Melatonin Inhibits mTOR-Dependent Autophagy during Liver Ischemia/Reperfusion
Cellular Physiology and Biochemistry
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. ; a These authors contributed equally to this work. Abstract Background: Autophagy is a self-digestion system responsible for maintaining cellular homeostasis and interacts with reactive oxygen species produced during
... produced during ischemia/reperfusion liver I/R injury. In this study, we examined the cytoprotective mechanisms of MLT in liver I/R, Methods: Male C57BL/6 mice were subjected to 70% liver ischemia for 60 min followed by reperfusion. MLT (10 mg/kg, i.p.) was injected 15 min prior to ischemia and again immediately before reperfusion. Rapamycin (Rapa, 1 mg/kg, i.p.), which induces autophagy, was injected 1.5 h before ischemia. Results: of sequestosome1/p62. This increase was attenuated by MLT. Likewise, electron microscopic analysis showed that autophagic vacuoles were increased in livers of mice exposed to I/R, which was attenuated by MLT. I/R decreased phosphorylation of mammalian target of rapamycin (mTOR) and 4E-BP1 and 70S6K, downstream molecules of the mTOR pathway, but increased expression of calpain 1 and calpain 2. MLT attenuated the decrease in mTOR, 4E-BP1 and 70S6K phosphorylation. Pretreatment of Rapa reversed the effect of MLT on autophagic Conclusion: autophagy via activation of mTOR signaling, which may in turn contribute to its protective effects in liver I/R injury. Introduction Liver ischemia/reperfusion (I/R) injury is a complex and multifactorial pathophysiological process that is a commonly encountered in a variety of clinical events such as liver transplantation, tumor resection, and traumatic shock. Due to their high metabolic rate, liver cells are vulnerable to the deleterious effects of ischemia including ATP depletion and anoxia. A plethora of studies have shown that excessive production of reactive oxygen species (ROS) followed by reperfusion is the earliest and most important factor for cellular damage in liver I/R [1, 2], because ROS directly attack cellular molecules as well as indirectly recruit and activate pro-in lammatory mediators. ltimately, these detrimental events during I/R lead to both necrotic and apoptotic cell death  . Autophagy is a self-digestion process that is important for maintaining basal homeostasis against intrinsic and extrinsic stress. Autophagic lux is the dynamic process of autophagy in which cytosolic organelles and proteins are sequestered by double membrane structures termed autophagosomes, which are then delivered to lysosomes for the formation of autolysosomes that are subsequently degraded by proteases  . Autophagy functions as an adaptive response by which amino acids and fatty acids are recycled for ATP generation and cellular components leading to malfunction are removed. However, excessive activation of autophagy can be toxic and may cause cell death, which is called type II programmed cell death  . Oxidative stress was recently shown to activate starvation-induced autophagy, and conversely, autophagy can also play an important role in suppressing ROS production [6, 7] . This conundrum is further complicated by cross-talk and coordinated regulation between autophagy and other types of cell death under oxidative environments. Although the role of autophagy in I/R injury of various organs has recently been investigated, the exact function of autophagy appears to be model and organ dependent. In the kidneys and heart, autophagy is activated by I/R, with the function of increased autophagy having been reported as both protective and deleterious [8, 9] . In the liver, warm reperfusion injury is involved in the degeneration of hepatocytes, a process that is stimulated by autophagy  . Moreover, induction of autophagy is evident in normothermic liver I/R, observed in the form of simultaneous apoptosis and necrosis  . In contrast, Kim et al.  reported that impaired autophagy occurs in calpain 2-dependent mechanisms that lead to mitochondrial dysfunction in anoxic rat hepatocytes. Most recently, autophagy was shown to suppress ischemic liver damage by reducing ROS-induced necrosis  . Melatonin (MLT, N-acetyl-5-methoxytryptamine) is a lipophilic indole secreted by pineal and non-pineal cells that has potent anti-oxidant, anti-in lammatory, and anti-apoptotic properties  . We previously showed that MLT inhibits necrotic and apoptotic cell death in a rat model of liver I/R by alleviating levels of oxidative stress  . Recently, a protective role of MLT has been reported in cold liver I/R through suppression of endoplasmic reticulum (ER) stress and enhanced autophagy  . Although the effects of MLT on autophagy have been actively studied, the precise mechanism by which MLT regulates autophagy in liver I/R remains unclear. This study was designed to investigate the protective mechanism of MLT in liver I/R, focusing particularly on autophagic lux and associated signaling pathways. Materials and Methods Chemicals and antibodies MLT (M5250) and chloroquine (CQ) (C6628) were purchased from Sigma-Aldrich (St. Louis, MO, SA). Rapamycin (Rapa, 53123-88-9) was purchased from Calbiochem ( illerica, MA, SA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, SA) eclin-1 (3738), Atg3 (3415), Atg7 (2631), p-mammalian target of rapamycin (mTOR) (Ser2448) (2971), p-4E-P1 (Ser65) (9451) and p-70S6K (Thr389) (9205). The following antibodies were purchased from Abcam (Cambridge, K) sequestosome 1 (SQSTM1)/p62 (ab56416), calpain 1 (ab49652) and calpain 2 (ab39168). Microtubule-25 Kang et al.: Melatonin Inhibits Autophagy in Ischemic Liver Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry associated protein 1 light chain 3 (LC3)-II antibody (N 100-2220) was purchased from Novus iologicals (Littleton, CO, SA) and the -actin antibody (A5441) was purchased from Sigma-Aldrich. Animals All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Institutes of Health (NIH publication No.86-23, revised 1985) and the guidelines of the Sungkyunkwan niversity Animal Care Committee. Male C57 L/6 mice weighing 22 to 24 g were obtained from Orient io Inc. (Seongnam, Korea) and were acclimati ed to the laboratory conditions at Sungkyunkwan niversity for at least one week prior to initiating experiments. Mice were maintained in a room with controlled temperature and humidity (25 ± 1°C and 55 ± 5%, respectively) and a 12-h light-dark cycle. The mice were fasted for 18 h prior to experiments and were provided with tap water ad libitum. 