Aerobic exercise improved liver steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway

Aerobic exercise improved liver steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway | 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 Aerobic exercise improved liver steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway Baoai Wu, Zhibin Zhang, Chong Xu, Jinfeng Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6207434/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract MicroRNA-34a (miR-34a) was closely associated with liver steatosis. However, the link between changes in miR-34a and the progression of liver steatosis remained unclear. In the work, sixty mice were randomly and equally selected into six groups: normal control group (NC), normal exercise group (NE), high-fat diet group (HFD), high-fat diet plus exercise group (HFE), miR-34a overexpression group (OE), and miR-34a overexpression plus exercise group (OEE). Live morphology showed that treadmill exercise intervention for 8 weeks reduced high-fat diet-induced liver steatosis in mice. 8-week treadmill exercise directly decreased mir-34a expression of mice in HFD group, confirmed in OE group. More, treadmill exercise enhanced the expression of PPARα and SIRT1, thereby affecting the downstream hepatic steatosis-associated target genes, including CPT1, CPT2, SLC27A4, SLC27A1, in addition to activating the expression of the central metabolic sensor AMPK. Following aerobic exercise intervention, miR-34a was upregulated, thereby affecting the expression of genes associated with hepatic steatosis, and this mechanism was confirmed in miR-34a overexpression mice. This study contributed to our understanding of the pathogenesis of hepatic steatosis and may provide new therapeutic approaches. Aerobic exercise liver steatosis miR-34a PPARα SIRT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Liver steatosis was the appearance of fat drips in the cytoplasm of liver cells whose long-term development may cause hepatocyte necrosis, liver fibrosis, steatohepatitis which may develop into liver cirrhosis and hepatocellular carcinoma 1 , 2 . In recent years, with improving of the people's living standard and decreasing exercise time, liver steatosis has become a serious public health problem in the world, affecting 30–40% of the total population which was still increasing 3 . However, the mechanism of hepatic steatosis was extremely limited and has not been determined. Therefore, the investigation of the molecular mechanisms underlying the progression of hepatic steatosis was urgent in order to obtain effective therapeutic approaches. MicroRNA (miRNA) was a small single-stranded non-coding RNA of about 19–25 nucleotides in length, which may regulate the expression of its target gene through reverse complementary pairing with the partial sequence of the 3' untranslated region (3'UTR) of its target gene 4 . In many diseases, miRNAs have received increasing attention due to their imbalance and their potential as both diagnostic and therapeutic targets 5 , 6 . miR-34a, a specific miRNA, has been reported to show a high level of expression in the hepatic disease patients 7 – 9 . Besides, studies have reported that inhibition of miR-34a may increase the levels of its downstream targets, including peroxisome proliferator-activated receptor-α (PPARα) and Sirtuin protein family-Sirtuin1 (SIRT1) that were related to fatty acid oxidation thereby alleviating liver steatosis 10 . As a member of the nuclear receptor superfamily, PPARα bound to the ligand form a heterodimer with the retinoid X receptor (RXR), and then the heterodimer bound to the PPAR response element of the target genes promoting the transcription of its downstream genes related to fatty acid oxidation 11 . So PPARα had great effects on the regulation of hepatic lipid metabolism. The key target genes of PPARα downstream that were involved in fatty acid oxidation and fatty acid transport consisted of solute carrier family, including SLC27A and CPTs. SIRT1 was an essential member of the Sirtuin protein family, and also was a NAD-dependent deacetylase, closely associating with hepatic steatosis 12 . AMP kinase (AMPK), a known regulator of energy metabolism, had a significant role in the development and progression of metabolic diseases, and SIRT1 may activate its activity thus promoting its phosphorylation 13 . Then phosphorylation of AMPK up-regulated β-fatty acid oxidation gene and down-regulated fatty acid transport-related proteins, thereby reducing liver steatosis 14 . Therefore, the SIRT1-AMPK phosphorylation signaling pathway functions in the regulation of lipid metabolic homeostasis and may be a new therapeutic target for hepatic steatosis. Studies have reported that aerobic exercise was a low-cost, low-risk intervention that may effectively reduce the occurrence and development of liver steatosis and has been adopted by most people 15 , 16 . The purpose of this study was to determine whether aerobic exercise may improve liver steatosis by adjusting the miR-34a-PPARα/SIRT1 signal pathway. 2. Materials and methods 2.1 Ethical Permission All operations in this experiment were performed under the rules of research ethics of Shanxi University with approval number of CIRP-IACUC-(R)2019014, in line with local and international animal ethics guide and with the utmost efforts to minimize animal suffering. 2.2 Animal Sixty SPF-grade C57BL/6J male mice (8 week old), weighing 21.4 ± 0.92 g, were bought from Beijing Life River Laboratory Animal Technology Co. (Animal license NO. SCXK (Beijing) 2016-0006). After adaptive feeding for one week, mice were randomly and equally selected into six groups: normal control group (NC), normal exercise group (NE), miR-34a overexpression group (OE), miR-34a overexpression plus exercise group (OEE), high-fat diet group (HFD, 60% fat, 20% carbohydrate, and 20% protein), and high-fat diet plus exercise group (HFE). All mice were housed under standard laboratory conditions with 12/12 dark light cycles, 20–26 ºC, 40–60% relative humidity, and free access to food and water. Mice body weights were recorded weekly. After the last treadmill exercise, mice were fasted overnight, anesthetized intraperitoneally with a dose of 80 mg/kg pentobarbital sodium, and then executed. Blood samples were collected in EDTA-containing tube, and centrifuged at 4 ºC and 3500 rpm for 15 min, then the supernatant was transferred to a new tube and stored at -80 ºC. The livers were rapidly taken out and washed with cold phosphate-buffered saline (PBS), then some of the liver tissues were cut into the fixative and the remaining liver tissues were saved at -80 ºC. The protocol was shown in Fig. 1 . After adaptive feeding, TBG promoter-driven overexpression-miR-34a adeno-associated viral vector TBG (AAV9) (100 µL, 1.95 × 1012 vg/ml) (Sangon Biotech, Shanghai, China) was injected into the tail vein to specifically promote miR-34a expression in the mouse liver. After 4 weeks of a normal or high-fat diet, mice in the NE, OEE, and HFE groups performed 1 week of adaptive treadmill exercise, followed by 8 weeks of treadmill exercise starting at 7 pm every day. 2.3 Triglyceride assay Triglycerides (TG) was measured at 510 nm on a spectrophotometer (UV-6100s, Mapada, Shanghai, China) by using the kit and strictly following the instructions of the kit. 2.4 Histologic analysis Liver tissue was first fixed in 10% neutral buffered formalin and then stained with hematoxylin-eosin (H&E) to view the level of hepatic steatosis by the microscope. 