Exercise protects against AMLN diet-induced lipid deposition in hepatocytes during MAFLD progression by regulating the UPRmt and FGF21 secretion

Exercise protects against AMLN diet-induced lipid deposition in hepatocytes during MAFLD progression by regulating the UPRmt and FGF21 secretion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exercise protects against AMLN diet-induced lipid deposition in hepatocytes during MAFLD progression by regulating the UPRmt and FGF21 secretion Xinmeng Yuan, Mengqi Xiang, Yaran Gao, Wanyu Feng, Wen Sun, Ye Xu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4446826/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background A high-calorie diet and lack of exercise are the primary risk factors contributing to metabolic associated fatty liver disease (MAFLD) initiation and progression. Although mitochondrial dysfunction in MAFLD has been widely recognized, the precise molecular mechanisms of mitochondrial function alteration during MAFLD development remain to be fully elucidated. Methods A total of sixty male C57/black mice were maintained on a normal or amylin liver NASH (AMLN) diet for 6 and 10 weeks. Half of the AMLN diet mice were then subjected to 8 weeks of voluntary wheel running with an AMLN diet persistently, while the other AMLN diet mice were sedentary until 14 and 18 weeks. After the experimental intervention, the mice were sacrificed under anesthesia, blood and liver tissue were collected for further analysis. Changes in biochemical parameters, histopathology, lipid accumulation, endoplasmic reticulum stress, mitochondrial function and mitochondrial unfolded protein response-related proteins were assessed and correlation analysis of serum FGF21 and mitochondrial unfolded genes expression was also performed. Results The results showed that the hepatic lipid deposition and PERK-eIF2α-ATF4 pathway activation were significant increased with prolonged duration of AMLN diet. However, expression of mitochondrial unfolded protein response (UPRmt) genes, such as LONP1, HSP60, and HSP70, as well as mitokine FGF21 secretion were significantly enhanced in the 14-week AMLN diet mice, but were markedly reduced with the excessive lipid deposition induced by the 18-week AMLN diet. In addition, there is a significant positive correlation between circulating FGF21 and the amount of mitochondrial unfolded genes expression during MAFLD progression. Moreover, exercise intervention significantly rescued the hepatic phenotype through improving mitochondrial function, regulating UPRmt activation pattern and increasing FGF21 secretion. Conclusions During the development of AMLN diet-induced MAFLD, the relationship between the degree of lipid deposition and mitochondrial function is not a linear model of negatively correlation. Instead, mitochondria could experience self-remodeling at the earlier stage of lipid accumulation, then lose their self-repair ability due to lipid overload. Exercise effectively prevents excessive lipid deposition, through regulating UPRmt, remodeling mitochondrial protein homeostasis and promoting the secretion of mitokine FGF21, which plays an essential role in delaying the MAFLD occurrence and progression. mitochondrial unfolded protein response hepatic lipid accumulation exercise intervention AMLN diet MAFLD progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The manifestations of metabolic associated fatty liver disease (MAFLD) are becoming a growing challenge for public health. MAFLD progression is caused by an imbalance between lipid acquisition and lipid disposal due to the overwhelmed metabolic capacity in the liver. This progression of NAFLD is currently explained by the “multiple-hit” hypothesis, which proposes that multiple concurrent insults, such as insulin resistance, oxidative stress, intracellular stress response and mitochondrial dysfunction, eventually contribute to liver injury and NAFLD progression [ 1 , 2 ]. Hepatocytes have abundant ER, which is crucial in regulating calcium homeostasis and lipid metabolism in cells. Many genetic or dietary models of fatty liver in recent studies have demonstrated that free fatty acid (FFA) overload and lipotoxicity are critical factors that lead to ER homeostasis disequilibrium in the liver [ 3 ]. ER stress can induce multiple effects by activating its downstream pathways, including the unfolded protein response (UPR), apoptotic [ 4 ] integrated stress response (ISR) [ 5 ], and inflammatory pathways [ 6 ], contributing to the progression from initial steatosis to non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is closely connected with the onset and progression of NAFLD, and the use of mitochondria as a target for NAFLD therapy has been gaining traction based on rodent models and human studies [ 7 ]. With increased lipid deposition in the liver, hepatic metabolism might shift to protect hepatocytes from the lipid burden at the initial stage. For example, studies have shown that hepatic metabolic adaptation and mitochondrial flexibility at the early stages of NAFLD development, such as increased mitochondrial fatty acid oxidation (FAO), enhanced the tricarboxylic acid (TCA) cycle, and raised oxidative phosphorylation (OXPHOS) [ 8 ]. However, at the advanced stages of MAFLD development, the imbalance between mitochondrial FAO and the electron transport chain (ETC) will cause reactive oxygen species (ROS) overproduction, which not only threatens the OXPHOS machinery but also other mitochondrial proteins, lipids, and mtDNA. In response to increased ROS production during MAFLD development, unfolded or misfolded proteins can aggregate in the mitochondrial matrix, leading to the initiation of the mitochondrial unfolded protein response (UPRmt). The UPRmt is an important mechanism for maintaining mitochondrial proteostasis during stress. Under mitochondrial stress, UPRmt process is initiated by inducing nuclear gene-encoded proteins transcription, such as chaperones heat shock protein 10 (HSP10) and heat shock protein 60 (HSP60), proteases caseinolytic protease (ClpP) and Lon peptidase 1 (LONP1) to maintain mitochondrial proteostasis. Indeed, increasing studies have documented that the UPRmt exerts a protective effect in liver metabolic related diseases [ 9 , 10 ], while an abnormal UPRmt triggers a series of aging-related diseases. Interestingly, evidence shows that the increased expression of UPRmt related proteases and chaperone proteins is accompanied by elevated expression of mitokines such as fibroblast growth factor 21 (FGF21) and GDF15 [ 11 ]. FGF21 is a protein that is highly synthesized in the liver and was the first cytokine reported in mammalian systems. Abundance of evidences have confirmed that circulating FGF21 content is implicated in the pathobiology of NAFLD [ 12 – 14 ], and abnormal FGF21 signaling is a major pathological changes in the development and progression of NAFLD [ 15 ]. Indeed, evidences have reported that FGF21 expression can be stimulated by the ER stress response via the PKR-like ER kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) axis [ 16 ]. In addition, some of the previously described that UPRmt and UPRER have interactive effects under homeostasis disruption, and PERK-ATF4 axis is the core hub collaborating UPR ER and UPRmt, hence regulating proteostasis by promoting ER and mitochondrial interactivities and improving their functions [ 17 ]. Exercise-induced mitochondrial adaptation not only favourable for improving muscle health, but also plays a vital role in mitochondrial dysfunction related metabolic alterations associated with liver diseases. Although the main mechanisms of exercise effect on NAFLD treatment include the reduction in intrahepatic fat content, attenuation of inflammation and oxidative stress, maintenance of ER homeostasis, and improvement of insulin signaling and mitochondrial function [ 18 , 19 ], the underlying molecular mechanism are not completely elucidated. Acute and chronic forms of physical exercise was shown to modulate mitochondrial metabolism and hepatic redox state [ 20 ]. In particular, exercise intervention can improve the antioxidant defense system capacity, regulate the PERK-eIF2α-ATF4 pathway, inhibit hepatocyte apoptosis and ameliorate hepatic lipid deposition [ 21 ]. Moreover, novel exercise-inducible cytokines are hypothesized to play a vital role in lipid metabolism such as myokines, hepatokines, and adipokines. Some researches in human and animal models have demonstrated the mechanisms for ameliorating MAFLD by detecting FGF21 during exercise. Others also suggested that FGF21 may play a crucial role in exercise to improve NAFLD [ 22 ], and serum FGF21 is largely secreted from the liver during aerobic exercise [ 23 ]. It is therefore postulated that the exercise could alleviates MAFLD occurrence and progression via regulating livermitochondrial function and FGF21 secretion, to further elucidate the specific mechanisms underlying the advantage of exercise intervention during different stages of MAFLD progression. In this study, it highlighted the effect of exercise on protecting against AMLN diet-induced lipid accumulation in hepatocytes during MAFLD progression in mice. According to results, 8 weeks of voluntary wheel running significant restrains MAFLD development via upregulation of UPRmt signaling mechanisms at the earlier stages of MAFLD development, and restoration of the impaired UPRmt at the later stages of MAFLD development. Furthermore, the variation tendency for FGF21 secretion and the UPRmt activation during MAFLD progression is alike, which may be a novel potential strategy for diagnosing the developmental stages of MAFLD. Materials and methods Animals Sixty male eight-week-old C57BL/6J mice were purchased from Jiangsu Gempharmatech Co., Ltd, Nanjing, China and were individually housed in cages in the Jiangsu Academy of Agricultural Sciences at an immobile temperature and humidity environment with freely access to food and water. Sixty C57BL/6J mice were divided equally into two groups, feed with normal diet (ND), contains 14.9% fat, 22.7% protein, and 62.4% carbohydrate (XIETONG.ORGANISM, 1010084, Nanjing, China) and Amylin Liver NASH (AMLN) diet, contains fat (40%), fructose (20%), and cholesterol (2%) (Research Diets, #D09100301, USA) [ 24 ]. After 6 and 10 weeks of AMLN diet feeding, half of the mice were divided into voluntary wheel running and sedentary groups with AMLN diet (Fig. 1 ). This study adhered to all guidelines for animal experiments issued by Nanjing Sport Institute’s Animal Ethics and Welfare Committee (Approval No. GZRDW-2020-03). After 18 weeks, the mice were anesthetized and sacrificed to collect retro-orbital blood, liver tissues for subsequent analysis. Training protocol Mice in the exercise intervention group were housed in cages equipped with a 14-cm-diameter voluntary running wheel. The number of runners’ revolutions were recorded by using a magnetic counter, and then converted into distance (kilometers) run per day according to a conversion formula. In this study, mice exercised an average of 6–11 km per day, reaching the standard of voluntary wheel running [ 25 ]. Biochemical measurements in serum Blood samples were centrifuged in 4°C, 5000 g for 10 min after standing on ice for 30 min before. The content of serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT), cholesterol (CHOL), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were detected according to the prescriptive protocol. Serum FGF-21 concentration was tested by mouse FGF-21 Elisa kit (Bio-techne China Co., Ltd, MF2100, Shanghai, China) following the manufacture’s instructions. Histological staining 3 mm cubes of liver tissues were collected after fixing with paraformaldehyde. Next, embedded tissue were cut into 4-µm slices before deparaffinizing and staining with Oil Red O (SIGMA-ALDRICH, #SHBL1039, USA) or H&E (Biosharp, BL702A, Hefei, China). Finally, the pathological morphology of the liver were observed by microscope (Zeiss, Axio Imager A2). Immunohistochemical analysis Dewaxed liver sections were applied to 3% H 2 O 2 treatment for 10 min at room temperature and washed with phosphate buffer (PBS). Then, the tissue slices were treated with citrate buffer at 95°C, and blocked. The samples were refrigerated overnight at 4°C by adding GRP78 (1:1000, Bioworld, USA). Next, slices were added HRP-labeled anti-rabbit IgG (Boster Biological Technology, Wuhan, China) and incubated at 37°C. Diaminobenzidine (DAB) was performed for 10 min at room temperature, counterstaining was performed using hematoxylin, 1% alcohol hydrochloric acid was differentiated and neutral gum was used for sealing. Finally, the the pathological morphology of the liver were observed by microscope (Zeiss, Axio Imager A2) and quantified by using Image J. NAFLD activity score evaluation The morphological changes in the liver tissue were evaluated by using the NAFLD activity score (NAS) standard [ 26 ]. The criterion of the NAS system indicated three degrees of fatty liver: steatosis (> 66% = 3, 33–66% = 2, 5–33% = 1, 4 foci = 3, 2–4 foci = 2, < 2 foci = 1, none = 0), and locular ballooning (prominent = 2, few = 1, none = 0). Determination of the hepatic TG level Liver TG was detected with Triglyceride (TG) Content Assay kit (Boxbio, AKFA003M, Beijing, China). TG was extracted from live tissue with normal heptane/dimethylcarbinol (1:1), evaporated. Then the liver TG concentration was determined and calculated according to the instructions. Electron microscopy Animals were perfused with physiological sodium and 4% paraformaldehyde for pre-fixation before transmission electron microscopy (TEM). Livers from C57BL/6J mice were harvested, and liver samples were cut into 1-mm cubes and fixed in 2.5% glutaraldehyde. Then samples were treated with 1% osmium tetroxide, dehydrated in alcohols, embedded in araldite resin. Tissues were cut into 1-µm slices, stained with methylene blue and so on. The images were collected by JEM-1400 electron microscope. Mitochondrial isolation Fresh liver tissue were minced and homogenized, then isolated by differential centrifugation, as described previously [ 27 ]. Samples were resuspended in Buffer I (70 mM sucrose, 210 mM mannitol, 1 mM ethylene glycol tetraacetic acid (EGTA) 5 mM HEPES, ,0.5% BSA, pH 7.4). After multi-step centrifugation, the pellet was resuspended in Buffer II, which include 210 mM mannitol, 70 mM sucrose, 10 mM MgCl 2 , 5 mM K 2 HPO 4 , 10 mM MOPS, 1 mM EGTA, pH 7.4, and the mitochondrial samples were obtained. Mitochondrial oxygen consumption Clarke-type electrode (Oxytherm, Hansatech, UK) was used for measuring mitochondrial oxygen consumption. Respiration buffer includes 20 mmol/L Tris-HCl, 125 mmol/L KCl, and 0.1 mmol/L EGTA, pH 7.4 and a volume of 75 ul mitochondrial sample were added to the reaction chamber, and determination of the proportion of dissolved oxygen consumption in fresh mitochondria with adding the presence of glutamate and ADP (state III respiration) or glutamate (state IV respiration). The ratio of state III to state IV is defined as the mitochondrial respiratory control rate (RCR). The oxygen consumption was valued by normalizing the oxygen consumption rate (nmole/s) to the total protein content (mg). The data were collected and analyzed by using O 2 view software (Hansatech, UK). Measurement of mitochondrial membrane potential Liver mitochondrial membrane potential were determined by using enhanced mitochondrial membrane potential assay kit (Beyotime, C2003S, Shanghai, China). The JC-1 working solution was configured as indicated, and the fluorescence intensity was detected by flow cytometry (ACEA NovoCyte™, USA) (excitation spectra at 490 and 525 nm and emission spectra at 530 and 590 nm). The phycoerythrin (PE) channel was set to detect JC-1 aggregates with orange–red fluorescence, and the fluoresceine isothiocyanate (FITC) channel was set to detect JC-1 monomers with green fluorescence. Data were collected and analyzed using Novo Express™ software (ACEA NovoCyteT M , USA). Western blotting Whole tissue proteins and mitochondrial proteins were extracted from liver tissue, then were quantified by using BCA detection kit (Epizyme, Shanghai, China). SDS-PAGE gels were used to separate proteins by electrophoresing under certain conditions, and transferred to a PVDF membrane (Millipore, Shanghai, China). After the membranes were washed with TBST and blocked with milk or BSA, the primary antibodies (Proteintech, USA): ATF4 (1:1000, 10835-1-AP), LONP1 (1:3000, 15440-1-AP), mtHSP60 (1:1000, 12165S), VDAC (1:1000, 10866-1-AP), GAPDH (1:20000, 6004-1-Ig); (Affinity, UK): p-PERK (1:1000, DF7576), p-eIF2α (1:1000, AF3087), and GRP78 (1:1000, Bioworld, USA), ,mtHSP70 (1:1000, SANTA, SC-66048, USA), ,ClpP (1:1000, absin, abs137925, USA), FGF-21 (1:3000, bioss, USA) were added to incubate overnight. The next day, the membrane was incubated with secondary antibodies: anti-rabbit IgG H&L (HRP) (1:10000, Bioworld, BS13278, USA) and goat anti-mouse IgG H&L (HRP) (1:2000, Cell Signaling, 7076S, USA), after washing with TBST. The blots were visualized by ECL system and were analyzed by using the Image Lab detection system (BioRad, Hercules, CA). Statistical analysis The statistical analysis were carried out using two-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test and t tests with GraphPad Prism 5.01 software (San Diego, CA, USA). The significant differences were considered at P < 0.05. The results are presented as the mean ± standard error of means (SEM). Results Exercise protects against AMLN diet-induced body weight gain and dyslipidemia during MAFLD progression During the experiment, animals fed with an AMLN diet exhibited a greater increase in body weight than those fed with a ND diet, and the effect of different dietary on mice body weight showed a significant difference from the 10th week (Fig. 2 A). Considering the effect of exercise, results revealed that mice in AMLN14 + E8 and AMLN18 + E8 groups gained less weight than AMLN diet mice ( P < 0.05, Fig. 2 B-D). On this basis, this study further examined the liver index, revealing that the mice in the AMLN diet group displayed an increased liver index compared to mice in the ND group, whereas the higher liver index level in the AMLN18 group was improved by the exercise intervention ( P < 0.01, Fig. 2 E). Next, the serum biochemistry profile from different groups at 14 and 18 weeks were characterized. As shown in Fig. 2 F and 2 H, 14-week AMLN treatment prominently increased the serum levels of CHOL and LDL-C compared to ND mice. In addition, compared to ND18 mice, the levels of CHOL, TG, LDL-C, and HDL-C in the mice of 18-week AMLN group were markedly raised ( P < 0.05, Fig. 2 F–I). It exists significant differences in the serum concentrations of CHOL, TG, and LDL-C when compared between the AMLN18 and AMLN14 groups. Notably, the 18-week AMLN diet-induced dyslipidemia mice showed significant improvement after 8 weeks of exercise intervention, with significant attenuation of serum CHOL, TG, and LDL-C levels ( P < 0.01, Fig. 2 F–H). In addition, the AMLN diet effect on liver injury was further evaluated, and found that 18-week AMLN diet contribute to liver damage as shown by significantly increased ALT and AST levels, and significantly higher than AMLN14 group. Similarly, 8-week exercise intervention was effective in suppressing liver injury induced by chronic AMLN diet ( P < 0.05, Fig. 2 J). Exercise alleviates AMLN diet-induced hepatic lipid accumulation during MAFLD progression To evaluate the biochemical evidence of liver lipid deposition, the liver tissue morphology and liver histological characteristics were quantified. From the liver tissue, it was observed that the AMLN diet resulted in a fatty color change and increased liver volume (Fig. 3 A). Liver sections of ND group mice exhibited normal hepatic cells each with prominent nucleus, well-arranged in plates or cords, and a branch of hepatic artery along the centrilobular vein. However, numerous lipid droplets and disordered liver structures appeared in the hepatocytes of 14- and 18-week AMLN diet mice (Fig. 3 B). Moreover, histological examination was used to indicated that 14- and 18-week AMLN diet significantly increased the score of hepatic steatosis, inflammation, locular ballooning, and NAFLD activity score. Furthermore, lipid accumulation between the AMLN14 and AMLN18 groups appeared a significant difference. Compare to the AMLN14 and AMLN18 groups, the lipid content was significantly declined in AMLN14 + E8 and AMLN18 + E8 groups ( P < 0.05, Fig. 3 D–G), suggesting that the longer the AMLN diet feeding duration, the more significant the lipid deposition, and that exercise can positively regulate AMLN dietary-induced lipid accumulation in different MAFLD developmental stages. To further measure the intrahepatic fat content, the hepatic lipid droplets were evaluated by Oil Red O staining. The areas of lipid droplets (LDs) in the 14-week and 18-week AMLN groups were increased significantly compared to those in the ND group, and mice in the AMLN18 group developed greater intrahepatic fat content than mice in the AMLN14 group ( P < 0.05, Fig. 3 C, H). Consistent with the above findings, AMLN diet mice indicated that the number and area of hepatic LDs were significantly decreased after exercise intervention, suggesting that exercise prevented aberrant LD deposition during MAFLD development ( P < 0.05, Fig. 3 H). Liver TG content measurements also showed the same trend with AMLN dietary and exercise intervention ( P < 0.05, Fig. 3 I). Exercise attenuates AMLN diet-induced hepatic PERK-eIF2α-ATF4 signaling activation especially at the later stages of MAFLD progression To further illuminate the mechanism by which different degrees of lipid deposition affect ER stress, especially the PERK-eIF2α-ATF4 signaling arm. Expression of GRP78 and its downstream target genes were next examined. As shown in Fig. 4 A and 4 B, 14- and 18-week AMLN diet mice were noted to have significantly increased GRP78 expression, as detected by Immunohistochemistry and western blotting compared to normal diet mice, suggesting the activation of ER stress. Present results also revealed the beneficial effects of exercise on alleviating ER stress, as demonstrated by decreased GRP78 expression in mice from the AMLN18 + E8 group compared to AMLN18 group ( P < 0.05, Fig. 4 B). Additionally, the GRP78 downstream pathways, including PERK, elF2α, ATF4, and CHOP expression were observed after AMLN diet with or without exercise intervention. The western blotting results indicated that the PERK and elF2α phosphorylation levels were highly increased by AMLN diet feeding, both at 14 and 18 weeks, where the longer the AMLN feeding period, the higher the level of pathway activation ( P < 0.05, Fig. 4 E, F). Furthermore, compared to ND diet mice, ATF4 and CHOP expression levels in the 14- and 18-week AMLN diet mice were also significantly increased associated with the upstream signaling PERK-eIF2α activation ( P < 0.05, Fig. 4 G, H). Notably, this AMLN-induced ER stress activation after 18 weeks was mitigated when mice were exercised for 8 weeks, as evidenced by decreased GRP78 and PERK-eIF2α-ATF4-CHOP pathway expression in AMLN18 + E8 group mice. Exercise restores AMLN diet-induced mitochondrial dysfunction and UPRmt impairment at the later stages of MAFLD development To study the effect of ATF4 activated signaling pathway on mitochondrial function, this study examined mitochondrial morphology by using electron microscopy. The results showed that hepatocytes from AMLN diet mice exhibited accumulation of large lipid droplets, as well as mitochondrial swelling and cristae fragmentation, particularly in the AMLN18 group. These striking features of damaged mitochondria were significantly improved and exhibited normalized mitochondrial structure after an 8-week voluntary wheel running intervention in both AMLN14 + E8 and AMLN18 + E8 groups (Fig. 5 A). Next, mitochondrial function indicators were tested, including mitochondrial respiration function, membrane potential, and mitochondrial respiratory chain complexes subunits. As shown in Fig. 5 B and C, compared to ND mice, 14- and 18-week AMLN diet mice exhibited decreased mitochondrial state III oxygen consumption and a reduced mitochondrial RCR, respectively. Moreover, the expression of mitochondrial respiratory chain complex subunits in AMLN diet mice were significantly declined ( P < 0.05, Fig. 5 D). Similar results were appeared to mitochondrial membrane potential ( P < 0.05, Fig. 5 E), showing that 18 weeks of AMLN feeding significantly increased the proportion of mitochondria with lower membrane potential. In contrast to the negative effect of AMLN diet on mitochondrial function, exercise intervention was able to protect the mitochondria of hepatocytes in AMLN diet mice to some extent, especially the effect on mitochondrial respiratory function and OXPHOS protein expression ( P < 0.05, Fig. 5 C, D). Given that exercise rescued the lipid accumulation-associated liver phenotype and improved mitochondrial function, hepatic UPRmt activation in this experimental model was further investigated. As shown in Fig. 5 F–J, western blot revealed that hepatic UPRmt component expression, including that of LONP1, HSP70, and HSP60 in the mitochondrial matrix was significantly elevated in 14-week AMLN diet mice when compared to the 14-week ND mice. Interestingly, compared to the AMLN14 group, mice on an AMLN diet for 18 weeks exhibited a sharp decline in UPRmt gene content and ClpP protein expression, highlighting a disconnect between hepatic lipid deposition and UPRmt activation because mitochondrial function is closely related with the degree of liver damage. In addition, the 8-week wheel running intervention significantly improved mitochondrial chaperone proteins and proteases and reversed the suppression of mtHSP70 ( P < 0.01, Fig. 5 G) and mtHSP60 ( P < 0.01, Fig. 5 H) expression in AMLN18 + E8 group mice. Positive correlation between FGF21 secretion and the UPRmt activation level during AMLN diet and exercise intervention Circulating FGF21 is highly correlated with mitochondrial function. Further analysis of serum and hepatic FGF21 levels revealed that the serum FGF21 levels were markedly higher in the 14-week AMLN diet group than in the ND14 group, whereas these levels were dramatically reduced in the 18-week AMLN diet group ( P < 0.05, Fig. 6 A, B). Thus, a apparent difference in serum and hepatic FGF21 level was observed between the 14- and 18-week AMLN groups (Fig. 6 A, B), which agreed with previous UPRmt-related data. Next, to determine whether FGF21 secretion is involved in the regulation of the hepatic UPRmt mechanism during MAFLD progression, this study performed linear regression analysis between the circulating FGF21 level and the value of UPRmt protein expression in all samples. As shown in Figs. 6 C–F, there were positive correlations between the serum FGF21 level and UPRmt protein content, including LONP1, mtHSP70, mtHSP60, and ClpP, suggesting a positive correlation between FGF21 secretion and the UPRmt activation level. Discussion Overnutrition and physical inactivity are precipitating factors that cannot be ignored in the occurrence of metabolic complications such as insulin resistance, type2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and MAFLD. MAFLD is a progressive disease, and excessive consumption of fat and/or sugar, particularly fructose, can cause hepatic steatosis and dyslipidemia. A diet constituting 40% fat, 20% fructose, and 2% cholesterol is widely known as the AMLN diet (Research diet, #D09100301) and is the preferred diet to induce many clinically relevant characteristics of NASH. Different periods of AMLN diet will result in different levels of lipid deposition in liver tissue, with consequent changes in oxidative and ER stress levels, as well as mitochondrial function [ 28 ]. Interestingly, a recent study revealed that mitochondrial FAO capacity in liver was decreased after 4weeks high fat diet (HFD) feeding, however, this effect was restored at 8 weeks [ 29 ], indicating that mitochondria function in liver tissue are highly plastic in diet-induced metabolic alteration. Exercise has also been confirmed to have significant effects on improving hepatic lipid metabolism, mitochondrial function, and the oxidative stress level. Zou et al. [ 30 ] recently showed that moderate-intensity exercise induced a greater improvement in antioxidant ability and reduced stress status via regulation of ER stress pathway-related proteins. Therefore, this study first to compare the changes concerning lipid deposition, ER stress signaling pathway, UPRmt, and mitokine secretion after 14 and 18 weeks of AMLN diet intervention, which are two time points during the progression of MAFLD. On this basis, this study further explored the role of an 8-week voluntary wheel running intervention in regulating MAFLD occurrence and progression, focusing mainly on the molecular mechanism involved in the PERK-eIF2α-ATF4 aix and UPRmt. The relationship between UPRmt levels and FGF21 secretion was also investigated. The AMLN diet is a common dietary formula that induces liver damage in a manner similar to that observed in the human liver diseases. NAFLD development in three stages, including steatosis, steatohepatitis with fibrosis, and cirrhosis [ 31 ]. Trevaskis et al. reported that animals had AMLN diet for 12 weeks demonstrated increased body weight and liver fat content without fibrosis, but showed progression toward fibrosis when fed for a chronic 30-week period [ 32 ]. Here, mice were fed an AMLN diet for 14 and 18 weeks to establish a model of hepatic steatosis and prefibrosis and found that hepatic lipid deposition occurred significantly in both time periods. Moreover, these changes were associated with abnormal blood lipid levels in the 18-week AMLN diet animals, but not in the AMLN14 group. As the circulating fatty acids uptake is the major way for liver to acquire lipids [ 33 ], the absence of dyslipidemia in the 14-week AMLN diet mice may be a result of the increased fatty acid intake and FA storage as TGs in the liver, which normalize serum lipid levels. However, this regulatory mechanisms were disappeared in the 18-week AMLN diet animals. These data suggest that prolonged administration of an AMLN diet not only leads to an abnormal increase in blood lipids but also induces massive macro- and microsteatosis. This may even aggravate oxidative stress and damage mitochondrial function, which contribute to cellular damage and disease progression. In addition, numerous evidence supports that exercise exerts many of its metabolic benefits in liver, adipose tissue, and pancreas [ 33 ]. Although the mechanisms by which exercise reduces liver fat are still largely unknown, the inner mechanism involves β-oxidation [ 34 ], lipogenesis, and lipid export. In addition, an improvement in insulin resistance with exercise intervention is thought to reduce the uptake of circulating FFAs to the liver. Therefore, exercises can be helpful in orchestrating the lipid synthesis, export [ 35 ], and their use as energy substrates. The results showed that liver lipid deposition in AMLN-fed animals at two stages improved significantly after an 8-week voluntary wheel running exercise intervention. However, this study failed to observe significantly decreased blood lipid levels in the early stages mediated by exercise, which may be due to the relatively lower blood lipid levels in the early stages than in the late. To further understand the alleviated AMLN diet-induced lipid deposition displayed by exercise intervention and with a focus on mitochondria-related metabolic mechanisms, the PERK-eIF2α-ATF4 signaling pathway was first examined, which is ER stress branch pathway and has been shown to be closely related to hepatic lipogenesis and steatosis regulation [ 36 ]. The activation of this ER stress or UPR mainly relays on the luminal chaperone GRP78 activation. In terms of its downstream genes, a study showed that decreased hepatic lipogenesis and steatosis in HFD fed mice is accompanied by long-term dephosphorylation of PERK downstream: the elongation initiation factor eIF2α [ 37 ]. In this study, the PERK-eIF2α-ATF4 signaling pathway was significantly awakened in both 14- and 18-week AMLN diet mice compared to the ND diet mice, which indicated that abnormal lipid accumulation often coincides with perturbed ER proteostasis in hepatocytes. The more severe the hepatic lipid deposition, the more obvious the PERK-eIF2α-ATF4 signaling pathway. Importantly, after 8 weeks of exercise intervention, the significant elevation of GRP78 and its downstream target gene expression was apparently improved in AMLN diet mice. These results indicant that this improvement induced by exercise in the ER stress signaling pathway is a beneficial adaptive mechanism. However, although ER stress responses were markedly activated by AMLN diets fed for different durations in this experiment and attenuated after exercise intervention, the underlying mechanisms may not be consistent. One clinical research showed that there is a variable degress of ER stress activation in the patients with NAFLD [ 38 ]. Prolonged phosphorylation of