Fas apoptotic inhibitor molecule 2 mitigates metabolic dysfunction-associated fatty liver disease through autophagic CRTC2 degradation

Fas apoptotic inhibitor molecule 2 mitigates metabolic dysfunction-associated fatty liver disease through autophagic CRTC2 degradation | 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 Fas apoptotic inhibitor molecule 2 mitigates metabolic dysfunction-associated fatty liver disease through autophagic CRTC2 degradation Peng Zhang, Guangnian Zhao, Hong Yu, Yongjie Yu, Sha Hu, Tuo Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6110029/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Lysosomal membrane proteins (LMPs) play fundamental roles in the lysosomal degradation of proteins and are attractive drug targets for metabolic dysfunction-associated fatty liver disease (MAFLD). Fas apoptotic inhibitory molecule 2 (FAIM2), an LMP, is previously recognized as an inhibitor of apoptosis in a variety of diseases. Here, we reveal that FAIM2 is an inhibitor of fatty acid synthesis and suppresses MAFLD. FAIM2 protein expression is decreased in MAFLD. Moreover, FAIM2 is degraded by the E3 ubiquitin ligase NEDD4L through catalyzing K48-linked ubiquitination. High-fat or high-fat and high-cholesterol diet-induced hepatic steatosis, inflammation and fibrosis was aggravated in FAIM2 knockout (KO) mice and alleviated in FAIM2 overexpressing mice. Furthermore, in hepatocytes, FAIM2 KO increased the expression of genes related to fatty acid synthesis, whereas over-expressing FAIM2 exhibits the opposite effect. Mechanistically, FAIM2 directly interacts with CREB-regulated transcription coactivator 2 (CRTC2), a prominent regulator of lipid metabolism, and medicates its degradation through autophagy. Specifically, we find that the N-terminus of FAIM2 which interacts with CRCT2 and LC3B is required for autophagic CRTC2 degradation. Collectively, our findings reveal that FAIM2 acts as a fatty acid synthesis inhibitor in MAFLD by promoting the autophagic degradation of CRTC2, and FAIM2-CRTC2 may be a promising therapeutic target. Biological sciences/Molecular biology/Proteolysis Health sciences/Diseases/Metabolic disorders MAFLD FAIM2 Fatty acid synthesis CRTC2 Degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease, has become the most prevalent chronic liver disease, with a global prevalence rate of up to 30%, and its prevalence is increasing [ 1 ] . Moreover, MAFLD can progress from simple hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which puts individuals at risk of end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma [ 2 ] . Despite the large number of individuals with MAFLD, poor adherence to lifestyle interventions is observed in clinical practice [ 3 ] , and only limited pharmacological therapy has been approved for MASH [ 4 ] . Therefore, the identification of effective therapeutic targets and strategies for MAFLD is urgently needed. Lysosomal degradation, including the endosome‒lysosome pathway and the autophagy‒lysosome pathway (also known as autophagy), is a pivotal physiological activity essential for maintaining cellular protein homeostasis and has been an appealing platform for drug discovery [ 5 – 7 ] . The membrane of lysosomes harbors a variety of proteins that play critical roles in maintaining the operation of lysosomal degradation (preserving the integrity of the lysosome structure; regulating enzyme activity) and mediating interactions with other organelles or molecules [ 8 – 11 ] . The dysregulation of lysosomal membrane proteins can disrupt cellular protein homeostasis, which in turn can cause a spectrum of diseases, including MAFLD [ 12 – 14 ] . The ablation of glycosylated lysosomal membrane protein in mice promote lipid deposition and causes fibrosis and hepatic cell death in the liver [ 15 , 16 ] . Moreover, lysosomal-associated protein transmembrane 5 is negatively correlated with NAFLD activity score, and the hepatocyte-specific depletion of lysosomal-associated protein transmembrane 5 exacerbates MASH symptoms in mice [ 17 ] . Our previous studies revealed that transmembrane BAX inhibitor motif-containing protein 1 (TMBIM1) protects against MAFLD by promoting the lysosomal degradation of Toll-like receptor 4 [ 18 ] . Given the crucial role of lysosomal membrane proteins in lysosomal degradation and their connections in MAFLD, lysosomal membrane proteins could be promising therapeutic targets for MAFLD treatment. Fas apoptotic inhibitory molecule 2 (FAIM2), also known as TMBIM2, belongs to the TMBIM family, which is characterized by a UPF0005 motif that encodes either six or seven transmembrane structures. FAIM2 has seven transmembrane structures and locate on lysosomes [ 19 ] ; it was initially identified in a study seeking genes involved in the development and maintenance of the nervous system [ 20 ] . FAIM2 protects hippocampal cells from death in the acute phase of bacterial meningitis [ 21 ] , reduces stroke volume and alleviates dopaminergic neuron degeneration in Parkinson’s disease [ 22 , 23 ] . Recently, multiple studies have suggested that SNP mutations at the FAIM2 gene locus are associated with obesity and type 2 diabetes, which are critical risk factors for MAFLD [ 24 – 27 ] . However, to date, the role of FAIM2 in MAFLD remains unknown. Here, we identified FAIM2 as an effective suppressor of MAFLD. A marked decrease in FAIM2 protein levels was observed in MAFLD. FAIM2 depletion exacerbated lipid deposition, inflammation, and fibrosis. However, overexpressing FAIM2 in mice presented the opposite effects. Moreover, in hepatocyte, FAIM2 overexpression suppress fatty acid synthesis related genes. Mechanistically, FAIM2 directly interact with CREB-regulated transcription coactivator 2 (CRTC2), a key regulator of lipid metabolism, and facilitated the degradation of CRTC2 through autophagy. Collectively, our findings suggest that FAIM2 suppresses MAFLD by promoting the autophagic degradation of CRTC2 and further suppressing fatty acid synthesis, which could be utilized as a therapeutic target for MAFLD. 2. Materials and Methods 2.1. Animal treatment . All animal protocols were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (grant no.:20230404G). The animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. 2.2. Human liver samples . All procedures involving the collection of human samples have been approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (grant no.:2018010), and the principles of the Helsinki Declaration have been followed to obtain written informed consent from the participants or their family members. Human steatotic liver samples were obtained from patients with simple steatosis or MASH who had undergone either liver biopsy or liver transplantation. Non-steatotic liver samples were collected from healthy regions of the livers from donors who had undergone liver resection because of a liver hemangioma or hepatic cyst. 2.3. Statistical Analysis . In brief, all statis. tical analyses were performed using SPSS software (version 25, USA). For data showing a Gaussian distribution, parametric statistical analysis was performed using the two-tailed Student’s t test for two groups. One-way ANOVA was applied to three or more groups, followed by either Bonferroni post hoc analysis for data meeting homogeneity of variance requirements or Tamhane’s T2(M) post hoc analysis for heteroscedastic data. For datasets with skewed distributions, nonparametric statistical analysis was performed using the Mann-Whitney U test for two groups and Kruskal-wallis test for three or more groups. All values were presented as the mean ± SD. P values were specified as follows: ∗ p < 0.05, ∗∗ p < 0.01. Data from animal studies were collected in a blinded fashion. No data were excluded when performing the final statistical analysis. A randomization process was performed in grouping mice with the same phenotypes. 2.4. Supplementary methods. Detailed materials and methods are described in the Supplementary Materials and Methods sections. 3. Results 3.1. FAIM2 expression is downregulated in fatty livers To investigate whether FAIM2 is involved in MAFLD, we first examined FAIM2 expression profiles in MAFLD livers. We found that the FAIM2 protein level was markedly lower in the livers of mice that were fed a high-fat diet (HFD) or a high-fat and high-cholesterol diet (HFHC) (Figure. 1A, B) . Moreover, FAIM2 protein levels were markedly lower in the livers of individuals with simple hepatic steatosis and MASH than in the livers of nonsteatotic control individuals (Figure. 1C) . The protein level of FAIM2 was also decreased in primary hepatocytes stimulated with prosteatotic stimuli (palmitate and oil acid, PAOA) (Figure. 1D) . And immunofluorescence analyses suggested that FAIM2 was decreased primarily in the cytoplasm of hepatocytes (Figure. 1E) . Although protein levels varied, FAIM2 mRNA levels did not significantly change under any of the conditions tested, as well as protein level in sinusoidal endothelial cells or Kupffer cells (Figure. 1F-H) . Taken together, the remarkable downregulation of FAIM2 expression observed in fatty livers suggests that FAIM2 may play a role in the occurrence and development of MAFLD and that FAIM2 may undergo posttranslational modification. 3.2. NEDD4L facilitates FAIM2 degradation through catalyzing its K48-linked ubiquitination Next, we explored the mechanism involved in the downregulation of FAIM2 protein in MAFLD. It has been reported that the ubiquitin-proteasome system (UPS) and autophagy were two major systems for intracellular protein degradation. We found that the downregulation of FAIM2 was regulated by the UPS (Figure. 2A, B) . We further explored whether ubiquitin E3 ligase (bioGRID) might participate in the degradation of FAIM2. And the result of Co-IP test showed that TRIM21, NEDD4, and NEDD4L can interact with FAIM2, while NEDD4L interacted stronger (Figure. 2B) . What’s more, the protein level of FAIM2 was significantly downregulated with the overexpression of NEDD4L (Figure. 