@article{c9dae5973eb148eb9287f6d407808903,
title = "Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion",
abstract = "We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.",
keywords = "Autophagy, Desipramine, Ischemia and reperfusion injury, Liver",
author = "Shin, {Jun Kyu} and Kim, {Jae Sung}",
note = "Funding Information: The manuscript was edited by Dr. Paul S. Cassidy, the Scientific Editor of the Institute of Clinical and Translational Sciences at Washington University, which is supported by an NIH Clinical and Translational Science Award ( UL1 TR002345 ). Funding Information: A key finding in the present study is the modulatory effect of DMI on ATG4B expression levels. ATG4B is a cysteine protease with a catalytic triad of Cys74, Asp278, and His280 residues [14]. LC3 is processed by three distinct steps, (1) proteolytic cleavage of pro-LC3 into LC3-I by ATG4B, (2) generation of LC3-II through the lipidation of LC3-I with phosphatidylethanolamine (PE) by ATG3 and ATG7, and (3) removal of the PE moiety from LC3-II and recycling of LC3-I by ATG4B [14?16]. We found that DMI treatment caused a significant increase in both ATG4B and ATG7 levels. Moreover, immunoblot analysis of LC3-I/II revealed unique changes in LC3-I levels induced by DMI during I/R. LC3-I levels remained unaltered during 4 h of ischemia in both groups, but there were noticeable reductions in LC3-I after reperfusion. While LC3-1 levels in control cells were considerably reduced after 120 min of reperfusion, DMI-treated cells showed a significant loss of LC3-I from 20 min after commencement of reperfusion. After 120 min, LC3-I expression was barely detectable in DMI-treated cells. When CQ was added to block the degradation of cargo molecules in autophagosomes, reductions in LC3-I expression were also observed in DMI-treated cells at 20 and 60 min after commencement of reperfusion. Intriguingly, CQ treatment significantly increased LC3-I levels at 120 min. The reduction in LC3-I levels could occur (1) if PE mediated lipidation of LC3-I to LC3-II outpaces de novo synthesis of LC3, or (2) if LC3-I is recycled back to LC3-II by ATG4B [17]. The former scenario is less likely as the changes in LC3-II levels from 60 min to 120 min of reperfusion were minimal in both groups. In addition, ischemic hepatocytes lose 97% of ATP during 60 min of reperfusion [18]. Hence, a highly ATP-consuming de novo synthesis of LC3 is energetically unfavorable in reperfused cells. The idea that DMI treatment induces enhanced recycling of LC3-I was also supported by our observation that the total LC3 levels during 60 min of reperfusion, as estimated by the sum of LC3-I and LC3-II on immunoblots, remained similar before and after the addition of CQ.This work was supported by the National Institutes of Health (R01 DK079879); Mid-America Transplant Foundation (07201903); and Foundation for Barnes-Jewish Hospital (4776, 5153). The manuscript was edited by Dr. Paul S. Cassidy, the Scientific Editor of the Institute of Clinical and Translational Sciences at Washington University, which is supported by an NIH Clinical and Translational Science Award (UL1 TR002345). Funding Information: This work was supported by the National Institutes of Health ( R01 DK079879 ); Mid-America Transplant Foundation ( 07201903 ); and Foundation for Barnes-Jewish Hospital ( 4776 , 5153 ). Publisher Copyright: {\textcopyright} 2021 The Authors",
year = "2021",
month = sep,
doi = "10.1016/j.bbrep.2021.101075",
language = "English",
volume = "27",
journal = "Biochemistry and Biophysics Reports",
issn = "2405-5808",
}