eIF-2α via activated PERK could specifically increase the downstream effectors ATF4 and CHOP, which enable the cell restore proteostasis. Conversely, there are other potential mechanisms induced by eIF-2α phosphorylation, several studies have shown that ER eIF2a phosphorylated downstream elements, which lead to the severeg oxidative stress by downregulating NFE2-related factor 2 (Nrf2) and depleting glutathione (GSH), eventually leading to apoptosis [ 39 – 41 ]. It has also been shown eIF2α phosphorylation is a key molecular event in mammalian UPRmt activation and is closely connection of the ISR [ 42 ]. Under stress conditions, the phosphorylation of eIF2 α triggers ATF4 activation, induces CHOP expression, and promotes UPRmt activation. Elevated transcript levels of ATF4 and CHOP in AMLN diet mice may be related to mitochondrial stress. Thus, next step was to investigate whether mitochondrial function and the UPRmt molecular pathways were altered under different durations of AMLN diet, and the influence mechanism of exercise on it. The UPRmt intimately associated with mitochondrial quality control system and plays an important role on protein homeostasis by stabilizing mitochondrial function against several pathologies. Although the UPRmt mechanism remains unclear, it promotes development during mild mitochondrial dysfunction. A recent study showed that the hepatic mitochondrial is closely related to the pathogenesis of NAFLD [ 43 ]. Results in this study demonstrated that mitochondrial membrane potential was significantly reduced in both 14- and 18-week AMLN diet mice, whereas mitochondrial respiration function, detected by the RCR, and OXPHOS gene expression were markedly decreased only in the 18-week AMLN diet group. These data suggest that mitochondria in excessive lipid deposition hepatocytes display an uncoupling of respiration from ATP production, indicating a reduced ability of ATP generation of mitochondria. However, this alteration did not occur at the early stages of hepatic lipid deposition. It is perhaps reasonable that mitochondrial function was disrupted during MAFLD development, but the changes in UPRmt-associated gene expression were unexpected. In terms of UPRmt, the expression of its downstream effectors (LONP1, mtHSP70, and mtHSP60) were elevated in 14 weeks AMLN diet mice in the presence of UPRmt compared to those fed an AMLN diet for 18 weeks in the absence of UPRmt. This suggests that UPRmt activation was triggered at the early stages of MAFLD progression and was impaired during higher-grade hepatic lipid deposition. It is speculated that the impairment in UPRmt associated with excessive lipid deposition could be due to imbalanced redox homeostasis or the heavily activated PERK-eIF2α-ATF4 pathway, which causes mitochondria to lose their ability to repair themselves. Indeed, a previous study reported that ATF4 negatively regulates TFAM expression and disrupts mitochondrial biogenesis and respiratory function in hepatocytes [ 44 ]. Another possible explanation could be that dysfunctional mitochondrial protein import due to the prolonged AMLN diet induced the overall decline in mitochondrial function, which further impeded the process of transporting nuclear genome-encoded mitochondrial chaperones or proteases to the mitochondrial matrix. Taken together, these data point out the possibility that UPRmt may have dual effects: the 14-week AMLN diet associated with slight stress can initiate UPRmt and restore mitochondrial proteostasis, conversely, chronic UPRmt activation may be harmful to cell survival. It is worth noting that, the decreased expression of some UPRmt components in 18-week AMLN diet mice, such as Hsp60, Hsp70, ClpP and LONP1 were restored with exercise intervention, as shown in AMLN18 + E8 group animals. These results highlighted the role of exercise in relieving mitochondrial stress and improving the UPRmt process, which coordinated to ensure mitochondrial function and viability. Future study is needed to probe the deep regulation mechanisms of UPRmt induction after exercise intervention. Mitochondria have been considered to crosstalk with other distant tissues through serum cytokine non-cell autonomously [ 44 ]. This study next focused on FGF21, a mitokine, mediated by the UPRmt-related eIF2α-ATF4-CHOP ER stress response axis, and is also a well-known exercise-induced hepatokine that stimulates lipolysis and fatty acid oxidation and suppresses lipogenesis in the liver [ 45 , 46 ]. Myung-Shik Lee’s group demonstrated that ATF4-dependent FGF21 induction was accompanied by impaired mitochondrial function, which contribute to suppress the diet-induced hepatic steatosis in animals [ 47 ]. The present study found that compared to ND mice, the FGF21 levels in serum and liver tissue were significantly enhanced after 14-week AMLN diet intervention, but the content was markedly reduced in 18-week AMLN diet mice, which is consistent with the previous UPRmt alteration. This observation suggests a relationship between UPRmt and FGF21 secretion. Yu-Wei Cheng recently reported that the ISR/ATF4-dependent induction of FGF21 was triggered by mitochondrial stress, which can mediate beneficial effects systemically [ 48 ]. Surprisingly, it found a positive correlation between UPRmt-related gene expression levels, including LONP1, HSP60, HSP70, and ClpP, and hepatic FGF21 levels as well as serum FGF21 content, suggesting that FGF21 is secreted by cells experiencing mitochondrial stress. Different from linear correlation, the relationship between mitochondrial perturbations-mediated UPRmt and hepatic lipid accumulation demonstrated a mitohormetic response. This mitohormesis concept suggests that mild mitochondrial stress initiates a diverse set of retrograde stress responses from mitonuclear interaction, whereas higher doses of stress can have harmful effects on cellular function [ 49 ]. Notably, this study also demonstrated that the decrease in circulating and hepatic FGF21 associated with excessive lipid accumulation was improved in response to exercise intervention. Based on the evidence presented above, this result may be due to the effect of exercise on regulating PERK-eIF2α-ATF4 or the UPRmt-related ISR/ATF4 pathway, both of which induce adaptive responses by triggering mitochondrial to nuclear communications, thereby recovering mitochondrial UPR and enabling mitochondrial self-healing. Previous studies have proposed an important role of cytokine factors as regulators of systemic energy metabolism. Secretion of mitokine during a specific period of disease progression has been proved to be a vital adaptive response that occurs [ 50 ]. In line with the concept of mitohormesis, exercise-mediated FGF21 secretion may be largely dependent on mitohormesis effect. Under normal conditions, long-term exercise can enhance cellular adaptations to an extent by triggering mitochondrial stress and mitonuclear communication. Moreover, in a state of severe lipid deposition in liver cells, exercise intervention could induce the mitohormetic response by improving the levels of oxidative stress and mitochondrial function, enhancing mitochondrial chaperones and proteases expression, and secreting mitokines. Study strengths and limitations This study demonstrated how an additional 4 weeks on the AMLN diet can progress the development of steatohepatitis, as well as the effects it has on the proteins involved in ER stress signaling pathway, and UPRmt. Moreover, this study further explored the effectiveness of exercise to reduce the development of MAFLD and to normalize the protein content and activation of proteins involved in ER signaling and UPRmt. However, several limitations are existed in this study. First, this study is limited to animal experiments. Although serum FGF21 levels are showed closely related to liver UPRmt level here, whether it can be used for clinical diagnosis of MAFLD still needs further confirmation from clinical experiments. Second, owing to the complex of the pathogenesis of MAFLD, this study mainly focus on the mitochondira, and the depth mechanisms related to UPRmt, such as mitonuclear communication and the interaction of mitochondria with the endoplasmic reticulum during MAFLD development deserves further investigation. Conclusion In summary, this study have demonstrated alterations in the PERK-eIF2α-ATF4 signaling pathway, mitochondrial UPR gene expression, and mitokine secretion during different developmental stages of MAFLD pathogenesis. Different from the linear correlation between lipid deposition and the PERK branch of ER stress in the liver, the changes in reduced mitochondrial function, downregulated UPRmt component expression, and decreased FGF21 secretion were found at the later stages of fatty liver development, indicating that severe lipid deposition leads to impaired UPRmt through overactivating the ISR/ATF4 pathway, resulting in mitochondria losing their self-repair ability and miscommunication with the nucleus. Importantly, exercise intervention rescued the 18-week AMLN diet-induced hepatic phenotype and reversed mitochondrial function, UPRmt activation pattern, and FGF21 secretion (Fig. 7 ). These results highlight the pivotal role of exercise in regulating the UPRmt and its related mitokine secretion to delay MAFLD progression. This study not only suggests that serum FGF21 content may be an important indicator for clinical diagnosis of the occurrence and development of MAFLD, but also confirms the significance of implementing exercise interventions as the primary prevention of MAFLD in clinical practice. Abbreviations Abbreviations Full name AMLN amylin liver NASH ATF4 activating transcription factor 4 ATF5 activating transcription factor 5 AST aspartate aminotransferase ALT alanine aminotransferase ANOVE analysis of variance BSA bovine serum albumin BiP binding protein ClpP casein lytic proteinase P CHOP C/EBP homologous protein CHOL cholesterol CVD cardiovascular disease DAB Diaminobenzidine ER endoplasmic reticulum ETC electron transport chain eIF2α eukaryotic initiation factor 2α FFA free fatty acid FAO fatty acid oxidation FGF21 fibroblast growth factor 21 FITC fluoresceine isothiocyanate GAPDH glyceraldehyde-3-phosphate dehydrogenase GRP78 glucose regulated protein 78 GSH glutathione HSP60 heatshockprotein 60 GDF15 growth differentiation factor 15 HSP70 heatshockprotein 70 HDL-C high-density lipoprotein cholesterol HFD high fat diet ISR integrated stress response JC-1 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine LONP1 lonpeptidase 1 LDL-C low-density lipoprotein cholesterol LDs lipid droplets MAFLD metabolic associated fatty liver disease mtHSP60 mitochondrial heatshockprotein 60 mtHSP70 mitochondrial heatshockprotein 70 mtDNA mitochondrial DNA MMP mitochondrial membrane potential NAFLD non-alcoholic fatty liver disease ND normal diet Nrf2 NFE2-related factor 2 NASH non-alcoholic steatohepatitis OXPHOS oxidative phosphorylation p-eIF2α phospho-eukaryotic initiation factor 2α PERK PKR-like ER kinase p-PERK phospho-PKR-like ER kinase PBS phosphate buffer saline PE phycoerythrin ROS reactive oxygen species RCR respiratory control rate SEM standard error of means TAC tricarboxylic acid TG triglyceride TEM transmission electron microscopy T2DM diabetes mellitus type2 TFAM Recombinant Transcription Factor A, Mitochondrial UPR unfolded protein response UPR ER endoplasmic reticulum unfolded protein reaction VDAC recombinant voltage dependent Anion channel protein Declarations Acknowledgments The authors would like to thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Graphical abstract was created with BioRender.com. Authors’ contributions Yuan Zhang, Jiao Lu, Qiang Tang conceived and designed research; Xinmeng Yuan, Mengqi Xiang, Wen Sun, Ye Xu, Yaran Gao, Wanyu Feng performed experiments; Xinmeng Yuan, Mengqi Xiang, Yuan Zhang, Liumei Zhang analyzed data; Xinmeng Yuan, Mengqi Xiang, Wen Sun, Ye Xu interpreted results of experiments; Xingmeng Yuan, Jingyi Wu prepared figures; Xinmeng Yuan, Yuan Zhang drafted manuscript; Yuan Zhang, Jiao Lu, Qiang Tang edited and revised manuscript; Yuan Zhang, approved final version of manuscript. Funding This work was supported by the Youth Project of National Natural Science Foundation of China (32000839), Qing Lan Project of Jiangsu Province of China ([2021]11), The National Key R&D Program of China (No. 2020YFC2007002), Graduate Research and Innovation Projects of Jiangsu Province (KYCX22_2247, KYCX22_2248, KYCX23_2363 and KYCX23_2380), The Innovation and Entrepreneurship Training Program for Undergraduates of Jiangsu Province of China (202310330011Z). Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate The protocol of animal experiments was complied with the Guide for National Institute of Health guidelines (NIH Publications No. 8023, revised 1978) and approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Approval No. GZRDW-2020-03). Consent for publication All authors agree to publish. Competing interests The authors declare no competing interests. References Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24(7):908–22. Takaki A, Kawai D, Yamamoto K. Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH). Int J Mol Sci. 2013;14(10):20704–28. Ajoolabady A, Kaplowitz N, Lebeaupin C, et al. Endoplasmic reticulum stress liver Dis Hepatol. 2023;77(2):619–39. Urano F, Wang X, Bertolotti A et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000;287(5453):664–6. Marciniak SJ, Chambers JE, Ron D. Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov. 2022;21(2):115–40. Willy JA, Young SK, Stevens JL, Masuoka HC, Wek RC. CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis. Mol Biol Cell. 2015;26(12):2190–204. Mansouri A, Gattolliat CH, Asselah T. Mitochondrial Dysfunction and Signaling. Chronic Liver Dis Gastroenterol. 2018;155(3):629–47. Verma AK, Sharma A, Subramaniyam N, Gandhi CR. Augmenter of liver regeneration. Mitochondrial function steatohepatitis J Hepatol. 2022;77(5):1410–21. Yi HS. Implications of Mitochondrial Unfolded Protein Response and Mitokines: A Perspective on Fatty Liver Diseases. Endocrinol Metab (Seoul). 2019;34(1):39–46. Kleinridders A, Lauritzen HP, Ussar S, et al. Leptin regulation of Hsp60 impacts hypothalamic insulin signaling. J Clin Invest. 2013;123(11):4667–80. Lee HY, Nga HT, Tian J, Yi HS. Mitochondrial Metabolic Signatures Hepatocellular Carcinoma Cells. 2021;10(8):1901. He L, Deng L, Zhang Q, et al. Diagnostic Value of CK-18, FGF-21, and Related Biomarker Panel in Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis. Biomed Res Int. 2017;2017:9729107. Tucker B, Li H, Long X, Rye KA, Ong KL. Fibroblast growth factor 21 in non-alcoholic fatty liver disease.Metabolism. 2019;101:153994. Liu J, Xu Y, Hu Y, Wang G. The role of fibroblast growth factor 21 in the pathogenesis of non-alcoholic fatty liver disease and implications for therapy.Metabolism. 2015;64(3):380–90. Tillman EJ, Rolph T. FGF21: An Emerging Therapeutic Target for Non-Alcoholic Steatohepatitis and Related Metabolic Diseases.Front Endocrinol. (Lausanne). 2020;11:601290. Kim SH, Kim KH, Kim HK et al. Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.Diabetologia. 2015;58(4):809–18. Liu X, Kwak D, Lu Z, et al. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling. Hypertension. 2014;64(4):738–44. Guo R, Liong EC, So KF, Fung ML, Tipoe GL. Beneficial mechanisms of aerobic exercise on hepatic lipid metabolism in non-alcoholic fatty liver disease. Hepatobiliary Pancreat Dis Int. 2015;14(2):139–44. Passos E, Ascensão A, Martins MJ, Magalhães J. Endoplasmic Reticulum Stress Response in Non-alcoholic Steatohepatitis: The Possible Role of. Phys Exerc Metabolism. 2015;64(7):780–92. Ascensão A, Martins MJ, Santos-Alves E et al. Modulation of hepatic redox status and mitochondrial metabolism by exercise: therapeutic strategy for liver diseases.Mitochondrion. 2013;13(6):862–70. Zhang Y, Liu Y, Liu X, et al. Exercise and Metformin Intervention Prevents Lipotoxicity-Induced Hepatocyte Apoptosis by Alleviating Oxidative and ER Stress and Activating the AMPK/Nrf2/HO-1 Signaling Pathway in db/db Mice. Oxid Med Cell Longev. 2022;2022:2297268. Wang Z, Sun T, Yu J, Li S, Gong L, Zhang Y. FGF21: A Sharp Weapon in the Process of Exercise to Improve NAFLD.Front Biosci (Landmark Ed). 2023;28(12):351. Li XH, Liu LZ, Chen L, et al. Aerobic exercise regulates FGF21 and NLRP3 inflammasome-mediated pyroptosis and inhibits atherosclerosis in mice. PLoS ONE. 2022;17(8):e0273527. Perakakis N, Joshi A, Peradze N, et al. The Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator CHS-131 Improves Liver Histopathology and Metabolism in a Mouse Model of Obesity and Nonalcoholic Steatohepatitis. Hepatol Commun. 