Oleic Acid-Induced Hepatic Steatosis is Coupled with Downregulation of Aquaporin 3 and Upregulation of Aquaporin 9 via Activation of p38 Signaling

Abstract

Excess lipid deposition in hepatocytes is a hallmark feature of nonalcoholic fatty liver disease (NAFLD). The present study was designed to explore the expression and regulation of aquaporin (AQP) 3 and AQP9 in oleic acid-induced hepatic steatosis. HepG2 cells were incubated with oleic acid at different concentrations and time points. Oil-Red-O staining and triglyceride content measurement were done to assess the extent of hepatic steatosis. The expression of AQP3 and AQP9 was assessed using quantitative real-time PCR and Western blot analyses. The mitogen-activated protein kinase (MAPK) pathways involved in the regulation of AQP3 and AQP9 expression were checked. Compared to untreated control cells, oleic acid treatment significantly (p<0.05) induced hepatic steatosis in HepG2 cells in a dose- and time-dependent fashion. Oleic acid-treated cells showed a significant reduction in the AQP3 expression and a concomitant increase in the AQP9 expression. Oleic acid exposure led to enhanced phosphorylation of p38, but not ERK1/2 or JNK MAPK. Pharmacological inhibition of p38 rather than ERK1/2 signaling significantly blocked the regulation of AQP3 and AQP9 expression by oleic acid. Oleic acid-induced hepatic steatosis in HepG2 cells is associated with the coordinated regulation of AQP3 and AQP9 via activation of p38 signaling. These findings warrant functional studies of aquaglyceroporins in NAFLD.

Key words

channel proteins - lipid accumulation - nonalcoholic fatty liver disease - signaling pathway

