GADD45β Inhibits Hepatic Lipogenesis through the AMPK/SREBP1 Pathway via Reducing the ubiquitination-mediated Degradation of SIRT1

GADD45β Inhibits Hepatic Lipogenesis through the AMPK/SREBP1 Pathway via Reducing the ubiquitination-mediated Degradation of SIRT1 | 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 Article GADD45β Inhibits Hepatic Lipogenesis through the AMPK/SREBP1 Pathway via Reducing the ubiquitination-mediated Degradation of SIRT1 Yuanyuan Xiao, Renjie Wang, Chaoyu Zhu, Qianqian Wang, Xinyi Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6769255/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Nonalcoholic fatty liver disease (NAFLD) is a globally increasing metabolic disorder associated with serious health complications. The molecular mechanisms linking stress-response proteins to hepatic lipogenesis in NAFLD remain poorly understood. Here, we identify GADD45β as a key suppressor of de novo lipogenesis through stabilization of SIRT1. In both methionine-choline-deficient (MCD) diet-fed mice and palmitic acid (PA)-treated hepatocytes, GADD45β deficiency exacerbated lipid accumulation and upregulated lipogenic genes (SREBP1, FASN, ACC). Mechanistically, GADD45β directly bound to SIRT1 and inhibited its ubiquitination, thereby prolonging SIRT1 protein stability. Enhanced SIRT1 stability increased AMPK phosphorylation, which suppressed SREBP1-mediated transcription of lipogenic targets. Crucially, hepatic overexpression of GADD45β reversed PA-induced steatosis in vitro. Our study uncovers a GADD45β-SIRT1-AMPK axis as a central regulator of hepatic lipogenesis, proposing GADD45β as a therapeutic target for NAFLD. Health sciences/Endocrinology Health sciences/Medical research Health sciences/Molecular medicine Health sciences/Pathogenesis GADD45β NAFLD Lipogenesis Ubiquitination SIRT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The increasing prevalence of nonalcoholic fatty liver disease (NAFLD) is a significant global health concern. Over the last decade, NAFLD has become the predominant cause of chronic liver disease in many parts of the world and affects more than 25% of the global population [ 1 ]. NAFLD is often linked to obesity, insulin resistance, and metabolic syndrome [ 2 ]. This condition is characterized by excessive lipid accumulation in hepatocytes, which can progress from simple steatosis to more severe forms, such as steatohepatitis and cirrhosis. The intricate mechanisms underlying hepatic lipid metabolism are critical for understanding the pathophysiology of NAFLD, yet they remain inadequately explored. De novo lipogenesis may play a crucial role in the development of NAFLD[ 3 ]. Lipogenesis converts excessive glucose or fructose into fatty acids and triglycerides[ 4 ] and is a normal process for maintaining homeostasis in the body; increased activation of this process may cause hepatic steatosis[ 5 ]. Therefore, the inhibition of lipogenesis is highly desirable as a therapeutic target for lipid metabolism-related diseases. The literature has focused primarily on various signaling pathways involved in lipid regulation, such as the roles of SIRT1 and AMPK, while the specific contributions of GADD45β in this context are not well characterized[6; 7]. Growth arrest and DNA damage-inducible 45β (GADD45β), a member of the GADD45 family, is known to participate in cellular stress responses, DNA repair, and apoptosis[ 8 ]. Recent studies have highlighted its potential involvement in glucose and lipid metabolism, suggesting that GADD45β may be a new key regulator in the pathogenesis of NAFLD[ 9 ]. However, whether GADD45β takes part in the process of lipogenesis to affect NAFLD remains unclear. The current research landscape reveals a significant gap regarding the mechanistic role of GADD45β in hepatic lipid metabolism, particularly its interactions with established pathways such as SIRT1/AMPK/SREBP1 signaling, which are crucial for maintaining metabolic homeostasis[10; 11]. In this study, we provide evidence that SIRT1 is the target substrate of GADD45β and identify an important correlation between them in NAFLD. GADD45β interacts with SIRT1 and deubiquitylates it, inhibits its proteasomal degradation and increases its stability. GADD45β deficiency decreased the activity of the SIRT1 target AMPK, resulting in lipogenesis exacerbation. Our findings highlight that GADD45β potentially plays a critical role in regulating the stability of SIRT1 and functions to reduce lipogenesis progression, providing new insights and potential therapeutic strategies for NAFLD in the future. Materials and methods Reagents and antibodies Cycloheximide (CHX, Cat# M4879) was purchased from AbMole Bioscience, Inc. MG132 (Cat# S2619) was purchased from Selleck Chemicals. Palmitic acid (PA, Cat# S-A9165-5G) was purchased from Sigma‒Aldrich. Compound C (CC, Cat# HY-13418) was purchased from Med ChemExpress. Antibodies against FASN (Cat# ARG55898, 1:1000), HSP90 (Cat# ARG55781, 1:1000), Flag Tag (Cat# ARG62342, 1:1000) and β-Actin (Cat# ARG65683, 1:1000) were purchased from Arigo. Antibodies against ubiquitin (Cat# 3936, 1:400), AMPK (Cat# 2532S, 1:1000) and p-AMPK (Cat# 2531S, 1:1000) were purchased from Cell Signaling Technology. Antibodies against Sirt1 (Cat# ab189494, 1:1000), SREBP1 (Cat# ab28481, 1:1000), and ACC1 (Cat# ab45174, 1:1000) were purchased from Abcam. Antibodies against GADD5β (Cat#sc377311, 1:1000) was purchased from Santa Cruz. HRP-conjugated secondary antibodies against mouse (Servicebio, GB23301) or rabbit (Servicebio, GB23303) were used. Animal treatment The experiments involving mice were approved by the Animal Research Ethics Committee of Shanghai Sixth Peoples’ Hospital in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments were conducted in accordance with the guidelines of Animal Research and the study adhered to the ARRIVE guidelines ensuring compliance with ethical standards for animal research[ 12 ]. Eight-week-old C57BL/6J mice weighing 23–28g were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd. (Shanghai, China). All the animals were maintained in a specific pathogen-free barrier facility under a 12 h light/dark cycle. After a week of adaptation, the mice were fed an MCD diet (A02082002B, Research Diets, USA) for 4 weeks to establish NASH models, and their corresponding normal chow diet (CON) served as a control. Adeno-associated virus (AAV)-delivered short hairpin RNA pscAAV-U6-shNC-CMV-EGFP-tWPA was constructed via an AAV-8 vector system (OBiO Technology, Shanghai, China) as a control group (AAV-shNC) in the liver. Briefly, AAV-shGADD45β particles targeting GADD5β (shGADD5β) were generated via pscAAV-U6-shGADD45β-CMV-EGFP-tWPA vectors (AAV-shGADD45β). The shRNAs had the following sequence: GADD5β (59-GCGACAATGACATTGACATCG-39). To knock down hepatic GADD5β expression, 2×10 11 PFU per mouse were delivered into MCD mice for 14 d. All viruses were purified via the cesium chloride method, dialyzed in PBS containing 10% glycerol, and administered to the mice via tail vein injection. Finally, all the mice were euthanized using an overdose anesthesia method of intraperitoneal injection anesthesia with 3% sodium pentobarbital 60 mg/kg body weight after 12 h of fasting, and the liver were dissected and snap-frozen in liquid nitrogen. Tissues and serum were kept at − 80°C until lysates were obtained and western blot or RNA analyses performed. The animal experiments were strictly adhered to the 3Rs principles (Replacement, Reduction, and Refinement). Additionally, predefined humane endpoints are established to promptly euthanize animals showing severe distress or irreversible suffering, ensuring their welfare is prioritized throughout the study. Biochemical assays Blood samples collected from the mice were allowed to stand at room temperature for 30 minutes, followed by centrifugation at 4°C and 5000 rcf for 5 minutes. The supernatant was subsequently centrifuged at 10000 rcf at 4°C for 5 minutes. The serum was carefully transferred into clean 1.5 mL EP tubes and stored at -20°C for further use. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), and gamma-glutamyltransferase (γ-GT) were measured via a fully automated biochemical analyzer. H&E and Oil Red O staining Liver tissues were fixed overnight in 10% formalin and then embedded in paraffin. Sections with a thickness of 4 µm were stained with hematoxylin and eosin (H&E). For Oil Red O staining, frozen liver sections (7 µm) were fixed in 4% paraformaldehyde for 10 min, washed with deionized water, stained with oil red O (SigmaAldrich) for 10 min at room temperature, washed in deionized water twice, and finally stained with hematoxylin for 30 s. Histological assessment was performed via an Olympus light microscope. Cell culture and treatment HepG2 cells were cultured in DMEM/HIGH GLUCOSE (Cat# SH30243.01, HyClone) supplemented with 10% fetal bovine serum (Cat#04-001-1ACS, Biological Industries) and 1% penicillin‒streptomycin (Cat#C0222, Beyotime) in a 5% CO2 incubator at 37°C. HepG2 cells were treated with fatty acid-free bovine serum albumin (BSA) or PA at a concentration of 400 µM for 24 h. Oil Red O was used for intracellular lipid droplet staining. Lipid staining was observed under a light microscope (Olympus, Tokyo, Japan). Cell transfection pSLenti-GADD5β-3xFlag (GADD5β) and control pSLenti-MCS-3xFlag (vector), pLenti-U6-siGADD5β-CMV (siGADD5β:GCATACTCCTTCCACGTTA) and control pSLenti-U6-shNC2-CMV (siNC:TTCTCCGAACGTGTCACGT) were constructed by OBiO Technology (Shanghai, China). When HepG2 cells were cultured in 35 mm plates at 70–80%, solutions A and B were prepared, respectively, with 2.5 µg of plasmid, 2.5 µl of PLUS and 125 µl of Opti-Bertani medium for Solution A, and 5 µl of LTX and 125 µl of Opti-Bertani medium for Solution B. The A and B solutions were gently mixed and left to stand for 5 min. The solution was added dropwise to the medium of the HepG2 cells, which could be changed after 6–8 h. The cells were collected for real-time qPCR and western blotting after transfection for 48 h. In this study, two lentivirus (LV) constructs, LentiCRISPRv2-SIRT1-sgRNA (LV-shSIRT1) and a blank vector (LV-shNC), were added to the medium of the HepG2 cells, which were subsequently fenghbio Biotechnology Co., Ltd. (Hunan, China). Upon reaching 80% confluence, the culture medium was supplemented with the virus mixture at an MOI of 50 for transfection. After 24 h of transfection, the serum-free Opti-MEM was replaced with fresh medium containing FBS, and puromycin (30 µg/mL) (HY-K1057, MedChemExpress) was applied to select successfully transfected cells. The culture was continued for 2 days, after which the cells were harvested and lysed for western blotting. Western blot analysis To prepare total protein extracts, liver tissues and HepG2 cells were lysed in RIPA buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1% NP40, and 0.1% SDS. Equivalent amounts of protein samples were denatured in loading buffer, resolved by 10–12% SDS‒PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 h before being incubated with primary antibodies overnight at 4°C. The membranes were washed with PBS containing Tween-20 five