ABSTRACT

Impaired macroautophagy/autophagy has been implicated in experimental and human nonalcoholic steatohepatitis (NASH). However, the mechanism underlying autophagy dysregulation in NASH is largely unknown. Here, we investigated the role and mechanism of TXNIP/VDUP1 (thioredoxin interacting protein), a key mediator of cellular stress responses, in the pathogenesis of NASH. Hepatic TXNIP expression was upregulated in nonalcoholic fatty liver disease (NAFLD) patients and in methionine choline-deficient (MCD) diet-fed mice, as well as in palmitic acid (PA)-treated hepatocytes. Upregulation of hepatic TXNIP was positively correlated with impaired autophagy, as evidenced by a decreased number of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) puncta and increased SQSTM1/p62 (sequestosome 1) expression. Deletion of the Txnip gene enhanced hepatic steatosis, inflammation, and fibrosis, accompanied by impaired autophagy and fatty acid oxidation (FAO) in MCD diet-fed mice. Mechanistically, TXNIP directly interacted with and positively regulated p-PRKAA, leading to inactivation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) and nuclear translocation of TFEB (transcription factor EB), which in turn promoted autophagy. Inhibition of MTORC1 by rapamycin induced autophagy and increased the expression levels of FAO-related genes and concomitantly attenuated lipid accumulation in PA-treated txnip-knockout (KO) hepatocytes, which was further abolished by silencing of Atg7. Rapamycin treatment also attenuated MCD diet-induced steatosis, inflammation, and fibrosis with increased TFEB nuclear translocation and restored FAO in txnip-KO mice. Our findings suggest that elevated TXNIP ameliorates steatohepatitis by interacting with PRKAA and thereby inducing autophagy and FAO. Targeting TXNIP may be a potential therapeutic approach for NASH.

Abbreviations: ACOX1: acyl-Coenzyme A oxidase 1, palmitoyl; ACSL1: acyl-CoA synthetase long-chain family member 1; ACTA2/α-SMA: actin, alpha 2, smooth muscle, aorta; ACTB: actin beta; ADGRE1/F4/80: adhesion G protein-coupled receptor E1; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BafA1: bafilomycin A1; COL1A1/Col1α1: collagen, type I, alpha 1; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; DGAT1: diacylglycerol O-acyltransferase 1; DGAT2: diacylglycerol O-acyltransferase 2; ECI2/Peci: enoyl-Coenzyme A isomerase 2; EHHADH: enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase; FAO: fatty acid oxidation; FASN: fatty acid synthase; FFA: free fatty acids; GFP: green fluorescent protein; GK/GYK: glycerol kinase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPAM: glycerol-3-phosphate acyltransferase, mitochondrial; GPT/ALT: glutamic pyruvic transaminase, soluble; H&E: hematoxylin and eosin; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; IOD: integral optical density; KO: knockout; Leu: leupeptin; LPIN1: lipin 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MCD: methionine choline-deficient; MMP9: matrix metallopeptidase 9; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver diseases; NASH: nonalcoholic steatohepatitis; PA: palmitic acid; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; qRT-PCR: quantitative real-time PCR; RPS6KB1/p70S6K1: ribosomal protein S6 kinase, polypeptide 1; RPTOR: regulatory associated protein of MTOR complex 1; SCD1: stearoyl-Coenzyme A desaturase 1; SEM: standard error of the mean; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TG: triglyceride; TGFB/TGF-β: transforming growth factor, beta; TIMP1: tissue inhibitor of metalloproteinase 1; TNF/TNF-α: tumor necrosis factor; TXNIP/VDUP1: thioredoxin interacting protein; WT: wild-type

