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.

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.

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.

Increased Circulating Cortisol After Vaginal Birth Is Associated With Increased FGF19 Secretion in Neonatal Pigs.

The influence of birth modality (scheduled cesarean or spontaneous vaginal) on the development of the newborn has been a source of controversy in neonatology. The impact of cesarean vs vaginal birth on the development of bile acid and fibroblast growth factor 19 (FGF19) signaling is unknown. Our aim was to determine the effect of birth modality and gestational age (preterm vs term) on plasma hormone levels, bile acid pool distribution, expression of genes in the bile acid-FXR-FGF19 pathway, and plasma levels of FGF19 at birth and on day 3 of life in neonatal pigs. Four sows underwent cesarean delivery on gestation day 105 (n = 2) and 114 (n = 2; term = 115 days), and 2 additional sows were allowed to farrow at term (gestation days 112 and 118). Piglets were euthanized at birth (Term-Vaginal n = 6; Term-Cesarean n = 8; Preterm n = 10) for tissue and blood collection, and the remaining pigs received total parenteral nutrition then were fed enterally on day 3 (Term-Vaginal n = 8; Term-Cesarean n = 10; Preterm n = 8), before blood and tissue were collected. Piglets born vaginally had a markedly (30-fold) higher plasma FGF19 at birth than term pigs born via cesarean delivery, and 70-fold higher than preterm pigs (P < 0.001). However, distal ileum FGF19 gene expression was similar in all groups (P > 0.05). Plasma FGF19 positively correlated with plasma cortisol (r = 0.58; P < 0.05) and dexamethasone treatment increased ileal FGF19 expression in cultured pig tissue explants and human enteroids. Our findings suggest that exposure to maternal or endogenous glucocorticoids in the perinatal period may upregulate the development of the bile acid-FGF19 pathway.

Impact of Parenteral Lipid Emulsion Components on Cholestatic Liver Disease in Neonates.

Total parenteral nutrition (TPN) is a life-saving intervention for infants that are unable to feed by mouth. Infants that remain on TPN for extended periods of time are at risk for the development of liver injury in the form of parenteral nutrition associated cholestasis (PNAC). Current research suggests the lipid component of TPN is a factor in the development of PNAC. Most notably, the fatty acid composition, vitamin E concentration, and presence of phytosterols are believed key mediators of lipid emulsion driven PNAC development. New emulsions comprised of fish oil and medium chain triglycerides show promise for reducing the incidence of PNAC in infants. In this review we will cover the current clinical studies on the benefit of fish oil and medium chain triglyceride containing lipid emulsions on the development of PNAC, the current constituents of lipid emulsions that may modulate the prevalence of PNAC, and potential new supplements to TPN to further reduce the incidence of PNAC.

Rifampicin, not vitamin E, suppresses parenteral nutrition-associated liver disease development through the pregnane X receptor pathway in piglets.

Infants receiving long-term parenteral nutrition (PN) develop PN-associated liver disease (PNALD). We previously (Ng K et al. JPEN J Parenter Enteral Nutr 40: 656-671, 2016. doi:10.1177/0148607114567900.) showed that PN containing soy-based lipid supplemented with vitamin E (α-tocopherol) prevents the development of PNALD. We hypothesize that this occurs via vitamin E activation of pregnane X receptor (PXR)-mediated pathways involved in bile acid metabolism. Neonatal piglets received PN for 14 days containing Intralipid (IL; soy-based lipid emulsion), IL supplemented with 12.6 mg·kg-1·day-1 vitamin E (VITE), or IL with 10 mg·kg-1·day-1 Rifadin IV (RIF), a PXR agonist. Pigs treated with IL and VITE, but not RIF, developed cholestasis and hyperbilirubinemia, markers of liver disease. The hepatic PXR target genes CYP3A29 and UGT1A6 increased during RIF treatment. RIF also modestly increased metabolism of chenodeoxycholic acid to the more hydrophilic bile acid hyocholic acid. Serum fibroblast growth factor (FGF)-19, a key regulator in suppressing hepatic bile acid synthesis, significantly increased in the RIF group. We conclude rifampicin modified markers of PNALD development by increased metabolism of bile acids and potentially suppressed bile acid synthesis. Vitamin E was ineffective at high lipid doses in preventing PNALD.NEW & NOTEWORTHY Intravenous vitamin E and rifampicin were administered to neonatal piglets receiving parenteral nutrition to determine their efficacy in reducing the progression of parenteral nutrition-associated liver disease (PNALD). Rifampicin increased serum FGF-19 concentrations and synthesis of the bile acid hyocholic acid which led to a reduction of PNALD parameters at 2 wk of administration. This result has potential clinical implications for the use of rifampicin as a safe and inexpensive treatment for short-term development of PNALD.

