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

2016; Elsevier BV; Volume: 57; Issue: 9 Linguagem: Inglês

10.1194/jlr.m069526

ISSN

1539-7262

Autores

Gregory Guthrie, Madhulika Kulkarni, Hester Vlaardingerbroek, Barbara J. Stoll, Kenneth Ng, Camilia R. Martin, John W. Belmont, Darryl L. Hadsell, William C. Heird, Christopher B. Newgard, Oluyinka O. Olutoye, Johannes B. van Goudoever, Charlotte Lauridsen, Xingxuan He, Edward H. Schuchman, Douglas G. Burrin,

Tópico(s)

Diet and metabolism studies

Resumo

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 13C-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. 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 13C-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. Many premature infants cannot tolerate full enteral (ENT) feeding due to an immature gastrointestinal tract. Parenteral nutrition (PN) is critical in these babies to avoid nutritional deficiencies, promote growth, and to achieve normal maturation. However, PN can lead to hepatic metabolic complications, such as cholestasis, within 2 weeks of PN administration (1.Kelly D.A. Liver complications of pediatric parenteral nutrition–epidemiology.Nutrition. 1998; 14: 153-157Crossref PubMed Scopus (280) Google Scholar), and 90% of infants who receive PN for greater than 3 months develop PN-associated liver disease (PNALD) (2.Beale E.F. Nelson R.M. Bucciarelli R.L. Donnelly W.H. Eitzman D.V. Intrahepatic cholestasis associated with parenteral nutrition in premature infants.Pediatrics. 1979; 64: 342-347Crossref PubMed Google Scholar). Limiting the lipid load administered to premature infants has been shown to be effective in preventing the onset of cholestasis (3.Sanchez S.E. Braun L.P. Mercer L.D. Sherrill M. Stevens J. Javid P.J. The effect of lipid restriction on the prevention of parenteral nutrition-associated cholestasis in surgical infants.J. Pediatr. Surg. 2013; 48: 573-578Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, infants require sufficient caloric intake to achieve appropriate growth and neurodevelopment (4.Ehrenkranz R.A. Dusick A.M. Vohr B.R. Wright L.L. Wrage L.A. Poole W.K. Growth in the neonatal intensive care unit influences neurodevelopmental and growth outcomes of extremely low birth weight infants.Pediatrics. 2006; 117: 1253-1261Crossref PubMed Scopus (975) Google Scholar), so the option to limit the PN lipid to prevent PNALD is not ideal. For these reasons, it is important to understand the process of PNALD development in this neonatal population. The lipid fraction of PN solutions has been a target of research to understand the development of PNALD. Soybean-based lipid emulsions are the prevalent lipid emulsion used in the US and are approved by the Food and Drug Administration for clinical use in neonates, but have been associated with PNALD (5.Waitzberg D.L. Torrinhas R.S. Jacintho T.M. New parenteral lipid emulsions for clinical use.JPEN J. Parenter. Enteral Nutr. 2006; 30: 351-367Crossref PubMed Scopus (178) Google Scholar). Total PN (TPN) solutions that contain fish oil-based emulsions have been associated with prevention of cholestasis and reversal of cholestasis in some limited studies (6.de Meijer V.E. Gura K.M. Le H.D. Meisel J.A. Puder M. Fish oil-based lipid emulsions prevent and reverse parenteral nutrition-associated liver disease: the Boston experience.JPEN J. Parenter. Enteral Nutr. 2009; 33: 541-547Crossref PubMed Scopus (144) Google Scholar, 7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), but the basis for these observations is not clear. Some research suggests that phytosterols, a component of soy lipid emulsions, can suppress bile acid synthesis and impact bile acid efflux target genes in the liver through inhibition of the nuclear hormone receptor, farnesoid X receptor. A recent report by El Kasmi et al. (8.El Kasmi K.C. Anderson A.L. Devereaux M.W. Vue P.M. Zhang W. Setchell K.D. Karpen S.J. Sokol R.J. Phytosterols promote liver Injury and Kupffer cell activation in parenteral nutrition-associated liver disease.Sci. Transl. Med. 2013; 5: 206ra137Crossref PubMed Scopus (144) Google Scholar) suggests that phytosterols taken up by Kupffer cells mediate an inflammation-induced injury to hepatocytes. However, our recent study in PN-fed piglets showed that a lipid emulsion blend of soy, medium-chain triglycerides (MCTs), olive oil, and fish oil, which contains less than one-half the total phytosterol content as soy-lipid emulsions [207 mg/dl vs. 439 mg/dl (9.Xu Z. Harvey K.A. Pavlina T. Dutot G. Hise M. Zaloga G.P. Siddiqui R.A. Steroidal compounds in commercial parenteral lipid emulsions.Nutrients. 