Mammalian Ste20-Like Kinase 1 Regulates AMPK to Mitigate the Progression of Non-Alcoholic Fatty Liver Disease

Mammalian Ste20-Like Kinase 1 Regulates AMPK to Mitigate the Progression of Non-Alcoholic Fatty Liver Disease | 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 Mammalian Ste20-Like Kinase 1 Regulates AMPK to Mitigate the Progression of Non-Alcoholic Fatty Liver Disease Yi Yang, Lijuan Wang, Chenglei Zhang, Jie Ma, Jiarui Li, Yuanyuan Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4443517/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Non-alcoholic steatohepatitis (NASH) escalates adverse liver-related outcomes, with its progression linked to hepatic lipotoxicity induced by excess hepatic free cholesterol (FC) MST1 has been identified as a potential regulator of hepatic lipid metabolism, potentially ameliorating NAFLD. This study aims to delineate the role of MST1 in the progression of NASH. Wild-type (WT) and MST1 gene knockout (MST1 KO) mice were induced into NASH using a high-fat, high-sugar, high-cholesterol Western diet (WD). In vivo overexpression of MST1 was conducted using lentivirus in WD-fed WT mice. In vitro, HepG2 cells were subjected to MST1 knockdown and overexpression treatments, cultured in a medium induced by a mixture of palmitic acid and oleic acid as free fatty acids (FFA). The NASH model activates the hepatic cholesterol synthesis pathway, leading to an overload of hepatic free cholesterol and downregulation of MST1 expression. Knocking out MST1 exacerbates hepatic FC accumulation and inflammatory damage, activating the cholesterol synthesis pathway. Conversely, upregulating MST1 expression improves hepatic FC deposition, alleviating hepatic damage and inflammation. We found that AMPKα is a substrate of MST1, and MST1 can phosphorylate AMPKα at Thr172. Phosphorylation of AMPKα at Thr172 inhibits the cholesterol synthesis pathway, significantly reversing hepatic FC overload and inflammation caused by MST1 deficiency. Further mechanistic studies indicate that MST1 inhibits cholesterol synthesis by targeting the AMPK/SREBP2 pathway, thereby improving hepatic inflammatory damage caused by FC overload. MST1 targeting AMPK in regulating hepatic cholesterol synthesis metabolism serves as an attractive therapeutic target for preventing the progression of NASH-associated inflammation and fibrosis. mammalian sterile 20-like kinase 1 AMP-activated protein kinase cholesterol synthesis hepatic free cholesterol non-alcoholic steatohepatitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Approximately one-fourth of the global adult population suffers from non-alcoholic fatty liver disease (NAFLD). 1 The disease manifests as a spectrum of liver abnormalities ranging from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). 2 Nearly 30% of NAFLD patients progress to NASH, with potential eventual development into cirrhosis, hepatocellular carcinoma (HCC), and liver failure. 3 , 4 NASH typically occurs against a background of hepatic steatosis, and while abnormal accumulation of triglycerides (TG) in the liver is a significant factor contributing to the onset and progression of NAFLD, mounting evidence suggests that hepatic cholesterol accumulation, particularly the lipotoxicity induced by excess free cholesterol (FC), serves as a primary driving factor for the necroinflammation and fibrosis observed in NASH. 5 The liver serves as the primary site for cholesterol synthesis metabolism, which is tightly regulated by the membrane-bound transcription factor SREBP2. SREBPs undergo a vesicular trafficking to the endoplasmic reticulum (ER), where their N- and C-terminal regions extend into the cytoplasm. Upon SREBPs' exit from the ER to the Golgi apparatus, they undergo two-step proteolytic processing by membrane-bound transcription factor site-1 protease (S1P) and site-2 protease (S2P). This process generates soluble N-terminal cleavage transcription factors, allowing SREBPs to form homodimers and translocate into the nucleus, where they bind to SRE sequences, stimulating the transcription of target genes . 6 When cholesterol homeostasis is disrupted, excessive cholesterol accumulation triggers endoplasmic reticulum stress, stimulates the unfolded protein response (UPR), activates the NLRP3 inflammasome, generates interleukin (IL)-1β, leading to cellular pyroptosis and hepatic inflammation. 7 – 10 It also decreases mitochondrial membrane fluidity, causing membrane protein dysfunction, resulting in ROS generation, lipid peroxidation, cytochrome c release, and triggering cellular apoptosis. 11 , 12 Exposure of Kupffer cells (KCs) to cholesterol crystals from residual lipid droplets (LDs) of deceased steatotic hepatocytes activates the release of pro-inflammatory factors such as IL-1β, TNF-α, TGF-β, and MCP1. This leads to the recruitment of immune cells to the liver and the transformation of hepatic stellate cells (HSCs) into myofibroblasts, resulting in liver fibrosis. 5 Considering hepatic cholesterol as a pivotal factor driving the progression of NASH, identifying the etiology behind excessive cholesterol deposition in the liver would be an effective strategy for treating NASH. Mammalian sterile 20-like kinase 1 (MST1) serves as a core component of the mammalian Hippo signaling pathway, known to regulate cellular apoptosis and proliferation, as well as maintain organ size and tissue homeostasis. 13 , 14 Recent studies have revealed that MST1 is involved in the progression of metabolic disorders such as cardiovascular diseases, obesity, and type 2 diabetes mellitus (T2DM) by regulating cellular apoptosis, autophagy, and immune-inflammatory responses. 15 In studies related to NAFLD, it was observed that MST −/− mice subjected to high-fat feeding exhibited notably increased hepatic steatosis and ballooning compared to the control group. Upregulation of MST1 activity can ameliorate hepatic injury and alleviate NAFLD by suppressing SREBP-1c expression while enhancing the expression of antioxidant genes and SIRT1. 16 In another mouse study, the concurrent deletion of the AKT negative regulator PTEN and the MST1-associated protein SAV1 amplified YAP/TAZ and AKT/IRS2 pathways, leading to the onset of NAFLD and subsequent hepatocarcinogenesis. Conversely, activating MST1 and inhibiting AKT reduced hepatic steatosis and inflammation, preventing the occurrence of liver tumors. 17 The aforementioned studies suggest that MST1 may be closely associated with the progression of NAFLD. Given that increased lipid and cholesterol synthesis are crucial drivers of NAFLD initiation and progression, many genes encoding cholesterol and fatty acid synthesis enzymes are transcriptional targets of SREBPs. Additionally, studies suggest that due to its significant association with p53, MST1 might regulate SREBPs activity through interaction with p53. 18 , 19 We propose that MST1 could be involved in the NAFLD disease process by modulating SREBPs. In earlier studies, we found that MST1 might enhance AMPK activation, increase SREBP-1c Ser372 phosphorylation, decrease the gene expression of key lipogenic enzymes, and ameliorate hepatic TG accumulation in NAFLD model mice. 20 In this study, we elucidated the association between MST1 and hepatic FC deposition, along with hepatic inflammation, underscoring its pivotal role in the progression of NASH. Additionally, we presented supplementary evidence supporting MST1's capability to phosphorylate AMPKα at the Thr172 site. This phosphorylation event inhibits the nuclear activation of SREBP2 and the expression of genes involved in cholesterol synthesis, ultimately mitigating hepatic FC deposition and inflammatory damage. In summary, our findings suggest that MST1 represents a significant therapeutic target for NAFLD, and the modulation of MST1 expression has the potential to influence the transition from NAFL to NASH. Materials and methods Animal experiments MST1 knockout (KO) homozygous male mice on an SPF C57BL6/J background were obtained from Beijing ViewSolid Biotechnology Co., Ltd. Wild-type C57BL6/J male mice from the same litter were used as controls. SPF-grade male C57BL6/J mice for lentiviral treatment were purchased from the Experimental Animal Center of Ningxia Medical University. The mice were housed at the Experimental Animal Center of Ningxia Medical University (Yinchuan, China), under controlled temperature and humidity, with a 12-hour light/dark cycle. At 6–8 weeks of age, MST1-KO homozygous and wild-type mice were fed a normal chow diet (NCD, Xietong Shengwu, China) and tap water ad libitum or a Western diet (WD, TD120528, Xietong Shengwu, China) containing 21.1% fat, 41% sucrose, and 1.25% cholesterol (by weight) along with a high-sugar solution (23.1g/L fructose and 18.9g/L sucrose, Xietong Shengwu, China) for 18 weeks. MST1 KO mice were subjected to intraperitoneal injections of AMPK activator (AICAR, APExBIO, USA, 500mg/kg, n = 6) three times a week for two weeks starting at week 16 of feeding. Blood and tissue samples were concurrently collected from mice after an 8-hour fast. This study strictly adhered to the 'Guide for the Care and Use of Laboratory Animals,' and all experimental protocols were approved by the Medical Ethics Review Committee of Ningxia Medical University. Lentiviral transfection Lentiviral vectors possess advantages such as a broad host range, large genetic capacity, sustained expression, and low immunogenicity. 21 – 23 Upon injection of a concentrated stock of lentiviral vectors carrying a CMV-driven GFP transgene into the livers of adult rats, efficient and sustained expression of GFP was observed without diminution from 2 to 22 weeks. 