酮病是奶牛产后常发生的一种代谢性疾病,是由脂质代谢紊乱引起的。乙酰辅酶A(CoA)乙酰转移酶2(ACAT2)对于平衡胆固醇和甘油三酯(TG)代谢很重要;然而,其在亚临床酮症奶牛中的作用尚不清楚。本研究旨在通过体内外实验探讨ACAT2与亚临床酮症奶牛脂代谢紊乱的潜在相关性。在体内实验中,从健康奶牛收集肝组织和血液样本(CON,n=6,β-羟基丁酸[BHBA]浓度<1.0mM)和亚临床酮症奶牛(亚临床酮症[SCK],n=6,BHBA浓度=1.2-3.0mM),以探讨ACAT2对SCK奶牛脂质代谢紊乱的影响。对于体外实验,以牛肝细胞(BHEC)为模型。通过BHBA浓度梯度实验研究了BHBA对ACAT2和脂质代谢的影响。随后,通过转染ACAT2的siRNA探讨ACAT2与脂质代谢紊乱的关系。转录组学显示,在脂质代谢过程中差异表达基因(DEGs)上调,并显着降低SCK组的ACAT2mRNA水平。与体内CON组相比,SCK组过氧化物酶体增殖物激活受体γ(PPARγ)和甾醇调节元件结合蛋白1c(SREBP1c)表达水平显著升高,过氧化物酶体增殖物激活受体α(PPARα)表达水平显著降低,肉碱棕榈酰转移酶1A(CPT1A),甾醇调节元件结合转录因子2(SREBP2),和3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)。此外,SCK组肝TG含量显著升高,血浆总胆固醇(TC)和游离胆固醇(FC)含量显著降低。这些结果指示具有SCK的奶牛肝脏中的TG和胆固醇代谢紊乱。此外,SCK组显示Perilipin-2(PLIN2)的表达增加,载脂蛋白B(APOB)的表达降低,通过下调ACAT2降低极低密度脂蛋白(VLDL)和低密度脂蛋白胆固醇(LDL-C)的血浆浓度,这表明肝脏中TG的积累。体外实验表明,BHBA诱导BHECTG含量增加,TC含量降低,PPARγ和SREBP1c的表达增加,和PPARα的表达减少,CPT1A,SREBP2和HMGCR。此外,BHBA增加PLIN2在BHEC中的表达,ACAT2的表达和荧光强度降低,VLDL和LDL-C含量降低。此外,沉默ACAT2表达增加TG含量;降低TC,VLDL,和LDL-C含量;降低HMGCR和SREBP2的表达;增加SREBP1c的表达;但对PLIN2的表达无影响。这些结果表明,BHEC中ACAT2下调促进TG积累并抑制胆固醇合成,导致TG和胆固醇代谢紊乱。总之,SCK组ACAT2下调抑制胆固醇合成,增加TG合成,降低了VLDL和LDL-C的含量,最终导致TG和胆固醇代谢紊乱。
Ketosis is a metabolic disease that often occurs in dairy cows postpartum and is a result of 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 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 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, 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.