Dileucine-supplemented essential amino acids support whole-body anabolism after resistance exercise and serum-stimulated cell-based anabolism

Background: Essential (EAA) and branched chain (BCAA) amino acid ingestion support whole-body anabolism after resistance exercise and can attenuate markers of postexercise myofibrillar protein breakdown (i.e. urinary 3-methylhistidine; 3MH). Leucine is often considered a primary anabolic EAA through its ability to activate the mechanistic target of rapamycin complex 1 (mTORC1) and stimulate muscle protein synthesis. The dipeptide leucine (dileucine) has been shown to more effectively stimulate myofibrillar protein synthesis than leucine in young males at rest. Therefore, we aimed to determine the effect of a dileucine-containing essential amino acid formula (DIEAA; 2 g dileucine, 1 g leucine, 9.15 g total EAA) on the anabolic and catabolic responses following resistance exercise in young recreationally active adults when compared with ingesting branched chain amino acids (BCAA; 3 g leucine, 1.5 g isoleucine, 1.5 g valine) or isonitrogenous (to DIEAA) collagen hydrolysate (COL). Methods: In a randomized, double-blind, crossover design, 12 healthy adults (8 M, 4F, aged 24 ± 3 y) performed a 60 min bout of whole-body resistance exercise, after which they ingested DIEAA, BCAA, or COL protein beverages containing 100 mg L-[1-¹³C]leucine (#NCT05754125). Total exogenous leucine retention (as an estimate of whole-body anabolism) was assessed over the 6 h postprandial period by determining total leucine oxidation from ¹³CO₂ enrichment (isotope ratio mass spectrometry) in repeated breath samples. A urinary 3MH:creatinine ratio (3MH:Cr) over 6 h was used as an estimate of skeletal muscle myofibrillar protein breakdown. To further assess the anabolic potential of nutrients, C2C12 myotubes were treated with a subset (n = 7) of human serum-conditioned media for 4 h to measure downstream mTORC1 substrate phosphorylation, protein synthesis (puromycin and L-ring-[D₅]phenylalanine incorporation) and breakdown (ubiquitinated protein), and myotube hypertrophy. Results: Total exogenous leucine retention were similar (p = 0.68) between DIEAA (215.72 ± 42.45 μmol·kg⁻¹) and BCAA conditions (219.15 ± 45.26 μmol·kg⁻¹), with both DIEAA and BCAA being greater (p < 0.0001) than COL (37.25 ± 8.16 μmol·kg⁻¹). There were no differences (p = 0.58) in 3MH:Cr between supplement conditions. There was no effect of condition ex vivo on puromycin incorporation into nascent peptides (p = 0.31), total protein ubiquitination as an estimate of protein breakdown (p = 0.59), phosphorylation of downstream mTORC1 substrates p-RPS6^S240/244 (p = 0.39) or p-4E-BP1^T37/46 (p = 0.50), and myotube diameter (p = 0.55). Stable isotope-derived rates of mixed muscle protein synthesis (MPS) demonstrated a trend toward a main effect (p = 0.086) with pairwise comparisons revealing a large effect of DIEAA compared to COL (dz = 1.47), a medium effect of DIEAA compared to BCAA (dz = 0.81), and a trivial effect of BCAA comapred to COL (dz = 0.002). Conclusions: Dileucine-supplemented EAA and BCAA support greater whole-body anabolism compared with COL after resistance exercise independent of attenuation in urinary estimates of myofibrillar protein breakdown. Exploratory ex vivo experiments reveal a potential anabolic effect of DIEAA in stimulating MPS. Collectively, these findings suggest that consuming dileucine with sufficient EAA and BCAA increases exogenous leucine retention to support whole-body anabolism during postexercise recovery in individuals performing resistance training.