24 Therefore, we opted for lentiviral transduction in mice. C57BL/6J mice were fed a Western diet for 16 weeks, followed by intravenous injection of MST1-overexpressing lentivirus LV-MST1(VL3721-PDS402_pL-CMV-luc-puro-stk4 (mst1), 1×10^8 TU/ml) and a control virus (LV-GFP), both packaged by Tsingke Biotechnology Co., Ltd (Beijing, China). Mouse tissues were collected two weeks after injection. Reagents and materials Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS) were supplied by Thermo Scientific (Waltham, MA, USA), penicillin/ streptomycin was supplied by Seven Biotech (Beijing, China). Advanced DNA RNA Transfection Reagent was supplied by Zeta Life (Menlo Park, CA, USA). Antibodies against MST1 (#3682), AMPK (#5831) were purchased from Cell Signaling (Danvers, MA, USA). SREBP2 (ab30682) was purchased from Abcam (Cambridge, UK). p-AMPKα (Thr172) (#AF3423) was purchased from Affinity Biosciences (Jiangsu, China). Mouse Anti-Rabbit IgG HRP (Avoiding Heavy Chain) (M21006) was purchased from Abmart (Shanghai, China). Total cholesterol detection kit was purchased from Elabscience (Wuhan, China). Free cholesterol detection kit was purchased from Solarbio (Beijing, China). AMPK activator (AICAR) was purchased from APExBIO (Houston, USA). FILIPIN staining kit for cells and tissues were purchased from Haling Biology (Shanghai, China). Cell culture and treatment The human hepatoma cell line HepG2 were obtained from ATCC (Manassas, VA, USA). Cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2. To determine the impact of MST1 gain and loss of function, HepG2 cells were transfected with a human MST1-overexpressing lentiviral vector, NM_006282 (Fenghui, Hunan, China), and siRNA (Tsingke, Beijing, China) for 24 hours, followed by treatment with a mixture of palmitic and oleic acid (1:2 ratio) free fatty acids (FFA) at 1mM concentration for another 24 hours. Immunoprecipitation analysis The immunoprecipitation (IP) experiments were conducted using the Classic Magnetic Protein A/G IP/Co-IP Kit (YJ201, EpiZyme, Shanghai, China). After washing HepG2 cells with PBS, cells were scraped and collected in 1.5 mL centrifuge tubes. Cell lysates were prepared by adding lysis/wash buffer and a protease inhibitor (GRF101, EpiZyme, Shanghai, China) in the appropriate proportions. The supernatant was collected after centrifugation (4°C, 12000g, 10min), and the protein concentration was quantified using the BCA assay (Thermo Fisher, Waltham, MA, USA). The cell lysate (0.5-1 mg) was incubated with the antibody overnight at 4°C on a rotator. Subsequently, Protein A/G magnetic beads were employed to capture the antibody-antigen complexes at 4°C for an additional 4 hours. The antigen-antibody-magnetic bead complexes were washed with wash buffer, and protein expression was assessed through immunoblotting experiments. Total RNA isolation and real-time PCR Total RNA was extracted from liver tissue and cells following the RNA extraction kit instructions (Omega Biotek, Georgia, USA). Subsequently, 500 ng of RNA was reverse-transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (#6210A, TaKaRa, Japan). RT-PCR reactions were carried out using TB Green Premix Ex Taq II (Tli RNaseH Plus) (#RR820A, TaKaRa, Japan) on a Q-PCR instrument (qTOWER3G, Analytik Jena, Germany). Target gene expression levels were quantified utilizing the double-delta method (2-ΔΔCt). Statistical analyses The results are presented as mean ± SEM. Statistical analyses were conducted using the unpaired Student's t-test (GraphPad Prism 8) between the two groups. p-values below 0.05 were considered statistically significant. Results Excessive cholesterol accumulation in NASH livers activates cholesterol synthesis pathways while downregulating MST1 expression The 'Western Diet' (WD), high in fat, fructose (or sucrose), and cholesterol, has been widely employed to establish NASH models in mice, correlating with the development of human NASH. After 18 weeks of the WD diet in the mouse model, we observed a significant increase in body weight and liver weight (Fig. S1 A, B), accompanied by impaired glucose tolerance and insulin resistance (Fig. S1 C). Serum ALT and AST levels showed a marked elevation (Fig. S1 D). H&E and Masson staining revealed extensive hepatic steatosis, inflammatory infiltration, localized hepatocyte damage, and early fibrotic changes (Fig. S1 E). In this dietary model, the model mice exhibited obesity, insulin resistance, and hepatic steatohepatitis, consistent with previous research findings. 25 – 29 Further examination in NASH mice revealed a significant increase in serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) (Fig. 1 A), as well as hepatic TC and FC levels (Fig. 1 B). Hepatic FC deposition was evident as shown by Filipin staining (Fig. 1 C), suggesting disrupted cholesterol homeostasis in NASH liver. Increased liver macrophages expressing F4/80 (Fig. 1 C) were observed, along with elevated expression of pro-inflammatory and pro-fibrotic cytokines secreted by these cells, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, chemokine ligand 2 (CCL2), and transforming growth factor-beta (TGF-β) (Fig. 1 D). This suggests that there is a close correlation between the deposition of free cholesterol in the NASH liver and the activation of liver macrophages, which promotes the expression of inflammatory factors. Our research also confirms the association of this disruption with the activation of the hepatic cholesterol synthesis pathway, as evidenced by the significant hydrolysis of the non-active precursor form of SREBP2 (pre-SREBP2) into its N-terminal mature form (n-SREBP2) in NASH liver (Fig. 1 E). Moreover, the transcriptional expression of SREBP2 and its downstream target genes HMGCR and HMGCS1 is upregulated (Fig. 1 F). The same conclusions were further validated in vitro. In HepG2 cells induced by FFA, the levels of TC and FC were significantly higher compared to the non-induced group (Ctrl) (Fig. S2A), with FC accumulating in the cell membrane (Fig. S2B). Enhanced nuclear SREBP2 expression was observed, as demonstrated by immunoblotting results (Fig. 1 G), along with upregulation of SREBP2, HMGCR, and HMGCS1 mRNA levels (Fig. 1 H). Meanwhile, both MST1 protein and mRNA levels were downregulated in the NASH in vivo and in vitro models (Fig. 1 E- H). These findings indicate that the activation of the hepatic cholesterol synthesis pathway in NASH leads to FC accumulation in the liver, further corroborating the previous research conclusion of a negative correlation between MST1 and NAFLD. 16 , 17 MST1 deficiency results in hepatic cholesterol accumulation, exacerbating liver damage and inflammation Although MST1 deficiency exacerbates hepatic steatosis in high-fat-fed mice, activating MST1 can alleviate steatosis. 16 However, the role of MST1 in the pathogenesis of NASH remains uncertain. To assess the in vivo effects of MST1 deficiency, we generated MST1 gene knockout (MST1 KO) mice and compared them with their wild-type (WT) littermates. The lack of MST1 does not significantly affect the body weight or liver wet weight of mice on a normal diet (Fig. S3A, B); however, serum levels of ALT and AST are significantly elevated (Fig. S3C), indicating a relationship between MST1 deficiency and liver injury. Further assessment of serum TC, LDL-C, and HDL-C levels revealed disruptions in cholesterol homeostasis in MST1-deficient mice fed a normal diet (Fig. 2 A). Hepatic TC and FC contents were significantly elevated (Fig. 2 B), with evident FC deposition in liver tissues (Fig. 2 C). Kupffer cell activation (Fig. 2 C) stimulated increased expression of TNF-α, CCL2, and TGF-β (Fig. 2 D). While hepatic steatosis was not significantly observed, hepatocellular ballooning, localized inflammatory cell infiltration, and mild perisinusoidal fibrosis were visible, as demonstrated by H&E and Masson's trichrome staining (Fig. 2 C). This indicates that the lack of MST1 can lead to the deposition of free cholesterol in the liver of mice on a normal diet, causing liver inflammation and damage. We induced NASH in MST1 KO mice by feeding them a WD. MST1 KO mice exhibited a slight increase in both body weight and liver wet weight compared to the control group, although the differences were not statistically significant (Fig. S3A, B). Markedly elevated serum ALT levels indicated aggravated liver injury due to MST1 deficiency (Fig. S3C). Serum TC and LDL-C were further increased, while HDL-C decreased compared to the control group (Fig. 2 A). There was an increase in hepatic TC and FC deposition (Fig. 2 B, C). Massive infiltration of Kupffer cells was observed (Fig. 2 C), stimulating the expression of pro-inflammatory factors TNF-α, IL-1β, IL-6, CCL2, and pro-fibrotic factor TGF-β (Fig. 2 D). Both groups of mice exhibited extensive hepatic steatosis; however, MST1 KO mice fed a Western diet (WD) displayed more pronounced hepatocellular ballooning, inflammatory infiltration, and fibrosis compared to wild-type mice (Fig. 2 C), indicating that MST1 deficiency induced liver free cholesterol overload, further amplifying the liver inflammatory damage induced by diet in NASH mice. Furthermore, we confirmed by immunoblotting that in vivo MST1 deficiency induces SREBP2 maturation (Fig. 2 E, G), leading to increased transcription of SREBP2 and its downstream targets HMGCR, HMGCS1 (Fig. 2 F, H) in both NCD and WD-fed mouse models. In vitro, silencing MST1 using siRNA in FFA-induced and non-induced HepG2 cells also demonstrated that downregulation of MST1 induces SREBP2 nuclear activation (Fig. S4A, C), upregulates SREBP2, HMGCR, HMGCS1 gene expression (Fig. S4B, D), and results in increased TC, FC levels in FFA-induced HepG2 cells (Fig. S4E), exacerbating FC deposition on the cell membrane in the NASH cell model (Fig. S4F). Therefore, MST1 deficiency induces SREBP2 maturation, elevates the expression of cholesterol synthesis genes, resulting in FC deposition in the liver, triggering the release of pro-inflammatory factors from hepatic macrophages, amplifying NASH-associated liver injury and inflammation. MST1 ameliorates cholesterol deposition and inflammation in NASH liver The impact of MST1 on cholesterol accumulation and inflammation in NASH liver was further evaluated through gain-of-function approaches. In FFA-treated HepG2 cells, transfection with an MST1 overexpression plasmid led to increased MST1 expression, resulting in decreased cellular TC and FC levels (Fig. 3 A) and an improvement in membrane FC deposition (Fig. 3 B). C57BL/6J mice fed a Western diet were treated with an MST1-overexpressing lentiviral vector; two weeks later, an upregulation of MST1 expression in the liver was observed (Fig. S5A). There was a slight decrease in body weight and a reduction in liver wet weight compared to the control group (Fig. S5B, C). Upregulation of MST1 function reduced serum TC levels in the NASH model mice (Fig. 3 C) as well as hepatic TC and FC levels (Fig. 3 D), significantly ameliorating hepatic FC accumulation (Fig. 3 E). This led to reduced Kupffer cell infiltration (Fig. 3 E), resulting in a downregulation of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, CCL2, and fibrotic factor TGF-β expression (Fig. 3 F). The severity of hepatic steatosis, liver injury, inflammatory cell infiltration, and fibrosis were improved to varying degrees (Fig. 3 E), with a decrease in serum ALT, AST levels in mice (Fig. 3 G). These findings suggest that restoring MST1 reverses hepatic inflammation and damage caused by excessive free cholesterol overload in the NASH model mice. MST1 activates AMPKα through phosphorylation at Thr172 AMPK, a critical regulatory factor in energy metabolism, requires phosphorylation of Thr172 in its α subunit for complete activation. 30 Studies have shown that AMPK-mediated phosphorylation can inhibit the transcriptional activity of nuclear SREBP2. 