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DOI:
10.1007/s00018-019-03263-6
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Citation for published version (APA):
Zhao, T., Wu, K., Hogstrand, C., Xu, Y. H., Chen, G. H., Wei, C. C., & Luo, Z. (2020). Lipophagy mediated
carbohydrate-induced changes of lipid metabolism via oxidative stress, endoplasmic reticulum (ER) stress and
ChREBP/PPAR pathways. Cellular and Molecular Life Sciences, 77, 1987–2003.
https://doi.org/10.1007/s00018-019-03263-6
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Download date: 10. Aug. 2022
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Lipophagy mediated carbohydrate-induced changes of lipid metabolism via
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oxidative stress, endoplasmic reticulum (ER) stress and ChREBP/PPARγ
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pathways
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Tao Zhao
1
, Kun Wu
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, Christer Hogstrand
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, Yi-Huan Xu
1
, Guang-Hui Chen
1
,
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Chuan-Chuan Wei
1
, Zhi Luo
1,2,*
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1
Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, Fishery
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College, Huazhong Agricultural University, Wuhan 430070, China
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2
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao
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National Laboratory for Marine Science and Technology, Qingdao 266237, China
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3
Diabetes and Nutritional Sciences Division, School of Medicine, King’s College
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London, United Kingdom
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*Corresponding author. Prof. Zhi Luo, Tel.: +86-27-8728-2113; Fax:
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+86-27-8728-2114; Email address: luozhi99@mail.hzau.edu.cn;
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luozhi99@aliyun.com (Z. Luo).
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Abstract:
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High carbohydrate diets (HCD) can induce the occurrence of nonalcoholic fatty liver
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disease (NAFLD), characterized by dramatic accumulation of hepatic lipid droplets
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(LDs). However, the potential molecular mechanisms are still largely unknown. In
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this study, we investigated the role of autophagy in the process of HCD-induced
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changes of hepatic lipid metabolism, and to examine the process of underlying
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mechanisms during these molecular contexts. We found that HCD significantly
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increased hepatic lipid accumulation and activated autophagy. Using primary
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hepatocytes, we found that HG increased lipid accumulation and stimulated the
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release of NEFA by autophagy-mediated lipophagy, and that lipophagy significantly
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alleviated high glucose (HG)-induced lipid accumulation. Oxidative and endoplasmic
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reticulum (ER) stress pathways played crucial regulatory roles in HG-induced
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lipophagy activation and HG-induced changes of lipid metabolism. Further
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investigation found that HG-activated lipophagy and HG-induced changes of lipid
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metabolism were via enhancing carbohydrate response element binding protein
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(ChREBP) DNA binding capacity at PPARγ promoter region, which in turn induced
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transcriptional activation of the key genes related to lipogenesis and autophagy. The
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manuscript-20190705 Click here to access/download;Manuscript;20190705.doc
Click here to view linked References
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present study, for the first time, revealed the novel mechanism for lipophagy
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mediating HCD-induced changes of lipid metabolism by oxidative stress and ER
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stress, and ChREBP/ PPARγ pathways. Our study provided innovative evidence for
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the direct relationship between carbohydrate and lipid metabolism via ChREBP/
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PPARγ pathway.
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Keywords: Dietary carbohydrate; Lipid deposition; Lipid metabolism; Regulatory
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pathways; Lipophagy
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Abbreviations: 3-MA, 3-methyl adenine; 4-PBA, 4-Phenylbutyric acid; 6PGD,
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6-phosphogluconate dehydrogenase; ACCa, acetyl-CoA carboxylase a; ACSL,
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acyl-CoA synthetase long-chain; AO, acridine orange; ATF, activating transcription
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factor; ATG, autophagy related gene; BSA, bovine serum albumin; CF, condition
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factor; ChREBP, carbohydrate response element-binding protein; CPT-1, carnitine
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palmitoyltransferase-1;CQ, chloroquine; DCFH2-DA,2′ 7′-dichlorodihydrofluorescein
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diacetate; eIF2α, eukaryotic translation initiation factor 2α; ERS, endoplasmic
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reticulum stress; FABPL, fatty acid-binding protein liver; FAS, fatty acid synthase;
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FBW, final mean body weight; FCR, feed conversion rate; FFA, free fatty acids; FI,
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feed intake; G6PD, glucose 6-phospate dehydrogenase; GLUT, glucose transporter;
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GRP78, Glucose-regulated protein 78; GSH, glutathione; GSSG, glutathione disulfide;
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HCD, high carbohydrate diet; H&E, hematoxylin and eosin; HG, high glucose; HSI,
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hepatosomatic index; HSL, hormone-sensitive lipase; ICD, intermediate carbohydrate
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diet; ICDH, isocitrate dehydrogenase; IRE1α, inositol requiring 1α; LCD, low
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carbohydrate diet; LD, lipid droplet; LXR a, liver x receptor a; MAP1LC3B,
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microtubule-associated proteins 1A/1B light chain 3B; IBW, initial mean body weight;
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M199, medium-199; MDA, malondialdehyde; MDC, monodansylcadaverine; ME,
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malic enzyme; NAC, N-acetyl-L-cysteine; NAFLD, nonalcoholic fatty liver disease;
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NEFA, nonesterified fatty acid; OD, optical density; ORO, oil red O; PERK, protein
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kinase R (PKR)-like ER kinase; PPAR, peroxisome proliferator-activated receptor;
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PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; S.E.M, standard
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error of the mean; SGR, specific growth rate; SOD, superoxide dismutase; SREBP-1,
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sterol regulatory element binding proteins-1; TG, triglyceride; UPR, unfolded protein
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response; VAI, visceral adipose index; VSI, viscerosomatic index; WG, weight gain;
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XBP1, x-box binding protein 1.
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Introduction
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Non-alcoholic fatty liver disease (NAFLD), characterized by the excessive
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triglyceride (TG) accumulation in the livers, has been increasing in recent years [1].
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The spectrum of NAFLD ranges from simple fatty liver to nonalcoholic
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steatohepatitis (NASH), which may result in liver fibrosis and eventually the
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development of hepatocellular carcinoma [2]. At present, treatment approaches are
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limited because of the unclear pathological mechanism of NAFLD. It is well known
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that the diet rich in carbohydrate along with other factors can induce hepatic NAFLD
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and NASH [3, 4]. However, the additional mechanisms of dietary carbohydrate
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leading to the NAFLD are not fully elucidated.
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Autophagy is a highly conserved self-renewal process in eukaryotic cells,
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characterized by the engulfment of cytoplasmic materials into double-membrane
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vesicles (autophagosomes) for subsequent degradation in lysosomes [5]. Studies
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suggested that autophagy regulates lipid metabolism by eliminating TG and its
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activation plays a key inhibitory role in the development of NAFLD [6]. Moreover,
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lipophagy, one kind of autophagy, regulates lipid metabolism by breaking lipid
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droplets down and enhancing the rate of mitochondrial β-oxidation [6-8]. Impaired
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lipophagy will increase cellular lipid storage and result in the occurrence of NAFLD
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[9, 10].
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Several signaling pathways were reported to regulate the autophagic process,
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such as oxidative stress, endoplasmic reticulum (ER) stress and PPARγ pathways.
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Oxidative stress, which results from an imbalance between free radical production and
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scavenging, induces the formation of autophagy [11]. Oxidative stress also accelerates
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lipid accumulation by disrupting mitochondrial functions and reducing oxidation of
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fatty acids [12-14]. The ER is the intracellular important organelle for the synthesis
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and folding of proteins. ER stress will occur if ER homeostasis is disrupted and
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unfolded /unprocessed proteins are accumulated within the ER. Increasing evidences
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demonstrated that ER stress might be associated with autophagy [15, 16], and
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involved in the development of NAFLD [17, 18]. The PPARγ is the important
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transcriptional factor which regulates autophagic process and lipid metabolism [19,
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20].
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Thus, in view of the important role of autophagy in regulating lipid metabolism,
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and the relevant signaling pathways can activate autophagy, we hypothesis that these
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molecular events mediated high carbohydrate diets-induced changes of lipid
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accumulation in the liver. If our hypothesis can be confirmed, our results will provide
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insight into key pathological mechanisms contributing towards fatty liver occurrence.
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Fish are the largest group of vertebrates in the world. During the evolution, fish
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were considered to experience the fish-specific genome duplication event (FSGD)
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[21]. By analyzing whole-genome sequence information, Gong et al. [22] found the
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FSGD in yellow catfish Pelteobagrus fulvidraco, an important freshwater omnivorous
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fish in China and other countries [23]. Some duplicated genes evolve new functions
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that in turn result in novel regulatory mechanism [21]. Therefore, using yellow catfish
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as a model, we hope to find some novel regulatory mechanism of metabolism.
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Moreover, previous study in our laboratory pointed that high dietary carbohydrate
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increased liver lipid deposition and developed fatty liver symptom in juvenile yellow
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catfish [23]. Accordingly, the present study investigated the mechanism of dietary
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carbohydrate influencing lipid metabolism and dissected the roles of lipophagy
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mediating carbohydrate-induced changes of lipid metabolism via oxidative stress, ER
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stress and ChREBP/PPARγ pathways.
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Materials and Methods
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Animals feeding and sampling
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The protocols of all animal and cells experiments were approved by the ethical
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guidelines of Huazhong Agricultural University for the care and use of laboratory
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animals. The experimental protocols were similar to those in our recent study [24], as
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described in Yang et al. [25]. Briefly, 270 uniformly-sized yellow catfish (4.1 ± 0.01g,
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mean ± SEM) were randomly stocked in 9 circular fiberglass tanks, 30 fish for each
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tank. They were fed with three experimental diets with dietary carbohydrate levels at
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17.2% (low carbohydrate diet, LCD), 22.8% (intermediate carbohydrate diet, ICD)
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and 30.2% (high carbohydrate diet, HCD), respectively, and corn starch was used as
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the carbohydrate source (Supplementary Table 1). Each diet was assigned to 3 tanks
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in a completely randomized manner and fed to apparent satiation twice daily at 8:00
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am and 4:00 pm for 10 weeks. During the experiment, water temperature ranged from
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25.7°C to 28.6°C; dissolved oxygen and NH
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-N were 6.07±0.01 mg/L and 0.11±0.01
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mg/L, respectively.
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At the end of the 10-week feeding trial, all fish were fasted for 24h before
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sampling. Fish were anesthetized with MS-222 (100 mg/L water), and then, counted
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and weighed to determine survival, WG (weight gain) and SGR (specific growth rate).
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