- Email: [email protected]
Oleic acid-induced defective autolysosome shows impaired lipid degradation
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Oleic acid-induced defective autolysosome shows impaired lipid degradation Da-Hye Lee a, Jiyun Ahn a, b, Young Jin Jang b, Tae-Youl Ha a, b, Chang Hwa Jung a, b, * a b
Department of Food Biotechnology, University of Science and Technology, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea Research Group of Natural Materials and Metabolism, Korea Food Research Institute, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2019 Accepted 4 April 2019 Available online xxx
Recent studies suggest an alternative pathway of lipid breakdown called lipophagy, which delivers lipid droplets (LDs) to lysosomes for degradation of LDs. However, molecular mechanisms regulating lipophagy are still largely unknown. In this study, we evaluated the effect of oleic acid (OA) on lipophagy in cells. We found that OA treatment results in accumulation of p62 and LC3-II proteins and reduces red fluorescence in cells stably expressing mCherry-GFP-LC3. In addition, OA inhibits the co-localization of LC3 with LAMP1 under serum-deprived condition, suggesting that OA blocks autophagosome-lysosome fusion. In the cells with ATG5 or ULK1 gene deletion, LDs did not increase upon OA treatment more than in wild type cells. However, cell starvation following OA removal resulted in reduced lipid accumulation by lipophagy and recovery of autophagy flux, suggesting that the specific condition of OA treatment and cell starvation are important for lipophagy flux activity. © 2019 Elsevier Inc. All rights reserved.
Keywords: Oleic acid Lipid droplets Lipophagy Autophagy Lysosome
1. Introduction Lipid droplets (LDs) are storage organelles for neutral lipid. They are composed of a neutral lipid core, mainly containing triglycerides and sterol esters, surrounded by a monolayer of phospholipid and various associated proteins [1]. LDs are important for protection against environmental fluctuations, which can lead to an energy surplus or starvation [2]. At times of increased energy needs, like under conditions of nutrient deprivation, free fatty acids are released from LDs by the action of lipolytic enzymes and used as energy substrates [3]. LDs breakdown is initiated by various hormones, resulting in activation of protein kinase A (PKA), which subsequently activates lipases such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL) [3e5]. Recently, a type of selective autophagy, called lipophagy, has received attention as alternative lipolytic pathway.
Abbreviations: OA, oleic acid; LDs, lipid droplets; mTORC1, mechanistic target of rapamycin complex1; ATG, Autophagy-related gene; LC3, microtubule-associated protein 1 light chain 3; ULK1, Unc-51-like kinase 1; MEF, mouse embryonic fibroblast; S6K1, S6 kinase 1; shRNA, short hairpin RNA; LAMP-1/2, lysosomal-associated membrane protein 1/2. * Corresponding author. Department of Food Biotechnology, University of Science and Technology, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea. E-mail address: [email protected] (C.H. Jung).
The autophagy degradative pathway delivers material into the lysosome through the autophagosome, a double-membraned vesicle. The autophagy pathway plays a critical role in physiology; protecting cells against starvation, recycling nutrients from digested organelles, and maintain cellular homeostasis by removing damaged organelles and misfolded proteins [6e8]. Recent findings suggest that autophagy-related pathways vary with the organelles, such as mitophagy (mitochondria), ribophagy (ribosome), lipophagy (LD), lysophagy (lysosome), nucleophagy (nycleus) and ERphagy (endoplasmic reticulum, ER) [9]. Singh and colleagues have demonstrated a role of autophagy for degradation of LDs [10]. They showed that autophagy inhibition using pharmacological inhibitor or genetic modification increased hepatocellular TG accumulation and impaired LDs-LC3 co-localization in response to a lipid stimulus, however, the molecular mechanisms involved in lipophagy are still unclear. LD degradation may occur “in bulk” under basal condition via macroautophagy. Lipids and lipid structural proteins have been found within the autophagosome, indicating that autophagy is involved in LD degradation [10]. Nevertheless, the general understanding of the proteins responsible for the turnover of specific cargo in lipophagy is limited. LC3 is one of best-characterized structural component of the autophagosome and is associated with the surface of LDs. The ATG conjugation system (ATG7, ATG5, ATG12, ATG16) is involved in the formation of not only
https://doi.org/10.1016/j.bbrc.2019.04.040 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
2
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
autophagosome but also LDs [11,12]. The specific mechanism by which the limiting autophagosome membrane grows form the LDs surface remains poorly characterized. The mammalian proteins p62 and NBR1 act as cargo receptors or adaptors for the autophagic degradation of ubquitinated substrates. These proteins interact with LC3 and may recruit autophagy-related proteins for autophagosome formation on the surface of LDs. Unc51-like kinase 1 (ULK1) is a serine/threonine kinase that involved in the early stages of autophagy process. ULK1 is negatively regulated by mechanistic target of rapamycin complex1 (mTORC1), the master controller of cell growth [13,14], which also targets other proteins, such as ATG13 [8,13,15], ATG14 [16], and Ambra1 [16]. Further, ULK1 is positively regulated by AMPK [17] and activates the lipid kinase VPS34 by phosphorylating BECN1 at Ser 14, a step that is necessary for Ptdins3P production and the recruitment of the core autophagic machinery [18]. However, the roles of the ATG conjugation system in the regulation of the lipophagic machinery are still unknown. The intracellular lipophagic events are mainly studied in oleic acid (OA)-treated hepatocytes [10,19,20]. In the present study, we investigated whether OA affects intracellular lipophagy flux. In contrast to previous reports, we found that OA negatively affects the lipophagy flux involved in the breakdown of LDs.
2. Materials and methods 2.1. Materials Antibodies against b-actin (sc-47778) and GAPDH (sc-25778) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p-70-kDa ribosomal protein S6 kinase 1 (S6K1) (#9205), S6K1 (#9202), and Alexa 488 were purchased from Cell Signaling Technology (Danvers, MA). Antibody against LC3 (NB600-1384) was purchased from Novus Biologicals (Luzern, Switzerland). Antibodies against ULK1 (A7481) and p62 (P0067), Oil Red O (O0625), Oleic acid (O3008), 40 6-Diamidino-2-phenylindole (DAPI), and 3-(4,5-dime thylthiazol2-yl)-2,5 diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). LAMP1 (ab24170) and LAMP2 (ab13524) were purchased from Abcam (Cambridge, MA). Rapamycin (A-275) was obtained from Enzo life sciences (Farmingdale, NY). BODYPI (D3922), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillinestreptomycin, and phosphate buffered saline (PBS) were obtained from Gibco BRL (Grand Island, NY).
