Abstract
When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 mM NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and β-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 mM) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 mM) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B, IL6, and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows.