Silibinin Ameliorates Fructose-induced Lipid Accumulation and Activates Autophagy in HepG2 Cells

Author(s): Yang Li, Luping Ren*, Guangyao Song, Pu Zhang, Liying Yang, Xinwei Chen, Xiaolei Yu, Shuchun Chen.

Journal Name: Endocrine, Metabolic & Immune Disorders - Drug Targets
(Formerly Current Drug Targets - Immune, Endocrine & Metabolic Disorders)

Volume 19 , Issue 5 , 2019

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Graphical Abstract:


Background: Autophagy was recently regarded as a potential mechanism in nonalcoholic fatty liver disease. Silibinin (SIL), a natural flavonoid, has been used to prevent nonalcoholic fatty liver disease, however, the underlying mechanism is unclear. The aim of the present study was to explore the effect of SIL on hepatic steatosis and the possible link with autophagy.

Methods: The degree of hepatic steatosis in HepG2 cells was observed by oil-red O staining and triglyceride content. The effect of SIL on autophagy was tested by the Autophagy Detection Kit, and the expression of sterol regulatory element binding protein 1 (srebp-1), Fatty Acid Synthase (Fas), light chain 3, beclin-1, p62, AMP-activated Kinase (AMPK), and mammalian Target Of Rapamycin (mTOR) was examined by western blots.

Results: The lipid accumulation of HepG2 cells increased significantly in the high-fructose group compared to the control group. After SIL intervention, lipid accumulation was decreased. Using a fluorescence microscope, SIL was found to induce autophagy. Compared to control, the expressions of srebp-1, Fas, and phosphorylated-mTOR were increased by high-fructose, while the expressions of light chain 3 and beclin-1 decreased and srebp-1, Fas, and p62 were increased by autophagy inhibition. In contrast, opposite results were found in the SIL intervention group. The protein content of phosphorylated- mTOR was decreased, while phosphorylated-AMPK was increased in the SIL group compared to the high-fructose group.

Conclusion: SIL can reduce lipid accumulation in HepG2 cells exposed to high-fructose by inducing autophagy. The AMPK/mTOR signaling pathway could be one of the underlying molecular mechanisms.

Keywords: Silibinin, autophgay, fructose, lipid metabolism, AMPK, mTOR.