2D-F) . In order to further explore the mechanism by which NEDD4L mediated FAIM2 degradation, we detected the ubiquitination of FAIM2 through IP assays. Our results showed that NEDD4L mediated FAIM2 degradation through K48-linked ubiquitination (Figure. 2G-I) . These results indicate that ubiquitin-proteasome degradation of FAIM2 was mediated by NEDD4L under metabolic stress. 3.3. FAIM2 knockout aggravates HFD-induced metabolic disorders Given the conspicuous changes in FAIM2 expression observed in fatty livers, we fed Faim2 -knockout ( Faim2 -KO) mice and wild type (WT) littermate control with a normal chow or HFD for 24 weeks to assess the function of FAIM2 in MAFLD (Figure. 3A) . FAIM2 deletion did not significantly affect the body weight of mice with NC, but increase the body weight of mice with HFD (HFD-fed Faim2 -KO mice, KO-HFD) (Figure. 3B) . Compared with HFD-fed WT mice, KO-HFD mice presented higher fasting blood glucose levels and more severe glucose intolerance (Figure. 3C, D, E) . Moreover, KO-HFD mice exhibited increased liver weight and white adipose weight, greater serum total triglyceride, total cholesterol and low-density lipoprotein contents, and aggravated accumulation of hepatic lipids (Figure. 3F-J) . In addition to steatosis, higher serum ALT and AST concentrations in KO-HFD mice indicated exacerbated liver injury (Figure. 3K) . RNA-seq analyses were performed to further assess the influence of FAIM2 on MAFLD. Principal component analysis clearly separated the HFD-treated liver samples into two subgroups: HFD-fed WT mice and KO-HFD livers (Figure. 3L) . Notably, as shown by the GSEA analyses, KEGG pathways related to lipid metabolism, including fatty acid synthesis and elongation, were enriched in the KO-HFD group (Figure. 3M) . Moreover, a heatmap revealed that FAIM2 deletion activated genes related to fatty acid synthesis (Figure. 3N) , that were confirmed by quantitative PCR (Figure. 3O) . Taken together, these findings suggest that FAIM2 deficiency exacerbates HFD-induced metabolic disorders may through activating fatty acid synthesis. 3.4. FAIM2 knockout aggravates HFHC-induced MASH We next examined whether FAIM2 knockout accelerated the development of HFHC-induced MASH, a dietary model that exhibits considerable fidelity to human MASH with features that included steatosis, advanced hepatic inflammation and fibrosis [ 28 ] . HFHC-fed Faim2 -KO mice (KO-HFHC) presented increased body weights compared with those in HFHC-fed WT mice (Figure. 4A) . In addition, KO-HFHC mice displayed glycometabolism disorder, greater liver weight and white adipose weight, increased serum TG, TC, ALT and AST contents, and more severe liver steatosis (Figure. 4B-H, J) . Furthermore, KO-HFHC mice presented more severe inflammatory responses (Figure. 4I, J) , and greater collagen deposition and expression of genes involved in fibrogenesis ( Col1a1 and Col4a1 ) (Figure. 4I, J) . Collectively, the above findings suggest that FAIM2 deficiency exacerbates HFHC-induced steatosis, inflammation and collagen deposition. 3.5. FAIM2 overexpression alleviates HFHC-induced MASH To further assess the function of FAIM2 in MAFLD, we generated FAIM2-overexpressing mice by injecting AAV8-FAIM2, and fed them and corresponding control mice with HFHC (Figure. 5A) . Noticeably, FAIM2-overexpressing mice presented lower body weights, improved glycometabolism, decreased liver and white adipose weight, and decreased serum TG, TC, LDL, ALT and AST contents (Figure. 5B-J) . Moreover, FAIM2 overexpression significant alleviated hepatic steatosis, inflammation, and fibrogenesis (Figure. 5K) . Above results suggested FAIM2 overexpression mitigates HFHC-induced MASH and FAIM2 is a protective factor under metabolic stress. 3.6. FAIM2 inhibits fatty acid synthesis in hepatocytes We observed FAIM2 deletion activated gene and pathway related to fatty acid synthesis in vivo , and hepatocytes are the predominant cell type in the liver and the main executor of lipid synthesis [ 29 ] . Thus, we further assayed the function of FAIM2 in hepatocytes from Faim2 -KO mice ( Figure. 6A ). The lipid content and total triglyceride content were greater in FAIM2-deficient primary hepatocytes stimulated with PAOA than in the corresponding WT controls (Figure. 6B, C) . Moreover, FAIM2 deletion promoted the expression of genes related to fatty acid synthesis and inflammation (Figure. 6D) . Next, we constructed an adenovirus expressing FAIM2 (Ad Faim2 ) to investigate the effects of FAIM2 overexpression in primary hepatocytes (Figure. 6E) . Notably, FAIM2 overexpression reduced lipid and TG accumulation and inhibited fatty acid synthesis and inflammation (Figure. 6F-H) . Above findings demonstrate that FAIM2 inhibits fatty acid synthesis and the inflammatory response in hepatocytes. 3.7. FAIM2 interacts with CRTC2 and reduces its expression We next explored the mechanistic link between FAIM2 and its function. The above results revealed that FAIM2 knockout significantly and consistently increased lipid accumulation and upregulated fatty acid synthesis-related gene expression in in vivo and in vitro MAFLD models, thus we speculated that FAIM2 may function by affecting lipid metabolism regulators. We applied immunoprecipitation-mass spectrometry to identify proteins that physically interact with FAIM2 and screened out lipid metabolism regulators. Among them, CRTC2 was reported to be a lipid metabolism regulator that promoted MAFLD ( Figure. 7A ) [ 30 – 32 ] . The interaction between FAIM2 and CRTC2 was verified, and the direct interaction between them was further confirmed via a GST pull-down assay (Figure. 7B-E) . Remarkably, although the Crtc2 mRNA level did not significantly change in hepatocytes overexpressing or lacking FAIM2 (Figure. 7F, H) , the CRTC2 protein level was decreased in FAIM2-overexpressing hepatocytes and increased in FAIM2-deficient hepatocytes (Figure. 7G, I) . In liver of mice fed with either HFD or HFHC, CRTC2 change in the same pattern (Figure. 7J-M) . Moreover, CRTC2 expression was increased in the liver of MAFLD individuals (Figure. 7N-O) . Collectively, these findings suggest that FAIM2 directly interacts with CRTC2 and decreases its protein level, indicating that FAIM2 promote CRTC2 posttranslational modification. 3.8. FAIM2 regulates lipid metabolism via CRTC2 To evaluate whether CRTC2 is required for FAIM2-regulated lipid metabolism in hepatocytes, we constructed an adenovirus to overexpress CRTC2 ( Figure. 8A ). The inhibitory effect of FAIM2 overexpression on lipid accumulation was blunted by CRTC2 overexpression (Figure. 8B, C) . Overexpressing CRTC2 also reversed the inhibitory effects of FAIM2 on fatty acid synthesis and inflammatory gene expression (Figure. 8D) . Next, we constructed adenovirus short hairpin RNA targeting Crtc2 (Adsh Crtc2 ) to knockdown CRTC2 in primary hepatocytes isolated from Faim2 -KO mice (Figure. 8E) . Interestingly, CRTC2 knockdown also reversed the effect of FAIM2 knockout (Figure. 8F-H) . These data indicate that the inhibitory effect of FAIM2 on fatty acid synthesis required CRTC2. 3.9. FAIM2 facilitates CRTC2 degradation through autophagy We next sought to determine how FAIM2 decreased the protein level of CRTC2. The protein level of CRTC2 decreased with increasing FAIM2 expression in a dose-dependent manner ( Figure. 9A ). Significantly, CRTC2 degradation occurred faster in FAIM2-overexpressed hepatocytes under cycloheximide (CHX) treatment (Figure. 9B) , and FAIM2-promoted CRTC2 protein degradation was blocked under CQ treatment (Figure. 9C) . Moreover, FAIM2 promoted the colocalization of CRTC2 and lysosomal marker LAMP1 (Figure. 9D) . These results suggest that FAIM2 promotes the degradation of CRTC2 via lysosomal pathway. The lysosomal pathway including the endosome‒lysosome pathway and autophagy [ 33 ] . Considering that autophagy is principally responsible for the degradation of cytoplasmic components [ 34 , 35 ] and FAIM2 and CRTC2 primarily colocalize in the cytoplasm under PAOA stimulation, we further investigated whether FAIM2 degrades CRTC2 through autophagy. The results revealed that FAIM2 overexpression in hepatocytes led to a reduction in CRTC2 levels concomitant with an increase in the expression of the LC3 and a decrease in the expression of p62 (Figure. 9E) . Compared with hepatocytes treated with bafilomycin A1, hepatocytes with bafilomycin A1 and overexpressing FAIM2, exhibited further LC3, and p62 accumulation. In addition, the FAIM2-promoted CRTC2 degradation was suppressed under bafilomycin A1 treatment (Figure. 9F) . Moreover, FAIM2 overexpression resulted in greater red fluorescence of GFP-RFP-LC3B in hepatocytes (representing autolysosomes) (Figure. 9G) . These results indicate that FAIM2 promotes autophagy, which in turn facilitates CRTC2 degradation. Next, we investigated how FAIM2 promotes autophagy. The deletion of FAIM2 led to an increase in LC3 and p62 levels (Figure. 9H) . Compared with that treated with rapamycin, the expression of LC3 was higher in Faim2 -KO hepatocytes stimulated with rapamycin (Figure. 9I) . In addition, Faim2 -KO hepatocytes presented more intense yellow fluorescence of GFP-RFP-LC3B (representing autophagosomes) (Figure. 9J). These results suggest that FAIM2 is required for autolysosome formation in hepatocytes. Taken together, these results suggest that FAIM2 promotes autophagy and mediates CRTC2 degradation through autophagy. 3.10. The N-terminal domain of FAIM2 is required for CRTC2 degradation To further investigate the detailed mechanism by which FAIM2 mediates CRTC2 degradation, the interaction domain between FAIM2 and CRTC2 was investigated. The results of a domain mapping analysis revealed that the N-terminal domain (cytoplasmic domain) [ 19 ] of FAIM2 interacts with the CREB binding domain (CBD) of CRTC2 (Figure. 