摘要

受损的巨自噬/自噬与实验性和人类非酒精性脂肪性肝炎 (NASH) 有关。然而，NASH 中自噬失调的潜在机制在很大程度上是未知的。在这里，我们研究了细胞应激反应的关键介质 TXNIP/VDUP1（硫氧还蛋白相互作用蛋白）在 NASH 发病机制中的作用和机制。在非酒精性脂肪性肝病 (NAFLD) 患者和蛋氨酸胆碱缺乏 (MCD) 饮食喂养的小鼠以及棕榈酸 (PA) 治疗的肝细胞中，肝脏 TXNIP 表达上调。肝脏 TXNIP 的上调与自噬受损呈正相关，这可以通过 MAP1LC3B/LC3B（微管相关蛋白 1 轻链 3 β）斑点数量减少和 SQSTM1/p62（sequestosome 1）表达增加来证明。删除Txnip基因增强了 MCD 饮食喂养小鼠的肝脏脂肪变性、炎症和纤维化，并伴有自噬和脂肪酸氧化 (FAO) 受损。在机制上，TXNIP 直接与 p-PRKAA 相互作用并正向调节，导致 MTOR（雷帕霉素激酶的机械靶点）复合物 1（MTORC1）失活和 TFEB（转录因子 EB）的核易位，进而促进自噬。雷帕霉素对 MTORC1 的抑制诱导自噬，增加了 FAO 相关基因的表达水平，同时减弱了 PA 处理的txnip敲除 (KO) 肝细胞中的脂质积累，而Atg7的沉默进一步消除了这种情况. 雷帕霉素治疗还减轻了 MCD 饮食诱导的脂肪变性、炎症和纤维化，增加了 TFEB 核易位，并在txnip -KO 小鼠中恢复了FAO。我们的研究结果表明，升高的 TXNIP 通过与 PRKAA 相互作用从而诱导自噬和 FAO 来改善脂肪性肝炎。靶向 TXNIP 可能是 NASH 的一种潜在治疗方法。

缩写：ACOX1：酰基辅酶 A 氧化酶 1，棕榈酰；ACSL1：酰基辅酶A合成酶长链家族成员1；ACTA2/α-SMA：肌动蛋白、α2、平滑肌、主动脉；ACTB：肌动蛋白β；ADGRE1/F4/80：粘附 G 蛋白偶联受体 E1；AMPK：AMP激活的蛋白激酶；ATG：自噬相关；BafA1：巴弗洛霉素A1；COL1A1/Col1α1：胶原蛋白，I 型，α 1；CPT1A：肉碱棕榈酰转移酶 1a，肝脏；CQ：氯喹；DGAT1：二酰基甘油O-酰基转移酶1；DGAT2：二酰基甘油O-酰基转移酶2；ECI2/Peci：烯酰辅酶A异构酶2；EHHADH：烯酰辅酶A、水合酶/3-羟酰基辅酶A脱氢酶；粮农组织：脂肪酸氧化；FASN：脂肪酸合酶；FFA：游离脂肪酸；GFP：绿色荧光蛋白；GK/GYK：甘油激酶；GOT1/AST：谷氨酸-草酰乙酸转氨酶 1，可溶性；GPAM：3-磷酸甘油酰基转移酶，线粒体; GPT/ALT：谷氨酸丙酮酸转氨酶，可溶性；H&E：苏木精和伊红；IL1B/IL-1β：白细胞介素 1 β；IL6：白细胞介素 6；IOD：积分光密度；KO：淘汰赛；Leu：亮肽素；LPIN1：脂蛋白1；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MCD：蛋氨酸胆碱缺乏；MMP9：基质金属肽酶9；mRNA：信使RNA；MTORC1：雷帕霉素激酶复合物 1 的机械靶点；NAFLD：非酒精性脂肪肝；NASH：非酒精性脂肪性肝炎；PA：棕榈酸；PPRA/PPARα：过氧化物酶体增殖物激活受体α；PPARG/PPARγ：过氧化物酶体增殖物激活受体γ；qRT-PCR：定量实时 PCR；RPS6KB1/p70S6K1：核糖体蛋白 S6 激酶，多肽 1；RPTOR：MTOR复合物1的调节相关蛋白；SCD1：硬脂酰辅酶A去饱和酶1；SEM：均值的标准误；siRNA：小干扰RNA；SQSTM1/p62：隔离体 1；TFEB：转录因子EB；TG：甘油三酯；TGFB/TGF-β：转化生长因子，β；TIMP1：金属蛋白酶 1 的组织抑制剂；TNF/TNF-α：肿瘤坏死因子；TXNIP/VDUP1：硫氧还蛋白相互作用蛋白；WT：野生型