Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53 - PubMed (original) (raw)
Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53
Young Mi Song et al. Int J Mol Sci. 2016.
Abstract
Metformin is known to alleviate hepatosteatosis by inducing 5' adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.
Keywords: Parkin; metformin; mitochondrial spheroid; mitophagy; p53.
Figures
Figure 1
Metformin induced mitophagy. Electron microscopy was performed on hepatocytes from ob/ob mice under ad libitum feeding of chow diet (A-a), caloric restriction (A-b), and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-c). In the ad libitum group, spheroid mitochondria were readily detected. However, in the metformin-treated group, autophagic double membranes were detected, indicating mitophagy; (B) co-localization between a marker of autophagosomes (LC3) and a mitochondrial marker (MitoTracker Red) was assessed in HepG2 cells stably transfected with green fluorescent protein (GFP)-LC3. Although there was little co-localization between mitochondria and autophagosomes in the 0.25 mM palmitate-treated HepG2 cells, LC3-labeled structures were seen surrounding the fragmented mitochondria in cells treated with 0.5 mM metformin and 0.25 mM palmitate. Scale bar = 20 μm.
Figure 2
Metformin restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced cytosolic p53. Western blotting analysis was performed to assess cytosolic p53 levels with the cytosolic fractions isolated from palmitate- (A-a) or metformin- (A-b) and 35 mM glucose-treated HepG2 cells; the expression of cytosolic p53 protein was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (B-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (B-b); (C) Expression of Parkin (green) on mitochondria (MitoTracker Red) was observed with a confocal microscopy. The bottom panels show enlarged views of the boxed areas. The yellow dots indicate the mitochondria that co-localize with Parkin. The cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria depending on the conditions used in the HepG2 cells; a co-immunoprecipitation assay was conducted to investigate the interaction between p53 and Parkin, depending on the conditions used in the HepG2 cells (D-a) or in hepatocytes from mice (D-b). GAPDH or COX IV was used for normalization. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). Scale bar: white = 20 μm, yellow = 10 μm. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1.
Figure 2
Metformin restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced cytosolic p53. Western blotting analysis was performed to assess cytosolic p53 levels with the cytosolic fractions isolated from palmitate- (A-a) or metformin- (A-b) and 35 mM glucose-treated HepG2 cells; the expression of cytosolic p53 protein was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (B-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (B-b); (C) Expression of Parkin (green) on mitochondria (MitoTracker Red) was observed with a confocal microscopy. The bottom panels show enlarged views of the boxed areas. The yellow dots indicate the mitochondria that co-localize with Parkin. The cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria depending on the conditions used in the HepG2 cells; a co-immunoprecipitation assay was conducted to investigate the interaction between p53 and Parkin, depending on the conditions used in the HepG2 cells (D-a) or in hepatocytes from mice (D-b). GAPDH or COX IV was used for normalization. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). Scale bar: white = 20 μm, yellow = 10 μm. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1.
Figure 3
Metformin restored Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced ER stress. The expression of ER stress markers was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (A-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, or ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-b) using Western blotting analysis. Values displayed are mean ± SEM (n = 3 independent experiments, respectively); (B) the expression of ER stress markers and p53 protein was assessed in response to thapsigargin in HepG2 cells; (C-a) cell viability was assessed in HepG2 cells exposed to 0.1 μM thapsigargin and/or 0.5 mM metformin using CCK-8. Values displayed are mean ± SEM (n = 5 independent experiments); (C-b) the expression of an ER stress marker was also examined under these conditions; (C-c) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). GAPDH or COX IV was used for normalization. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1; CCK-8, cell counting kit-8.
Figure 3
Metformin restored Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced ER stress. The expression of ER stress markers was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (A-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, or ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-b) using Western blotting analysis. Values displayed are mean ± SEM (n = 3 independent experiments, respectively); (B) the expression of ER stress markers and p53 protein was assessed in response to thapsigargin in HepG2 cells; (C-a) cell viability was assessed in HepG2 cells exposed to 0.1 μM thapsigargin and/or 0.5 mM metformin using CCK-8. Values displayed are mean ± SEM (n = 5 independent experiments); (C-b) the expression of an ER stress marker was also examined under these conditions; (C-c) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). GAPDH or COX IV was used for normalization. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1; CCK-8, cell counting kit-8.
Figure 4
A p53 inhibitor restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity. (A-a) the expression of cytosolic p53 was assessed in HepG2 cells treated with 50 μM PFT-α, a p53 inhibitor, and/or 0.25 mM palmitate with 35 mM glucose using Western blotting analysis; (A-b) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Exposure time of Western blotting was long, compared to Figure 2C; (B) co-localization was assessed between a marker of autophagosomes (LC3) and a mitochondrial marker (MitoTracker Red) in HepG2 cells stably transfected with GFP-LC3, depending on the conditions used. LC3-labeled structures were observed surrounding the fragmented mitochondria in cells treated with PFT-α and palmitate, although there was little co-localization between mitochondria and autophagosomes in cells treated with 0.25 mM palmitate alone. Scale bar = 20 μm. Abbreviation: Mfn1, mitofusin1; PFT-α, pifithrin-α.
Figure 5
Summary of the working thesis of this study. See the text for details. Black and red arrows indicate the effects of glucolipotoxicity and the drugs (metformin or PFTα), respectively.
References
- Song Y.M., Lee Y.H., Kim J.W., Ham D.S., Kang E.S., Cha B.S., Lee H.C., Lee B.W. Metformin alleviates hepatosteatosis by restoring sirt1-mediated autophagy induction via an amp-activated protein kinase-independent pathway. Autophagy. 2015;11:46–59. doi: 10.4161/15548627.2014.984271. - DOI - PMC - PubMed
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