Yousef, M.H.; Al, J.A.; Albarrak, A.A.; Ibdah, J.A.; Tahan, V. Fatty liver without a large “belly”: Magnified review of non-alcoholic fatty liver disease in non-obese patients. World J. Gastrointest. Pathophysiol., 2017, 8(3), 100-107.
Namekawa, J.; Takagi, Y.; Wakabayashi, K.; Nakamura, Y.; Watanabe, A.; Nagakubo, D.; Shirai, M.; Asai, F. Effects of high-fat diet and fructose-rich diet on obesity, dyslipidemia and hyperglycemia in the WBN/Kob-Leprfa rat, a new model of type 2 diabetes mellitus. J. Vet. Med. Sci., 2017, 79(6), 988-991.
Johnson, R.J.; Nakagawa, T.; Sanchez-Lozada, L.G.; Shafiu, M.; Sundaram, S.; Le, M.; Ishimoto, T.; Sautin, Y.Y.; Lanaspa, M.A. Sugar, uric acid, and the etiology of diabetes and obesity. Diabetes, 2013, 62(10), 3307-3315.
Jegatheesan, P.; De Bandt, J.P. Fructose and NAFLD: the multifaceted aspects of fructose metabolism. Nutrients, 2017, 9(3), 230.
Baena, M.; Sanguesa, G.; Hutter, N.; Sanchez, R.M.; Roglans, N.; Laguna, J.C.; Alegret, M. Fructose supplementation impairs rat liver autophagy through mTORC activation without inducing endoplasmic reticulum stress. Biochim. Biophys. Acta, 2015, 1851(2), 107-116.
Debosch, B.J.; Chen, Z.; Saben, J.L.; Finck, B.N.; Moley, K.H. Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis. J. Biol. Chem., 2014, 289(16), 10989-10998.
Ezquerro, S.; Mendez-Gimenez, L.; Becerril, S.; Moncada, R.; Valenti, V.; Catalan, V.; Gomez-Ambrosi, J.; Fruhbeck, G.; Rodriguez, A. Acylated and desacyl ghrelin are associated with hepatic lipogenesis, beta-oxidation and autophagy: Role in NAFLD amelioration after sleeve gastrectomy in obese rats. Sci. Rep., 2016, 6(39942)
Rotman, Y.; Sanyal, A.J. Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease. Gut, 2017, 66(1), 180-190.
Abenavoli, L.; Bellentani, S. Milk thistle to treat non-alcoholic fatty liver disease: dream or reality? Expert Rev. Gastroenterol. Hepatol., 2013, 7(8), 677-679.
Gabrielova, E.; Zholobenko, A.V.; Bartosikova, L.; Necas, J.; Modriansky, M. Silymarin constituent 2,3-dehydrosilybin triggers reserpine-sensitive positive inotropic effect in perfused rat heart. PLoS One, 2015, 10(9)e139208
Gu, M.; Zhao, P.; Huang, J.; Zhao, Y.; Wang, Y.; Li, Y.; Li, Y.; Fan, S.; Ma, Y.M.; Tong, Q.; Yang, L.; Ji, G.; Huang, C. Silymarin ameliorates metabolic dysfunction associated with diet-induced obesity via activation of farnesyl x receptor. Front. Pharmacol., 2016, 7(345)
Stiuso, P.; Scognamiglio, I.; Murolo, M.; Ferranti, P.; De Simone, C.; Rizzo, M.R.; Tuccillo, C.; Caraglia, M.; Loguercio, C.; Federico, A. Serum oxidative stress markers and lipidomic profile to detect NASH patients responsive to an antioxidant treatment: A pilot study. Oxid. Med. Cell. Longev., 2014, 2014(2)169216
Polimeni, L.; Del, B.M.; Baratta, F.; Perri, L.; Albanese, F.; Pastori, D.; Violi, F.; Angelico, F. Oxidative stress: New insights on the association of non-alcoholic fatty liver disease and atherosclerosis. World J. Hepatol., 2015, 7(10), 1325-1336.
Song, X.; Liu, B.; Cui, L.; Zhou, B.; Liu, W.; Xu, F.; Hayashi, T.; Hattori, S.; Ushiki-Kaku, Y.; Tashiro, S.I.; Ikejima, T. Silibinin ameliorates anxiety/depression-like behaviors in amyloid beta-treated rats by upregulating BDNF/TrkB pathway and attenuating autophagy in hippocampus. Physiol. Behav., 2017, 179, 487-493.
Zheng, N.; Liu, L.; Liu, W.W.; Li, F.; Hayashi, T.; Tashiro, S.I.; Onodera, S.; Ikejima, T. Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro. Acta Pharmacol. Sin., 2017, 38(2), 277-289.
Wang, M.; Li, Y.J.; Ding, Y.; Zhang, H.N.; Sun, T.; Zhang, K.; Yang, L.; Guo, Y.Y.; Liu, S.B.; Zhao, M.G.; Wu, Y.M. Silibinin prevents autophagic cell death upon oxidative stress in cortical neurons and cerebral ischemia-reperfusion injury. Mol. Neurobiol., 2016, 53(2), 932-943.
Ghareghani, P.; Shanaki, M.; Ahmadi, S.; Khoshdel, A.R.; Rezvan, N.; Meshkani, R.; Delfan, M.; Gorgani-Firuzjaee, S. Aerobic endurance training improves nonalcoholic fatty liver disease (NAFLD) features via miR-33 dependent autophagy induction in high fat diet fed mice. Obes. Res. Clin. Pract., 2017, 12(2), 80-89.
Singh, R.; Kaushik, S.; Wang, Y.; Xiang, Y.; Novak, I.; Komatsu, M.; Tanaka, K.; Cuervo, A.M.; Czaja, M.J. Autophagy regulates lipid metabolism. Nature, 2009, 458(7242), 1131-1135.