times and incubated with secondary antibody for 1 h. The signals of the proteins were then visualized via an electrochemiluminescence system. Coimmunoprecipitation (Co-IP) The cells were lysed on ice for 30 min with IP lysis solution (Beyotime, China) containing phosphatase inhibitors as well as protease inhibitors, and the supernatant was obtained by centrifugation. After quantification and analysis, the cells were incubated overnight at 4°C with the corresponding antibodies or control IgG, and then 20 µl of A/G agarose beads was added and incubated at 4°C for 4 h. After sequential elution with different buffers, the supernatant was collected by instantaneous centrifugation for immunoprecipitation experiments. Quantitative real-time polymerase chain reaction (qRT‒PCR) Total RNA was extracted from cells or liver tissues via TRIzol reagent and reverse transcribed into cDNA via PrimeScript™ RT Master Mix (Cat# R433-01, Vazyme) according to the manufacturer’s instructions. qRT‒PCR was performed with SYBR Premix Ex Taq™ (Cat#Q312-02, Vazyme) reagents, and the results were analyzed via Light Cycler 480 software (Roche Diagnostics GmbH, Mannheim, Germany). The relative levels of target gene expression were calculated via the 2 − ΔΔCt method, with GAPDH used as a control. The primers used for RT‒PCR are shown in Table S1 . Statistics The calculations were carried out with Graph Pad InStat Software (San Diego, CA, USA). All the data are presented as the means ± SEMs. For animal and cellular experiments, a two-tailed unpaired Student’s t test was performed to compare two groups. The western blot and morphological images are representative of at least three experiments with similar results. Statistical significance was determined by nonparametric procedures via Student’s t test or ANOVA for analysis of variance. The normality of the distributions of the quantitative variables was assessed via the Kolmogorov‒Smirnov test. P values < 0.05, P < 0.01, and P < 0.001, respectively. Results Hepatic GADD45β expression decreased in NAFLD By accessing the human GEO database, two NAFLD datasets, GSE33814 and GSE48452, were included in this study, which included 37 healthy controls, 39 NAFLD subjects and 31 NASH subjects. Notably, GADD45β was consistently downregulated in NAFLD and NASH patients in both datasets at the mRNA level (Fig. 1 A, B). To further confirm the downregulation of GADD45β in NASH, GADD45β mRNA and protein expression levels in mouse liver samples were evaluated by western blot and qPCR. Compared with those in the control group, GADD45β levels were substantially lower in the livers of MCD diet-fed mice (Fig. 1 C, D). Hepatic knockdown of GADD45β exacerbates steatohepatitis in MCD mice Further study of the dysregulation of hepatic GADD45β in C57BL/6J mice and human subjects revealed that GADD45β could affect steatohepatitis. MCD mice were injected with GADD45β-specific adenoviral associate shRNA via the tail vein, which inhibited endogenous GADD45β expression in the liver (Fig. 2 . A). At 4 weeks after injection, GADD45β expression in the liver was significantly lower in GADD45β-knockdown mice than in control mice (Fig. 2 . B). Notably, there was no significant difference in the serum levels of TC, TG, AST and ALT between GADD45β-knockdown and control MCD diet-fed mice (Fig. 2 . F-I), whereas the serum levels of γGT in the AAV-shGADD45β-MCD mice were greater than those in the control mice (Fig. 2 . J), indicating that GADD45β knockdown might aggravate liver injury. Although there was no significant change in weight, the liver weight, liver index and liver TG levels were significantly increased in the GADD45β-knockdown group (Fig. 2 C-E). H&E staining and Oil Red O staining also revealed significant hepatic steatosis in GADD45β-knockdown MCD mice (Fig. 2 K-L). All of the above data suggest that the knockdown of hepatic GADD45β aggravates liver steatohepatitis. Hepatic knockdown of GADD45β increased hepatic lipogenesis gene expression in MCD diet-fed mice Comparison of the transcription of lipogenesis genes in the livers of GADD45β hepatic knockdown MCD diet-fed mice. MCD feeding significantly upregulated hepatic Srebp1 mRNA expression, and liver-specific knockdown of GADD45β further increased Srebp1 mRNA expression. Liver-specific knockdown of GADD45β significantly upregulated the mRNA levels of two other hepatic lipogenesis genes, Fasn and Acc (Fig. 3 A). However, the hepatic mRNA levels of genes related to fatty acid oxidation, fatty acid transport and lipolysis were unaffected (Fig. 3 A). In addition, the increase in the hepatic protein levels of SREBP1, FASN and ACC1 in the GADD45β hepatic knockdown mice shown by Western blotting was consistent with the increase in the mRNA levels (Fig. 3 .B). These results showed that liver-specific knockdown of GADD45β significantly upregulated hepatic lipogenesis gene expression in MCD diet-fed mice. GADD45β inhibited palmitic acid-induced steatosis and lipogenesis in hepatocytes To confirm the function of GADD45β in hepatocytes, GADD45β expression was upregulated in HepG2 cells via GADD45β plasmid transfection. Western blot analysis revealed a significant increase in GADD45β protein levels in HepG2 cells after transfection (Fig. 4 . F), HepG2 cells were treated with 400 µM palmitic acid (PA) for 24 h to simulate hepatocyte steatosis, and Oil Red O staining revealed that GADD45β overexpression significantly decreased intracellular lipid accumulation. In addition, PA stimulation significantly elevated the intracellular TG content. Specifically, GADD45β overexpression blocked the effects of PA on the intracellular TG content (Fig. 4 . A-C). These results indicate that under PA-induced hepatocyte steatosis, increased GADD45β levels play an important role in regulating hepatosteatosis. Moreover, the effect of lipogenesis via GADD45β in PA-treated hepatocytes was also evaluated. As expected, the results of the quantitative RT‒PCR and WB experiments confirmed that the overexpression of GADD45β significantly reduced the PA-induced levels of lipogenesis regulators, such as SREBP1, ACC1, and FASN, in HepG2 cells (Fig. 4 F‒H). Additionally, we validated the conclusions from the opposite perspective. In HepG2 cells, selected siRNAs were used to downregulate the expression level of GADD45β, after which hepatic steatosis was induced with PA. The results revealed that GADD45β knockdown significantly increased the expression levels of lipogenesis-related genes after PA induction (Fig. 4 A. I-K), thereby exacerbating lipid accumulation and steatosis in hepatocytes (Fig. 4 A, D, E). These findings collectively suggest that GADD45β exerts a protective effect against hepatic steatosis by inhibiting lipogenesis within hepatocytes. GADD45β mediated lipogenesis via the SIRT1/AMPK/SREBP1 pathway in hepatocytes Sirtuin 1 (SIRT1) is a member of the mammalian histone deacetylase sirtuin family and plays a pivotal role in hepatic steatosis and lipogenesis via the phosphorylation of AMPK. To study whether the inhibition of GAADD45β could influence the Sirt1/AMPK pathway, GADD45β hepatic knockdown MCD mice were used in vivo to further examine whether it had effects on the SIRT1/AMPK pathway. The immunohistochemical results revealed that the expression levels of SIRT1 and p-AMPK were significantly lower in the shGADD45β MCD mice than in the shNC mice (Fig. 5 D-E). The western blot results revealed that SIRT1 expression and the p-AMPK/AMPK ratio were significantly lower in the GADD45β hepatic knockdown group than in the control group (Fig. 5 A). In addition, the suppression of GAADD45β had negative effects on the SIRT1/AMPK pathway in PA-treated HepG2 cells and reduced the protein levels of Sirt1 and p-AMPK/AMPK in parallel with SREBP1 to a certain extent (Fig. 5 B). In contrast, in HepG2 cells overexpressing GADD45β, the SIRT1/AMPK pathway was significantly increased after treatment with PA, and SREBP1 levels were decreased (Fig. 5 C). These results indicate that GADD45β expression could mediate lipogenesis via the SIRT1/AMPK pathway in hepatocyte steatosis. It was hypothesized that SIRT1 may be a major component of the process by which GADD45β regulates the p-AMPK/AMPK signaling pathway. Western blotting analyses revealed that GADD45β rescued the reduction in SIRT1 protein levels in HepG2 cells stimulated with PA. In HepG2 cells, lentiviruses were used to knock down SIRT1 protein levels (Fig. 5 . F), increased the p-AMPK/AMPK ratio promoted by GADD45β, and reversed the downregulatory effects of GADD45β on SREBP1 (Fig. 5 G). To assess whether AMPK inhibition could diminish the protective effect of GADD45β, studies were also conducted in which HepG2 cells were treated with compound C, an inhibitor of AMPK, for 4 hours (Fig. 5 H). The results showed that Compound C significantly reduced the phosphorylation of AMPK. GADD45β overexpression in the Compound C- and PA-induced groups failed to prevent the increase in SREBP1 and did not rescue the decrease in p-AMPK (Fig. 5 I). These findings underscore the significant impairment of the SIRT1/AMPK signaling pathway in lipogenesis. These data indicate that SIRT1/AMPK mediate lipogenesis and that GADD45β has a defensive effect through this signaling pathway in hepatocytes in NAFLD. GADD45β directly interacted with SIRT1 Although the overexpression of GADD45β increased the SIRT1 protein content, as indicated by the data above, it did not alter the SIRT1 mRNA levels, as determined by RT‒qPCR analysis (Fig. 6 A). To elucidate the molecular mechanisms by which GADD45β regulates SIRT1, we predicted that GADD45β might be a binding partner of SIRT1. To test this hypothesis, coimmunoprecipitation tests were used to investigate the interaction of GADD45β and SIRT1 in PA-induced HepG2 cells. As shown in Fig. 6 . B), GADD45β was pulled down by SIRT1. In a mutual pulldown experiment using an anti-GADD45β antibody, GADD45β also pulled down SIRT1. Indeed, when GADD45β was pulled down by an anti-GADC45β antibody, SIRT1 was detected via coimmunoprecipitation via western blotting. Taken together, these data suggest that GADD45β may directly interact with SIRT1 and further regulate exercise. GADD45β upregulated SIRT1 levels by inhibiting its ubiquitin‒proteasome degradation To exclude the influence of protein synthesis on SIRT1 levels and because SIRT1 can be regulated through ubiquitination to promote its stabilization, the possible role of GADD45β-mediated SIRT1 was further investigated. To determine whether GADD45β could inhibit SIRT1 degradation, GADD45β-encoding plasmids were transfected into HepG2 cells, and the SIRT1 protein levels were measured after treatment with CHX, a protein synthesis inhibitor. As expected, GADD45β significantly shortened the half-life of SIRT1, which was decreased by CHX (Fig. 7 A). Conversely, the knockdown of GADD45β decreased SIRT1 protein stability in PA-induced HepG2 cells (Fig. 7 . B), suggesting that GADD45β-mediated inhibition of SIRT1 degradation, rather than promoting protein synthesis, contributes to the increase in SIRT1 levels in response to GADD45β. The K48-linked ubiquitin‒proteasome system (UPS) is a major intracellular protein