Fig. 1. Oleic acid blocks autophagy flux (A) HepG2 cells were treated with indicated OA concentrations in normal medium. (B) HepG2 cells were treated with indicated OA concentrations in serum-free medium. (C) Huh7 cells stably expressing mCherry-EGFP-LC3 were treated in the presence or absence of OA under serum-free medium. (D) Quantification of red and yellow fluorescence dots of mCherry-EGFP-LC3. Data are shown as mean ± SD. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.2. Cell culture HepG2 and Huh7 cells were purchased from the American Type Culture Collection (ATCC). Atg5 / and matched wild type immortalized MEF cell lines were kindly provided by Dr. N. Mizushima. ULK1 / and matched wild type immortalized MEF cells lines were purchased from Cancer Research Technology Ltd (London, UK). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C under 5% CO2. 2.3. Oil Red O staining Cells were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 1 h, washed with phosphate-buffered saline and then stained for 1 h with 0.5% Oil Red O in 60% isopropanol. After the stained cells were washed with distilled water, they were observed under a fluorescence microscope (IX71, Olympus Co.). Oil Red O was extracted from cells with 100% isopropanol. Absorbance was determined at an excitation wavelength of 490 nm. 2.4. Autophagy flux analysis Autophagy flux was measured using a tandem-tagged mCherryGFP-LC3 vector. The retroviral pBABE-EGFP-LC3B vector was purchased from Addgene (www.addgene.org, 22418). The vector was transfected into 293T cells (ATCC) with pCMV-VSV-G (Addgene,
3
8454) and pUMVC (Addgene, 8449) using the FuGENE6® reagent (Roche Applied Science, Indianapolis, IN). Huh7 cells were infected with retroviruses to generate stable cell lines and the infected cells were then selected with puromycin. The percentage of red and yellow puncta were quantified. 2.5. Immunoblotting Cells were harvested using lysis buffer containing 40 mM Hepes (pH 7.4), 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na3VO4, 10 mM beglycerophosphate, and 1% Triton X-100, supplemented with protease and phosphatase inhibitor cocktails (78440, Thermo scientific-Pierce, Rockford, IL). The proteins were separated by SDSPAGE, transferred to a PVDF membrane, blotted with the aforementioned antibodies, and visualized by chemiluminescence (Pierce ECL substrate from Thermo Fisher Scientific Inc.). 2.6. Image analysis Cells were seeded on a 0.2% gelatin coated slide chamber at a concentration of 5 104. The next day, cells were treated with 200 mM oleic acid in serum free media for 24 h. Cells were fixed with 4%formaldehyde for 15min and incubated with 0.05% saponin for 30 min at room temperature to enhance the permeability of the antibodies across the cell membrane. Cells were then treated a 1% BSA solution for 30min and incubated with the first antibody overnight at 4 C. After incubation, the cells were stained with the
Fig. 2. Oleic acid inhibits autophagosome-lysosome fusion xdza0073(A) HepG2 cells were treated with OA under serum-free and stained for co-localization of lipid droplets (BODIPY stain, green) with LC3 (red). (B) HepG2 cells were treated with OA under serum-free and stained for co-localization of lipid droplets (BODIPY stain, green) with LAMP1 (red). (C) Image shows co-localization of LC3 and LAMP2, as indicated by yellow dots in the merged image. (D) Co-localization was analyzed and quantified using imageJ. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
4
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
secondary antibody for 30 min and with 4, 6-diamidinophenylindole (DAPI) for 1min. The wells are washed with PBS three times, and then dried and covered with glass. Microscopy was performed at 408 nm (blue), 488 nm (green), 543 nm (red) using Nikon ECLIPSE TI/C1 confocal microscope (Tokyo, Japan). 2.7. Statistical analysis All data are expressed as mean ± standard deviations. Significant differences were established by one-way analysis of variance using GraphPad Prism 7 software (GraphPad software, Inc., CA, USA) and differences between groups were considered significant for values of p < 0.05. 3. Results
OA readily induced lipid accumulation in hepatocytes compared to PA (Supplementary Fig. 1). OA itself did not increased the lipidated form of Intracellular microtubule-associated protein 1 light chain 3 (LC3) in hepatocytes in a dose-dependent manner (Fig. 1A). Western blot analysis showed that levels of LC3-II and p62/SQSTM1, which are markers of autophagy flux, were increased in OA-treated HepG2 cells in a dose-dependent manner (Fig. 1B), suggesting that OA treatment may alter the autophagy flux. We further confirmed the autophagy flux using tandem-tagged mCherry-GFP-LC3, which displays yellow or red fluorescence according to autophagosome and autolysosome conditions. Huh7 cells stably expressing mCherry-GFP-LC3 were treated with OA in serum-free medium. The autophagosome significantly increased in OA-treated cells, whereas autolysosome increased in OA-untreated cells (Fig. 1C and D), suggesting that OA blocks autophagy flux in hepatocytes.
3.1. Oleic acid blocks autophagy flux
3.2. Oleic acid inhibits autophagosome-lysosome fusion
We have checked whether saturated (palmitic acid, PA) and unsaturated (oleic acid, OA) fatty acids differentially regulate lipid accumulation in hepatocytes. Consistent with previous study [21],
To further determine whether OA indeed inhibits autophagy flux, cells were stained with BOPIPY 493/503 and LC3 antibody for co-localization analysis of autophagosome with LDs in OA-treated
Fig. 3. OA treatment in ATG5 or ULK1 deficient cells did not lead to increased lipid accumulation (A) HepG2 cells were treated with rapamycin (RM) for 24 h in normal medium and lipid droplets stained with Oil Red O dye. (B, C) ATG5/ULK1þ/þ or / MEFs were treated with indicated OA concentration under serum-free conditions and the stained lipid droplets were quantified. (D) BODIPY stain and (E) immunoblot in OA-treated ULK1þ/þ or / MEF cells under serum-free condition. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
serum-free medium. LC3 partially co-localized with LD (Fig. 2A), although LD and the lysozyme marker, LAMP1 did not co-localize (Fig. 2B). LC3 and LAMP2 also showed lower co-localization in OA-treated cells than in OA untreated cells (Fig. 2C and D). Consistent with the blocked autophagy flux, rapamycin treatment to induce autophagy did not affect the lipid breakdown (Fig. 3A). Upon treating ATG5 þ/þ or / MEFs with OA in serum-free medium, we observed that LDs did not significantly increase in ATG5 / MEF compared with ATG5þ/þ MEF (Fig. 3B). Similar to observations with ATG5, lipid accumulation in ULK1/ cells was not significantly affected compared with ULK1þ/þ cells (Fig. 3C). In addition, ULK1 deficiency did not show further increase in the lipid stained with BOPIPY 493/503 than the ULK1þ/þ MEF cells (Fig. 3D).
