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.

中文翻译：

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自噬的增加介导了高酮血症奶牛的乳腺适应机制。

高酮血症是奶牛的一种代谢性疾病，与低干物质摄入量（DMI）和营养需求增加引起的负营养平衡（NNB）相关。高血钾症可引起代谢应激，可能间接影响乳腺组织。自噬是一种高度保守的生理过程，其导致细胞内物质的更新，并在NNB诱导的代谢应激的挑战下参与维持细胞稳态。这项研究的目的是调查高酮血症奶牛乳腺中AMP激活激酶α（AMPKα）的自噬状态和自噬相关途径以及雷帕霉素（mTOR）的机制靶标。高酮血症奶牛[CWH，n = 10，血液中β-羟基丁酸酯（BHB）浓度为1.2至3.0 mmol / L]和无高酮血症奶牛（CWOH，n = 10，从牛群中随机选择3至12 DIM的BHB <1.2 mmol / L）。在喂奶前的3至12天，在牛奶中于0630至0800 h之间收集了这些母牛的乳腺组织和血液样本。喂食前，在0630至0800 h之间使用带有商业试剂盒的自动分析仪测定血清葡萄糖，BHB和脂肪酸的浓度。脂肪酸，BHB（中位数和四分位数范围：CWH，2.44和1.3，2.82 mM； CWOH，0.49和0.41，0.57 mM）和乳脂的浓度在CWH中较高。CWH中的DMI，葡萄糖浓度，牛奶产量和牛奶蛋白质水平较低。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型（PIK3C3），自噬相关基因（ATG）5，ATG7，ATG12，微管相关蛋白1轻链3（ MAP1LC3，CWH的乳腺中也称为LC3）和sequestosome-1（SQSTM1，也称为p62）。CWH中LC3-II的蛋白丰度和Unc-51-like激酶1（ULK1）的磷酸化水平较高，而泛素化蛋白总量和p62的蛋白丰度较低。透射电子显微镜显示在CWH的乳腺中自噬体的数量增加。此外，在CWH的乳腺中，AMPKα的磷酸化较大，而mTOR的磷酸化较低。这些结果表明，mTOR途径的活性和自噬活性以及AMPKα的上调可能是缓解奶牛乳腺高酮血症引起的代谢应激的反应机制。CWH中LC3-II的蛋白丰度和Unc-51-like激酶1（ULK1）的磷酸化水平较高，而泛素化蛋白总量和p62的蛋白丰度较低。透射电子显微镜显示在CWH的乳腺中自噬体的数量增加。此外，在CWH的乳腺中，AMPKα的磷酸化较大，而mTOR的磷酸化较低。这些结果表明，mTOR通路的活性和自噬活性以及AMPKα的上调，可能是缓解奶牛乳腺高酮血症引起的代谢应激的反应机制。CWH中LC3-II的蛋白丰度和Unc-51-like激酶1（ULK1）的磷酸化水平较高，而泛素化蛋白总量和p62的蛋白丰度较低。透射电子显微镜显示在CWH的乳腺中自噬体的数量增加。此外，在CWH的乳腺中，AMPKα的磷酸化较大，而mTOR的磷酸化较低。这些结果表明，mTOR通路的活性和自噬活性以及AMPKα的上调，可能是缓解奶牛乳腺高酮血症引起的代谢应激的反应机制。透射电子显微镜显示在CWH的乳腺中自噬体的数量增加。此外，在CWH的乳腺中，AMPKα的磷酸化较大，而mTOR的磷酸化较低。这些结果表明，mTOR通路的活性和自噬活性以及AMPKα的上调，可能是缓解奶牛乳腺高酮血症引起的代谢应激的反应机制。透射电子显微镜显示在CWH的乳腺中自噬体的数量增加。此外，在CWH的乳腺中，AMPKα的磷酸化较大，而mTOR的磷酸化较低。这些结果表明，mTOR通路的活性和自噬活性以及AMPKα的上调，可能是缓解奶牛乳腺高酮血症引起的代谢应激的反应机制。