Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, P.R. China.
Department of Animal Science and Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801.
J Dairy Sci. 2023 Dec;106(12):9868-9878. doi: 10.3168/jds.2023-23452. Epub 2023 Sep 9.
Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.
在围产期补充瘤胃保护性胆碱（RPC）在某些情况下可以预防和缓解奶牛的脂肪肝。然而，从机制上讲，胆碱如何防止肝细胞中脂质滴（LD）的积累尚不清楚。在这项研究中，使用了从 3 头非妊娠泌乳中期的荷斯坦奶牛（产后约 160 天）通过穿刺活检获得的肝组织中分离的原代肝细胞。在含有 150 μmol/L 亚油酸的胆碱缺乏培养基中培养 24 小时后，通过油红 O 染色分析 LD，通过 Western 印迹分析蛋白质丰度，通过薄层色谱法和 HPLC/质谱法测量磷脂含量和组成。在随后的实验中，在培养有 30、60 或 90 μmol/L 胆碱-氯化物的肝细胞中评估脂噬作用。所有数据均使用 SPSS 20.0 通过 t 检验或单因素方差分析进行统计学分析。与单独在 Dulbecco's Modified Eagle Medium 中培养的肝细胞相比，胆碱缺乏增加了 LD 的平均直径（1.59 对 2.10 µm），将<2 µm 的小 LD 的比例从 75.3%降低到 56.6%，并将>4 µm 的大 LD 的比例从 5.6%增加到 15.0%。此外，胆碱的缺乏加速了 LD 的融合。在磷脂种类中，肝细胞中的卵磷脂（PC）含量下降了 34.5%。17 种 PC（PC [18:2_22:6]、PC [15:0_16:1]、PC [14:0_20:4] 等）和 6 种溶血磷脂酰胆碱（LPC；LPC [15:0/0:0]），PC（22:2/0:0），LPC（20:2/0:0）等）减少，而 PC（14:1_16:1）和 LPC（0:0/20:1）增加。胆碱缺乏增加了肝细胞中的甘油三酯（TAG）含量（0.57 对 0.39 μmol/mg），并使固醇调节元件结合蛋白 1、固醇调节元件结合蛋白切割激活蛋白和脂肪酸合酶的蛋白质丰度分别增加了 23.5%、17%和 36.1%。在用胆碱重新补充后，LD 的表型（TAG 含量、大小、比例和磷脂谱）得到逆转，并且自噬标志物 LC3II/LC3I 蛋白的比例呈剂量依赖性显著上调。总的来说，至少在泌乳中期奶牛的体外实验中，这些数据表明 PC 合成对于正常 LD 的形成是必要的，并且两者都依赖于胆碱的可用性。根据所使用的肝细胞来源的限制，应进一步开展工作，以确定这些效应是否适用于产后奶牛的肝细胞，这是使用 RPC 预防和治疗脂肪肝的生理阶段。