10A, B) . To determine whether the interaction between FAIM2 and CRTC2 is necessary for the protective role of FAIM2 against MAFLD, an adenovirus containing FAIM2 lacking the N-terminal domain (Ad Faim2 -▲N) was constructed. Interestingly, after the N-terminal domain was deleted, FAIM2 lost its ability to decrease CRTC2, inhibit lipid accumulation and downregulate the expression of fatty acid synthesis- and inflammation-related genes (Figure. 10C-F) . In addition, the ability of Faim2 -▲N to promote CRTC2 and lysosome colocalization, as well as the response to CQ, was lost (Figure. 10G, H) . These results indicate that the inhibitory effects of FAIM2 on CRTC2 and metabolic stress are required the N-terminal domain of FAIM2. It has been reported that the N-terminal domain of FAIM2 contains an LC3-interacting region (LIR) motif which is responsible for the autolysosome formation [ 19 ] . Therefore, we further investigated whether the interaction of FAIM2 and CRTC2 is dependent on the LIR motif. We constructed plasmid and adenovirus to overexpress FAIM2 lacking the LIR domain (FAIM2-▲LIR). FAIM2-▲LIR lost its ability to promote autophagy and suppress CRTC2 in primary hepatocytes (Figure. 10I) . FAIM2 was able to interact with both CRTC2 and LC3B, whereas FAIM2-▲LIR was unable to bind with LC3B (Figure. 10J, K) . However, FAIM2-▲LIR was still able to interact with CRTC2 (Figure. 10L) , indicating that the interaction of FAIM2 and CRTC2 did not dependent on the LIR motif. Taken together, these results suggest that the N-terminal domain of FAIM2 interacts with both CRTC2 and LC3B, a process that is required for the suppression of CRTC2 by FAIM2. 4. Discussion In this study, we demonstrate that FAIM2 is ubiquitinated and degraded by NEDD4L under conditions of metabolic stresses. FAIM2 deficiency significantly increase lipid deposition and upregulatedpathways and genes related to fatty acid synthesis. Whereas, overexpression of FAIM2 presents the opposite effect. These findings reveal that FAIM2 plays a protective role in MAFLD by mitigating fatty acid synthesis in hepatocytes, and highlight the important role of LMPs in MAFLD or other metabolism-related diseases. We speculated that FAIM2 may mitigate fatty acid synthesis by affecting lipid metabolism regulators. Through immunoprecipitation‒mass spectrometry, we identified lipid metabolism regulators that interact with FAIM2. Among these regulators, CRTC2 is a crucial lipid metabolism regulator in the liver. Liver-specific knockout of Crtc2 reduces fatty acid synthesis genes expression ( Fasn , Scd1 , Pparg and Acaca ), alleviates diet-induced liver lipid accumulation, inhibits inflammation [ 30 ] , and leads to reduced blood lipid and blood glucose concentrations [ 31 ] . Additionally, CRTC2 overexpression enhances liver cholesterol synthesis by promoting the transcription of SREBP-2 [ 32 ] . CRTC2 regulate fatty acid, glucose and cholesterol metabolism, making it a nonnegligible target. We verified that FAIM2 facilitates CRTC2 degradation through autophagy, and the N-terminal domain of FAIM2 is required for this process, which elucidates the underlying mechanism of FAIM2 in MAFLD and provides a potential therapeutic target for further clinical translation. We propose that the targeting of CRTC2 for degradation is the primary cellular mechanism underlying the function of FAIM2 in MAFLD. However, CRTC2 regulates not only fatty acid biosynthesis [ 30 ] , but also blood glucose levels and cholesterol synthesis [ 32 , 36 ] , and participates in the processes of type 2 diabetes, stroke, tumors, circadian rhythm, etc [ 37 – 40 ] . Indeed, the complete disruption of an important target that has such fundamental roles in physiological activity might lead to serious side effects, thus greatly hindering the development of new therapeutic agents. Our results demonstrated that FAIM2 facilitated the autophagic degradation of CRTC2, thereby preventing its excessive activation under prosteatotic stimulus, which works by balancing CRTC2 dynamics to maintain CRTC2 expression at an appropriate level rather than severely blocking its function. This working mode would likely be beneficial for treating MAFLD without inducing serious side effects. Our previous studies reported that TMBIM1 is an effective inhibitor of MAFLD. TMBIM1 collaborates with the ESCRT-mediated endosomal sorting complex to promote multivesicular body formation, thereby facilitating the lysosomal degradation of TLR4 and exerting its protective function [ 18 ] . Although both TMBIM1 and FAIM2 belong to the TMBIM family, we found that FAIM2 protects against MAFLD by mediating the autophagic degradation of CRTC2. The different underlying mechanisms of TMBIM1 and FAIM2 indicated that they do not simply serve as functional substitutes. Although we have employed various models and experimental approaches to elucidate the function and mechanism of FAIM2 in MAFLD, there remains room for refinement in our study. We have demonstrated that FAIM2 can act through primary hepatocytes, but the potential effects of hepatic nonparenchymal cells—such as Kupffer cells, endothelial cells, and stellate cells—require further investigation. Additionally, the use of a molecular docking assay to predict and validate the specific amino acid sequence within the N-terminus of FAIM2 where CRTC2 binds and the use of AI technology to design small-molecule compounds for therapeutic testing may pave the way for clinical translation. In summary, this study reveals the role and mechanism of FAIM2 in MAFLD. Under metabolic stresses, FAIM2 mediates the degradation of CRTC2 via autophagy, and further suppresses fatty acid synthesis and alleviates MAFLD. Thereby, FAIM2-CRTC2 may holds promise as a novel therapeutic target for MAFLD, and targeting LMPs to control protein homeostasis may provide new strategies and promising therapeutic targets. Declarations Acknowledgments This work was supported by grants from the Henan Charity Federation Hepatobiliary Fund (grant no.: GDXZ2021002), National Science Foundation of China (grant no.: 82370882), Hubei Provincial Natural Science Foundation of China (grant no.: 2023AFB670), the Key R&D Program of Jiangxi Province of China (20223BBG71008) and Excellent Youth Foundation of Jiangxi Province (grant no.: 20242BAB23074). Conflict of Interest statement The authors declare no competing interests. Authorship contribution statement Yongjie Yu: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Sha Hu: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Tuo Zhang: Writing – original draft, Investigation, Formal analysis, Data curation. Dajun Li: Methodology, Investigation, Data curation. Yongping Huang : Visualization, Software, Formal analysis. Hong Yu: review & editing, Supervision, Conceptualization. Guang-nian Zhao: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. Peng Zhang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Wong VW, Ekstedt M, Wong GL & Hagström H. Changing epidemiology, global trends and implications for outcomes of NAFLD. J Hepatol 79 , 842-852 (2023). Cohen JC, Horton JD & Hobbs HH. Human fatty liver disease: old questions and new insights. Science 332 , 1519-1523 (2011). Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 67 , 328-357 (2018). Mironova M, Sherker AH. 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Additional Declarations There is no conflict of interest Supplementary Files Supplementarymaterial.docx Supplementary material Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 21 Apr, 2025 Review # 2 received at journal 18 Apr, 2025 Review # 1 received at journal 07 Apr, 2025 Reviewer # 2 agreed at journal 01 Apr, 2025 Reviewer # 1 agreed at journal 24 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Submission checks completed at journal 26 Feb, 2025 Editor assigned by journal 26 Feb, 2025 First submitted to journal 26 Feb, 2025 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-6110029","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431853155,"identity":"a4d182c4-6623-4772-bcf8-bd593c335a3a","order_by":0,"name":"Peng Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAp0lEQVRIiWNgGAWjYDACCQbGB4wNIFYC8VqYDUjWwiZBmhb52b3Hqnl3HGbgZ88xYPi5gwgtjHPOpd3mPXOYQbLnjQFj7xkitDBL5Jjdzm07zGBwI8eAmbGNCC1sQC3FIC32RGvhAWphBtsiQawWCYm8ZOm/bek8EmeeFRzsJUaL/Izcgx9ntlnL8bcnb3zwkxgtQKchyANEaYBpGQWjYBSMglGAGwAA0n4w3kF724IAAAAASUVORK5CYII=","orcid":"","institution":"Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhang","suffix":""},{"id":431853156,"identity":"71d3ff27-db4f-4353-8fdd-2645172a75de","order_by":1,"name":"Guangnian Zhao","email":"","orcid":"","institution":"Tongji Medical College of HUST, Affiliated Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guangnian","middleName":"","lastName":"Zhao","suffix":""},{"id":431853157,"identity":"3db4cf6f-9a20-4711-bc67-5ad4423dacc3","order_by":2,"name":"Hong Yu","email":"","orcid":"","institution":"School of Basic Medical Sciences, Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Yu","suffix":""},{"id":431853158,"identity":"0bdf3a4d-6bcd-45f6-ab00-3b19d8923c17","order_by":3,"name":"Yongjie Yu","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Yongjie","middleName":"","lastName":"Yu","suffix":""},{"id":431853159,"identity":"d227191a-0036-430b-be67-0fb166fb7247","order_by":4,"name":"Sha Hu","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Hu","suffix":""},{"id":431853160,"identity":"73406ec7-dbe6-4d4a-a03d-83fa5d893612","order_by":5,"name":"Tuo Zhang","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Tuo","middleName":"","lastName":"Zhang","suffix":""},{"id":431853161,"identity":"d2a8fac7-7116-4dd4-932e-a8c31a396b84","order_by":6,"name":"Dajun Li","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Dajun","middleName":"","lastName":"Li","suffix":""},{"id":431853162,"identity":"b7306fa2-00d7-4905-8b6e-1353035a0a91","order_by":7,"name":"Yongping Huang","email":"","orcid":"","institution":"Wuhan university","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-02-26 