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 autophagy-related diagnostic biomarkers. MAIN METHODS: 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis.

Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD.

Suppression of autophagy sensitizes Kupffer cells to endotoxin.

AIM: Recent evidence suggests that protein degradation system autophagy is implicated in a component of innate immunity. We report here that suppression of autophagy in Kupffer cells due to hepatic steatosis enhances an inflammatory response to endotoxin. METHODS: Kupffer cells were isolated from C57BL/6J mice fed chow diet (control) or high-fat diet (HFD) for 12 weeks, liver-specific autophagy-deficient mice (Atg7(F/F) :Mx1-Cre) and wild-type mice (Atg7(F/F) ). Kupffer cells were incubated with 100 ng/mL lipopolysaccharide (LPS). The concentration of tumor necrosis factor (TNF)-α in media was measured by enzyme-linked immunoassay. Expression of Toll-like receptor (TLR)4, IκB kinase (IKK)-α/ß, p38, p62 and LC3 in Kupffer cells was evaluated by western blot analysis. RESULTS: Incubation with LPS increased LC3-II expression of Kupffer cells from control mice; however, an increase in LC3-II expression due to LPS was suppressed in Kupffer cells from HFD mice. Moreover, both p62 expression and TNF-α production in Kupffer cells from HFD mice was higher than control mice. On the other hand, LPS exposure increased TNF-α production from autophagy-deficient Kupffer cells more than wild type. There was no significant difference in expression of TLR4 between wild and autophagy-deficient Kupffer cells. Nevertheless, activation of p38 or IKK in Kupffer cells due to LPS was augmented by autophagy deficiency. The addition of the p38 inhibitor SB203580 attenuated TNF-α production in both wild and autophagy-deficient Kupffer cells. CONCLUSION: These results suggest that suppression of autophagy observed in Kupffer cells from steatotic liver sensitizes to endotoxin. In conclusion, suppression of autophagy may play a pivotal role on progression of NAFLD.

Loss of autophagy promotes murine acetaminophen hepatotoxicity.

BACKGROUND: Previous reports indicate that mitochondrial dysfunction is essential for the development of liver injury due to acetaminophen. On the other hand, autophagy, which is a major catabolic pathway, plays a critical role in removing protein aggregates, as well as damaged or excess mitochondria in order to maintain intracellular homeostasis. The aim of this study was to clarify if autophagy is linked to liver injury due to acetaminophen. METHODS: Acetaminophen was administered intraperitoneally to liver-specific Atg7-deficient mice and wild-type mice. A variety of cellular and molecular approaches were used to evaluate the role of autophagy in acetaminophen-induced cell death. RESULTS: Treatment with acetaminophen induced formation of autophagosomes in hepatocytes from wild-type mice, but not in Atg7-deficient mice. Autophagy deficiency enhanced acetaminophen-induced liver injury and activation of caspase-3 and -7 in the liver. Acetaminophen-induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, and JNK activation in hepatocytes were accelerated by autophagy deficiency. The combination of cyclosporin A or JNK inhibitor (SP600125) with acetaminophen blunted both acetaminophen-induced apoptotic and necrotic cell death in autophagy-deficient hepatocytes. CONCLUSIONS: Induction of autophagy during acetaminophen treatment plays a pivotal role in the protection against acetaminophen-induced hepatotoxicity through the removal of damaged mitochondria and oxidative stress.

Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.