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.

Phytosterols Synergize With Endotoxin to Augment Inflammation in Kupffer Cells but Alone Have Limited Direct Effect on Hepatocytes.

INTRODUCTION: Phytosterols are implicated in the development of parenteral nutrition-associated liver disease. A newly proposed mechanism for phytosterol-mediated parenteral nutrition-associated liver disease is through phytosterol-facilitated hepatic proinflammatory cytokine release following exposure to intestinally derived bacteria. Whether the proinflammatory effects are liver cell specific is not known. AIM: To determine if phytosterols cause inflammation in hepatocytes or Kupffer cells independently or require costimulation by lipopolysaccharide (LPS). METHODS: In an in vivo study, neonatal piglets on parenteral nutrition for 11 days received an 8-hour infusion of LPS. In the in vitro studies, neonatal piglet Kupffer cells and hepatocytes were treated with media, media + 1% soy oil, or media + 1% soy oil + 100µM phytosterols. After 24-hour incubation, cells were treated with farnesoid X receptor (FXR) agonist obeticholic acid or liver X receptor (LXR) agonist GW3965 and challenged with LPS or interleukin 1ß. RESULTS: LPS administration in piglets led to transient increases in proinflammatory cytokines and suppression of the transporters bile salt export pump and ATP-binding cassette transporter G5. In hepatocytes, phytosterols did not activate inflammation. Phytosterol treatment alone did not activate inflammation in Kupffer cells but, combined with LPS, synergistically increased interleukin 1ß production. FXR and LXR agonists increased transporter expression in hepatocytes. GW3965 suppressed proinflammatory cytokine production in Kupffer cells, but obeticholic acid did not. CONCLUSIONS: LPS suppresses transporters that control bile acid and phytosterol clearance. Phytosterols alone do not cause inflammatory response. However, with costimulation by LPS, phytosterols synergistically maximize the inflammatory response in Kupffer cells.

Emerging Clinical Benefits of New-Generation Fat Emulsions in Preterm Neonates.

Soybean oil-based intravenous fat emulsions (IVFEs) have been the predominant parenteral nutrition IVFE used in the United States for neonates over the past 45 years. Even though this emulsion has proven useful in supplying infants with energy for growth and essential fatty acids, there have been concerns over its composition in the development of several morbidities, ranging from sepsis to liver disease, bronchopulmonary dysplasia, and impaired neurodevelopment and growth. The exact mechanisms that drive these morbidities in preterm infants are multifactorial, but potential contributors include high ω-6 (n-6) fatty acid composition, low docosahexaenoic acid and antioxidant supplementation, and the presence of potentially harmful nonnutritive components (eg, phytosterols). To address these issues, new-generation IVFEs with various types and amounts of fat have been developed containing greater amounts of the medium-chain fatty acids, long-chain polyunsaturated fatty acid, docosahexaenoic acid, lower concentrations of ω-6 polyunsaturated fatty acids, supplemental vitamin E, and low or negligible amounts of phytosterols. This review examines the clinical outcomes associated with different morbidities of parenteral nutrition in neonates who have received either soybean oil-based or new-generation IVFEs and addresses whether the proposed benefits of new-generation IVFEs have improved outcomes in the neonatal population.

Gene Expression Patterns Are Altered in Athymic Mice and Metabolic Syndrome Factors Are Reduced in C57BL/6J Mice Fed High-Fat Diets Supplemented with Soy Isoflavones.

