The effect of L-arginine supplementation on model in vitro cultured cellular metabolism that underpins metabolic disorders

L-arginine (Arg) is a semi-essential amino acid in mammals. L-Arg metabolism in the cell can give rise to urea production and L-ornithine via arginase activity or nitric oxide (NO) and L-citrulline production via nitric oxide synthase (NOS). NO is produced by three isoenzymes of NOS, neuronal NOS (nNOS) and endothelial NOS (eNOS) are isoforms constitutively expressed, inducible NOS (iNOS) is mainly expressed during inflammatory responses. NO is an important intra- and inter-cellular signalling molecule, that regulates lipid and glucose metabolism. Synthesis of NO requires a number of co-factors including tetrahydrobiopterin (BH4). The biological availability of NO is affected by the NOS inhibitor; NG-nitro-L-arginine methyl ester (L-NAME) and the NO donor; S-nitroso-N-acetyl-DL-penicillamine (SNAP). This study was conducted to investigate the impact of addition of different concentrations of exogenous L-Arg to cultured model cell systems and on the NOS signalling pathway. The experiments were conducted with cell models to define the direct effects of L-Arg and its catabolic products on specific cell signalling pathway in insulin-sensitive cells; mouse liver epithelial cultured model BNL CL2 (a hepatocyte cell model) and mouse adipocyte cells; 3T3 L1. The cells were cultured in two different additional exogenous concentrations of L-Arg (400 and 800 µM) in L-Arg deficient media or control complete DMEM media (contains 250 µM L-Arg itself and maintained without excess L-Arg treatment) with 10% FBS and the cellular response investigated 24 and 72 h after the L-Arg additions. qRT-PCR was used to determine the mRNA levels of key transcripts for enzymes involved in the metabolism of L-Arg and downstream metabolic pathways whilst Western blotting was undertaken for proteins analysis. NO production was also determined with Griess reagent and residual cell culture supernatant amino acid concentrations (L-Arg, L-Cit and L-Orn) measured by HPLC. To further investigate NO production, the impact of the NOS inhibitor L-NAME (4 mM), external NO donor SNAP (100 µM), and NOS co-factor BH4 (40 µM) was also analysed. Finally, stably constitutively over-expressing iNOS 3T3 L1 cells were generated and the impact of culturing in elevated exogenous L-Arg (0, 400 and 800 µM) investigated.

The culture viability and number of viable cells were similar upon the addition of exogenous L-Arg whilst the mRNA levels of AMPK and ACC-1 were increased in both cell types, whilst that of CPT-1 was increased in 3T3 L1 cells and decreased in BNL CL2 cells. The protein expression and activation of AMPK and ACC-1 was increased in liver cells in response to increased extracellular L-Arg in a concentration and time dependent manner, explaining the increased energy metabolism in arginine-treated BNL CL2 cells. In this cell model phosphorylated ACC-1, a downstream target of AMPK, increased in response to L-Arg supplementation, resulting in inactivation of ACC-1 and an increase in the activity of CPT-1. The activation of AMPK and ACC-1 was decreased in 3T3 L1 adipose cells in response to L-Arg supplementation, however, there was an increase in the activity of CPT-1 that reportedly facilitates the transport of long-chain fatty acids from the cytosol to mitochondria for oxidation. L-Arg addition also impacted on the level of post-translational modification of proteins involved in cholesterol synthesis, HMGCR and SREBP-2 and a lipogenic regulator SREBP-1 in liver cells (BNL CL2, increased) and adipose cells (3T3 L1, decreased) compared to the control. A hall mark of L-Arg metabolism; NO in the form of NO2- in cell culture supernatant, was elevated in 800 µM L-Arg cultured samples after 24 h in BNL CL2 cells. However, in 3T3 L1 cells, the amount of NO2- was elevated in 400 µM (at 72 h) and 800 µM L-Arg (at 24 h) cultures. L-NAME significantly inhibited NO production from liver and adipose cells in a time-dependent manner and subsequently impacted AMPK and ACC expression. L-Arg supplementation also affected mRNA and protein levels of AMPK and ACC-1 in the cells grown in the presence of BH4 and SNAP. Associated with these changes were changes in the concentration of L-Arg, L-Cit and L-Orn in the culture media. Interestingly there were elevated iNOS mRNA levels in the control, 0 µM and untreated at T=0 samples in comparison to low mRNA levels in the L-Arg supplemented samples. Construction of iNOS in-frame with a V5 tag enabled generation of cell pools over-expressing the iNOS-V5 tagged protein but surprisingly there appeared to be little impact on cell growth or L-Arg metabolism.In conclusion, this study provides new insights into how excess L-Arg in cell culture media impacts NO production and cell signalling pathways in model in-vitro cultured liver and adipocyte cells. Collectively, these results show that excess L-Arg is sensed by the cell which then regulates AMPK and ACC-1 expression in response. The findings could have implications in modulation of signalling pathways for treating obesity and obesity induced diabetic mellitus. Inhibition of NO synthesis moderately attenuated the arginine-stimulated increases of AMPK and ACC-1 in BNL CL2 cells whilst regulation of the L-Arg/NOS/NO pathway by BH4 and SNAP impacted the mRNA and protein expression of AMPK and ACC-1, and consequently culture supernatant nitrite. These data suggest that modulation of the L-Arg-NO pathway may provide a potentially novel means to impact metabolism that underpins generation of fat mass and such a hypothesis could be tested in the future.