Abstract Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide. Numerous evidence has demonstrated that metabolic reprogramming serves as a hallmark associated with an elevated risk of NAFLD progression. Selenoprotein W (SelW) is an extensively expressed hepatic selenoprotein that plays a crucial role in antioxidant function. Here, we first demonstrated that SelW is a significantly distinct factor in the liver tissue of NAFLD patients through the Gene Expression Omnibus (GEO) database. Additionally, loss of SelW alleviated hepatic steatosis induced by a high-fat diet (HFD), and was accompanied by the regulation of metabolic and inflammatory pathways as verified by transcriptomic analysis. Moreover, co-immunoprecipitation (CO-IP), liquid chromatography-tandem mass spectrometry (LC-MS), laser scanning confocal microscopy (LSCM) and molecular docking analysis were subsequently implemented to identify Pyruvate Kinase M2 (PKM2) as a potential interacting protein of SelW. Meanwhile, SelW modulated PKM2 translocation into the nucleus to trigger transactivation of the HIF-1α, in further mediating mitochondrial apoptosis, eventually resulting in mitochondrial damage, ROS excessive production and mtDNA leakage. Additionally, mito-ROS accumulation induced the activation of the NLRP3 inflammasome-mediated pyroptosis, thereby facilitating extracellular leakage of mtDNA. The escaped mtDNA then evokes the cGAS-STING signaling pathway in macrophage, thus inducing a shift in macrophage phenotype. Together, our results suggest SelW promotes hepatocyte apoptosis and pyroptosis by regulating metabolic reprogramming to activate cGAS/STING signaling of macrophages, thereby exacerbating the progression of NAFLD. Keywords: Selenoprotein W, Non-alcoholic fatty liver disease, Metabolism, Glycolysis, Pyruvate kinase M2, Pyroptosis Graphical abstract [31]Image 1 [32]Open in a new tab Highlights * • SelW expression was upregulated in the liver tissue of NAFLD patients. * • SelW interacted with PKM2 to regulate metabolic reprogramming. * • SelW damaged mitochondria, resulting in the mito-ROS production and mtDNA leakage. * • SelW activated pyroptosis by mito-ROS, promoting extracellular leakage of mtDNA. * • SelW induced mtDNA to trigger alterations in macrophage polarization by cGAS/STING. Abbreviations: NAFLD Non-alcoholic fatty liver disease SelW Selenoprotein W NCD Normal chow diet HFD High fat diet GEO Gene Expression Omnibus HbA1C Hemoglobin A1C PKM2 Pyruvate Kinase M2 HK2 Hexokinase 2 LDHA Lactate dehydrogenase A PFKM Phosphofructokinase ACACA Acetyl-CoA carboxylase alpha FASN Fatty acid synthase ACLY ATP citrate lyase ACOX1 Acyl-Coenzyme A oxidase 1 CPT1A Carnitine palmitoyltransferase 1A PPARγ Peroxisome proliferator activated receptor gamma HIF-1α Hypoxia inducible factor 1 subunit alpha NLRP3 NLR family, pyrin domain containing 3 GSDMD Gasdermin D IL-1β Interleukin 1 beta BAX BCL2 associated X, apoptosis regulator BCL2 B cell leukemia/lymphoma 2 cGAS Cyclic GMP-AMP synthase STING Stimulator of interferon gene CXCL9 C-X-C motif chemokine ligand 9 ARG1 Arginase 1 IL-10 Interleukin 10 1. Introduction Non-alcoholic fatty liver disease (NAFLD) is an the immensely prevalent chronic liver condition that is characterized by abnormal aggregation of hepatic lipids in the absence of significant alcohol consumption and comprises the progression from steatosis or non-alcoholic steatotic liver (NAFL) to non-alcoholic steatohepatitis (NASH) [[33]1,[34]2]. The metabolic abnormality has been considered a central driver of NAFLD pathogenesis, which is often accompanied by exceeding lipid droplet accumulation [[35]3,[36]4]. In addition, accumulated evidence has widely publicized that reprogramming of metabolism reprogramming of metabolism in liver diseases is associated with the activity of glycolysis [[37][5], [38][6], [39][7], [40][8]]. An adaptive metabolic switch has been observed in a variety of liver diseases that preferential transition of energy production from oxidative phosphorylation to glycolysis, resulting in the partial conversion of pyruvate to lactic acid [[41]6]. NAFLD and NASH patients have notably enhanced glycolysis, accompanied by elevated lactate levels [[42]7,[43]8]. Moreover, previous study has confirmed that alternated glycolysis contributed to the further deterioration of NAFLD and eventually the progression to cirrhosis and HCC [[44]8], elucidating the crucial role of glycolysis in the occurrence and progression of liver diseases. Furthermore, high-fat diet (HFD)-induced NAFLD mice exhibited significantly elevated plasma levels of glycolysis-associated metabolites, including glucose, lactic acid, and pyruvate, in comparison with the wild type mice [[45]9]. As one of the well-known rate-limiting metabolic enzymes of glycolysis, Pyruvate Kinase M2 (PKM2) has been considered to be strongly associated with obesity and NAFLD [[46]10]. It has been demonstrated that PKM2 can convert metabolic reprogramming from glycolysis to oxidative phosphorylation in macrophages by interacting with Annexin A5, alleviating of