5
The p62/SQSTM1 is decreased in the autophagic degradation when autophagosome is fused with lysozyme [22]. The p62/SQSTM1 levels was also not changed in OA-treated cells under starvation (Fig. 3E). These results indicate that OA inhibits autophagosomelysosome fusion and thus cannot degrade LDs through lipophagy. 3.3. Lipophagy is restored by removal of oleic acid In order to observe lipophagy, we examined whether the treatment conditions of OA and serum starvation affect the lipophagy flux when the conditions are changed (Fig. 4A). Immunoblotting showed a decrease in the level of p62/SQSTM1 and increase in the phosphorylation of ATG14 under starvation after OA
Fig. 4. OA elimination recovered autophagy flux (A) Cells were serum starved after OA removal. (B) Degradation of p62/SQSTM1 in serum-starved cells after OA removal. (C) Autophagy flux was analyzed using Huh7 cells stably expressing mCherry-EGFP-LC3 according to method #1 and method #2. (D) Quantification of red and yellow fluorescence dots of mCherry-EGFP-LC3. . Data are shown as mean ± SD. (E) Intracellular triglyceride (TG) levels. (F) ULK1þ/þ or / MEFs were serum starved after OA removal. Lipid droplets were stained with Oil Red O dye and quantified. (G) BODIPY stain, immunoblot showing LC3 conversion in Atg5 (H) ULK1 (I) þ/þ or / MEF cells under method #2. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
6
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
elimination, indicating that autophagy occurred normally (Fig. 4B). Autophagy flux was also monitored using tandem fluorescenttagged LC3 (mCherry-GFP-LC3) based on different pH stability. The method #2 enhanced red fluorescent protein compared with the method #1 (Fig. 4C and D), suggesting that OA interferes with the fusion between autophagosome and autolysosome. Intracellular TG levels were also lower in the method #2 than in the method #1 (Fig. 4E). LDs were significantly decreased in ATG5þ/þ MEF cells by serum deprivation after OA elimination and there was no significant change in ATG5/ MEF cells (Fig. 4F). Lipids stained with BOPIPY 493/503 were decreased in ATG5þ/þ MEF cells compared with ATG5/ MEF cells (Fig. 4G). Chloroquine treatment increased LC3-II levels in ATG5þ/þ MEF cells and ULK1þ/þ MEF cells, whereas ATG5 or ULK1 deletion did not (Fig. 3H and I). Taken together, these results suggests that autolysosome formation occurred under method #2 condition.
In conclusion, although autophagy is involved in the degradation of LD, OA suppresses the fusion of autophagosome and lysosome, and autophagy flux is restored when OA is removed. Therefore, OA treatment and starvation might be important to confirm the effect of lipophagy cells. Author contributions D.H.L. and C.H.J. wrote the manuscript. D.H.L. performed the image experiment. C.H.J. designed this study. J.A. and Y.J.J. performed the in vitro assay. T.Y.H. contributed to discussion. Conflicts of interest The authors declare no competing interests. Conflicts of interest
4. Discussion The authors declare no competing financial interests. Lipophagy is the selective autophagy for LDs degradation through the lysosomal-autophagic pathway [10]. Lipophagy has been reported to play an important role in non-alcoholic fatty liver disease (NAFLD) [23]. However, it is not known how lipophagy is regulated or altered in fatty liver. In order for the LDs to become targets of autophagy, chaperone-mediated autophagy (CMA) firstly degrades perilipin to facilitate LD catabolism, allowing access for lipases and subsequent lipophagy [24]. Lipases also promote autophagy to facilitate LDs catabolism, which leads to the generation of free fatty acids [25]. Recently, it was reported that the Huntingtin protein acts as a receptor of LD and is involved in lipophagy [26]. In addition, p62/SQSTM1 [27], CALCOCO2 [28], optineurin [29], and NBR1 [30] may play potential scaffolding roles in lipophagy. ULK1-Atg13-FIP200 complex is the most upstream components in the autophagy process and regulates BECN1-VPS34 complex to promote autophagy induction and maturation [18]. ATG5 is involved in the lipidation of LC3 in the formation of autophagosome [31]. These genes have also been reported to be involved in lipophagy [10,32]. It was thought that lipid accumulation would be increased by OA treatment of cells with ATG5 or ULK1 KO cells. ULK1/ or ATG5/ MEFs treated with OA did not show significant change in LD accumulation compared with OA-treated ULK1þ/þ or ATG5þ/þ MEF (Fig. 2 A &B). In addition, OA-treated cells showed low co-localization with autophagy and lysozyme markers, LC3 and LAMP-1. On the other hand, LC3 partially colocalized with BODIPY, indicate that OA blocks autolysosome and inhibits lipid breakdown by lipophagy. The prevention of OA-mediated fusion of autophagosome and lysosome is likely to be due to various factors such as decrease in vesicle traffic, changes in lysosomal acidification, or damage to protease activity. Recently, the analysis of autophagosome and lysosome in the liver of mice fed with high-fat diet showed that the activity of lysosome is important in the fusion machinery [33]. Thus, precise monitoring of lysosomal activity may be required in OA-treated cells. To determine the effect of OA on fusion of autophagosome and lysosome, OA was removed by serum-free medium exchange and autophagy flux was analyzed. We found that p62/SQSTM1 expression was decreased, and in cells expressing mCherry-GFPLC3, the red fluorescence protein increased in starved cells after OA elimination. These results suggest that the fusion of autophagosome and lysosome is suppressed in presence of OA. In the cells lacking the autophagy gene, the lipid accumulation tended to increase more in the ATG5 and ULK1 KO cells, and the autophagy flux also recovered. ULK1-silenced HepG2 cells also gave the same results (data not shown).