2.5 Real-time quantitative PCR Total RNA was extracted from livers using a spin column animal total RNA purification kit (Sangon Biotech, Shanghai, China) and was reverse-transcribed using M-MuLV first-strand cDNA synthesis kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. Real-time quantitative PCR (qPCR) was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq Ⅱ mix (TaKaRa, Dalian, China). Target gene expression was calculated by the 2^-△△CT method. Gene-specific primers were presented in Table 1 , with internal control of GAPDH. 2.6 Western blot The liver tissues were lysed in RIPA lysis buffer, and the protein concentration was determined by the BCA method (Beyotime, Shanghai, China). Proteins of 40 µg were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then cropped and transferred to PVDF membranes, blocked for 1 hour with 5% nonfat milk. After overnight incubation with primary antibody (1:1000 anti-PPARα, anti-SIRT1, anti-AMPK, anti-CPT1, anti-CPT2, anti-SLC27A1, anti-SLC27A4) (Proteintech Group Inc, Wuhan, China), membranes were washed and incubated with HRP-conjugated secondary antibody (1:5000) (Boster Biotech, Wuhan, China). Then the ECL detection kit (Applygen Technologies Inc, Beijing, China) was used for detection, and imaging was performed on a chemiluminescence meter (ChemiDoc XRS+, Bio-Rad, USA). 2.7 Statistical analysis All data are shown as mean ± SD ( n = 10). Statistical analysis was performed using SPSS 25.0 software. One-way ANOVA was used for multiple comparisons. A two-tailed t-test was used for comparison between two groups. p < 0.05 was considered to be statistically significant. 3. Results 3.1 Treadmill exercise alleviated liver steatosis in mice Morphological observation of the liver was showed in Fig. 2 A. There were no obvious changes in the liver of NC and NE mice, which was fresh red in color with a smooth envelope and clear edges. In The liver of the HFD group mice was yellowish-brown with blunted edges, soft texture, and a greasy surface. After treadmill exercise, the livers of mice in the HFE group recovered to a state close to that of the NC group. H&E staining of liver tissue may directly reflect whether the liver steatosis occurred. As shown in Fig. 2 B, there were no significant changes in the NE group and NC mice, while the HFD group showed a significant increase in the number and size of hepatic lipid droplets with inflammatory cell infiltration. In contrast, mice in the HFE group showed a reduced number of hepatic lipid droplets, neatly aligned hepatocytes, and significantly improved hepatic steatosis. Besides, the liver weight (Fig. 2 C) and the ratio of liver weight to body weight (Fig. 2 D) of mice in the HFD group were significantly increased to (1.53 ± 0.25) g ( p < 0.05) and (4.87 ± 0.53)% ( p < 0.05) from (1.00 ± 0.10) g and (3.3 ± 0.24)% in the NC group, respectively, but after aerobic exercise intervention moderately rescued to (1.23 ± 0.15) g ( p < 0.05 vs HFD group) and (3.60 ± 0.28)% ( p < 0.05 vs HFD group) in the HFE group, correspondingly. One of the major features of liver steatosis was the significant change of liver TG levels, so liver TG levels were detected (Fig. 2 E). TG levels in the HFD group was significantly increased to 67.67 ± 2.52 (mg/g liver) ( p < 005) compared with the NC group (28.67 ± 2.08), but after aerobic exercise intervention restored to 55.3 ± 4.53 (mg/g liver) ( p < 005 vs HFD group) in the HFE group. These observations suggested that high-fat diet-induced hepatic steatosis may be attenuated by treadmill exercise. 3.2 Treadmill exercise inhibited miR-34a expression Next, the effects of treadmill exercise on hepatic miR-34a expression were examined, and the results were showed in Fig. 3 A. miR-34a expression levels were not significantly changed in NE group mice compared with NC group mice, whereas miR-34a expression was significantly up-regulated to 2.04 ± 0.08 ( p < 0.05) compared with the NC group in the liver of HFD mice, and treadmill exercise reversed this rise to 1.39 ± 0.05 ( p < 0.05). This result revealed that treadmill training could improve liver steatosis by inhibiting miR-34a. To further clarify the effect of aerobic exercise on miR-34a, we injected miR-34a adenovirus vector into mice through the tail vein and performed treadmill intervention. the miR-34a level in the liver of over-expression mice was markedly enhanced to 2.98 ± 0.20 ( p < 0.05) compared with the NC group, but which was significantly restored to 1.78 ± 0.14 ( p < 0.05) by aerobic exercise intervention compared with the the Overexpression of miR-34a group, implying that treadmill exercise may down-regulate the expression of miR-34a. 3.3 Changes in PPARα and its downstream target genes caused by treadmill exercise PPARα played an important role in the regulation of hepatic lipid metabolism. The protein expression levels (Fig. 3 B) and mRNA (Fig. 3 C) of PPARα in the NE group were basically the same as the NC group, and were decreased to 0.66 ± 0.07 ( p < 0.05) and 0.69 ± 0.09 ( p < 0.05) in the HFD group, respectively, while were increased to levels comparable to NC group with 0.96 ± 0.06 ( p < 0.05) and 1.21 ± 0.05 ( p < 0.05) after treadmill exercise. To gain insight into the possible involvement of the miR-34a-PPARα pathway in hepatic steatosis alleviation induced by treadmill exercise, several key target genes of PPARα downstream that involved in fatty acid oxidation and fatty acid transport were examined by qPCR and western blot, including CPT1, CPT2, SLC27A1, and SLC27A4, and the results were shown in Fig. 3 D. The expression levels of these downstream target genes in NE group mice were similar to those in NC group mice, while the expression of CPT1 and CPT2 was lower in HFD mice than in NC mice (0.53 ± 0.05 vs. 1.00 ± 0.09, p < 0.05 and 0.31 ± 0.04 vs. 1.00 ± 0.07, p < 0.05) and the expression of SLC27A1 and SLC27A4 was higher (1.8 ± 0.13 vs. 1.00 ± 0.08, p < 0.05 and 1.68 ± 0.15 vs. 1.00 ± 0.09, p < 0.05), finally, in the HFE group, the expression of CPT1 and CPT2 was reversed up to 0.81 ± 0.08 and 0.55 ± 0.04 ( p < 0.05), and the expression of SLC27A1 and SLC27A4 was reversed down to 1.26 ± 0.11 and 1.43 ± 0.12 ( p < 0.05). Additionally, the mRNA changes of all the above-mentioned genes were confirmed by qPCR (Fig. 3 E). Thus, the change in PPARα and its target genes could explain the alleviating effect of aerobic exercise on HFD-induced liver steatosis. 