31 Given MST1's identity as a protein kinase, we hypothesized its potential to activate the AMPK pathway by stimulating phosphorylation of the AMPKα protein. In the NASH model characterized by increased nuclear translocation of SREBP2 and upregulated transcriptional activity, we observed a concomitant downregulation of AMPK Thr172 phosphorylation and MST1 expression (Fig. 1 E, 1 G, 4 A). Further confirmation of the interaction between MST1 and AMPK was obtained through immunoprecipitation experiments (Fig. 4 B). We hypothesized that AMPK might serve as a substrate for MST1. Evaluation of the livers from MST1 knockout mice fed with both NCD and WD revealed a reduction in AMPK Thr172 phosphorylation (Fig. 4 C). Similarly, in vitro experiments involving siRNA-mediated knockdown of MST1 in HepG2 cells induced with and without FFA demonstrated a decrease in AMPK Thr172 phosphorylation (Fig. 4 D). These results provide further support for the notion that AMPK is a substrate of MST1, suggesting that MST1 may activate the AMPK pathway by promoting the phosphorylation of AMPKα protein. Activation of AMPK reversed the hepatic cholesterol accumulation and liver damage induced by MST1 deficiency Based on our findings, we propose that AMPK may act as a downstream substrate involved in MST1-mediated regulation of cholesterol synthesis and metabolism. To investigate further whether AMPK activation can mitigate the effects of MST1 deficiency, we administered intraperitoneal injections of an AMPK activator (AICAR) to MST1 KO mice under both NCD and WD conditions for a 2-week treatment period. Remarkably, AICAR improved disrupted cholesterol metabolism in MST1 KO mice, evidenced by reduced body weight (Figure S3a), decreased serum TC, LDL-C levels (Fig. 5 A), lowered hepatic TC and FC contents (Fig. 5 B), and notably reduced hepatic FC deposition (Fig. 5 C), regardless of the diet. AICAR significantly ameliorated hepatic damage and inflammation in MST1 KO mice, both under NCD and WD feeding conditions. Serum ALT and AST levels decreased notably, particularly in MST1 KO mice fed a WD (Figure S3c), accompanied by reduced activity of hepatic macrophages and downregulation of pro-inflammatory and pro-fibrotic factors (Fig. 5 C, D). NASH-related hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, and liver fibrosis were markedly improved (Fig. 5 C). Subsequent research further confirmed that AMPK independently regulates SREBP2, separate from MST1. AICAR treatment upregulated AMPKα Thr172 phosphorylation in MST1-deficient liver tissues, inhibiting SREBP2 nuclear translocation. This resulted in reduced active n-SREBP2 expression and increased expression of inactive pre-SREBP2 (Fig. 5 E, G). Moreover, there was a significant downregulation in mRNA expression of SREBP2, HMGCR, and HMGCS1 (Fig. 5 F, H). These findings indicate that AMPK, downstream of MST1, modulates hepatic cholesterol synthesis metabolism through its control over SREBP2 nuclear translocation and transcriptional activity. Activating AMPK can reverse hepatic cholesterol deposition and damage caused by MST1 deficiency. MST1 participates in regulating cholesterol synthesis metabolism through the AMPK/SREBP2 signaling pathway Given the clear regulatory role of the AMPK/SREBP2 signaling pathway in cholesterol metabolism, we have also confirmed the phosphorylation of AMPKα by MST1 kinase. Consequently, we investigated whether MST1 modulates cholesterol synthesis metabolism through the AMPK/SREBP2 signaling pathway. Protein immunoblot analysis of the livers from C57BL6/J mice treated with LV-MST1 under a WD regimen revealed enhanced MST1 function, increased AMPKα Thr172 phosphorylation, AMPK activation, and inhibition of SREBP2 maturation, leading to reduced n-SREBP2 expression (Fig. 6 A). Additionally, there was an upregulation in AMPK transcription levels along with a downregulation in transcription levels of SREBP2, HMGCR, and HMGCS1 (Fig. 6 B). In vitro, we transfected HepG2 cells with overexpressed MST1 plasmids under both FFA-induced and non-induced conditions. Consistent with in vivo findings, MST1 facilitated AMPKα Thr172 phosphorylation, activated AMPK, and suppressed SREBP2 cleavage for nuclear activation, consequently downregulating nuclear SREBP2 expression (Fig. 6 C, E). Concurrently, MST1 overexpression upregulated AMPK mRNA levels and downregulated transcriptional expression of SREBP2, HMGCR, and HMGCS1 (Fig. 6 D, F). Overall, both in vivo and in vitro findings demonstrate that MST1 activates AMPK, inhibits SREBP2 cleavage into its active form, and suppresses the transcription of SREBP2 and its target genes, thereby downregulating the activity of the cholesterol synthesis pathway. Discussion Our study reveals that in both in vivo and in vitro models of NASH, the cholesterol synthesis pathway is activated, accompanied by hepatic FC deposition and concurrent inflammation and fibrosis, while MST1 expression is reduced. MST1 deficiency promotes SREBP2 nuclear translocation, upregulates the expression of key cholesterol synthesis genes, enhances hepatic FC accumulation, and exacerbates NASH-related liver damage, inflammation, and fibrosis. Our findings indicate that MST1 regulates SREBP2 through the phosphorylation of AMPKα at the Thr172 site. MST1 overexpression upregulates AMPKα Thr172 phosphorylation, inhibits SREBP2 nuclear translocation, downregulates cholesterol synthesis gene expression, reduces hepatic FC deposition, and ameliorates NASH-related liver inflammation and fibrosis. NASH, as a progressive stage of NAFLD, has been widely associated with hepatic accumulation of toxic lipid-free cholesterol (FC). Lipidomic analyses in human liver tissues indicate significantly elevated FC levels in NASH patients compared to NAFL individuals. 32 In NAFLD patients, the co-occurrence of NASH and fibrosis parallels the hepatic aggregation of FC. Experimental induction of hepatic FC accumulation accelerates liver inflammation and fibrosis, while correcting excessive hepatic FC load can alleviate the severity of NASH. 33 In NASH patients and NASH mouse models induced by high fat and high cholesterol (HFHC) diets, cholesterol crystals formed in the LDs of steatotic hepatocytes can activate Kupffer cells, 34 KCs secrete proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which recruit neutrophils and circulating monocytes through the interaction of CCL2 and CCR2. Monocytes differentiate into proinflammatory macrophages, further amplifying liver inflammation, while stimulating hepatic stellate cells through TGF-β, promoting their transformation into activated myofibroblasts, thereby promoting fibrosis. 35 , 36 In the study of NASH model mice induced by a high-fat and high cholesterol diet, we also observed a correlation consistent with the above research results, further clarifying the correlation between liver FC overload and NASH inflammatory injury. As an important site for cholesterol metabolism, the liver is known to have many disruptions in liver cholesterol metabolism throughout the progression of NAFLD, which can lead to liver cholesterol deposition. Hyperinsulinemia and inflammation lead to the loss of SCAP/SREBP2 inhibition, causing excessive cholesterol accumulation in the liver of mice. 37 – 39 NAFLD/NASH patients exhibit increased nuclear SREBP2, HMGCR mRNA, HMGCR protein, and phosphorylation levels alongside decreased LDLR, ABCA1, ABCG1, and ABCG5 mRNA levels. This imbalance results in heightened cholesterol synthesis and reduced excretion, contributing to increased hepatic cholesterol accumulation. 32 , 33 , 37 – 40 In our findings from the NASH model mice, serum TC, LDL, hepatic TC, and FC levels were notably elevated. However, SREBP2 activity remained unimpeded, leading to increased nuclear SREBP2 expression and enhanced transcriptional expression of cholesterol synthesis genes HMGCR and HMGCS1. These results align with the previously mentioned disruptions in cholesterol synthesis metabolism. As a core kinase in the Hippo signaling pathway, MST1 has been associated with hepatic lipid metabolism disorders. Significant lipid metabolism disturbances are observed in MST1 gene knockout mice, believed to be directly linked to decreased liver Sirt1 expression, enhanced ubiquitination degradation pathways, and increased expression of SREBP-1c. 16 However, studies investigating its correlation with hepatic cholesterol metabolism are scarce. Existing studies have prominently highlighted the interplay between core components of the Hippo pathway, such as LATS1/2, Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ), with hepatic cholesterol metabolism. Research by Xiaobo Wang et al. demonstrates that excessive hepatic cholesterol induces downstream calcium signaling, triggering RhoA activity and inhibiting LATS1/2, leading to increased protein levels of TAZ and YAP at the transcriptional level. Elevated TAZ in liver cells can induce NASH fibrosis. 41 , 42 Zhiping Shu and colleagues confirmed YAP as a crucial coactivator of SREBP-1c and SREBP-2, controlling the expression of FAS and HMGCR genes in hepatocytes. Overexpression of LATS1 inhibits YAP dephosphorylation and nuclear translocation, improving systemic insulin resistance and hepatic steatosis in diabetic mice. 43 Yael Aylon's research demonstrates that LATS2 binds to the endoplasmic reticulum-linked precursors of SREBP-1 and SREBP-2, inhibiting their processing and subsequently dampening the nuclear transcriptional activity of SREBPs. 44 These studies collectively suggest a close association between the core component MST1 within the Hippo signaling pathway and hepatic lipid metabolism, cholesterol metabolism, as well as the occurrence and progression of NASH. Building upon our previous work, 20 we propose the potential of MST1 to regulate hepatic cholesterol synthesis metabolism through AMPK. Our study elucidates the interaction between MST1 and AMPK, providing further validation that MST1 can phosphorylate the Thr172 site of AMPK. Treatment of MST1 knockout mice with AICAR confirmed AMPK as a substrate of MST1 and demonstrated its ability to reverse hepatic FC overload and liver damage caused by MST1 deficiency. Additional mechanistic investigations revealed that AMPK regulates the nuclear activation of SREBP2 and, at the transcriptional level, modulates the expression of SREBP2 and its downstream targets HMGCR and HMGCS1. These findings are consistent with prior research by other investigators. 