Soy isoflavones exert beneficial health effects; however, their potential to ameliorate conditions associated with the metabolic syndrome (MetS) has not been studied in detail. In vitro and in vivo models were used to determine the effect of isoflavones on lipid metabolism, inflammation, and oxidative stress. In nude mice, consumption of Novasoy (NS) increased cholesterol and lipid metabolism gene expression, including Scd-1 (27.7-fold), Cyp4a14 (35.2-fold), and Cyp4a10 (9.5-fold), and reduced anti-inflammatory genes, including Cebpd (16.4-fold). A high-fat (HF) diet containing 0.4% (w/w) NS for 10 weeks significantly reduced percent weight gain (74.6 ± 2.5 vs 68.6 ± 3.5%) and hepatic lipid accumulation (20 ± 1.2 vs 27 ± 1.5%), compared to HF alone (p < 0.05) in C57BL/6J mice. NS also increased lipid oxidation and antioxidant gene expression while decreasing inflammatory cytokines. In vitro analysis in HepG2 cells revealed that genistein dose-dependently decreases oleic acid-induced lipid accumulation. Soy isoflavones may ameliorate symptoms associated with MetS via anti-inflammatory, antioxidant, and hypolipidemic modulation.

Multi-omic profiles of hepatic metabolism in TPN-fed preterm pigs administered new generation lipid emulsions.

We aimed to characterize the lipidomic, metabolomic, and transcriptomic profiles in preterm piglets administered enteral (ENT) formula or three parenteral lipid emulsions [parenteral nutrition (PN)], Intralipid (IL), Omegaven (OV), or SMOFlipid (SL), for 14 days. Piglets in all parenteral lipid groups showed differential organ growth versus ENT piglets; whole body growth rate was lowest in IL piglets, yet there were no differences in either energy expenditure or (13)C-palmitate oxidation. Plasma homeostatic model assessment of insulin resistance demonstrated insulin resistance in IL, but not OV or SL, compared with ENT. The fatty acid and acyl-CoA content of the liver, muscle, brain, and plasma fatty acids reflected the composition of the dietary lipids administered. Free carnitine and acylcarnitine (ACT) levels were markedly reduced in the PN groups compared with ENT piglets. Genes associated with oxidative stress and inflammation were increased, whereas those associated with alternative pathways of fatty acid oxidation were decreased in all PN groups. Our results show that new generation lipid emulsions directly enrich tissue fatty acids, especially in the brain, and lead to improved growth and insulin sensitivity compared with a soybean lipid emulsion. In all total PN groups, carnitine levels are limiting to the formation of ACTs and gene expression reflects the stress of excess lipid on liver function.

Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function.

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.

Gastric and colonic zinc transporter ZIP11 (Slc39a11) in mice responds to dietary zinc and exhibits nuclear localization.

Zinc transporters have been characterized to further understand the absorption and metabolism of dietary zinc. Our goal was to characterize zinc transporter Slc39a11 (ZIP11) expression and its subcellular localization within cells of the murine gastrointestinal tract of mice and to determine if dietary zinc regulates ZIP11. The greatest ZIP11 expression was in the stomach, cecum, and colon. Both Zip11 mRNA and ZIP11 protein were shown to be downregulated during dietary zinc restriction (<1 mg Zn/kg) in the murine stomach tissue but were unaffected in the colon. Acute repletion with zinc did not restore Zip11 mRNA levels in the stomach. Immunohistochemistry (IHC) revealed high ZIP11 levels in the lower regions of gastric glands and parietal cells of the stomach. IHC analysis of the colon showed a marked ZIP11 abundance within the cytoplasm of the colonic epithelial cells. IHC also showed an increase in ZIP11 expression in the colon during zinc restriction. There is a robust abundance of ZIP11 in the nuclei of cells of both stomach and colon. Our experiments suggest that when dietary zinc intake is compromised, the colon may increase zinc transporter expression to improve the efficiency for absorption via increased expression of specific zinc transporters, including ZIP11 and also zinc transporter Slc39a4. In conclusion, ZIP11 is highly expressed within the murine stomach and colon and appears to be partially regulated by dietary zinc intake within these tissues. ZIP11 may play a specialized role in zinc homeostasis within these tissues, helping to maintain mucosal integrity and function.

Zinc transporter ZIP14 functions in hepatic zinc, iron and glucose homeostasis during the innate immune response (endotoxemia).

ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn(2+) and non-transferrin-bound Fe(2+) in vitro. Using a Zip14(-/-) mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/-) mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/-) mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/-) phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14(-/-) mice and their phenotype shows defects in glucose homeostasis.

Proteomic analysis shows the upregulation of erythrocyte dematin in zinc-restricted human subjects.

BACKGROUND: Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool. OBJECTIVE: The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans. DESIGN: A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins. RESULTS: Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet. CONCLUSIONS: The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as NCT01221129.