Subclinical ketosis leads to lipid metabolism disorder by down-regulating the expression of Acetyl-CoA acetyltransferase 2 (ACAT2) in dairy cows.

Ketosis is a metabolic disease that often occurs in dairy cows postpartum and results from disordered lipid metabolism. Acetyl-coenzyme A (CoA) acetyltransferase 2 (ACAT2) is important for balancing cholesterol and triglyceride (TG) metabolism; however, its role in subclinical ketotic dairy cows is unclear. This study aimed to explore the potential correlation between ACAT2 and lipid metabolism disorders in subclinical ketotic cows through in vitro and in vivo experiments. In the in vivo experiment, liver tissue and blood samples were collected from healthy cows (CON, n = 6, β-hydroxybutyric acid [BHBA] concentration < 1.0 mM) and subclinical ketotic cows (subclinical ketosis [SCK], n = 6, BHBA concentration = 1.2-3.0 mM) to explore the effect of ACAT2 on lipid metabolism disorders in SCK cows. For the in vitro experiment, bovine hepatocytes (BHEC) were used as the model. The effects of BHBA on ACAT2 and lipid metabolism were investigated via BHBA concentration gradient experiments. Subsequently, the relation between ACAT2 and lipid metabolism disorder was explored by transfection with siRNA of ACAT2. Transcriptomics showed an upregulation of differentially expression genes (DEGs) during lipid metabolism and significantly lower ACAT2 mRNA levels in the SCK group. Compared with the CON group in vivo, the SCK group showed significantly higher expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulator element binding protein 1c (SREBP1c) and significantly lower expression levels of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyl-transferase 1A (CPT1A), sterol regulatory element binding transcription factor 2 (SREBP2), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Moreover, the SCK group had a significantly higher liver TG content and significantly lower plasma total cholesterol (TC) and free cholesterol (FC) content. These results were indicative of TG and cholesterol metabolism disorders in the liver of dairy cows with SCK. Additionally, the SCK group showed an increased expression of Perilipin-2 (PLIN2), decreased expression of apolipoprotein B (APOB), and decreased plasma concentration of very low-density lipoproteins (VLDL) and low-density lipoproteins-cholesterol (LDL-C) by downregulating ACAT2, which indicated an accumulation of TG in liver. In vitro experiments showed that BHBA induced an increase in the TG content of BHEC, decreased content TC, increased expression of PPARγ and SREBP1c, and decreased expression of PPARα, CPT1A, SREBP2, and HMGCR. Additionally, BHBA increased the expression of PLIN2 in BHEC, decreased the expression and fluorescence intensity of ACAT2, and decreased the VLDL and LDL-C contents. Furthermore, silencing ACAT2 expression increased the TG content; decreased the TC, VLDL, and LDL-C contents; decreased the expression of HMGCR and SREBP2; and increased the expression of SREBP1c; but had no effect on the expression of PLIN2. These results suggest that ACAT2 downregulation in BHEC promotes TG accumulation and inhibits cholesterol synthesis, leading to TG and cholesterol metabolic disorders. In conclusion, ACAT2 downregulation in the SCK group inhibited cholesterol synthesis, increased TG synthesis, and reduced the contents of VLDL and LDL-C, eventually leading to disordered TG and cholesterol metabolism.