Acknowledgements This study was supported by Main Research Program of the Korea Food Research Institute funded by the Ministry of Science and ICT. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.040. References [1] K. Liu, M.J. Czaja, Regulation of lipid stores and metabolism by lipophagy, Cell Death Differ. 20 (2013) 3e11. [2] R.V. Farese Jr., T.C. Walther, Lipid droplets finally get a little R-E-S-P-E-C-T, Cell 139 (2009) 855e860. [3] R.E. Duncan, M. Ahmadian, K. Jaworski, E. Sarkadi-Nagy, H.S. Sul, Regulation of lipolysis in adipocytes, Annu. Rev. Nutr. 27 (2007) 79e101. [4] A. Pol, S.P. Gross, R.G. Parton, Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites, J. Cell Biol. 204 (2014) 635e646. [5] T.C. Walther, R.V. Farese Jr., Lipid droplets and cellular lipid metabolism, Annu. Rev. Biochem. 81 (2012) 687e714. [6] C. Dall'Armi, K.A. Devereaux, G. Di Paolo, The role of lipids in the control of autophagy, Curr. Biol. 23 (2013) R33eR45. [7] H. Dong, M.J. Czaja, Regulation of lipid droplets by autophagy, Trends Endocrinol. Metabol. 22 (2011) 234e240. [8] I.G. Ganley, H. Lam du, J. Wang, X. Ding, S. Chen, X. Jiang, ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy, J. Biol. Chem. 284 (2009) 12297e12305. [9] K. Okamoto, Organellophagy: eliminating cellular building blocks via selective autophagy, J. Cell Biol. 205 (2014) 435e445. [10] R. Singh, S. Kaushik, Y. Wang, Y. Xiang, I. Novak, M. Komatsu, K. Tanaka, A.M. Cuervo, M.J. Czaja, Autophagy regulates lipid metabolism, Nature 458 (2009) 1131e1135. [11] M. Shibata, K. Yoshimura, N. Furuya, M. Koike, T. Ueno, M. Komatsu, H. Arai, K. Tanaka, E. Kominami, Y. Uchiyama, The MAP1-LC3 conjugation system is involved in lipid droplet formation, Biochem. Biophys. Res. Commun. 382 (2009) 419e423. [12] M. Shibata, K. Yoshimura, H. Tamura, T. Ueno, T. Nishimura, T. Inoue, M. Sasaki, M. Koike, H. Arai, E. Kominami, Y. Uchiyama, LC3, a microtubuleassociated protein1A/B light chain 3, is involved in cytoplasmic lipid droplet formation, Biochem. Biophys. Res. Commun. 393 (2010) 274e279. [13] C.H. Jung, C.B. Jun, S.H. Ro, Y.M. Kim, N.M. Otto, J. Cao, M. Kundu, D.H. Kim, ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery, Mol. Biol. Cell 20 (2009) 1992e2003. [14] N. Hosokawa, T. Hara, T. Kaizuka, C. Kishi, A. Takamura, Y. Miura, S. Iemura, T. Natsume, K. Takehana, N. Yamada, J.L. Guan, N. Oshiro, N. Mizushima, Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy, Mol. Biol. Cell 20 (2009) 1981e1991. [15] Y.Y. Chang, T.P. Neufeld, An Atg1/Atg13 complex with multiple roles in TORmediated autophagy regulation, Mol. Biol. Cell 20 (2009) 2004e2014. [16] H.X. Yuan, R.C. Russell, K.L. Guan, Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy, Autophagy 9 (2013) 1983e1995. [17] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132e141. [18] R.C. Russell, Y. Tian, H. Yuan, H.W. Park, Y.Y. Chang, J. Kim, H. Kim, T.P. Neufeld, A. Dillin, K.L. Guan, ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase, Nat. Cell Biol. 15 (2013) 741e750. [19] C.I. Cheng, Y.H. Lee, P.H. Chen, Y.C. Lin, M.H. Chou, Y.H. Kao, Free fatty acids induce autophagy and LOX-1 upregulation in cultured aortic vascular smooth muscle cells, J. Cell. Biochem. 118 (2017) 1249e1261. [20] M. Niso-Santano, S.A. Malik, F. Pietrocola, J.M. Bravo-San Pedro, G. Marino, V. Cianfanelli, A. Ben-Younes, R. Troncoso, M. Markaki, V. Sica, V. Izzo, K. Chaba, C. Bauvy, N. Dupont, O. Kepp, P. Rockenfeller, H. Wolinski, F. Madeo, S. Lavandero, P. Codogno, F. Harper, G. Pierron, N. Tavernarakis, F. Cecconi, M.C. Maiuri, L. Galluzzi, G. Kroemer, Unsaturated fatty acids induce noncanonical autophagy, EMBO J. 34 (2015) 1025e1041. [21] S. Mei, H.M. Ni, S. Manley, A. Bockus, K.M. Kassel, J.P. Luyendyk, B.L. Copple, W.X. Ding, Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes, J. Pharmacol. Exp. Ther. 339 (2011) 487e498. [22] G. Bjorkoy, T. Lamark, S. Pankiv, A. Overvatn, A. Brech, T. Johansen, Monitoring autophagic degradation of p62/SQSTM1, Methods Enzymol. 452 (2009) 181e197. [23] R.J. Schulze, K. Drizyte, C.A. Casey, M.A. McNiven, Hepatic lipophagy: new insights into autophagic catabolism of lipid droplets in the liver, Hepatol. Commun. 1 (2017) 359e369. [24] S. Kaushik, A.M. Cuervo, Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis, Nat. Cell Biol. 17 (2015) 759e770. [25] R.J. Schulze, A. Sathyanarayan, D.G. Mashek, Breaking fat: the regulation and
[26]
[27]
[28]
[29] [30]
[31]
[32]
[33]
7
mechanisms of lipophagy, Biochimica et biophysica acta, Mol. Cell Biol. Lipids 1862 (2017) 1178e1187. Y.N. Rui, Z. Xu, B. Patel, Z. Chen, D. Chen, A. Tito, G. David, Y. Sun, E.F. Stimming, H.J. Bellen, A.M. Cuervo, S. Zhang, Huntingtin functions as a scaffold for selective macroautophagy, Nat. Cell Biol. 17 (2015) 262e275. T. Lam, R. Harmancey, H. Vasquez, B. Gilbert, N. Patel, V. Hariharan, A. Lee, M. Covey, H. Taegtmeyer, Reversal of intramyocellular lipid accumulation by lipophagy and a p62-mediated pathway, Cell Death Discov. 2 (2016) 16061. X. Xie, F. Li, Y. Wang, Y. Wang, Z. Lin, X. Cheng, J. Liu, C. Chen, L. Pan, Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2, Autophagy 11 (2015) 1775e1789. T.A. Ryan, D.A. Tumbarello, Optineurin: a coordinator of membraneassociated cargo trafficking and autophagy, Front. Immunol. 9 (2018) 1024. A. Werner, B. Herzog, O. Voigt, O. Valerius, G.H. Braus, S. Poggeler, NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog, Autophagy (2018) 1e20. T. Hanada, N.N. Noda, Y. Satomi, Y. Ichimura, Y. Fujioka, T. Takao, F. Inagaki, Y. Ohsumi, The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy, J. Biol. Chem. 282 (2007) 37298e37302. B.L. Farah, D.J. Landau, R.A. Sinha, E.D. Brooks, Y. Wu, S.Y.S. Fung, T. Tanaka, M. Hirayama, B.H. Bay, D.D. Koeberl, P.M. Yen, Induction of autophagy improves hepatic lipid metabolism in glucose-6-phosphatase deficiency, J. Hepatol. 64 (2016) 370e379. H. Koga, S. Kaushik, A.M. Cuervo, Altered lipid content inhibits autophagic vesicular fusion, FASEB J. : Off. Publ. Fed. Am. Soc. Exp. Biol. 24 (2010) 3052e3065.
