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

Aims: 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 autophagyrelated diagnostic biomarkers.& nbsp;Main methods: The insoluble nuclear protein fraction was extracted from the livers of Atg7(F/F), Atg7(F/F):alb-Cre (hepatocyte-specific autophagy-deficient mice), C57BL/6 J, and KKA(y) (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.& nbsp;& nbsp;Key findings: The levels of insoluble nuclear proteins 14-3-3 zeta and importin alpha 4 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 14-3-3 zeta expression in the nuclear insoluble fraction; in contrast, the addition of rapamycin downregulated 14-3-3 zeta expression. Importin alpha 4 expression was increased in the nuclear insoluble fraction after incubation with tunicamycin or hydrogen peroxide.& nbsp;Significance: Accumulation of 14-3-3 zeta and importin alpha 4 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.