31 , 45 – 47 By inducing MST1 overexpression in in vivo and in vitro models, we have elucidated that MST1 phosphorylates AMPKα at Thr172, leading to a cascade of changes in the AMPK/SREBP2 signaling pathway. This modulation affects SREBP2 nuclear translocation and alters the expression of SREBP2 and downstream target genes HMGCR and HMGCS1. The reduction in cholesterol synthesis alleviates hepatic free cholesterol load, resulting in an improvement in the degree of NASH-related liver inflammation and fibrosis. In conclusion, our study demonstrates that MST1 can alleviate hepatic free cholesterol load and thus mitigate the progression of NASH through the regulation of AMPK/SREBP2 signaling. This highlights MST1 as a promising target for the treatment of NAFLD/NASH. Abbreviations AICAR, AMPK activator; ALT, alanine aminotransferase; AMPK, 5′ AMP-activated protein kinase; AST, aspartate aminotransferase; CCL2, chemokine C-C motif ligand 2; FC, free cholesterol; FFA, free fatty acids; H&E, haematoxylin and eosin; HDL-C, High-density lipoprotein cholesterol; HMGCR, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; HMGCS1, 3-hydroxy-3-methylglutaryl coenzyme A synthetase1; HSCs, hepatic stellate cells; IL-1β, interleukin-1β; IL-6, interleukin-6; KCs, Kupffer cells; LDs, lipid droplets; LDL-C, Low density lipoprotein cholesterol; MST1, mammalian sterile 20-like kinase 1; NASH, non-alcoholic steatohepatitis; NCD , normal chow diet ; SREBP2, sterol regulatory element-binding transcription factor 2; TC, total cholesterol; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; WD, Western diet; WT, Wild-type. Declarations Author contributions Yi Yang and Lijuan Wang conceived the study. Lijuan Wang, Chenglei Zhang, and Jie Ma conducted experiments and performed data analysis. Jiarui Li, Yuanyuan Wu, Yanru Ren, and Jianning Li conducted animal experiments and analyzed data. Lijuan Wang and Yan Li drafted and edited the manuscript. Yi Yang revised the paper. All authors approved the final version of the manuscript. Funding information This work was supported by the National Natural Science Foundation of China (No. 82160171) Conflict of interests The authors declare that they have no conflict of interest. Ethical approval This study strictly followed the Guidelines for the Care and Use of Experimental Animals published by the National Institutes of Health, and all experimental protocols were approved by the Medical Ethics Review Committee of Ningxia Medical University. References Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-Analytic assessment of prevalence, incidence, and outcomes. 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Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 2011;141(4):1393-1403. Simonen P, Kotronen A, Hallikainen M, et al. Cholesterol synthesis is increased and absorption decreased in non-alcoholic fatty liver disease independent of obesity. J Hepatol 2011;54(1): 153-159. Wang XB, Cai BS, Yang XM, et al. Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic Steatohepatitis. Cell Metab 2020;31(5): 969-986.e7. Wang XB, Zheng Z, Caviglia JM, et al. Hepatocyte TAZ/WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis. Cell Metab 2016;24(6): 848-862. Shu ZP, Gao Y, Zhang GP, et al. A functional interaction between Hippo-YAP signaling and SREBPs mediates hepatic steatosis in diabetic mice. J Cell Mol Med 2019;23(5): 3616-3628. Aylon Y, Gershoni A, Rotkopf R, et al. The LATS2 tumor suppressor inhibits SREBP and suppresses hepatic cholesterol accumulation. Genes Dev 2016;30(7): 786-797. Tang H, Yu R, Liu S, et al. Irisin Inhibits Hepatic Cholesterol Synthesis via AMPK-SREBP2 Signaling. EBioMedicine 2016;6: 139-148. Gopoju R, Panangipalli S, Kotamraju S. Metformin treatment prevents SREBP2-mediated cholesterol uptake and improves lipid homeostasis during oxidative stress-induced atherosclerosis. Free Radic Biol Med 2018;118:85-97. Wang X, Dong LY, Gai QJ. Lack of Augmenter of Liver Regeneration Disrupts Cholesterol Homeostasis of Liver in Mice by Inhibiting the AMPK Pathway. Hepatol Commun 2020;4(8): 1149-1167. Additional Declarations There is NO conflict of interest to disclose Supplementary Files Supplementaryfigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4443517","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":314921047,"identity":"25d0bf53-7b3f-42f1-b9a9-adbea0a318b7","order_by":0,"name":"Yi Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYNACAxsog414LWkka2E4TIIWg+NnD7/mKTifZ3DtjAHDh7LDDPyzGwhoOZOXZs1jcLtYcnaOAeOMc4cZJO4cIKDlQI6ZMVBLYr90jgEzb9thBgOJBAJazr8BaTmX2AbS8pcoLTdyjB/zGByA2MJIjBbJG2/MGOcYJCfOnJ1WcLDnXDqPxA0CWvjO5xh/ePPHLnHD7eSND36UWcvxzyCgReEAA5sUD5RzAIh58CiGAPkGBuaPPwgqGwWjYBSMghENACOXQ92Qmig7AAAAAElFTkSuQmCC","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""},{"id":314921048,"identity":"925d724f-8e10-4508-9b6e-c1f9f5b0f204","order_by":1,"name":"Lijuan Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Wang","suffix":""},{"id":314921049,"identity":"c2339c45-73a5-4bf6-81f1-c7990a0f2e82","order_by":2,"name":"Chenglei Zhang","email":"","orcid":"https://orcid.org/0000-0001-6494-0040","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chenglei","middleName":"","lastName":"Zhang","suffix":""},{"id":314921050,"identity":"edb32a3c-405e-4106-9ec5-a5e925e1baaa","order_by":3,"name":"Jie Ma","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Ma","suffix":""},{"id":314921051,"identity":"ce01124e-035e-4755-8864-f1221bd96bb0","order_by":4,"name":"Jiarui Li","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiarui","middleName":"","lastName":"Li","suffix":""},{"id":314921052,"identity":"f93657e8-8569-49ef-bbc6-a571d95c8240","order_by":5,"name":"Yuanyuan Wu","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Wu","suffix":""},{"id":314921053,"identity":"701d92c4-dadf-4990-bf60-9b3ef6baf370","order_by":6,"name":"Yanru Ren","email":"","orcid":"","institution":"General Hospital of Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanru","middleName":"","lastName":"Ren","suffix":""},{"id":314921054,"identity":"985e56a3-c38a-4bbe-a7b1-d20139c26474","order_by":7,"name":"Jianning Li","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianning","middleName":"","lastName":"Li","suffix":""},{"id":314921055,"identity":"d255792f-570d-46fd-b0f8-91cb4083e59d","order_by":8,"name":"Yan Li","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-05-19 08:25:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4443517/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4443517/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59464106,"identity":"e4860895-61b9-4e72-96f1-394abad7bd9f","added_by":"auto","created_at":"2024-07-02 06:06:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":856276,"visible":true,"origin":"","legend":"\u003cp\u003eExcessive accumulation of hepatic cholesterol in NASH, activation of cholesterol synthesis pathway, and downregulation of MST1 expression. Wild-type C57BL6/J mice were fed NCD and WD diets for 18 weeks. (A) Serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). (B) Hepatic levels of TC and free cholesterol (FC). (C) Representative Filipin staining (100×) and F4/80 immunohistochemical sections of liver tissues (200×), fluorescence intensity, and positive cell area quantified. (D) Expression of pro-inflammatory and pro-fibrotic genes in liver tissues. (E) Protein blots and densitometry analysis of n-SREBP2, pre-SREBP2, and MST1 in mouse liver tissues. (F) mRNA expression of MST1, SREBP2, HMGCR, and HMGCS1 in mouse liver tissues. (G) Protein blots and densitometry analysis of n-SREBP2, pre-SREBP2, and MST1 in FFA induced HepG2 cells. (H) mRNA expression of MST1, SREBP2, HMGCR, and HMGCS1 in FFA-induced HepG2 cells. Data are presented as mean ± SEM. n = 6 mice/group. Results are representative of three independent experiments. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; .001, \u003csup\u003e****\u003c/sup\u003ep \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/cd7efd3aede9c8be86156267.png"},{"id":59464104,"identity":"c6777245-4343-4589-9710-f0e288cd1882","added_by":"auto","created_at":"2024-07-02 06:06:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1979345,"visible":true,"origin":"","legend":"\u003cp\u003eMST1 deletion induces hepatic cholesterol accumulation, aggravates liver injury and inflammation, and activates the cholesterol synthesis pathway. MST1 knockout (MST1 KO) mice were fed NCD and WD diets for 18 weeks. (A) Serum levels of TC, LDL-C, and HDL-C. (B) Hepatic levels of TC and FC. (C) Representative Filipin staining (100x), F4/80 immunohistochemistry, H\u0026amp;E staining, and Masson's trichrome sections of liver tissues (200×). Fluorescence intensity, F4/80 positive cell area and collagen fiber area quantified, NAFLD activity score. (D) Expression of pro-inflammatory and pro-fibrotic genes in mouse liver tissues. (E) Protein blots and densitometry analysis of n-SREBP2, pre-SREBP2, and MST1 in NCD-fed mouse liver tissues. (F) mRNA expression of MST1, SREBP2, HMGCR, and HMGCS1 in NCD-fed mouse liver tissues. (G) Protein blots and densitometry analysis of n-SREBP2, pre-SREBP2, and MST1 in WD-fed mouse liver tissues. (H) mRNA expression of MST1, SREBP2, HMGCR, and HMGCS1 in WD-fed mouse liver tissues. Data are presented as mean ± SEM. n = 6 mice/group. Results are representative of 3 independent experiments.\u003csup\u003e *\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; .001, \u003csup\u003e****\u003c/sup\u003ep \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/56be351c8910a82f3195fe3b.png"},{"id":59464561,"identity":"9f0b42f7-2c31-4b58-8351-6a862282bc7f","added_by":"auto","created_at":"2024-07-02 06:14:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1615622,"visible":true,"origin":"","legend":"\u003cp\u003eMST1 improves hepatic cholesterol deposition and inflammation in NASH. HepG2 cells were induced with 1mM FFA for 24 hours followed by transfection with MST1 overexpression. (A) Cellular TC and FC levels. (B) Representative Filipin staining of cells (scale bars, 20 μm). C57BL/6J mice were fed a Western diet for 16 weeks, followed by treatment with an MST1-overexpressing lentivirus. (C) Serum levels of TC, LDL-C, and HDL-C in mice. (D) Hepatic TC and FC levels. (E) Representative Filipin staining (100x), F4/80 immunohistochemistry, H\u0026amp;E staining, and Masson's trichrome sections of liver tissues (200×). Fluorescence intensity, F4/80 positive cell area and collagen fiber area quantified, NAFLD activity score. (F) Expression of pro-inflammatory and pro-fibrotic genes in mouse liver tissues. (G) Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) in mice. Data are presented as the mean ± SEM. n = 6 mice/group. Results are representative of 3 independent experiments. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; .001, \u003csup\u003e****\u003c/sup\u003ep \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/42cb7403fbb355c8d238d207.png"},{"id":59464563,"identity":"20c7286a-3e3a-4803-aa9b-1c288f7af154","added_by":"auto","created_at":"2024-07-02 06:14:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":239858,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphorylation of AMPKα at Thr172 by MST1. (A) In vivo and in vitro NASH model liver p-AMPKα and AMPKα protein immunoblotting and grayscale analysis. (B) The immunoprecipitation of AMPK and MST1 was conducted separately utilizing antibodies specific to MST1 and AMPK, respectively. (C) Liver p-AMPKα and AMPKα protein immunoblotting and grayscale analysis in NCD and WD-fed MST1 KO mice. (D) Immunoblotting and grayscale analysis of p-AMPKα and AMPKα proteins in FFA-induced or non-induced HepG2 cells following MST1 knockdown. Data are presented as the mean ± SEM. Results represent three independent experiments. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e****\u003c/sup\u003ep \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/894e1963deaecc951d76da70.png"},{"id":59465201,"identity":"1e64b832-b12e-4e5b-9768-f67eecb56140","added_by":"auto","created_at":"2024-07-02 06:22:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1877510,"visible":true,"origin":"","legend":"\u003cp\u003eAMPK activation reverses hepatic cholesterol accumulation and liver injury caused by MST1 deficiency. MST1 KO mice were fed NCD and WD for 16 weeks followed by intraperitoneal injection of AICAR for 2 weeks. (A) Serum levels of TC, LDL-C, HDL-C in mice. (B) Hepatic TC, FC content. (C) Representative Filipin staining (100x), F4/80 immunohistochemistry, H\u0026amp;E staining, and Masson's trichrome sections of liver tissues (200×). Fluorescence intensity, F4/80 positive cell area and collagen fiber area quantified, NAFLD activity score. (D) Expression of pro-inflammatory and pro-fibrotic genes in mouse liver tissue. (E) Protein immunoblots and grayscale values of p-AMPKα, AMPKα, n-SREBP2, pre-SREBP2, and MST1 in NCD mouse liver. (F) mRNA expression of AMPK, SREBP2, HMGGCR, and HMGCS1 in NCD mouse liver. (G) Protein immunoblots and grayscale values of p-AMPKα, AMPKα, n-SREBP2, pre-SREBP2, and MST1 in WD mouse liver. (H) mRNA expression of AMPK, SREBP2, HMGGCR, and HMGCS1 in WD mouse liver. Data are presented as the mean ± SEM. n = 6 mice/group, Data are representative of 3 independent experiments. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; .001, \u003csup\u003e****\u003c/sup\u003eP \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/3ab5e9cdff84392787ab626d.png"},{"id":59464108,"identity":"e6e6dbcf-4b45-48da-895e-780f2ec20a4f","added_by":"auto","created_at":"2024-07-02 06:06:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":319255,"visible":true,"origin":"","legend":"\u003cp\u003eMST1 participates in cholesterol synthesis metabolism via the AMPK/SREBP2 signaling pathway. (A) Protein immunoblots and grayscale values of p-AMPKα, AMPKα, n-SREBP2, pre-SREBP2, and MST1 in the liver of C57BL6/J mice fed a WD and transduced with MST1 overexpressing lentivirus. (B) mRNA expression of MST1, AMPK, SREBP2, HMGGCR, and HMGCS1 in mouse liver. (C) Protein immunoblots and grayscale values of p-AMPKα, AMPKα, n-SREBP2, pre-SREBP2, and MST1 in non-induced HepG2 cells overexpressing MST1. (D) mRNA expression of MST1, AMPK, SREBP2, HMGGCR, and HMGCS1 in non-induced HepG2 cells overexpressing MST1. (E) Protein immunoblots and grayscale values of p-AMPKα, AMPKα, n-SREBP2, pre-SREBP2, and MST1 in FFA-induced HepG2 cells overexpressing MST1. (F) mRNA expression of MST1, AMPK, SREBP2, HMGGCR, and HMGCS1 in FFA-induced HepG2 cells overexpressing MST1. Data are presented as the mean ± SEM. Data are representative of 3 independent experiments. \u003csup\u003e*\u003c/sup\u003ep \u0026lt; .05, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; .01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; .001, \u003csup\u003e****\u003c/sup\u003eP \u0026lt; .0001 vs the respective control groups.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/ccb6f99c8abe37f252344f91.png"},{"id":66458487,"identity":"59d23294-19f1-415a-81f0-7cf171f213cb","added_by":"auto","created_at":"2024-10-12 09:58:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8062530,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/2e08aca2-74b5-4c30-b8bc-ae7d6e386a2b.pdf"},{"id":59464110,"identity":"f890e692-3c8c-42bc-b2e2-41f074bfa39a","added_by":"auto","created_at":"2024-07-02 06:06:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1917693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4443517/v1/bfbe670324a2749c5c1ae46a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Mammalian Ste20-Like Kinase 1 Regulates AMPK to Mitigate the Progression of Non-Alcoholic Fatty Liver Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApproximately one-fourth of the global adult population suffers from non-alcoholic fatty liver disease (NAFLD).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The disease manifests as a spectrum of liver abnormalities ranging from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Nearly 30% of NAFLD patients progress to NASH, with potential eventual development into cirrhosis, hepatocellular carcinoma (HCC), and liver failure.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e NASH typically occurs against a background of hepatic steatosis, and while abnormal accumulation of triglycerides (TG) in the liver is a significant factor contributing to the onset and progression of NAFLD, mounting evidence suggests that hepatic cholesterol accumulation, particularly the lipotoxicity induced by excess free cholesterol (FC), serves as a primary driving factor for the necroinflammation and fibrosis observed in NASH.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe liver serves as the primary site for cholesterol synthesis metabolism, which is tightly regulated by the membrane-bound transcription factor SREBP2. SREBPs undergo a vesicular trafficking to the endoplasmic reticulum (ER), where their N- and C-terminal regions extend into the cytoplasm. Upon SREBPs' exit from the ER to the Golgi apparatus, they undergo two-step proteolytic processing by membrane-bound transcription factor site-1 protease (S1P) and site-2 protease (S2P). This process generates soluble N-terminal cleavage transcription factors, allowing SREBPs to form homodimers and translocate into the nucleus, where they bind to SRE sequences, stimulating the transcription of target genes .\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e When cholesterol homeostasis is disrupted, excessive cholesterol accumulation triggers endoplasmic reticulum stress, stimulates the unfolded protein response (UPR), activates the NLRP3 inflammasome, generates interleukin (IL)-1β, leading to cellular pyroptosis and hepatic inflammation.\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e It also decreases mitochondrial membrane fluidity, causing membrane protein dysfunction, resulting in ROS generation, lipid peroxidation, cytochrome c release, and triggering cellular apoptosis.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Exposure of Kupffer cells (KCs) to cholesterol crystals from residual lipid droplets (LDs) of deceased steatotic hepatocytes activates the release of pro-inflammatory factors such as IL-1β, TNF-α, TGF-β, and MCP1. This leads to the recruitment of immune cells to the liver and the transformation of hepatic stellate cells (HSCs) into myofibroblasts, resulting in liver fibrosis.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Considering hepatic cholesterol as a pivotal factor driving the progression of NASH, identifying the etiology behind excessive cholesterol deposition in the liver would be an effective strategy for treating NASH.\u003c/p\u003e \u003cp\u003eMammalian sterile 20-like kinase 1 (MST1) serves as a core component of the mammalian Hippo signaling pathway, known to regulate cellular apoptosis and proliferation, as well as maintain organ size and tissue homeostasis.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Recent studies have revealed that MST1 is involved in the progression of metabolic disorders such as cardiovascular diseases, obesity, and type 2 diabetes mellitus (T2DM) by regulating cellular apoptosis, autophagy, and immune-inflammatory responses.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In studies related to NAFLD, it was observed that MST\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice subjected to high-fat feeding exhibited notably increased hepatic steatosis and ballooning compared to the control group. Upregulation of MST1 activity can ameliorate hepatic injury and alleviate NAFLD by suppressing SREBP-1c expression while enhancing the expression of antioxidant genes and SIRT1.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In another mouse study, the concurrent deletion of the AKT negative regulator PTEN and the MST1-associated protein SAV1 amplified YAP/TAZ and AKT/IRS2 pathways, leading to the onset of NAFLD and subsequent hepatocarcinogenesis. Conversely, activating MST1 and inhibiting AKT reduced hepatic steatosis and inflammation, preventing the occurrence of liver tumors.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The aforementioned studies suggest that MST1 may be closely associated with the progression of NAFLD.\u003c/p\u003e \u003cp\u003eGiven that increased lipid and cholesterol synthesis are crucial drivers of NAFLD initiation and progression, many genes encoding cholesterol and fatty acid synthesis enzymes are transcriptional targets of SREBPs. Additionally, studies suggest that due to its significant association with p53, MST1 might regulate SREBPs activity through interaction with p53.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e We propose that MST1 could be involved in the NAFLD disease process by modulating SREBPs. In earlier studies, we found that MST1 might enhance AMPK activation, increase SREBP-1c Ser372 phosphorylation, decrease the gene expression of key lipogenic enzymes, and ameliorate hepatic TG accumulation in NAFLD model mice.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e In this study, we elucidated the association between MST1 and hepatic FC deposition, along with hepatic inflammation, underscoring its pivotal role in the progression of NASH. Additionally, we presented supplementary evidence supporting MST1's capability to phosphorylate AMPKα at the Thr172 site. This phosphorylation event inhibits the nuclear activation of SREBP2 and the expression of genes involved in cholesterol synthesis, ultimately mitigating hepatic FC deposition and inflammatory damage. In summary, our findings suggest that MST1 represents a significant therapeutic target for NAFLD, and the modulation of MST1 expression has the potential to influence the transition from NAFL to NASH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eMST1 knockout (KO) homozygous male mice on an SPF C57BL6/J background were obtained from Beijing ViewSolid Biotechnology Co., Ltd. Wild-type C57BL6/J male mice from the same litter were used as controls. SPF-grade male C57BL6/J mice for lentiviral treatment were purchased from the Experimental Animal Center of Ningxia Medical University. The mice were housed at the Experimental Animal Center of Ningxia Medical University (Yinchuan, China), under controlled temperature and humidity, with a 12-hour light/dark cycle. At 6\u0026ndash;8 weeks of age, MST1-KO homozygous and wild-type mice were fed a normal chow diet (NCD, Xietong Shengwu, China) and tap water ad libitum or a Western diet (WD, TD120528, Xietong Shengwu, China) containing 21.1% fat, 41% sucrose, and 1.25% cholesterol (by weight) along with a high-sugar solution (23.1g/L fructose and 18.9g/L sucrose, Xietong Shengwu, China) for 18 weeks. MST1 KO mice were subjected to intraperitoneal injections of AMPK activator (AICAR, APExBIO, USA, 500mg/kg, n\u0026thinsp;=\u0026thinsp;6) three times a week for two weeks starting at week 16 of feeding. Blood and tissue samples were concurrently collected from mice after an 8-hour fast. This study strictly adhered to the 'Guide for the Care and Use of Laboratory Animals,' and all experimental protocols were approved by the Medical Ethics Review Committee of Ningxia Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLentiviral transfection\u003c/h2\u003e \u003cp\u003eLentiviral vectors possess advantages such as a broad host range, large genetic capacity, sustained expression, and low immunogenicity.