Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis.
Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis.
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DOI：
10.3168/jds.2021-20892
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发表时间：
2022-03
影响因子：
3.5
通讯作者：
Zhiyuan Fang;Xinwei Li;Shu Wang;Q. Jiang;J. Loor;Xiuhuan Jiang;Lingxue Ju;Hao Yu;Taiyu Shen-T
中科院分区：
文献类型：
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作者：
Zhiyuan Fang;Xinwei Li;Shu Wang;Q. Jiang;J. Loor;Xiuhuan Jiang;Lingxue Ju;Hao Yu;Taiyu Shen-T
酮病最常发生在围产期，并与肝损伤和脂肪变性有关。溶酶体作为终末降解站，通过其在消化功能障碍的细胞器和脂滴中的作用，促进肝脏的动态平衡。转录因子EB(TFEB)是溶酶体功能的主要调节因子。因此，本研究的目的是研究非反刍动物体内溶酶体功能和TFEB转录活性的状况以及TFEB上游效应因子丰度的潜在变化，包括机制靶标雷帕霉素激酶复合体1(MTORC 1)、蛋白激酶B(AKT)、糖原合成酶β(GSK3β)和细胞外信号调节蛋白1/2(ERK1/2)，并探讨是什么因素导致了上述变化。取自健康奶牛(n=10)和酮症奶牛(n=10)的肝脏和血液样本，这些奶牛的哺乳次数(中位数=3，范围=2-4)和哺乳天数(中位数=6d，范围=3-9d)相似。用10 ng/mLGH、3.0 mMβ-羟丁酸酯、1.5 ng/mLIL-18、0.15 ng/mL肿瘤坏死因子-α、1.2 mM游离脂肪酸处理12 h后，发现酮症奶牛血清游离脂肪酸水平和丙氨酸氨基转移酶、天冬氨酸氨基转移酶活性高于酮症奶牛，血糖低于酮症奶牛。此外，酮症奶牛的血清GH、IL-18和肿瘤坏死因子-α浓度较高，而血清胰岛素浓度较低。溶酶体相关膜蛋白1(LAMP1)的蛋白丰度和LAMP1的信使核糖核酸丰度均较低，说明酮症奶牛的肝脏溶酶体质量较低。此外，β-N-乙酰氨基葡萄糖苷酶活性降低，组织蛋白酶D蛋白丰度降低，空泡ATPase的CTSD和V0结构域基因丰度降低，提示酮病引起的肝脏溶酶体功能受损。酮症奶牛肝脏中TFEB和过氧化物酶体增殖物激活受体γ共激活因子1α的核丰度、总蛋白和m RNA丰度较低，而磷酸化(P)-TFEB较高，表明肝脏TFEB转录活性受损。在酮症奶牛肝脏中，磷酸化mTOR及其下游效应蛋白核糖体蛋白S6K B和真核细胞因子4E结合蛋白1的蛋白丰度较高，而p-Akt、p-GSK3β和p-ERK1/2的蛋白丰度较低。重要的是，在GH、BHB、IL-18、肿瘤坏死因子-α和游离脂肪酸处理的小牛肝细胞中，观察到mTOR、RPS6KB和EIF4EBP1的磷酸化水平升高。此外，牛血清白蛋白、肿瘤坏死因子-α和游离脂肪酸，而不是生长激素和IL-18，降低了TFEB转录活性并损害了小牛肝细胞的溶酶体功能。综上所述，这些数据表明，BHB、肿瘤坏死因子-α和游离脂肪酸在酮症过程中过度激活了肝脏mTORC1信号通路，进一步削弱了TFEB的转录活性和溶酶体功能，这可能是肝脏损伤和脂肪变性的原因之一。
Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of β-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.