3.4 SIRT1 was involved in the miR-34a-induced lipid metabolism pathway As one of miR-34a downstream target gene, SIRT1 was able to modulate the activity of AMP kinase (AMPK), a regulator of energy metabolism. The increase of the AMPK phosphorylation level was closely related to the enhancement of lipid metabolism. Then SIRT1 and AMPK were examined, and Fig. 3FG showed the results. SIRT1 protein levels and mRNA in NE mice were observed no significant changes, and exhibited a significant decrease in mice fed a high-fat diet were found compared to NC mice, with 0.52 ± 0.04 and 0.55 ± 0.05, respectively ( p <0.05). Then, this reduction was again up-regulated to 0.82 ± 0.08 and 1.2 ± 0.09, respectively ( p < 0.05 vs HFD group) after aerobic exercise intervention. As was shown in Fig. 3 H, the level of p-AMPKα (Thr-172) was lower in the HFD group (0.35 ± 0.10, p <0.05) than that in the NC group, while in the HFE group treadmill training partially abolished the decrease of HFD-induced p-AMPK (thr-172) expression (0.61 ± 0.09, p <0.05). Thus, these observations indicated that SIRT1 may involve in the miR-34a-induced lipid metabolism pathway and aerobic exercise may also improve liver steatosis via the miR-34a-SIRT1-AMPK pathway. 3.5 Aerobic exercise improved liver steatosis via miR-34a Changes in miR-34a expression showed a profound regulation of the PPARα/SIRT1-AMPK signaling pathway, which is closely associated with disorders of hepatic lipid metabolism. This relationship prompted us to further investigate whether the PPARα/SIRT1-AMPK pathway was involved in miR-34a-regulated hepatic steatosis and its associated pathology. This was investigated by injecting miR-34a adenovirus vector into mice through the tail vein and then performing 5 weeks of treadmill intervention. As expected, it was observed that treadmill exercise up-regulated the protein expression levels and mRNA of PPARα which both were decreased by miR-34a over-expression (Fig. 4AB). Also the protein expression levels and mRNA of CPT1 and CPT2 were significantly reduced in liver samples from miR-34a overexpressing mice, and those of SLC27A1 and SLC27A4 were significantly increased, and these changes were altered after the exercise intervention (Fig. 4CD). It was found in Fig. 4EF that SIRT1 showed the same trend as PPARα. In addition, AMPK phosphorylation levels (Fig. 4 G) were reduced in miR-34a overexpressing mice, and the reduction was significantly reversed after 5 weeks of aerobic exercise. In a word, PPARα/SIRT1-AMPK pathway was involved in miR-34a-regulated hepatic steatosis. The regulatory role of miR-34a-PPARα/SIRT1 in liver steatosis was summarized in Fig. 5 . 4. Discussion In the work, after 8 weeks of treadmill exercise, mice in HFD group showed significant improvement in liver weight and liver pathology. Recently, many studies have revealed that miR-34a was a specifically regulated miRNA in liver diseases and has a significant effect on the development and process of hepatic steatosis 10 . The expression level of miR-34a was high in patients with nonalcoholic fatty liver1 7,18 as well as in animal models of fatty liver 19 , 20 . Similarly, our hepatic steatosis mouse model showed increased hepatic expression of miR-34a, which was in agreement with previous findings. Numerous studies have shown that the expression of miRNA may be affected by aerobic exercise 21 . miR-34a over-expression disrupted lipid normal metabolism and may also be another critical marker for exercise to improve hepatic steatosis. In the work, we showed that treadmill exercise inhibited miR-34a over-expression in HFD mice to reduce hepatic lipid droplet accumulation. Consistent with the morphological findings, aerobic exercise also reduced the level of TG in the liver tissue of mice in the HFE group. Besides, the liver weight/body weight ratio was also markedly reduced in the HFE group when compared to the HFD group. In a word, aerobic exercise inhibited increase of miR-34a, TG, and the liver weight in HFD mice. miR-34a can potentially post-transcriptionally regulate PPARα through specific binding with the PPARα wild-type luciferase construct 10 . By observing the mRNA and protein levels of PPARα in mouse liver, we found that PPARα mRNA and protein levels were significantly lower in the HFD group compared to the NC group, and 8 weeks of aerobic exercise reversed this decline. As one of the direct downstream target genes of miR-34a, SIRT1 has taken a pivotal role in regulating cell lipid metabolism and reducing inflammation 22 – 24 . We found that the mRNA and protein levels of SIRT1 were significantly increased in the liver of the HFE group after aerobic exercise. AMPK was an AMP-dependent protein kinase which was widely expressed in various organs related to metabolism and can be activated by exercise, also being a biological energy key molecule of metabolic regulation 11 , 12 . Our research showed that treadmill exercise increased the expression of SIRT1 through inhibiting miR-34a, thereby stimulating AMPK signals which was to monitor the fuel meter of cell energy status and activate the gene that was related to fatty acid oxidation. Therefore, we supposed that miR-34a-mediated modulation of hepatic lipid metabolism by PPARα and SIRT1-AMPK pathways may be two independent regulatory mechanisms in fatty liver. CPT1 catalyzed the esterification of long-chain acyl-CoA into L-carnitine which was used as a carrier to transfer long-chain fatty acids from outside the mitochondria to the inside of the mitochondria in the form of an acyl-carnitine conjugate. Then acyl-carnitine conjugates were transformed back to acyl-CoA esters by CPT2 in the mitochondria to promote the oxidation and decomposition of fatty acids 25 . In the work, it was found that both the activation of AMPK and the rise in PPARα expression increased the levels of CPT1 and CPT2, leading to increased transport of fatty acids into the mitochondria, and facilitated the oxidation of mitochondrial palmitoyl-CoA. These observations helped us to understand the mechanism of enhanced fatty acid oxidation after aerobic exercise inhibits miR-34a, with AMPK as a medium for the increased hepatic oxidation of miR-34a after aerobic exercise inhibition. SLC27 was an integral transmembrane protein that promoted the uptake of long-chain fatty acids into cells and also served as a key player in lipid metabolism 26 , 27 . Our study showed that the mRNA and protein levels of SLC27A1 and SLC27A4 were significantly elevated in HFD mice, which was significantly reversed after aerobic exercise. This result suggested that aerobic exercise may improve hepatic steatosis by affecting fatty acid transporter proteins. In conclusion, our research showed that aerobic exercise down-regulated miR-34a, and up-regulated the levels of PPARα and SIRT1 which then affected fatty acid oxidation-related genes CPT1, CPT2, and fatty acid transportation-related genes SLC27A1, SLC27A4 transcription, thereby ameliorated hepatic steatosis. Notably, SIRT1 influenced downstream target genes after activation of the AMPK pathway. This study provided new insights into the pathogenesis of liver steatosis and had the potential to suggest new approaches to treat