Casein Kinase 2α Ablation Confers Protection Against Metabolic Dysfunction-Associated Steatotic Liver Disease: Role of FUN14 Domain Containing 1-Dependent Regulation of Mitophagy and Ferroptosis

Abstract

Mitochondrial dyshomeostasis provokes the onset of metabolic dysfunction-associated steatotic liver disease (MASLD) although its precise involvement in particular mitophagy in MASLD remains elusive. This work evaluated the role of casein kinase 2α (CK2α) and FUNDC1 in high-fat diet (HFD)-evoked MASLD. WT and CK2α deletion (CK2α^-/-) mice were subjected to low fat or HFD for 20 weeks. Global metabolism, AST, ALT, cholesterol, triglycerides, hepatic steatosis, fibrosis, inflammation, mitochondrial injury, mitophagy and ferroptosis were examined. Bioinformatics analysis enriched mitochondria-related pathways in MASLD. Hepatic CK2α and FUNDC1 were upregulated and downregulated, respectively, in MASLD patients and HFD-fed mice. HFD led to adiposity, hepatomegaly, hepatic steatosis, fibrosis, inflammation, ferroptosis, mitochondrial injury, elevated hepatic tissue Fe²⁺, FAS, CHREBP, SREBP1, PGC1α, PPARα, PPARγ, SCD1, PEPCK, G6Pase, and DGAT1 as well as downregulated FUNDC1, GPx4, SLC7A11 and NCOA4, the effects (except for NCOA4) were nullified by CK2α deletion. FUNDC1 deletion nullified CK2α deletion-evoked benefit on hepatic ferroptosis and lipid enzymes. In vitro study using palmitic acid indicated an obligatory role for CK2α, FUNDC1 and ferroptosis in hepatocyte steatosis. Collectively, our results demonstrated that CK2α activation by HFD serves as a trigger for mitochondrial damage, hepatic injury, and pathogenesis of MASLD through FUNDC1 disruption and ferroptosis.

Keywords

casein kinase 2α / ferroptosis / FUN14 domain containing 1 / MASLD / steatosis

References

[1] N. Stefan, H. Yki-Jarvinen, and B. A. Neuschwander-Tetri, “Metabolic Dysfunction-Associated Steatotic Liver Disease: Heterogeneous Pathomechanisms and Effectiveness of Metabolism-Based Treatment,” Lancet Diabetes & Endocrinology 13, no. 2 (2024): P134-148.

[2] A. Do, F. Zahrawi, and W. Z. Mehal, “Therapeutic Landscape of Metabolic Dysfunction-Associated Steatohepatitis (MASH),” Nature Reviews Drug Discovery 24, no. 3 (2024): 171-189.

[3] R. J. Wong, B. Liu, and T. Bhuket, “Significant Burden of Nonalcoholic Fatty Liver Disease With Advanced Fibrosis in the US: A Cross-Sectional Analysis of 2011-2014 National Health and Nutrition Examination Survey,” Alimentary Pharmacology & Therapeutics 46, no. 10 (2017): 974-980.

[4] S. P. Singh, T. Madke, and P. Chand, “Global Epidemiology of Hepatocellular Carcinoma,” Journal of Clinical and Experimental Hepatology 15, no. 2 (2025): 102446.

[5] P. Chen, L. Yao, M. Yuan, et al., “Mitochondrial Dysfunction: A Promising Therapeutic Target for Liver Diseases,” Genes and Diseases 11, no. 3 (2024): 101115.

[6] R. Liu, M. P. Qian, and Y. Y. Cui, “Protein Kinases: The Key Contributors in Pathogenesis and Treatment of Nonalcoholic Fatty Liver Disease-Derived Hepatocellular Carcinoma,” Metabolism 147 (2023): 155665.

[7] S. Iturbe-Rey, C. Maccali, M. Arrese, et al., “Lipotoxicity-Driven Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD),” Atherosclerosis 400 (2025): 119053.

[8] Y. Zhang and J. Ren, “Epigenetics and Obesity Cardiomyopathy: From Pathophysiology to Prevention and Management,” Pharmacology & Therapeutics 161 (2016): 52-66.

[9] S. Jin, Y. Li, T. Xia, et al., “Mechanisms and Therapeutic Implications of Selective Autophagy in Nonalcoholic Fatty Liver Disease,” Journal of Advanced Research 67 (2024): 317-329.

[10] Q. Ren, Q. Sun, and J. Fu, “Dysfunction of Autophagy in High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease,” Autophagy 20 (2024): 221-241.

[11] H. Cho, J. Mu, and J. K. Kim, “Insulin Resistance and a Diabetes Mellitus-Like Syndrome in Mice Lacking the Protein Kinase Akt2 (PKB beta),” Science 292 (2001): 1728-1731.

