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: FUN14 domain containing 1; MASLD; casein kinase 2α; ferroptosis; steatosis.

FIGURE 1

Bioinformatic analysis of mitochondrial function, expression of CK2α and FUNDC1 in the pathogenesis of metabolic dysfunction‐associated steatotic liver disease (MASLD) in human, mice, and primary mouse hepatocytes. (A–G): Bioinformatic analysis of mitochondrial function; A: GO analysis of differentially expressed genes (DEGs) among control and MASLD human liver samples; B,C: Mitochondrial pathways significantly enriched in GSEA analysis; D: PCA analysis of pathways quantified by GSVA (normalized quantification pathway score); E: Interaction between GSVA score of mitochondria related pathway and clinical features in partial correlation analysis; F: Heat map exhibiting the relationship between multifactorial clinical features and GSVA score of mitochondrial pathway; G: Heatmap displaying relationship between clinical features and enriched mitochondrial genes; H–J: Expression of CK2α in livers from MASLD patients, high‐fat diet (HFD)‐fed mice (60% fat diet for 20 weeks) and palmitic acid (0.5 mM for 72 h)‐challenged primary mouse hepatocytes; K–M: Expression of mitophagy protein FUNDC1 in livers from MASLD patients, HFD‐fed mice and palmitic acid (0.5 mM for 72 h)‐challenged primary mouse hepatocytes. Mean ± SEM, sample size (n) is indicated within the closed circle on each bar, *p < 0.05 vs. respective control or low‐fat diet (LFD) group.

FIGURE 2

Biometric properties in 5‐week–old WT, CK2α knockout (CK2α–^/–), FUNDC1 knockout (FUNDC1–^/–) and CK2α‐FUNDC1 double knockout (CK2α–^/—FUNDC1–^/–) mice placed on a 60% high fat (HF) or nutritionally matched low fat (LF) diet for 20 weeks. A: Representative photographs of mice (prone and supine positions), liver and white adipose tissues; B: Weekly trajectory of body weight in mice from various groups; C: Liver weight; D: Kidney weight; E: Inguinal white adipose tissue (WAT) weight; F: Epididymal WAT weight; G: Intraperitoneal glucose tolerance test (IPGTT); H: Area under curve (AUC) for IPGTT; and I: Food intake (per mouse daily). Mean ± SEM, sample size (n) is indicated within the closed circle on each bar, *p < 0.05 vs. WT mice, #p < 0.05 vs. WT‐HF mice, †p < 0.05 vs. CK2α–^/—HF mice.

FIGURE 3

Metabolic properties (24‐hr cycle) of 5‐week–old WT, CK2α knockout (CK2α–^/–), FUNDC1 knockout (FUNDC1–^/–) and CK2α‐FUNDC1 double knockout (CK2α–^/—FUNDC1–^/–) mice placed on a 60% high fat (HF) or nutritionally matched low fat (LF) diet for 20 weeks. A: O₂ consumption in WT and CK2α^‐/‐mice; B: O₂ consumption in FUNDC1^‐/‐ and CK2α^‐/–FUNDC1^‐/‐ mice; C: Pooled O₂ consumption (light); D: Pooled O₂ consumption (dark); E: Pooled O₂ consumption (combined); F: CO₂ production in WT and CK2α^‐/‐ mice; G: CO₂ production in FUNDC1^‐/‐ and CK2α^‐/–FUNDC1^‐/‐ mice; H: Pooled CO₂ production (light); I: Pooled CO₂ production (dark); J: Pooled CO₂ production (combined); K: Respiratory exchange ratio (RER) in WT and CK2α^‐/‐ mice; L: RER ratio in FUNDC1^‐/‐ and CK2α^‐/–FUNDC1^‐/‐ mice; M: Pooled data of RER (light); N: Pooled data of RER (dark); O: Pooled data of RER (combined); P: Heat production; Q: Total physical activity; R: Pooled data of heat production (24 h); S: Pooled data of total physical activity; and T: Aconitase activity. Mean ± SEM, sample size (n) is indicated within the closed circle on each bar. *p < 0.05 vs. WT mice, #p < 0.05 vs. WT‐HF mice, †p < 0.05 vs. CK2α–^/—HF mice.

FIGURE 4

Hematoxylin and eosin (HE) staining and oil red O staining of liver sections of WT, CK2α knockout (CK2α–^/–), FUNDC1 knockout (FUNDC1–^/–) and CK2α‐FUNDC1 double knockout (CK2α–^/—FUNDC1–^/–) mice placed on a 60% high fat (HF) or nutritionally matched low fat (LF) diet for 20 weeks. A: Representative HE and oil red O staining of liver sections from mice fed LF or HF diet for 20 weeks; B: Serum AST levels; C: Serum ALT levels; D: Serum albumin levels; E: Blood triglyceride levels; F: Blood total cholesterol level; G: Lipid droplet area (% total field) from oil red O staining; H: Lipid droplet size (oil red O staining); I: Plasma insulin levels; J: Plasma HOMA‐IR index; and K: Liver DHE levels. Means ± SEM, sample size (n) is indicated within the closed circle on each bar. *p < 0.05 vs. WT mice, #p < 0.05 vs. WT‐HF mice, †p < 0.05 vs. CK2α–^/—HF mice.