liver steatosis at the miRNA level. Declarations Ethical Approval All animal experiments were approved by the Ethics Committee of Shanxi University (approval number [SXULL2021055]). Declaration of competing interest The authors declare no competing interests. Funding This research was supported by Fundamental Research Program of Shanxi Province (NO. 202203021221022). Author Contribution B.AW.: Project administration, funding acquisition, conception and design of the study, experimental instruction, revising the article, and final approval of the version to be submitted. Z.B.Z. and C.X.: Conception and design of the study, acquisition, analysis and interpretation of data, drafting the article, and final approval of the version to be submitted. J.F.Z.: Conception and design of the study, experimental instruction, and final approval of the version to be submitted. Acknowledgements The authors thank the Institute of Biological Sciences Shanxi University for professional technical assistance and the China Institute of Radiation Protection for animal care. References Castro RE, Ferreira DM, Afonso MB, et al. miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease. J. Hepatol. 2013;58:119–125. Wang H, Wang L, Li Y, et al. The HIF-2α/PPARα pathway is essential for liraglutide-alleviated, lipid-induced hepatic steatosis. Biomed. Pharmacother. 2021;140:111778. Idilman IS, Ozdeniz I, Karcaaltincaba M. Hepatic Steatosis: etiology, patterns, and quantification, semin. Ultrasound CT. MR. 2016;37:501–510. Churov A, Summerhill V, GrechKD A, et al. MicroRNAs as potential biomarkers in atherosclerosis. Int. J. Mol. Sci. 2019;20:5547. 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J. Lipid Res. 2009;50:491–500. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6207434","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434684681,"identity":"372e1623-029c-4bad-a86d-149ebd4cde29","order_by":0,"name":"Baoai Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYDACCTBpw8PP30CaljQZyRkHSNNy2MagIYFIHQa3mx9/5m07z2PAcIDxw8ccYrTcOWYmObPtNo85cwOz5MxtRGgxu5FgxvARqMWy4QAbMy9xWtI/f0hsO8djcCCBaC05BhIf2w6QoMX+zpkyyRnnknkkZxxsJs4vkrPbN3/mKbOz5+dvPvjhIzFakABjA2nqR8EoGAWjYBTgBgDz+Dcs7qumBwAAAABJRU5ErkJggg==","orcid":"","institution":"Shanxi University","correspondingAuthor":true,"prefix":"","firstName":"Baoai","middleName":"","lastName":"Wu","suffix":""},{"id":434684682,"identity":"3d516bb5-328b-479a-969b-ff8c739156a6","order_by":1,"name":"Zhibin Zhang","email":"","orcid":"","institution":"Shanxi University","correspondingAuthor":false,"prefix":"","firstName":"Zhibin","middleName":"","lastName":"Zhang","suffix":""},{"id":434684683,"identity":"78fd46b4-f434-4005-ab44-e16e7a7f35d0","order_by":2,"name":"Chong Xu","email":"","orcid":"","institution":"Shanxi University","correspondingAuthor":false,"prefix":"","firstName":"Chong","middleName":"","lastName":"Xu","suffix":""},{"id":434684684,"identity":"c6eeb049-0daf-46aa-b0ef-9db160c4ccc1","order_by":3,"name":"Jinfeng Zhao","email":"","orcid":"","institution":"Shanxi University","correspondingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-03-12 00:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6207434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6207434/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79463962,"identity":"66e7e608-01d3-4064-bb31-7ed4b9c5f1fc","added_by":"auto","created_at":"2025-03-28 18:23:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":273936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAerobic exercise prevented HFD-induced hepatic steatosis. \u003c/strong\u003e(A) An overview of the experimental design of the treadmill exercise. (B) Liver morphology. (C) Hepatic lipid droplets. (D) Liver weight. (E) The ratio of liver weight to body weight. (F) Liver TG levels. All data are shown as mean±SD. NC: Normal control group, NE: Normal exercise group, OE: miR-34a overexpression group, OEE: miR-34a overexpression plus exercise group, HFD: High-fat diet group, and HFE: High-fat diet plus exercise group. * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs NC, and # \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs HFD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/f5ceccf552f2f02fa888db41.png"},{"id":79463964,"identity":"72f9dfee-cbad-43ba-bbe6-9149cfbe9f03","added_by":"auto","created_at":"2025-03-28 18:23:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreadmill training improved hepatic steatosis by inhibiting miR-34a and its downstream targets.\u003c/strong\u003e(A) Treadmill training inhibited miR-34a mRNA levels increased by high-fat diet and AAV9 injection. (B) The PPARα protein expression was detected in liver tissues by western blotting, with β-actin as a loading control. (C) The mRNA levels of PPARα in mouse liver tissue by qPCR. Internal control: GAPDH. The blots were cropped and the gels were run under the same experimental conditions. (D) Western blotting analysis of PPARα downstream target gene proteins (including CPT1, CPT2, SLC27A4, SLC27A1) in liver tissues. (E) The mRNA levels of CPT1, CPT2, SLC27A4, SLC27A1 in mouse liver tissue by qPCR. (F) The SIRT1 protein expression was detected by western blot. (G) The mRNA levels of SIRT1 in mice liver tissues by qPCR. (H) Western blotting for p-AMPK in liver extracts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/9c5cc879fae485180661f48d.png"},{"id":79463977,"identity":"4334963c-32d5-4786-ac35-8c10a6fe23ae","added_by":"auto","created_at":"2025-03-28 18:23:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAerobic exercise improved hepatic steatosis via miR-34a induced by AAV9 injection.\u003c/strong\u003e (A) Western blot analysis of PPARα protein expression levels and (B) the mRNA level of PPARα. (C) Protein expression levels of downstream fatty acid oxidation and transport-related target genes. (D) mRNA levels of downstream fatty acid oxidation and transport-related target genes. (E) The protein expression levels SIRT1 and (F) the mRNA levels of SIRT1 were assessed by western blot and qPCR, respectively. (G) The phosphorylation levels of AMPK.