• References

• 1 Cheung O, Sanyal AJ. Abnormalities of lipid metabolism in nonalcoholic fatty liver disease. Semin Liver Dis 2008; 28: 351-359
• 2 Tiniakos DG, Vos MB, Brunt EM. Nonalcoholic fatty liver disease: pathology and pathogenesis. Annu Rev Pathol 2010; 5: 145-171
• 3 Gao D, Nong S, Huang X, Lu Y, Zhao H, Lin Y, Man Y, Wang S, Yang J, Li J. The effects of palmitate on hepatic insulin resistance are mediated by NADPH Oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways. J Biol Chem 2010; 285: 29965-29973
• 4 Verkman AS. Aquaporins in clinical medicine. Annu Rev Med 2012; 63: 303-316
• 5 Ishibashi K, Kondo S, Hara S, Morishita Y. The evolutionary aspects of aquaporin family. Am J Physiol Regul Integr Comp Physiol 2011; 300: R566-R576
• 6 Buffoli B. Aquaporin biology and nervous system. Curr Neuropharmacol 2010; 8: 97-104
• 7 Ryu HM, Oh EJ, Park SH, Kim CD, Choi JY, Cho JH, Kim IS, Kwon TH, Chung HY, Yoo M, Kim YL. Aquaporin 3 expression is up-regulated by TGF-β1 in rat peritoneal mesothelial cells and plays a role in wound healing. Am J Pathol 2012; 181: 2047-2057
• 8 Rodríguez A, Catalán V, Gómez-Ambrosi J, García-Navarro S, Rotellar F, Valentí V, Silva C, Gil MJ, Salvador J, Burrell MA, Calamita G, Malagón MM, Frühbeck G. Insulin- and leptin-mediated control of aquaglyceroporins in human adipocytes and hepatocytes is mediated via the PI3K/Akt/mTOR signaling cascade. J Clin Endocrinol Metab 2011; 96: E586-E597
• 9 Rojek AM, Skowronski MT, Füchtbauer EM, Füchtbauer AC, Fenton RA, Agre P, Frøkiaer J, Nielsen S. Defective glycerol metabolism in aquaporin 9 (AQP9) knockout mice. Proc Natl Acad Sci USA 2007; 104: 3609-3614
• 10 Cai C, Wang C, Ji W, Liu B, Kang Y, Hu Z, Jiang Z. Knockdown of hepatic aquaglyceroporin-9 alleviates high fat diet-induced non-alcoholic fatty liver disease in rats. Int Immunopharmacol 2013; 15: 550-556
• 11 Wang C, Lv ZL, Kang YJ, Xiang TX, Wang PL, Jiang Z. Aquaporin-9 downregulation prevents steatosis in oleic acid-induced non-alcoholic fatty liver disease cell models. Int J Mol Med 2013; 32: 1159-1165
• 12 van der Vusse GJ. Albumin as fatty acid transporter. Drug Metab Pharmacokinet 2009; 24: 300-307
• 13 Pagliassotti MJ. Endoplasmic reticulum stress in nonalcoholic fatty liver disease. Annu Rev Nutr 2012; 32: 17-33
• 14 Nehra V, Angulo P, Buchman AL, Lindor KD. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis. Dig Dis Sci 2001; 46: 2347-2352
• 15 Soardo G, Donnini D, Domenis L, Catena C, De Silvestri D, Cappello D, Dibenedetto A, Carnelutti A, Bonasia V, Pagano C, Sechi LA. Oxidative stress is activated by free fatty acids in cultured human hepatocytes. Metab Syndr Relat Disord 2011; 9: 397-401
• 16 Lian Z, Li Y, Gao J, Qu K, Li J, Hao L, Wu S, Zhu H. A novel AMPK activator, WS070117, improves lipid metabolism discords in hamsters and HepG2 cells. Lipids Health Dis 2011; 10: 67
• 17 Vidyashankar S, Sandeep Varma R, Patki PS. Quercetin ameliorate insulin resistance and up-regulates cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells. Toxicol In Vitro 2013; 27: 945-953
• 18 Tilg H. Adipocytokines in nonalcoholic fatty liver disease: key players regulating steatosis, inflammation and fibrosis. Curr Pharm Des 2010; 16: 1893-1895
• 19 Vidyashankar S, Sandeep Varma R, Patki PS. Quercetin ameliorate insulin resistance and up-regulates cellular antioxidants during oleic acid induced hepatic steatosis in HepG2 cells. Toxicol In Vitro 2013; 27: 945-953
• 20 Vidyashankar S, Sharath Kumar LM, Barooah V, Sandeep Varma R, Nandakumar KS, Patki PS. Liv.52 up-regulates cellular antioxidants and increase glucose uptake to circumvent oleic acid induced hepatic steatosis in HepG2 cells. Phytomedicine 2012; 19: 1156-1165
• 21 Song CY, Shi J, Zeng X, Zhang Y, Xie WF, Chen YX. Sophocarpine alleviates hepatocyte steatosis through activating AMPK signaling pathway. Toxicol In Vitro 2013; 27: 1065-1071
• 22 Wu HT, Chen W, Cheng KC, Ku PM, Yeh CH, Cheng JT. Oleic acid activates peroxisome proliferator-activated receptor δ to compensate insulin resistance in steatotic cells. J Nutr Biochem 2012; 23: 1264-1270
• 23 Lee DH, Park DB, Lee YK, An CS, Oh YS, Kang JS, Kang SH, Chung MY. The effects of thiazolidinedione treatment on the regulations of aquaglyceroporins and glycerol kinase in OLETF rats. Metabolism 2005; 54: 1282-1289
• 24 Cai C, Wang C, Ji W, Liu B, Kang Y, Hu Z, Jiang Z. Knockdown of hepatic aquaglyceroporin-9 alleviates high fat diet-induced non-alcoholic fatty liver disease in rats. Int Immunopharmacol 2013; 15: 550-556
• 25 Rodríguez A, Catalán V, Gómez-Ambrosi J, Frühbeck G. Aquaglyceroporins serve as metabolic gateways in adiposity and insulin resistance control. Cell Cycle 2011; 10: 1548-1556
• 26 Calamita G, Gena P, Ferri D, Rosito A, Rojek A, Nielsen S, Marinelli RA, Frühbeck G, Svelto M. Biophysical assessment of aquaporin-9 as principal facilitative pathway in mouse liver import of glucogenetic glycerol. Biol Cell 2012; 104: 342-351
• 27 Hibuse T, Maeda N, Funahashi T, Yamamoto K, Nagasawa A, Mizunoya W, Kishida K, Inoue K, Kuriyama H, Nakamura T, Fushiki T, Kihara S, Shimomura I. Aquaporin 7 deficiency is associated with development of obesity through activation of adipose glycerol kinase. Proc Natl Acad Sci USA 2005; 102: 10993-10998
• 28 Sinha-Hikim I, Sinha-Hikim AP, Shen R, Kim HJ, French SW, Vaziri ND, Crum AC, Rajavashisth TB, Norris KC. A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice. Exp Mol Pathol 2011; 91: 419-428
• 29 Aghazadeh S, Yazdanparast R. Inhibition of JNK along with activation of ERK1/2 MAPK pathways improve steatohepatitis among the rats. Clin Nutr 2010; 29: 381-385
• 30 Patsouris D, Mandard S, Voshol PJ, Escher P, Tan NS, Havekes LM, Koenig W, März W, Tafuri S, Wahli W, Müller M, Kersten S. PPARalpha governs glycerol metabolism. J Clin Invest 2004; 114: 94-103