2020;4(9):1302–15. Manzanares G, Brito-da-Silva G, Gandra PG. Voluntary wheel running: patterns and physiological effects in mice.Braz. J Med Biol Res. 2018;52(1):e7830. Kleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005;41(6):1313–21. Hynes J, Swiss RL, Will Y. High-Throughput Analysis of Mitochondrial Oxygen Consumption. Methods Mol Biol. 2018;1782:71–87. Carneros D, López-Lluch G, Bustos M. Physiopathology of Lifestyle Interventions in Non-Alcoholic Fatty Liver Disease (NAFLD).Nutrients. 2020;12(11):3472. Chen Z, Tian R, She Z, Cai J, Li H. Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease [published correction appears in Free. Radic Biol Med 2020;:] Free Radic Biol Med. 2020;152:116–41. Zou Y, Qi Z. Understanding the Role of Exercise in Nonalcoholic Fatty Liver Disease. ERS-Linked Mol Pathways Mediators Inflamm. 2020;2020:6412916. Yang Y, Li X, Liu Z et al. Moderate Treadmill Exercise Alleviates NAFLD by Regulating the Biogenesis and Autophagy of Lipid Droplet.Nutrients. 2022;14(22):4910. Trevaskis JL, Griffin PS, Wittmer C, et al. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol. 2012;302(8):G762–72. Clapper JR, Hendricks MD, Gu G, et al. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol. 2013;305(7):G483–95. Ipsen DH, Lykkesfeldt J, Tveden-Nyborg P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci. 2018;75(18):3313–27. Ferré P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.Diabetes Obes Metab. 2010;12 Suppl 2:83–92. Lebeaupin C, Vallée D, Hazari Y, Hetz C, Chevet E, Bailly-Maitre B. Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol. 2018;69(4):927–47. Melber A, Haynes CM. UPRmt regulation and output: a stress response mediated by mitochondrial-nuclear communication. Cell Res. 2018;28(3):281–95. Puri P, Mirshahi F, Cheung O et al. Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease.Gastroenterology. 2008;134(2):568–76. Ashraf NU, Sheikh TA. Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of Non-alcoholic fatty liver disease. Free Radic Res. 2015;49(12):1405–18. Fuchs CD, Claudel T, Scharnagl H, Stojakovic T, Trauner M. FXR controls CHOP expression in steatohepatitis. FEBS Lett. 2017;591(20):3360–8. Song B, Scheuner D, Ron D, Pennathur S, Kaufman RJ. Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes. J Clin Invest. 2008;118(10):3378–89. Derisbourg MJ, Hartman MD, Denzel MS, Perspective. Modulating the integrated stress response to slow aging and ameliorate age-related pathology. Nat Aging. 2021;1(9):760–8. Fromenty B, Roden M. Mitochondrial alterations in fatty liver diseases. J Hepatol. 2023;78(2):415–29. Mottis A, Herzig S, Auwerx J. Mitocellular communication: Shaping health and disease.Science. 2019;366(6467):827–32. Flippo KH, Potthoff MJ. Metabolic Messengers: FGF21.Nat Metab. 2021;3(3):309–317. Lewis JE, Ebling FJP, Samms RJ, Tsintzas K. Going Back to the Biology of FGF21: New Insights. Trends Endocrinol Metab. 2019;30(8):491–504. Kim KH, Jeong YT, Oh H, et al. Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine. Nat Med. 2013;19(1):83–92. Cheng YW, Liu J, Finkel T. Mitohormesis Cell Metab. 2023;35(11):1872–86. Bárcena C, Mayoral P, Quirós PM. Mitohormesis, an Antiaging Paradigm. Int Rev Cell Mol Biol. 2018;340:35–77. Yi HS, Chang JY, Shong M. The mitochondrial unfolded protein response and mitohormesis: a perspective on metabolic diseases. J Mol Endocrinol. 2018;61(3):R91–105. Additional Declarations No competing interests reported. Supplementary Files Dateofwesternblotting.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviews received at journal 01 Jun, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers invited by journal 22 May, 2024 Editor assigned by journal 20 May, 2024 Submission checks completed at journal 20 May, 2024 First submitted to journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4446826","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307886574,"identity":"77fe42b2-18ea-418f-b6ce-57e8e95245de","order_by":0,"name":"Xinmeng Yuan","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Xinmeng","middleName":"","lastName":"Yuan","suffix":""},{"id":307886575,"identity":"3e1c1d99-83eb-427b-be3a-b75433899433","order_by":1,"name":"Mengqi Xiang","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Xiang","suffix":""},{"id":307886576,"identity":"62b747ef-9113-4701-bf96-14bbd5c6d3f6","order_by":2,"name":"Yaran Gao","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Yaran","middleName":"","lastName":"Gao","suffix":""},{"id":307886577,"identity":"fa34af18-df4a-46a6-9fac-6e5ef4687c64","order_by":3,"name":"Wanyu Feng","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Wanyu","middleName":"","lastName":"Feng","suffix":""},{"id":307886578,"identity":"683ecf17-ae87-4635-9a9c-bac968a13aad","order_by":4,"name":"Wen Sun","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Sun","suffix":""},{"id":307886579,"identity":"cbcabceb-f1cb-4f21-bd92-fee2f93416fc","order_by":5,"name":"Ye Xu","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Xu","suffix":""},{"id":307886580,"identity":"ce712c6a-4808-44c5-a289-4fcff04609bf","order_by":6,"name":"Liumei Zhang","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Liumei","middleName":"","lastName":"Zhang","suffix":""},{"id":307886581,"identity":"52e32c8b-793f-4254-9b8c-442a73f10bce","order_by":7,"name":"Jingyi Wu","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Wu","suffix":""},{"id":307886582,"identity":"927e0cab-5945-4ab0-8cd4-a282e26737bb","order_by":8,"name":"Qiang Tang","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Tang","suffix":""},{"id":307886583,"identity":"377a1354-2cda-45a1-8c10-816d316fb579","order_by":9,"name":"Jiao Lu","email":"","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Lu","suffix":""},{"id":307886584,"identity":"0b90909e-5725-4573-996e-06e1b694247d","order_by":10,"name":"Yuan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCRiDvYGBIYGBgbGBeC08B0jWIpEApghrkZ/d/Ozh17bD8uaSbw9/eMBgI7vhAPOzB/i0MM45Zm4s23bYcOfsvDSgRWnGGw6wmRvg08IskWAmLdl2mHHD7RwzoF8OJ244wMMmgU8Lm0T6N5AW+w03zxh/SGD4T1gLj0SOmeTHNqDhN3gMgA47QFiLhEROmTTDufTkDWdyzCQSDJKNZx5mM8OrRX5G+jbJH2XWthuOnzH++KPCTrbvePMzvFpAgJmXDcYEBRUzIfVAwPjjDxGqRsEoGAWjYOQCAM+PSXKF7/KAAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Sport Institute","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-20 05:46:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4446826/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4446826/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57416205,"identity":"c4527126-71dd-4f9e-bb90-521c0c38e225","added_by":"auto","created_at":"2024-05-30 11:44:26","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":599549,"visible":true,"origin":"","legend":"\u003cp\u003eStudy design. The mice were fed with normal diet or AMLN diet for 14 or 18 weeks. Some of the mice were randomly selected and given 8-week voluntary wheel running intervention after 6 weeks (n=10) or 10 weeks (n=10) of AMLN dietary feeding.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/d1617af43352d3406ff54b37.jpeg"},{"id":57416192,"identity":"3911a0f9-7119-4772-a398-da4996bb1d70","added_by":"auto","created_at":"2024-05-30 11:44:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1258689,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AMLN diet and exercise on the mice body weight, liver index, and serum lipid levels. A: Mice body weight in different weeks. B: Representative time course curves of 14-week mice body weight. C: Representative time course curves of 18-week mice body weight. D: Mice body weight at the time of execution. E: Liver index, liver weight to body weight ratio. F-I: serum CHOL, TG, LDL-C and HDL-C concentrations. J: ALT and AST levels in serum. All data are shown as means ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, effect of diet; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, effect of exercise.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/1b9288f5053a586bbe887393.jpg"},{"id":57416204,"identity":"bcfc0d82-2367-4a23-8d90-9e4095f92435","added_by":"auto","created_at":"2024-05-30 11:44:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2754633,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AMLN diet and exercise on liver lipid deposition in mice. A: Representative liver morphology. B: H\u0026amp;E staining of mice liver tissue; scale bar: 20 μm. C: Oil Red O staining of mice liver tissue; scale bar: 20 μm. D-G: The analysis of NAFLD activity score. H: Liver lipid droplet area (%) as assessed by Image J. I: Hepatic triglyceride content as assessed by enzyme-labeled instrument. All data are shown as means ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, effect of diet; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, effect of exercise.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/145f2096b8ada05ec69d5304.jpg"},{"id":57416200,"identity":"f968ece7-6c0c-4bb6-aadf-78e4403f9d87","added_by":"auto","created_at":"2024-05-30 11:44:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1713760,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AMLN diet and exercise on PERK-eIF2α-ATF4 signaling pathway during MAFLD development. A: Representative images of hepatic GRP78 IHC staining; scale bar: 20 μm. B: GRP78 staining quantification in \u0026gt;5 fields per animal using Image J. C: Western blot results for GRP78, p-PERK, p-eIF2α, ATF4, CHOP and GAPDH. D-H: Western blotting quantification of relative protein expression using Image J. All data are shown as means ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, effect of diet; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, effect of exercise.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/188d92956926d625c5dbdad8.jpg"},{"id":57417047,"identity":"162e25d4-238c-4f90-a47f-2941dc745519","added_by":"auto","created_at":"2024-05-30 11:52:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2647188,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of AMLN diet and exercise on mitochondrial function and UPRmt marker gene expression. A: Representative TEM images of mitochondrial morphology; scale bar: 1 μm. B: Mitochondrial oxygen consumption was examined at 30℃ by treating mitochondria with glutamate without ADP(state IV) or with ADP (state III). C: Mitochondrial RCR: state III/state IV. D: Western blot results for the upper five enzyme complexes of the electron transport chain (ETC) and VDAC. E: Mitochondrial membrane potential. F: Western blot results for LONP1, mtHSP70, mtHSP60, ClpP and VDAC. G-J: western blotting quantification of relative protein expression using Image J. All data are shown as means ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, effect of diet; # \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, effect of exercise.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/48346f2537d4ca58ccb1e1d1.jpg"},{"id":57416202,"identity":"a821e570-aefa-4103-a411-469bffac6620","added_by":"auto","created_at":"2024-05-30 11:44:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214814,"visible":true,"origin":"","legend":"\u003cp\u003eThe FGF21 expression was closely correlated with UPRmt genes expression level. A: Serum FGF21 concentrations. B: Western blot results for hepatic FGF21 expression. C-F: Correlation tests were performed between the UPRmt genes expression level and serum FGF21. All data are shown as means ± SEM. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, effect of diet.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/ef201110f3b30c3a87cdc320.jpg"},{"id":57416195,"identity":"a82223a1-7150-4a4d-89e5-69dc0a3946cc","added_by":"auto","created_at":"2024-05-30 11:44:23","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2372545,"visible":true,"origin":"","legend":"\u003cp\u003ePossible mechanism of AMLN diet and exercise on mitochondrial homeostasis during MAFLD development. Under normal circumstances, the liver is ruddy in color and the mitochondria function properly. However, alterations were found in PERK-eIF2α-ATF4 signaling pathway, mitochondrial UPR gene expression, and mitokine secretion at different stages of MAFLD pathogenesis development. A. Early stage of MAFLD development: a 14-week AMLN diet induced hepatic steatosis and ER stress. This mild mitochondrial stress initiated a diverse set of retrograde stress responses, including mitonuclearcommunication, UPRmt, and FGF21 secretion, which contribute to maintaining mitochondrial function, enabling mitochondrial self-healing, and alleviating liver damage. B. Late stage of MAFLD development: an 18-week AMLN diet aggravated the accumulation of hepatic lipids. Severe lipid deposition led to disconnect communication between mitochondria and nuclear, impaired UPRmt through overactivating the ISR/ATF4 pathway, then resulted in mitochondria losing their self-repair ability. Moreover, exercise intervention significantly improved the imbalanced mitochondrial homeostasis due to chronic AMLN diet, which plays a pivotal role in reversing the progression of MAFLD. Red arrows: increase effect; Black arrows: decreased effect.\u003c/p\u003e","description":"","filename":"Figure7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/c9adce7e151f86e46a47f270.jpeg"},{"id":57417547,"identity":"dfe18916-16e5-4959-9f81-46528eaedd46","added_by":"auto","created_at":"2024-05-30 12:00:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12314563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/6db18a14-e30e-491d-bd2c-ebce015cb642.pdf"},{"id":57417046,"identity":"8113dde3-6fb9-491e-8a3d-07a24d2c8094","added_by":"auto","created_at":"2024-05-30 11:52:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":283026,"visible":true,"origin":"","legend":"","description":"","filename":"Dateofwesternblotting.docx","url":"https://assets-eu.researchsquare.com/files/rs-4446826/v1/8cb5aa5c97da0f4da98b4e26.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exercise protects against AMLN diet-induced lipid deposition in hepatocytes during MAFLD progression by regulating the UPRmt and FGF21 secretion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe manifestations of metabolic associated fatty liver disease (MAFLD) are becoming a growing challenge for public health. MAFLD progression is caused by an imbalance between lipid acquisition and lipid disposal due to the overwhelmed metabolic capacity in the liver. This progression of NAFLD is currently explained by the \u0026ldquo;multiple-hit\u0026rdquo; hypothesis, which proposes that multiple concurrent insults, such as insulin resistance, oxidative stress, intracellular stress response and mitochondrial dysfunction, eventually contribute to liver injury and NAFLD progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Hepatocytes have abundant ER, which is crucial in regulating calcium homeostasis and lipid metabolism in cells. Many genetic or dietary models of fatty liver in recent studies have demonstrated that free fatty acid (FFA) overload and lipotoxicity are critical factors that lead to ER homeostasis disequilibrium in the liver [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. ER stress can induce multiple effects by activating its downstream pathways, including the unfolded protein response (UPR), apoptotic [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] integrated stress response (ISR) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and inflammatory pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], contributing to the progression from initial steatosis to non-alcoholic steatohepatitis (NASH).