FIGURE 5

Hepatic fibrosis, ultrastructure, and apoptosis along with adipocyte size in WT, CK2α knockout (CK2α–^/–), FUNDC1 knockout (FUNDC1–^/–) and CK2α‐FUNDC1 double knockout (CK2α–^/—FUNDC1–^/–) mice placed on a 60% high fat (HF) or nutritionally matched low fat (LF) diet for 20 weeks. A: Representative Masson trichrome staining of liver sections from mice fed LF or HF diet for 20 weeks; B: Pooled hepatic fibrosis; C: Representative adipocyte HE staining from mice fed LF or HF diet for 20 weeks; D: Pooled adipocyte area; E: Representative TEM images exhibiting mitochondrial injury. Damaged mitochondria are shown by disorganization and reduced crista, and signs of vacuolation; F: Representative TUNEL staining of liver sections from mice fed LF or HF diet for 20 weeks; and G: Pooled TUNEL staining manifested as percent TUNEL‐positive cells. Means ± SEM, sample size (n) is indicated within the closed circle on each bar. *p < 0.05 vs. WT mice, #p < 0.05 vs. WT‐HF mice, †p < 0.05 vs. CK2α–^/—HF mice.

FIGURE 6

Levels of lipid regulating enzymes in livers from WT, CK2α knockout (CK2α–^/–), FUNDC1 knockout (FUNDC1–^/–) and CK2α‐FUNDC1 double knockout (CK2α–^/—FUNDC1–^/–) mice placed on a 60% high fat (HF) or nutritionally matched low fat (LF) diet for 20 weeks. A: Representative immunoblots depicting FAS, ACC (pan and phosphorylated), ChREBP, SREBP1, PGC1α, PPARα, PPARγ, SCD1, DGAT, PEPCK and G6Pase (GAPDH as loading control); B: FAS levels; C: pACC‐to‐ACC ratio; D: ChREBP levels; E: SREBP1 levels; F: PGC1α levels; G: PPARα levels; H: PPARγ levels; I: SCD1 levels; J: DGAT levels; K: PEPCK levels; and L: G6Pase levels. Mean ± SEM, sample size (n) is indicated within the closed circle on each bar. *p < 0.05 vs. WT mice, #p < 0.05 vs. WT‐HF mice, †p < 0.05 vs. CK2α–^/—HF mice.

FIGURE 7

Effect of CK2α and FUNDC1 knockdown as well as CK2α selective inhibitor CX4945 (Silmitasertib) on palmitic acid (PA)‐induced changes in mitoKeima mitophagy in HepG2 cells and primary murine hepatocytes. HepG2 cells or primary murine hepatocytes were transfected with siCK2α or siFUNDC1 prior to exposure to PA (0.5 mM) in the presence or absence of the ferroptosis inducer erastin (ERA, 20 µM), the ferroptosis inhibitor LIP1 (200 nM) or the mitophagy inhibitor liensinine (LIEN, 20 µM) for 72 h. A subset of PA‐treated HepG2 cells or primary hepatocytes were incubated with the CK2α selective inhibitor CX4945 (10 µM). Scramble (scr) RNA serves as the negative control. A: Representative mitoKeima images depicting mitophagy from indicated HepG2 groups; B: Quantification of mitoKeima staining exhibiting HepG2 cellular mitophagy levels; C: Representative mitoKeima images depicting mitophagy from indicated primary murine hepatocyte groups; and D: Quantification of mitoKeima staining exhibiting primary hepatocyte mitophagy levels. Mean ± SEM, sample size (n) is indicated within the closed circle on each bar, *p < 0.05 vs. scrCK2α‐scrFUNDC1 group, #p < 0.05 vs. scrCK2α‐scrFUNDC1‐PA group, †p < 0.05 vs. siCK2α‐PA group.

FIGURE 8

Effect of CK2α and FUNDC1 knockdown as well as CK2α selective inhibitor CX4945 (Silmitasertib) on palmitic acid (PA)‐induced changes in lipid peroxidation and steatosis in primary murine hepatocytes and HepG2 cells, respectively. Primary murine hepatocytes and HepG2 cells were transfected with siCK2α or siFUNDC1 prior to exposure to PA (0.5 mM) in the presence or absence of the ferroptosis inducer erastin (ERA, 20 µM), the ferroptosis inhibitor LIP1 (200 nM) or the mitophagy inhibitor liensinine (LIEN, 20 µM) for 72 hrs. A subset of PA‐treated HepG2 cells were incubated with the CK2α selective inhibitor CX4945 (10 µM). Scramble (scr) RNA serves as the negative control. A: Representative Bodipy C11 fluorescence images depicting lipid peroxidation from indicated primary hepatocyte groups; B: Quantification of Bodipy C11 fluorescence exhibiting lipid peroxidation in primary murine hepatocytes; C: Representative oil red O staining images from indicated HepG2 groups; D: Quantification of oil red O exhibiting intracellular triglyceride levels in HepG2 cells; E‐F: Disease and function analysis of CK2α knockout versus WT mice under HFD. This network illustrates differentially expressed proteins identified in this study. Red Nodes: Upregulated proteins; Green Nodes: Downregulated proteins. Orange Inner Ring: Predicted activation of associated functions; Blue Inner Ring: Predicted inhibition of associated functions. The box inset in panel F offers a denotations for these predictive indicators; and G: Schematic diagram exhibiting possible mechanisms for the role of CK2α and FUNDC1 in high fat diet‐induced hepatic steatosis. Long‐term high fat diet insult dampened FUNDC1‐mediated mitophagy through upregulation of CK2α, prompting disturbed lipid metabolism and lipid accumulation due to compromised mitophagy. Lipotoxicity then promotes onset of lipotoxic ferroptosis, imperiling hepatic steatosis. Created in BioRender. Lin, L. (2025) https://BioRender.com/i28p846. Mean ± SEM, sample size (n) is indicated within the closed circle on each bar, *p < 0.05 vs. scrCK2α‐scrFUNDC1 group, #p < 0.05 vs. scrCK2α‐scrFUNDC1‐PA group, †p < 0.05 vs. siCK2α‐PA group.