inflammation, steatosis, and fibrosis in NASH mice [[47]11]. Recent literature has evidenced that PKM2 performed a mitigation effect on glycolysis to markedly inhibit M1 macrophage polarization, as the key singling pathway in the therapeutic effect of Celastrol on the NALFD [[48]12]. Selenium (Se), a crucial micronutrient, is indispensable for the biosynthesis of selenoproteins and closely associated with the pathogenesis of NAFLD [[49][13], [50][14], [51][15]]. The results of the National Health and Nutrition Examination Survey (NHANES) 2017-18 statistics reveal a positive correlation between elevated levels of blood Se and liver steatosis [[52]15], contradicting with other conclusions. The meta-analysis results suggested that a negative association between Se status in the body and Se intake with cirrhosis, hepatitis, and liver cancer [[53]16]. Therefore, the controversial aspect of Se's potential in preventing chronic liver diseases persists. Selenoprotein W (SelW) is a small molecule selenoprotein (9.32 kDa) with significant expressed in the liver, associating with antioxidant activity [[54]17,[55]18]. It regulates the redox tone of macrophages during inflammation, further affecting the cellular redox processes and bioenergetics [[56]13]. However, recent investigation has indicated that SelW deficiency in the skeletal muscle of obese mice fed with HFD performs has no influence on oxidative stress and insulin sensitivity [[57]19]. Hence, whether SelW participates in the HFD-induced NAFLD progression remains unknown. In the present study, we found that SelW was significantly upregulated in the livers of patients with NAFLD. SelW ablation effectively alleviate hepatic steatosis and concurrently regulated metabolic and inflammatory pathways in HFD-induced NAFLD mice. In particular, we elucidated that SelW modulates PKM2 translocation and triggers HIF-1α to induce mitochondrial apoptosis and damage, while activating the NLRP3 inflammasome-mediated pyroptosis, thereby initiating the cGAS-STING signaling pathway in macrophages and altering the macrophage phenotype. Thus, we revealed an association between SelW and PKM2 in metabolic reprogramming as well as the progression of NAFLD This provides evidence for further studying the biological function of SelW and potential strategies for treating NAFLD. 2. Materials and methods 2.1. Animals and experimental design SelW-knockout (KO) C57BL/6 mice were purchased from Cyagen Biosciences (Jiangsu, China), and the control wild-type (WT) mice were obtained from our laboratory. The procedure employed in this study were conducted in accordance with the Northeast Agricultural University's Institutional Animal Care and Use Committee (certification No. NEAU- [2011]-9). All mice were kept in pairs per cage, following a 12 h light/dark cycle, at 24–27 °C. Four groups were formed by randomly assigning eight-week-old male WT and KO mice. Twenty-five WT fed normal chow diet (NCD) as the NCD WT group, or fed HFD as the HFD WT group. Twenty-five KO mice fed a NCD as the NCD KO group, or fed HFD as the HFD KO group. During the period of experiment, mice were respectively administered with NCD or HFD for 12 weeks, replacing the fresh feed and water daily, and changing bedding material weekly. 2.2. Samples collection At the conclusion of the experiment, the blood glucose levels were detected by obtaining samples from the tail veins of mice. Liver tissue was treated with 4% paraformaldehyde (PFA) solution or fixed in 2.5% glutaraldehyde solution, preserving at 4 °C. The remaining tissue was rapidly frozen using liquid nitrogen, and subsequently preserved at −80 °C for subsequent analysis. The HbA1C contents in the blood of mice were quantified using an HbA1C ELISA kit, following the manufacturer's protocols (Huamei Biological Engineering, China). 2.3. Histological detection of liver tissue The liver of mice was examined using transmission electron microscopy (TEM) and light microscopy to observe the pathological alterations. Additionally, H&E and ORO staining techniques were employed to evaluate the pathological changes [[58]20]. For specific procedures, please refer to the supplementary materials. 2.4. Transcriptome sequencing The transcriptome sequencing experiment was completed by Wuhan Bioacme biotechnology Co., Ltd. TRIzol reagent (Invitrogen, USA) were used to extract total RNA from the liver tissues. The concentration of RNA was determined using the Qubit® RNA Assay Kit in Qubit® 3.0 Flurometer (Life Technologies, CA, U.S.A.). mRNA in total RNA were obtained by Oligo (dT) magnetic beads, and sequencing libraries were generated using the RNA Library Preparation Kit (NEB, USA). The sequencing libraries were sequenced using Hiseq 4000SBS Kit, and applying HISAT2 to map the mice genome. The data were performed using DESeq 2 combined with the difference ratio (|log2 (Fold change) | > 2) and significance level (P value < 0.01) to determine the differentially expression genes (DEGs). 