Abstract
Hepatosteatosis is a common metabolic disorder of dairy cows, especially during early lactation. Currently, there are a few models of bovine hepatic steatosis available, including primary hepatocytes, liver slices, and animal models. Studies that elucidate the influence of single fatty acids on lipid classes, fatty acid pattern, gene expression, and phenotypic changes are still limited. Hence, we investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis. To create a steatotic environment, we treated BFH12 with stearic acid, palmitic acid, or oleic acid in non-toxic doses. Thin-layer chromatography and gas chromatography were used to analyze lipid classes and fatty acid pattern, and qPCR was used to quantify gene expression of relevant target genes. Lipid droplets were visualized with confocal laser scanning microscopy and evaluated for number and size. Treatment with oleic acid increased triglycerides, as well as lipid droplet count per cell and upregulated carnitine palmitoyl transferase 1, which correlates with findings of in vivo models. Oleic acid was largely incorporated into triglycerides, phospholipids, and non-esterified fatty acids. Stearic acid was found mainly in non-esterified fatty acids and triglycerides, whereas palmitic acid was mainly desaturated to palmitoleic acid. All three fatty acids downregulated stearyl-CoA-desaturase 1. In conclusion, BFH12 can acquire a steatotic phenotype by incorporating and accumulating fatty acids. Oleic acid is particularly suitable to produce hepatosteatosis. Therefore, BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.
Highlights
cell line from bovine fetal hepatocytes (BFH12) was cultured in 75-cm3 cell culture flasks with Williams’ Medium E (WME; BIO&SELL, Nuremberg, Germany) containing 600 mg/l glucose, supplemented with 5% heat-inactivated fetal bovine serum (FBS superior, Biochrom/Merck, Berlin, Germany), 1% penicillin/streptomycin (Biochrom/Merck, Berlin, Germany), 2 mM L-alanyl-Lglutamine (Biochrom/Merck, Berlin, Germany), 100 nM dexamethasone (Sigma-Aldrich, Taufkirchen, Germany), and 0.2 U/ml insulin (Sigma-Aldrich, Taufkirchen, Germany)
Total amounts of fatty acid (FA) increased after treatment with oleic acid (OA) (Table 2)
We investigated how the FA added on top influences the proportion of lipid classes and the fatty acid pattern in the lipid classes, respectively
Summary
Diseases associated with parturition are gaining more and more attention in the dairy industry. Up to 50% of dairy cows show elevated triglyceride (TAG) levels in early lactation, leading to mild or severe fatty liver. This hepatosteatosis (HS) is associated with a higher risk for metabolic diseases and reproductive problems [1]. The diagnosis and treatment of HS is difficult and expensive. The diagnostic gold standard is liver biopsy, while the only proven treatment is long-term intravenous infusion of glucagon [2]. The main cause of HS is a negative energy balance (NEB) after parturition. With the onset of lactation, whenever dry matter intake lacks behind energy demand for subsistence and milk production, the organism massively mobilizes fat from the peripheral organs, mainly adipose tissue.
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