Effect of pivalate-induced secondary carnitine deficiency on hepatic transcriptome and plasma metabolome in growing pigs

Administration of pivalate has been demonstrated to be suitable for induction of secondary car-nitine deficiency (CD) in pigs, as model objects for humans. In order to comprehensively charac-terize the metabolic effects of secondary CD in the liver of pigs, the present study aimed to carry out comparative analysis of hepatic transcriptome and plasma metabolome of a total of 12, male 5-weeks-old pigs administered either pivalate (group PIV, n = 6) or vehicle (group CON, n = 6) for 28 days. Pigs of group PIV had approximately 40-60% lower concentrations of free carnitine and acetylcarnitine in plasma, liver and different skeletal muscles than pigs of group CON (p < 0.05). Transcript profiling of the liver revealed 140 differentially expressed genes (DEGs) between group PIV and group CON (fold change > 1.2 or < −1.2, p-value < 0.05). Biological process terms dealing with the innate immune response were found to be enriched with the DEGs (p < 0.05). Using a targeted metabolomics approach for the simultaneous quantification of 630 metabolites, 13 me-tabolites were identified to be lower and 5 metabolites to be higher in group PIV than in group CON (p < 0.05). Despite pivalate-induced CD caused only weak alterations of the hepatic tran-scriptome and the plasma metabolome, the changes observed indicate that secondary CD modu-lates the innate immune response of pigs. For the experiment a total of 12, 5-weeks-old, male cross-bred piglets (Pietrain x (German Landrace x German Edelschwein)) were used, which were kept in pairs in flat-deck pens under controlled conditions (23 ± 2°C room tem-perature, 50-60 % relative humidity, light from 06.00 a.m. to 07.00 p.m.). The piglets were randomly allocated to two groups (group CON, group PIV) of n = 6 piglets each, and fed identical nutrient-adequate diets [48] according to a phase feeding system (phase I: until a body weight of 15 kg; phase II: until the end of the experiment) throughout the experimental period of 28 days. The diet in phase I consisted of (g/kg): wheat, 381.7; barley, 315; soybean meal (44% crude protein), 250; soybean oil, 15; min-eral and vitamin premix, 33.5; L-lysin, 2.6; DL-methionine, 1.0 and L-threonine, 1.2. The diet of phase II consisted of (g/kg): wheat, 401.9; barley, 302; soybean meal (44% crude protein), 240; soybean oil, 15; mineral and vitamin premix, 33.4; L-lysin, 1.5; DL-methionine, 0.5 and L-threonine, 0.7 and a mixture of organic acids (containing 45% formic acid, 7.7% lactic acid, 5% sodium acetate), 5. The carnitine concentration in the diets was below 10 mg/kg diet as analyzed by LC-MS/MS according to [49]. In ad-dition, piglets were given orally either (per kg body weight (BW)): NaHCO3 (Sig-ma-Aldrich, Steinheim, Germany) solution (60 mg/100 mL) (group CON) or 30 mg so-dium pivalate (95% purity; Sigma-Aldrich, Steinheim, Germany) dissolved in NaHCO3 solution (group PIV) each day throughout the experimental period of 28 days. Water was constantly available ad libitum from nipple drinkers during both experiments. At the end of the experiment, the pigs were killed in the post-prandial period (2 h after their last meal) by electronarcosis followed by exsanguination. Blood was col-lected into heparinized polyethylene tubes (Sarstedt, Nümbrecht, Germany) and plasma obtained by centrifugation at 1100 x g for 10 min at 4°C and subsequently stored at -80°C. Aliquots from liver and selected muscles (longissimus dorsi, semitendi-nosus, superficial biceps femoris) were collected, snap-frozen in liquid nitrogen and stored at -80°C pending analysis.