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.

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.

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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-d-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 weeks: 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 pro-fibrotic 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.

Background Fructose consumption has been linked to nonalcoholic fatty liver disease (NAFLD) in children. However, the effect of high-fructose corn syrup (HFCS) compared with sucrose in pediatric NAFLD has not been investigated. Objectives We tested whether the isocaloric substitution of dietary sucrose by HFCS would increase the severity of NAFLD in juvenile pigs, and whether this effect would be associated with changes in gut histology, SCFA production, and microbial diversity. Methods Iberian pigs, 53-d-old and pair-housed in pens balanced for weight and sex, were randomly assigned to receive a mash diet top-dressed with increasing amounts of sucrose (SUC; n = 3 pens; 281.6–486.8 g/kg diet) or HFCS (n = 4; 444.3–724.8 g/kg diet) during 16 wk. Diets exceeded the animal's energy requirements by providing sugars in excess, but met the requirements for all other nutrients. Animals were killed at 165 d of age after blood sampling, and liver, muscle, and gut were collected for histology, metabolome, and microbiome analyses. Data were analyzed by multivariate and univariate statistics. Results Compared with SUC, HFCS increased subcutaneous fat, triacylglycerides in plasma, and butyrate in colon (P ≤ 0.05). In addition, HFCS decreased UMP and short-chain acyl carnitines in liver, and urea nitrogen and creatinine in serum (P ≤ 0.05). Microbiome analysis showed a 24.8% average dissimilarity between HFCS and SUC associated with changes in SCFA-producing bacteria. Body weight gain, intramuscular fat, histological and serum markers of liver injury, and circulating hormones, glucose, and proinflammatory cytokines did not differ between diets. Conclusions Fructose consumption derived from HFCS promoted butyrate synthesis, triglyceridemia, and subcutaneous lipid deposition in juvenile Iberian pigs, but did not increase serum and histological markers of NAFLD compared with a sucrose-enriched diet. Longer studies could be needed to observe differences in liver injury among sugar types.

Objectives Non-alcoholic fatty liver disease (NAFLD) represents the major cause of pediatric chronic liver pathology in the United States. The objective of this study was to compare the relative effect of inclusion of isocaloric amounts of saturated medium-chain fatty acids (hydrogenated coconut oil), saturated long-chain fatty acids (lard) and unsaturated long-chain fatty acids (olive oil) on endpoints of NAFLD and insulin resistance. Methods Thirty-eight 15-d-old Iberian pigs were fed 1 of 4 diets containing (g/kg body weight × d) 1) control (CON; n = 8): 0 g fructose, 10.5 g fat, and 187 kcal metabolizable energy (ME), 2) lard (LAR; n = 10): 21.6 g fructose, 17.1 g fat (100% lard) and 299 kcal ME, 3) hydrogenated coconut oil (COCO; n = 10): 21.6 g fructose, 16.9 g fat (42.5% lard and 57.5% coconut oil) and 299 kcal ME, and 4) olive oil (OLV, n = 10): 21.6 g fructose, 17.1 g fat (43.5% lard and 56.5% olive oil) and 299 kcal ME, for 9 consecutive weeks. Body weight was recorded every 3 d. Serum markers of liver injury and dyslipidemia were measured on d 60 at 2 h post feeding, with all other serum measures assessed on d 70. Liver tissue was collected on d 70 for histology, triacylglyceride (TG) quantification, and metabolomics analysis. Results Tissue histology indicated the presence of steatosis in LAR, COCO and OLV compared with CON (P ≤ 0.001), with a further increase in in non-alcoholic steatohepatitis (NASH) in OLV and COCO compared with LAR (P ≤ 0.01). Alanine and aspartate aminotransferases were higher in COCO and OLV (P ≤ 0.01) than CON. All treatment groups had lower liver concentrations of methyl donor's choline and betaine versus CON, while bile acids were differentially changed (P ≤ 0.05). COCO had higher levels of TGs with less carbons (Total carbons &lt; 52) than all other groups (P ≤ 0.05). Several long-chain acylcarnitines involved in fat oxidation were higher in OLV versus all other groups (P ≤ 0.05). Conclusions Inclusion of fats enriched in medium-chain saturated and long-chain unsaturated fatty acids in a high-fructose high-fat diet increased liver injury, compared with fats with a long-chain saturated fatty acid profile. Further research is required to investigate the mechanisms causing this difference in physiological response to these dietary fat sources. Funding Sources ARI, AcornSeekers.