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Oleic acid-induced defective autolysosome shows impaired lipid degradation Da-Hye Lee a, Jiyun Ahn a, b, Young Jin Jang b, Tae-Youl Ha a, b, Chang Hwa Jung a, b, * a b
Department of Food Biotechnology, University of Science and Technology, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea Research Group of Natural Materials and Metabolism, Korea Food Research Institute, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2019 Accepted 4 April 2019 Available online xxx
Recent studies suggest an alternative pathway of lipid breakdown called lipophagy, which delivers lipid droplets (LDs) to lysosomes for degradation of LDs. However, molecular mechanisms regulating lipophagy are still largely unknown. In this study, we evaluated the effect of oleic acid (OA) on lipophagy in cells. We found that OA treatment results in accumulation of p62 and LC3-II proteins and reduces red fluorescence in cells stably expressing mCherry-GFP-LC3. In addition, OA inhibits the co-localization of LC3 with LAMP1 under serum-deprived condition, suggesting that OA blocks autophagosome-lysosome fusion. In the cells with ATG5 or ULK1 gene deletion, LDs did not increase upon OA treatment more than in wild type cells. However, cell starvation following OA removal resulted in reduced lipid accumulation by lipophagy and recovery of autophagy flux, suggesting that the specific condition of OA treatment and cell starvation are important for lipophagy flux activity. © 2019 Elsevier Inc. All rights reserved.
Keywords: Oleic acid Lipid droplets Lipophagy Autophagy Lysosome
1. Introduction Lipid droplets (LDs) are storage organelles for neutral lipid. They are composed of a neutral lipid core, mainly containing triglycerides and sterol esters, surrounded by a monolayer of phospholipid and various associated proteins [1]. LDs are important for protection against environmental fluctuations, which can lead to an energy surplus or starvation [2]. At times of increased energy needs, like under conditions of nutrient deprivation, free fatty acids are released from LDs by the action of lipolytic enzymes and used as energy substrates [3]. LDs breakdown is initiated by various hormones, resulting in activation of protein kinase A (PKA), which subsequently activates lipases such as adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase (MGL) [3e5]. Recently, a type of selective autophagy, called lipophagy, has received attention as alternative lipolytic pathway.
Abbreviations: OA, oleic acid; LDs, lipid droplets; mTORC1, mechanistic target of rapamycin complex1; ATG, Autophagy-related gene; LC3, microtubule-associated protein 1 light chain 3; ULK1, Unc-51-like kinase 1; MEF, mouse embryonic fibroblast; S6K1, S6 kinase 1; shRNA, short hairpin RNA; LAMP-1/2, lysosomal-associated membrane protein 1/2. * Corresponding author. Department of Food Biotechnology, University of Science and Technology, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea. E-mail address: [email protected] (C.H. Jung).
The autophagy degradative pathway delivers material into the lysosome through the autophagosome, a double-membraned vesicle. The autophagy pathway plays a critical role in physiology; protecting cells against starvation, recycling nutrients from digested organelles, and maintain cellular homeostasis by removing damaged organelles and misfolded proteins [6e8]. Recent findings suggest that autophagy-related pathways vary with the organelles, such as mitophagy (mitochondria), ribophagy (ribosome), lipophagy (LD), lysophagy (lysosome), nucleophagy (nycleus) and ERphagy (endoplasmic reticulum, ER) [9]. Singh and colleagues have demonstrated a role of autophagy for degradation of LDs [10]. They showed that autophagy inhibition using pharmacological inhibitor or genetic modification increased hepatocellular TG accumulation and impaired LDs-LC3 co-localization in response to a lipid stimulus, however, the molecular mechanisms involved in lipophagy are still unclear. LD degradation may occur “in bulk” under basal condition via macroautophagy. Lipids and lipid structural proteins have been found within the autophagosome, indicating that autophagy is involved in LD degradation [10]. Nevertheless, the general understanding of the proteins responsible for the turnover of specific cargo in lipophagy is limited. LC3 is one of best-characterized structural component of the autophagosome and is associated with the surface of LDs. The ATG conjugation system (ATG7, ATG5, ATG12, ATG16) is involved in the formation of not only
https://doi.org/10.1016/j.bbrc.2019.04.040 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
2
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
autophagosome but also LDs [11,12]. The specific mechanism by which the limiting autophagosome membrane grows form the LDs surface remains poorly characterized. The mammalian proteins p62 and NBR1 act as cargo receptors or adaptors for the autophagic degradation of ubquitinated substrates. These proteins interact with LC3 and may recruit autophagy-related proteins for autophagosome formation on the surface of LDs. Unc51-like kinase 1 (ULK1) is a serine/threonine kinase that involved in the early stages of autophagy process. ULK1 is negatively regulated by mechanistic target of rapamycin complex1 (mTORC1), the master controller of cell growth [13,14], which also targets other proteins, such as ATG13 [8,13,15], ATG14 [16], and Ambra1 [16]. Further, ULK1 is positively regulated by AMPK [17] and activates the lipid kinase VPS34 by phosphorylating BECN1 at Ser 14, a step that is necessary for Ptdins3P production and the recruitment of the core autophagic machinery [18]. However, the roles of the ATG conjugation system in the regulation of the lipophagic machinery are still unknown. The intracellular lipophagic events are mainly studied in oleic acid (OA)-treated hepatocytes [10,19,20]. In the present study, we investigated whether OA affects intracellular lipophagy flux. In contrast to previous reports, we found that OA negatively affects the lipophagy flux involved in the breakdown of LDs.
2. Materials and methods 2.1. Materials Antibodies against b-actin (sc-47778) and GAPDH (sc-25778) and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p-70-kDa ribosomal protein S6 kinase 1 (S6K1) (#9205), S6K1 (#9202), and Alexa 488 were purchased from Cell Signaling Technology (Danvers, MA). Antibody against LC3 (NB600-1384) was purchased from Novus Biologicals (Luzern, Switzerland). Antibodies against ULK1 (A7481) and p62 (P0067), Oil Red O (O0625), Oleic acid (O3008), 40 6-Diamidino-2-phenylindole (DAPI), and 3-(4,5-dime thylthiazol2-yl)-2,5 diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). LAMP1 (ab24170) and LAMP2 (ab13524) were purchased from Abcam (Cambridge, MA). Rapamycin (A-275) was obtained from Enzo life sciences (Farmingdale, NY). BODYPI (D3922), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillinestreptomycin, and phosphate buffered saline (PBS) were obtained from Gibco BRL (Grand Island, NY).