[12] C. Mu, S. Wang, Z. Wang, et al., “Mechanisms and Therapeutic Targets of Mitochondria in the Progression of Metabolic Dysfunction-Associated Steatotic Liver Disease,” Annals of Hepatology 30, no. 1 (2025): 101774.

[13] A. Ajoolabady, C. Lebeaupin, N. N. Wu, R. J. Kaufman, and J. Ren, “ER Stress and Inflammation Crosstalk in Obesity,” Medicinal Research Reviews 43 (2023): 5-30.

[14] A. Ajoolabady, M. Chiong, S. Lavandero, D. J. Klionsky, and J. Ren, “Mitophagy in Cardiovascular Diseases: Molecular Mechanisms, Pathogenesis, and Treatment,” Trends in Molecular Medicine 28 (2022): 836-849.

[15] C. Shi, Y. Cai, Y. Li, et al., “Yap Promotes Hepatocellular Carcinoma Metastasis and Mobilization via Governing Cofilin/F-actin/Lamellipodium Axis by Regulation of JNK/Bnip3/SERCA/CaMKII Pathways,” Redox Biology 14 (2018): 59-71.

[16] M. Tian, J. Hou, Z. Liu, et al., “BNIP3 in Hypoxia-Induced Mitophagy: Novel Insights and Promising Target for Non-Alcoholic Fatty Liver Disease,” International Journal of Biochemistry & Cell Biology 168 (2024): 106517.

[17] H. He, Y. Tang, L. Zhuang, Y. Zheng, and X. Huang, “PINK1/Park2-Mediated Mitophagy Relieve Non-Alcoholic Fatty Liver Disease,” Physiological Research 73 (2024): 253-263.

[18] B. Pan, X. Ma, S. Zhou, et al., “Predicting Mitophagy-Related Genes and Unveiling Liver Endothelial Cell Heterogeneity in Hepatic Ischemia-Reperfusion Injury,” Frontiers in Immunology 15 (2024): 1370647.

[19] J. Deng, Q. Liu, L. Ye, et al., “The Janus Face of Mitophagy in Myocardial Ischemia/Reperfusion Injury and Recovery,” Biomedicine & Pharmacotherapy 173 (2024): 116337.

[20] J. Wang, P. Zhu, R. Li, J. Ren, and H. Zhou, “Fundc1-Dependent Mitophagy Is Obligatory to Ischemic Preconditioning-Conferred Renoprotection in Ischemic AKI via Suppression of Drp1-Mediated Mitochondrial Fission,” Redox Biology 30 (2020): 101415.

[21] M. Yang, B. S. Linn, Y. Zhang, and J. Ren, “Mitophagy and Mitochondrial Integrity in Cardiac Ischemia-Reperfusion Injury,” Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1865 (2019): 2293-2302.

[22] H. Zhou, P. Zhu, J. Wang, H. Zhu, J. Ren, and Y. Chen, “Pathogenesis of Cardiac Ischemia Reperfusion Injury Is Associated With CK2alpha-Disturbed Mitochondrial Homeostasis via Suppression of FUNDC1-Related Mitophagy,” Cell Death and Differentiation 25 (2018): 1080-1093.

[23] Y. Mao, J. Ren, and L. Yang, “FUN14 Domain Containing 1 (FUNDC1): A Promising Mitophagy Receptor Regulating Mitochondrial Homeostasis in Cardiovascular Diseases,” Frontiers in Pharmacology 13 (2022): 887045.

[24] W. Li, Y. Li, S. Siraj, et al., “FUN14 Domain-Containing 1-Mediated Mitophagy Suppresses Hepatocarcinogenesis by Inhibition of Inflammasome Activation in Mice,” Hepatology 69, no. 2 (2019): 604-621.

[25] R. Undamatla, O. G. Fagunloye, J. Chen, et al., “Reduced Mitophagy Is an Early Feature of NAFLD and Liver-Specific PARKIN Knockout Hastens the Onset of Steatosis, Inflammation and Fibrosis,” Scientific Reports 13 (2023): 7575.

[26] M. Chen, Z. Chen, Y. Wang, et al., “Mitophagy Receptor FUNDC1 Regulates Mitochondrial Dynamics and Mitophagy,” Autophagy 12 (2016): 689-702.

[27] S. E. Choi, S. Kwon, S. Seok, et al., “Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver,” Molecular and Cellular Biology 37, no. 15 (2017): e00006-17.

[28] H. Deng, X. Rao, S. Zhang, et al., “Protein Kinase CK2: An Emerging Regulator of Cellular Metabolism,” Biofactors 50, no. 4 (2024): 624-633.

[29] C. Y. Lian, H. J. Li, W. H. Xia, et al., “Insufficient FUNDC1-Dependent Mitophagy due to Early Environmental Cadmium Exposure Triggers Mitochondrial Redox Imbalance to Aggravate Diet-Induced Lipotoxicity,” Environmental Pollution 361 (2024): 124724.