degradation mechanism. As shown in (Fig. 7 . C), intervention with MG132, a proteasome inhibitor, and GADD45β overexpression significantly reversed the reduction in SIRT1 protein levels after exposure to PA. These data seem to favor the involvement of the UPS in SIRT1 degradation. To address this issue, the ubiquitination levels of SIRT1 were also examined through immunoprecipitation. The results revealed increased ubiquitination of SIRT1 in HepG2 cells upon exposure to PA, whereas total SIRT1 protein expression was downregulated. However, GADD45β transfection reversed this effect. These results indicate that GADD45β increases SIRT1 levels by inhibiting its ubiquitin‒proteasome degradation. These experimental results suggest that the expression of GADD45β in the hepatocytes of NASH patients and MCD-fed mice is decreased, which reduces the interaction between GADD45β and SIRT1. Subsequently, SIRT1 is degraded via the ubiquitin–proteasome pathway. The reduction in SIRT1 prevents the activation of phosphorylated AMPK protein, which in turn exacerbates SREBP1-mediated de novo fatty acid synthesis. This leads to increased lipogenesis, thereby synergistically aggravating fatty acid accumulation and hepatic steatosis (Fig. 8 ). Discussion Nonalcoholic fatty liver disease (NAFLD) has emerged as a significant global health concern due to its complex pathogenesis, which involves multiple factors, such as insulin resistance, oxidative stress, and inflammatory pathways. With the increasing prevalence of obesity and metabolic syndrome, NAFLD is now recognized not only as a hepatic disorder but also as a major risk factor for cardiovascular diseases, type 2 diabetes, and other metabolic disorders. This underscores the urgent need to understand its mechanisms and develop effective therapeutic strategies to address this condition [13; 14]. In this study, we identified a novel factor, GADD45β, which plays a significant role in the development and progression of NAFLD. GADD45β is a member of the GADD45 family and is an acidic protein that responds to cellular stress. It plays crucial regulatory roles in cellular functions such as DNA repair, cell cycle regulation, apoptosis, inflammation, and stress responses through protein‒protein interactions [ 15 ]. Furthermore, numerous studies have demonstrated the involvement of GADD45β in lipid metabolism. Dong et al. demonstrated that GADD45β levels were downregulated in multiple clinical databases and a murine model of nonalcoholic fatty liver disease (NAFLD). They elucidated the beneficial role of GADD45β in mitigating excessive lipid accumulation and insulin resistance in NAFLD induced by a high-fat diet (HFD) in mice [ 6 ]. Fuhrmeister et al. reported that under fasting conditions, GADD45β facilitates the cytoplasmic retention of fatty acid binding protein 1 by binding to it, thereby inhibiting the uptake of fatty acids by hepatocytes and enhancing lipid metabolism [ 16 ]. Additionally, research by Kim et al. revealed that GADD45β significantly suppresses hepatic gluconeogenesis by enhancing the protein stability and transcriptional activity of forkhead box protein O1 (FoxO1). They also noted an increase in the expression of genes associated with lipogenesis in GADD45β knockout mice. Furthermore, GADD45β has been identified as an inducible coactivator of the constitutive androstane receptor (CAR), promoting rapid liver growth. Cai et al. reported that the effect of CAR on energy metabolism also depends on Gadd45β. In HFD-induced obese GADD45β knockout mice, the reduction in body weight gain and improvement in insulin sensitivity caused by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) were markedly blunted by Gadd45β knockout. Mechanistically, these effects are related to the inhibition of lipogenesis in hepatocytes[ 17 ], however no further mechanistic studies have been conducted. In the present study, we observed that both the mRNA and protein expression levels of GADD45β gradually decreased in human and mouse NAFLD livers as the disease progressed from NAFLD to NASH. By constructing a liver-specific GADD45β gene-knockdown methionine‒choline-deficient (MCD) diet mouse model, we found that, compared with that in control diet-fed mice, liver steatosis in the GADD45β-knockdown group was exacerbated. Further studies revealed that hepatic lipid deposition was related to the upregulation of lipogenesis genes, including SREBP-1, FASN and ACC, which is consistent with the findings of other previous studies. In vitro experiments revealed that the overexpression of GADD45β in HepG2 cells led to a significant decrease in the expression of these lipogenesis genes upon fatty acid treatment. Conversely, with GADD45β knockout, the expression of these genes increased inversely. These data indicate that GADD45β is involved in the occurrence and development of hepatic lipid metabolism and fat deposition in NAFLD. To clarify how GADD45β influences hepatic lipid metabolism, we further investigated the interaction between GADD45β and a key regulator of lipogenesis, adenosine monophosphate (AMP)-activated protein kinase (AMPK). AMPK is a heterologous trimeric protein kinase that provides energy for cells via lipid metabolism. Studies have demonstrated that AMPK plays a key role in adjusting hepatic fatty acid oxidation, inhibiting cholesterol and TG synthesis, and repressing lipogenesis[ 18 ]. Among these, sterol regulatory element binding protein-1c (SREBP-1c) is one of the key lipogenesis transcription factors in the liver that is regulated by AMPK [ 19 ]. AMPK mainly reduces lipogenic gene expression by inhibiting the activation of SREBP-1c through phosphorylation at the Ser372 residue and preventing the cleavage process by proteases[ 20 ]. Furthermore, AMPK might suppress SREBP-1c expression through mTOR and LXRa [ 21 ]. After being regulated by AMPK, SREBP-1 binds to the sterol regulatory element in the nucleus and activates the transcription of other target genes, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD1), which are associated with fatty acid synthesis [ 22 – 24 ]. Although numerous studies have demonstrated that AMPK significantly influences lipogenesis genes, whether GADD45β regulates lipid metabolism through AMPK has not yet been reported. Our study revealed that protein levels associated with lipid synthesis in the AMPK pathway were significantly altered in vitro and in vivo under NAFLD conditions and that changes in GADD45β significantly augmented these changes. As Compound C (CC) is an ATP-competitive inhibitor of AMPK kinase activity[ 25 ], we further found that CC significantly blocked GADD45β-induced activation of the AMPK pathway, thereby blocking the role of GADD45β in inhibiting lipogenesis. These results revealed that GADD45β may ameliorate lipid metabolism dysfunction via the AMPK/SREBP-1 pathway in NAFLD. Even though GADD45β affects the AMPK signaling pathway, the activity of AMPK is regulated primarily by phosphorylation, and GADD45β does not possess phosphatase activity. In addition, since GADD45β is only a scaffold protein and does not directly participate in the transcriptional regulation of any targets, we further sought to identify the downstream factors that might be influenced by GADD45β. Additional research revealed that SIRT1 is one of the targets bound by GADD45β and further influences the AMPK/SREBP-1 pathway, which is the key finding of our study to discuss how GADD45β regulates lipogenesis in NAFLD. SIRT1 is a class III family of histone deacetylases whose reactions require nicotinamide adenine (NAD+) to concurrently deacetylate histones and nonhistones from proteins involved in multiple metabolic processes and stress responses[26; 27]. It is widely expressed in several organ cells, including the brain, adipose tissue, kidneys, pancreas, endothelium, spleen, skeletal muscle and liver. Furthermore, its expression is known to be involved in several diseases, including metabolic diseases and agerelated diseases[28]. SIRT1 can regulate multiple metabolic processes, including fat cell accumulation and maturation, lipid metabolism in the liver, systemic inflammation, nutrient sensing, circadian rhythms and especially lipogenesis [29]. It has been characterized as the ‘master of metabolic regulators’ because of its pivotal role in maintaining the homeostasis of lipid metabolism by affecting several proteins involved[30; 31]. Previous studies have demonstrated that SIRT1 can inhibit many lipogenesis enzymes, such as SREBP-1c, which act as key regulators and abolish the perturbation of hepatic lipid metabolism[32–34]. SIRT1 can also inhibit the activity of ACC and FASN to regulate lipogenesis in NAFLD[35]. More importantly, AMPK, the natural regulator of SREBP-1, is also affected by SIRT1 through an indirect mechanism involving the deacetylation of its upstream kinase[36]. SIRT1 can activate the AMPK pathway to amplify the ability of AMPK to maintain the homeostasis of lipid metabolism[37; 38]. These findings are in line with several studies demonstrating that SIRT1 activators can alleviate fatty liver in NAFLD patients, and demonstrate that SIRT1 plays a prominent role in the development of lipid-related diseases through the AMPK signaling pathway[39; 40]. In this study, by disrupting the expression of SIRT1, we determined that the impact of GADD45β on hepatic lipid synthesis is mediated through the SIRT1/AMPK signaling pathway. To further elucidate the role of GADD45β in the progression of NAFLD, we confirmed the interaction between GADD45β and SIRT1 in both animal and cellular models of hepatic steatosis. Additionally, the MCD diet significantly downregulated the expression level of SIRT1 in hepatocytes, which is consistent with previous findings. Interestingly, in GADD45β liver-specific knockdown mice, the expression level of SIRT1 was inhibited, and lipogenesis genes were significantly increased. In vitro, we also revealed that the overexpression of GADD45β significantly reversed the reduction in SIRT1 and the increase in lipogenesis gene levels caused by fatty acid treatment in HepG2 cells. These results suggest that GADD45β may be involved in the progression of hepatic steatosis by influencing the expression of SIRT1 and subsequently affecting key lipogenesis genes downstream. Another novel finding of our study is that GADD45β is linked to the ubiquitination and stabilization of SIRT1. To better understand how GADD45β influences the SIRT1/AMPK pathway, we found that GADD45β not only interacts with SIRT1 but also significantly modulates its expression levels and activity states. Previous studies have shown that SIRT1 can be regulated through ubiquitin‒proteasome degradation and that the regulation of SIRT1 ubiquitination plays a crucial role in its stability. Ubiquitination is a key signal for protein degradation. Ubiquitin ligases catalyze the covalent linkage of ubiquitin to various substrates. Multiple ubiquitin ligases have been shown to directly interact with specific substrate proteins to initiate and extend polyubiquitin chains. In NAFLD, SIRT1 acts as a substrate of E3 ubiquitin ligases, which can be subjected to K48-linked ubiquitination and