Fig. 1. Oleic acid blocks autophagy flux (A) HepG2 cells were treated with indicated OA concentrations in normal medium. (B) HepG2 cells were treated with indicated OA concentrations in serum-free medium. (C) Huh7 cells stably expressing mCherry-EGFP-LC3 were treated in the presence or absence of OA under serum-free medium. (D) Quantification of red and yellow fluorescence dots of mCherry-EGFP-LC3. Data are shown as mean ± SD. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
2.2. Cell culture HepG2 and Huh7 cells were purchased from the American Type Culture Collection (ATCC). Atg5 / and matched wild type immortalized MEF cell lines were kindly provided by Dr. N. Mizushima. ULK1 / and matched wild type immortalized MEF cells lines were purchased from Cancer Research Technology Ltd (London, UK). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 C under 5% CO2. 2.3. Oil Red O staining Cells were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 1 h, washed with phosphate-buffered saline and then stained for 1 h with 0.5% Oil Red O in 60% isopropanol. After the stained cells were washed with distilled water, they were observed under a fluorescence microscope (IX71, Olympus Co.). Oil Red O was extracted from cells with 100% isopropanol. Absorbance was determined at an excitation wavelength of 490 nm. 2.4. Autophagy flux analysis Autophagy flux was measured using a tandem-tagged mCherryGFP-LC3 vector. The retroviral pBABE-EGFP-LC3B vector was purchased from Addgene (www.addgene.org, 22418). The vector was transfected into 293T cells (ATCC) with pCMV-VSV-G (Addgene,
3
8454) and pUMVC (Addgene, 8449) using the FuGENE6® reagent (Roche Applied Science, Indianapolis, IN). Huh7 cells were infected with retroviruses to generate stable cell lines and the infected cells were then selected with puromycin. The percentage of red and yellow puncta were quantified. 2.5. Immunoblotting Cells were harvested using lysis buffer containing 40 mM Hepes (pH 7.4), 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na3VO4, 10 mM beglycerophosphate, and 1% Triton X-100, supplemented with protease and phosphatase inhibitor cocktails (78440, Thermo scientific-Pierce, Rockford, IL). The proteins were separated by SDSPAGE, transferred to a PVDF membrane, blotted with the aforementioned antibodies, and visualized by chemiluminescence (Pierce ECL substrate from Thermo Fisher Scientific Inc.). 2.6. Image analysis Cells were seeded on a 0.2% gelatin coated slide chamber at a concentration of 5 104. The next day, cells were treated with 200 mM oleic acid in serum free media for 24 h. Cells were fixed with 4%formaldehyde for 15min and incubated with 0.05% saponin for 30 min at room temperature to enhance the permeability of the antibodies across the cell membrane. Cells were then treated a 1% BSA solution for 30min and incubated with the first antibody overnight at 4 C. After incubation, the cells were stained with the
Fig. 2. Oleic acid inhibits autophagosome-lysosome fusion xdza0073(A) HepG2 cells were treated with OA under serum-free and stained for co-localization of lipid droplets (BODIPY stain, green) with LC3 (red). (B) HepG2 cells were treated with OA under serum-free and stained for co-localization of lipid droplets (BODIPY stain, green) with LAMP1 (red). (C) Image shows co-localization of LC3 and LAMP2, as indicated by yellow dots in the merged image. (D) Co-localization was analyzed and quantified using imageJ. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
4
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
secondary antibody for 30 min and with 4, 6-diamidinophenylindole (DAPI) for 1min. The wells are washed with PBS three times, and then dried and covered with glass. Microscopy was performed at 408 nm (blue), 488 nm (green), 543 nm (red) using Nikon ECLIPSE TI/C1 confocal microscope (Tokyo, Japan). 2.7. Statistical analysis All data are expressed as mean ± standard deviations. Significant differences were established by one-way analysis of variance using GraphPad Prism 7 software (GraphPad software, Inc., CA, USA) and differences between groups were considered significant for values of p < 0.05. 3. Results
OA readily induced lipid accumulation in hepatocytes compared to PA (Supplementary Fig. 1). OA itself did not increased the lipidated form of Intracellular microtubule-associated protein 1 light chain 3 (LC3) in hepatocytes in a dose-dependent manner (Fig. 1A). Western blot analysis showed that levels of LC3-II and p62/SQSTM1, which are markers of autophagy flux, were increased in OA-treated HepG2 cells in a dose-dependent manner (Fig. 1B), suggesting that OA treatment may alter the autophagy flux. We further confirmed the autophagy flux using tandem-tagged mCherry-GFP-LC3, which displays yellow or red fluorescence according to autophagosome and autolysosome conditions. Huh7 cells stably expressing mCherry-GFP-LC3 were treated with OA in serum-free medium. The autophagosome significantly increased in OA-treated cells, whereas autolysosome increased in OA-untreated cells (Fig. 1C and D), suggesting that OA blocks autophagy flux in hepatocytes.
3.1. Oleic acid blocks autophagy flux
3.2. Oleic acid inhibits autophagosome-lysosome fusion
We have checked whether saturated (palmitic acid, PA) and unsaturated (oleic acid, OA) fatty acids differentially regulate lipid accumulation in hepatocytes. Consistent with previous study [21],
To further determine whether OA indeed inhibits autophagy flux, cells were stained with BOPIPY 493/503 and LC3 antibody for co-localization analysis of autophagosome with LDs in OA-treated
Fig. 3. OA treatment in ATG5 or ULK1 deficient cells did not lead to increased lipid accumulation (A) HepG2 cells were treated with rapamycin (RM) for 24 h in normal medium and lipid droplets stained with Oil Red O dye. (B, C) ATG5/ULK1þ/þ or / MEFs were treated with indicated OA concentration under serum-free conditions and the stained lipid droplets were quantified. (D) BODIPY stain and (E) immunoblot in OA-treated ULK1þ/þ or / MEF cells under serum-free condition. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
serum-free medium. LC3 partially co-localized with LD (Fig. 2A), although LD and the lysozyme marker, LAMP1 did not co-localize (Fig. 2B). LC3 and LAMP2 also showed lower co-localization in OA-treated cells than in OA untreated cells (Fig. 2C and D). Consistent with the blocked autophagy flux, rapamycin treatment to induce autophagy did not affect the lipid breakdown (Fig. 3A). Upon treating ATG5 þ/þ or / MEFs with OA in serum-free medium, we observed that LDs did not significantly increase in ATG5 / MEF compared with ATG5þ/þ MEF (Fig. 3B). Similar to observations with ATG5, lipid accumulation in ULK1/ cells was not significantly affected compared with ULK1þ/þ cells (Fig. 3C). In addition, ULK1 deficiency did not show further increase in the lipid stained with BOPIPY 493/503 than the ULK1þ/þ MEF cells (Fig. 3D).