2.5. Culturing of cell and transfection The Hepa1-6 mouse hepatoma cell line and AML12 mouse hepatoparenchymal cell line were maintained in Dulbecco modified Eagle medium (DMEM) (GIBCO, USA) medium, in which AML12 cells were supplemented with 1% ITS (100 × purchased from Sigma) [[59]21,[60]22]. The bone marrow-derived macrophages (BMDMs) were obtained from WT mice according to the previously reported protocol [[61]23]. For SelW overexpression and knockdown, please refer to the supplementary materials. 2.6. Co-immunoprecipitation (CO-IP) and liquid chromatography tandem mass spectrometry (LC-MS) We used FLAG® Immunoprecipitation Kit provided by Sigma Aldrich (St. Louis, USA) to detect potential interaction proteins. Hepa1-6 cells were respectively transfected with pCDNA-W plasmid and pCDNA 3.1 (+) vector (Negative control) for 72 h. The collected cells were lysed with cell lysate contained a protease inhibitor cocktail on ice for 30 min, following the manufacturer's protocol for subsequent operations. Finally, the immunoprecipitated proteins weas detected by western blot and LC-MS analysis by Sangon Biotech (Shanghai, China). 2.7. Molecular docking The possible binding pattern of SelW and PKM2 protein was predicted by protein-protein molecular docking. Swiss-Model ([62]https://swissmodel.expasy.org/) were utilized for performing homology modeling based on the protein sequences of SelW and PKM2. Then protein-protein docking was conducted using AutoDocktools 1.5.6 software for measurement purposes. Finally, the most optimal interaction mode of SelW and PKM2 was analyzed using PyMoL 2.3.0 software. 2.8. Nuclear and cytoplasmic fractionation We used Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) to separated cellular cytoplasmic and nuclear fractions based on previous studies [[63]24]. Subsequently, western blot analysis was performed separately on the nuclear and cytoplasmic proteins. 2.9. Disuccinimidyl suberate (DSS) cross-linking assay We used DSS crosslinkers provided by MedChemExpress (New Jersey, USA) to determine the structural morphology of PKM2. AML 12 cells were transfected by pCDNA-W, Si–W and pCDNA 3.1 vector (+) (as Vehicle group) plasmids for 72 h, and respectively collected cells to rinsed with cold PBS. The samples were reacted with 500 μM DSS solution for 30 min at room temperature, and added 10 mM Tris to incubated for 15 min. Lysates of the sample were analysis by western blot. 2.10. Apoptosis analysis The TdT mediated dUTP nick-end labeling (TUNEL) were employed to illustrate the apoptosis in liver of mouse according to the method described by Sun et al. [[64]25]. The paraffin sections were permeabilized and incubated with dUTP buffer at 37 °C for 2 h. DAPI (Beyotime, China) were implemented to stain and localize the nuclei of cells. For Annexin V-FITC/PI staining (KENGEN, China), the post-transfection cells were labelled with Annexin V-FITC and PI base on the established instructions [[65]26]. The amount of apoptosis cells was detected by flow cytometry. For Hoechst staining (Beyotime, China), cells labelled with Hoechst working solution (10 μg/mL) for 5 min after transfected with plasmids, and the images of fluorescence signal were captured by fluorescence microscope (Thermo Fisher, USA). 2.11. Mitochondrial function assessment and ATP detection Both 10 μg/ml JC-1 fluorescent probe (Beyotime, China) and 20 nm MitoTracker Green probe (Beyotime, China) were performed in present study to accurately determine the mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. In addition, 10 μM 2,7-dichlorofuorescin diacetate (DCFH-DA) (Nanjing Jianchang, China) fluorescent probe were used to assess intracellular ROS levels by fluorescence microscopy (Thermo Fisher Scientific, USA). While mitochondrial ROS (mito-ROS) levels were loaded with 5 μM MitoSOX Red (ABclonal Technology, China) and visualized by Laser confocal microscopy (Olympus Optical, Japan) and fluorescence microplate reader [[66]27]. For ATP detection, the cellular ATP content and NA^+-K^+ -ATPase activity were assessed by ATP assay kit (Product No.A095-1-1, Nanjing Jianchang, China). The measurement of mitochondrial ATP (mito-ATP), mitochondria were isolated using a mitochondria isolation kit (Beyotime, China) according to the instruction manual and then detected using an ATP assay kit (Product No.A095-1-1, Nanjing Jianchang, China) [[67]28]. 2.12. mtDNA analysis Base on the described previously [[68]29], the TIANamp Genomic DNA Kit (Tiangen, China) were extracted and purified the DNA from both whole cells and the culture supernatant of cells. For cytosolic DNA, we used 0.1% NP-40 (Beyotime, China) to cytoplasmically lysis for 20 min on ice. the supernatant was also utilized TIANamp Genomic DNA Kit to extract and purify cytosolic DNA after centrifuged at 15000 rpm at 4 °C for 20 min. Subsequently, the mtDNA primers (Dloop1-3) and nuclear DNA primers (Tert) were used to quantify the levels of mtDNA by qRT-PCR, following the sequence described by Ma et al. [[69]30]. The primers for Tert and mtDNA (Dloop1-3) were synthesized as shown in [70]Table S2. The 2^−ΔΔCt method was used to calculation. The mtDNA of whole cells were used as internal references.