced intestinal villi heights and peptidase activities, but only at 5 and 9 days. In preterm pigs, IGF-1 reduced mortality primarily occurring from gastrointestinal complications and with a tendency towards salvaging smaller pigs. IGF-1 supplementation also increased spleen and kidney weights, small intestine length and maltase to lactase activity, reflecting gut maturation. CONCLUSION: Preterm birth affects body composition and gut maturation in the first 1-2 weeks, but differences are marginal thereafter. Supplemental IGF-1 may improve gut health in pigs and infants in the first few weeks after preterm birth. IMPACT: Insulin-like growth factor 1 (IGF-1) supplementation may improve gut health and development in prematurity, but whether the effects are sustained beyond the immediate postnatal period is unclear. In preterm pigs, the prematurity effects on IGF-1 and gut health deficiencies are most pronounced during the first week of life and diminishes thereafter. In preterm pigs, IGF-1 supplementation beyond the first week of life reduced mortality. The present study provides evidence of a sustained effect of IGF-1 supplementation on the gastrointestinal tract after the immediate postnatal period.

Decreased FXR Agonism in the Bile Acid Pool Is Associated with Impaired FXR Signaling in a Pig Model of Pediatric NAFLD.

The objective of this study was to investigate whether the impairment of farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in juvenile pigs with non-alcoholic fatty liver disease (NAFLD) is associated with changes in the composition of the enterohepatic bile acid pool. Eighteen 15-day-old Iberian pigs, pair-housed in pens, were allocated to receive either a control (CON) or high-fructose, high-fat (HFF) diet. Animals were euthanized in week 10, and liver, blood, and distal ileum (DI) samples were collected. HFF-fed pigs developed NAFLD and had decreased FGF19 expression in the DI and lower FGF19 levels in the blood. Compared with the CON, the HFF diet increased the total cholic acid (CA) and the CA to chenodeoxycholic acid (CDCA) ratio in the liver, DI, and blood. CA and CDCA levels in the DI were negatively and positively correlated with ileal FGF19 expression, respectively, and blood levels of FGF19 decreased with an increasing ileal CA to CDCA ratio. Compared with the CON, the HFF diet increased the gene expression of hepatic 12-alpha-hydrolase, which catalyzes the synthesis of CA in the liver. Since CA species are weaker FXR ligands than CDCA, our results suggest that impairment of FXR-FGF19 signaling in NAFLD pigs is associated with a decrease in FXR agonism in the bile acid pool.

Preterm Pigs Fed Donor Human Milk Have Greater Liver ß-Carotene Concentrations than Pigs Fed Infant Formula.

BACKGROUND: Milk carotenoids may support preterm infant health and neurodevelopment. Infants fed human milk often have higher blood and tissue carotenoid concentrations than infants fed carotenoid-containing infant formula (IF). Donor human milk (DHM) is a supplement to mother's own milk, used to support preterm infant nutrition. OBJECTIVES: We tested whether tissue and plasma ß-carotene concentrations would be higher in preterm pigs fed pasteurized DHM versus premature IF. METHODS: This is a secondary analysis of samples collected from a study of the effects of enteral diet composition on necrotizing enterocolitis incidence. Preterm pigs received partial enteral feeding of either DHM (n = 7) or premature IF (n = 7) from 2 to 7 d of age. The diets provided similar ß-carotene (32 nM), but DHM had higher lutein, zeaxanthin, and lycopene, whereas IF had higher total vitamin A. Plasma, liver, and jejunum carotenoid and vitamin A concentrations were measured by HPLC-PDA. Jejunal expression of 12 genes associated with carotenoid and lipid metabolism were measured. RESULTS: Liver ß-carotene concentrations were higher in DHM- than IF-fed piglets (23 ± 4 compared with 16 ± 2 µg/g, respectively, P = 0.0024), whereas plasma and jejunal ß-carotene concentrations were similar between diets. Liver vitamin A stores were higher in piglets fed IF than DHM (50.6 ± 10.1 compared with 30.9 ± 7.2 µg/g, respectively, P=0.0013); however, plasma vitamin A was similar between groups. Plasma, liver, and jejunum concentrations of lutein, zeaxanthin, and lycopene were higher with DHM than IF feeding. Relative to piglets fed DHM, jejunal low density lipoprotein receptor (Ldlr) expression was higher (61%, P = 0.018) and cluster determinant 36 (Cd36) expression (-27%, P = 0.034) was lower in IF-fed piglets. CONCLUSIONS: Preterm pigs fed DHM accumulate more liver ß-carotene than IF-fed pigs. Future studies should further investigate infant carotenoid bioactivity and bioavailability.