05:50:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6110029/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6110029/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-025-01559-1","type":"published","date":"2025-10-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79087574,"identity":"4545c156-4bef-4d27-9920-59953a9cce0e","added_by":"auto","created_at":"2025-03-24 09:29:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":533883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of FAIM2 expression with fatty liver diseases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B)\u003c/strong\u003e Western blot images and quantification of FAIM2 expression in the livers from C57BL/6J mice that were fed a HFD or NC for 24 weeks (n = 4) (A), or mice were fed a HFHC or NC for 16 weeks (n = 4) (B). \u003cstrong\u003e(C)\u003c/strong\u003e Western blots images and quantification of FAIM2 expression from non-steatotic (NS) donors, individuals with NAFL and individuals with MASH (n = 4). \u003cstrong\u003e(D)\u003c/strong\u003e Western blots for FAIM2 expression in primary hepatocytes that were treated with palmitate and oil acid (PAOA, PA:OA = 0.5 mM:1 mM) (n = 3). \u003cstrong\u003e(E)\u003c/strong\u003e Images of FAIM2 immunofluorescence staining in primary hepatocytes treated with PBS or PAOA. Scale bars, 20 μm. (\u003cstrong\u003eF) \u003c/strong\u003eqPCR analyses of the relative mRNA levels of \u003cem\u003eFAIM2 \u003c/em\u003ein fatty livers of human, HFD-fed mice, HFHC-fed mice and primary hepatocytes of mice (n = 5). \u003cstrong\u003e(G and H)\u003c/strong\u003e Western blots for FAIM2 expression in endothelial cells treated with PAOA or kuffer cells treated with LPS (n = 3).\u003cstrong\u003e \u003c/strong\u003e**\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/be9ca74fcd67d76940b27f50.png"},{"id":79089430,"identity":"72f7faba-ff9d-4c6e-bb00-9406e16785ef","added_by":"auto","created_at":"2025-03-24 09:45:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":876139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNEDD4L medicated FAIM2 degradation through catalyzing K48-linked ubiquitination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blot images of FAIM2 expression in mouse hepatocytes treated with MG132 (50 μM), CQ (50 μM), or DMSO. \u003cstrong\u003e(B) \u003c/strong\u003eUbiquitination assays determining the ubiquitination of FAIM2 in primary hepatocytes. \u003cstrong\u003e(C) \u003c/strong\u003eInteraction between FAIM2 and TRIM21, NEDD4, NEDD4L in HEK293T cells. \u003cstrong\u003e(D) \u003c/strong\u003eWestern blot images of FAIM2 expression after transfected with indicated plasmids. \u003cstrong\u003e(E-F) \u003c/strong\u003eWestern blot images of FAIM2 expression of indicated groups. \u003cstrong\u003e(G) \u003c/strong\u003eUbiquitination assays determining the ubiquitination of FAIM2 after transfected with indicated plasmids. \u003cstrong\u003e(H) \u003c/strong\u003eUbiquitination screening of FAIM2 by NEDD4L with the indicated types of ubiquitin. \u003cstrong\u003e(I) \u003c/strong\u003eUbiquitination of FAIM2 in HEK293T cellstransfected with indicated plasmids.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/18f0eae3134277266960f8b9.png"},{"id":79087575,"identity":"64c50fb3-9a4e-4e11-a0ab-10f5bfb20b0a","added_by":"auto","created_at":"2025-03-24 09:29:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1794105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 deletion exacerbates HFD-induced metabolic disorders.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blots images of FAIM2 protein expression in liver from FAIM2 knockout and control mice (n = 3).\u003cstrong\u003e (B) \u003c/strong\u003eBody weight of FAIM2-Flox and FAIM2-KO mice that were fed NC or HFD (n = 7). \u003cstrong\u003e(C-E)\u003c/strong\u003e Mice blood glucose levels (C), blood glucose levels in a glucose tolerance test (GTT) (D) and GTT were under curve (AUC) (E) of indicated groups (n = 7).\u003cstrong\u003e (F-H) \u003c/strong\u003eMice liver weight, liver weight/body weight(F), white adipose weight(G), white adipose/ body weight(H) of indicated groups (n = 7). \u003cstrong\u003e(I)\u003c/strong\u003e Mice serum TG, TC, and LDL level of indicated groups (n = 7). \u003cstrong\u003e(J) \u003c/strong\u003eImages and statistics of H\u0026amp;E-stained (top) and Oil Red O–stained (bottom) liver sections of indicated groups (n = 5). Scale bars, 50 µm. \u003cstrong\u003e(K) \u003c/strong\u003eSerum ALT and AST levels of indicated groups (n = 7).\u003cstrong\u003e (L-N) \u003c/strong\u003ePCA analyses (L), GSEA analyses (M) and heat map analyses (N) of liver in FAIM2-KO mice fed with HFD (n = 4).\u003cstrong\u003e (O) \u003c/strong\u003eRelative mRNA levels of genes related to fatty acid metabolism in the livers of indicated groups. Gene expression was normalized to \u003cem\u003eActb\u003c/em\u003e mRNA levels (n = 5). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/039b98dd9f243822b5d933c1.png"},{"id":79088220,"identity":"240e3b68-43dd-45ad-9989-7c6263bafe11","added_by":"auto","created_at":"2025-03-24 09:37:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1740800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 deletion exacerbates HFHC-induced MASH.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blots images of FAIM2 protein expression in liver from AVV8-Faim2 and control mice (n = 3).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB)\u003c/strong\u003e Body weight of AVV8-Ctrl and AVV8-Faim2 mice that were fed NC or HFHC (n = 8). (\u003cstrong\u003eC)\u003c/strong\u003e Mice blood glucose level of indicated groups (n = 8). (\u003cstrong\u003eD and E)\u003c/strong\u003e Mice blood glucose level of GTT and GTT AUC of indicated groups (n = 8). (\u003cstrong\u003eF)\u003c/strong\u003e Mice liver weight and liver weight/body weight of indicated groups (n = 8). (\u003cstrong\u003eG and H)\u003c/strong\u003e White adipose weight and white adipose/ body weight of indicated groups (n = 8). (\u003cstrong\u003eI and J)\u003c/strong\u003e Mice serum TG, TC, LDL (I), ALT and AST level (J) of indicated groups (n = 8).\u0026nbsp; (\u003cstrong\u003eK)\u003c/strong\u003e Images of H\u0026amp;E-stained, Oil Red O–stained, Cd11b–stained and PSR–stained liver sections of indicated groups (n = 5). Scale bars, 50 µm. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/34f67795e73403ad7bb5c5b5.png"},{"id":79087581,"identity":"49485c24-06ff-4197-86ba-cbeafe3cca8e","added_by":"auto","created_at":"2025-03-24 09:29:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1604086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 overexpression alleviated HFHC-induced MASH.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA)\u003c/strong\u003e Body weight of FAIM2 -WT and FAIM2-KO mice that were fed NC or HFHC (n = 8). (\u003cstrong\u003eB)\u003c/strong\u003e Mice blood glucose level of indicated groups (n = 8). (\u003cstrong\u003eC)\u003c/strong\u003e Mice blood glucose level of GTT and GTT AUC of indicated groups (n = 8). (\u003cstrong\u003eD-E)\u003c/strong\u003e Mice liver weight, liver weight/body weight, white adipose weight and white adipose/ body weight of indicated groups (n = 8). (\u003cstrong\u003eF and G)\u003c/strong\u003e Mice serum TG, serum TC (F), serum ALT and serum AST level (G) of indicated groups (n = 8).\u0026nbsp; (\u003cstrong\u003eH and I)\u003c/strong\u003e Images of H\u0026amp;E-stained (H left), Oil Red O–stained (H right), Cd11b–stained (I left) and PSR–stained (I right) liver sections of indicated groups (n = 5). Scale bars, 50 µm. (\u003cstrong\u003eJ)\u003c/strong\u003e Relative mRNA levels of genes related to fatty acid synthesis (\u003cem\u003eScd1\u003c/em\u003eand \u003cem\u003eFasn\u003c/em\u003e), inflammation (\u003cem\u003eCcl2\u003c/em\u003e and \u003cem\u003eCxcl10\u003c/em\u003e) and fibrosis (\u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eCol4a1\u003c/em\u003e) in the livers of indicated groups. (n = 5). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/2ba055ed84bf66867cd4683d.png"},{"id":79087585,"identity":"c3f45836-e148-413d-b9bb-47f5c68d822a","added_by":"auto","created_at":"2025-03-24 09:29:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":559623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 inhibits lipid synthesis in hepatocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blots images of FAIM2 protein expression in hepatocytes from FAIM2 knockout and control mice (n = 3). \u003cstrong\u003e(B)\u003c/strong\u003e Nile Red staining image of hepatocytes treatment with PAOA or BSA vehicle of indicated groups (n = 3). Scale bar, 25 μm. \u003cstrong\u003e(C)\u003c/strong\u003e TG content of hepatocytes treatment with PAOA or BSA vehicle of indicated groups (n = 5). \u003cstrong\u003e(D)\u003c/strong\u003e Relative mRNA levels of genes related to fatty acid synthesis and inflammation in the primary hepatocytes from FAIM2-knockout and control mice with PAOA treatment (n = 5).\u003cstrong\u003e (E)\u003c/strong\u003e Western blots images of FAIM2 protein expression in hepatocytes infected with AdFlag-FAIM2 or its control AdGFP (n = 3). \u003cstrong\u003e(F) \u003c/strong\u003eNile Red staining image of indicated groups (n = 3). Scale bar, 25 μm. \u003cstrong\u003e(G)\u003c/strong\u003e TG content of indicated groups (n = 5). \u003cstrong\u003e(H)\u003c/strong\u003eRelative mRNA levels of indicated groups (n = 5). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/19d252335d196997361e8420.png"},{"id":79087586,"identity":"9d0b2f87-53fd-4ac9-8567-beeeac962ebb","added_by":"auto","created_at":"2025-03-24 09:29:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":841414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 binds to CRTC2 and reduces its protein levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSchematic diagram showing IP-MS analysis to identify the specific target of FAIM2. \u003cstrong\u003e(B and C)\u003c/strong\u003e Immunoprecipitation and western blot analyses showing the binding of FAIM2 to CRTC2. \u003cstrong\u003e(D)\u003c/strong\u003e Confocal microscopy images showing co-localization between FAIM2 (red) and CRTC2 (green). Scale bar, 20 µm. \u003cstrong\u003e(E)\u003c/strong\u003e GST pull down assay and western blot analyses showing the directly binding of FAIM2 to CRTC2 in HEK293T. \u003cstrong\u003e(F and G)\u003c/strong\u003e CRTC2 mRNA level (F) and protein level (G) of indicated groups (n = 3). \u003cstrong\u003e(H and I)\u003c/strong\u003e CRTC2 mRNA level (H) and protein level (I) of indicated groups (n = 3). \u003cstrong\u003e(J and K)\u003c/strong\u003e CRTC2 mRNA level (J) and protein level (K) in the liver of NC- or HFD-fed FAIM2-KO and FAIM2-Flox mice (n = 3). \u003cstrong\u003e(L and M)\u003c/strong\u003e CRTC2 mRNA level (L) and protein level (M) of indicated groups (n = 3). \u003cstrong\u003e(O and P)\u003c/strong\u003e CRTC2 mRNA level (O) and protein level (P) in the liver of non-steatotic donors and individuals MAFLD (n = 4). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; n.s., not significant. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/881375258214dfda16e58d54.png"},{"id":79088227,"identity":"74e3cced-0aba-42bd-a8b7-6fa886b47f5e","added_by":"auto","created_at":"2025-03-24 09:37:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":767935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 inhibits lipid synthesis via CRTC2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blots images of Flag-FAIM2 and HA-CRTC2 in primary hepatocytes infected with AdFlag-FAIM2 or AdHA-CRTC2. \u003cstrong\u003e(B)\u003c/strong\u003e Nile Red staining images of indicated groups (n = 3). Scale bar, 10 mm. \u003cstrong\u003e(C)\u003c/strong\u003e TG content of hepatocytes of indicated groups (n = 5). \u003cstrong\u003e(D)\u003c/strong\u003e Relative mRNA levels of indicated groups (n = 5). \u003cstrong\u003e(E)\u003c/strong\u003e Western blots images of FAIM2 and CRTC2 in primary hepatocytes from FAIM2-KO or its control mice infected with AdshCRTC2. \u003cstrong\u003e(F)\u003c/strong\u003eNile Red staining images of indicated groups (n = 3). Scale bar, 10 mm. \u003cstrong\u003e(G)\u003c/strong\u003eTG content of hepatocytes of indicated groups (n = 5). \u003cstrong\u003e(H)\u003c/strong\u003e Relative mRNA levels of indicated groups (n = 5). **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA and Two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/090c382acad246177a55b56f.png"},{"id":79088228,"identity":"3ecf4fc9-ce0f-4ea9-b4b5-9b3f262e951d","added_by":"auto","created_at":"2025-03-24 09:37:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1484961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAIM2 promotes CRTC2 degradation via autophagy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blots images of CRTC2 and Flag-FAIM2 of primary hepatocytes infected with AdFlag-FAIM2 and treated with PAOA. \u003cstrong\u003e(B)\u003c/strong\u003e Western blots images of indicated groups with PAOA and cycloheximide (CHX) (50 μM, 6 h). \u003cstrong\u003e(C)\u003c/strong\u003e Western blots images of indicated groups with DMSO, MG132 (50 μM) or CQ (50 μM) for 6 h. \u003cstrong\u003e(D)\u003c/strong\u003eConfocal microscopy images showing the distribution of CRTC2 (Green) and LAMP1 (Red) of indicated groups. Scale bar, 7.5 µm. \u003cstrong\u003e(E and F)\u003c/strong\u003e Western blots images of LC3, P62, CRTC2, Flag-FAIM2 of indicated groups treated with or without Bafilomycin A1 (Baf A1, 75 nM, 2 h). \u003cstrong\u003e(G)\u003c/strong\u003e Images of primary hepatocytes that infected with AdGFP-RFP-LC3B of indicated groups. Scale bar, 7.5 µm. \u003cstrong\u003e(H and I)\u003c/strong\u003e Western blots images of indicated groups treated with or without rapamycin (10 uM, 6 h). \u003cstrong\u003e(J)\u003c/strong\u003e Images of indicated groups that infected with AdGFP-RFP-LC3B. Scale bar, 7.5 µm. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/713418ba6af833eea957e2ba.png"},{"id":79087614,"identity":"e468ddab-39be-47b0-87c3-7c825838707b","added_by":"auto","created_at":"2025-03-24 09:29:02","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1138832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe N-terminal domain of FAIM2 is required for CRTC2 degradation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B)\u003c/strong\u003eSchematic showing full-length and truncated FAIM2 (A, top) and CRTC2 (B, top) with co-IP assays (bottom) for the mapping analyses of the domains responsible for the FAIM2–CRTC2 interaction in HEK293T cells. ▲N, missing N-terminal domain; TM, transmembrane domain; ▲C, missing C-terminal domain; CREB binding domain (CBD), CREB binging domain; RD, regulation domain; TAD, transactivation domain. (\u003cstrong\u003eC)\u003c/strong\u003e Western blots images of indicated groups (\u003cstrong\u003eD) \u003c/strong\u003eNile Red staining images of hepatocytes from indicated groups. (n = 3). Scale bar, 50 mm. (\u003cstrong\u003eE and F)\u003c/strong\u003e TG content and relative mRNA levels of indicated groups (n = 5). (\u003cstrong\u003eG)\u003c/strong\u003e Confocal microscopy images of indicated groups. Scale bar, 7.5 µm. (\u003cstrong\u003eH-I)\u003c/strong\u003e Western blots images of indicated groups. (LIR, LC3-interacting region) (\u003cstrong\u003eJ-L)\u003c/strong\u003e Immunoprecipitation and western blot analyses showing the binding relationship between FAIM2, FAIM2-▲LIR, CRTC2 and LC3B in HEK293T cells transfected with indicated plasmid. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; n.s., not significant. Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way ANOVA.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/7ea11d8994cc4b9130e18c81.png"},{"id":93010037,"identity":"92ec412e-7e6c-4c53-a088-bba6559f2a00","added_by":"auto","created_at":"2025-10-08 07:10:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15223545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/2147c0c3-4b50-457b-ad2d-dbb56ae1bf1b.pdf"},{"id":79087573,"identity":"b6cb8f65-1a16-48ff-a28a-585b5d8648ea","added_by":"auto","created_at":"2025-03-24 09:29:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32189,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6110029/v1/c0faa0a41d7f19f0e9ee15fe.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Fas apoptotic inhibitor molecule 2 mitigates metabolic dysfunction-associated fatty liver disease through autophagic CRTC2 degradation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMetabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease, has become the most prevalent chronic liver disease, with a global prevalence rate of up to 30%, and its prevalence is increasing \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Moreover, MAFLD can progress from simple hepatic steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which puts individuals at risk of end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Despite the large number of individuals with MAFLD, poor adherence to lifestyle interventions is observed in clinical practice \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, and only limited pharmacological therapy has been approved for MASH \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Therefore, the identification of effective therapeutic targets and strategies for MAFLD is urgently needed.\u003c/p\u003e \u003cp\u003eLysosomal degradation, including the endosome‒lysosome pathway and the autophagy‒lysosome pathway (also known as autophagy), is a pivotal physiological activity essential for maintaining cellular protein homeostasis and has been an appealing platform for drug discovery \u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. The membrane of lysosomes harbors a variety of proteins that play critical roles in maintaining the operation of lysosomal degradation (preserving the integrity of the lysosome structure; regulating enzyme activity) and mediating interactions with other organelles or molecules \u003csup\u003e[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The dysregulation of lysosomal membrane proteins can disrupt cellular protein homeostasis, which in turn can cause a spectrum of diseases, including MAFLD \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The ablation of glycosylated lysosomal membrane protein in mice promote lipid deposition and causes fibrosis and hepatic cell death in the liver \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Moreover, lysosomal-associated protein transmembrane 5 is negatively correlated with NAFLD activity score, and the hepatocyte-specific depletion of lysosomal-associated protein transmembrane 5 exacerbates MASH symptoms in mice \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Our previous studies revealed that transmembrane BAX inhibitor motif-containing protein 1 (TMBIM1) protects against MAFLD by promoting the lysosomal degradation of Toll-like receptor 4 \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Given the crucial role of lysosomal membrane proteins in lysosomal degradation and their connections in MAFLD, lysosomal membrane proteins could be promising therapeutic targets for MAFLD treatment.\u003c/p\u003e \u003cp\u003eFas apoptotic inhibitory molecule 2 (FAIM2), also known as TMBIM2, belongs to the TMBIM family, which is characterized by a UPF0005 motif that encodes either six or seven transmembrane structures. FAIM2 has seven transmembrane structures and locate on lysosomes \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e; it was initially identified in a study seeking genes involved in the development and maintenance of the nervous system \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. FAIM2 protects hippocampal cells from death in the acute phase of bacterial meningitis \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, reduces stroke volume and alleviates dopaminergic neuron degeneration in Parkinson\u0026rsquo;s disease \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Recently, multiple studies have suggested that SNP mutations at the FAIM2 gene locus are associated with obesity and type 2 diabetes, which are critical risk factors for MAFLD \u003csup\u003e[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. However, to date, the role of FAIM2 in MAFLD remains unknown.