\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Upon injection of a concentrated stock of lentiviral vectors carrying a CMV-driven GFP transgene into the livers of adult rats, efficient and sustained expression of GFP was observed without diminution from 2 to 22 weeks.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Therefore, we opted for lentiviral transduction in mice. C57BL/6J mice were fed a Western diet for 16 weeks, followed by intravenous injection of MST1-overexpressing lentivirus LV-MST1(VL3721-PDS402_pL-CMV-luc-puro-stk4 (mst1), 1\u0026times;10^8 TU/ml) and a control virus (LV-GFP), both packaged by Tsingke Biotechnology Co., Ltd (Beijing, China). Mouse tissues were collected two weeks after injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eReagents and materials\u003c/h2\u003e \u003cp\u003eDulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS) were supplied by Thermo Scientific (Waltham, MA, USA), penicillin/ streptomycin was supplied by Seven Biotech (Beijing, China). Advanced DNA RNA Transfection Reagent was supplied by Zeta Life (Menlo Park, CA, USA). Antibodies against MST1 (#3682), AMPK (#5831) were purchased from Cell Signaling (Danvers, MA, USA). SREBP2 (ab30682) was purchased from Abcam (Cambridge, UK). p-AMPKα (Thr172) (#AF3423) was purchased from Affinity Biosciences (Jiangsu, China). Mouse Anti-Rabbit IgG HRP (Avoiding Heavy Chain) (M21006) was purchased from Abmart (Shanghai, China). Total cholesterol detection kit was purchased from Elabscience (Wuhan, China). Free cholesterol detection kit was purchased from Solarbio (Beijing, China). AMPK activator (AICAR) was purchased from APExBIO (Houston, USA). FILIPIN staining kit for cells and tissues were purchased from Haling Biology (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eThe human hepatoma cell line HepG2 were obtained from ATCC (Manassas, VA, USA). Cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin at 37\u0026deg;C in a humidified atmosphere with 5% CO2. To determine the impact of MST1 gain and loss of function, HepG2 cells were transfected with a human MST1-overexpressing lentiviral vector, NM_006282 (Fenghui, Hunan, China), and siRNA (Tsingke, Beijing, China) for 24 hours, followed by treatment with a mixture of palmitic and oleic acid (1:2 ratio) free fatty acids (FFA) at 1mM concentration for another 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation analysis\u003c/h2\u003e \u003cp\u003eThe immunoprecipitation (IP) experiments were conducted using the Classic Magnetic Protein A/G IP/Co-IP Kit (YJ201, EpiZyme, Shanghai, China). After washing HepG2 cells with PBS, cells were scraped and collected in 1.5 mL centrifuge tubes. Cell lysates were prepared by adding lysis/wash buffer and a protease inhibitor (GRF101, EpiZyme, Shanghai, China) in the appropriate proportions. The supernatant was collected after centrifugation (4\u0026deg;C, 12000g, 10min), and the protein concentration was quantified using the BCA assay (Thermo Fisher, Waltham, MA, USA). The cell lysate (0.5-1 mg) was incubated with the antibody overnight at 4\u0026deg;C on a rotator. Subsequently, Protein A/G magnetic beads were employed to capture the antibody-antigen complexes at 4\u0026deg;C for an additional 4 hours. The antigen-antibody-magnetic bead complexes were washed with wash buffer, and protein expression was assessed through immunoblotting experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation and real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from liver tissue and cells following the RNA extraction kit instructions (Omega Biotek, Georgia, USA). Subsequently, 500 ng of RNA was reverse-transcribed into cDNA using the PrimeScript\u0026trade; II 1st Strand cDNA Synthesis Kit (#6210A, TaKaRa, Japan). RT-PCR reactions were carried out using TB Green Premix Ex Taq II (Tli RNaseH Plus) (#RR820A, TaKaRa, Japan) on a Q-PCR instrument (qTOWER3G, Analytik Jena, Germany). Target gene expression levels were quantified utilizing the double-delta method (2-ΔΔCt).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eThe results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were conducted using the unpaired Student's t-test (GraphPad Prism 8) between the two groups. p-values below 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExcessive cholesterol accumulation in NASH livers activates cholesterol synthesis\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003epathways while downregulating MST1 expression\u003c/h2\u003e \u003cp\u003eThe 'Western Diet' (WD), high in fat, fructose (or sucrose), and cholesterol, has been widely employed to establish NASH models in mice, correlating with the development of human NASH. After 18 weeks of the WD diet in the mouse model, we observed a significant increase in body weight and liver weight (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B), accompanied by impaired glucose tolerance and insulin resistance (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Serum ALT and AST levels showed a marked elevation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD). H\u0026amp;E and Masson staining revealed extensive hepatic steatosis, inflammatory infiltration, localized hepatocyte damage, and early fibrotic changes (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). In this dietary model, the model mice exhibited obesity, insulin resistance, and hepatic steatohepatitis, consistent with previous research findings.\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Further examination in NASH mice revealed a significant increase in serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), as well as hepatic TC and FC levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Hepatic FC deposition was evident as shown by Filipin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting disrupted cholesterol homeostasis in NASH liver. Increased liver macrophages expressing F4/80 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) were observed, along with elevated expression of pro-inflammatory and pro-fibrotic cytokines secreted by these cells, including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, chemokine ligand 2 (CCL2), and transforming growth factor-beta (TGF-β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). This suggests that there is a close correlation between the deposition of free cholesterol in the NASH liver and the activation of liver macrophages, which promotes the expression of inflammatory factors. Our research also confirms the association of this disruption with the activation of the hepatic cholesterol synthesis pathway, as evidenced by the significant hydrolysis of the non-active precursor form of SREBP2 (pre-SREBP2) into its N-terminal mature form (n-SREBP2) in NASH liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the transcriptional expression of SREBP2 and its downstream target genes HMGCR and HMGCS1 is upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe same conclusions were further validated in vitro. In HepG2 cells induced by FFA, the levels of TC and FC were significantly higher compared to the non-induced group (Ctrl) (Fig. S2A), with FC accumulating in the cell membrane (Fig. S2B). Enhanced nuclear SREBP2 expression was observed, as demonstrated by immunoblotting results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), along with upregulation of SREBP2, HMGCR, and HMGCS1 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Meanwhile, both MST1 protein and mRNA levels were downregulated in the NASH in vivo and in vitro models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE- H). These findings indicate that the activation of the hepatic cholesterol synthesis pathway in NASH leads to FC accumulation in the liver, further corroborating the previous research conclusion of a negative correlation between MST1 and NAFLD.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMST1 deficiency results in hepatic cholesterol accumulation, exacerbating liver damage and inflammation\u003c/h2\u003e \u003cp\u003eAlthough MST1 deficiency exacerbates hepatic steatosis in high-fat-fed mice, activating MST1 can alleviate steatosis.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e However, the role of MST1 in the pathogenesis of NASH remains uncertain. To assess the in vivo effects of MST1 deficiency, we generated MST1 gene knockout (MST1 KO) mice and compared them with their wild-type (WT) littermates. The lack of MST1 does not significantly affect the body weight or liver wet weight of mice on a normal diet (Fig. S3A, B); however, serum levels of ALT and AST are significantly elevated (Fig. S3C), indicating a relationship between MST1 deficiency and liver injury. Further assessment of serum TC, LDL-C, and HDL-C levels revealed disruptions in cholesterol homeostasis in MST1-deficient mice fed a normal diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Hepatic TC and FC contents were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), with evident FC deposition in liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Kupffer cell activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) stimulated increased expression of TNF-α, CCL2, and TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). While hepatic steatosis was not significantly observed, hepatocellular ballooning, localized inflammatory cell infiltration, and mild perisinusoidal fibrosis were visible, as demonstrated by H\u0026amp;E and Masson's trichrome staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This indicates that the lack of MST1 can lead to the deposition of free cholesterol in the liver of mice on a normal diet, causing liver inflammation and damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe induced NASH in MST1 KO mice by feeding them a WD. MST1 KO mice exhibited a slight increase in both body weight and liver wet weight compared to the control group, although the differences were not statistically significant (Fig. S3A, B). Markedly elevated serum ALT levels indicated aggravated liver injury due to MST1 deficiency (Fig. S3C). Serum TC and LDL-C were further increased, while HDL-C decreased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). There was an increase in hepatic TC and FC deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Massive infiltration of Kupffer cells was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), stimulating the expression of pro-inflammatory factors TNF-α, IL-1β, IL-6, CCL2, and pro-fibrotic factor TGF-β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Both groups of mice exhibited extensive hepatic steatosis; however, MST1 KO mice fed a Western diet (WD) displayed more pronounced hepatocellular ballooning, inflammatory infiltration, and fibrosis compared to wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that MST1 deficiency induced liver free cholesterol overload, further amplifying the liver inflammatory damage induced by diet in NASH mice.