Metformin acts to suppress β-hydroxybutyric acid-mediated inflammatory responses through activation of AMPK signalling in bovine hepatocytes

The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signalling. Bovine hepatocytes isolated from cows at around 160 days in milk (DIM) were used in this study. Moreover, primary bovine hepatocytes were used for the treatment with BHBA and pretreatment of metformin at different doses. The results demonstrated that BHBA at 1.2 mM triggered the activation of NF-κB signalling and pro-inflammatory cytokines expression. Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signalling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, the pretreatment with metformin increased the BHBA-inhibited cells proliferation. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared to BHBA treated cells, the expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The NF-κB displayed higher binding activity onto IL1B promoter, and this was suppressed by pretreatment with metformin. Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signalling in bovine hepatocytes.