2012; 4: 904-921Crossref PubMed Scopus (47) Google Scholar)], also protects against PNALD (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Pure fish oil and fish oil blend emulsions, unlike soybean oil-only emulsions, contain considerable amounts of long-chain omega-3 PUFAs. The administration of fish oil high in omega-3 fatty acids, DHA, and EPA, can suppress both insulin- and carbohydrate-mediated triglyceride synthesis through repression of sterol regulatory element-binding protein (SREBP) (10.Xu J. Nakamura M.T. Cho H.P. Clarke S.D. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats.J. Biol. Chem. 1999; 274: 23577-23583Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) and carbohydrate responsive element-binding protein (11.Dentin R. Benhamed F. Pegorier J.P. Foufelle F. Viollet B. Vaulont S. Girard J. Postic C. Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation.J. Clin. Invest. 2005; 115: 2843-2854Crossref PubMed Scopus (237) Google Scholar), respectively. Omega-3 fatty acids are also capable of activating the nuclear hormone receptor, PPARα, to upregulate downstream target genes of fat oxidation in both mitochondria and peroxisomes (12.Pawar A. Jump D.B. Unsaturated fatty acid regulation of peroxisome proliferator-activated receptor alpha activity in rat primary hepatocytes.J. Biol. Chem. 2003; 278: 35931-35939Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13.Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (1045) Google Scholar). In addition, omega-3 fatty acids promote the formation of anti-inflammatory prostaglandins, eicosanoids, and resolvins (14.Calder P.C. n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases.Am. J. Clin. Nutr. 2006; 83: 1505S-1519SCrossref PubMed Google Scholar). It is likely that improved steatohepatitis and glucose sensitivity observed in adults administered omega-3 treatments are due to a combination of these targeted effects (15.Parker H.M. Johnson N.A. Burdon C.A. Cohn J.S. O'Connor H.T. George J. Omega-3 supplementation and non-alcoholic fatty liver disease: a systematic review and meta-analysis.J. Hepatol. 2012; 56: 944-951Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 16.Scorletti E. Byrne C.D. Omega-3 fatty acids, hepatic lipid metabolism, and nonalcoholic fatty liver disease.Annu. Rev. Nutr. 2013; 33: 231-248Crossref PubMed Scopus (221) Google Scholar). However, there is limited evidence in preterm infant studies describing how these markedly different fatty acids present in new generation parenteral lipid emulsions alter tissue fatty acid profiles and metabolic function, especially in the liver. As with omega-3 fatty acids, clinical trials in adults have shown that vitamin E protects against nonalcoholic fatty liver disease (NAFLD) (17.Sanyal A.J. Chalasani N. Kowdley K.V. McCullough A. Diehl A.M. Bass N.M. Neuschwander-Tetri B.A. Lavine J.E. Tonascia J. Unalp A. et al.Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis.N. Engl. J. Med. 2010; 362: 1675-1685Crossref PubMed Scopus (2243) Google Scholar) and chronic hepatitis C (18.Ji H.F. Sun Y. Shen L. Effect of vitamin E supplementation on aminotransferase levels in patients with NAFLD, NASH, and CHC: Results from a meta-analysis.Nutrition. 2014; 30: 986-991Crossref PubMed Scopus (50) Google Scholar). In NAFLD, free fatty acids can induce reactive oxygen species causing mitochondrial dysfunction, including reduced fatty acid β-oxidation (19.Pérez-Carreras M. Del Hoyo P. Martin M.A. Rubio J.C. Martin A. Castellano G. Colina F. Arenas J. Solis-Herruzo J.A. Defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis.Hepatology. 2003; 38: 999-1007Crossref PubMed Scopus (555) Google Scholar). This reduced β-oxidation capacity can trigger a compensatory increase in activity of hepatic peroxisomal and microsomal fatty acid oxidizing enzymes, like cytochrome P450 2E1 (CYP2E1), that generate large amounts of hydrogen peroxide (20.Weltman M.D. Farrell G.C. Hall P. Ingelman-Sundberg M. Liddle C. Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis.Hepatology. 1998; 27: 128-133Crossref PubMed Scopus (564) Google Scholar). A key protective function of vitamin E is its strong antioxidant activity that confers defense against reactive oxygen species-mediated lipid peroxidation (21.Zamin Jr., I. Mattos A.A. Mattos A.Z. Coral G. Santos D. Rhoden C. The vitamin E reduces liver lipoperoxidation and fibrosis in a model of nonalcoholic steatohepatitis.Arq. Gastroenterol. 2010; 47: 86-92Crossref PubMed Scopus (14) Google Scholar). In addition, there is evidence suggesting that natural vitamin E (RRR-α-tocopherol and δ- and γ-tocopherol) can act as ligand for the pregnane X receptor and activate xenobiotic metabolic pathways (22.Landes N. Pfluger P. Kluth D. Birringer M. Ruhl R. Bol G.F. Glatt H. Brigelius-Flohe R. Vitamin E activates gene expression via the pregnane X receptor.Biochem. Pharmacol. 2003; 65: 269-273Crossref PubMed Scopus (207) Google Scholar). Treatment with the pregnane X receptor agonist, rifampicin, has been shown to be beneficial in the treatment of pruritus of cholestasis (23.Khurana S. Singh P. Rifampin is safe for treatment of pruritus due to chronic cholestasis: a meta-analysis of prospective randomized-controlled trials.Liver Int. 2006; 26: 943-948Crossref PubMed Scopus (132) Google Scholar). The effect of new generation lipid emulsions on the vitamin E status in preterm infants and enrichment in tissue, such as the liver, is poorly understood. In our previous report, we established that new generation lipid emulsions containing fish oil and vitamin E lead to decreased indices of liver damage and lipid accumulation (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In this report, we have taken a multi-omics approach to examine the effect of those lipid emulsions on whole body metabolism, fatty acid utilization, and lipidomic and metabolomic profiles in the liver and muscle tissue in our PN-fed piglet model. Our second aim was to characterize the impact of these different lipid emulsions on hepatic gene networks using a genome-wide transcriptomic analysis. The study protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Division of Receipt and Referral/National Institutes of Health, Bethesda, MD). Sows were obtained from the Texas Department of Criminal Justice (Huntsville, TX) and housed in the Children's Nutrition Research Center. Our surgical protocol has been previously described (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, domestic crossbred piglets were delivered 7 days preterm on gestation day 108 by caesarian section. Piglets were administered injections of iron dextran (100 mg/ml), ampicillin (125 mg/ml), and buprenorphine (0.3 mg/ml) prior to surgery. Piglets received either ENT nutrition or three forms of TPN. Saline or TPN was administered via surgically implanted jugular and umbilical arterial catheters. Enterally fed piglets were implanted with an orogastric feeding tube and TPN groups received a sham puncture. TPN consisted of a solution containing a complete nutrient mixture of amino acids, glucose, electrolytes, vitamins, and trace minerals, administered via jugular catheter, and a parenteral lipid emulsion, which was infused separately via an umbilical arterial catheter. Piglets in the TPN groups randomly received one of the following lipid emulsions: 100% soybean oil [20% Intralipid (IL)], 100% fish oil [Omegaven (OV)], or a mixture of 30% soybean oil, 30% MCTs, 25% olive oil, and 15% fish oil [SMOFlipid (SL)]; all three lipid emulsions were provided by Fresenius Kabi (Bad Homburg, Germany). ENT piglets were fed a milk-based formula (Litter Life; Merrick, Middletown, WI) at 240 ml/kg in eight feeds per day (24.Stoll B. Horst D.A. Cui L. Chang X. Ellis K.J. Hadsell D.L. Suryawan A. Kurundkar A. Maheshwari A. Davis T.A. et al.Chronic parenteral nutrition induces hepatic inflammation, steatosis, and insulin resistance in neonatal pigs.J. Nutr. 2010; 140: 2193-2200Crossref PubMed Scopus (57) Google