\u003c/p\u003e \u003cp\u003eHere, we identified FAIM2 as an effective suppressor of MAFLD. A marked decrease in FAIM2 protein levels was observed in MAFLD. FAIM2 depletion exacerbated lipid deposition, inflammation, and fibrosis. However, overexpressing FAIM2 in mice presented the opposite effects. Moreover, in hepatocyte, FAIM2 overexpression suppress fatty acid synthesis related genes. Mechanistically, FAIM2 directly interact with CREB-regulated transcription coactivator 2 (CRTC2), a key regulator of lipid metabolism, and facilitated the degradation of CRTC2 through autophagy. Collectively, our findings suggest that FAIM2 suppresses MAFLD by promoting the autophagic degradation of CRTC2 and further suppressing fatty acid synthesis, which could be utilized as a therapeutic target for MAFLD.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Animal treatment\u003c/strong\u003e. All animal protocols were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (grant no.:20230404G). The animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Human liver samples\u003c/strong\u003e. All procedures involving the collection of human samples have been approved by the Ethics Committee of Zhongnan Hospital of Wuhan University (grant no.:2018010), and the principles of the Helsinki Declaration have been followed to obtain written informed consent from the participants or their family members. Human steatotic liver samples were obtained from patients with simple steatosis or MASH who had undergone either liver biopsy or liver transplantation. Non-steatotic liver samples were collected from healthy regions of the livers from donors who had undergone liver resection because of a liver hemangioma or hepatic cyst.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Statistical Analysis\u003c/strong\u003e. In brief, all statis. tical analyses were performed using SPSS software (version 25, USA). For data showing a Gaussian distribution, parametric statistical analysis was performed using the two-tailed Student\u0026rsquo;s t test for two groups. One-way ANOVA was applied to three or more groups, followed by either Bonferroni post hoc analysis for data meeting homogeneity of variance requirements or Tamhane\u0026rsquo;s T2(M) post hoc analysis for heteroscedastic data. For datasets with skewed distributions, nonparametric statistical analysis was performed using the Mann-Whitney U test for two groups and Kruskal-wallis test for three or more groups. All values were presented as the mean \u0026plusmn; SD. \u003cem\u003eP\u003c/em\u003e values were specified as follows:\u0026nbsp;\u0026lowast;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,\u0026nbsp;\u0026lowast;\u0026lowast;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. Data from animal studies were collected in a blinded fashion. No data were excluded when performing the final statistical analysis. A randomization process was performed in grouping mice with the same phenotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Supplementary methods.\u0026nbsp;\u003c/strong\u003eDetailed materials and methods are described in the Supplementary Materials and Methods sections.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1. FAIM2 expression is downregulated in fatty livers\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo investigate whether FAIM2 is involved in MAFLD, we first examined FAIM2 expression profiles in MAFLD livers. We found that the FAIM2 protein level was markedly lower in the livers of mice that were fed a high-fat diet (HFD) or a high-fat and high-cholesterol diet (HFHC) \u003cb\u003e(Figure. 1A, B)\u003c/b\u003e. Moreover, FAIM2 protein levels were markedly lower in the livers of individuals with simple hepatic steatosis and MASH than in the livers of nonsteatotic control individuals \u003cb\u003e(Figure. 1C)\u003c/b\u003e. The protein level of FAIM2 was also decreased in primary hepatocytes stimulated with prosteatotic stimuli (palmitate and oil acid, PAOA) \u003cb\u003e(Figure. 1D)\u003c/b\u003e. And immunofluorescence analyses suggested that FAIM2 was decreased primarily in the cytoplasm of hepatocytes \u003cb\u003e(Figure. 1E)\u003c/b\u003e. Although protein levels varied, \u003cem\u003eFAIM2\u003c/em\u003e mRNA levels did not significantly change under any of the conditions tested, as well as protein level in sinusoidal endothelial cells or Kupffer cells \u003cb\u003e(Figure. 1F-H)\u003c/b\u003e. Taken together, the remarkable downregulation of FAIM2 expression observed in fatty livers suggests that FAIM2 may play a role in the occurrence and development of MAFLD and that FAIM2 may undergo posttranslational modification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2. NEDD4L facilitates FAIM2 degradation through catalyzing its K48-linked ubiquitination\u003c/h2\u003e \u003cp\u003eNext, we explored the mechanism involved in the downregulation of FAIM2 protein in MAFLD. It has been reported that the ubiquitin-proteasome system (UPS) and autophagy were two major systems for intracellular protein degradation. We found that the downregulation of FAIM2 was regulated by the UPS \u003cb\u003e(Figure. 2A, B)\u003c/b\u003e. We further explored whether ubiquitin E3 ligase (bioGRID) might participate in the degradation of FAIM2. And the result of Co-IP test showed that TRIM21, NEDD4, and NEDD4L can interact with FAIM2, while NEDD4L interacted stronger \u003cb\u003e(Figure. 2B)\u003c/b\u003e. What\u0026rsquo;s more, the protein level of FAIM2 was significantly downregulated with the overexpression of NEDD4L \u003cb\u003e(Figure. 2D-F)\u003c/b\u003e. In order to further explore the mechanism by which NEDD4L mediated FAIM2 degradation, we detected the ubiquitination of FAIM2 through IP assays. Our results showed that NEDD4L mediated FAIM2 degradation through K48-linked ubiquitination \u003cb\u003e(Figure. 2G-I)\u003c/b\u003e. These results indicate that ubiquitin-proteasome degradation of FAIM2 was mediated by NEDD4L under metabolic stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3. FAIM2 knockout aggravates HFD-induced metabolic disorders\u003c/h2\u003e \u003cp\u003eGiven the conspicuous changes in FAIM2 expression observed in fatty livers, we fed \u003cem\u003eFaim2\u003c/em\u003e-knockout (\u003cem\u003eFaim2\u003c/em\u003e-KO) mice and wild type (WT) littermate control with a normal chow or HFD for 24 weeks to assess the function of FAIM2 in MAFLD \u003cb\u003e(Figure. 3A)\u003c/b\u003e. FAIM2 deletion did not significantly affect the body weight of mice with NC, but increase the body weight of mice with HFD (HFD-fed \u003cem\u003eFaim2\u003c/em\u003e-KO mice, KO-HFD) \u003cb\u003e(Figure. 3B)\u003c/b\u003e. Compared with HFD-fed WT mice, KO-HFD mice presented higher fasting blood glucose levels and more severe glucose intolerance \u003cb\u003e(Figure. 3C, D, E)\u003c/b\u003e. Moreover, KO-HFD mice exhibited increased liver weight and white adipose weight, greater serum total triglyceride, total cholesterol and low-density lipoprotein contents, and aggravated accumulation of hepatic lipids \u003cb\u003e(Figure. 3F-J)\u003c/b\u003e. In addition to steatosis, higher serum ALT and AST concentrations in KO-HFD mice indicated exacerbated liver injury \u003cb\u003e(Figure. 3K)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eRNA-seq analyses were performed to further assess the influence of FAIM2 on MAFLD. Principal component analysis clearly separated the HFD-treated liver samples into two subgroups: HFD-fed WT mice and KO-HFD livers \u003cb\u003e(Figure. 3L)\u003c/b\u003e. Notably, as shown by the GSEA analyses, KEGG pathways related to lipid metabolism, including fatty acid synthesis and elongation, were enriched in the KO-HFD group \u003cb\u003e(Figure. 3M)\u003c/b\u003e. Moreover, a heatmap revealed that FAIM2 deletion activated genes related to fatty acid synthesis \u003cb\u003e(Figure. 3N)\u003c/b\u003e, that were confirmed by quantitative PCR \u003cb\u003e(Figure. 3O)\u003c/b\u003e. Taken together, these findings suggest that FAIM2 deficiency exacerbates HFD-induced metabolic disorders may through activating fatty acid synthesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FAIM2 knockout aggravates HFHC-induced MASH\u003c/h2\u003e \u003cp\u003eWe next examined whether FAIM2 knockout accelerated the development of HFHC-induced MASH, a dietary model that exhibits considerable fidelity to human MASH with features that included steatosis, advanced hepatic inflammation and fibrosis \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. HFHC-fed \u003cem\u003eFaim2\u003c/em\u003e-KO mice (KO-HFHC) presented increased body weights compared with those in HFHC-fed WT mice \u003cb\u003e(Figure. 4A)\u003c/b\u003e. In addition, KO-HFHC mice displayed glycometabolism disorder, greater liver weight and white adipose weight, increased serum TG, TC, ALT and AST contents, and more severe liver steatosis \u003cb\u003e(Figure. 4B-H, J)\u003c/b\u003e. Furthermore, KO-HFHC mice presented more severe inflammatory responses \u003cb\u003e(Figure. 4I, J)\u003c/b\u003e, and greater collagen deposition and expression of genes involved in fibrogenesis (\u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eCol4a1\u003c/em\u003e) \u003cb\u003e(Figure. 4I, J)\u003c/b\u003e. Collectively, the above findings suggest that FAIM2 deficiency exacerbates HFHC-induced steatosis, inflammation and collagen deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5. FAIM2 overexpression alleviates HFHC-induced MASH\u003c/h2\u003e \u003cp\u003eTo further assess the function of FAIM2 in MAFLD, we generated FAIM2-overexpressing mice by injecting AAV8-FAIM2, and fed them and corresponding control mice with HFHC \u003cb\u003e(Figure. 