Autophagic dysfunction in the liver enhances the expression of insoluble nuclear proteins 14-3-3ζ and importin α4

Autophagic dysfunction is associated with the progression of various liver diseases, including nonalcoholic fatty liver disease (NAFLD). However, serum markers for evaluating autophagic function have not been reported. Highly insoluble nuclear proteins participate in many cellular functions and are potential diagnostic markers for cancer. We performed a proteomic analysis of the hepatic nuclear insoluble fraction to identify novel autophagy-related diagnostic biomarkers.The insoluble nuclear protein fraction was extracted from the livers of Atg7F/F, Atg7F/F:alb-Cre (hepatocyte-specific autophagy-deficient mice), C57BL/6 J, and KKAy (NAFLD model) mice. Proteins were separated by two-dimensional electrophoresis and visualized by silver staining. Protein spots were identified using mass spectrometry. The localization of proteins in hepatocytes was verified by immunofluorescence using a confocal microscope.The levels of insoluble nuclear proteins 14-3-3ζ and importin α4 were upregulated following hepatic autophagy dysfunction and were detectable in serum. Under normal conditions, these proteins are mainly distributed in the cytoplasm, whereas autophagic dysfunction induces their translocation to the nucleus. Incubation with an autophagy inhibitor up-regulated these proteins expression in the insoluble nuclear fraction of primary hepatocytes. Treatment with EGF or insulin enhanced 14-3-3ζ expression in the nuclear insoluble fraction; in contrast, the addition of rapamycin downregulated 14-3-3ζ expression. Importin α4 expression was increased in the nuclear insoluble fraction after incubation with tunicamycin or hydrogen peroxide.Accumulation of 14-3-3ζ and importin α4 as nuclear-insoluble proteins may be associated with autophagic dysfunction. Our findings indicate that these proteins might be useful diagnostic biomarkers for liver diseases with autophagic disorders.