Insulin-like growth factor 1 supplementation supports motor coordination and affects myelination in preterm pigs.

Introduction: Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants. Methods: Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements. Results: The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus. Conclusion: Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

Towards a model of biliary atresia - Pilot feasibility study in newborn piglets.

Biliary atresia (BA) is a rare congenital liver disease with unknown etiology, and it is the most common indication for liver transplantation in children. As BA infants suffer from intestinal malabsorption and neurodevelopmental deficits, it is necessary to identify optimal medical and nutritional strategies using appropriate neonatal animal models. We aim to determine the feasibility of using newborn piglets with surgically induced cholestasis (bile duct ligation (BDL)) to mimic clinical features of BA. Six piglets were subjected to abdominal surgery on day 4 after birth. The bile ducts were ligated, and the piglet were followed for up to 12 days. On day 12 the piglets were subjected to a hepatobiliary scintigraphy using the tracer radiolabeled Technetium(99m-tc)-mebrofenin, and blood samples were collected for biochemical profiling. Of the six piglets, hepatobiliary scintigraphy verified that two piglets (BDL) had no excretion of bile into the duodenum, i.e. full cholestasis with a hepatic extraction fraction of 84-87% and clearance time of 230-318 min. One piglet (SHAM) had bile excretion to the duodenum. In accordance with this, the BDL piglets had steatorrhea, and increased levels of bilirubin and gammaglutamyl transferase (GGT). The last three piglets were euthanized due to bile leakage or poor growth. Surgically induced cholestasis in young piglets, may offer an animal model that displays clinical characteristics of biliary atresia, including malabsorption, hyperbilirubinaemia, increased GGT and reduced hepatic excretory function. Following refinement, this animal model may be used to optimize feeding strategies to secure optimal nutrition and neurodevelopment for neonatal cholestasis/BA patients.

Probiotics and Human Milk Differentially Influence the Gut Microbiome and NEC Incidence in Preterm Pigs.

Necrotizing enterocolitis (NEC) is the leading cause of death caused by gastrointestinal disease in preterm infants. Major risk factors include prematurity, formula feeding, and gut microbial colonization. Microbes have been linked to NEC, yet there is no evidence of causal species, and select probiotics have been shown to reduce NEC incidence in infants. In this study, we evaluated the effect of the probiotic Bifidobacterium longum subsp. infantis (BL. infantis), alone and in combination with a human milk oligosaccharide (HMO)-sialylactose (3'SL)-on the microbiome, and the incidence of NEC in preterm piglets fed an infant formula diet. We studied 50 preterm piglets randomized between 5 treatments: (1) Preterm infant formula, (2) Donor human milk (DHM), (3) Infant formula + 3'SL, (4) Infant formula + BL. infantis, and (5) Infant formula and BL. infantis + 3'SL. NEC incidence and severity were assessed through the evaluation of tissue from all the segments of the GI tract. The gut microbiota composition was assessed both daily and terminally through 16S and whole-genome sequencing (WGS) of rectal stool samples and intestinal contents. Dietary BL. infantis and 3'SL supplementation had no effect, yet DHM significantly reduced the incidence of NEC. The abundance of BL. infantis in the gut contents negatively correlated with disease severity. Clostridium sensu stricto 1 and Clostridium perfringens were significantly more abundant in NEC and positively correlated with disease severity. Our results suggest that pre- and probiotics are not sufficient for protection from NEC in an exclusively formula-based diet. The results highlight the differences in microbial species positively associated with both diet and NEC incidence.

Insulin-Like Growth Factor-1 Supplementation Promotes Brain Maturation in Preterm Pigs.