degradation, thereby exacerbating hepatic steatosis[ 41 ]. A large-scale proteomic study revealed that SIRT1 could be ubiquitinated by MDM2 in response to oxidative stress-induced cell senescence[ 42 ], ubiquitinated by SMURF2 to inhibit cell proliferation and tumor formation[ 43 ], ubiquitinated by CUL4 to promote cancer cell autophagy[ 44 ], and ubiquitinated by COP1 to exacerbate lipid toxicity[ 45 ]. Our study first confirmed that GADD45β acts as a regulator of SIRT1 ubiquitination. GADD45β intervention effectively attenuated the increase in SIRT1 ubiquitination, thereby increasing SIRT1 protein expression and preventing its proteasomal degradation, which has never been reported before. Although our study revealed that GADD45β can bind to SIRT1 and influence its ubiquitination process, GADD45β itself is not a ubiquitin ligase. Previous studies have suggested that the binding of specific adaptor proteins can add multiple regulatory possibilities to ubiquitin ligase activity[ 46 ]. Ubiquitin ligases form ubiquitin ligase complexes with substrate receptors or adaptor proteins, which then catalyze the ubiquitination and degradation of substrate proteins[ 47 ]. Therefore, we speculate that GADD45β may act as an adaptor protein to promote ubiquitin ligase-mediated ubiquitination of SIRT1. However, the specific ubiquitin ligases involved in this process require further investigation. Additionally, the specific ubiquitination site on SIRT1 that is affected by GADD45β warrants further exploration. In conclusion, this investigation underscores the pivotal role of GADD45β in modulating hepatic lipogenesis, specifically through its interaction with SIRT1, increasing SIRT1 deubiquitylation and activation of the SIRT1 signaling pathway. These results indicate that GADD45β acts as a critical regulator, enhancing the stability of SIRT1 and subsequently inhibiting lipogenesis. These findings not only provide novel insights into the molecular mechanisms underlying lipogenesis in the liver but also highlight GADD45β as a potential therapeutic target for the management of MAFLD. Given the increasing incidence of metabolic liver disorders, further exploration of GADD45β function may yield significant advancements in the development of targeted interventions aimed at mitigating the impact of these conditions. Declarations Funding This work was supported by the Shanghai Sixth People’s Hospital Clinic Research Project (ynhg202103) to Yuanyuan Xiao; Foundation of Shanghai University of Medicine and Health Sciences (SSF-23-14-001) to Chaoyu Zhu; Fundamental Research Funds for the Central Universities (24X010301321) to Qianqian Wang; Institutional Project of Shanghai Sixth People’s Hospital (ynhglg202405) to Xinyi Wang; Shanghai Municipal Science and Technology Commission Project (20ZR1442500) to Li Wei. All the funding sources were not involved in the study design; collection, analysis, and interpretation of data; writing of the manuscript; and decision to submit the article for publication. Author Contribution Conceptualization, Yuanyuan Xiao,Renjie Wang, Chaoyu Zhu,Jun Yi and Li Wei; Data curation, Yuanyuan Xiao,Renjie Wang and Li Wei; Formal analysis, Yuanyuan Xiao,Renjie Wang and Chaoyu Zhu; Funding acquisition,Yuanyuan Xiao, Chaoyu Zhu,Qianqian Wang, Xinyi Wang and Li Wei ; Investigation,Yuanyuan Xiao,Renjie Wang, Chaoyu Zhu, Qianqian Wang, Xinyi Wang, Wenjing Song, Shouxia Li and Fusong Jiang ; Methodology,Yuanyuan Xiao,Renjie Wang, Jun Yin and Li Wei; Project administration,Jun Yin and Li Wei ; Supervision, Dingkun Gui and Youhua Xu; Writing—original draft, Yuanyuan Xiao and Renjie Wang; Writing—review & editing, Jun Yin and Li Wei. All authors have read and agreed to the published version of the manuscript. Data Availability RNA sequencing data were obtained from the GEO database (GSE33814, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE33814 and GSE48452, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE48452). 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14:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6769255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6769255/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24864-1","type":"published","date":"2025-11-07T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85574372,"identity":"51c99bed-7041-4261-b721-9ce8160aaea7","added_by":"auto","created_at":"2025-06-27 17:40:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4201409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatic GADD45β expression is decreased in NAFLD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) GADD45β mRNA expression levels in two different NAFLD GEO datasets. (C) GADD45β mRNA expression levels in the livers of control diet-fed (CON) and methionine- and choline-deficient (MCD) diet-fed mice (n=6). (D) GADD45β protein expression levels in the livers of control diet-fed mice (CON) and methionine- and choline-deficient diet-fed mice (MCD) (n=6). \u003csup\u003ens\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05, *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Reslut1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/e00682abd87882567fd7aa27.jpg"},{"id":85574381,"identity":"fcd90608-e121-49e7-b969-fc9f54f93f73","added_by":"auto","created_at":"2025-06-27 17:40:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13830608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatic knockdown of GADD45β aggravated steatohepatitis in MCD diet-fed mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic Diagram of Animal Experimental Procedures (B, C) GADD45βexpression in liver tissues of GADD45β hepaticknockdown mice comparedwith the control group (n=6). (C-E) Liver weight, liver weight/body weight ratio(liver index) and liver TG levels of each group (n=6). (F-J) Serum TG, TC ALT, AST and γGT levels in each group (n=5). (K, L) Representative images of H\u0026amp;E and Oil Red O staining of liver sections from each group (n=5); scale bar, 100 mm. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Reslut2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/fd5a2eaa0ea2b8fe03eb4122.jpg"},{"id":85574375,"identity":"9f80478f-f86d-4a62-a676-77c832aabfe2","added_by":"auto","created_at":"2025-06-27 17:40:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5139196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatic knockdown of GADD45βincreased hepatic lipogenesis gene expression in MCD diet-fed mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Quantitative PCR analysis of the mRNA expression levels of lipid metabolism-related genes in the liver tissue of different groups (n=6). (B) Western blot of ACC1, FASN, nuclear SREBP1(SREBP1-N) and cytoplasmic SREBP1(SREBP1-P) in the liver tissues of different groups (n=6). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Reslut3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/617201c7551b6d73a3c517de.jpg"},{"id":85574377,"identity":"03cb35e6-a789-44c1-944b-dc6cbfc7e035","added_by":"auto","created_at":"2025-06-27 17:40:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9448980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGADD45β inhibited palmitic acid-induced steatosis and lipogenesis in hepatocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Oil Red O staining and area quantification of GADD45β-overexpressing or GADD45β-knockdown HepG2 cells after treatment with PA. (B-E) Lipid drops, intracellular TG levels and relative quantification of GADD45β-overexpressing or GADD45β-knockdown HepG2 cells after treatment with PA. (F-H) Protein and mRNA expression levels of FASN, ACC1, and SREBP1 in PA-induced GADD45β-overexpressing HepG2 cells (n=3). (I-K) Protein and mRNA expression levels of FASN, ACC1,and SREBP1 in PA-induced GADD45β-knockdown HepG2 cells (n=3).\u003c/p\u003e","description":"","filename":"Reslut4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/d7ac5b0068b3b696d7f5e9e0.jpg"},{"id":85574460,"identity":"0d9cbcd2-19fe-4f41-810d-da40913d3c61","added_by":"auto","created_at":"2025-06-27 17:48:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGADD45β mediated lipogenesis via the SIRT1/AMPK/SREBP1 pathway in hepatocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of p-AMPK, AMPK and SIRT1 expression in liver tissues from GADD45β-knockdown and control mice (n=6). (B) Western blot of p-AMPK, AMPK and SIRT1 expression in GADD45β-knockdown HepG2 cells treated with PA (n=6).(C) Western blot of p-AMPK, AMPK and SIRT1 expression in GADD45β-overexpressing HepG2 cells treated with PA (n=6). (D-E) Representative IHC staining and relative quantification of p-AMPK and SIRT1 in liver sections from theindicated individuals. (n=6). (F) Western blot analysis of SIRT1 expression in PA-induced HepG2 cells infected with LV-shSIRT1 or LV-shControl. (G) SIRT1, p-AMPK/AMPK,and SREBP-1 expression in PA-induced SIRT1 knockdown and GADD45β-overexpressing HepG2 cells (n=6). (H) Western blot of p-AMPK/AMPK expression in PA-induced HepG2 cells treated withmultiple doses of Compound C. (I) p-AMPK/AMPK and SREBP-1 expression in PA-induced GADD45β-overexpressing and Compound C-treated HepG2 cells (n=6).\u003c/p\u003e","description":"","filename":"Reslut5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/164b941be201d709ff13d74f.jpg"},{"id":85574374,"identity":"8248b545-ba7f-40ec-81ae-76fd9f877cdb","added_by":"auto","created_at":"2025-06-27 17:40:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2193847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGADD45β directly interacted with SIRT1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GADD45βand SIRT1 mRNA expression in HepG2 cells after the overexpression of GADD45β. (B) Coimmunoprecipitation (co-IP) of GADD45β with SIRT1 proteins in HepG2 cells. The co-IP and western blot data shown are representative of at least 3 independent experiments with consistent results.\u003c/p\u003e","description":"","filename":"Reslut6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/6e6e69ffb8ec60f0858434f3.jpg"},{"id":85574462,"identity":"bec100f2-4d9a-4a41-8c10-f1420f7b1e15","added_by":"auto","created_at":"2025-06-27 17:48:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6307269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGADD45β facilitates SIRT1 stability by inhibiting its ubiquitin‒proteasome degradation. \u003c/strong\u003e(A) Western blots of PA-induced GADD45β-overexpressing HeG2 cells treated with cycloheximide (CHX; 20 μg/ml) for the indicated time periods and semiquantification of SIRT1 levels. (B) Western blots of PA-induced GADD45β-knockdown HeG2 cells treated with cycloheximide (CHX; 20 μg/ml) for the indicated time periods and semiquantification of SIRT1 levels. (C) Coimmunoprecipitation (co-IP) of SIRT1 with ubiquitin proteins in PA-induced GADD45β-overexpressing HepG2 cells after treatment with 10 µMMG132 for 8 hours (n=3). The Co-IP and western blot data shown are representative of 3 independent experiments with consistent results.