5
The p62/SQSTM1 is decreased in the autophagic degradation when autophagosome is fused with lysozyme [22]. The p62/SQSTM1 levels was also not changed in OA-treated cells under starvation (Fig. 3E). These results indicate that OA inhibits autophagosomelysosome fusion and thus cannot degrade LDs through lipophagy. 3.3. Lipophagy is restored by removal of oleic acid In order to observe lipophagy, we examined whether the treatment conditions of OA and serum starvation affect the lipophagy flux when the conditions are changed (Fig. 4A). Immunoblotting showed a decrease in the level of p62/SQSTM1 and increase in the phosphorylation of ATG14 under starvation after OA
Fig. 4. OA elimination recovered autophagy flux (A) Cells were serum starved after OA removal. (B) Degradation of p62/SQSTM1 in serum-starved cells after OA removal. (C) Autophagy flux was analyzed using Huh7 cells stably expressing mCherry-EGFP-LC3 according to method #1 and method #2. (D) Quantification of red and yellow fluorescence dots of mCherry-EGFP-LC3. . Data are shown as mean ± SD. (E) Intracellular triglyceride (TG) levels. (F) ULK1þ/þ or / MEFs were serum starved after OA removal. Lipid droplets were stained with Oil Red O dye and quantified. (G) BODIPY stain, immunoblot showing LC3 conversion in Atg5 (H) ULK1 (I) þ/þ or / MEF cells under method #2. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
6
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
elimination, indicating that autophagy occurred normally (Fig. 4B). Autophagy flux was also monitored using tandem fluorescenttagged LC3 (mCherry-GFP-LC3) based on different pH stability. The method #2 enhanced red fluorescent protein compared with the method #1 (Fig. 4C and D), suggesting that OA interferes with the fusion between autophagosome and autolysosome. Intracellular TG levels were also lower in the method #2 than in the method #1 (Fig. 4E). LDs were significantly decreased in ATG5þ/þ MEF cells by serum deprivation after OA elimination and there was no significant change in ATG5/ MEF cells (Fig. 4F). Lipids stained with BOPIPY 493/503 were decreased in ATG5þ/þ MEF cells compared with ATG5/ MEF cells (Fig. 4G). Chloroquine treatment increased LC3-II levels in ATG5þ/þ MEF cells and ULK1þ/þ MEF cells, whereas ATG5 or ULK1 deletion did not (Fig. 3H and I). Taken together, these results suggests that autolysosome formation occurred under method #2 condition.
In conclusion, although autophagy is involved in the degradation of LD, OA suppresses the fusion of autophagosome and lysosome, and autophagy flux is restored when OA is removed. Therefore, OA treatment and starvation might be important to confirm the effect of lipophagy cells. Author contributions D.H.L. and C.H.J. wrote the manuscript. D.H.L. performed the image experiment. C.H.J. designed this study. J.A. and Y.J.J. performed the in vitro assay. T.Y.H. contributed to discussion. Conflicts of interest The authors declare no competing interests. Conflicts of interest
4. Discussion The authors declare no competing financial interests. Lipophagy is the selective autophagy for LDs degradation through the lysosomal-autophagic pathway [10]. Lipophagy has been reported to play an important role in non-alcoholic fatty liver disease (NAFLD) [23]. However, it is not known how lipophagy is regulated or altered in fatty liver. In order for the LDs to become targets of autophagy, chaperone-mediated autophagy (CMA) firstly degrades perilipin to facilitate LD catabolism, allowing access for lipases and subsequent lipophagy [24]. Lipases also promote autophagy to facilitate LDs catabolism, which leads to the generation of free fatty acids [25]. Recently, it was reported that the Huntingtin protein acts as a receptor of LD and is involved in lipophagy [26]. In addition, p62/SQSTM1 [27], CALCOCO2 [28], optineurin [29], and NBR1 [30] may play potential scaffolding roles in lipophagy. ULK1-Atg13-FIP200 complex is the most upstream components in the autophagy process and regulates BECN1-VPS34 complex to promote autophagy induction and maturation [18]. ATG5 is involved in the lipidation of LC3 in the formation of autophagosome [31]. These genes have also been reported to be involved in lipophagy [10,32]. It was thought that lipid accumulation would be increased by OA treatment of cells with ATG5 or ULK1 KO cells. ULK1/ or ATG5/ MEFs treated with OA did not show significant change in LD accumulation compared with OA-treated ULK1þ/þ or ATG5þ/þ MEF (Fig. 2 A &B). In addition, OA-treated cells showed low co-localization with autophagy and lysozyme markers, LC3 and LAMP-1. On the other hand, LC3 partially colocalized with BODIPY, indicate that OA blocks autolysosome and inhibits lipid breakdown by lipophagy. The prevention of OA-mediated fusion of autophagosome and lysosome is likely to be due to various factors such as decrease in vesicle traffic, changes in lysosomal acidification, or damage to protease activity. Recently, the analysis of autophagosome and lysosome in the liver of mice fed with high-fat diet showed that the activity of lysosome is important in the fusion machinery [33]. Thus, precise monitoring of lysosomal activity may be required in OA-treated cells. To determine the effect of OA on fusion of autophagosome and lysosome, OA was removed by serum-free medium exchange and autophagy flux was analyzed. We found that p62/SQSTM1 expression was decreased, and in cells expressing mCherry-GFPLC3, the red fluorescence protein increased in starved cells after OA elimination. These results suggest that the fusion of autophagosome and lysosome is suppressed in presence of OA. In the cells lacking the autophagy gene, the lipid accumulation tended to increase more in the ATG5 and ULK1 KO cells, and the autophagy flux also recovered. ULK1-silenced HepG2 cells also gave the same results (data not shown).
