(P15-007-25) Short-term Citrulline Supplementation Has Minimal Effects on Skeletal Muscle Protein Synthesis and mTORC1 Signaling in Neonatal Preterm Pigs

Objectives: Postnatal growth of lean mass is commonly impaired in preterm infants and may contribute to long-term morbidities. In neonates, arginine is an indispensable amino acid that can activate mTORC1 and promote protein anabolism in skeletal muscle. 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 citrulline supplementation enhances skeletal muscle protein synthesis by increasing arginine availability and activating mTORC1-dependent translation initiation signaling.

Methods: Pigs were delivered 9 days preterm by cesarean section. They received total parenteral nutrition for 3 days and, on day 4, were fed a protein- and energy-balanced milk replacer (ME = 174 kcal/(kg/d); protein = 11.5 g/(kg/d)) via orogastric tube. They were assigned to receive either citrulline (CIT; 600 µmol/(kg BW/h); n = 14) or alanine (ALA; 600 µmol/(kg BW/h); n = 13). After 60 minutes, they received a large pulse of L-[ring-²H₅]-Phe to measure protein synthesis. Pigs were euthanized 30 minutes later, and longissimus dorsi and gastrocnemius muscles were collected. ²H₅-Phe enrichment of muscle proteins and the precursor pool was measured by LC-MS/MS, plasma amino acids by reverse-phase HPLC, and mTORC1 signaling markers by immunoprecipitation and immunoblotting.

Results: Compared to ALA, CIT supplementation increased plasma arginine 3.2-fold (P < 0.01) and ornithine 2.7-fold (P < 0.01) but reduced plasma glutamine (P = 0.02). No significant changes were observed in other plasma amino acids. Despite the increase in plasma arginine, there was only a trend toward increased fractional protein synthesis in the longissimus dorsi muscle of the CIT pigs (P = 0.09). CIT supplementation had no effect on S6K1 and 4EBP phosphorylation or on eIF4E-eIF4G complex formation.

Conclusions: Short-term enteral citrulline supplementation effectively raises plasma arginine levels in preterm piglets but does not stimulate mTORC1 signaling or protein synthesis in skeletal muscle.

Funding Sources: Research was supported by NIH and USDA.