\u003c/p\u003e","description":"","filename":"Reslut7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/715f6bc44e8f1d96ba97fbd6.jpg"},{"id":85574461,"identity":"4b57e8ab-f128-4284-a5ea-49f91a1d003b","added_by":"auto","created_at":"2025-06-27 17:48:04","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":79997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model of GADD45β in the regulation of hepatic steatosis. GADD45β expression is decreased in hepatocytes fed an MCD diet. \u003c/strong\u003eA decrease in GADD45β does not interact with Sirtuin 1 (SIRT1) and is subsequently degraded by ubiquitination. This results in increased lipogenesis and thereby synergistically aggravates fatty acid accumulation and hepatic steatosis.\u003c/p\u003e","description":"","filename":"Reslut8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/81e00b5432aa52901426601c.jpg"},{"id":95564078,"identity":"698b860a-a486-4f4c-8da1-cdde62161415","added_by":"auto","created_at":"2025-11-10 16:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":42713811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/e5f7b79b-d450-4819-b9b2-05e4708b9a90.pdf"},{"id":85574750,"identity":"f9418b57-9aab-4614-96b6-01a0fe9782f7","added_by":"auto","created_at":"2025-06-27 17:56:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":567635,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6769255/v1/a20d75c129946cf0b8982d03.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"GADD45β Inhibits Hepatic Lipogenesis through the AMPK/SREBP1 Pathway via Reducing the ubiquitination-mediated Degradation of SIRT1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing prevalence of nonalcoholic fatty liver disease (NAFLD) is a significant global health concern. Over the last decade, NAFLD has become the predominant cause of chronic liver disease in many parts of the world and affects more than 25% of the global population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. NAFLD is often linked to obesity, insulin resistance, and metabolic syndrome [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This condition is characterized by excessive lipid accumulation in hepatocytes, which can progress from simple steatosis to more severe forms, such as steatohepatitis and cirrhosis. The intricate mechanisms underlying hepatic lipid metabolism are critical for understanding the pathophysiology of NAFLD, yet they remain inadequately explored. De novo lipogenesis may play a crucial role in the development of NAFLD[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Lipogenesis converts excessive glucose or fructose into fatty acids and triglycerides[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and is a normal process for maintaining homeostasis in the body; increased activation of this process may cause hepatic steatosis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the inhibition of lipogenesis is highly desirable as a therapeutic target for lipid metabolism-related diseases. The literature has focused primarily on various signaling pathways involved in lipid regulation, such as the roles of SIRT1 and AMPK, while the specific contributions of GADD45β in this context are not well characterized[6; 7].\u003c/p\u003e \u003cp\u003eGrowth arrest and DNA damage-inducible 45β (GADD45β), a member of the GADD45 family, is known to participate in cellular stress responses, DNA repair, and apoptosis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recent studies have highlighted its potential involvement in glucose and lipid metabolism, suggesting that GADD45β may be a new key regulator in the pathogenesis of NAFLD[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, whether GADD45β takes part in the process of lipogenesis to affect NAFLD remains unclear. The current research landscape reveals a significant gap regarding the mechanistic role of GADD45β in hepatic lipid metabolism, particularly its interactions with established pathways such as SIRT1/AMPK/SREBP1 signaling, which are crucial for maintaining metabolic homeostasis[10; 11].\u003c/p\u003e \u003cp\u003eIn this study, we provide evidence that SIRT1 is the target substrate of GADD45β and identify an important correlation between them in NAFLD. GADD45β interacts with SIRT1 and deubiquitylates it, inhibits its proteasomal degradation and increases its stability. GADD45β deficiency decreased the activity of the SIRT1 target AMPK, resulting in lipogenesis exacerbation. Our findings highlight that GADD45β potentially plays a critical role in regulating the stability of SIRT1 and functions to reduce lipogenesis progression, providing new insights and potential therapeutic strategies for NAFLD in the future.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eCycloheximide (CHX, Cat# M4879) was purchased from AbMole Bioscience, Inc. MG132 (Cat# S2619) was purchased from Selleck Chemicals. Palmitic acid (PA, Cat# S-A9165-5G) was purchased from Sigma‒Aldrich. Compound C (CC, Cat# HY-13418) was purchased from Med ChemExpress. Antibodies against FASN (Cat# ARG55898, 1:1000), HSP90 (Cat# ARG55781, 1:1000), Flag Tag (Cat# ARG62342, 1:1000) and β-Actin (Cat# ARG65683, 1:1000) were purchased from Arigo. Antibodies against ubiquitin (Cat# 3936, 1:400), AMPK (Cat# 2532S, 1:1000) and p-AMPK (Cat# 2531S, 1:1000) were purchased from Cell Signaling Technology. Antibodies against Sirt1 (Cat# ab189494, 1:1000), SREBP1 (Cat# ab28481, 1:1000), and ACC1 (Cat# ab45174, 1:1000) were purchased from Abcam. Antibodies against GADD5β (Cat#sc377311, 1:1000) was purchased from Santa Cruz. HRP-conjugated secondary antibodies against mouse (Servicebio, GB23301) or rabbit (Servicebio, GB23303) were used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal treatment\u003c/h3\u003e\n\u003cp\u003e The experiments involving mice were approved by the Animal Research Ethics Committee of Shanghai Sixth Peoples\u0026rsquo; Hospital in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experiments were conducted in accordance with the guidelines of Animal Research and the study adhered to the ARRIVE guidelines ensuring compliance with ethical standards for animal research[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEight-week-old C57BL/6J mice weighing 23\u0026ndash;28g were purchased from Shanghai Sippe-Bk Lab Animal Co., Ltd. (Shanghai, China). All the animals were maintained in a specific pathogen-free barrier facility under a 12 h light/dark cycle. After a week of adaptation, the mice were fed an MCD diet (A02082002B, Research Diets, USA) for 4 weeks to establish NASH models, and their corresponding normal chow diet (CON) served as a control. Adeno-associated virus (AAV)-delivered short hairpin RNA pscAAV-U6-shNC-CMV-EGFP-tWPA was constructed via an AAV-8 vector system (OBiO Technology, Shanghai, China) as a control group (AAV-shNC) in the liver. Briefly, AAV-shGADD45β particles targeting GADD5β (shGADD5β) were generated via pscAAV-U6-shGADD45β-CMV-EGFP-tWPA vectors (AAV-shGADD45β). The shRNAs had the following sequence: GADD5β (59-GCGACAATGACATTGACATCG-39). To knock down hepatic GADD5β expression, 2\u0026times;10\u003csup\u003e11\u003c/sup\u003e PFU per mouse were delivered into MCD mice for 14 d. All viruses were purified via the cesium chloride method, dialyzed in PBS containing 10% glycerol, and administered to the mice via tail vein injection. Finally, all the mice were euthanized using an overdose anesthesia method of intraperitoneal injection anesthesia with 3% sodium pentobarbital 60 mg/kg body weight after 12 h of fasting, and the liver were dissected and snap-frozen in liquid nitrogen. Tissues and serum were kept at \u0026minus;\u0026thinsp;80\u0026deg;C until lysates were obtained and western blot or RNA analyses performed. The animal experiments were strictly adhered to the 3Rs principles (Replacement, Reduction, and Refinement). Additionally, predefined humane endpoints are established to promptly euthanize animals showing severe distress or irreversible suffering, ensuring their welfare is prioritized throughout the study.\u003c/p\u003e\n\u003ch3\u003eBiochemical assays\u003c/h3\u003e\n\u003cp\u003eBlood samples collected from the mice were allowed to stand at room temperature for 30 minutes, followed by centrifugation at 4\u0026deg;C and 5000 rcf for 5 minutes. The supernatant was subsequently centrifuged at 10000 rcf at 4\u0026deg;C for 5 minutes. The serum was carefully transferred into clean 1.5 mL EP tubes and stored at -20\u0026deg;C for further use. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), and gamma-glutamyltransferase (γ-GT) were measured via a fully automated biochemical analyzer.\u003c/p\u003e\n\u003ch3\u003eH\u0026E and Oil Red O staining\u003c/h3\u003e\n\u003cp\u003eLiver tissues were fixed overnight in 10% formalin and then embedded in paraffin. Sections with a thickness of 4 \u0026micro;m were stained with hematoxylin and eosin (H\u0026amp;E). For Oil Red O staining, frozen liver sections (7 \u0026micro;m) were fixed in 4% paraformaldehyde for 10 min, washed with deionized water, stained with oil red O (SigmaAldrich) for 10 min at room temperature, washed in deionized water twice, and finally stained with hematoxylin for 30 s. Histological assessment was performed via an Olympus light microscope.\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eHepG2 cells were cultured in DMEM/HIGH GLUCOSE (Cat# SH30243.01, HyClone) supplemented with 10% fetal bovine serum (Cat#04-001-1ACS, Biological Industries) and 1% penicillin‒streptomycin (Cat#C0222, Beyotime) in a 5% CO2 incubator at 37\u0026deg;C. HepG2 cells were treated with fatty acid-free bovine serum albumin (BSA) or PA at a concentration of 400 \u0026micro;M for 24 h. Oil Red O was used for intracellular lipid droplet staining. Lipid staining was observed under a light microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003epSLenti-GADD5β-3xFlag (GADD5β) and control pSLenti-MCS-3xFlag (vector), pLenti-U6-siGADD5β-CMV (siGADD5β:GCATACTCCTTCCACGTTA) and control pSLenti-U6-shNC2-CMV (siNC:TTCTCCGAACGTGTCACGT) were constructed by OBiO Technology (Shanghai, China). When HepG2 cells were cultured in 35 mm plates at 70\u0026ndash;80%, solutions A and B were prepared, respectively, with 2.5 \u0026micro;g of plasmid, 2.5 \u0026micro;l of PLUS and 125 \u0026micro;l of Opti-Bertani medium for Solution A, and 5 \u0026micro;l of LTX and 125 \u0026micro;l of Opti-Bertani medium for Solution B. The A and B solutions were gently mixed and left to stand for 5 min. The solution was added dropwise to the medium of the HepG2 cells, which could be changed after 6\u0026ndash;8 h. The cells were collected for real-time qPCR and western blotting after transfection for 48 h. In this study, two lentivirus (LV) constructs, LentiCRISPRv2-SIRT1-sgRNA (LV-shSIRT1) and a blank vector (LV-shNC), were added to the medium of the HepG2 cells, which were subsequently fenghbio Biotechnology Co., Ltd. (Hunan, China). Upon reaching 80% confluence, the culture medium was supplemented with the virus mixture at an MOI of 50 for transfection. After 24 h of transfection, the serum-free Opti-MEM was replaced with fresh medium containing FBS, and puromycin (30 \u0026micro;g/mL) (HY-K1057, MedChemExpress) was applied to select successfully transfected cells. The culture was continued for 2 days, after which the cells were harvested and lysed for western blotting.