Acetoacetyl-CoA synthetase modulates lipid partitioning and cholesterol-related gene expression in NEFA-challenged neonatal bovine hepatocytes.

Ketosis is a common metabolic disorder in dairy cows, typically occurring during early postpartum negative energy balance and characterized by hyperketonemia, hepatic lipid accumulation, and oxidative stress. Although acetoacetyl-CoA synthetase (AACS) plays a key role in ketone body metabolism, its association with acetoacetate (AcAc) utilization during ketosis remains unclear. This study aimed to assess the effects of AACS on fatty acid and cholesterol metabolic pathways in neonatal bovine hepatocytes exposed to high concentrations of non-esterified fatty acids (NEFA). Liver tissues were collected from healthy (n = 6; BHBA < 1.0 mM) and ketotic (n = 6; BHBA > 3.0 mM) dairy cows. Additionally, hepatocytes isolated from neonatal calves were treated with 1.2 mM NEFA to establish an in vitro ketotic model. To evaluate the role of AACS, two separate experimental approaches were employed using NEFA-challenged hepatocytes: siRNA-mediated AACS knockdown and exogenous AcAc supplementation. Results demonstrated that liver tissues from ketotic cows and NEFA-treated cells exhibited upregulation of AACS and HMGCS2, along with key lipogenic proteins (SREBF1, ACACA, and FASN). In contrast, the expression of cholesterol synthesis and efflux factors (SREBF2, HMGCR, ABCA1, ABCG5, and ACAT2) was downregulated. NEFA challenge also reduced BDH1 and CPT1A levels, decreased intracellular total cholesterol, increased TAG accumulation, and induced oxidative stress and mitochondrial dysfunction. Silencing AACS partially attenuated NEFA-induced lipid accumulation but further suppressed cholesterol synthesis and efflux-related gene expression. Conversely, AcAc supplementation upregulated the expression of cholesterol synthesis and efflux-related genes but exacerbated lipid deposition, oxidative stress, and mitochondrial dysfunction. Overall, these findings indicate that NEFA-induced upregulation of AACS abundance in hepatocytes may modulate the partitioning of acetoacetate toward de novo lipogenesis and alter cholesterol synthesis and efflux-related gene expression.