Scholar). Postsurgery, TPN was started at 5 ml/(kg·h) and gradually increased to 10 ml/(kg·h). ENT piglets also received TPN with IL following surgery, but were started on ENT feeds on day of life 2 and gradually weaned off TPN. On day 7, all TPN and enterally fed piglets received full amounts of nutrition per kilogram body weight: fluid, 240 ml; energy, 195 kcal; carbohydrate, 25 g; protein, 14 g; and lipid, 5 g, as described (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Piglets were weighed every other day and blood samples were drawn during surgery (day 0) and at day 14. Immediately after the last blood sample was taken on day 14, the animals were anesthetized with isoflurane and euthanized with injection of Beuthanasia (pentobarbital sodium, phenytoin sodium). Organs were isolated and weighed. Liver tissue samples were frozen in liquid nitrogen and stored at −80°C until analysis. Liver samples were also fixed in OCT for histopathology. Blood samples were collected in Na2EDTA tubes and centrifuged for 10 min at 2,000 g in 4°C. Samples were flash-frozen in liquid nitrogen and stored at −80°C until analysis. Glucose and insulin were analyzed as previously described (24.Stoll B. Horst D.A. Cui L. Chang X. Ellis K.J. Hadsell D.L. Suryawan A. Kurundkar A. Maheshwari A. Davis T.A. et al.Chronic parenteral nutrition induces hepatic inflammation, steatosis, and insulin resistance in neonatal pigs.J. Nutr. 2010; 140: 2193-2200Crossref PubMed Scopus (57) Google Scholar). As piglets were on continuous TPN infusion, they received a constant glucose infusion rate. Utilizing this, we performed homeostatic model assessment of insulin resistance (HOMA-IR) calculations with the HOMA2 formula (25.Matthews D.R. Hosker J.P. Rudenski A.S. Naylor B.A. Treacher D.F. Turner R.C. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man.Diabetologia. 1985; 28: 412-419Crossref PubMed Scopus (25498) Google Scholar): HOMA-IR = [glucose (mmol/l) × insulin (μU/ml)]/22.5. For serum cholesterol, triglyceride, and lactate dehydrogenase, analysis was performed on a Cobas Integra 400 Plus analyzer (Roche). For measurements of respiratory exchange ratio (RER), heat production, and 13CO2, piglets were placed in air-tight calorimetry chambers (Columbus Instruments) and measurements were performed as described previously (26.Bauchart-Thevret C. Stoll B. Benight N.M. Olutoye O. Lazar D. Burrin D.G. Supplementing monosodium glutamate to partial enteral nutrition slows gastric emptying in preterm pigs(1–3).J. Nutr. 2013; 143: 563-570Crossref PubMed Scopus (12) Google Scholar). Specifically for 13CO2 analysis, on day 11, piglets were placed in a calorimetry chamber and administered [1-13C]palmitate. Prior to infusion, arterial and breath samples were taken to determine background enrichment of 13C. After background sampling, a primed continuous 4 h co-infusion of [1-13C]palmitate (15 μmol/(kg·h)) with respective lipid treatment was administered. Expired breath samples from the calorimetry chamber were collected every 30 min for analysis of 13CO2 enrichment during the 4 h infusion. Whole-body [1-13C]palmitate oxidation was calculated using standard steady state equations (26.Bauchart-Thevret C. Stoll B. Benight N.M. Olutoye O. Lazar D. Burrin D.G. Supplementing monosodium glutamate to partial enteral nutrition slows gastric emptying in preterm pigs(1–3).J. Nutr. 2013; 143: 563-570Crossref PubMed Scopus (12) Google Scholar). Total fatty acids were isolated from plasma and tissues using a modified method from Folch (27.Freedman S.D. Blanco P.G. Zaman M.M. Shea J.C. Ollero M. Hopper I.K. Weed D.A. Gelrud A. Regan M.M. Laposata M. et al.Association of cystic fibrosis with abnormalities in fatty acid metabolism.N. Engl. J. Med. 2004; 350: 560-569Crossref PubMed Scopus (333) Google Scholar). Briefly, 20–150 mg tissue samples were homogenized in 1–2 ml PBS. Plasma samples (200 μl) were diluted with 500 μl PBS. Samples were extracted from plasma and tissues (500 μl) by the addition of chloroform-methanol (2:1 v/v). The samples were then incubated for 10 min on ice, and then spun at 2,500 rpm for 10 min. The top layer was discarded and the remaining sample was dried under nitrogen gas. Methanolic NaOH (0.5 ml, 0.05 M) was added to the dried sample and incubated for 3 min at 100°C, followed by addition of 0.5 ml BF3-methanol. The samples were heated for an additional 1 min at 100°C, and then 1 ml hexane and 6.5 ml saturated NaCl were added. Samples were vortexed and spun at 1,700 rpm for 4 min. The upper hexane phase was used for analysis. Quantification of fatty acid methyl esters was performed on a gas chromatograph (HP5890 Series II, Hewlett Packard) equipped with a SP-10 capillary column (Supelco) attached to a mass spectrometer (HP-5971, Hewlett Packard). Vitamin E (α-tocopherol and γ-tocopherol) in plasma and liver was analyzed as described previously (28.Jensen S.K. Engberg R.M. Hedemann M.S. All-rac-alpha-tocopherol acetate is a better vitamin E source than all-rac-alpha-tocopherol succinate for broilers.J. Nutr. 1999; 129: 1355-1360Crossref PubMed Scopus (75) Google Scholar, 29.Jensen S.K. Nielsen K.N. Tocopherols, retinol, beta-carotene and fatty acids in fat globule membrane and fat globule core in cows' milk.J. Dairy Res. 1996; 63: 565-574Crossref PubMed Google Scholar). In brief, liver tissue was homogenized in two volumes of ethanol with an Ultra-Turrax homogenizer on ice. Aliquots of the homogenates corresponding to 200 mg liver were saponified in a mixture of ethanol, methanol, ascorbic acid (20% w/v), and KOH-water (1:1 w/v) at 80°C for 30 min, subsequently cooled, and extracted twice with 5 ml of heptane. The HPLC column for determination of tocopherol consisted of a 4.0 × 125 mm Perkin-Elmer HS-5-Silica column (Perkin-Elmer GmbH, Überlingen, Germany). The mobile phase consisted of heptane containing 2-propanol (3.0 ml/l) and degassed with helium. The flow rate was 3.0 ml/min. A comparison of retention time and peak areas with Merck (Damstadt, Germany) external standards was used to obtain the identification and quantification of the tocopherol. RNA was extracted from liver tissue using TruSeq RNA sample preparation kit v2 (Illumina) and sequenced using Illumina RNA Hi-seq 2000. Sequencing data was analyzed using a series of software tools, which included Bowtie, tophat, and the cufflinks package [cufflinks, cuffcompare, cuffmerge, and cuffdiff (30.Trapnell C. Roberts A. Goff L. Pertea G. Kim D. Kelley D.R. Pimentel H. Salzberg S.L. Rinn J.L. Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.Nat. Protoc. 2012; 7: 562-578Crossref PubMed Scopus (168) Google Scholar)]. These generated lists of differentially expressed and regulated genes and transcripts. Piglet genome Sus scrofa 10.2.68 from Ensembl (http://www.ensembl.org/Sus_scrofa/Info/Index) was used as a reference. Cutoff values for significance were: fold change greater than ±1.5, P < 0.05, and q < 0.1. Analyzed data was subjected to Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com). Acyl-CoAs (ACs), acylcarnitines (ACTs), and organic acids (OAs) were analyzed by targeted MS. For preparation of tissue samples for AC and OA content, tissues were homogenized in 50% aqueous acetonitrile containing 0.3% formic acid. OAs were analyzed by Trace Ultra GC coupled to a Trace DSQ mass spectrometer using a stable isotope dilution method described previously (31.Jensen M.V. Joseph J.W. Ilkayeva O. Burgess S. Lu D. Ronnebaum S.M. Odegaard M. Becker T.C. Sherry A.D. Newgard C.B. Compensatory responses to pyruvate carboxylase suppression in islet beta-cells. Preservation of glucose-stimulated insulin secretion.J. Biol. Chem. 2006; 281: 22342-22351Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 32.Ng K. Stoll B. Chacko S. Saenz de Pipaon M. Lauridsen C. Gray M. Squires E.J. Marini J. Zamora I.J. Olutoye O.O. et al.Vitamin E in new-generation lipid emulsions protects against parenteral nutrition-associated liver disease in parenteral nutrition-fed preterm pigs.JPEN J. Parenter. Enteral Nutr. 2016; 40: 656-671Crossref PubMed Scopus (57) Google Scholar). For analysis of long-chain ACs, chloroacetaldehyde was added to the homogenate and samples were separated by HPLC and then detection of individual ACs was done fluorometrically (33.Larson T.R. Graham I.A. Technical advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues.Plant J. 2001; 25: 115-125Crossref PubMed Google Scholar). For ACT analysis, samples were homogenized in water and analyzed by flow injection MS/MS, as described previously (34.An J. Muoio D.M. Shiota M. Fujimoto Y. Cline G.W. Shulman G.I. Koves T.R. Stevens R. Millington D. Newgard C.B. Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance.Nat. Med. 2004; 10: 268-274Crossref PubMed Scopus (364) Google Scholar). Mean absolute concentrations of each metabolite were then transformed using base package in R (http://www.R-project.org). Clustering heatmaps were created using the heatmap.2 function in gplot (35.Warnes G.R. Bolker B. Bonebakker L. Gentleman R. Huber W. Liaw A. Lumley T. Maechler M. Magnusson A. Moeller S. Gplots: Various R programming tools for plotting data..https://cran.r-project.org/web/packages/gplots/index.html. 2019; 10Google Scholar) in conjunction with Mkmisc (36.Kohl M. MKmisc: Miscellaneous functions from M. Kohl.J. Clin. Invest. 2013; 10Google Scholar) and Rcolor (37.Neuwirth E. RColorBrewer: ColorBrewer palettes... 2017; 10Google Scholar). Real-time quantitative (q)PCR was performed on frozen liver samples. The cDNA was generated from RNA extracted from 100 to 150 mg of frozen liver tissue, as described previously (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Real-time qPCR was performed with SYBR Green chemistry (Applied Biosystems) on a Bio-Rad CFX96. Primers were designed using software from NCBI Primer Blast. The primers for porcine Aox1, Cyp2e1, Cyp1a2, Fmo5, interleukin (Il)-1β, Il-6, Tnf, and Il-8 (supplemental Table S1) were designed using the predicted porcine sequence available on Ensemble Genome Browser [Gene identification (ID): ENSSSCG00000012871]. Amplification efficiency was controlled by the use of an internal control (GAPDH or actin). Relative quantification of target mRNA expression was calculated and normalized to GAPDH or actin expression. All reactions were performed under the following thermal cycling conditions: 10 min at 95°C followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The 2−ΔΔCT method was used to compare gene expression levels between samples, which were analyzed to determine the fold induction of mRNA expression. Statistical analyses were performed using SPSS 16 software (Armonk, NY). Differences among the four groups were first analyzed using one-way ANOVA, and post hoc analysis was done using Tukey's test, as described in the figure legends. For results that were not of normal distribution, the nonparametric Kruskal-Wallis ANOVA on Ranks followed by pairwise comparisons using Dunn's test was used to determine significance. P < 0.05 was considered significant. Results are presented as mean ± SEM. Following 14 days of ENT or TPN administration, body weight and tissue weights differed in piglets between experimental treatments (Table 1). Body and liver weights were previously published, but included in this analysis for continuity and comparison with other tissue weights (7.Vlaardingerbroek H. Ng K. Stoll B. Benight N. Chacko S. Kluijtmans L.A. Kulik W. Squires E.J. Olutoye O. Schady D. et al.New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs.J. Lipid Res. 2014; 55: 466-477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Growth rates [g/(kg·h)] were significantly decreased (P < 0.05) in the IL group versus other treatments. Jejunum and ileum weights were significantly lower, whereas liver and spleen weights were higher in the TPN-administered groups compared with ENT (P < 0.05). The pancreas weight was lower (P < 0.05) in the OV and SL groups compared with ENT, but IL did not differ from ENT. The heart tissue weight was significantly higher (P < 0.05) in the TPN versus ENT piglets.TABLE 1Body and tissue weights of preterm piglets after 14 days with ENT and TPN treatmentsENT (n = 14)IL (n = 12)OV (n = 13)SL (n = 7)Body weightDay 0 (g)1,224 ± 84.01,257 ± 115.51,280 ± 72.421,289 ± 120Day 14 (g)2,914 ± 1632,562 ± 198.22,790 ± 106.92,844 ± 184Growth rate [g/(kg·day)]62.79 ± 3.0047.51 ± 1.24aP < 0.05 versus ENT.56.08 ± 2.0657.32 ± 2.60Tissue weights (g/kg body weight)Liver32.38 ± 1.2747.80 ± 1.75aP < 0.05 versus ENT.47.64 ± 1.48aP < 0.05 versus ENT.41.59 ± 1.26aP < 0.

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