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eTo prepare total protein extracts, liver tissues and HepG2 cells were lysed in RIPA buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1% NP40, and 0.1% SDS. Equivalent amounts of protein samples were denatured in loading buffer, resolved by 10\u0026ndash;12% SDS‒PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 h before being incubated with primary antibodies overnight at 4\u0026deg;C. The membranes were washed with PBS containing Tween-20 five times and incubated with secondary antibody for 1 h. The signals of the proteins were then visualized via an electrochemiluminescence system.\u003c/p\u003e\n\u003ch3\u003eCoimmunoprecipitation (Co-IP)\u003c/h3\u003e\n\u003cp\u003eThe cells were lysed on ice for 30 min with IP lysis solution (Beyotime, China) containing phosphatase inhibitors as well as protease inhibitors, and the supernatant was obtained by centrifugation. After quantification and analysis, the cells were incubated overnight at 4\u0026deg;C with the corresponding antibodies or control IgG, and then 20 \u0026micro;l of A/G agarose beads was added and incubated at 4\u0026deg;C for 4 h. After sequential elution with different buffers, the supernatant was collected by instantaneous centrifugation for immunoprecipitation experiments.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (qRT‒PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells or liver tissues via TRIzol reagent and reverse transcribed into cDNA via PrimeScript\u0026trade; RT Master Mix (Cat# R433-01, Vazyme) according to the manufacturer\u0026rsquo;s instructions. qRT‒PCR was performed with SYBR Premix Ex Taq\u0026trade; (Cat#Q312-02, Vazyme) reagents, and the results were analyzed via Light Cycler 480 software (Roche Diagnostics GmbH, Mannheim, Germany). The relative levels of target gene expression were calculated via the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method, with GAPDH used as a control. The primers used for RT‒PCR are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eThe calculations were carried out with Graph Pad InStat Software (San Diego, CA, USA). All the data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. For animal and cellular experiments, a two-tailed unpaired Student\u0026rsquo;s t test was performed to compare two groups. The western blot and morphological images are representative of at least three experiments with similar results. Statistical significance was determined by nonparametric procedures via Student\u0026rsquo;s t test or ANOVA for analysis of variance. The normality of the distributions of the quantitative variables was assessed via the Kolmogorov‒Smirnov test. P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHepatic GADD45β expression decreased in NAFLD\u003c/h2\u003e \u003cp\u003eBy accessing the human GEO database, two NAFLD datasets, GSE33814 and GSE48452, were included in this study, which included 37 healthy controls, 39 NAFLD subjects and 31 NASH subjects. Notably, GADD45β was consistently downregulated in NAFLD and NASH patients in both datasets at the mRNA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). To further confirm the downregulation of GADD45β in NASH, GADD45β mRNA and protein expression levels in mouse liver samples were evaluated by western blot and qPCR. Compared with those in the control group, GADD45β levels were substantially lower in the livers of MCD diet-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHepatic knockdown of GADD45β exacerbates steatohepatitis in MCD mice\u003c/h2\u003e \u003cp\u003eFurther study of the dysregulation of hepatic GADD45β in C57BL/6J mice and human subjects revealed that GADD45β could affect steatohepatitis. MCD mice were injected with GADD45β-specific adenoviral associate shRNA via the tail vein, which inhibited endogenous GADD45β expression in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A). At 4 weeks after injection, GADD45β expression in the liver was significantly lower in GADD45β-knockdown mice than in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. B). Notably, there was no significant difference in the serum levels of TC, TG, AST and ALT between GADD45β-knockdown and control MCD diet-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. F-I), whereas the serum levels of γGT in the AAV-shGADD45β-MCD mice were greater than those in the control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. J), indicating that GADD45β knockdown might aggravate liver injury. Although there was no significant change in weight, the liver weight, liver index and liver TG levels were significantly increased in the GADD45β-knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). H\u0026amp;E staining and Oil Red O staining also revealed significant hepatic steatosis in GADD45β-knockdown MCD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L). All of the above data suggest that the knockdown of hepatic GADD45β aggravates liver steatohepatitis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHepatic knockdown of GADD45β increased hepatic lipogenesis gene expression in MCD diet-fed mice\u003c/h2\u003e \u003cp\u003eComparison of the transcription of lipogenesis genes in the livers of GADD45β hepatic knockdown MCD diet-fed mice. MCD feeding significantly upregulated hepatic Srebp1 mRNA expression, and liver-specific knockdown of GADD45β further increased Srebp1 mRNA expression. Liver-specific knockdown of GADD45β significantly upregulated the mRNA levels of two other hepatic lipogenesis genes, Fasn and Acc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, the hepatic mRNA levels of genes related to fatty acid oxidation, fatty acid transport and lipolysis were unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, the increase in the hepatic protein levels of SREBP1, FASN and ACC1 in the GADD45β hepatic knockdown mice shown by Western blotting was consistent with the increase in the mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.B). These results showed that liver-specific knockdown of GADD45β significantly upregulated hepatic lipogenesis gene expression in MCD diet-fed mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGADD45β inhibited palmitic acid-induced steatosis and lipogenesis in hepatocytes\u003c/h2\u003e \u003cp\u003eTo confirm the function of GADD45β in hepatocytes, GADD45β expression was upregulated in HepG2 cells via GADD45β plasmid transfection. Western blot analysis revealed a significant increase in GADD45β protein levels in HepG2 cells after transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. F), HepG2 cells were treated with 400 \u0026micro;M palmitic acid (PA) for 24 h to simulate hepatocyte steatosis, and Oil Red O staining revealed that GADD45β overexpression significantly decreased intracellular lipid accumulation. In addition, PA stimulation significantly elevated the intracellular TG content. Specifically, GADD45β overexpression blocked the effects of PA on the intracellular TG content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A-C). These results indicate that under PA-induced hepatocyte steatosis, increased GADD45β levels play an important role in regulating hepatosteatosis. Moreover, the effect of lipogenesis via GADD45β in PA-treated hepatocytes was also evaluated. As expected, the results of the quantitative RT‒PCR and WB experiments confirmed that the overexpression of GADD45β significantly reduced the PA-induced levels of lipogenesis regulators, such as SREBP1, ACC1, and FASN, in HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF‒H).\u003c/p\u003e \u003cp\u003eAdditionally, we validated the conclusions from the opposite perspective. In HepG2 cells, selected siRNAs were used to downregulate the expression level of GADD45β, after which hepatic steatosis was induced with PA. The results revealed that GADD45β knockdown significantly increased the expression levels of lipogenesis-related genes after PA induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. I-K), thereby exacerbating lipid accumulation and steatosis in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, D, E). These findings collectively suggest that GADD45β exerts a protective effect against hepatic steatosis by inhibiting lipogenesis within hepatocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGADD45β mediated lipogenesis via the SIRT1/AMPK/SREBP1 pathway in hepatocytes\u003c/h2\u003e \u003cp\u003eSirtuin 1 (SIRT1) is a member of the mammalian histone deacetylase sirtuin family and plays a pivotal role in hepatic steatosis and lipogenesis via the phosphorylation of AMPK. To study whether the inhibition of GAADD45β could influence the Sirt1/AMPK pathway, GADD45β hepatic knockdown MCD mice were used in vivo to further examine whether it had effects on the SIRT1/AMPK pathway. The immunohistochemical results revealed that the expression levels of SIRT1 and p-AMPK were significantly lower in the shGADD45β MCD mice than in the shNC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). The western blot results revealed that SIRT1 expression and the p-AMPK/AMPK ratio were significantly lower in the GADD45β hepatic knockdown group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, the suppression of GAADD45β had negative effects on the SIRT1/AMPK pathway in PA-treated HepG2 cells and reduced the protein levels of Sirt1 and p-AMPK/AMPK in parallel with SREBP1 to a certain extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, in HepG2 cells overexpressing GADD45β, the SIRT1/AMPK pathway was significantly increased after treatment with PA, and SREBP1 levels were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results indicate that GADD45β expression could mediate lipogenesis via the SIRT1/AMPK pathway in hepatocyte steatosis.