\u003c/p\u003e \u003cp\u003eFurthermore, we confirmed by immunoblotting that in vivo MST1 deficiency induces SREBP2 maturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, G), leading to increased transcription of SREBP2 and its downstream targets HMGCR, HMGCS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, H) in both NCD and WD-fed mouse models. In vitro, silencing MST1 using siRNA in FFA-induced and non-induced HepG2 cells also demonstrated that downregulation of MST1 induces SREBP2 nuclear activation (Fig. S4A, C), upregulates SREBP2, HMGCR, HMGCS1 gene expression (Fig. S4B, D), and results in increased TC, FC levels in FFA-induced HepG2 cells (Fig. S4E), exacerbating FC deposition on the cell membrane in the NASH cell model (Fig. S4F).\u003c/p\u003e \u003cp\u003eTherefore, MST1 deficiency induces SREBP2 maturation, elevates the expression of cholesterol synthesis genes, resulting in FC deposition in the liver, triggering the release of pro-inflammatory factors from hepatic macrophages, amplifying NASH-associated liver injury and inflammation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMST1 ameliorates cholesterol deposition and inflammation in NASH liver\u003c/h2\u003e \u003cp\u003eThe impact of MST1 on cholesterol accumulation and inflammation in NASH liver was further evaluated through gain-of-function approaches. In FFA-treated HepG2 cells, transfection with an MST1 overexpression plasmid led to increased MST1 expression, resulting in decreased cellular TC and FC levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and an improvement in membrane FC deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). C57BL/6J mice fed a Western diet were treated with an MST1-overexpressing lentiviral vector; two weeks later, an upregulation of MST1 expression in the liver was observed (Fig. S5A). There was a slight decrease in body weight and a reduction in liver wet weight compared to the control group (Fig. S5B, C). Upregulation of MST1 function reduced serum TC levels in the NASH model mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) as well as hepatic TC and FC levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), significantly ameliorating hepatic FC accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This led to reduced Kupffer cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), resulting in a downregulation of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, CCL2, and fibrotic factor TGF-β expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The severity of hepatic steatosis, liver injury, inflammatory cell infiltration, and fibrosis were improved to varying degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), with a decrease in serum ALT, AST levels in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). These findings suggest that restoring MST1 reverses hepatic inflammation and damage caused by excessive free cholesterol overload in the NASH model mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMST1 activates AMPKα through phosphorylation at Thr172\u003c/h2\u003e \u003cp\u003eAMPK, a critical regulatory factor in energy metabolism, requires phosphorylation of Thr172 in its α subunit for complete activation.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Studies have shown that AMPK-mediated phosphorylation can inhibit the transcriptional activity of nuclear SREBP2.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Given MST1's identity as a protein kinase, we hypothesized its potential to activate the AMPK pathway by stimulating phosphorylation of the AMPKα protein.\u003c/p\u003e \u003cp\u003eIn the NASH model characterized by increased nuclear translocation of SREBP2 and upregulated transcriptional activity, we observed a concomitant downregulation of AMPK Thr172 phosphorylation and MST1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Further confirmation of the interaction between MST1 and AMPK was obtained through immunoprecipitation experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe hypothesized that AMPK might serve as a substrate for MST1. Evaluation of the livers from MST1 knockout mice fed with both NCD and WD revealed a reduction in AMPK Thr172 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Similarly, in vitro experiments involving siRNA-mediated knockdown of MST1 in HepG2 cells induced with and without FFA demonstrated a decrease in AMPK Thr172 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results provide further support for the notion that AMPK is a substrate of MST1, suggesting that MST1 may activate the AMPK pathway by promoting the phosphorylation of AMPKα protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eActivation of AMPK reversed the hepatic cholesterol accumulation and liver damage induced by MST1 deficiency\u003c/h2\u003e \u003cp\u003eBased on our findings, we propose that AMPK may act as a downstream substrate involved in MST1-mediated regulation of cholesterol synthesis and metabolism. To investigate further whether AMPK activation can mitigate the effects of MST1 deficiency, we administered intraperitoneal injections of an AMPK activator (AICAR) to MST1 KO mice under both NCD and WD conditions for a 2-week treatment period. Remarkably, AICAR improved disrupted cholesterol metabolism in MST1 KO mice, evidenced by reduced body weight (Figure S3a), decreased serum TC, LDL-C levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), lowered hepatic TC and FC contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and notably reduced hepatic FC deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), regardless of the diet.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAICAR significantly ameliorated hepatic damage and inflammation in MST1 KO mice, both under NCD and WD feeding conditions. Serum ALT and AST levels decreased notably, particularly in MST1 KO mice fed a WD (Figure S3c), accompanied by reduced activity of hepatic macrophages and downregulation of pro-inflammatory and pro-fibrotic factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). NASH-related hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, and liver fibrosis were markedly improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eSubsequent research further confirmed that AMPK independently regulates SREBP2, separate from MST1. AICAR treatment upregulated AMPKα Thr172 phosphorylation in MST1-deficient liver tissues, inhibiting SREBP2 nuclear translocation. This resulted in reduced active n-SREBP2 expression and increased expression of inactive pre-SREBP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, G). Moreover, there was a significant downregulation in mRNA expression of SREBP2, HMGCR, and HMGCS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, H). These findings indicate that AMPK, downstream of MST1, modulates hepatic cholesterol synthesis metabolism through its control over SREBP2 nuclear translocation and transcriptional activity. Activating AMPK can reverse hepatic cholesterol deposition and damage caused by MST1 deficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMST1 participates in regulating cholesterol synthesis metabolism through the AMPK/SREBP2 signaling pathway\u003c/h2\u003e \u003cp\u003eGiven the clear regulatory role of the AMPK/SREBP2 signaling pathway in cholesterol metabolism, we have also confirmed the phosphorylation of AMPKα by MST1 kinase. Consequently, we investigated whether MST1 modulates cholesterol synthesis metabolism through the AMPK/SREBP2 signaling pathway. Protein immunoblot analysis of the livers from C57BL6/J mice treated with LV-MST1 under a WD regimen revealed enhanced MST1 function, increased AMPKα Thr172 phosphorylation, AMPK activation, and inhibition of SREBP2 maturation, leading to reduced n-SREBP2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, there was an upregulation in AMPK transcription levels along with a downregulation in transcription levels of SREBP2, HMGCR, and HMGCS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn vitro, we transfected HepG2 cells with overexpressed MST1 plasmids under both FFA-induced and non-induced conditions. Consistent with in vivo findings, MST1 facilitated AMPKα Thr172 phosphorylation, activated AMPK, and suppressed SREBP2 cleavage for nuclear activation, consequently downregulating nuclear SREBP2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, E). Concurrently, MST1 overexpression upregulated AMPK mRNA levels and downregulated transcriptional expression of SREBP2, HMGCR, and HMGCS1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, F).\u003c/p\u003e \u003cp\u003eOverall, both in vivo and in vitro findings demonstrate that MST1 activates AMPK, inhibits SREBP2 cleavage into its active form, and suppresses the transcription of SREBP2 and its target genes, thereby downregulating the activity of the cholesterol synthesis pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study reveals that in both in vivo and in vitro models of NASH, the cholesterol synthesis pathway is activated, accompanied by hepatic FC deposition and concurrent inflammation and fibrosis, while MST1 expression is reduced. MST1 deficiency promotes SREBP2 nuclear translocation, upregulates the expression of key cholesterol synthesis genes, enhances hepatic FC accumulation, and exacerbates NASH-related liver damage, inflammation, and fibrosis. Our findings indicate that MST1 regulates SREBP2 through the phosphorylation of AMPKα at the Thr172 site. MST1 overexpression upregulates AMPKα Thr172 phosphorylation, inhibits SREBP2 nuclear translocation, downregulates cholesterol synthesis gene expression, reduces hepatic FC deposition, and ameliorates NASH-related liver inflammation and fibrosis.\u003c/p\u003e \u003cp\u003eNASH, as a progressive stage of NAFLD, has been widely associated with hepatic accumulation of toxic lipid-free cholesterol (FC). Lipidomic analyses in human liver tissues indicate significantly elevated FC levels in NASH patients compared to NAFL individuals.