[30] A. Tsuji, S. Yoshikawa, Y. Ikeda, et al., “Tactics With Prebiotics for the Treatment of Metabolic Dysfunction-Associated Fatty Liver Disease via the Improvement of Mitophagy,” International Journal of Molecular Sciences 24, no.6 (2023): 5465.

[31] E. Ferrannini, “The Theoretical Bases of Indirect Calorimetry: A Review,” Metabolism 37, no. 3 (1988): 287-301.

[32] N. Subramaiyam, “Insights of Mitochondrial Involvement in Alcoholic Fatty Liver Disease,” Journal of Cellular Physiology 238, no. 10 (2023): 2175-2190.

[33] T. Ueno and M. Komatsu, “Autophagy in the Liver: Functions in Health and Disease,” Nature Reviews Gastroenterology & Hepatology 14 (2017): 170-184.

[34] S. Wang, J. Tao, H. Chen, et al., “Ablation of Akt2 and AMPKalpha2 Rescues High Fat Diet-Induced Obesity and Hepatic Steatosis Through Parkin-Mediated Mitophagy,” Acta Pharmaceutica Sinica B 11 (2021): 3508-3526.

[35] P. Liu, H. Lin, Y. Xu, et al., “Frataxin-Mediated PINK1-Parkin-Dependent Mitophagy in Hepatic Steatosis: The Protective Effects of Quercetin,” Molecular Nutrition & Food Research 62 (2018): e1800164.

[36] L. Wang, X. Liu, J. Nie, et al., “ALCAT1 Controls Mitochondrial Etiology of Fatty Liver Diseases, Linking Defective Mitophagy to Steatosis,” Hepatology 61 (2015): 486-496.

[37] R. Singh, S. Kaushik, Y. Wang, et al., “Autophagy Regulates Lipid Metabolism,” Nature 458 (2009): 1131-1135.

[38] M. Shimano, N. Ouchi, R. Shibata, et al., “Adiponectin Deficiency Exacerbates Cardiac Dysfunction Following Pressure Overload Through Disruption of an AMPK-Dependent Angiogenic Response,” Journal of Molecular and Cellular Cardiology 49 (2010): 210-220.

[39] T. Fu, Z. Xu, L. Liu, et al., “Mitophagy Directs Muscle-Adipose Crosstalk to Alleviate Dietary Obesity,” Cell Reports 23 (2018): 1357-1372.

[40] L. R. Martins, P. Lucio, A. Melao, et al., “Activity of the Clinical-Stage CK2-Specific Inhibitor CX-4945 Against Chronic Lymphocytic Leukemia,” Leukemia 28 (2014): 179-182.

[41] M. J. Borad, L.-Y. Bai, M.-H. Chen, et al., “Silmitasertib (CX-4945) in Combination With Gemcitabine and Cisplatin as First-Line Treatment for Patients With Locally Advanced or Metastatic Cholangiocarcinoma: A Phase Ib/II Study,” Journal of Clinical Oncology 39 (2021): 312.

[42] Y. Bi, S. Liu, X. Qin, et al., “FUNDC1 Interacts With GPx4 to Govern Hepatic Ferroptosis and Fibrotic Injury Through a Mitophagy-Dependent Manner,” Journal of Advanced Research 55 (2024): 45-60.

[43] Y. Bi, H. Xu, X. Wang, et al., “FUNDC1 Protects Against Doxorubicin-Induced Cardiomyocyte PANoptosis Through Stabilizing mtDNA via Interaction With TUFM,” Cell Death & Disease 13 (2022): 1020.

[44] X. Xu, Y. Hua, S. Nair, Y. Zhang, and J. Ren, “Akt2 Knockout Preserves Cardiac Function in High-Fat Diet-Induced Obesity by Rescuing Cardiac Autophagosome Maturation,” Journal of Molecular Cell Biology 5 (2013): 61-63.

[45] S. Turdi, M. R. Kandadi, J. Zhao, A. F. Huff, M. Du, and J. Ren, “Deficiency in AMP-Activated Protein Kinase Exaggerates High Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction,” Journal of Molecular and Cellular Cardiology 50 (2011): 712-722.

[46] E. Clough and T. Barrett, “The Gene Expression Omnibus Database,” Methods in Molecular Biology 1418 (2016): 93-110.

[47] M. Ahrens, O. Ammerpohl, W. von Schönfels, et al., “DNA Methylation Analysis in Nonalcoholic Fatty Liver Disease Suggests Distinct Disease-Specific and Remodeling Signatures After Bariatric Surgery,” Cell Metabolism 18 (2013): 296-302.