Objectives Non-alcoholic fatty liver disease (NAFLD) is a leading nutrition-linked liver disease in children. We have recently identified the neonatal Iberian pig as a novel model of pediatric NAFLD. Interestingly, feeding a high fructose-high fat (HFF) diet to neonatal Iberian pigs for 10 weeks did not induce obesity and decreased high and low density lipoproteins in serum, while the liver exhibited many of the histological features of pediatric NAFLD. In this study we utilized a metabolomics approach to characterize the metabolic phenotype of this pig model. Methods Plasma and liver samples collected from Iberian pigs fed control, HFF, control + probiotic and HFF + probiotic diets were analyzed by LC-MS using targeted assays for primary metabolomics, aminomics, and lipidomics. Data was analyzed with multivariate statistics and 2-way ANOVA with t-tests adjusted for false discovery rate. Results Results from multivariate tests indicated no diet × probiotic interaction or probiotic effect, so only the main effect of diet is shown. A total of 224 and 218 metabolites were identified in the plasma and liver, respectively. In agreement with previous NAFLD models, the HFF diet increased hepatic branched chain amino acids, cholesteryl esters, hydroxyproline, plasma acylcarnitines and primary bile acids, and altered hepatic triacylglycerol-species composition (P < 0.0001). In addition, HFF pigs had increased homocysteine and methionine levels combined with a decrease in pyridoxate in liver (P < 0.0001), suggesting a disruption in the one carbon cycle and its methyl donation capability. HFF decreased hepatic choline and betaine (P < 0.0001), which may indicate a compensatory use of the methyl groups of these compounds. The aforementioned reduction of choline used for the CDP-choline pathway and the potential one carbon cycle impact on SAM availability for the phosphatidylethanolamine N-methyltransferase (PEMT) pathway may explain the observed differential changes in hepatic phosphatidylcholines (P < 0.0001), which are required for the production of very low density lipoproteins (VLDL). Conclusions Unique features of this model such as reduced plasma lipoproteins and lack of weight gain in the presence of hepatic steatosis may be related to an altered one carbon cycle impacting phosphatidylcholine production. Funding Sources ARI, AcornSeekers.

Objectives Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder and the most common liver disease in pediatric populations. Epidemiological studies have observed a parallel increase in fructose consumption and incidence of NAFLD among children. The objective of this study was to compare the relative effect of inclusion of isocaloric amounts of high fructose corn syrup (66.5% fructose, 33.5% glucose) versus sucrose (50% fructose, 50% glucose) in the diet for 16 weeks on endpoints of NAFLD and insulin resistance. Methods 30-d-old Iberian pigs were housed in pairs and randomly assigned to receive solid diets (g/kg body weight × d) of 1) control (CON; n = 6): 0 g HFCS, 0 g SUC, and 174.03 kcal metabolizable energy (ME), 2) high-fructose corn syrup (HFCS; n = 8): 31.20 g high-fructose corn syrup, 0 g sucrose and 261 kcal ME, and 3) Sucrose (SUC; n = 6): 0 g high-fructose corn syrup, 24.04 g sucrose and 261 kcal ME for 16 consecutive weeks. Results Compared to CON, both HFCS and SUC diets increased body weight gain (P ≤ 0.001), relative liver weight (P ≤ 0.01) and leptin levels (P ≤ 0.01), and decreased percentage of lean mass composition in the animals (P ≤ 0.001). In addition, HFCS increased fasting insulin levels compared to CON (P ≤ 0.05), and decreased percentage lean mass compared to SUC (P ≤ 0.05). 75% of HFCS and 66.6% of SUC pigs showed histopathological lesions consistent with microvesicular steatosis with periportal or diffuse distribution. Serum markers of liver injury did not differ between diets, and none of the animals developed inflammation, hepatocellular ballooning, Mallory hyaline or necrosis in the liver. Metabolomics analysis revealed liver sorbitol and monosaccharide concentrations were higher in both the HFCS and SUC groups versus CON (P ≤ 0.05), while adenosine monophosphate (AMP) were higher and adenosine diphosphate levels lower in the HFCS and SUC in comparison to CON (P ≤ 0.05). Numerous phosphatidylcholines and sphingomyelins were differentially changed in the HFCS group versus CON (P ≤ 0.05). Conclusions Feeding diets high in either sucrose or high fructose corn syrup promoted obesity and steatosis in the animals. Further research is needed to investigate the mechanisms leading to increased insulin resistance in the HFCS group. Funding Sources ARI #58,873, AcornSeekers, STRIDE.