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

Eid, Nabil; Ito, Yuko; Maemura, Kentaro; Otsuki, Yoshinori

doi:10.1007/s10735-013-9483-x

Cited by 98 publications

(109 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Finally, autophagy has recently been linked to the delivery of free fatty acids to mitochondria and peroxisomes from triglyceride-rich vesicles in epithelial and mesenchymal cells, a process known as lipophagy. 21 It will be important to understand whether lipophagy is similarly disrupted in this model.…”

Section: Discussionmentioning

confidence: 99%

Deficient Autophagy Results in Mitochondrial Dysfunction and FSGS

Kondo

Gomez

Ren

et al. 2015

Journal of the American Society of Nephrology

144

124

View full text Add to dashboard Cite

FSGS is a heterogeneous fibrosing disease of the kidney, the cause of which remains poorly understood. In most cases, there is no effective treatment to halt or retard progression to renal failure. Increasing evidence points to mitochondrial dysfunction and the generation of reactive oxygen species in the pathogenesis of CKD. Autophagy, a major intracellular lysosomal degradation system, performs homeostatic functions linked to metabolism and organelle turnover. We prevented normal autophagic pathways in nephrons of mice by mutating critical autophagy genes ATG5 or ATG7 during nephrogenesis. Mutant mice developed mild podocyte and tubular dysfunction within 2 months, profound glomerular and tubular changes bearing close similarity to human disease by 4 months, and organ failure by 6 months. Ultrastructurally, podocytes and tubular cells showed vacuolization, abnormal mitochondria, and evidence of endoplasmic reticulum stress, features that precede the appearance of histologic or clinical disease. Similar changes were observed in human idiopathic FSGS kidney biopsy specimens. Biochemical analysis of podocytes and tubules of 2-month-old mutant mice revealed elevated production of reactive oxygen species, activation of endoplasmic reticulum stress pathways, phosphorylation of p38, and mitochondrial dysfunction. Furthermore, cultured proximal tubule cells isolated from mutant mice showed marked mitochondrial dysfunction and elevated mitochondrial reactive oxygen species generation that was suppressed by a mitochondrial superoxide scavenger. We conclude that mitochondrial dysfunction and endoplasmic reticulum stress due to impaired autophagic organelle turnover in podocytes and tubular epithelium are sufficient to cause many of the manifestations of FSGS in mice.

show abstract

Section: Discussionmentioning

confidence: 99%

Deficient Autophagy Results in Mitochondrial Dysfunction and FSGS

Kondo

Gomez

Ren

et al. 2015

Journal of the American Society of Nephrology

144

124

View full text Add to dashboard Cite

show abstract

“…Cells were fixed by 4 % paraformaldehyde for 15 min for immunofluorescence study and LD staining by LipidTOX TM . The methods for immunofluorescence labeling of LC3, LAMP-2, CHOP, and mTOR were performed as previously reported and according to the manufacturers' protocols [8,31].…”

Section: Reagents Kits and Antibodiesmentioning

confidence: 99%

“…The effects of ER stress blocking on LC3 dot formation (marker of autophagy) and LD accumulation were observed under an immunofluorescence microscope and quantified [8,33].…”

Section: Blocking Autophagy With 3-ma and Cell Viability Assaymentioning

confidence: 99%

“…This pathway is regulated by several autophagy proteins and is morphologically characterized by the formation of isolation membranes, which sequester a region of the cell cytoplasm or selectively an organelle forming autophagosomes mediated by microtubule-associated protein light chain 3 (LC3). The autophagosomes then fuse with lysosomes via lysosomal-associated membrane protein 2 (LAMP-2), forming autolysosomes, where the contents of the cargo are digested by lysosomal cathepsins [1][2][3][4][5][6][7][8]. Excessive accumulation of LDs in lean organs such as the pancreas, heart, liver, kidney, and small intestine may induce a lipotoxic response, characterized by destruction of organelle membranes, activation of stress pathways as ER stress, and apoptosis (lipoapoptosis) [9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Indomethacin suppresses LAMP-2 expression and induces lipophagy and lipoapoptosis in rat enterocytes via the ER stress pathway

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Although the higher autophagosome content in EtOH‐fed rats may have occurred due to either increased autophagic flux or a block in lysosomal degradation,35, 36 the finding that autophagosomes were physically associated with LDs of EtOH‐fed rats suggests that autophagosomes were trafficked/directed to LDs and they attempted or perhaps sequestered the LDs for degradation. Using electron microscope imaging of liver tissue, others have shown that LDs were the predominant cargo of autophagosomes in livers of EtOH‐fed rats,37 suggesting that at least a subset of autophagosomes in EtOH‐fed rats sequestered LDs for subsequent degradation in lysosomes.…”

Section: Discussionmentioning

confidence: 99%

Ethanol‐induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity

Rasineni

Donohue

Thomes

et al. 2017

Hepatology Communications

View full text Add to dashboard Cite

Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down‐regulating dynamin 2 (Dyn2), a protein that facilitates lysosome re‐formation, contributing to hepatocellular steatosis. Primary hepatocytes were isolated from male Wistar rats fed Lieber–DeCarli control or ethanol (EtOH) liquid diets for 6‐8 weeks. Hepatocytes were incubated in complete medium (fed) or nutrient‐free medium (fasting) with or without the Dyn2 inhibitor dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2 and markers of autophagy were quantified using western blot analysis. Colocalization of LDs with autophagic machinery was determined using confocal microscopy. In hepatocytes from pair‐fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower triglyceride (TG) content when compared with hepatocytes in complete media. Fasting‐induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared with controls, hepatocytes from EtOH‐fed rats had 66% and 40% lower content of phosphorylated Src (pSrc) and phosphorylated Dyn2 (pDyn2), respectively, coupled with a lower rate of fasting‐induced TG loss. This slower rate of fasting‐induced TG loss was blocked in cells coincubated with Dynasore. Microscopic examination of EtOH‐fed rat hepatocytes revealed increased colocalization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH‐fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy. Conclusion: Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown. (Hepatology Communications 2017;1:501–512)

show abstract

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

Cited by 98 publications

References 65 publications

Deficient Autophagy Results in Mitochondrial Dysfunction and FSGS

Deficient Autophagy Results in Mitochondrial Dysfunction and FSGS

Indomethacin suppresses LAMP-2 expression and induces lipophagy and lipoapoptosis in rat enterocytes via the ER stress pathway

Ethanol‐induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity

Contact Info

Product

Resources

About