Xing Dongmei, Wang Baogen, Lu Hong, Peng Tao, Su Jianming, Lei Hongyu, He Jianhua, Zhou Yingfang, Liu Lei
Hunan Provincial Key Laboratory of Protein Engineering in Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China.
College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China.
Vet Sci. 2021 Jun 30;8(7):121. doi: 10.3390/vetsci8070121.
Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 m NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE ( 0.01), and raised VLDL contents in the supernatants ( < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE ( < 0.01), and increased VLDL contents in the supernatants ( < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.
脂肪肝与奶牛血液中游离脂肪酸（NEFA）浓度升高以及极低密度脂蛋白（VLDL）水平降低密切相关。高浓度的NEFA会抑制VLDL的合成与组装，并导致肝脏三酰甘油（TAG）沉积。沉默调节蛋白3（SIRT3）是一种线粒体脱乙酰酶，通过调节奶牛肝细胞中脂肪酸合成和氧化基因的表达，拮抗NEFA诱导的TAG积累。然而，SIRT3在VLDL合成与组装中的作用在很大程度上尚不清楚。在此，我们旨在测试SIRT3是否能恢复高NEFA诱导的奶牛肝细胞中VLDL的合成与组装。从3头荷斯坦奶牛中分离出原代奶牛肝细胞。肝细胞分别用SIRT3过表达腺病毒（Ad-SIRT3）、SIRT3短发夹RNA（shRNA）感染，或者先感染Ad-SIRT3，然后与1.0 mM NEFA共同孵育（Ad-SIRT3 + NEFA）。检测VLDL合成关键基因的表达以及细胞培养上清液中的VLDL含量。SIRT3过表达显著增加了微粒体甘油三酯转移蛋白（MTP）、载脂蛋白B100（ApoB100）和载脂蛋白E（ApoE）的mRNA丰度（P < 0.01），并提高了上清液中的VLDL含量（P < 0.01）。然而，与SIRT3过表达相比，SIRT3沉默呈现出相反的效果。与单独的NEFA处理相比，Ad-SIRT3 + NEFA显著上调了MTP、ApoB100和ApoE的mRNA丰度（P < 0.01），并增加了上清液中的VLDL含量（P < 0.01）。我们的数据表明，SIRT3恢复了NEFA刺激的奶牛肝细胞中VLDL的合成与组装，为进一步研究其在围产期奶牛中对抗肝脏TAG积累的可行性提供了体外依据。