\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction is closely connected with the onset and progression of NAFLD, and the use of mitochondria as a target for NAFLD therapy has been gaining traction based on rodent models and human studies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. With increased lipid deposition in the liver, hepatic metabolism might shift to protect hepatocytes from the lipid burden at the initial stage. For example, studies have shown that hepatic metabolic adaptation and mitochondrial flexibility at the early stages of NAFLD development, such as increased mitochondrial fatty acid oxidation (FAO), enhanced the tricarboxylic acid (TCA) cycle, and raised oxidative phosphorylation (OXPHOS) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, at the advanced stages of MAFLD development, the imbalance between mitochondrial FAO and the electron transport chain (ETC) will cause reactive oxygen species (ROS) overproduction, which not only threatens the OXPHOS machinery but also other mitochondrial proteins, lipids, and mtDNA. In response to increased ROS production during MAFLD development, unfolded or misfolded proteins can aggregate in the mitochondrial matrix, leading to the initiation of the mitochondrial unfolded protein response (UPRmt). The UPRmt is an important mechanism for maintaining mitochondrial proteostasis during stress. Under mitochondrial stress, UPRmt process is initiated by inducing nuclear gene-encoded proteins transcription, such as chaperones heat shock protein 10 (HSP10) and heat shock protein 60 (HSP60), proteases caseinolytic protease (ClpP) and Lon peptidase 1 (LONP1) to maintain mitochondrial proteostasis. Indeed, increasing studies have documented that the UPRmt exerts a protective effect in liver metabolic related diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while an abnormal UPRmt triggers a series of aging-related diseases. Interestingly, evidence shows that the increased expression of UPRmt related proteases and chaperone proteins is accompanied by elevated expression of mitokines such as fibroblast growth factor 21 (FGF21) and GDF15 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. FGF21 is a protein that is highly synthesized in the liver and was the first cytokine reported in mammalian systems. Abundance of evidences have confirmed that circulating FGF21 content is implicated in the pathobiology of NAFLD [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and abnormal FGF21 signaling is a major pathological changes in the development and progression of NAFLD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Indeed, evidences have reported that FGF21 expression can be stimulated by the ER stress response via the PKR-like ER kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) axis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, some of the previously described that UPRmt and UPRER have interactive effects under homeostasis disruption, and PERK-ATF4 axis is the core hub collaborating UPR\u003csup\u003eER\u003c/sup\u003e and UPRmt, hence regulating proteostasis by promoting ER and mitochondrial interactivities and improving their functions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExercise-induced mitochondrial adaptation not only favourable for improving muscle health, but also plays a vital role in mitochondrial dysfunction related metabolic alterations associated with liver diseases. Although the main mechanisms of exercise effect on NAFLD treatment include the reduction in intrahepatic fat content, attenuation of inflammation and oxidative stress, maintenance of ER homeostasis, and improvement of insulin signaling and mitochondrial function [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the underlying molecular mechanism are not completely elucidated. Acute and chronic forms of physical exercise was shown to modulate mitochondrial metabolism and hepatic redox state [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In particular, exercise intervention can improve the antioxidant defense system capacity, regulate the PERK-eIF2α-ATF4 pathway, inhibit hepatocyte apoptosis and ameliorate hepatic lipid deposition [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, novel exercise-inducible cytokines are hypothesized to play a vital role in lipid metabolism such as myokines, hepatokines, and adipokines. Some researches in human and animal models have demonstrated the mechanisms for ameliorating MAFLD by detecting FGF21 during exercise. Others also suggested that FGF21 may play a crucial role in exercise to improve NAFLD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and serum FGF21 is largely secreted from the liver during aerobic exercise [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is therefore postulated that the exercise could alleviates MAFLD occurrence and progression via regulating livermitochondrial function and FGF21 secretion, to further elucidate the specific mechanisms underlying the advantage of exercise intervention during different stages of MAFLD progression.\u003c/p\u003e \u003cp\u003eIn this study, it highlighted the effect of exercise on protecting against AMLN diet-induced lipid accumulation in hepatocytes during MAFLD progression in mice. According to results, 8 weeks of voluntary wheel running significant restrains MAFLD development via upregulation of UPRmt signaling mechanisms at the earlier stages of MAFLD development, and restoration of the impaired UPRmt at the later stages of MAFLD development. Furthermore, the variation tendency for FGF21 secretion and the UPRmt activation during MAFLD progression is alike, which may be a novel potential strategy for diagnosing the developmental stages of MAFLD.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSixty male eight-week-old C57BL/6J mice were purchased from Jiangsu Gempharmatech Co., Ltd, Nanjing, China and were individually housed in cages in the Jiangsu Academy of Agricultural Sciences at an immobile temperature and humidity environment with freely access to food and water. Sixty C57BL/6J mice were divided equally into two groups, feed with normal diet (ND), contains 14.9% fat, 22.7% protein, and 62.4% carbohydrate (XIETONG.ORGANISM, 1010084, Nanjing, China) and Amylin Liver NASH (AMLN) diet, contains fat (40%), fructose (20%), and cholesterol (2%) (Research Diets, #D09100301, USA) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After 6 and 10 weeks of AMLN diet feeding, half of the mice were divided into voluntary wheel running and sedentary groups with AMLN diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This study adhered to all guidelines for animal experiments issued by Nanjing Sport Institute\u0026rsquo;s Animal Ethics and Welfare Committee (Approval No. GZRDW-2020-03). After 18 weeks, the mice were anesthetized and sacrificed to collect retro-orbital blood, liver tissues for subsequent analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTraining protocol\u003c/h2\u003e \u003cp\u003eMice in the exercise intervention group were housed in cages equipped with a 14-cm-diameter voluntary running wheel. The number of runners\u0026rsquo; revolutions were recorded by using a magnetic counter, and then converted into distance (kilometers) run per day according to a conversion formula. In this study, mice exercised an average of 6\u0026ndash;11 km per day, reaching the standard of voluntary wheel running [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical measurements in serum\u003c/h2\u003e \u003cp\u003eBlood samples were centrifuged in 4\u0026deg;C, 5000 g for 10 min after standing on ice for 30 min before. The content of serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT), cholesterol (CHOL), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were detected according to the prescriptive protocol.\u003c/p\u003e \u003cp\u003eSerum FGF-21 concentration was tested by mouse FGF-21 Elisa kit (Bio-techne China Co., Ltd, MF2100, Shanghai, China) following the manufacture\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological staining\u003c/h2\u003e \u003cp\u003e3 mm cubes of liver tissues were collected after fixing with paraformaldehyde. Next, embedded tissue were cut into 4-\u0026micro;m slices before deparaffinizing and staining with Oil Red O (SIGMA-ALDRICH, #SHBL1039, USA) or H\u0026amp;E (Biosharp, BL702A, Hefei, China). Finally, the pathological morphology of the liver were observed by microscope (Zeiss, Axio Imager A2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e \u003cp\u003eDewaxed liver sections were applied to 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment for 10 min at room temperature and washed with phosphate buffer (PBS). Then, the tissue slices were treated with citrate buffer at 95\u0026deg;C, and blocked. The samples were refrigerated overnight at 4\u0026deg;C by adding GRP78 (1:1000, Bioworld, USA). Next, slices were added HRP-labeled anti-rabbit IgG (Boster Biological Technology, Wuhan, China) and incubated at 37\u0026deg;C. Diaminobenzidine (DAB) was performed for 10 min at room temperature, counterstaining was performed using hematoxylin, 1% alcohol hydrochloric acid was differentiated and neutral gum was used for sealing. Finally, the the pathological morphology of the liver were observed by microscope (Zeiss, Axio Imager A2) and quantified by using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNAFLD activity score evaluation\u003c/h2\u003e \u003cp\u003eThe morphological changes in the liver tissue were evaluated by using the NAFLD activity score (NAS) standard [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The criterion of the NAS system indicated three degrees of fatty liver: steatosis (\u0026gt;\u0026thinsp;66% = 3, 33\u0026ndash;66% = 2, 5\u0026ndash;33% = 1, \u0026lt;\u0026thinsp;5% = 0), lobular inflammation (\u0026gt;\u0026thinsp;4 foci\u0026thinsp;=\u0026thinsp;3, 2\u0026ndash;4 foci\u0026thinsp;=\u0026thinsp;2, \u0026lt;\u0026thinsp;2 foci\u0026thinsp;=\u0026thinsp;1, none\u0026thinsp;=\u0026thinsp;0), and locular ballooning (prominent\u0026thinsp;=\u0026thinsp;2, few\u0026thinsp;=\u0026thinsp;1, none\u0026thinsp;=\u0026thinsp;0).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the hepatic TG level\u003c/h2\u003e \u003cp\u003eLiver TG was detected with Triglyceride (TG) Content Assay kit (Boxbio, AKFA003M, Beijing, China). TG was extracted from live tissue with normal heptane/dimethylcarbinol (1:1), evaporated. Then the liver TG concentration was determined and calculated according to the instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eElectron microscopy\u003c/h2\u003e \u003cp\u003eAnimals were perfused with physiological sodium and 4% paraformaldehyde for pre-fixation before transmission electron microscopy (TEM). Livers from C57BL/6J mice were harvested, and liver samples were cut into 1-mm cubes and fixed in 2.5% glutaraldehyde. Then samples were treated with 1% osmium tetroxide, dehydrated in alcohols, embedded in araldite resin. Tissues were cut into 1-\u0026micro;m slices, stained with methylene blue and so on. The images were collected by JEM-1400 electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial isolation\u003c/h2\u003e \u003cp\u003eFresh liver tissue were minced and homogenized, then isolated by differential centrifugation, as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Samples were resuspended in Buffer I (70 mM sucrose, 210 mM mannitol, 1 mM ethylene glycol tetraacetic acid (EGTA) 5 mM HEPES, ,0.5% BSA, pH 7.4). After multi-step centrifugation, the pellet was resuspended in Buffer II, which include 210 mM mannitol, 70 mM sucrose, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 10 mM MOPS, 1 mM EGTA, pH 7.4, and the mitochondrial samples were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial oxygen consumption\u003c/h2\u003e \u003cp\u003eClarke-type electrode (Oxytherm, Hansatech, UK) was used for measuring mitochondrial oxygen consumption. Respiration buffer includes 20 mmol/L Tris-HCl, 125 mmol/L KCl, and 0.1 mmol/L EGTA, pH 7.4 and a volume of 75 ul mitochondrial sample were added to the reaction chamber, and determination of the proportion of dissolved oxygen consumption in fresh mitochondria with adding the presence of glutamate and ADP (state III respiration) or glutamate (state IV respiration). The ratio of state III to state IV is defined as the mitochondrial respiratory control rate (RCR). The oxygen consumption was valued by normalizing the oxygen consumption rate (nmole/s) to the total protein content (mg). The data were collected and analyzed by using O\u003csub\u003e2\u003c/sub\u003e view software (Hansatech, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003eLiver mitochondrial membrane potential were determined by using enhanced mitochondrial membrane potential assay kit (Beyotime, C2003S, Shanghai, China). The JC-1 working solution was configured as indicated, and the fluorescence intensity was detected by flow cytometry (ACEA NovoCyte\u0026trade;, USA) (excitation spectra at 490 and 525 nm and emission spectra at 530 and 590 nm). The phycoerythrin (PE) channel was set to detect JC-1 aggregates with orange\u0026ndash;red fluorescence, and the fluoresceine isothiocyanate (FITC) channel was set to detect JC-1 monomers with green fluorescence. Data were collected and analyzed using Novo Express\u0026trade; software (ACEA NovoCyteT\u003csup\u003eM\u003c/sup\u003e, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eWhole tissue proteins and mitochondrial proteins were extracted from liver tissue, then were quantified by using BCA detection kit (Epizyme, Shanghai, China). SDS-PAGE gels were used to separate proteins by electrophoresing under certain conditions, and transferred to a PVDF membrane (Millipore, Shanghai, China). After the membranes were washed with TBST and blocked with milk or BSA, the primary antibodies (Proteintech, USA): ATF4 (1:1000, 10835-1-AP), LONP1 (1:3000, 15440-1-AP), mtHSP60 (1:1000, 12165S), VDAC (1:1000, 10866-1-AP), GAPDH (1:20000, 6004-1-Ig); (Affinity, UK): p-PERK (1:1000, DF7576), p-eIF2α (1:1000, AF3087), and GRP78 (1:1000, Bioworld, USA), ,mtHSP70 (1:1000, SANTA, SC-66048, USA), ,ClpP (1:1000, absin, abs137925, USA), FGF-21 (1:3000, bioss, USA) were added to incubate overnight. The next day, the membrane was incubated with secondary antibodies: anti-rabbit IgG H\u0026amp;L (HRP) (1:10000, Bioworld, BS13278, USA) and goat anti-mouse IgG H\u0026amp;L (HRP) (1:2000, Cell Signaling, 7076S, USA), after washing with TBST. The blots were visualized by ECL system and were analyzed by using the Image Lab detection system (BioRad, Hercules, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e The statistical analysis were carried out using two-way analysis of variance (ANOVA), followed by Dunnett\u0026rsquo;s multiple comparison test and t tests with GraphPad Prism 5.01 software (San Diego, CA, USA). The significant differences were considered at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of means (SEM).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExercise protects against AMLN diet-induced body weight gain and dyslipidemia during MAFLD progression\u003c/h2\u003e \u003cp\u003eDuring the experiment, animals fed with an AMLN diet exhibited a greater increase in body weight than those fed with a ND diet, and the effect of different dietary on mice body weight showed a significant difference from the 10th week (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Considering the effect of exercise, results revealed that mice in AMLN14\u0026thinsp;+\u0026thinsp;E8 and AMLN18\u0026thinsp;+\u0026thinsp;E8 groups gained less weight than AMLN diet mice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). On this basis, this study further examined the liver index, revealing that the mice in the AMLN diet group displayed an increased liver index compared to mice in the ND group, whereas the higher liver index level in the AMLN18 group was improved by the exercise intervention (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eNext, the serum biochemistry profile from different groups at 14 and 18 weeks were characterized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, 14-week AMLN treatment prominently increased the serum levels of CHOL and LDL-C compared to ND mice. In addition, compared to ND18 mice, the levels of CHOL, TG, LDL-C, and HDL-C in the mice of 18-week AMLN group were markedly raised (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;I). It exists significant differences in the serum concentrations of CHOL, TG, and LDL-C when compared between the AMLN18 and AMLN14 groups. Notably, the 18-week AMLN diet-induced dyslipidemia mice showed significant improvement after 8 weeks of exercise intervention, with significant attenuation of serum CHOL, TG, and LDL-C levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;H). In addition, the AMLN diet effect on liver injury was further evaluated, and found that 18-week AMLN diet contribute to liver damage as shown by significantly increased ALT and AST levels, and significantly higher than AMLN14 group. Similarly, 8-week exercise intervention was effective in suppressing liver injury induced by chronic AMLN diet (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExercise alleviates AMLN diet-induced hepatic lipid accumulation during MAFLD progression\u003c/h2\u003e \u003cp\u003eTo evaluate the biochemical evidence of liver lipid deposition, the liver tissue morphology and liver histological characteristics were quantified. From the liver tissue, it was observed that the AMLN diet resulted in a fatty color change and increased liver volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Liver sections of ND group mice exhibited normal hepatic cells each with prominent nucleus, well-arranged in plates or cords, and a branch of hepatic artery along the centrilobular vein. However, numerous lipid droplets and disordered liver structures appeared in the hepatocytes of 14- and 18-week AMLN diet mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, histological examination was used to indicated that 14- and 18-week AMLN diet significantly increased the score of hepatic steatosis, inflammation, locular ballooning, and NAFLD activity score. Furthermore, lipid accumulation between the AMLN14 and AMLN18 groups appeared a significant difference. Compare to the AMLN14 and AMLN18 groups, the lipid content was significantly declined in AMLN14\u0026thinsp;+\u0026thinsp;E8 and AMLN18\u0026thinsp;+\u0026thinsp;E8 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;G), suggesting that the longer the AMLN diet feeding duration, the more significant the lipid deposition, and that exercise can positively regulate AMLN dietary-induced lipid accumulation in different MAFLD developmental stages.