[48] A. G. Baltz, M. Munschauer, B. Schwanhäusser, et al., “The mRNA-Bound Proteome and Its Global Occupancy Profile On Protein-Coding Transcripts,” Molecular Cell 46 (2012): 674-690.

[49] M. E. Ritchie, B. Phipson, D. Wu, et al., “limma Powers Differential Expression Analyses for RNA-Sequencing and Microarray Studies,” Nucleic Acids Research 43 (2015): e47.

[50] L. Gautier, L. Cope, B. M. Bolstad, and R. A. Irizarry, “affy-analysis of Affymetrix GeneChip Data at the Probe Level,” Bioinformatics 20 (2004): 307-315.

[51] T. Wu, E. Hu, S. Xu, et al. “clusterProfiler 40: A Universal Enrichment Tool for Interpreting Omics Data,” Innovation 2 (2021): 100141.

[52] S. Hänzelmann, R. Castelo, and J. Guinney, “GSVA: Gene Set Variation Analysis for Microarray and RNA-seq Data,” BMC Bioinformatics 14 (2013): 7.

[53] R. K. Powers, A. Goodspeed, H. Pielke-Lombardo, A. C. Tan, and J. C. Costello, “GSEA-InContext: Identifying Novel and Common Patterns in Expression Experiments,” Bioinformatics 34 (2018): i555-i564.

[54] G. Shan, E. Bayram, J. Z. K. Caldwell, J. B. Miller, J. J. Shen, and S. Gerstenberger, “Partial Correlation Coefficient for a Study With Repeated Measurements,” Statistics in Biopharmaceutical Research 13 (2021): 448-454.

[55] R. C. M. van Aert and C. Goos, “A Critical Reflection on Computing the Sampling Variance of the Partial Correlation Coefficient,” Research Synthesis Methods 14 (2023): 520-525.

[56] K. Ben Salem and A. Ben Abdelaziz, “Principal Component Analysis (PCA),” La Tunisie Medicale 99 (2021): 383-389.

[57] A. Giuliani, “The Application of Principal Component Analysis to Drug Discovery and Biomedical Data,” Drug Discovery Today 22 (2017): 1069-1076.

[58] X. Xu, L. K. Hueckstaedt, and J. Ren, “Deficiency of Insulin-Like Growth Factor 1 Attenuates Aging-Induced Changes in Hepatic Function: Role of Autophagy,” Journal of Hepatology 59 (2013): 308-17.

[59] W. Yu, X. Qin, Y. Zhang, et al., “Curcumin Suppresses Doxorubicin-Induced Cardiomyocyte Pyroptosis via a PI3K/Akt/mTOR-Dependent Manner,” Cardiovascular Diagnosis and Therapy 10 (2020): 752-769.

[60] W. Yu, W. Zha, H. Peng, Q. Wang, S. Zhang, and J. Ren, “Trehalose Protects Against Insulin Resistance-Induced Tissue Injury and Excessive Autophagy in Skeletal Muscles and Kidney,” Current Pharmaceutical Design 25 (2019): 2077-2085.

[61] H. Peng, J. Zhang, Z. Zhang, et al., “Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Cardiac Anomalies Through Reconciliation of Autophagy and Ferroptosis,” Life Sciences 328 (2023): 121821.

[62] W. Wu, W. Bu, Y. Tan, and Y. Wang, “Effect of Sulfasalazine on Ferroptosis During Intestinal Injury in Rats After Liver Transplantation,” Scientific Reports 14 (2024): 7349.

[63] K. M. Kalemba, Y. Wang, H. Xu, et al., “Glycerol Induces G6pc in Primary Mouse Hepatocytes and Is the Preferred Substrate for Gluconeogenesis both in Vitro and in Vivo,” Journal of Biological Chemistry 294 (2019): 18017-18028.

[64] H. Xu, X. Wang, W. Yu, et al., “Syntaxin 17 Protects Against Heart Failure through Recruitment of CDK1 to Promote DRP1-Dependent Mitophagy,” JACC Basic to Translational Science 8 (2023): 1215-1239.

[65] N. Zhou, L. Ma, W. Shi, et al., “Akt Mitigates ER Stress-Instigated Cardiac Dysfunction via Regulation of Ferroptosis and Mitochondrial Integrity in a DHODH-Dependent Manner,” Life Sciences 371 (2025): 123591.

[66] X. Zhu, H. Yan, M. Xia, et al., “Metformin Attenuates Triglyceride Accumulation in HepG2 Cells Through Decreasing Stearyl-Coenzyme A Desaturase 1 Expression,” Lipids in Health and Disease 17 (2018): 114.

[67] R. Li, J. Li, Y. Huang, et al., “Polydatin Attenuates Diet-Induced Nonalcoholic Steatohepatitis and Fibrosis in Mice,” International Journal of Biological Sciences 14 (2018): 1411-1425.

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