68 A single citrulline-supplemented meal increases arginine availability but not mTORC1 signaling or protein synthesis in skeletal muscle of preterm piglets.

Abstract Low birth weight piglets often exhibit poor postnatal growth and increased mortality rates, particularly before weaning. During the perinatal period, arginine is an indispensable amino acid that can activate mechanistic target of rapamycin complex 1 (mTORC1)-dependent translation initiation signaling and promote protein synthesis. However, due to extensive first-pass hepatic metabolism, dietary arginine has limited systemic availability. Citrulline, a metabolic precursor of arginine, bypasses hepatic metabolism and is converted to arginine in tissues. We hypothesized that early citrulline supplementation would enhance skeletal muscle protein synthesis in preterm piglets by increasing arginine availability and activating mTORC1-dependent translation initiation signaling. Preterm piglets (Yorkshire × Landrace sows bred to Duroc boars; 1.03 ± 0.16 kg BW) were delivered by cesarean section at gestational day 105 (9 d preterm) and received total parenteral nutrition for 3 d. On d 4, following a 4 h fast, piglets were continuously fed a milk formula (ME = 174 kcal/kg/d; protein = 11.5 g/kg/d) supplemented with either alanine (ALA; 600 µmol/kg BW/h; n = 13) or citrulline (CIT; 600 µmol/kg BW/h; n = 14) via an orogastric tube. After 60 minutes, L-[ring-2H5]-phenylalanine was administered to measure fractional protein synthesis rates. Piglets were euthanized 30 min later, and longissimus dorsi and gastrocnemius muscles were collected. Enrichment of ²H₅-phenylalanine in muscle proteins and precursor pools, as well as free amino acid concentrations in plasma and muscle, was quantified by LC-MS/MS. mTORC1 signaling markers were analyzed by immunoprecipitation and immunoblotting. CIT supplementation increased plasma concentrations of arginine (3.1-fold; P < 0.01) and ornithine (2.5-fold; P < 0.01), while reducing plasma glutamine levels (P = 0.02), compared to ALA controls. No significant changes were observed in other plasma amino acids. These plasma changes were mirrored in skeletal muscle, where CIT-treated piglets exhibited higher levels of arginine (1.6-fold; P < 0.01) and ornithine (1.5-fold; P < 0.01) in the longissimus dorsi muscle. Despite the increased plasma and muscle availability of arginine, CIT supplementation resulted in only a modest, non-significant trend toward increased fractional protein synthesis in the longissimus dorsi (16.8 vs. 20.1 %/d; P = 0.09). Markers of mTORC1 pathway activation for translation initiation, including phosphorylation of S6K1 and 4EBP1, and formation of the eIF4E-eIF4G complex, did not differ between CIT- and ALA-supplemented piglets. Our findings demonstrate that short-term enteral citrulline supplementation effectively raises plasma and skeletal muscle arginine levels in preterm piglets but does not stimulate mTORC1 signaling to translation initiation or protein synthesis in skeletal muscle. Funding Sources: NIH and USDA.