β-Catenin regulates hepatic mitochondrial function and energy balance in mice

Abstract

Background & aims: Wnt signaling regulates hepatic function and nutrient homeostasis. However, little is known about the roles of β-catenin in cellular respiration or mitochondria of hepatocytes.

Methods: We investigated β-catenin's role in the metabolic function of hepatocytes under homeostatic conditions and in response to metabolic stress using mice with hepatocyte-specific deletion of β-catenin and their wild-type littermates, given either saline (sham) or ethanol (as a model of binge drinking and acute ethanol intoxication).

Results: Under homeostatic conditions, β-catenin-deficient hepatocytes demonstrated mitochondrial dysfunctions that included impairments to the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) and decreased production of adenosine triphosphate (ATP). There was no evidence for redox imbalance or oxidative cellular injury in the absence of metabolic stress. In mice with β-catenin-deficient hepatocytes, ethanol intoxication led to significant redox imbalance in the hepatocytes and further deterioration in mitochondrial function that included reduced OXPHOS, fatty acid oxidation (FAO), and ATP production. Ethanol feeding significantly increased liver steatosis and oxidative damage, compared with wild-type mice, and disrupted the ratio of nicotinamide adenine dinucleotide. β-catenin-deficient hepatocytes also had showed disrupted signaling of Sirt1/peroxisome proliferator-activated receptor-α signaling.

Conclusions: β-catenin has an important role in the maintenance of mitochondrial homeostasis, regulating ATP production via the tricarboxylic acid cycle, OXPHOS, and fatty acid oxidation; β-catenin function in these systems is compromised under conditions of nutrient oxidative stress. Reagents that alter Wnt-β-catenin signaling might be developed as a useful new therapeutic strategy for treatment of liver disease.

Fig 1.. β-catenin KO mice demonstrate an energy deficit despite sufficient nutrient availability.

(A) Efficient β-catenin deletion in KO mice is demonstrated for mRNA and protein by qRT-PCR and western blot analysis. (B) Reduced baseline ATP levels are detected in β-catenin deficient livers. (C) Equivalent pyruvate levels in β-catenin KO and WT mice. (D-E) Glycolytic gene expression (GK and LPK) is similar during homeostasis in WT and KO livers as measured by qRT-PCR. (F) KO mice show increased serum lactate levels under homeostatic conditions compared to WT controls. RLU, relative light units. n=8. *p<0.05, **p<0.01; n.s., not significant.

Fig 2.. β-catenin deficient hepatocytes have impaired mitochondrial function.

Mitochondria, isolated from WT and KO livers, were (A) stained with JC-1 and analyzed by fluorescent plate reader. KO mice demonstrate lower mitochondrial membrane potential as detected by JC-1 ratio (fluorescence intensity 560 nm / 485 nm). KO mice show impaired TCA cycle as measured by (B) citrate synthase and (C) aconitase activity as well as OXPHOS dysfunction as evidenced by (D) reduced Complex IV activity. (E) Decreased oxygen consumption rate in primary β-catenin KO hepatocytes is measured using a Clark electrode. (F) Mitochondrial OXPHOS gene expression is detected in WT and KO livers by qRT-PCR and validated at protein level by western blot. mOD, mean optical density. n=5. *p<0.05.

Fig 3.. β-catenin deficient liver demonstrates redox imbalance after ethanol intoxication.

Increased ROS levels were detected in ethanol-treated KO mice by (A) DHE staining and (B) MDA assay. (C-E) KO mice treated with ethanol demonstrate increased expression of the antioxidant genes (SOD1, GPX1, GST-M1) by qRT-PCR. Note: β-catenin KO mice show no redox imbalance at baseline. n=5. *p<0.05; n.s., not significant.

Fig 4.. β-catenin KO mice are more susceptible to ethanol-induced liver steatosis.

β-catenin null mice show increased liver injury and severe steatosis with lipid accumulation in response to ethanol intoxication as assessed by (A) serum ALT and (B) triglycerides or by (C) H&E and (D) oil red O staining in ethanol-treated WT and KO livers using the Binge drinking model. NAC treatment (200 mg/kg) prior to ethanol treatment reduces steatosis in WT mice, but has no effect on KO livers. (E) NAC treatment prior to ethanol intoxication did not change pyruvate levels in WT and KO mice. (F) Lower ATP levels were measured in β-catenin deficient liver despite NAC treatment. n=5. *p<0.05; n.s., not significant.

Fig 5.. Hepatic loss of β-catenin impairs fatty acid β-oxidation upon ethanol intoxication with reduced Sirt1/PPAR-α signaling.

(A) Elevated lipids in β-catenin KO sham- and ethanol-treated mice as measured by increased serum free fatty acids. (B) β-catenin deletion reduces PPAR-α mRNA expression level in sham- and ethanol-treated livers. (C) Reduced Sirt1, PPAR-α and MCAD proteins are detected in ethanol-treated KO liver lysates. (D) Decreased PPAR-α target genes (AOX, MCAD) involved in FAO in sham- and ethanol-treated livers as detected by qRT-PCR. (D) Reduced Sirt1 mRNA expression in sham- and ethanol-treated KO livers as detected by qRT-PCR. (F) β-catenin deficient mice show a decreased NAD⁺/NADH ratio after ethanol intoxication in comparison to WT controls. n=5. *p<0.05; n.s., not significant.

Fig 6.. Working model summarizing the role of β-catenin in mitochondrial function and energy metabolism.

In the absence of β-catenin, mitochondrial function is significantly impaired with decreased membrane potential, impaired TCA cycle, reduced OXPHOS, and reduced FAO leading to decreased energy production (ATP) in the presence of sufficient nutrients (oxygen, glucose, pyruvate) as a result of impaired Sirt1/PPAR-α signaling axis. Given nutrient stress (ethanol), mitochondrial impairment in the absence of β-catenin leads to increased ROS production and redox imbalance that further diminishes mitochondrial function and results in hepatosteatosis.