AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

Abstract

ABSTRACT Recent reports indicated that mitophagy protects against alcohol-induced liver injury, which helps remove damaged mitochondria to reduce the accumulation of reactive oxygen species (ROS). AMP-activated protein kinase (AMPK) has been recently used in ALD (alcoholic liver disease) and mitochondrial dysfunction research. However, the inner mechanism, whether AMPK can regulate mitophagy in ALD, remains unknown. Here we found that AMPK can significantly reduce alcohol-induced liver injury and enhances hepatocytes’ mitophagy level. Next, we identified that AMPK rescued alcohol-induced low expression of UQCRC2 (ubiquinol-cytochrome c reductase core protein 2). Interestingly, UQCRC2 knockdown (KD) treatment causes impaired mitophagy, whereas UQCRC2 overexpression (OE) can significantly increase mitophagy to attenuate liver injury. Also, we identified that AMPK indirectly upregulates UQCRC2 protein level, and RNA-seq, chromatin immunoprecipitation (ChIP) assay, bioinformatics, and luciferase assays helped us understand that AMPK enhanced UQCRC2 gene transcription through activating NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2). Our results demonstrate that AMPK regulating UQCRC2 is a significant mitochondrial event in mitophagy. It identifies a new signaling axis, AMPK-NFE2L2-UQCRC2, in the regulation of mitophagy levels in the liver, suggesting a possible therapeutic strategy to treat ALD. Abbreviations: AAV: AENO-associated virus; ALD: alcoholic liver disease; AMPK: AMP-activated protein kinase; BUN: blood urea nitrogen; H&E: hematoxylin and eosin; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ChIP: chromatin immunoprecipitation assay; CO-IP: co-immunoprecipitation; COPD: chronic obstructive pulmonary disease; EM: electron microscope; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic—pyruvic transaminase; IF: immunofluorescence; IHC: immunohistochemistry; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain protein 3; MTDR: MitoTracker Deep Red; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; mtDNA: mitochondrial DNA; MTRC: MitoTracker Red CMXRos; OCR: Oxygen consumption rate; OE: overexpress; PINK1: PTEN induced kinase 1; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SD: standard deviation; SOD2: superoxide dismutase 2; UQCRC2: ubiquinol-cytochrome c reductase core protein 2; WB: western blot; ΔΨ: mitochondrial membrane potential;