Regulation of inflammation, antioxidant production, and methyl-carbon metabolism during methionine supplementation in lipopolysaccharide-challenged neonatal bovine hepatocytes

Supplementation of methionine (Met) may improve immunometabolic status, specifically during a period of inflammatory stress. The aim of the present study was to establish an inflammation model using primary neonatal bovine hepatocytes and to examine the effects of increasing concentrations of dl-Met and a maintained Met to lysine (Lys) ratio on hepatocyte inflammatory responses, antioxidant production, and Met metabolism during lipopolysaccharide (LPS) challenge. Hepatocytes isolated from 4 calves were maintained as monolayer cultures and exposed to 0, 10, or 40 µMdl-Met and 100 µM Lys (0Met100Lys, 10Met100Lys, or 40Met100Lys) or 10 µMdl-Met and 25 µM Lys (10Met25Lys). Cells were exposed to each treatment for 16 h and then challenged with either 0 or 100 ng/mL of LPS for 8 h. In the absence of LPS, glutathione (GSH) was not altered by 10Met100Lys or 10Met25Lys but was increased by 40Met100Lys. With LPS challenge, GSH concentration was decreased with 40Met100Lys and tended to be decreased with 10Met100Lys. Hepatocytes receiving 10Met100Lys treated with 100 ng/mL of LPS showed an inflammatory response with increased mRNA expression of tumor necrosis factor (TNFα), IL-6, IL-1β, and interferon gamma, which was accompanied by increased nuclear factor κB inhibitor and serum amyloid A3 mRNA. The treatment 40Met100Lys was effective for preventing the LPS-induced increase in expression of the above genes except TNFα. Similar preventative effects were observed for 10Met25Lys; however, it did not prevent the LPS-induced increase in TNFα or IL-6 mRNA. Lipopolysaccharide challenge decreased mRNA expression of key genes controlling the transmethylation and Met regeneration pathways, which was not prevented by Met supplementation. The data suggest that bovine hepatocyte cultures can be used as a biological model to study the inflammatory cascade via an LPS challenge. Supplementation of Met prevents the LPS-induced hepatocyte cytokine expression and is associated with elevated intracellular GSH concentration.