Acknowledgements This study was supported by Main Research Program of the Korea Food Research Institute funded by the Ministry of Science and ICT. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.040. References [1] K. Liu, M.J. Czaja, Regulation of lipid stores and metabolism by lipophagy, Cell Death Differ. 20 (2013) 3e11. [2] R.V. Farese Jr., T.C. Walther, Lipid droplets finally get a little R-E-S-P-E-C-T, Cell 139 (2009) 855e860. [3] R.E. Duncan, M. Ahmadian, K. Jaworski, E. Sarkadi-Nagy, H.S. Sul, Regulation of lipolysis in adipocytes, Annu. Rev. Nutr. 27 (2007) 79e101. [4] A. Pol, S.P. Gross, R.G. Parton, Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites, J. Cell Biol. 204 (2014) 635e646. [5] T.C. Walther, R.V. Farese Jr., Lipid droplets and cellular lipid metabolism, Annu. Rev. Biochem. 81 (2012) 687e714. [6] C. Dall'Armi, K.A. Devereaux, G. Di Paolo, The role of lipids in the control of autophagy, Curr. Biol. 23 (2013) R33eR45. [7] H. Dong, M.J. Czaja, Regulation of lipid droplets by autophagy, Trends Endocrinol. Metabol. 22 (2011) 234e240. [8] I.G. Ganley, H. Lam du, J. Wang, X. Ding, S. Chen, X. Jiang, ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy, J. Biol. Chem. 284 (2009) 12297e12305. [9] K. Okamoto, Organellophagy: eliminating cellular building blocks via selective autophagy, J. Cell Biol. 205 (2014) 435e445. [10] R. Singh, S. Kaushik, Y. Wang, Y. Xiang, I. Novak, M. Komatsu, K. Tanaka, A.M. Cuervo, M.J. Czaja, Autophagy regulates lipid metabolism, Nature 458 (2009) 1131e1135. [11] M. Shibata, K. Yoshimura, N. Furuya, M. Koike, T. Ueno, M. Komatsu, H. Arai, K. Tanaka, E. Kominami, Y. Uchiyama, The MAP1-LC3 conjugation system is involved in lipid droplet formation, Biochem. Biophys. Res. Commun. 382 (2009) 419e423. [12] M. Shibata, K. Yoshimura, H. Tamura, T. Ueno, T. Nishimura, T. Inoue, M. Sasaki, M. Koike, H. Arai, E. Kominami, Y. Uchiyama, LC3, a microtubuleassociated protein1A/B light chain 3, is involved in cytoplasmic lipid droplet formation, Biochem. Biophys. Res. Commun. 393 (2010) 274e279. [13] C.H. Jung, C.B. Jun, S.H. Ro, Y.M. Kim, N.M. Otto, J. Cao, M. Kundu, D.H. Kim, ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery, Mol. Biol. Cell 20 (2009) 1992e2003. [14] N. Hosokawa, T. Hara, T. Kaizuka, C. Kishi, A. Takamura, Y. Miura, S. Iemura, T. Natsume, K. Takehana, N. Yamada, J.L. Guan, N. Oshiro, N. Mizushima, Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy, Mol. Biol. Cell 20 (2009) 1981e1991. [15] Y.Y. Chang, T.P. Neufeld, An Atg1/Atg13 complex with multiple roles in TORmediated autophagy regulation, Mol. Biol. Cell 20 (2009) 2004e2014. [16] H.X. Yuan, R.C. Russell, K.L. Guan, Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy, Autophagy 9 (2013) 1983e1995. [17] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040
D.-H. Lee et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132e141. [18] R.C. Russell, Y. Tian, H. Yuan, H.W. Park, Y.Y. Chang, J. Kim, H. Kim, T.P. Neufeld, A. Dillin, K.L. Guan, ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase, Nat. Cell Biol. 15 (2013) 741e750. [19] C.I. Cheng, Y.H. Lee, P.H. Chen, Y.C. Lin, M.H. Chou, Y.H. Kao, Free fatty acids induce autophagy and LOX-1 upregulation in cultured aortic vascular smooth muscle cells, J. Cell. Biochem. 118 (2017) 1249e1261. [20] M. Niso-Santano, S.A. Malik, F. Pietrocola, J.M. Bravo-San Pedro, G. Marino, V. Cianfanelli, A. Ben-Younes, R. Troncoso, M. Markaki, V. Sica, V. Izzo, K. Chaba, C. Bauvy, N. Dupont, O. Kepp, P. Rockenfeller, H. Wolinski, F. Madeo, S. Lavandero, P. Codogno, F. Harper, G. Pierron, N. Tavernarakis, F. Cecconi, M.C. Maiuri, L. Galluzzi, G. Kroemer, Unsaturated fatty acids induce noncanonical autophagy, EMBO J. 34 (2015) 1025e1041. [21] S. Mei, H.M. Ni, S. Manley, A. Bockus, K.M. Kassel, J.P. Luyendyk, B.L. Copple, W.X. Ding, Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes, J. Pharmacol. Exp. Ther. 339 (2011) 487e498. [22] G. Bjorkoy, T. Lamark, S. Pankiv, A. Overvatn, A. Brech, T. Johansen, Monitoring autophagic degradation of p62/SQSTM1, Methods Enzymol. 452 (2009) 181e197. [23] R.J. Schulze, K. Drizyte, C.A. Casey, M.A. McNiven, Hepatic lipophagy: new insights into autophagic catabolism of lipid droplets in the liver, Hepatol. Commun. 1 (2017) 359e369. [24] S. Kaushik, A.M. Cuervo, Degradation of lipid droplet-associated proteins by chaperone-mediated autophagy facilitates lipolysis, Nat. Cell Biol. 17 (2015) 759e770. [25] R.J. Schulze, A. Sathyanarayan, D.G. Mashek, Breaking fat: the regulation and
[26]
[27]
[28]
[29] [30]
[31]
[32]
[33]
7
mechanisms of lipophagy, Biochimica et biophysica acta, Mol. Cell Biol. Lipids 1862 (2017) 1178e1187. Y.N. Rui, Z. Xu, B. Patel, Z. Chen, D. Chen, A. Tito, G. David, Y. Sun, E.F. Stimming, H.J. Bellen, A.M. Cuervo, S. Zhang, Huntingtin functions as a scaffold for selective macroautophagy, Nat. Cell Biol. 17 (2015) 262e275. T. Lam, R. Harmancey, H. Vasquez, B. Gilbert, N. Patel, V. Hariharan, A. Lee, M. Covey, H. Taegtmeyer, Reversal of intramyocellular lipid accumulation by lipophagy and a p62-mediated pathway, Cell Death Discov. 2 (2016) 16061. X. Xie, F. Li, Y. Wang, Y. Wang, Z. Lin, X. Cheng, J. Liu, C. Chen, L. Pan, Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2, Autophagy 11 (2015) 1775e1789. T.A. Ryan, D.A. Tumbarello, Optineurin: a coordinator of membraneassociated cargo trafficking and autophagy, Front. Immunol. 9 (2018) 1024. A. Werner, B. Herzog, O. Voigt, O. Valerius, G.H. Braus, S. Poggeler, NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog, Autophagy (2018) 1e20. T. Hanada, N.N. Noda, Y. Satomi, Y. Ichimura, Y. Fujioka, T. Takao, F. Inagaki, Y. Ohsumi, The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy, J. Biol. Chem. 282 (2007) 37298e37302. B.L. Farah, D.J. Landau, R.A. Sinha, E.D. Brooks, Y. Wu, S.Y.S. Fung, T. Tanaka, M. Hirayama, B.H. Bay, D.D. Koeberl, P.M. Yen, Induction of autophagy improves hepatic lipid metabolism in glucose-6-phosphatase deficiency, J. Hepatol. 64 (2016) 370e379. H. Koga, S. Kaushik, A.M. Cuervo, Altered lipid content inhibits autophagic vesicular fusion, FASEB J. : Off. Publ. Fed. Am. Soc. Exp. Biol. 24 (2010) 3052e3065.
Please cite this article as: D.-H. Lee et al., Oleic acid-induced defective autolysosome shows impaired lipid degradation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.04.040