Very preterm infants show low levels of insulin-like growth factor-1 (IGF-1), which is associated with postnatal growth restriction and poor neurologic outcomes. It remains unknown whether supplemental IGF-1 may stimulate neurodevelopment in preterm neonates. Using cesarean-delivered preterm pigs as a model of preterm infants, we investigated the effects of supplemental IGF-1 on motor function and on regional and cellular brain development. Pigs were treated with 2.25 mg/kg/d recombinant human IGF-1/IGF binding protein-3 complex from birth until day 5 or 9 before the collection of brain samples for quantitative immunohistochemistry (IHC), RNA sequencing, and quantitative PCR analyses. Brain protein synthesis was measured using in vivo labeling with [2H5] phenylalanine. We showed that the IGF-1 receptor was widely distributed in the brain and largely coexisted with immature neurons. Region-specific quantification of IHC labeling showed that IGF-1 treatment promoted neuronal differentiation, increased subcortical myelination, and attenuated synaptogenesis in a region-dependent and time-dependent manner. The expression levels of genes involved in neuronal and oligodendrocyte maturation, and angiogenic and transport functions were altered, reflecting enhanced brain maturation in response to IGF-1 treatment. Cerebellar protein synthesis was increased by 19% at day 5 and 14% at day 9 after IGF-1 treatment. Treatment had no effect on Iba1+ microglia or regional brain weights and did not affect motor development or the expression of genes related to IGF-1 signaling. In conclusion, the data show that supplemental IGF-1 promotes brain maturation in newborn preterm pigs. The results provide further support for IGF-1 supplementation therapy in the early postnatal period in preterm infants.

Fibroblast growth factor 19 secretion and function in perinatal development.

Limited work has focused on fibroblast growth factor-19 (FGF19) secretion and function in the perinatal period. FGF19 is a potent growth factor that coordinates development of the brain, eye, inner ear, and skeletal system in the embryo, but after birth, FGF19 transitions to be an endocrine regulator of the classic pathway of hepatic bile acid synthesis. FGF19 has emerged as a mediator of metabolism and bile acid synthesis in aged animals and adults in the context of liver disease and metabolic dysfunction. FGF19 has also been shown to have systemic insulin-sensitizing and skeletal muscle hypertrophic effects when induced or supplemented at supraphysiological levels in adult rodent models. These effects could be beneficial to improve growth and nutritional outcomes in preterm infants, which are metabolically resistant to the anabolic effects of enteral nutrition. Existing clinical data on FGF19 secretion and function in the perinatal period in term and preterm infants has been equivocal. Studies in pigs show that FGF19 expression and secretion are upregulated with gestational age and point to molecular and endocrine factors that may be involved. Work focused on FGF19 in pediatric diseases suggests that augmentation of FGF19 secretion by activation of gut FXR signaling is associated with benefits in diseases such as short bowel syndrome, parenteral nutrition-associated liver disease, and biliary atresia. Future work should focus on characterization of FGF19 secretion and the mechanism underpinning the transition of FGF19 function as an embryological growth factor to metabolic and bile acid regulator.

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.

Bile acids and their receptors in regulation of gut health and diseases.

It is well established that bile acids play important roles in lipid metabolism. In recent decades, bile acids have also been shown to function as signaling molecules via interacting with various receptors. Bile acids circulate continuously through the enterohepatic circulation and go through microbial transformation by gut microbes, and thus bile acids metabolism has profound effects on the liver and intestinal tissues as well as the gut microbiota. Farnesoid X receptor and G protein-coupled bile acid receptor 1 are two pivotal bile acid receptors that highly expressed in the intestinal tissues, and they have emerged as pivotal regulators in bile acids metabolism, innate immunity and inflammatory responses. There is considerable interest in manipulating the metabolism of bile acids and the expression of bile acid receptors as this may be a promising strategy to regulate intestinal health and disease. This review aims to summarize the roles of bile acids and their receptors in regulation of gut health and diseases.

Clinical outcome and gut development after insulin-like growth factor-1 supplementation to preterm pigs.