\u003c/p\u003e \u003cp\u003eTo further measure the intrahepatic fat content, the hepatic lipid droplets were evaluated by Oil Red O staining. The areas of lipid droplets (LDs) in the 14-week and 18-week AMLN groups were increased significantly compared to those in the ND group, and mice in the AMLN18 group developed greater intrahepatic fat content than mice in the AMLN14 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, H). Consistent with the above findings, AMLN diet mice indicated that the number and area of hepatic LDs were significantly decreased after exercise intervention, suggesting that exercise prevented aberrant LD deposition during MAFLD development (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Liver TG content measurements also showed the same trend with AMLN dietary and exercise intervention (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExercise attenuates AMLN diet-induced hepatic PERK-eIF2α-ATF4 signaling activation especially at the later stages of MAFLD progression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further illuminate the mechanism by which different degrees of lipid deposition affect ER stress, especially the PERK-eIF2α-ATF4 signaling arm. Expression of GRP78 and its downstream target genes were next examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, 14- and 18-week AMLN diet mice were noted to have significantly increased GRP78 expression, as detected by Immunohistochemistry and western blotting compared to normal diet mice, suggesting the activation of ER stress. Present results also revealed the beneficial effects of exercise on alleviating ER stress, as demonstrated by decreased GRP78 expression in mice from the AMLN18\u0026thinsp;+\u0026thinsp;E8 group compared to AMLN18 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, the GRP78 downstream pathways, including PERK, elF2α, ATF4, and CHOP expression were observed after AMLN diet with or without exercise intervention. The western blotting results indicated that the PERK and elF2α phosphorylation levels were highly increased by AMLN diet feeding, both at 14 and 18 weeks, where the longer the AMLN feeding period, the higher the level of pathway activation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). Furthermore, compared to ND diet mice, ATF4 and CHOP expression levels in the 14- and 18-week AMLN diet mice were also significantly increased associated with the upstream signaling PERK-eIF2α activation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). Notably, this AMLN-induced ER stress activation after 18 weeks was mitigated when mice were exercised for 8 weeks, as evidenced by decreased GRP78 and PERK-eIF2α-ATF4-CHOP pathway expression in AMLN18\u0026thinsp;+\u0026thinsp;E8 group mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExercise restores AMLN diet-induced mitochondrial dysfunction and UPRmt impairment at the later stages of MAFLD development\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo study the effect of ATF4 activated signaling pathway on mitochondrial function, this study examined mitochondrial morphology by using electron microscopy. The results showed that hepatocytes from AMLN diet mice exhibited accumulation of large lipid droplets, as well as mitochondrial swelling and cristae fragmentation, particularly in the AMLN18 group. These striking features of damaged mitochondria were significantly improved and exhibited normalized mitochondrial structure after an 8-week voluntary wheel running intervention in both AMLN14\u0026thinsp;+\u0026thinsp;E8 and AMLN18\u0026thinsp;+\u0026thinsp;E8 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, mitochondrial function indicators were tested, including mitochondrial respiration function, membrane potential, and mitochondrial respiratory chain complexes subunits. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C, compared to ND mice, 14- and 18-week AMLN diet mice exhibited decreased mitochondrial state III oxygen consumption and a reduced mitochondrial RCR, respectively. Moreover, the expression of mitochondrial respiratory chain complex subunits in AMLN diet mice were significantly declined (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Similar results were appeared to mitochondrial membrane potential (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), showing that 18 weeks of AMLN feeding significantly increased the proportion of mitochondria with lower membrane potential. In contrast to the negative effect of AMLN diet on mitochondrial function, exercise intervention was able to protect the mitochondria of hepatocytes in AMLN diet mice to some extent, especially the effect on mitochondrial respiratory function and OXPHOS protein expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003eGiven that exercise rescued the lipid accumulation-associated liver phenotype and improved mitochondrial function, hepatic UPRmt activation in this experimental model was further investigated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;J, western blot revealed that hepatic UPRmt component expression, including that of LONP1, HSP70, and HSP60 in the mitochondrial matrix was significantly elevated in 14-week AMLN diet mice when compared to the 14-week ND mice. Interestingly, compared to the AMLN14 group, mice on an AMLN diet for 18 weeks exhibited a sharp decline in UPRmt gene content and ClpP protein expression, highlighting a disconnect between hepatic lipid deposition and UPRmt activation because mitochondrial function is closely related with the degree of liver damage. In addition, the 8-week wheel running intervention significantly improved mitochondrial chaperone proteins and proteases and reversed the suppression of mtHSP70 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and mtHSP60 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) expression in AMLN18\u0026thinsp;+\u0026thinsp;E8 group mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePositive correlation between FGF21 secretion and the UPRmt activation level during AMLN diet and exercise intervention\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCirculating FGF21 is highly correlated with mitochondrial function. Further analysis of serum and hepatic FGF21 levels revealed that the serum FGF21 levels were markedly higher in the 14-week AMLN diet group than in the ND14 group, whereas these levels were dramatically reduced in the 18-week AMLN diet group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Thus, a apparent difference in serum and hepatic FGF21 level was observed between the 14- and 18-week AMLN groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), which agreed with previous UPRmt-related data. Next, to determine whether FGF21 secretion is involved in the regulation of the hepatic UPRmt mechanism during MAFLD progression, this study performed linear regression analysis between the circulating FGF21 level and the value of UPRmt protein expression in all samples. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;F, there were positive correlations between the serum FGF21 level and UPRmt protein content, including LONP1, mtHSP70, mtHSP60, and ClpP, suggesting a positive correlation between FGF21 secretion and the UPRmt activation level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOvernutrition and physical inactivity are precipitating factors that cannot be ignored in the occurrence of metabolic complications such as insulin resistance, type2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and MAFLD. MAFLD is a progressive disease, and excessive consumption of fat and/or sugar, particularly fructose, can cause hepatic steatosis and dyslipidemia. A diet constituting 40% fat, 20% fructose, and 2% cholesterol is widely known as the AMLN diet (Research diet, #D09100301) and is the preferred diet to induce many clinically relevant characteristics of NASH. Different periods of AMLN diet will result in different levels of lipid deposition in liver tissue, with consequent changes in oxidative and ER stress levels, as well as mitochondrial function [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Interestingly, a recent study revealed that mitochondrial FAO capacity in liver was decreased after 4weeks high fat diet (HFD) feeding, however, this effect was restored at 8 weeks [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], indicating that mitochondria function in liver tissue are highly plastic in diet-induced metabolic alteration. Exercise has also been confirmed to have significant effects on improving hepatic lipid metabolism, mitochondrial function, and the oxidative stress level. Zou et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] recently showed that moderate-intensity exercise induced a greater improvement in antioxidant ability and reduced stress status via regulation of ER stress pathway-related proteins. Therefore, this study first to compare the changes concerning lipid deposition, ER stress signaling pathway, UPRmt, and mitokine secretion after 14 and 18 weeks of AMLN diet intervention, which are two time points during the progression of MAFLD. On this basis, this study further explored the role of an 8-week voluntary wheel running intervention in regulating MAFLD occurrence and progression, focusing mainly on the molecular mechanism involved in the PERK-eIF2α-ATF4 aix and UPRmt. The relationship between UPRmt levels and FGF21 secretion was also investigated.\u003c/p\u003e \u003cp\u003eThe AMLN diet is a common dietary formula that induces liver damage in a manner similar to that observed in the human liver diseases. NAFLD development in three stages, including steatosis, steatohepatitis with fibrosis, and cirrhosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Trevaskis et al. reported that animals had AMLN diet for 12 weeks demonstrated increased body weight and liver fat content without fibrosis, but showed progression toward fibrosis when fed for a chronic 30-week period [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Here, mice were fed an AMLN diet for 14 and 18 weeks to establish a model of hepatic steatosis and prefibrosis and found that hepatic lipid deposition occurred significantly in both time periods. Moreover, these changes were associated with abnormal blood lipid levels in the 18-week AMLN diet animals, but not in the AMLN14 group. As the circulating fatty acids uptake is the major way for liver to acquire lipids [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], the absence of dyslipidemia in the 14-week AMLN diet mice may be a result of the increased fatty acid intake and FA storage as TGs in the liver, which normalize serum lipid levels. However, this regulatory mechanisms were disappeared in the 18-week AMLN diet animals. These data suggest that prolonged administration of an AMLN diet not only leads to an abnormal increase in blood lipids but also induces massive macro- and microsteatosis. This may even aggravate oxidative stress and damage mitochondrial function, which contribute to cellular damage and disease progression. In addition, numerous evidence supports that exercise exerts many of its metabolic benefits in liver, adipose tissue, and pancreas [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although the mechanisms by which exercise reduces liver fat are still largely unknown, the inner mechanism involves β-oxidation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], lipogenesis, and lipid export. In addition, an improvement in insulin resistance with exercise intervention is thought to reduce the uptake of circulating FFAs to the liver. Therefore, exercises can be helpful in orchestrating the lipid synthesis, export [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and their use as energy substrates. The results showed that liver lipid deposition in AMLN-fed animals at two stages improved significantly after an 8-week voluntary wheel running exercise intervention. However, this study failed to observe significantly decreased blood lipid levels in the early stages mediated by exercise, which may be due to the relatively lower blood lipid levels in the early stages than in the late.\u003c/p\u003e \u003cp\u003eTo further understand the alleviated AMLN diet-induced lipid deposition displayed by exercise intervention and with a focus on mitochondria-related metabolic mechanisms, the PERK-eIF2α-ATF4 signaling pathway was first examined, which is ER stress branch pathway and has been shown to be closely related to hepatic lipogenesis and steatosis regulation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The activation of this ER stress or UPR mainly relays on the luminal chaperone GRP78 activation. In terms of its downstream genes, a study showed that decreased hepatic lipogenesis and steatosis in HFD fed mice is accompanied by long-term dephosphorylation of PERK downstream: the elongation initiation factor eIF2α [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, the PERK-eIF2α-ATF4 signaling pathway was significantly awakened in both 14- and 18-week AMLN diet mice compared to the ND diet mice, which indicated that abnormal lipid accumulation often coincides with perturbed ER proteostasis in hepatocytes. The more severe the hepatic lipid deposition, the more obvious the PERK-eIF2α-ATF4 signaling pathway. Importantly, after 8 weeks of exercise intervention, the significant elevation of GRP78 and its downstream target gene expression was apparently improved in AMLN diet mice. These results indicant that this improvement induced by exercise in the ER stress signaling pathway is a beneficial adaptive mechanism. However, although ER stress responses were markedly activated by AMLN diets fed for different durations in this experiment and attenuated after exercise intervention, the underlying mechanisms may not be consistent. One clinical research showed that there is a variable degress of ER stress activation in the patients with NAFLD [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Prolonged phosphorylation of eIF-2α via activated PERK could specifically increase the downstream effectors ATF4 and CHOP, which enable the cell restore proteostasis. Conversely, there are other potential mechanisms induced by eIF-2α phosphorylation, several studies have shown that ER eIF2a phosphorylated downstream elements, which lead to the severeg oxidative stress by downregulating NFE2-related factor 2 (Nrf2) and depleting glutathione (GSH), eventually leading to apoptosis [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It has also been shown eIF2α phosphorylation is a key molecular event in mammalian UPRmt activation and is closely connection of the ISR [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Under stress conditions, the phosphorylation of eIF2 α triggers ATF4 activation, induces CHOP expression, and promotes UPRmt activation. Elevated transcript levels of ATF4 and CHOP in AMLN diet mice may be related to mitochondrial stress. Thus, next step was to investigate whether mitochondrial function and the UPRmt molecular pathways were altered under different durations of AMLN diet, and the influence mechanism of exercise on it.