Volume 143, Issue 3, Pages 754-764 (September 2012) β-Catenin Regulates Hepatic Mitochondrial Function and Energy Balance in Mice  Nadja Lehwald, Guo–Zhong Tao, Kyu Yun Jang, Ioanna Papandreou, Bowen Liu, Bo Liu, Marybeth A. Pysz, Jürgen K. Willmann, Wolfram T. Knoefel, Nicholas C. Denko, Karl G. Sylvester  Gastroenterology  Volume 143, Issue 3, Pages 754-764 (September 2012) DOI: 10.1053/j.gastro.2012.05.048 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 β-catenin KO mice demonstrate an energy deficit despite sufficient nutrient availability. (A) Efficient β-catenin deletion in KO mice is demonstrated for messenger RNA (mRNA) and protein by quantitative reverse-transcription polymerase chain reaction (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 and E) Glycolytic gene expression (glucokinase [GK] and liver pyruvate kinase [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 with WT controls. RLU, relative light units. n = 8. *P < .05, **P < .01; n.s., not significant. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions

Figure 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 quantitative reverse-transcription polymerase chain reaction and validated at protein level by Western blot. mOD, mean optical density. n = 5. *P < .05. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions

Figure 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) malondialdehyde (MDA) assay. (C–E) KO mice treated with ethanol demonstrate increased expression of the antioxidant genes (superoxide dismutase 1 [SOD1], glutathione peroxidase 1 [GPX1], and glutathione S-transferase Mu1 [GST-M1]) by quantitative reverse-transcription polymerase chain reaction. Note: β-catenin KO mice show no redox imbalance at baseline. n = 5. *P < .05; n.s., not significant. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions

Figure 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 < .05; n.s., not significant. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions

Figure 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 messenger RNA (mRNA) expression level in sham- and ethanol-treated livers. (C) Reduced Sirt1, PPAR-α, and medium chain acyl CoA dehydrogenase (MCAD) proteins are detected in ethanol-treated KO liver lysates. (D) Decreased PPAR-α target genes (Acyl-CoA oxidase [AOX], MCAD) involved in FAO in sham- and ethanol-treated livers as detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). (E) 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 with WT controls. n = 5. *P < .05; n.s., not significant. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions

Figure 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. Gastroenterology 2012 143, 754-764DOI: (10.1053/j.gastro.2012.05.048) Copyright © 2012 AGA Institute Terms and Conditions