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/32160806f72507a46a11403d.png"},{"id":79463973,"identity":"3b903fd5-cf7b-49c1-bff8-852bd5e265bc","added_by":"auto","created_at":"2025-03-28 18:23:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36414,"visible":true,"origin":"","legend":"\u003cp\u003eSummarization of the regulatory role of miR-34a-PPARα/SIRT1 in liver steatosis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/c02ef341704a3f78a8b5e7df.png"},{"id":79543551,"identity":"2a8a6b6e-23b1-486b-9298-ec2158a77192","added_by":"auto","created_at":"2025-03-31 04:31:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1157214,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/a65d0e22-18e6-4779-80ce-71d388272b69.pdf"},{"id":79463965,"identity":"6a0decd6-0ec9-4313-b06c-04f2535b267d","added_by":"auto","created_at":"2025-03-28 18:23:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20216,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/e31d0e1231ccc50845e41b7c.docx"},{"id":79463989,"identity":"0c6af4d0-5648-4d43-a8c6-7a98ed9b5dff","added_by":"auto","created_at":"2025-03-28 18:23:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":981752,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6207434/v1/5683770a0615be517500d60d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aerobic exercise improved liver steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLiver steatosis was the appearance of fat drips in the cytoplasm of liver cells whose long-term development may cause hepatocyte necrosis, liver fibrosis, steatohepatitis which may develop into liver cirrhosis and hepatocellular carcinoma\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In recent years, with improving of the people's living standard and decreasing exercise time, liver steatosis has become a serious public health problem in the world, affecting 30\u0026ndash;40% of the total population which was still increasing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, the mechanism of hepatic steatosis was extremely limited and has not been determined. Therefore, the investigation of the molecular mechanisms underlying the progression of hepatic steatosis was urgent in order to obtain effective therapeutic approaches.\u003c/p\u003e \u003cp\u003eMicroRNA (miRNA) was a small single-stranded non-coding RNA of about 19\u0026ndash;25 nucleotides in length, which may regulate the expression of its target gene through reverse complementary pairing with the partial sequence of the 3' untranslated region (3'UTR) of its target gene\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In many diseases, miRNAs have received increasing attention due to their imbalance and their potential as both diagnostic and therapeutic targets\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. miR-34a, a specific miRNA, has been reported to show a high level of expression in the hepatic disease patients\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Besides, studies have reported that inhibition of miR-34a may increase the levels of its downstream targets, including peroxisome proliferator-activated receptor-α (PPARα) and Sirtuin protein family-Sirtuin1 (SIRT1) that were related to fatty acid oxidation thereby alleviating liver steatosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs a member of the nuclear receptor superfamily, PPARα bound to the ligand form a heterodimer with the retinoid X receptor (RXR), and then the heterodimer bound to the PPAR response element of the target genes promoting the transcription of its downstream genes related to fatty acid oxidation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. So PPARα had great effects on the regulation of hepatic lipid metabolism. The key target genes of PPARα downstream that were involved in fatty acid oxidation and fatty acid transport consisted of solute carrier family, including SLC27A and CPTs.\u003c/p\u003e \u003cp\u003eSIRT1 was an essential member of the Sirtuin protein family, and also was a NAD-dependent deacetylase, closely associating with hepatic steatosis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. AMP kinase (AMPK), a known regulator of energy metabolism, had a significant role in the development and progression of metabolic diseases, and SIRT1 may activate its activity thus promoting its phosphorylation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Then phosphorylation of AMPK up-regulated β-fatty acid oxidation gene and down-regulated fatty acid transport-related proteins, thereby reducing liver steatosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, the SIRT1-AMPK phosphorylation signaling pathway functions in the regulation of lipid metabolic homeostasis and may be a new therapeutic target for hepatic steatosis.\u003c/p\u003e \u003cp\u003eStudies have reported that aerobic exercise was a low-cost, low-risk intervention that may effectively reduce the occurrence and development of liver steatosis and has been adopted by most people\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The purpose of this study was to determine whether aerobic exercise may improve liver steatosis by adjusting the miR-34a-PPARα/SIRT1 signal pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethical Permission\u003c/h2\u003e \u003cp\u003e All operations in this experiment were performed under the rules of research ethics of Shanxi University with approval number of CIRP-IACUC-(R)2019014, in line with local and international animal ethics guide and with the utmost efforts to minimize animal suffering.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animal\u003c/h2\u003e \u003cp\u003eSixty SPF-grade C57BL/6J male mice (8 week old), weighing 21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 g, were bought from Beijing Life River Laboratory Animal Technology Co. (Animal license NO. SCXK (Beijing) 2016-0006). After adaptive feeding for one week, mice were randomly and equally selected into six groups: normal control group (NC), normal exercise group (NE), miR-34a overexpression group (OE), miR-34a overexpression plus exercise group (OEE), high-fat diet group (HFD, 60% fat, 20% carbohydrate, and 20% protein), and high-fat diet plus exercise group (HFE). All mice were housed under standard laboratory conditions with 12/12 dark light cycles, 20\u0026ndash;26 \u0026ordm;C, 40\u0026ndash;60% relative humidity, and free access to food and water. Mice body weights were recorded weekly. After the last treadmill exercise, mice were fasted overnight, anesthetized intraperitoneally with a dose of 80 mg/kg pentobarbital sodium, and then executed. Blood samples were collected in EDTA-containing tube, and centrifuged at 4 \u0026ordm;C and 3500 rpm for 15 min, then the supernatant was transferred to a new tube and stored at -80 \u0026ordm;C. The livers were rapidly taken out and washed with cold phosphate-buffered saline (PBS), then some of the liver tissues were cut into the fixative and the remaining liver tissues were saved at -80 \u0026ordm;C. The protocol was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAfter adaptive feeding, TBG promoter-driven overexpression-miR-34a adeno-associated viral vector TBG (AAV9) (100 \u0026micro;L, 1.95 \u0026times; 1012 vg/ml) (Sangon Biotech, Shanghai, China) was injected into the tail vein to specifically promote miR-34a expression in the mouse liver.