27 Kang et al.: Melatonin Inhibits Autophagy in Ischemic Liver Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry Statistical analysis All results are presented as the mean ± S.E.M. The overall signi icance of the data was tested by two-way analysis of variance using the SPSS v.12.0 statistical software package (SPSS, Chicago, IL, SA). Differences between groups were considered statistically signi icant at p < 0.05 with appropriate onferroni corrections made for multiple comparisons. Results Time course of autophagy changes and hepatocellular damage during liver I/R Serum ALT activity in sham-operated animals remained at basal levels throughout the experimental period. Serum ALT activity was not different from those of sham-operated animals at the end of the ischemic period or immediately after reperfusion (0 h of reperfusion). Fig. 2. Time course of hepatic injury and autophagy changes during liver I/R. Serum ALT activity was measured after ischemia for 60-min and reperfusion for 0, 1, 5 or 24 h (A). Western blot analysis was performed to measure proteins levels of beclin-1 ( ), LC3-II/I (C) and SQSTM1/p62 (D). Results are presented as the mean ± S.E.M. of 6 to 8 mice per group. For comparison of proteins in different gels, an internal control was used. ** Signi icantly different (p < 0.01) from sham-operated animals. However, after reperfusion for 1 h, serum ALT activity increased to 2243.9 ± 272.5 /L and reached a peak of 6043.1 ± 401.0 /L after reperfusion for 5 h. Serum ALT then gradually declined to 2251.7 ± 221.1 /L after reperfusion for 24 h ( Fig. 2A) . At the end of the ischemic period, the protein levels of beclin-1 were similar to those of sham-operated animals. However, the levels of beclin-1 protein expression increased Fig. 3 . Effect of MLT on liver injury induced by I/R. MLT was administered 15 min prior to ischemia and directly before reperfusion. After reperfusion for 5 h, serum ALT and AST activities were measured (A). Representative H E staining (x200) with Su uki score ( ) and T NEL staining images and caspase-3 activity (C) are shown. Arrow heads indicate necrotic area. Apoptosis in liver sections was quanti ied by counting the number of T NEL-positive cells in random microscopic high-power elds (x100). Caspase-3 activity was measured in the cytosolic fraction. Levels of lipid peroxidation and the SH/ SS ratio were measured in liver tissue (D). Results are presented as the mean ± S.E.M. of 6 to 8 mice per group. * , ** Signi icantly different (p < 0.05, p < 0.01) from sham-operated animals. + , ++ Signi icantly different (p < 0.05, p < 0.01) from I/R animals, respectively. 30 Kang et al.: Melatonin Inhibits Autophagy in Ischemic Liver Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry we analy ed the effect of MLT using a model of ischemia for 60 min followed by reperfusion for 5 h. Serum ALT and AST activities in sham-operated animals were 33.0 ± 3.1 /L and 74.5 ± 8.0 /L, respectively. MLT alone did not affect serum ALT and AST activities. After reperfusion for 5 h, serum ALT and AST activities signi icantly increased to 6504.1 ± 628.2 /L and 9706.3 ± 1000.1 /L, which were attenuated by MLT (3207.2 ± 450.8 /L and 5395.2 ± 820.2 /L, respectively) (Fig. 3A) . H E stained liver sections were evaluated for the degree of hepatocellular damage using the Su uki s criteria. The ischemic lobes in I/R animals showed severe necrosis, sinusoidal congestion and hepatocyte vacuoli ation (Su uki score 8.2 0.9). However, MLT treatment attenuated the Su uki score (5.3 0.4) (Fig. 3 ) . We also analy ed apoptosis by T NEL assay, analysis and representative images of which are shown in Fig. 3C . While no T NEL-positive cells were detected in sham-operated animals, the level of apoptosis induced by I/R was 62.0 ± 9.1%, which was decreased to 21.4 ± 3.7% after treatment with MLT. After 5 h of reperfusion, caspase-3 activity in the cytosolic fraction signi icantly increased (235.5 11.8 % of sham) and MLT attenuated this increase (157.9 14.6 % of sham). In the sham group, the level of MDA was 0.45 ± 0.06 nmol/mg protein. Following I/R, the level of MDA was signi icantly increased to 1.08 ± 0.13 nmol/mg protein. MLT attenuated the increase in MDA levels (0.69 ± 0.05 nmol/mg protein). The SH/ SS ratio in the sham animals were 23.4 ± 1.9. After reperfusion, the SH/ SS ratio decreased signi icantly to 6.5 ± 0.7, which was attenuated by MLT (15.7 ± 0.2) (Fig. 3D) . Effects of MLT on autophagic lux during liver I/R As shown in Fig. 4A , I/R caused a signi icant increase in the LC3-II/LC3-I ratio compared to that of sham-operated animals (549.5% of sham). MLT augmented the I/R-induced Fig. 5. Effect of MLT on calpains and the mTOR pathway during liver I/R. MLT was administered 15 min prior to ischemia and again directly before reperfusion. 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