L-Arginine supplementation modulates L-Arg/NO metabolic processes and AMPK/ACC-1 signalling in BNL CL2 hepatocytes

Abstract The enzyme nitric oxide synthase (NOS) breaks down the semi-essential amino acid L-arginine (L-Arg) in the cell to produce citrulline and nitric oxide (NO). NO is a crucial signalling molecule in cells that controls the metabolism of fats and carbohydrates. The aim of this study was to investigate two important genes in the L-Arg-NOS-NO signalling pathway, AMPK and ACC-1, as markers of the molecular mechanisms that are triggered when liver cells sense elevated L-Arg. Mouse liver epithelial insulin-sensitive BNL CL2 cells were used as a model system and cultured with 0, 400 or 800 µM L-Arg. Cell growth parameters were analysed alongside qRT-PCR based analysis of target transcripts involved in lipid and glucose metabolic pathways. In a further experiment, NOS inhibitor; L-NAME (40 mM) and external NO donor; SNAP (100 µM) were added and the effect on target gene expression analysed. L-Arg addition impacted culture viability and cell growth. AMP-activated protein kinase (AMPK) was regulated in response to L-Arg addition with increasing extracellular concentrations elevating AMPK mRNA and protein expressions. L-NAME decreased target gene expression in an L-Arg addition dependent manner. SNAP (100 µM) addition increased target gene expression after 6 and 24 h. NO, produced as a result of L-Arg addition and the factors L-NAME and SNAP, that regulate NO bioavailability, impacted BNL CL2 cell NO/AMPK/ACC-1 signalling pathways via regulating mRNA expression and subsequently protein expression. Competing Interest Statement The authors have declared no competing interest. Abbreviations - BNL CL2 - Mouse hepatocytes spontaneously immortalized cell line - ACC - Acetyl-CoA carboxylase - AMPK - 5’-Adenosine monophosphate-activated protein kinase - HPLC - High-performance liquid chromatography - L-Arg - L-arginine - L-Cit - L-citrulline - L-NAME - NG-nitro-L-Arg methylester - L-Orn - L-ornithine - NOS - Nitric oxide synthase - NO - Nitric oxide - NO2- - Nitrite - SILAC DMEM - Stable isotope labeling with amino acids in cell culture DMEM - SNAP - S-nitroso-N-acetyl-DL-penicillamine