Elevated autophagic sequestration of mitochondria and lipid droplets in steatotic hepatocytes of chronic ethanol-treated rats: an immunohistochemical and electron microscopic study

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.

[1]  M. Kojima,et al.  Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome , 2008, Cancer science.

[2]  Y. Ohsaki,et al.  Cytoplasmic Lipid Droplets , 2006, Annals of the New York Academy of Sciences.

[3]  D. Stolz,et al.  Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. , 2010, Gastroenterology.

[4]  A. Cuervo,et al.  Autophagy and lipids: tightening the knot , 2010, Seminars in Immunopathology.

[5]  R. Youle,et al.  Mechanisms of mitophagy , 2010, Nature Reviews Molecular Cell Biology.

[6]  M. Czaja.  Autophagy in health and disease. 2. Regulation of lipid metabolism and storage by autophagy: pathophysiological implications. , 2010, American journal of physiology. Cell physiology.

[7]  R. Youle,et al.  Targeting mitochondrial dysfunction: role for PINK1 and Parkin in mitochondrial quality control. , 2011, Antioxidants & redox signaling.

[8]  J. Teckman,et al.  Mitochondrial autophagy and injury in the liver in α1-antitrypsin deficiency , 2004 .

[9]  M. Czaja.  Functions of autophagy in hepatic and pancreatic physiology and disease. , 2011, Gastroenterology.

[10]  Robert V Farese,et al.  The life of lipid droplets. , 2009, Biochimica et biophysica acta.

[11]  N. Mizushima,et al.  Methods in Mammalian Autophagy Research , 2010, Cell.

[12]  S. Barth,et al.  Autophagy: assays and artifacts , 2010, The Journal of pathology.

[13]  R. Youle,et al.  PINK1- and Parkin-mediated mitophagy at a glance , 2012, Journal of Cell Science.

[14]  Yucui Zhu,et al.  Resveratrol‐Mediated Downregulation of Rictor Attenuates Autophagic Process and Suppresses UV‐Induced Skin Carcinogenesis † , 2012, Photochemistry and photobiology.

[15]  Ted M. Dawson,et al.  PINK1-dependent recruitment of Parkin to mitochondria in mitophagy , 2009, Proceedings of the National Academy of Sciences.

[16]  H. Fox,et al.  Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment. , 2012, Biochemical and biophysical research communications.

[17]  M. Czaja,et al.  Lipases in lysosomes, what for? , 2009, Autophagy.

[18]  O. Koch,et al.  Ultrastructural and biochemical aspects of liver mitochondria during recovery from ethanol-induced alterations. Experimental evidence of mitochondrial division. , 1978, The American journal of pathology.

[19]  Ming Yan,et al.  Ethanol induced mitochondria injury and permeability transition pore opening: role of mitochondria in alcoholic liver disease. , 2007, World journal of gastroenterology.

[20]  A. Dolganiuc,et al.  Autophagy in alcohol-induced liver diseases. , 2012, Alcoholism, clinical and experimental research.

[21]  N. Osna,et al.  Involvement of autophagy in alcoholic liver injury and hepatitis C pathogenesis. , 2011, World journal of gastroenterology.

[22]  N. Mizushima,et al.  Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy , 2012, Journal of Cell Science.

[23]  E. Gordon.  Mitochondrial functions in an ethanol-induced fatty liver. , 1973, The Journal of biological chemistry.

[24]  R. Wilkinson,et al.  The Development of an Immunohistochemical Method to Detect the Autophagy-Associated Protein LC3-II in Human Tumor Xenografts , 2011, Toxicologic pathology.

[25]  R. Gottlieb.  Autophagy in Health and Disease , 2004, Science.

[26]  K. von Figura,et al.  Role of LAMP-2 in lysosome biogenesis and autophagy. , 2002, Molecular biology of the cell.

[27]  Syed F. Ali,et al.  Methamphetamine Induces Autophagy and Apoptosis in a Mesencephalic Dopaminergic Neuronal Culture Model , 2006, Annals of the New York Academy of Sciences.

[28]  Xiao-Ming Yin,et al.  Mitophagy: mechanisms, pathophysiological roles, and analysis , 2012, Biological chemistry.

[29]  P. Navas,et al.  Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease , 2010, Arthritis research & therapy.

[30]  E. Eskelinen.  To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells , 2008, Autophagy.

[31]  C. Lieber,et al.  Experimental methods of ethanol administration , 1989, Hepatology.

[32]  J. Teckman,et al.  Mitochondrial Autophagy and Injury in the Liver in Alpha-1-antitrypsin Deficiency , 2022 .

[33]  P. Kim,et al.  ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy , 2012, Autophagy.

[34]  A. Cuervo,et al.  Lipophagy: Connecting Autophagy and Lipid Metabolism , 2012, International journal of cell biology.

[35]  T. Pan,et al.  Rapamycin protects against rotenone-induced apoptosis through autophagy induction , 2009, Neuroscience.

[36]  John L Cleveland,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes , 2008, Autophagy.

[37]  I. Novak,et al.  Mitophagy: a complex mechanism of mitochondrial removal. , 2012, Antioxidants & redox signaling.