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e In NAFLD patients, the co-occurrence of NASH and fibrosis parallels the hepatic aggregation of FC. Experimental induction of hepatic FC accumulation accelerates liver inflammation and fibrosis, while correcting excessive hepatic FC load can alleviate the severity of NASH.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e In NASH patients and NASH mouse models induced by high fat and high cholesterol (HFHC) diets, cholesterol crystals formed in the LDs of steatotic hepatocytes can activate Kupffer cells,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e KCs secrete proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which recruit neutrophils and circulating monocytes through the interaction of CCL2 and CCR2. Monocytes differentiate into proinflammatory macrophages, further amplifying liver inflammation, while stimulating hepatic stellate cells through TGF-β, promoting their transformation into activated myofibroblasts, thereby promoting fibrosis.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e In the study of NASH model mice induced by a high-fat and high cholesterol diet, we also observed a correlation consistent with the above research results, further clarifying the correlation between liver FC overload and NASH inflammatory injury.\u003c/p\u003e \u003cp\u003eAs an important site for cholesterol metabolism, the liver is known to have many disruptions in liver cholesterol metabolism throughout the progression of NAFLD, which can lead to liver cholesterol deposition. Hyperinsulinemia and inflammation lead to the loss of SCAP/SREBP2 inhibition, causing excessive cholesterol accumulation in the liver of mice.\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e NAFLD/NASH patients exhibit increased nuclear SREBP2, HMGCR mRNA, HMGCR protein, and phosphorylation levels alongside decreased LDLR, ABCA1, ABCG1, and ABCG5 mRNA levels. This imbalance results in heightened cholesterol synthesis and reduced excretion, contributing to increased hepatic cholesterol accumulation.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e In our findings from the NASH model mice, serum TC, LDL, hepatic TC, and FC levels were notably elevated. However, SREBP2 activity remained unimpeded, leading to increased nuclear SREBP2 expression and enhanced transcriptional expression of cholesterol synthesis genes HMGCR and HMGCS1. These results align with the previously mentioned disruptions in cholesterol synthesis metabolism.\u003c/p\u003e \u003cp\u003eAs a core kinase in the Hippo signaling pathway, MST1 has been associated with hepatic lipid metabolism disorders. Significant lipid metabolism disturbances are observed in MST1 gene knockout mice, believed to be directly linked to decreased liver Sirt1 expression, enhanced ubiquitination degradation pathways, and increased expression of SREBP-1c.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e However, studies investigating its correlation with hepatic cholesterol metabolism are scarce. Existing studies have prominently highlighted the interplay between core components of the Hippo pathway, such as LATS1/2, Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ), with hepatic cholesterol metabolism. Research by Xiaobo Wang et al. demonstrates that excessive hepatic cholesterol induces downstream calcium signaling, triggering RhoA activity and inhibiting LATS1/2, leading to increased protein levels of TAZ and YAP at the transcriptional level. Elevated TAZ in liver cells can induce NASH fibrosis.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Zhiping Shu and colleagues confirmed YAP as a crucial coactivator of SREBP-1c and SREBP-2, controlling the expression of FAS and HMGCR genes in hepatocytes. Overexpression of LATS1 inhibits YAP dephosphorylation and nuclear translocation, improving systemic insulin resistance and hepatic steatosis in diabetic mice.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Yael Aylon's research demonstrates that LATS2 binds to the endoplasmic reticulum-linked precursors of SREBP-1 and SREBP-2, inhibiting their processing and subsequently dampening the nuclear transcriptional activity of SREBPs.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e These studies collectively suggest a close association between the core component MST1 within the Hippo signaling pathway and hepatic lipid metabolism, cholesterol metabolism, as well as the occurrence and progression of NASH.\u003c/p\u003e \u003cp\u003eBuilding upon our previous work,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e we propose the potential of MST1 to regulate hepatic cholesterol synthesis metabolism through AMPK. Our study elucidates the interaction between MST1 and AMPK, providing further validation that MST1 can phosphorylate the Thr172 site of AMPK. Treatment of MST1 knockout mice with AICAR confirmed AMPK as a substrate of MST1 and demonstrated its ability to reverse hepatic FC overload and liver damage caused by MST1 deficiency. Additional mechanistic investigations revealed that AMPK regulates the nuclear activation of SREBP2 and, at the transcriptional level, modulates the expression of SREBP2 and its downstream targets HMGCR and HMGCS1. These findings are consistent with prior research by other investigators.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e By inducing MST1 overexpression in in vivo and in vitro models, we have elucidated that MST1 phosphorylates AMPKα at Thr172, leading to a cascade of changes in the AMPK/SREBP2 signaling pathway. This modulation affects SREBP2 nuclear translocation and alters the expression of SREBP2 and downstream target genes HMGCR and HMGCS1. The reduction in cholesterol synthesis alleviates hepatic free cholesterol load, resulting in an improvement in the degree of NASH-related liver inflammation and fibrosis.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that MST1 can alleviate hepatic free cholesterol load and thus mitigate the progression of NASH through the regulation of AMPK/SREBP2 signaling. This highlights MST1 as a promising target for the treatment of NAFLD/NASH.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAICAR, AMPK activator; ALT, alanine aminotransferase; AMPK, 5\u0026prime; AMP-activated protein kinase; AST, aspartate aminotransferase; CCL2, chemokine C-C motif ligand 2; FC, free cholesterol; FFA, free fatty acids; H\u0026amp;E, haematoxylin and eosin; HDL-C, High-density lipoprotein cholesterol; HMGCR, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; HMGCS1, 3-hydroxy-3-methylglutaryl coenzyme A synthetase1; HSCs, hepatic stellate cells; IL-1\u0026beta;, interleukin-1\u0026beta;; IL-6, interleukin-6; KCs, Kupffer cells; LDs, lipid droplets; LDL-C, Low density lipoprotein cholesterol; MST1, mammalian sterile 20-like kinase 1; NASH, non-alcoholic steatohepatitis; NCD , normal chow diet ; SREBP2, sterol regulatory element-binding transcription factor 2; TC, total cholesterol; TGF-\u0026beta;, transforming growth factor-beta; TNF-\u0026alpha;, tumor necrosis factor-alpha; WD, Western diet; WT, Wild-type.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYi Yang and Lijuan Wang conceived the study. Lijuan Wang, Chenglei Zhang, and Jie Ma conducted experiments and performed data analysis. Jiarui Li, Yuanyuan Wu, Yanru Ren, and Jianning Li conducted animal experiments and analyzed data. Lijuan Wang and Yan Li drafted and edited the manuscript. Yi Yang revised the paper. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82160171)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study strictly followed the Guidelines for the Care and Use of Experimental Animals published by the National Institutes of Health, and all experimental protocols were approved by the Medical Ethics Review Committee of Ningxia Medical University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. 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Hepatol Commun 2020;4(8): 1149-1167.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mammalian sterile 20-like kinase 1, AMP-activated protein kinase, cholesterol synthesis, hepatic free cholesterol, non-alcoholic steatohepatitis","lastPublishedDoi":"10.21203/rs.3.rs-4443517/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4443517/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNon-alcoholic steatohepatitis (NASH) escalates adverse liver-related outcomes, with its progression linked to hepatic lipotoxicity induced by excess hepatic free cholesterol (FC) MST1 has been identified as a potential regulator of hepatic lipid metabolism, potentially ameliorating NAFLD. This study aims to delineate the role of MST1 in the progression of NASH. Wild-type (WT) and MST1 gene knockout (MST1 KO) mice were induced into NASH using a high-fat, high-sugar, high-cholesterol Western diet (WD). In vivo overexpression of MST1 was conducted using lentivirus in WD-fed WT mice. In vitro, HepG2 cells were subjected to MST1 knockdown and overexpression treatments, cultured in a medium induced by a mixture of palmitic acid and oleic acid as free fatty acids (FFA). The NASH model activates the hepatic cholesterol synthesis pathway, leading to an overload of hepatic free cholesterol and downregulation of MST1 expression. Knocking out MST1 exacerbates hepatic FC accumulation and inflammatory damage, activating the cholesterol synthesis pathway. Conversely, upregulating MST1 expression improves hepatic FC deposition, alleviating hepatic damage and inflammation. We found that AMPKα is a substrate of MST1, and MST1 can phosphorylate AMPKα at Thr172. Phosphorylation of AMPKα at Thr172 inhibits the cholesterol synthesis pathway, significantly reversing hepatic FC overload and inflammation caused by MST1 deficiency. Further mechanistic studies indicate that MST1 inhibits cholesterol synthesis by targeting the AMPK/SREBP2 pathway, thereby improving hepatic inflammatory damage caused by FC overload. MST1 targeting AMPK in regulating hepatic cholesterol synthesis metabolism serves as an attractive therapeutic target for preventing the progression of NASH-associated inflammation and fibrosis.\u003c/p\u003e","manuscriptTitle":"Mammalian Ste20-Like Kinase 1 Regulates AMPK to Mitigate the Progression of Non-Alcoholic Fatty Liver Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-02 06:05:59","doi":"10.21203/rs.3.rs-4443517/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65a0fd48-6fc4-40dd-b298-b422960d3ab6","owner":[],"postedDate":"July 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-16T10:38:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-02 06:05:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4443517","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4443517","identity":"rs-4443517","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}