\u003c/p\u003e \u003cp\u003eIt was hypothesized that SIRT1 may be a major component of the process by which GADD45β regulates the p-AMPK/AMPK signaling pathway. Western blotting analyses revealed that GADD45β rescued the reduction in SIRT1 protein levels in HepG2 cells stimulated with PA. In HepG2 cells, lentiviruses were used to knock down SIRT1 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. F), increased the p-AMPK/AMPK ratio promoted by GADD45β, and reversed the downregulatory effects of GADD45β on SREBP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). To assess whether AMPK inhibition could diminish the protective effect of GADD45β, studies were also conducted in which HepG2 cells were treated with compound C, an inhibitor of AMPK, for 4 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). The results showed that Compound C significantly reduced the phosphorylation of AMPK. GADD45β overexpression in the Compound C- and PA-induced groups failed to prevent the increase in SREBP1 and did not rescue the decrease in p-AMPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These findings underscore the significant impairment of the SIRT1/AMPK signaling pathway in lipogenesis. These data indicate that SIRT1/AMPK mediate lipogenesis and that GADD45β has a defensive effect through this signaling pathway in hepatocytes in NAFLD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGADD45β directly interacted with SIRT1\u003c/h2\u003e \u003cp\u003eAlthough the overexpression of GADD45β increased the SIRT1 protein content, as indicated by the data above, it did not alter the SIRT1 mRNA levels, as determined by RT‒qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To elucidate the molecular mechanisms by which GADD45β regulates SIRT1, we predicted that GADD45β might be a binding partner of SIRT1. To test this hypothesis, coimmunoprecipitation tests were used to investigate the interaction of GADD45β and SIRT1 in PA-induced HepG2 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. B), GADD45β was pulled down by SIRT1. In a mutual pulldown experiment using an anti-GADD45β antibody, GADD45β also pulled down SIRT1. Indeed, when GADD45β was pulled down by an anti-GADC45β antibody, SIRT1 was detected via coimmunoprecipitation via western blotting. Taken together, these data suggest that GADD45β may directly interact with SIRT1 and further regulate exercise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGADD45β upregulated SIRT1 levels by inhibiting its ubiquitin‒proteasome degradation\u003c/h2\u003e \u003cp\u003eTo exclude the influence of protein synthesis on SIRT1 levels and because SIRT1 can be regulated through ubiquitination to promote its stabilization, the possible role of GADD45β-mediated SIRT1 was further investigated. To determine whether GADD45β could inhibit SIRT1 degradation, GADD45β-encoding plasmids were transfected into HepG2 cells, and the SIRT1 protein levels were measured after treatment with CHX, a protein synthesis inhibitor. As expected, GADD45β significantly shortened the half-life of SIRT1, which was decreased by CHX (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Conversely, the knockdown of GADD45β decreased SIRT1 protein stability in PA-induced HepG2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. B), suggesting that GADD45β-mediated inhibition of SIRT1 degradation, rather than promoting protein synthesis, contributes to the increase in SIRT1 levels in response to GADD45β.\u003c/p\u003e \u003cp\u003eThe K48-linked ubiquitin‒proteasome system (UPS) is a major intracellular protein degradation mechanism. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. C), intervention with MG132, a proteasome inhibitor, and GADD45β overexpression significantly reversed the reduction in SIRT1 protein levels after exposure to PA. These data seem to favor the involvement of the UPS in SIRT1 degradation. To address this issue, the ubiquitination levels of SIRT1 were also examined through immunoprecipitation. The results revealed increased ubiquitination of SIRT1 in HepG2 cells upon exposure to PA, whereas total SIRT1 protein expression was downregulated. However, GADD45β transfection reversed this effect. These results indicate that GADD45β increases SIRT1 levels by inhibiting its ubiquitin‒proteasome degradation. These experimental results suggest that the expression of GADD45β in the hepatocytes of NASH patients and MCD-fed mice is decreased, which reduces the interaction between GADD45β and SIRT1. Subsequently, SIRT1 is degraded via the ubiquitin\u0026ndash;proteasome pathway. The reduction in SIRT1 prevents the activation of phosphorylated AMPK protein, which in turn exacerbates SREBP1-mediated de novo fatty acid synthesis. This leads to increased lipogenesis, thereby synergistically aggravating fatty acid accumulation and hepatic steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNonalcoholic fatty liver disease (NAFLD) has emerged as a significant global health concern due to its complex pathogenesis, which involves multiple factors, such as insulin resistance, oxidative stress, and inflammatory pathways. With the increasing prevalence of obesity and metabolic syndrome, NAFLD is now recognized not only as a hepatic disorder but also as a major risk factor for cardiovascular diseases, type 2 diabetes, and other metabolic disorders. This underscores the urgent need to understand its mechanisms and develop effective therapeutic strategies to address this condition [13; 14]. In this study, we identified a novel factor, GADD45β, which plays a significant role in the development and progression of NAFLD. GADD45β is a member of the GADD45 family and is an acidic protein that responds to cellular stress. It plays crucial regulatory roles in cellular functions such as DNA repair, cell cycle regulation, apoptosis, inflammation, and stress responses through protein‒protein interactions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, numerous studies have demonstrated the involvement of GADD45β in lipid metabolism. Dong et al. demonstrated that GADD45β levels were downregulated in multiple clinical databases and a murine model of nonalcoholic fatty liver disease (NAFLD). They elucidated the beneficial role of GADD45β in mitigating excessive lipid accumulation and insulin resistance in NAFLD induced by a high-fat diet (HFD) in mice [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Fuhrmeister et al. reported that under fasting conditions, GADD45β facilitates the cytoplasmic retention of fatty acid binding protein 1 by binding to it, thereby inhibiting the uptake of fatty acids by hepatocytes and enhancing lipid metabolism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, research by Kim et al. revealed that GADD45β significantly suppresses hepatic gluconeogenesis by enhancing the protein stability and transcriptional activity of forkhead box protein O1 (FoxO1). They also noted an increase in the expression of genes associated with lipogenesis in GADD45β knockout mice. Furthermore, GADD45β has been identified as an inducible coactivator of the constitutive androstane receptor (CAR), promoting rapid liver growth. Cai et al. reported that the effect of CAR on energy metabolism also depends on Gadd45β. In HFD-induced obese GADD45β knockout mice, the reduction in body weight gain and improvement in insulin sensitivity caused by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) were markedly blunted by Gadd45β knockout. Mechanistically, these effects are related to the inhibition of lipogenesis in hepatocytes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], however no further mechanistic studies have been conducted. In the present study, we observed that both the mRNA and protein expression levels of GADD45β gradually decreased in human and mouse NAFLD livers as the disease progressed from NAFLD to NASH. By constructing a liver-specific GADD45β gene-knockdown methionine‒choline-deficient (MCD) diet mouse model, we found that, compared with that in control diet-fed mice, liver steatosis in the GADD45β-knockdown group was exacerbated. Further studies revealed that hepatic lipid deposition was related to the upregulation of lipogenesis genes, including SREBP-1, FASN and ACC, which is consistent with the findings of other previous studies. In vitro experiments revealed that the overexpression of GADD45β in HepG2 cells led to a significant decrease in the expression of these lipogenesis genes upon fatty acid treatment. Conversely, with GADD45β knockout, the expression of these genes increased inversely. These data indicate that GADD45β is involved in the occurrence and development of hepatic lipid metabolism and fat deposition in NAFLD.\u003c/p\u003e \u003cp\u003eTo clarify how GADD45β influences hepatic lipid metabolism, we further investigated the interaction between GADD45β and a key regulator of lipogenesis, adenosine monophosphate (AMP)-activated protein kinase (AMPK). AMPK is a heterologous trimeric protein kinase that provides energy for cells via lipid metabolism. Studies have demonstrated that AMPK plays a key role in adjusting hepatic fatty acid oxidation, inhibiting cholesterol and TG synthesis, and repressing lipogenesis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among these, sterol regulatory element binding protein-1c (SREBP-1c) is one of the key lipogenesis transcription factors in the liver that is regulated by AMPK [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. AMPK mainly reduces lipogenic gene expression by inhibiting the activation of SREBP-1c through phosphorylation at the Ser372 residue and preventing the cleavage process by proteases[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, AMPK might suppress SREBP-1c expression through mTOR and LXRa [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After being regulated by AMPK, SREBP-1 binds to the sterol regulatory element in the nucleus and activates the transcription of other target genes, such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD1), which are associated with fatty acid synthesis [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although numerous studies have demonstrated that AMPK significantly influences lipogenesis genes, whether GADD45β regulates lipid metabolism through AMPK has not yet been reported. Our study revealed that protein levels associated with lipid synthesis in the AMPK pathway were significantly altered in vitro and in vivo under NAFLD conditions and that changes in GADD45β significantly augmented these changes. As Compound C (CC) is an ATP-competitive inhibitor of AMPK kinase activity[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], we further found that CC significantly blocked GADD45β-induced activation of the AMPK pathway, thereby blocking the role of GADD45β in inhibiting lipogenesis. These results revealed that GADD45β may ameliorate lipid metabolism dysfunction via the AMPK/SREBP-1 pathway in NAFLD.