\u003c/p\u003e \u003cp\u003eThe UPRmt intimately associated with mitochondrial quality control system and plays an important role on protein homeostasis by stabilizing mitochondrial function against several pathologies. Although the UPRmt mechanism remains unclear, it promotes development during mild mitochondrial dysfunction. A recent study showed that the hepatic mitochondrial is closely related to the pathogenesis of NAFLD [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Results in this study demonstrated that mitochondrial membrane potential was significantly reduced in both 14- and 18-week AMLN diet mice, whereas mitochondrial respiration function, detected by the RCR, and OXPHOS gene expression were markedly decreased only in the 18-week AMLN diet group. These data suggest that mitochondria in excessive lipid deposition hepatocytes display an uncoupling of respiration from ATP production, indicating a reduced ability of ATP generation of mitochondria. However, this alteration did not occur at the early stages of hepatic lipid deposition. It is perhaps reasonable that mitochondrial function was disrupted during MAFLD development, but the changes in UPRmt-associated gene expression were unexpected. In terms of UPRmt, the expression of its downstream effectors (LONP1, mtHSP70, and mtHSP60) were elevated in 14 weeks AMLN diet mice in the presence of UPRmt compared to those fed an AMLN diet for 18 weeks in the absence of UPRmt. This suggests that UPRmt activation was triggered at the early stages of MAFLD progression and was impaired during higher-grade hepatic lipid deposition. It is speculated that the impairment in UPRmt associated with excessive lipid deposition could be due to imbalanced redox homeostasis or the heavily activated PERK-eIF2α-ATF4 pathway, which causes mitochondria to lose their ability to repair themselves. Indeed, a previous study reported that ATF4 negatively regulates TFAM expression and disrupts mitochondrial biogenesis and respiratory function in hepatocytes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Another possible explanation could be that dysfunctional mitochondrial protein import due to the prolonged AMLN diet induced the overall decline in mitochondrial function, which further impeded the process of transporting nuclear genome-encoded mitochondrial chaperones or proteases to the mitochondrial matrix. Taken together, these data point out the possibility that UPRmt may have dual effects: the 14-week AMLN diet associated with slight stress can initiate UPRmt and restore mitochondrial proteostasis, conversely, chronic UPRmt activation may be harmful to cell survival. It is worth noting that, the decreased expression of some UPRmt components in 18-week AMLN diet mice, such as Hsp60, Hsp70, ClpP and LONP1 were restored with exercise intervention, as shown in AMLN18\u0026thinsp;+\u0026thinsp;E8 group animals. These results highlighted the role of exercise in relieving mitochondrial stress and improving the UPRmt process, which coordinated to ensure mitochondrial function and viability. Future study is needed to probe the deep regulation mechanisms of UPRmt induction after exercise intervention.\u003c/p\u003e \u003cp\u003eMitochondria have been considered to crosstalk with other distant tissues through serum cytokine non-cell autonomously [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This study next focused on FGF21, a mitokine, mediated by the UPRmt-related eIF2α-ATF4-CHOP ER stress response axis, and is also a well-known exercise-induced hepatokine that stimulates lipolysis and fatty acid oxidation and suppresses lipogenesis in the liver [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Myung-Shik Lee\u0026rsquo;s group demonstrated that ATF4-dependent FGF21 induction was accompanied by impaired mitochondrial function, which contribute to suppress the diet-induced hepatic steatosis in animals [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The present study found that compared to ND mice, the FGF21 levels in serum and liver tissue were significantly enhanced after 14-week AMLN diet intervention, but the content was markedly reduced in 18-week AMLN diet mice, which is consistent with the previous UPRmt alteration. This observation suggests a relationship between UPRmt and FGF21 secretion. Yu-Wei Cheng recently reported that the ISR/ATF4-dependent induction of FGF21 was triggered by mitochondrial stress, which can mediate beneficial effects systemically [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Surprisingly, it found a positive correlation between UPRmt-related gene expression levels, including LONP1, HSP60, HSP70, and ClpP, and hepatic FGF21 levels as well as serum FGF21 content, suggesting that FGF21 is secreted by cells experiencing mitochondrial stress. Different from linear correlation, the relationship between mitochondrial perturbations-mediated UPRmt and hepatic lipid accumulation demonstrated a mitohormetic response. This mitohormesis concept suggests that mild mitochondrial stress initiates a diverse set of retrograde stress responses from mitonuclear interaction, whereas higher doses of stress can have harmful effects on cellular function [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably, this study also demonstrated that the decrease in circulating and hepatic FGF21 associated with excessive lipid accumulation was improved in response to exercise intervention. Based on the evidence presented above, this result may be due to the effect of exercise on regulating PERK-eIF2α-ATF4 or the UPRmt-related ISR/ATF4 pathway, both of which induce adaptive responses by triggering mitochondrial to nuclear communications, thereby recovering mitochondrial UPR and enabling mitochondrial self-healing. Previous studies have proposed an important role of cytokine factors as regulators of systemic energy metabolism. Secretion of mitokine during a specific period of disease progression has been proved to be a vital adaptive response that occurs [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In line with the concept of mitohormesis, exercise-mediated FGF21 secretion may be largely dependent on mitohormesis effect. Under normal conditions, long-term exercise can enhance cellular adaptations to an extent by triggering mitochondrial stress and mitonuclear communication. Moreover, in a state of severe lipid deposition in liver cells, exercise intervention could induce the mitohormetic response by improving the levels of oxidative stress and mitochondrial function, enhancing mitochondrial chaperones and proteases expression, and secreting mitokines.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStudy strengths and limitations\u003c/h2\u003e \u003cp\u003eThis study demonstrated how an additional 4 weeks on the AMLN diet can progress the development of steatohepatitis, as well as the effects it has on the proteins involved in ER stress signaling pathway, and UPRmt. Moreover, this study further explored the effectiveness of exercise to reduce the development of MAFLD and to normalize the protein content and activation of proteins involved in ER signaling and UPRmt. However, several limitations are existed in this study. First, this study is limited to animal experiments. Although serum FGF21 levels are showed closely related to liver UPRmt level here, whether it can be used for clinical diagnosis of MAFLD still needs further confirmation from clinical experiments. Second, owing to the complex of the pathogenesis of MAFLD, this study mainly focus on the mitochondira, and the depth mechanisms related to UPRmt, such as mitonuclear communication and the interaction of mitochondria with the endoplasmic reticulum during MAFLD development deserves further investigation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study have demonstrated alterations in the PERK-eIF2α-ATF4 signaling pathway, mitochondrial UPR gene expression, and mitokine secretion during different developmental stages of MAFLD pathogenesis. Different from the linear correlation between lipid deposition and the PERK branch of ER stress in the liver, the changes in reduced mitochondrial function, downregulated UPRmt component expression, and decreased FGF21 secretion were found at the later stages of fatty liver development, indicating that severe lipid deposition leads to impaired UPRmt through overactivating the ISR/ATF4 pathway, resulting in mitochondria losing their self-repair ability and miscommunication with the nucleus. Importantly, exercise intervention rescued the 18-week AMLN diet-induced hepatic phenotype and reversed mitochondrial function, UPRmt activation pattern, and FGF21 secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results highlight the pivotal role of exercise in regulating the UPRmt and its related mitokine secretion to delay MAFLD progression. This study not only suggests that serum FGF21 content may be an important indicator for clinical diagnosis of the occurrence and development of MAFLD, but also confirms the significance of implementing exercise interventions as the primary prevention of MAFLD in clinical practice.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eAbbreviations\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eFull name\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eAMLN\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eamylin liver NASH\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eATF4\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eactivating transcription factor 4\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eATF5\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eactivating transcription factor\u0026nbsp;5\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eAST\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003easpartate aminotransferase\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eALT\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ealanine aminotransferase\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eANOVE\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eanalysis of variance\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eBSA\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ebovine serum albumin\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eBiP\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ebinding protein\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eClpP\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ecasein lytic proteinase P\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eCHOP\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eC/EBP homologous protein\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eCHOL\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003echolesterol\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eCVD\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ecardiovascular disease\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eDAB\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eDiaminobenzidine\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eER\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eendoplasmic reticulum\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eETC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eelectron transport chain\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eeIF2\u0026alpha;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eeukaryotic initiation factor 2\u0026alpha;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eFFA\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003efree fatty acid\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eFAO\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003efatty acid oxidation\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eFGF21\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003efibroblast growth factor 21\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eFITC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003efluoresceine isothiocyanate\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eGAPDH\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eglyceraldehyde-3-phosphate dehydrogenase\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eGRP78\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eglucose regulated protein 78\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eGSH\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eglutathione\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eHSP60\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eheatshockprotein 60\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eGDF15\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003egrowth\u0026nbsp;differentiation\u0026nbsp;factor 15\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eHSP70\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eheatshockprotein 70\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eHDL-C\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ehigh-density lipoprotein cholesterol\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eHFD\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ehigh fat diet\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eISR\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eintegrated stress response\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eJC-1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003e5,5\u0026prime;,6,6\u0026prime;-Tetrachloro-1,1\u0026prime;,3,3\u0026prime;-tetraethyl-imidacarbocyanine\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eLONP1\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003elonpeptidase 1\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eLDL-C\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003elow-density lipoprotein cholesterol\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eLDs\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003elipid droplets\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eMAFLD\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003emetabolic associated fatty liver disease\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003emtHSP60\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003emitochondrial heatshockprotein 60\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003emtHSP70\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003emitochondrial heatshockprotein 70\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003emtDNA\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003emitochondrial DNA\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eMMP\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003emitochondrial membrane potential\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eNAFLD\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003enon-alcoholic fatty liver disease\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eND\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003enormal diet\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eNrf2\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eNFE2-related factor 2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eNASH\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003enon-alcoholic steatohepatitis\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eOXPHOS\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eoxidative phosphorylation\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003ep-eIF2\u0026alpha;\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ephospho-eukaryotic initiation factor 2\u0026alpha;\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003ePERK\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ePKR-like ER kinase\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003ep-PERK\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ephospho-PKR-like ER kinase\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003ePBS\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ephosphate buffer saline\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003ePE\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ephycoerythrin\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eROS\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ereactive oxygen species\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eRCR\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003erespiratory control rate\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eSEM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003estandard error of means\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eTAC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003etricarboxylic acid\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eTG\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003etriglyceride\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eTEM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003etransmission electron microscopy\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eT2DM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003ediabetes mellitus type2\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eTFAM\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eRecombinant Transcription Factor A, Mitochondrial\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eUPR\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eunfolded protein response\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eUPR\u003csup\u003eER\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003eendoplasmic reticulum unfolded protein reaction\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.722627737226276%\"\u003eVDAC\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"76.27737226277372%\"\u003erecombinant voltage dependent Anion channel protein\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Graphical abstract was created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan Zhang, Jiao Lu, Qiang Tang conceived and designed research; Xinmeng Yuan, Mengqi Xiang, Wen Sun, Ye Xu, Yaran Gao, Wanyu Feng performed experiments; Xinmeng Yuan, Mengqi Xiang, Yuan Zhang, Liumei Zhang analyzed data; Xinmeng Yuan, Mengqi Xiang, Wen Sun, Ye Xu interpreted results of experiments; Xingmeng Yuan, Jingyi Wu prepared figures; Xinmeng Yuan, Yuan Zhang drafted manuscript; Yuan Zhang, Jiao Lu, Qiang Tang edited and revised manuscript; Yuan Zhang, approved final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Youth Project of National Natural Science Foundation\u0026nbsp;of China (32000839), Qing Lan Project of Jiangsu Province of China\u0026nbsp;([2021]11), The National Key R\u0026amp;D Program of China (No. 2020YFC2007002), Graduate Research and Innovation Projects of Jiangsu Province (KYCX22_2247, KYCX22_2248, KYCX23_2363 and KYCX23_2380), The Innovation and Entrepreneurship Training Program for Undergraduates of Jiangsu Province of China (202310330011Z).