\u003c/p\u003e \u003cp\u003eAfter 4 weeks of a normal or high-fat diet, mice in the NE, OEE, and HFE groups performed 1 week of adaptive treadmill exercise, followed by 8 weeks of treadmill exercise starting at 7 pm every day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Triglyceride assay\u003c/h2\u003e \u003cp\u003eTriglycerides (TG) was measured at 510 nm on a spectrophotometer (UV-6100s, Mapada, Shanghai, China) by using the kit and strictly following the instructions of the kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Histologic analysis\u003c/h2\u003e \u003cp\u003eLiver tissue was first fixed in 10% neutral buffered formalin and then stained with hematoxylin-eosin (H\u0026amp;E) to view the level of hepatic steatosis by the microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Real-time quantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from livers using a spin column animal total RNA purification kit (Sangon Biotech, Shanghai, China) and was reverse-transcribed using M-MuLV first-strand cDNA synthesis kit (Sangon Biotech, Shanghai, China) according to the manufacturer\u0026rsquo;s protocol. Real-time quantitative PCR (qPCR) was performed on a LightCycler480 system (Roche, Switzerland) using TB Green premix Ex Taq Ⅱ mix (TaKaRa, Dalian, China). Target gene expression was calculated by the 2^-△△CT method. Gene-specific primers were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with internal control of GAPDH.\u003c/p\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot\u003c/h2\u003e \u003cp\u003eThe liver tissues were lysed in RIPA lysis buffer, and the protein concentration was determined by the BCA method (Beyotime, Shanghai, China). Proteins of 40 \u0026micro;g were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then cropped and transferred to PVDF membranes, blocked for 1 hour with 5% nonfat milk. After overnight incubation with primary antibody (1:1000 anti-PPARα, anti-SIRT1, anti-AMPK, anti-CPT1, anti-CPT2, anti-SLC27A1, anti-SLC27A4) (Proteintech Group Inc, Wuhan, China), membranes were washed and incubated with HRP-conjugated secondary antibody (1:5000) (Boster Biotech, Wuhan, China). Then the ECL detection kit (Applygen Technologies Inc, Beijing, China) was used for detection, and imaging was performed on a chemiluminescence meter (ChemiDoc XRS+, Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10). Statistical analysis was performed using SPSS 25.0 software. One-way ANOVA was used for multiple comparisons. A two-tailed t-test was used for comparison between two groups. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Treadmill exercise alleviated liver steatosis in mice\u003c/h2\u003e \u003cp\u003eMorphological observation of the liver was showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. There were no obvious changes in the liver of NC and NE mice, which was fresh red in color with a smooth envelope and clear edges. In The liver of the HFD group mice was yellowish-brown with blunted edges, soft texture, and a greasy surface. After treadmill exercise, the livers of mice in the HFE group recovered to a state close to that of the NC group. H\u0026amp;E staining of liver tissue may directly reflect whether the liver steatosis occurred. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, there were no significant changes in the NE group and NC mice, while the HFD group showed a significant increase in the number and size of hepatic lipid droplets with inflammatory cell infiltration. In contrast, mice in the HFE group showed a reduced number of hepatic lipid droplets, neatly aligned hepatocytes, and significantly improved hepatic steatosis. Besides, the liver weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and the ratio of liver weight to body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) of mice in the HFD group were significantly increased to (1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25) g (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and (4.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53)% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) g and (3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24)% in the NC group, respectively, but after aerobic exercise intervention moderately rescued to (1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15) g (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs HFD group) and (3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28)% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs HFD group) in the HFE group, correspondingly. One of the major features of liver steatosis was the significant change of liver TG levels, so liver TG levels were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). TG levels in the HFD group was significantly increased to 67.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52 (mg/g liver) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;005) compared with the NC group (28.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08), but after aerobic exercise intervention restored to 55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53 (mg/g liver) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;005 vs HFD group) in the HFE group. These observations suggested that high-fat diet-induced hepatic steatosis may be attenuated by treadmill exercise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Treadmill exercise inhibited miR-34a expression\u003c/h2\u003e \u003cp\u003eNext, the effects of treadmill exercise on hepatic miR-34a expression were examined, and the results were showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. miR-34a expression levels were not significantly changed in NE group mice compared with NC group mice, whereas miR-34a expression was significantly up-regulated to 2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the NC group in the liver of HFD mice, and treadmill exercise reversed this rise to 1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This result revealed that treadmill training could improve liver steatosis by inhibiting miR-34a. To further clarify the effect of aerobic exercise on miR-34a, we injected miR-34a adenovirus vector into mice through the tail vein and performed treadmill intervention. the miR-34a level in the liver of over-expression mice was markedly enhanced to 2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with the NC group, but which was significantly restored to 1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) by aerobic exercise intervention compared with the the Overexpression of miR-34a group, implying that treadmill exercise may down-regulate the expression of miR-34a.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Changes in PPARα and its downstream target genes caused by treadmill exercise\u003c/h2\u003e \u003cp\u003ePPARα played an important role in the regulation of hepatic lipid metabolism. The protein expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) of PPARα in the NE group were basically the same as the NC group, and were decreased to 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the HFD group, respectively, while were increased to levels comparable to NC group with 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after treadmill exercise.\u003c/p\u003e \u003cp\u003eTo gain insight into the possible involvement of the miR-34a-PPARα pathway in hepatic steatosis alleviation induced by treadmill exercise, several key target genes of PPARα downstream that involved in fatty acid oxidation and fatty acid transport were examined by qPCR and western blot, including CPT1, CPT2, SLC27A1, and SLC27A4, and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. The expression levels of these downstream target genes in NE group mice were similar to those in NC group mice, while the expression of CPT1 and CPT2 was lower in HFD mice than in NC mice (0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the expression of SLC27A1 and SLC27A4 was higher (1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 vs. 