\u003c/p\u003e \u003cp\u003eEven though GADD45β affects the AMPK signaling pathway, the activity of AMPK is regulated primarily by phosphorylation, and GADD45β does not possess phosphatase activity. In addition, since GADD45β is only a scaffold protein and does not directly participate in the transcriptional regulation of any targets, we further sought to identify the downstream factors that might be influenced by GADD45β. Additional research revealed that SIRT1 is one of the targets bound by GADD45β and further influences the AMPK/SREBP-1 pathway, which is the key finding of our study to discuss how GADD45β regulates lipogenesis in NAFLD. SIRT1 is a class III family of histone deacetylases whose reactions require nicotinamide adenine (NAD+) to concurrently deacetylate histones and nonhistones from proteins involved in multiple metabolic processes and stress responses[26; 27]. It is widely expressed in several organ cells, including the brain, adipose tissue, kidneys, pancreas, endothelium, spleen, skeletal muscle and liver. Furthermore, its expression is known to be involved in several diseases, including metabolic diseases and agerelated diseases[28]. SIRT1 can regulate multiple metabolic processes, including fat cell accumulation and maturation, lipid metabolism in the liver, systemic inflammation, nutrient sensing, circadian rhythms and especially lipogenesis [29]. It has been characterized as the \u0026lsquo;master of metabolic regulators\u0026rsquo; because of its pivotal role in maintaining the homeostasis of lipid metabolism by affecting several proteins involved[30; 31]. Previous studies have demonstrated that SIRT1 can inhibit many lipogenesis enzymes, such as SREBP-1c, which act as key regulators and abolish the perturbation of hepatic lipid metabolism[32\u0026ndash;34]. SIRT1 can also inhibit the activity of ACC and FASN to regulate lipogenesis in NAFLD[35]. More importantly, AMPK, the natural regulator of SREBP-1, is also affected by SIRT1 through an indirect mechanism involving the deacetylation of its upstream kinase[36]. SIRT1 can activate the AMPK pathway to amplify the ability of AMPK to maintain the homeostasis of lipid metabolism[37; 38]. These findings are in line with several studies demonstrating that SIRT1 activators can alleviate fatty liver in NAFLD patients, and demonstrate that SIRT1 plays a prominent role in the development of lipid-related diseases through the AMPK signaling pathway[39; 40]. In this study, by disrupting the expression of SIRT1, we determined that the impact of GADD45β on hepatic lipid synthesis is mediated through the SIRT1/AMPK signaling pathway. To further elucidate the role of GADD45β in the progression of NAFLD, we confirmed the interaction between GADD45β and SIRT1 in both animal and cellular models of hepatic steatosis. Additionally, the MCD diet significantly downregulated the expression level of SIRT1 in hepatocytes, which is consistent with previous findings. Interestingly, in GADD45β liver-specific knockdown mice, the expression level of SIRT1 was inhibited, and lipogenesis genes were significantly increased. In vitro, we also revealed that the overexpression of GADD45β significantly reversed the reduction in SIRT1 and the increase in lipogenesis gene levels caused by fatty acid treatment in HepG2 cells. These results suggest that GADD45β may be involved in the progression of hepatic steatosis by influencing the expression of SIRT1 and subsequently affecting key lipogenesis genes downstream.\u003c/p\u003e \u003cp\u003eAnother novel finding of our study is that GADD45β is linked to the ubiquitination and stabilization of SIRT1. To better understand how GADD45β influences the SIRT1/AMPK pathway, we found that GADD45β not only interacts with SIRT1 but also significantly modulates its expression levels and activity states. Previous studies have shown that SIRT1 can be regulated through ubiquitin‒proteasome degradation and that the regulation of SIRT1 ubiquitination plays a crucial role in its stability. Ubiquitination is a key signal for protein degradation. Ubiquitin ligases catalyze the covalent linkage of ubiquitin to various substrates. Multiple ubiquitin ligases have been shown to directly interact with specific substrate proteins to initiate and extend polyubiquitin chains. In NAFLD, SIRT1 acts as a substrate of E3 ubiquitin ligases, which can be subjected to K48-linked ubiquitination and degradation, thereby exacerbating hepatic steatosis[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A large-scale proteomic study revealed that SIRT1 could be ubiquitinated by MDM2 in response to oxidative stress-induced cell senescence[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], ubiquitinated by SMURF2 to inhibit cell proliferation and tumor formation[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], ubiquitinated by CUL4 to promote cancer cell autophagy[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and ubiquitinated by COP1 to exacerbate lipid toxicity[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our study first confirmed that GADD45β acts as a regulator of SIRT1 ubiquitination. GADD45β intervention effectively attenuated the increase in SIRT1 ubiquitination, thereby increasing SIRT1 protein expression and preventing its proteasomal degradation, which has never been reported before.\u003c/p\u003e \u003cp\u003eAlthough our study revealed that GADD45β can bind to SIRT1 and influence its ubiquitination process, GADD45β itself is not a ubiquitin ligase. Previous studies have suggested that the binding of specific adaptor proteins can add multiple regulatory possibilities to ubiquitin ligase activity[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Ubiquitin ligases form ubiquitin ligase complexes with substrate receptors or adaptor proteins, which then catalyze the ubiquitination and degradation of substrate proteins[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, we speculate that GADD45β may act as an adaptor protein to promote ubiquitin ligase-mediated ubiquitination of SIRT1. However, the specific ubiquitin ligases involved in this process require further investigation. Additionally, the specific ubiquitination site on SIRT1 that is affected by GADD45β warrants further exploration.\u003c/p\u003e \u003cp\u003eIn conclusion, this investigation underscores the pivotal role of GADD45β in modulating hepatic lipogenesis, specifically through its interaction with SIRT1, increasing SIRT1 deubiquitylation and activation of the SIRT1 signaling pathway. These results indicate that GADD45β acts as a critical regulator, enhancing the stability of SIRT1 and subsequently inhibiting lipogenesis. These findings not only provide novel insights into the molecular mechanisms underlying lipogenesis in the liver but also highlight GADD45β as a potential therapeutic target for the management of MAFLD. Given the increasing incidence of metabolic liver disorders, further exploration of GADD45β function may yield significant advancements in the development of targeted interventions aimed at mitigating the impact of these conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Shanghai Sixth People\u0026rsquo;s Hospital Clinic Research Project (ynhg202103) to Yuanyuan Xiao; Foundation of Shanghai University of Medicine and Health Sciences (SSF-23-14-001) to Chaoyu Zhu; Fundamental Research Funds for the Central Universities (24X010301321) to Qianqian Wang; Institutional Project of Shanghai Sixth People\u0026rsquo;s Hospital (ynhglg202405) to Xinyi Wang; Shanghai Municipal Science and Technology Commission Project (20ZR1442500) to Li Wei. All the funding sources were not involved in the study design; collection, analysis, and interpretation of data; writing of the manuscript; and decision to submit the article for publication.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Yuanyuan Xiao,Renjie Wang, Chaoyu Zhu,Jun Yi and Li Wei; Data curation, Yuanyuan Xiao,Renjie Wang and Li Wei; Formal analysis, Yuanyuan Xiao,Renjie Wang and Chaoyu Zhu; Funding acquisition,Yuanyuan Xiao, Chaoyu Zhu,Qianqian Wang, Xinyi Wang and Li Wei ; Investigation,Yuanyuan Xiao,Renjie Wang, Chaoyu Zhu, Qianqian Wang, Xinyi Wang, Wenjing Song, Shouxia Li and Fusong Jiang ; Methodology,Yuanyuan Xiao,Renjie Wang, Jun Yin and Li Wei; Project administration,Jun Yin and Li Wei ; Supervision, Dingkun Gui and Youhua Xu; Writing\u0026mdash;original draft, Yuanyuan Xiao and Renjie Wang; Writing\u0026mdash;review \u0026amp; editing, Jun Yin and Li Wei. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRNA sequencing data were obtained from the GEO database (GSE33814, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE33814 and GSE48452, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE48452). All other data generated or analyzed during this study are included in the published manuscript and the Supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFriedman, S. L., Neuschwander-Tetri, B. A., Rinella, M. \u0026amp; Sanyal, A. J. Mechanisms of NAFLD development and therapeutic strategies. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (7), 908\u0026ndash;922 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEslam, M. et al. 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Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e362\u003c/b\u003e (6419), 1177\u0026ndash;1182 (2018).\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"GADD45β, NAFLD, Lipogenesis, Ubiquitination, SIRT1","lastPublishedDoi":"10.21203/rs.3.rs-6769255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6769255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNonalcoholic fatty liver disease (NAFLD) is a globally increasing metabolic disorder associated with serious health complications. The molecular mechanisms linking stress-response proteins to hepatic lipogenesis in NAFLD remain poorly understood. Here, we identify GADD45β as a key suppressor of de novo lipogenesis through stabilization of SIRT1. In both methionine-choline-deficient (MCD) diet-fed mice and palmitic acid (PA)-treated hepatocytes, GADD45β deficiency exacerbated lipid accumulation and upregulated lipogenic genes (SREBP1, FASN, ACC). Mechanistically, GADD45β directly bound to SIRT1 and inhibited its ubiquitination, thereby prolonging SIRT1 protein stability. Enhanced SIRT1 stability increased AMPK phosphorylation, which suppressed SREBP1-mediated transcription of lipogenic targets. Crucially, hepatic overexpression of GADD45β reversed PA-induced steatosis in vitro. Our study uncovers a GADD45β-SIRT1-AMPK axis as a central regulator of hepatic lipogenesis, proposing GADD45β as a therapeutic target for NAFLD.\u003c/p\u003e","manuscriptTitle":"GADD45β Inhibits Hepatic Lipogenesis through the AMPK/SREBP1 Pathway via Reducing the ubiquitination-mediated Degradation of SIRT1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 17:39:59","doi":"10.21203/rs.3.rs-6769255/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-24T05:16:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T00:39:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T14:43:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T14:41:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15214330871254709161833065667323378901","date":"2025-06-25T06:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263293120329618649942568439600531883387","date":"2025-06-25T05:53:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333838088038243235357843783890270607361","date":"2025-06-25T05:14:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-25T05:10:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-25T04:56:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-24T10:26:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-21T05:36:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-21T05:33:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11d72edf-bb6b-4f0e-bc88-d6463c56ec58","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50558104,"name":"Health sciences/Endocrinology"},{"id":50558105,"name":"Health sciences/Medical research"},{"id":50558106,"name":"Health sciences/Molecular medicine"},{"id":50558107,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-11-10T16:02:14+00:00","versionOfRecord":{"articleIdentity":"rs-6769255","link":"https://doi.org/10.1038/s41598-025-24864-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-07 15:57:45","publishedOnDateReadable":"November 7th, 2025"},"versionCreatedAt":"2025-06-27 17:39:59","video":"","vorDoi":"10.1038/s41598-025-24864-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24864-1","workflowStages":[]},"version":"v1","identity":"rs-6769255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6769255","identity":"rs-6769255","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}