Background: Elevation of circulating insulin-like growth factor-1 (IGF-1) within normal physiological levels may alleviate several morbidities in preterm infants but safety and efficacy remain unclear. We hypothesized that IGF-1 supplementation during the first 1-2 weeks after preterm birth improves clinical outcomes and gut development, using preterm pigs as a model for infants. Methods: Preterm pigs were given vehicle or recombinant human IGF-1/binding protein-3 (rhIGF-1, 2.25 mg/kg/d) by subcutaneous injections for 8 days (Experiment 1, n = 34), or by systemic infusion for 4 days (Experiment 2, n = 19), before collection of blood and organs for analyses. Results: In both experiments, rhIGF-1 treatment increased plasma IGF-1 levels 3-4 fold, reaching the values reported for term suckling piglets. In Experiment 1, rhIGF-1 treatment increased spleen and intestinal weights without affecting clinical outcomes like growth, blood biochemistry (except increased sodium and gamma-glutamyltransferase levels), hematology (e.g., red and white blood cell populations), glucose homeostasis (e.g., basal and glucose-stimulated insulin and glucose levels) or systemic immunity variables (e.g., T cell subsets, neutrophil phagocytosis, LPS stimulation, bacterial translocation to bone marrow). The rhIGF-1 treatment increased gut protein synthesis (+11%, p < 0.05) and reduced the combined incidence of all-cause mortality and severe necrotizing enterocolitis (NEC, p < 0.05), but had limited effects on intestinal morphology, cell proliferation, cell apoptosis, brush-border enzyme activities, permeability and levels of cytokines (IL-1ß, IL-6, IL-8). In Experiment 2, rhIGF-1 treated pigs had reduced blood creatine kinase, creatinine, potassium and aspartate aminotransferase levels, with no effects on organ weights (except increased spleen weight), blood chemistry values, clinical variables or NEC. Conclusion: Physiological elevation of systemic IGF-1 levels for 8 days after preterm birth increased intestinal weight and protein synthesis, spleen weight and potential overall viability of pigs, without any apparent negative effects on recorded clinical parameters. The results add further preclinical support for safety and efficacy of supplemental IGF-1 to hospitalized very preterm infants.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

Cholestasis alters brain lipid and bile acid composition and compromises motor function in neonatal piglets.

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more hydrophilic and conjugated bile acid profile in the brain. Additionally, comprehensive lipidomics of the cerebellum revealed a decrease in total lipids including phosphatidylinositols and phosphatidylserines and increases in lysophospholipid species. This was paralleled by elevated cerebellar expression of genes related to inflammation and tissue damage albeit without significant impact on the brain transcriptome. This study offers new insights into the developing brain's molecular response to neonatal cholestasis indicating that bile acids and lipids may contribute in mediating motor deficits.

Maternal and Fetal Bile Acid Homeostasis Regulated by Sulfated Progesterone Metabolites through FXR Signaling Pathway in a Pregnant Sow Model.

Abnormally elevated circulating bile acids (BA) during pregnancy endanger fetal survival and offspring health; however, the pathology and underlying mechanisms are poorly understood. A total of nineteen pregnant sows were randomly assigned to day 60 of gestation, day 90 of gestation (G60, G90), and the farrowing day (L0), to investigate the intercorrelation of reproductive hormone, including estradiol, progesterone and sulfated progesterone metabolites (PMSs), and BA in the peripheral blood of mother and fetuses during pregnancy. All data were analyzed by Student's t-test or one-way ANOVA of GraphPad Prism and further compared by using the Student-Newman-Keuls test. Correlation analysis was also carried out using the CORR procedure of SAS to study the relationship between PMSs and BA levels in both maternal and fetal serum at G60, G90, and L0. Allopregnanolone sulphate (PM4S) and epiallopregnanolone sulphate (PM5S) were firstly identified in the maternal and fetal peripheral blood of pregnant sows by using newly developed ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods. Correlation analysis showed that pregnancy-associated maternal BA homeostasis was correlated with maternal serum PM4S levels, whereas fetal BA homeostasis was correlated with fetal serum PM5S levels. The antagonist activity role of PM5S on farnesoid X receptor (FXR)-mediated BA homeostasis and fibroblast growth factor 19 (FGF19) were confirmed in the PM5S and FXR activator co-treated pig primary hepatocytes model, and the antagonist role of PM4S on FXR-mediated BA homeostasis and FGF19 were also identified in the PM4S-treated pig primary hepatocytes model. Together with the high relative expression of FGF19 in pig hepatocytes, the pregnant sow is a promising animal model to investigate the pathogenesis of cholestasis during pregnancy.

Tissue-specific mechanisms of bile acid homeostasis and activation of FXR-FGF19 signaling in preterm and term neonatal pigs.

