Reducing hepatic endoplasmic reticulum stress ameliorates the impairment in insulin signaling induced by high levels of β-hydroxybutyrate in bovine hepatocytes
Reducing hepatic endoplasmic reticulum stress ameliorates the impairment in insulin signaling induced by high levels of β-hydroxybutyrate in bovine hepatocytes
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DOI：
10.3168/jds.2021-20611
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发表时间：
2021-11-17
影响因子：
3.5
通讯作者：
Li, Xinwei
中科院分区：
文献类型：
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作者：
Lei, Lin;Gao, Wenwen;Li, Xinwei
酮症奶牛表现出负能量平衡（NEB）状态，其特征在于0-羟基丁酸（BHB）和脂肪酸的循环水平升高。奶牛肝脏胰岛素信号传导受损经常发生在向泌乳过渡期间，但其在此期间对肝功能的作用尚不清楚。在非反刍动物中，内质网（ER）应激是导致肝脏胰岛素信号转导受损的一个原因。因此，本研究的目的是调查肝脏胰岛素和内质网应激信号的状态，以及内质网应激是否有助于酮病奶牛胰岛素信号受损。在饲喂前，选择牛奶中3至10天的健康（对照奶牛，n = 10，BHB = 1.2 mM）奶牛进行肝活检和血液采样。用来自5头健康小牛（1日龄，禁食雌性，体重30-40 kg）的分离肝细胞进行体外实验。处理包括BHB（0、0.9、1.8、3.6 mM）、牛磺熊去氧胆酸（TUDCA，ER应激的典型抑制剂）和不同孵育时间（0.5、1、2、3、5、7、9或12 h）。酮病奶牛的日产奶量较低（中位数：29.50 vs 23.00 kg），血浆非酯化脂肪酸（NEFA）升高（中位数：0.33 vs. 1.17 mM），BHB（中位数：0.43 vs. 3.22 mM），天冬氨酸转氨酶（中位数：70.58 vs. 155.70 U/L），丙氨酸转氨酶（中位数：18.31 vs. 37.90 U/L），血糖较低（中位数：4.32 vs. 2.37 mg/ dL）和修订的定量胰岛素敏感性检查指数（中位数：0.39 vs. 0.37）。在酮症奶牛中，磷酸化胰岛素受体底物-1（IRS 1）丰度增加，磷酸化蛋白激酶B（AKT）和糖原合成酶激酶-30（GSK 30）丰度减少，表明胰岛素抵抗状态。此外，在酮病奶牛的肝脏中，磷酸化蛋白激酶RNA样ER激酶（PERK）和肌醇需要蛋白-1 α（IRE 1 α）的丰度以及激活转录因子-6（ATF 6）的裂解更高。在体外，在孵育的早期阶段，用BHB处理上调了IRE 1 α、PERK的磷酸化丰度和ATF 6的切割，以及几种未折叠的蛋白质应答基因[X盒结合蛋白-1（XBP 1）、78 kDa葡萄糖调节蛋白（GRP 78）和C/EBP同源蛋白（CHOP）]。此外，响应于BHB剂量的增加，PERK、IRE 1 α的磷酸化水平和ATF 6的切割以及XBP 1、GRP 78和CHOP的丰度增加。此外，BHB处理增加了IRS 1的磷酸化，降低了AKT和GSK 30的磷酸化，并上调了致凋亡基因（磷酸烯醇丙酮酸羧激酶和葡萄糖-6-磷酸酶）的丰度。重要的是，这些变化通过用TUDCA处理抑制ER应激而逆转。总的来说，本研究表明，逆转酮症期间的ER应激可能有助于减轻肝脏胰岛素抵抗。靶向内质网应激可能是控制酮症对肝功能的负面影响的潜在治疗靶点。
Ketotic dairy cows exhibit a state of negative energy balance (NEB) characterized by elevated circulating levels of 0-hydroxybutyrate (BHB) and fatty acids. Impaired hepatic insulin signaling in dairy cows occurs frequently during the transition into lactation, but its role on liver function during this period is not well known. In nonruminants, endoplasmic reticulum (ER) stress is a causal factor contributing to impaired insulin signaling in the liver. Thus, the aim of this study was to investigate the status of hepatic insulin and ER stress signaling and whether ER stress contributes to impaired insulin signaling in dairy cows with ketosis. Healthy (control cows, n = 10, BHB = 1.2 mM) cows at 3 to 10 d in milk were selected for liver biopsy and blood sampling before feeding. In vitro experiments were conducted with isolated hepatocytes from 5 healthy calves (1 d old, fasted female, 30-40 kg of body weight). Treatments included BHB (0, 0.9, 1.8, 3.6 mM), tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress), and different incubation times (0.5, 1, 2, 3, 5, 7, 9, or 12 h). Ketotic cows had lower daily milk yield (median: 29.50 vs. 23.00 kg), higher plasma nonesterified fatty acid (NEFA) (median: 0.33 vs. 1.17 mM), BHB (median: 0.43 vs. 3.22 mM), aspartate aminotransferase (median: 70.58 vs. 155.70 U/L), alanine aminotransferase (median: 18.31 vs. 37.90 U/L), lower plasma glucose (median: 4.32 vs. 2.37 mg/ dL), and revised quantitative insulin sensitivity check index (median: 0.39 vs. 0.37) compared with healthy cows. Increased abundance of phosphorylated insulin receptor substrate-1 (IRS1) and decreased abundance of phosphorylated protein kinase B (AKT) and gly cogen synthase kinase-30 (GSK30) in ketotic cows indicated a state of insulin resistance. In addition, abundance of phosphorylated protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1 alpha (IRE1 alpha), and cleavage of activating transcription factor-6 (ATF6) were greater in the liver of ketotic cows. In vitro, at the early stages of incubation, treatment with BHB upregulated abundance of phosphorylated of IRE1 alpha, PERK, and the cleavage of ATF6, as well as several unfolded protein response genes [X-box binding protein-1 (XBP1), 78 kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP)]. Furthermore, in response to increasing doses of BHB, the phosphorylation level of PERK, IRE1 alpha, and the cleavage of ATF6, and the abundance of XBP1, GRP78, and CHOP increased. In addition, BHB treatment increased phosphorylation of IRS1 and decreased phosphorylation of AKT and GSK30, and upregulated abundance of gluconeogenic genes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase). Importantly, these changes were reversed by inhibiting ER stress with TUDCA treatment. Overall, the present study indicated that reversing ER stress during ketosis might help alleviate hepatic insulin resistance. Targeting ER stress may represent a potential therapeutic target for controlling the negative aspects of ketosis on liver function.