Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Nutrition and Clinical Nutrition Department, Faculty of Veterinary Medicine, Cairo University, Egypt 12211.
J Dairy Sci. 2020 Mar;103(3):2545-2555. doi: 10.3168/jds.2019-16910. Epub 2020 Jan 9.
Hyperketonemia is a metabolic disease in dairy cows, associated with negative nutrition balance (NNB) induced by low dry matter intake (DMI) and increased nutrient requirements. Hyperketonemia could induce metabolic stress, which might indirectly affect mammary tissue. Autophagy is a highly conserved physiological process that results in the turnover of intracellular material, and is involved in maintaining cellular homeostasis under the challenge of metabolic stress induced by NNB. The aim of this study was to investigate the autophagy status and autophagy-related pathways AMP-activated kinase α (AMPKα) and mechanistic target of rapamycin (mTOR) in the mammary glands of dairy cows with hyperketonemia. Cows with hyperketonemia [CWH, n = 10, blood β-hydroxybutyrate (BHB) concentration 1.2 to 3.0 mmol/L] and cows without hyperketonemia (CWOH, n = 10, BHB < 1.2 mmol/L) from 3 to 12 DIM were randomly selected from the herd. The mammary tissue and blood samples were collected from these cows between 0630 and 0800 h, before feeding, at 3 to 12 d in milk. Serum concentrations of glucose, BHB, and fatty acids were determined using an autoanalyzer with commercial kits between 0630 and 0800 h, before feeding. Concentrations of fatty acids, BHB (median and interquartile range: CWH, 2.44 and 1.3, 2.82 mM; CWOH, 0.49 and 0.41, 0.57 mM), and milk fat were greater in CWH. The DMI, glucose concentration, milk production, and milk protein levels were lower in CWH. The mRNA abundance of autophagosome formation-related gene, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 5, ATG7, ATG12, microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and sequestosome-1 (SQSTM1, also called p62) were greater in the mammary glands of CWH. The protein abundance of LC3-II and phosphorylation level of Unc-51-like kinase 1 (ULK1) were greater in CWH, but the total ubiquitinated proteins and protein abundance of p62 were lower. Transmission electron microscopy showed an increased number of autophagosomes in the mammary glands of CWH. Furthermore, the phosphorylation of AMPKα was greater, but the phosphorylation of mTOR was lower in the mammary glands of CWH. These results indicate that activity of mTOR pathways and autophagy activity, and upregulation of AMPKα, may be response mechanisms to mitigate metabolic stress induced by hyperketonemia in the mammary glands of dairy cows.
奶牛酮血症是一种代谢疾病，与低干物质采食量（DMI）和营养需求增加导致的负营养平衡（NNB）有关。酮血症可诱导代谢应激，这可能间接影响乳腺组织。自噬是一种高度保守的生理过程，导致细胞内物质的周转，并且在由 NNB 诱导的代谢应激下参与维持细胞内环境稳定。本研究的目的是研究患有酮血症的奶牛乳腺中的自噬状态和自噬相关途径 AMP 激活的蛋白激酶α（AMPKα）和雷帕霉素靶蛋白（mTOR）。在 3 至 12 DIM 期间，从牛群中随机选择患有酮血症的奶牛[CWH，n = 10，血液β-羟丁酸（BHB）浓度为 1.2 至 3.0 mmol/L]和没有酮血症的奶牛（CWOH，n = 10，BHB < 1.2 mmol/L）。在 0630 至 0800 h，在饲喂前，在 3 至 12 天产奶期间，从这些奶牛中采集乳腺组织和血液样本。在 0630 至 0800 h，在饲喂前，使用自动分析仪和商业试剂盒测定血清中葡萄糖、BHB 和脂肪酸的浓度。CWH 中的脂肪酸、BHB（中位数和四分位距：CWH，2.44 和 1.3，2.82 mM；CWOH，0.49 和 0.41，0.57 mM）和乳脂浓度较高。CWH 的 DMI、葡萄糖浓度、产奶量和乳蛋白水平较低。自噬体形成相关基因 beclin 1（BECN1）、磷脂酰肌醇 3-激酶催化亚基 3（PIK3C3）、自噬相关基因（ATG）5、ATG7、ATG12、微管相关蛋白 1 轻链 3（MAP1LC3，也称为 LC3）和自噬相关蛋白 SQSTM1（也称为 p62）的 mRNA 丰度在 CWH 的乳腺中更高。CWH 中的 LC3-II 蛋白丰度和 UNC-51 样激酶 1（ULK1）的磷酸化水平更高，但总泛素化蛋白和 p62 的蛋白丰度较低。透射电子显微镜显示 CWH 乳腺中的自噬体数量增加。此外，CWH 乳腺中的 AMPKα 磷酸化增加，但 mTOR 磷酸化减少。这些结果表明，mTOR 通路和自噬活性的活性以及 AMPKα 的上调可能是奶牛乳腺应对酮血症引起的代谢应激的反应机制。
