DL-methionine increases glutathione concentration and alleviates inflammatory responses in primary bovine hepatocytes

Abstract Text:

Supplementation of rumen-protected methionine (Met) to dairy cows during the peripartal period improves postpartal performance and may decrease oxidative stress. The aim of the present study was to determine the effects of increasing concentrations of DL-Met on hepatic inflammatory responses and oxidative status. Hepatocytes isolated from 4 calves less than 7 days old were maintained as monolayer cultures for 24h prior to addition of treatments. Treatments included 0, 10, or 40 µM DL-Met added to Met-free media containing 100 µM Lys (0MET100LYS, 10MET100LYS, or 40MET100LYS), and 10 µM DL-Met added to Met-free media containing 25 µM Lys (10MET25LYS). Both 40MET100LYS and 10MET25LYS had a Met:Lys of 1:2.5. Cells were exposed to each treatment in triplicate for 16h and then challenged with either 0 or 100 ng/mL lipopolysaccharide (LPS) for 8 h. Cell lysates were collected for quantification of glutathione (GSH) by fluorometic assay and quantification of gene expression by quantitative PCR. Abundance of mRNA was normalized to the mean of 3 reference genes. Cell media was collected for quantification of reactive oxygen species (ROS) by fluorometric assay. Data were analyzed using PROC MIXED of SAS 9.3. The model included treatment, LPS, their interaction, and random effect of calf. Data are reported as LSMEANS±SE. There was an interaction (P<0.05) of treatment and LPS on GSH concentration which increased (P<0.01) as Met concentration increased (107.5, 114. 5 vs. 131. 5±23.5 µM) without LPS challenge, and 40MET100LYS had greater (P<0.01) GSH than 10MET25LYS (131. 5 vs. 97.5±23.5 µM). With LPS challenge, GSH concentration was not different (P>0.10) among treatments. Hepatocytes challenged by LPS showed an inflammatory response with increased (P<0.001) expression of tumor necrosis factor (1.425 vs. 2.257±0.344 arbitrary unit (AU)) independent of treatment. However, there was an interaction (P<0.01) of treatment and LPS on interleukin (IL)-6 expression, which was increased by LPS in cells receiving 10MET100LYS (1.086 vs. 3.851±0.643 AU) and 10MET25LYS (0.918 vs. 2.296±0.643 AU), but was not increased by LPS in cells receiving 40MET100LYS (0.912 vs. 1.770±0.643 AU). Cell culture media ROS concentration was not different (P>0.10) among treatments with or without LPS. The data suggest that a stress model can be established using primary bovine hepatocytes with LPS challenge. Increasing Met concentration enhances intracellular antioxidant production and alleviates inflammatory responses, although ROS released from the cells was not affected. The treatment effects were attributed to increase in Met concentration, not the Met:Lys. 

Keywords:

glutathione, interleukin, lipopolysaccharide