5A)\u003c/b\u003e. Noticeably, FAIM2-overexpressing mice presented lower body weights, improved glycometabolism, decreased liver and white adipose weight, and decreased serum TG, TC, LDL, ALT and AST contents \u003cb\u003e(Figure. 5B-J)\u003c/b\u003e. Moreover, FAIM2 overexpression significant alleviated hepatic steatosis, inflammation, and fibrogenesis \u003cb\u003e(Figure. 5K)\u003c/b\u003e. Above results suggested FAIM2 overexpression mitigates HFHC-induced MASH and FAIM2 is a protective factor under metabolic stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.6. FAIM2 inhibits fatty acid synthesis in hepatocytes\u003c/h2\u003e \u003cp\u003eWe observed FAIM2 deletion activated gene and pathway related to fatty acid synthesis \u003cem\u003ein vivo\u003c/em\u003e, and hepatocytes are the predominant cell type in the liver and the main executor of lipid synthesis \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Thus, we further assayed the function of FAIM2 in hepatocytes from \u003cem\u003eFaim2\u003c/em\u003e-KO mice (\u003cb\u003eFigure. 6A\u003c/b\u003e). The lipid content and total triglyceride content were greater in FAIM2-deficient primary hepatocytes stimulated with PAOA than in the corresponding WT controls \u003cb\u003e(Figure. 6B, C)\u003c/b\u003e. Moreover, FAIM2 deletion promoted the expression of genes related to fatty acid synthesis and inflammation \u003cb\u003e(Figure. 6D)\u003c/b\u003e. Next, we constructed an adenovirus expressing FAIM2 (Ad\u003cem\u003eFaim2\u003c/em\u003e) to investigate the effects of FAIM2 overexpression in primary hepatocytes \u003cb\u003e(Figure. 6E)\u003c/b\u003e. Notably, FAIM2 overexpression reduced lipid and TG accumulation and inhibited fatty acid synthesis and inflammation \u003cb\u003e(Figure. 6F-H)\u003c/b\u003e. Above findings demonstrate that FAIM2 inhibits fatty acid synthesis and the inflammatory response in hepatocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.7. FAIM2 interacts with CRTC2 and reduces its expression\u003c/h2\u003e \u003cp\u003eWe next explored the mechanistic link between FAIM2 and its function. The above results revealed that FAIM2 knockout significantly and consistently increased lipid accumulation and upregulated fatty acid synthesis-related gene expression in \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e MAFLD models, thus we speculated that FAIM2 may function by affecting lipid metabolism regulators. We applied immunoprecipitation-mass spectrometry to identify proteins that physically interact with FAIM2 and screened out lipid metabolism regulators. Among them, CRTC2 was reported to be a lipid metabolism regulator that promoted MAFLD (\u003cb\u003eFigure. 7A\u003c/b\u003e) \u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The interaction between FAIM2 and CRTC2 was verified, and the direct interaction between them was further confirmed via a GST pull-down assay \u003cb\u003e(Figure. 7B-E)\u003c/b\u003e. Remarkably, although the \u003cem\u003eCrtc2\u003c/em\u003e mRNA level did not significantly change in hepatocytes overexpressing or lacking FAIM2 \u003cb\u003e(Figure. 7F, H)\u003c/b\u003e, the CRTC2 protein level was decreased in FAIM2-overexpressing hepatocytes and increased in FAIM2-deficient hepatocytes \u003cb\u003e(Figure. 7G, I)\u003c/b\u003e. In liver of mice fed with either HFD or HFHC, CRTC2 change in the same pattern \u003cb\u003e(Figure. 7J-M)\u003c/b\u003e. Moreover, CRTC2 expression was increased in the liver of MAFLD individuals \u003cb\u003e(Figure. 7N-O)\u003c/b\u003e. Collectively, these findings suggest that FAIM2 directly interacts with CRTC2 and decreases its protein level, indicating that FAIM2 promote CRTC2 posttranslational modification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.8. FAIM2 regulates lipid metabolism via CRTC2\u003c/h2\u003e \u003cp\u003eTo evaluate whether CRTC2 is required for FAIM2-regulated lipid metabolism in hepatocytes, we constructed an adenovirus to overexpress CRTC2 (\u003cb\u003eFigure. 8A\u003c/b\u003e). The inhibitory effect of FAIM2 overexpression on lipid accumulation was blunted by CRTC2 overexpression \u003cb\u003e(Figure. 8B, C)\u003c/b\u003e. Overexpressing CRTC2 also reversed the inhibitory effects of FAIM2 on fatty acid synthesis and inflammatory gene expression \u003cb\u003e(Figure. 8D)\u003c/b\u003e. Next, we constructed adenovirus short hairpin RNA targeting Crtc2 (Adsh\u003cem\u003eCrtc2\u003c/em\u003e) to knockdown CRTC2 in primary hepatocytes isolated from \u003cem\u003eFaim2\u003c/em\u003e-KO mice \u003cb\u003e(Figure. 8E)\u003c/b\u003e. Interestingly, CRTC2 knockdown also reversed the effect of FAIM2 knockout \u003cb\u003e(Figure. 8F-H)\u003c/b\u003e. These data indicate that the inhibitory effect of FAIM2 on fatty acid synthesis required CRTC2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.9. FAIM2 facilitates CRTC2 degradation through autophagy\u003c/h2\u003e \u003cp\u003eWe next sought to determine how FAIM2 decreased the protein level of CRTC2. The protein level of CRTC2 decreased with increasing FAIM2 expression in a dose-dependent manner (\u003cb\u003eFigure. 9A\u003c/b\u003e). Significantly, CRTC2 degradation occurred faster in FAIM2-overexpressed hepatocytes under cycloheximide (CHX) treatment \u003cb\u003e(Figure. 9B)\u003c/b\u003e, and FAIM2-promoted CRTC2 protein degradation was blocked under CQ treatment \u003cb\u003e(Figure. 9C)\u003c/b\u003e. Moreover, FAIM2 promoted the colocalization of CRTC2 and lysosomal marker LAMP1 \u003cb\u003e(Figure. 9D)\u003c/b\u003e. These results suggest that FAIM2 promotes the degradation of CRTC2 via lysosomal pathway.\u003c/p\u003e \u003cp\u003eThe lysosomal pathway including the endosome‒lysosome pathway and autophagy \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Considering that autophagy is principally responsible for the degradation of cytoplasmic components \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e and FAIM2 and CRTC2 primarily colocalize in the cytoplasm under PAOA stimulation, we further investigated whether FAIM2 degrades CRTC2 through autophagy. The results revealed that FAIM2 overexpression in hepatocytes led to a reduction in CRTC2 levels concomitant with an increase in the expression of the LC3 and a decrease in the expression of p62 \u003cb\u003e(Figure. 9E)\u003c/b\u003e. Compared with hepatocytes treated with bafilomycin A1, hepatocytes with bafilomycin A1 and overexpressing FAIM2, exhibited further LC3, and p62 accumulation. In addition, the FAIM2-promoted CRTC2 degradation was suppressed under bafilomycin A1 treatment \u003cb\u003e(Figure. 9F)\u003c/b\u003e. Moreover, FAIM2 overexpression resulted in greater red fluorescence of GFP-RFP-LC3B in hepatocytes (representing autolysosomes) \u003cb\u003e(Figure. 9G)\u003c/b\u003e. These results indicate that FAIM2 promotes autophagy, which in turn facilitates CRTC2 degradation.\u003c/p\u003e \u003cp\u003eNext, we investigated how FAIM2 promotes autophagy. The deletion of FAIM2 led to an increase in LC3 and p62 levels \u003cb\u003e(Figure. 9H)\u003c/b\u003e. Compared with that treated with rapamycin, the expression of LC3 was higher in \u003cem\u003eFaim2\u003c/em\u003e-KO hepatocytes stimulated with rapamycin \u003cb\u003e(Figure. 9I)\u003c/b\u003e. In addition, \u003cem\u003eFaim2\u003c/em\u003e-KO hepatocytes presented more intense yellow fluorescence of GFP-RFP-LC3B (representing autophagosomes) \u003cb\u003e(Figure. 9J).\u003c/b\u003e These results suggest that FAIM2 is required for autolysosome formation in hepatocytes.\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that FAIM2 promotes autophagy and mediates CRTC2 degradation through autophagy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.10. The N-terminal domain of FAIM2 is required for CRTC2 degradation\u003c/h2\u003e \u003cp\u003eTo further investigate the detailed mechanism by which FAIM2 mediates CRTC2 degradation, the interaction domain between FAIM2 and CRTC2 was investigated. The results of a domain mapping analysis revealed that the N-terminal domain (cytoplasmic domain) \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e of FAIM2 interacts with the CREB binding domain (CBD) of CRTC2 \u003cb\u003e(Figure. 10A, B)\u003c/b\u003e. To determine whether the interaction between FAIM2 and CRTC2 is necessary for the protective role of FAIM2 against MAFLD, an adenovirus containing FAIM2 lacking the N-terminal domain (Ad\u003cem\u003eFaim2\u003c/em\u003e-▲N) was constructed. Interestingly, after the N-terminal domain was deleted, FAIM2 lost its ability to decrease CRTC2, inhibit lipid accumulation and downregulate the expression of fatty acid synthesis- and inflammation-related genes \u003cb\u003e(Figure. 10C-F)\u003c/b\u003e. In addition, the ability of \u003cem\u003eFaim2\u003c/em\u003e-▲N to promote CRTC2 and lysosome colocalization, as well as the response to CQ, was lost \u003cb\u003e(Figure. 10G, H)\u003c/b\u003e. These results indicate that the inhibitory effects of FAIM2 on CRTC2 and metabolic stress are required the N-terminal domain of FAIM2.\u003c/p\u003e \u003cp\u003eIt has been reported that the N-terminal domain of FAIM2 contains an LC3-interacting region (LIR) motif which is responsible for the autolysosome formation \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Therefore, we further investigated whether the interaction of FAIM2 and CRTC2 is dependent on the LIR motif. We constructed plasmid and adenovirus to overexpress FAIM2 lacking the LIR domain (FAIM2-▲LIR). FAIM2-▲LIR lost its ability to promote autophagy and suppress CRTC2 in primary hepatocytes \u003cb\u003e(Figure. 10I)\u003c/b\u003e. FAIM2 was able to interact with both CRTC2 and LC3B, whereas FAIM2-▲LIR was unable to bind with LC3B \u003cb\u003e(Figure. 10J, K)\u003c/b\u003e. However, FAIM2-▲LIR was still able to interact with CRTC2 \u003cb\u003e(Figure. 10L)\u003c/b\u003e, indicating that the interaction of FAIM2 and CRTC2 did not dependent on the LIR motif.