The tissue-specific molecular mechanisms involved in perinatal liver and intestinal farnesoid X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling are poorly defined. Our aim was to establish how gestational age and feeding status affect bile acid synthesis pathway, bile acid pool size, ileal response to bile acid stimulation, genes involved in bile acid-FXR-FGF19 signaling and plasma FGF19 in neonatal pigs. Term (n = 23) and preterm (n = 33) pigs were born via cesarean section at 100% and 90% gestation, respectively. Plasma FGF19, hepatic bile acid and oxysterol profiles, and FXR target gene expression were assessed in pigs at birth and after a bolus feed on day 3 of life. Pig ileal tissue explants were used to measure signaling response to bile acids. Preterm pigs had smaller, more hydrophobic bile acid pools, lower plasma FGF19, and blunted FXR-mediated ileal response to bile acid stimulation than term pigs. GATA binding protein 4 (GATA-4) expression was higher in jejunum than ileum and was higher in preterm than term pig ileum. Hepatic oxysterol analysis suggested dominance of the alternative pathway of bile acid synthesis in neonates, regardless of gestational age and persists in preterm pigs after feeding on day 3. These results highlight the tissue-specific molecular basis for the immature enterohepatic bile acid signaling via FXR-FGF19 in preterm pigs and may have implications for disturbances of bile acid homeostasis and metabolism in preterm infants.NEW & NOTEWORTHY Our results show that the lower hepatic bile acid synthesis and ileum FXR-FGF19 pathway responsiveness to bile acids contribute to low-circulating FGF19 in preterm compared with term neonatal pigs. The molecular mechanism explaining immature or low-ileum FXR-FGF19 signaling may be linked to developmental patterning effects of GATA-4.

High-Fructose, High-Fat Diet Alters Muscle Composition and Fuel Utilization in a Juvenile Iberian Pig Model of Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a serious metabolic condition affecting millions of people worldwide. A "Western-style diet" has been shown to induce pediatric NAFLD with the potential disruption of skeletal muscle composition and metabolism. To determine the in vivo effect of a "Western-style diet" on pediatric skeletal muscle fiber type and fuel utilization, 28 juvenile Iberian pigs were fed either a control diet (CON) or a high-fructose, high-fat diet (HFF), with or without probiotic supplementation, for 10 weeks. The HFF diets increased the total triacylglycerol content of muscle tissue but decreased intramyocellular lipid (IMCL) content and the number of type I (slow oxidative) muscle fibers. HFF diets induced autophagy as assessed by LC3I and LC3II, and inflammation, as assessed by IL-1α. No differences in body composition were observed, and there was no change in insulin sensitivity, but HFF diets increased several plasma acylcarnitines and decreased expression of lipid oxidation regulators PGC1α and CPT1, suggesting disruption of skeletal muscle metabolism. Our results show that an HFF diet fed to juvenile Iberian pigs produces a less oxidative skeletal muscle phenotype, similar to a detraining effect, and reduces the capacity to use lipid as fuel, even in the absence of insulin resistance and obesity.

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.

Potential Benefits of Bovine Colostrum in Pediatric Nutrition and Health.

Bovine colostrum (BC), the first milk produced from cows after parturition, is increasingly used as a nutritional supplement to promote gut function and health in other species, including humans. The high levels of whey and casein proteins, immunoglobulins (Igs), and other milk bioactives in BC are adapted to meet the needs of newborn calves. However, BC supplementation may improve health outcomes across other species, especially when immune and gut functions are immature in early life. We provide a review of BC composition and its effects in infants and children in health and selected diseases (diarrhea, infection, growth-failure, preterm birth, necrotizing enterocolitis (NEC), short-bowel syndrome, and mucositis). Human trials and animal studies (mainly in piglets) are reviewed to assess the scientific evidence of whether BC is a safe and effective antimicrobial and immunomodulatory nutritional supplement that reduces clinical complications related to preterm birth, infections, and gut disorders. Studies in infants and animals suggest that BC should be supplemented at an optimal age, time, and level to be both safe and effective. Exclusive BC feeding is not recommended for infants because of nutritional imbalances relative to human milk. On the other hand, adverse effects, including allergies and intolerance, appear unlikely when BC is provided as a supplement within normal nutrition guidelines for infants and children. Larger clinical trials in infant populations are needed to provide more evidence of health benefits when patients are supplemented with BC in addition to human milk or formula. Igs and other bioactive factors in BC may work in synergy, making it critical to preserve bioactivity with gentle processing and pasteurization methods. BC has the potential to become a safe and effective nutritional supplement for several pediatric subpopulations.