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), finally, in the HFE group, the expression of CPT1 and CPT2 was reversed up to 0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the expression of SLC27A1 and SLC27A4 was reversed down to 1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 and 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the mRNA changes of all the above-mentioned genes were confirmed by qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Thus, the change in PPARα and its target genes could explain the alleviating effect of aerobic exercise on HFD-induced liver steatosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SIRT1 was involved in the miR-34a-induced lipid metabolism pathway\u003c/h2\u003e \u003cp\u003eAs one of miR-34a downstream target gene, SIRT1 was able to modulate the activity of AMP kinase (AMPK), a regulator of energy metabolism. The increase of the AMPK phosphorylation level was closely related to the enhancement of lipid metabolism. Then SIRT1 and AMPK were examined, and Fig.\u0026nbsp;3FG showed the results. SIRT1 protein levels and mRNA in NE mice were observed no significant changes, and exhibited a significant decrease in mice fed a high-fat diet were found compared to NC mice, with 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, respectively (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Then, this reduction was again up-regulated to 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs HFD group) after aerobic exercise intervention.\u003c/p\u003e \u003cp\u003eAs was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, the level of p-AMPKα (Thr-172) was lower in the HFD group (0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) than that in the NC group, while in the HFE group treadmill training partially abolished the decrease of HFD-induced p-AMPK (thr-172) expression (0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Thus, these observations indicated that SIRT1 may involve in the miR-34a-induced lipid metabolism pathway and aerobic exercise may also improve liver steatosis via the miR-34a-SIRT1-AMPK pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Aerobic exercise improved liver steatosis via miR-34a\u003c/h2\u003e \u003cp\u003eChanges in miR-34a expression showed a profound regulation of the PPARα/SIRT1-AMPK signaling pathway, which is closely associated with disorders of hepatic lipid metabolism. This relationship prompted us to further investigate whether the PPARα/SIRT1-AMPK pathway was involved in miR-34a-regulated hepatic steatosis and its associated pathology. This was investigated by injecting miR-34a adenovirus vector into mice through the tail vein and then performing 5 weeks of treadmill intervention.\u003c/p\u003e \u003cp\u003eAs expected, it was observed that treadmill exercise up-regulated the protein expression levels and mRNA of PPARα which both were decreased by miR-34a over-expression (Fig.\u0026nbsp;4AB). Also the protein expression levels and mRNA of CPT1 and CPT2 were significantly reduced in liver samples from miR-34a overexpressing mice, and those of SLC27A1 and SLC27A4 were significantly increased, and these changes were altered after the exercise intervention (Fig.\u0026nbsp;4CD). It was found in Fig.\u0026nbsp;4EF that SIRT1 showed the same trend as PPARα. In addition, AMPK phosphorylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) were reduced in miR-34a overexpressing mice, and the reduction was significantly reversed after 5 weeks of aerobic exercise. In a word, PPARα/SIRT1-AMPK pathway was involved in miR-34a-regulated hepatic steatosis.\u003c/p\u003e \u003cp\u003eThe regulatory role of miR-34a-PPARα/SIRT1 in liver steatosis was summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the work, after 8 weeks of treadmill exercise, mice in HFD group showed significant improvement in liver weight and liver pathology. Recently, many studies have revealed that miR-34a was a specifically regulated miRNA in liver diseases and has a significant effect on the development and process of hepatic steatosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The expression level of miR-34a was high in patients with nonalcoholic fatty liver1\u003csup\u003e7,18\u003c/sup\u003e as well as in animal models of fatty liver\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Similarly, our hepatic steatosis mouse model showed increased hepatic expression of miR-34a, which was in agreement with previous findings.\u003c/p\u003e \u003cp\u003eNumerous studies have shown that the expression of miRNA may be affected by aerobic exercise\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. miR-34a over-expression disrupted lipid normal metabolism and may also be another critical marker for exercise to improve hepatic steatosis. In the work, we showed that treadmill exercise inhibited miR-34a over-expression in HFD mice to reduce hepatic lipid droplet accumulation. Consistent with the morphological findings, aerobic exercise also reduced the level of TG in the liver tissue of mice in the HFE group. Besides, the liver weight/body weight ratio was also markedly reduced in the HFE group when compared to the HFD group. In a word, aerobic exercise inhibited increase of miR-34a, TG, and the liver weight in HFD mice.\u003c/p\u003e \u003cp\u003emiR-34a can potentially post-transcriptionally regulate PPARα through specific binding with the PPARα wild-type luciferase construct\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. By observing the mRNA and protein levels of PPARα in mouse liver, we found that PPARα mRNA and protein levels were significantly lower in the HFD group compared to the NC group, and 8 weeks of aerobic exercise reversed this decline. As one of the direct downstream target genes of miR-34a, SIRT1 has taken a pivotal role in regulating cell lipid metabolism and reducing inflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We found that the mRNA and protein levels of SIRT1 were significantly increased in the liver of the HFE group after aerobic exercise. AMPK was an AMP-dependent protein kinase which was widely expressed in various organs related to metabolism and can be activated by exercise, also being a biological energy key molecule of metabolic regulation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Our research showed that treadmill exercise increased the expression of SIRT1 through inhibiting miR-34a, thereby stimulating AMPK signals which was to monitor the fuel meter of cell energy status and activate the gene that was related to fatty acid oxidation. Therefore, we supposed that miR-34a-mediated modulation of hepatic lipid metabolism by PPARα and SIRT1-AMPK pathways may be two independent regulatory mechanisms in fatty liver.