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol of animal experiments was complied with the Guide for National Institute of Health guidelines (NIH Publications No. 8023, revised 1978) and approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Approval No. GZRDW-2020-03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFriedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24(7):908\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakaki A, Kawai D, Yamamoto K. Multiple hits, including oxidative stress, as pathogenesis and treatment target in non-alcoholic steatohepatitis (NASH). Int J Mol Sci. 2013;14(10):20704\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjoolabady A, Kaplowitz N, Lebeaupin C, et al. Endoplasmic reticulum stress liver Dis Hepatol. 2023;77(2):619\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrano F, Wang X, Bertolotti A et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000;287(5453):664\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarciniak SJ, Chambers JE, Ron D. Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov. 2022;21(2):115\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilly JA, Young SK, Stevens JL, Masuoka HC, Wek RC. CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis. Mol Biol Cell. 2015;26(12):2190\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansouri A, Gattolliat CH, Asselah T. Mitochondrial Dysfunction and Signaling. Chronic Liver Dis Gastroenterol. 2018;155(3):629\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma AK, Sharma A, Subramaniyam N, Gandhi CR. Augmenter of liver regeneration. Mitochondrial function steatohepatitis J Hepatol. 2022;77(5):1410\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi HS. Implications of Mitochondrial Unfolded Protein Response and Mitokines: A Perspective on Fatty Liver Diseases. Endocrinol Metab (Seoul). 2019;34(1):39\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKleinridders A, Lauritzen HP, Ussar S, et al. Leptin regulation of Hsp60 impacts hypothalamic insulin signaling. J Clin Invest. 2013;123(11):4667\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee HY, Nga HT, Tian J, Yi HS. Mitochondrial Metabolic Signatures Hepatocellular Carcinoma Cells. 2021;10(8):1901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe L, Deng L, Zhang Q, et al. Diagnostic Value of CK-18, FGF-21, and Related Biomarker Panel in Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis. Biomed Res Int. 2017;2017:9729107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker B, Li H, Long X, Rye KA, Ong KL. Fibroblast growth factor 21 in non-alcoholic fatty liver disease.Metabolism. 2019;101:153994.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Xu Y, Hu Y, Wang G. The role of fibroblast growth factor 21 in the pathogenesis of non-alcoholic fatty liver disease and implications for therapy.Metabolism. 2015;64(3):380\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTillman EJ, Rolph T. FGF21: An Emerging Therapeutic Target for Non-Alcoholic Steatohepatitis and Related Metabolic Diseases.Front Endocrinol. (Lausanne). 2020;11:601290.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Kim KH, Kim HK et al. Fibroblast growth factor 21 participates in adaptation to endoplasmic reticulum stress and attenuates obesity-induced hepatic metabolic stress.Diabetologia. 2015;58(4):809\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Kwak D, Lu Z, et al. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling. Hypertension. 2014;64(4):738\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo R, Liong EC, So KF, Fung ML, Tipoe GL. Beneficial mechanisms of aerobic exercise on hepatic lipid metabolism in non-alcoholic fatty liver disease. Hepatobiliary Pancreat Dis Int. 2015;14(2):139\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePassos E, Ascens\u0026atilde;o A, Martins MJ, Magalh\u0026atilde;es J. Endoplasmic Reticulum Stress Response in Non-alcoholic Steatohepatitis: The Possible Role of. Phys Exerc Metabolism. 2015;64(7):780\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAscens\u0026atilde;o A, Martins MJ, Santos-Alves E et al. Modulation of hepatic redox status and mitochondrial metabolism by exercise: therapeutic strategy for liver diseases.Mitochondrion. 2013;13(6):862\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Liu Y, Liu X, et al. Exercise and Metformin Intervention Prevents Lipotoxicity-Induced Hepatocyte Apoptosis by Alleviating Oxidative and ER Stress and Activating the AMPK/Nrf2/HO-1 Signaling Pathway in db/db Mice. Oxid Med Cell Longev. 2022;2022:2297268.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Sun T, Yu J, Li S, Gong L, Zhang Y. FGF21: A Sharp Weapon in the Process of Exercise to Improve NAFLD.Front Biosci (Landmark Ed). 2023;28(12):351.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi XH, Liu LZ, Chen L, et al. Aerobic exercise regulates FGF21 and NLRP3 inflammasome-mediated pyroptosis and inhibits atherosclerosis in mice. PLoS ONE. 2022;17(8):e0273527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerakakis N, Joshi A, Peradze N, et al. The Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator CHS-131 Improves Liver Histopathology and Metabolism in a Mouse Model of Obesity and Nonalcoholic Steatohepatitis. Hepatol Commun. 2020;4(9):1302\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManzanares G, Brito-da-Silva G, Gandra PG. Voluntary wheel running: patterns and physiological effects in mice.Braz. J Med Biol Res. 2018;52(1):e7830.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKleiner DE, Brunt EM, Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005;41(6):1313\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHynes J, Swiss RL, Will Y. High-Throughput Analysis of Mitochondrial Oxygen Consumption. Methods Mol Biol. 2018;1782:71\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarneros D, L\u0026oacute;pez-Lluch G, Bustos M. Physiopathology of Lifestyle Interventions in Non-Alcoholic Fatty Liver Disease (NAFLD).Nutrients. 2020;12(11):3472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Tian R, She Z, Cai J, Li H. Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease [published correction appears in Free. Radic Biol Med 2020;:] Free Radic Biol Med. 2020;152:116\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou Y, Qi Z. Understanding the Role of Exercise in Nonalcoholic Fatty Liver Disease. ERS-Linked Mol Pathways Mediators Inflamm. 2020;2020:6412916.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Li X, Liu Z et al. Moderate Treadmill Exercise Alleviates NAFLD by Regulating the Biogenesis and Autophagy of Lipid Droplet.Nutrients. 2022;14(22):4910.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrevaskis JL, Griffin PS, Wittmer C, et al. Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice. Am J Physiol Gastrointest Liver Physiol. 2012;302(8):G762\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClapper JR, Hendricks MD, Gu G, et al. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment. Am J Physiol Gastrointest Liver Physiol. 2013;305(7):G483\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIpsen DH, Lykkesfeldt J, Tveden-Nyborg P. Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease. Cell Mol Life Sci. 2018;75(18):3313\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerr\u0026eacute; P, Foufelle F. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.Diabetes Obes Metab. 2010;12 Suppl 2:83\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebeaupin C, Vall\u0026eacute;e D, Hazari Y, Hetz C, Chevet E, Bailly-Maitre B. Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol. 2018;69(4):927\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelber A, Haynes CM. UPRmt regulation and output: a stress response mediated by mitochondrial-nuclear communication. Cell Res. 2018;28(3):281\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuri P, Mirshahi F, Cheung O et al. Activation and dysregulation of the unfolded protein response in nonalcoholic fatty liver disease.Gastroenterology. 2008;134(2):568\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshraf NU, Sheikh TA. Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of Non-alcoholic fatty liver disease. Free Radic Res. 2015;49(12):1405\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuchs CD, Claudel T, Scharnagl H, Stojakovic T, Trauner M. FXR controls CHOP expression in steatohepatitis. FEBS Lett. 2017;591(20):3360\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong B, Scheuner D, Ron D, Pennathur S, Kaufman RJ. Chop deletion reduces oxidative stress, improves beta cell function, and promotes cell survival in multiple mouse models of diabetes. J Clin Invest. 2008;118(10):3378\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDerisbourg MJ, Hartman MD, Denzel MS, Perspective. Modulating the integrated stress response to slow aging and ameliorate age-related pathology. Nat Aging. 2021;1(9):760\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFromenty B, Roden M. Mitochondrial alterations in fatty liver diseases. J Hepatol. 2023;78(2):415\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMottis A, Herzig S, Auwerx J. Mitocellular communication: Shaping health and disease.Science. 2019;366(6467):827\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlippo KH, Potthoff MJ. Metabolic Messengers: FGF21.Nat Metab. 2021;3(3):309\u0026ndash;317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewis JE, Ebling FJP, Samms RJ, Tsintzas K. Going Back to the Biology of FGF21: New Insights. Trends Endocrinol Metab. 2019;30(8):491\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim KH, Jeong YT, Oh H, et al. Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine. Nat Med. 2013;19(1):83\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng YW, Liu J, Finkel T. Mitohormesis Cell Metab. 2023;35(11):1872\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026aacute;rcena C, Mayoral P, Quir\u0026oacute;s PM. Mitohormesis, an Antiaging Paradigm. Int Rev Cell Mol Biol. 2018;340:35\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi HS, Chang JY, Shong M. The mitochondrial unfolded protein response and mitohormesis: a perspective on metabolic diseases. J Mol Endocrinol. 2018;61(3):R91\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mitochondrial unfolded protein response, hepatic lipid accumulation, exercise intervention, AMLN diet, MAFLD progression","lastPublishedDoi":"10.21203/rs.3.rs-4446826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4446826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA high-calorie diet and lack of exercise are the primary risk factors contributing to metabolic associated fatty liver disease (MAFLD) initiation and progression. Although mitochondrial dysfunction in MAFLD has been widely recognized, the precise molecular mechanisms of mitochondrial function alteration during MAFLD development remain to be fully elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of sixty male C57/black mice were maintained on a normal or amylin liver NASH (AMLN) diet for 6 and 10 weeks. Half of the AMLN diet mice were then subjected to 8 weeks of voluntary wheel running with an AMLN diet persistently, while the other AMLN diet mice were sedentary until 14 and 18 weeks. After the experimental intervention, the mice were sacrificed under anesthesia, blood and liver tissue were collected for further analysis. Changes in biochemical parameters, histopathology, lipid accumulation, endoplasmic reticulum stress, mitochondrial function and mitochondrial unfolded protein response-related proteins were assessed and correlation analysis of serum FGF21 and mitochondrial unfolded genes expression was also performed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that the hepatic lipid deposition and PERK-eIF2α-ATF4 pathway activation were significant increased with prolonged duration of AMLN diet. However, expression of mitochondrial unfolded protein response (UPRmt) genes, such as LONP1, HSP60, and HSP70, as well as mitokine FGF21 secretion were significantly enhanced in the 14-week AMLN diet mice, but were markedly reduced with the excessive lipid deposition induced by the 18-week AMLN diet. In addition, there is a significant positive correlation between circulating FGF21 and the amount of mitochondrial unfolded genes expression during MAFLD progression. Moreover, exercise intervention significantly rescued the hepatic phenotype through improving mitochondrial function, regulating UPRmt activation pattern and increasing FGF21 secretion.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eDuring the development of AMLN diet-induced MAFLD, the relationship between the degree of lipid deposition and mitochondrial function is not a linear model of negatively correlation. Instead, mitochondria could experience self-remodeling at the earlier stage of lipid accumulation, then lose their self-repair ability due to lipid overload. Exercise effectively prevents excessive lipid deposition, through regulating UPRmt, remodeling mitochondrial protein homeostasis and promoting the secretion of mitokine FGF21, which plays an essential role in delaying the MAFLD occurrence and progression.\u003c/p\u003e","manuscriptTitle":"Exercise protects against AMLN diet-induced lipid deposition in hepatocytes during MAFLD progression by regulating the UPRmt and FGF21 secretion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 11:44:16","doi":"10.21203/rs.3.rs-4446826/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-06-15T12:07:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163100532503447809747351120050292602992","date":"2024-06-04T15:49:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-01T13:09:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185207720271220375596047558509488430084","date":"2024-05-23T05:20:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-22T21:41:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-20T07:40:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-20T06:20:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lipids in Health and Disease","date":"2024-05-20T05:44:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lipids-in-health-and-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lhad","sideBox":"Learn more about [Lipids in Health and Disease](http://lipidworld.biomedcentral.com/)","snPcode":"12944","submissionUrl":"https://submission.nature.com/new-submission/12944/3","title":"Lipids in Health and Disease","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4194c638-64d0-445d-a0fa-eb6d3ddc56e3","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-30T11:44:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-30 11:44:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4446826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4446826","identity":"rs-4446826","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}