Shibata, M.; Yoshimura, K.; Furuya, N.; Koike, M.; Ueno, T.; Komatsu, M.; Arai, H.; Tanaka, K.; Kominami, E.; Uchiyama, Y. The MAP1-LC3 conjugation system is involved in lipid droplet formation. Biochem. Biophys. Res. Commun., 2009, 382(2), 419-423.
Park, H.W.; Lee, J.H. Calcium channel blockers as potential therapeutics for obesity-associated autophagy defects and fatty liver pathologies. Autophagy, 2014, 10(12), 2385-2386.
Nascimbeni, F.; Pais, R.; Bellentani, S.; Day, C.P.; Ratziu, V.; Loria, P.; Lonardo, A. From NAFLD in clinical practice to answers from guidelines. J. Hepatol., 2013, 59(4), 859-871.
Ren, L.P.; Yu, X.; Song, G.Y.; Zhang, P.; Sun, L.N.; Chen, S.C.; Hu, Z.J.; Zhang, X.M. Impact of activating transcription factor 4 signaling on lipogenesis in HepG2 cells. Mol. Med. Rep., 2016, 14(2), 1649-1658.
Zhang, Y.; Hai, J.; Cao, M.; Zhang, Y.; Pei, S.; Wang, J.; Zhang, Q. Silibinin ameliorates steatosis and insulin resistance during non-alcoholic fatty liver disease development partly through targeting IRS-1/PI3K/Akt pathway. Int. Immunopharmacol., 2013, 17(3), 714-720.
Gu, M.; Zhao, P.; Huang, J.; Zhao, Y.; Wang, Y.; Li, Y.; Li, Y.; Fan, S.; Ma, Y.M.; Tong, Q.; Yang, L.; Ji, G.; Huang, C. Silymarin ameliorates metabolic dysfunction associated with diet-induced obesity via activation of farnesyl x receptor. Front. Pharmacol., 2016, 7(345)
Anding, A.L.; Baehrecke, E.H. Autophagy in cell life and cell death. Curr. Top. Dev. Biol., 2015, 114, 67-91.
Sinha, R.A.; You, S.H.; Zhou, J.; Siddique, M.M.; Bay, B.H.; Zhu, X.; Privalsky, M.L.; Cheng, S.Y.; Stevens, R.D.; Summers, S.A.; Newgard, C.B.; Lazar, M.A.; Yen, P.M. Thyroid hormone stimulates hepatic lipid catabolism via activation of autophagy. J. Clin. Invest., 2012, 122(7), 2428-2438.
Sinha, R.A.; Farah, B.L.; Singh, B.K.; Siddique, M.M.; Li, Y.; Wu, Y.; Ilkayeva, O.R.; Gooding, J.; Ching, J.; Zhou, J.; Martinez, L.; Xie, S.; Bay, B.H.; Summers, S.A.; Newgard, C.B.; Yen, P.M. Caffeine stimulates hepatic lipid metabolism by the autophagy-lysosomal pathway in mice. Hepatology, 2014, 59(4), 1366-1380.
Ma, D.; Molusky, M.M.; Song, J.; Hu, C.R.; Fang, F.; Rui, C.; Mathew, A.V.; Pennathur, S.; Liu, F.; Cheng, J.X.; Guan, J.L.; Lin, J.D. Autophagy deficiency by hepatic FIP200 deletion uncouples steatosis from liver injury in NAFLD. Mol. Endocrinol., 2013, 27(10), 1643-1654.
Seo, Y.K.; Jeon, T.I.; Chong, H.K.; Biesinger, J.; Xie, X.; Osborne, T.F. Genome-wide localization of SREBP-2 in hepatic chromatin predicts a role in autophagy. Cell Metab., 2011, 13(4), 367-375.
Nalbandian, A.; Llewellyn, K.J.; Nguyen, C.; Yazdi, P.G.; Kimonis, V.E. Rapamycin and chloroquine: The in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy. PLoS One, 2015, (10)4 e122888
Tanaka, S.; Hikita, H.; Tatsumi, T.; Sakamori, R.; Nozaki, Y.; Sakane, S.; Shiode, Y.; Nakabori, T.; Saito, Y.; Hiramatsu, N.; Tabata, K.; Kawabata, T.; Hamasaki, M.; Eguchi, H.; Nagano, H.; Yoshimori, T.; Takehara, T. Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice. Hepatology, 2016, 64(6), 1994-2014.
Figarola, J.L.; Singhal, J.; Tompkins, J.D.; Rogers, G.W.; Warden, C.; Horne, D.; Riggs, A.D.; Awasthi, S.; Singhal, S.S. SR4 uncouples mitochondrial oxidative phosphorylation, modulates amp-dependent Kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling, and inhibits proliferation of hepg2 hepatocarcinoma cells. J. Biol. Chem., 2015, 290(51), 30321-30341.
Smith, B.K.; Marcinko, K.; Desjardins, E.M.; Lally, J.S.; Ford, R.J.; Steinberg, G.R. Treatment of nonalcoholic fatty liver disease: Role of AMPK. Am. J. Physiol. Endocrinol. Metab., 2016, 311(4), E730-E740.
Figarola, J.L.; Singhal, J.; Tompkins, J.D.; Rogers, G.W.; Warden, C.; Horne, D.; Riggs, A.D.; Awasthi, S.; Singhal, S.S. SR4 uncouples mitochondrial oxidative phosphorylation, modulates AMP-dependent kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling, and inhibits proliferation of HepG2 hepatocarcinoma cells. J. Biol. Chem., 2015, 51, 30321-30341.
Kim, Y.C.; Guan, K.L. mTOR: A pharmacologic target for autophagy regulation. J. Clin. Invest., 2015, 125(1), 25-32.

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Year: 2019
Page: [632 - 642]
Pages: 11
DOI: 10.2174/1871530319666190207163325
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