Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum

Abstract

Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 μmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/μg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/β, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/β-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.