\u003c/p\u003e \u003cp\u003eCPT1 catalyzed the esterification of long-chain acyl-CoA into L-carnitine which was used as a carrier to transfer long-chain fatty acids from outside the mitochondria to the inside of the mitochondria in the form of an acyl-carnitine conjugate. Then acyl-carnitine conjugates were transformed back to acyl-CoA esters by CPT2 in the mitochondria to promote the oxidation and decomposition of fatty acids\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In the work, it was found that both the activation of AMPK and the rise in PPARα expression increased the levels of CPT1 and CPT2, leading to increased transport of fatty acids into the mitochondria, and facilitated the oxidation of mitochondrial palmitoyl-CoA. These observations helped us to understand the mechanism of enhanced fatty acid oxidation after aerobic exercise inhibits miR-34a, with AMPK as a medium for the increased hepatic oxidation of miR-34a after aerobic exercise inhibition.\u003c/p\u003e \u003cp\u003eSLC27 was an integral transmembrane protein that promoted the uptake of long-chain fatty acids into cells and also served as a key player in lipid metabolism \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our study showed that the mRNA and protein levels of SLC27A1 and SLC27A4 were significantly elevated in HFD mice, which was significantly reversed after aerobic exercise. This result suggested that aerobic exercise may improve hepatic steatosis by affecting fatty acid transporter proteins.\u003c/p\u003e \u003cp\u003eIn conclusion, our research showed that aerobic exercise down-regulated miR-34a, and up-regulated the levels of PPARα and SIRT1 which then affected fatty acid oxidation-related genes CPT1, CPT2, and fatty acid transportation-related genes SLC27A1, SLC27A4 transcription, thereby ameliorated hepatic steatosis. Notably, SIRT1 influenced downstream target genes after activation of the AMPK pathway. This study provided new insights into the pathogenesis of liver steatosis and had the potential to suggest new approaches to treat liver steatosis at the miRNA level.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Ethics Committee of Shanxi University (approval number [SXULL2021055]).\u003c/p\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by Fundamental Research Program of Shanxi Province (NO. 202203021221022).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.AW.: Project administration, funding acquisition, conception and design of the study, experimental instruction, revising the article, and final approval of the version to be submitted. Z.B.Z. and C.X.: Conception and design of the study, acquisition, analysis and interpretation of data, drafting the article, and final approval of the version to be submitted. J.F.Z.: Conception and design of the study, experimental instruction, and final approval of the version to be submitted.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e The authors thank the Institute of Biological Sciences Shanxi University for professional technical assistance and the China Institute of Radiation Protection for animal care.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCastro RE, Ferreira DM, Afonso MB, et al. miR-34a/SIRT1/p53 is suppressed by ursodeoxycholic acid in the rat liver and activated by disease severity in human non-alcoholic fatty liver disease. J. Hepatol. 2013;58:119\u0026ndash;125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Wang L, Li Y, et al. 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Biochem. 2020;126:242\u0026ndash;249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi CX, Gao JG, Wan XY, et al. Allyl isothiocyanate ameliorates lipid accumulation and inflammation in nonalcoholic fatty liver disease \u003cem\u003evia\u003c/em\u003e the Sirt1/AMPK and NF-κB signaling pathways. World J. Gastroenterol. 2019;25:5120\u0026ndash;5133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerhart-Hines Z, Rodgers JT, Bare O, et al. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. EMBO. J. 2007;26:1913\u0026ndash;1923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi SE, Fu T, Seok s, et al. Elevated microRNA-34a in obesity reduces NAD\u0026thinsp;+\u0026thinsp;levels and SIRT1 activity by directly targeting NAMPT. Aging Cell. 2013;12:1062\u0026ndash;1072.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaffer JE, Lodish HF. Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell. 1994;79:427\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStahl A, Hirsch DJ, Gimeno RE, et al. Identification of the major intestinal fatty acid transport protein. Mol. Cell. 1999;4:299\u0026ndash;308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShim J, Moulson CL, Newberry EP, et al. Fatty acid transport protein 4 is dispensable for intestinal lipid absorption in mice. J. Lipid Res. 2009;50:491\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aerobic exercise, liver steatosis, miR-34a, PPARα, SIRT1","lastPublishedDoi":"10.21203/rs.3.rs-6207434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6207434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroRNA-34a (miR-34a) was closely associated with liver steatosis. However, the link between changes in miR-34a and the progression of liver steatosis remained unclear. In the work, sixty mice were randomly and equally selected into six groups: normal control group (NC), normal exercise group (NE), high-fat diet group (HFD), high-fat diet plus exercise group (HFE), miR-34a overexpression group (OE), and miR-34a overexpression plus exercise group (OEE). Live morphology showed that treadmill exercise intervention for 8 weeks reduced high-fat diet-induced liver steatosis in mice. 8-week treadmill exercise directly decreased mir-34a expression of mice in HFD group, confirmed in OE group. More, treadmill exercise enhanced the expression of PPARα and SIRT1, thereby affecting the downstream hepatic steatosis-associated target genes, including CPT1, CPT2, SLC27A4, SLC27A1, in addition to activating the expression of the central metabolic sensor AMPK. Following aerobic exercise intervention, miR-34a was upregulated, thereby affecting the expression of genes associated with hepatic steatosis, and this mechanism was confirmed in miR-34a overexpression mice. This study contributed to our understanding of the pathogenesis of hepatic steatosis and may provide new therapeutic approaches.\u003c/p\u003e","manuscriptTitle":"Aerobic exercise improved liver steatosis by modulating miR-34a-mediated PPARα/SIRT1-AMPK signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-28 18:23:04","doi":"10.21203/rs.3.rs-6207434/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c03e045e-bad4-4b85-93c6-2bf4fc75135c","owner":[],"postedDate":"March 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T04:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-28 18:23:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6207434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6207434","identity":"rs-6207434","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}