[38]  S. Navarro,et al.  PINK1 displays tissue-specific subcellular location and regulates apoptosis and cell growth in breast cancer cells. , 2011, Human pathology.

[39]  C. Chu,et al.  Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress , 2008, Autophagy.

[40]  G. D. De Meyer,et al.  In Situ Detection of Starvation-induced Autophagy , 2006, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[41]  L. Yerian,et al.  Histopathological evaluation of fatty and alcoholic liver diseases , 2011, Journal of digestive diseases.

[42]  M. Büchler,et al.  Impaired autolysosome formation correlates with Lamp-2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis. , 2009, Gastroenterology.

[43]  M. Sarwal,et al.  Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis. , 2010, Journal of the American Society of Nephrology : JASN.

[44]  K. Otsu,et al.  Discovery of Atg5/Atg7-independent alternative macroautophagy , 2009, Nature.

[45]  R. Dunstan,et al.  An Immunohistochemical Approach to Differentiate Hepatic Lipidosis from Hepatic Phospholipidosis in Rats , 2007, Toxicologic pathology.

[46]  R. Gottlieb,et al.  Autophagy in Atherosclerosis: A Cell Survival and Death Phenomenon With Therapeutic Potential , 2008 .

[47]  J. Puyal,et al.  Postischemic treatment of neonatal cerebral ischemia should target autophagy , 2009, Annals of neurology.

[48]  E. Schon,et al.  The kinase domain of mitochondrial PINK1 faces the cytoplasm , 2008, Proceedings of the National Academy of Sciences.

[49]  Xiao-Ming Yin,et al.  Selective taste of ethanol-induced autophagy for mitochondria and lipid droplets , 2011, Autophagy.

[50]  N. Jhala,et al.  Mitochondria‐targeted ubiquinone (MitoQ) decreases ethanol‐dependent micro and macro hepatosteatosis , 2011, Hepatology.

[51]  Robert Clarke,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy , 2012 .

[52]  A. Feldstein,et al.  Emerging role of redox dysregulation in alcoholic and nonalcoholic fatty liver disease. , 2011, Antioxidants & redox signaling.

[53]  I. Nonaka,et al.  Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy. , 2011, Human molecular genetics.

[54]  S. Ao,et al.  Ultrastructural and biochemical aspects of liver mitochondria during recovery from ethanol-induced alterations. Experimental evidence of mitochondrial division. , 1978 .

[55]  A. Ochiai,et al.  Enhancer of zeste homolog 2 downregulates E‐cadherin by mediating histone H3 methylation in gastric cancer cells , 2008, Cancer science.

[56]  R. Bataller,et al.  Alcoholic liver disease: pathogenesis and new therapeutic targets. , 2011, Gastroenterology.

[57]  Yuko Ito,et al.  Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance , 2011, Journal of Molecular Histology.

[58]  Guido Kroemer,et al.  Autophagy and the integrated stress response. , 2010, Molecular cell.

[59]  R. Nixon,et al.  Monitoring autophagy in Alzheimer's disease and related neurodegenerative diseases. , 2009, Methods in enzymology.

[60]  M. Czaja,et al.  Autophagy regulates lipid metabolism , 2009, Nature.

[61]  R. Hotchkiss,et al.  Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study , 2009, Laboratory Investigation.

[62]  W. Kouidhi,et al.  Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats , 2010, Journal of Molecular Histology.

[63]  C. Chu.  A pivotal role for PINK1 and autophagy in mitochondrial quality control: implications for Parkinson disease. , 2010, Human molecular genetics.

[64]  M. Czaja,et al.  Regulation of lipid droplets by autophagy , 2011, Trends in Endocrinology & Metabolism.

[65]  C. Chu,et al.  Mitochondrially localized ERK 2 regulates mitophagy and autophagic cell stress : Implications for Parkinson ’ s disease , 2008 .

[66]  L. Gottlieb,et al.  The ultrastructure of fatty liver induced by prolonged ethanol ingestion. , 1966, The American journal of pathology.

[67]  M. Matsui,et al.  In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. , 2003, Molecular biology of the cell.

[68]  X. Zhang,et al.  Gastrointestinal , Hepatobiliary , and Pancreatic Pathology Chronic Alcohol Exposure Stimulates Adipose Tissue Lipolysis in Mice Role of Reverse Triglyceride Transport in the Pathogenesis of Alcoholic Steatosis , 2012 .

[69]  J. S. Ahn,et al.  Cathepsin D inhibits oxidative stress-induced cell death via activation of autophagy in cancer cells. , 2012, Cancer letters.

[70]  Yuko Ito,et al.  Involvement of Fas system and active caspases in apoptotic signalling in testicular germ cells of ethanol-treated rats. , 2002, International journal of andrology.

[71]  W. Ding,et al.  The emerging role of autophagy in alcoholic liver disease , 2011, Experimental biology and medicine.

[72]  S. Rodríguez-Enríquez,et al.  Selective degradation of mitochondria by mitophagy. , 2007, Archives of biochemistry and biophysics.