\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that the N-terminal domain of FAIM2 interacts with both CRTC2 and LC3B, a process that is required for the suppression of CRTC2 by FAIM2.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we demonstrate that FAIM2 is ubiquitinated and degraded by NEDD4L under conditions of metabolic stresses. FAIM2 deficiency significantly increase lipid deposition and upregulatedpathways and genes related to fatty acid synthesis. Whereas, overexpression of FAIM2 presents the opposite effect. These findings reveal that FAIM2 plays a protective role in MAFLD by mitigating fatty acid synthesis in hepatocytes, and highlight the important role of LMPs in MAFLD or other metabolism-related diseases.\u003c/p\u003e \u003cp\u003eWe speculated that FAIM2 may mitigate fatty acid synthesis by affecting lipid metabolism regulators. Through immunoprecipitation‒mass spectrometry, we identified lipid metabolism regulators that interact with FAIM2. Among these regulators, CRTC2 is a crucial lipid metabolism regulator in the liver. Liver-specific knockout of Crtc2 reduces fatty acid synthesis genes expression (\u003cem\u003eFasn\u003c/em\u003e, \u003cem\u003eScd1\u003c/em\u003e, \u003cem\u003ePparg\u003c/em\u003e and \u003cem\u003eAcaca\u003c/em\u003e), alleviates diet-induced liver lipid accumulation, inhibits inflammation \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, and leads to reduced blood lipid and blood glucose concentrations \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Additionally, CRTC2 overexpression enhances liver cholesterol synthesis by promoting the transcription of SREBP-2 \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. CRTC2 regulate fatty acid, glucose and cholesterol metabolism, making it a nonnegligible target. We verified that FAIM2 facilitates CRTC2 degradation through autophagy, and the N-terminal domain of FAIM2 is required for this process, which elucidates the underlying mechanism of FAIM2 in MAFLD and provides a potential therapeutic target for further clinical translation.\u003c/p\u003e \u003cp\u003eWe propose that the targeting of CRTC2 for degradation is the primary cellular mechanism underlying the function of FAIM2 in MAFLD. However, CRTC2 regulates not only fatty acid biosynthesis \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, but also blood glucose levels and cholesterol synthesis \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, and participates in the processes of type 2 diabetes, stroke, tumors, circadian rhythm, etc \u003csup\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. Indeed, the complete disruption of an important target that has such fundamental roles in physiological activity might lead to serious side effects, thus greatly hindering the development of new therapeutic agents. Our results demonstrated that FAIM2 facilitated the autophagic degradation of CRTC2, thereby preventing its excessive activation under prosteatotic stimulus, which works by balancing CRTC2 dynamics to maintain CRTC2 expression at an appropriate level rather than severely blocking its function. This working mode would likely be beneficial for treating MAFLD without inducing serious side effects.\u003c/p\u003e \u003cp\u003eOur previous studies reported that TMBIM1 is an effective inhibitor of MAFLD. TMBIM1 collaborates with the ESCRT-mediated endosomal sorting complex to promote multivesicular body formation, thereby facilitating the lysosomal degradation of TLR4 and exerting its protective function \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Although both TMBIM1 and FAIM2 belong to the TMBIM family, we found that FAIM2 protects against MAFLD by mediating the autophagic degradation of CRTC2. The different underlying mechanisms of TMBIM1 and FAIM2 indicated that they do not simply serve as functional substitutes.\u003c/p\u003e \u003cp\u003eAlthough we have employed various models and experimental approaches to elucidate the function and mechanism of FAIM2 in MAFLD, there remains room for refinement in our study. We have demonstrated that FAIM2 can act through primary hepatocytes, but the potential effects of hepatic nonparenchymal cells\u0026mdash;such as Kupffer cells, endothelial cells, and stellate cells\u0026mdash;require further investigation. Additionally, the use of a molecular docking assay to predict and validate the specific amino acid sequence within the N-terminus of FAIM2 where CRTC2 binds and the use of AI technology to design small-molecule compounds for therapeutic testing may pave the way for clinical translation.\u003c/p\u003e \u003cp\u003eIn summary, this study reveals the role and mechanism of FAIM2 in MAFLD. Under metabolic stresses, FAIM2 mediates the degradation of CRTC2 via autophagy, and further suppresses fatty acid synthesis and alleviates MAFLD. Thereby, FAIM2-CRTC2 may holds promise as a novel therapeutic target for MAFLD, and targeting LMPs to control protein homeostasis may provide new strategies and promising therapeutic targets.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Henan Charity Federation Hepatobiliary Fund (grant no.: GDXZ2021002), National Science Foundation of China (grant no.: 82370882), Hubei Provincial Natural Science Foundation of China (grant no.: 2023AFB670), the Key R\u0026amp;D Program of Jiangxi Province of China (20223BBG71008) and Excellent Youth Foundation of Jiangxi Province (grant no.: 20242BAB23074).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYongjie Yu:\u003c/strong\u003e Writing \u0026ndash; original draft, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eSha Hu:\u003c/strong\u003e Writing \u0026ndash; original draft, Methodology, Investigation, Formal analysis, Data curation. \u003cstrong\u003eTuo Zhang:\u003c/strong\u003e Writing \u0026ndash; original draft, Investigation, Formal analysis, Data curation. \u003cstrong\u003eDajun Li:\u003c/strong\u003e Methodology, Investigation, Data curation. \u003cstrong\u003e\u0026nbsp;Yongping Huang\u003c/strong\u003e: Visualization, Software, Formal analysis. \u003cstrong\u003eHong Yu:\u0026nbsp;\u003c/strong\u003ereview \u0026amp; editing, Supervision, Conceptualization.\u003cstrong\u003e\u0026nbsp;Guang-nian Zhao:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization. \u003cstrong\u003ePeng Zhang:\u003c/strong\u003e Writing \u0026ndash; review \u0026amp; editing, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWong VW, Ekstedt M, Wong GL \u0026amp; Hagstr\u0026ouml;m H. 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J Biol Chem \u003cstrong\u003e290\u003c/strong\u003e, 2189-2197(2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MAFLD, FAIM2, Fatty acid synthesis, CRTC2, Degradation","lastPublishedDoi":"10.21203/rs.3.rs-6110029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6110029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLysosomal membrane proteins (LMPs) play fundamental roles in the lysosomal degradation of proteins and are attractive drug targets for metabolic dysfunction-associated fatty liver disease (MAFLD). Fas apoptotic inhibitory molecule 2 (FAIM2), an LMP, is previously recognized as an inhibitor of apoptosis in a variety of diseases. Here, we reveal that FAIM2 is an inhibitor of fatty acid synthesis and suppresses MAFLD. FAIM2 protein expression is decreased in MAFLD. Moreover, FAIM2 is degraded by the E3 ubiquitin ligase NEDD4L through catalyzing K48-linked ubiquitination. High-fat or high-fat and high-cholesterol diet-induced hepatic steatosis, inflammation and fibrosis was aggravated in FAIM2 knockout (KO) mice and alleviated in FAIM2 overexpressing mice. Furthermore, in hepatocytes, FAIM2 KO increased the expression of genes related to fatty acid synthesis, whereas over-expressing FAIM2 exhibits the opposite effect. Mechanistically, FAIM2 directly interacts with CREB-regulated transcription coactivator 2 (CRTC2), a prominent regulator of lipid metabolism, and medicates its degradation through autophagy. Specifically, we find that the N-terminus of FAIM2 which interacts with CRCT2 and LC3B is required for autophagic CRTC2 degradation. Collectively, our findings reveal that FAIM2 acts as a fatty acid synthesis inhibitor in MAFLD by promoting the autophagic degradation of CRTC2, and FAIM2-CRTC2 may be a promising therapeutic target.\u003c/p\u003e","manuscriptTitle":"Fas apoptotic inhibitor molecule 2 mitigates metabolic dysfunction-associated fatty liver disease through autophagic CRTC2 degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 09:28:54","doi":"10.21203/rs.3.rs-6110029/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-04-21T04:07:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-18T09:40:58+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-07T07:54:11+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-02T02:34:38+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-03-24T06:46:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-21T03:31:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-27T01:31:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-26T05:48:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-02-26T05:48:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b583e011-fcdd-41d6-a761-70a5989009e1","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45998883,"name":"Biological sciences/Molecular biology/Proteolysis"},{"id":45998884,"name":"Health sciences/Diseases/Metabolic disorders"}],"tags":[],"updatedAt":"2025-10-08T07:10:45+00:00","versionOfRecord":{"articleIdentity":"rs-6110029","link":"https://doi.org/10.1038/s12276-025-01559-1","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-10-07 04:00:00","publishedOnDateReadable":"October 7th, 2025"},"versionCreatedAt":"2025-03-24 09:28:54","video":"","vorDoi":"10.1038/s12276-025-01559-1","vorDoiUrl":"https://doi.org/10.1038/s12276-025-01559-1","workflowStages":[]},"version":"v1","identity":"rs-6110029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6110029","identity":"rs-6110029","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}