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Sulforaphane regulates abnormal lipid metabolism via both ERS-dependent XBP1/ACC &SCD1 and ERS-independent SREBP/FAS pathways Sicong Tian1,#, Baolong Li2,#, Peng Lei1, Xiuli Yang1, Xiaohong Zhang3,*, Yongping Bao4,*, Yujuan Shan1,* 1Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150090, China. 2Heilongjiang University of Chinese Medicine, Harbin, 150040, China. 3Institute of Preventative Medicine and Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, School of Medicine, Ningbo University, Zhejiang, 315211, China. 4Norwich Medical School, University of East Anglia, Norwich NR4 7UQ UK. #Contributed equally to this work *Corresponding author Abbreviations 4-PBA 4-Phenyl Butyric Acid ACC1 Acetyl CoA Carboxylase 1 ERS Endoplasmic Reticulum Stress FAS Fatty Acid Synthase Received: 25-Aug-2017; Revised: 14-Jan-2018; Accepted: 17-Jan-2018 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201700737. This article is protected by copyright. All rights reserved. www.mnf-journal.com Page 2 Molecular Nutrition & Food Research IRE1 Inositol-requiring Enzyme-1 LDs Lipid Droplets NAFLD Non-alcoholic Fat Liver Disease PERK Protein Kinase-like ER Kinase PPC Polyene Phosphatidylcholine SCD1 Stearoyl-CoA Desaturase 1 SFN Sulforaphane SREBP-1c Sterol Regulatory Element Binding Protein-1c TC Cholesterol TG Triglyceride UPR Unfolded Protein Response XBP1 X-Box Binding Protein 1 HFD High Fat Diet VLDL Very-Low-Density Lipoproteins HMGCS HMG-CoA synthase HMGCR HMG-CoA reductase Keywords: sulforaphane; lipid metabolism; sterol regulatory element binding protein-1c; endoplasmic reticulum stress; lipogenic enzymes Abstract Scope: To investigate the effect of sulforaphane (SFN) on the abnormal lipid metabolism and underlying mechanisms. This article is protected by copyright. All rights reserved. www.mnf-journal.com Page 3 Molecular Nutrition & Food Research Methods and results: Models with abnormal lipid metabolism were established both in rats and human hepatocytes. Hepatic steatosis was detected by H&E and oil red O staining. The structure of endoplasmic reticulum was visualized by transmission electron microscopy. The expressions of X-box binding protein 1 (XBP1), protein kinase-like ER kinase (PERK), sterol regulatory element binding protein-1c (SREBP1c) and lipogenic enzymes were determined by real-time PCR and western blot analysis. SFN lowered the content of triglyceride and cholesterol. SFN alleviated the swelling of endoplasmic reticulum (ER) and decreased the perimeter of ER. SFN significantly decreased the expressions of acetyl CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase 1 (SCD1) and fatty acid synthase. SFN inhibited SREBP1c by blocking the PERK. Meanwhile, SFN suppressed ACC1 and SCD1 via blocking the formation of splicing-type XBP1. The key roles of XBP1 and SREBP1c in SFN-reduced lipid droplets were confirmed by a timed sequence of measurement according to time points. Conclusion: SFN improved abnormal lipid metabolism via both ER stress -dependent and -independent pathways. Introduction The Westernization of dietary patterns in China may lead to the excessive intake of fats and accumulation of lipids in the body which can cause non-alcoholic fat liver disease (NAFLD), diabetes, obesity, cardiovascular disease, atherosclerosis, etc. [1-2]. In addition to the physical activities, the dietary habit is more critical way to be adjusted. NAFLD, characteristic of hepatic lipid accumulation, results from an imbalance between lipid synthesis, storage, oxidation, and/or secretion, and eventually triggers hepatitis hepatocirrhosis and even hepatoma [3]. Lipids such as triglyceride (TG) and cholesterol (TC) are stored in the form of lipid droplets (LDs) in hepatocytes. Lipids are synthesized in the endoplasmic reticulum (ER) together with the folding of membrane proteins and secretory proteins. An overload of unfolded or misfolded proteins may result in excessive fatty acids retention in the ER followed by the ER stress (ERS) which in turn aggravates the lipid accumulation. Long term lipid accumulation in the non-adipose cells i.e. hepatocytes, could exceed the capacity of the cells to manage lipids and cause further chronic ERS and lipotoxicity [4]. This article is protected by copyright. All rights reserved. www.mnf-journal.com Page 4 Molecular Nutrition & Food Research Mono-unsaturated fatty acids have the ability to attenuate acute ERS induced by palmitate, while excess fatty acid exposure over long periods of time can exceed the capacity, especially of non-adipose cells. For example, short-term treatment of cells with oleic acid increases very-low-density lipoproteins (VLDL) and triglyceride secretion, whereas prolonged incubation of cells with oleic acid can lead to ERS and of VLDL secretion [5]. When ERS occurs, the ER initiates the unfolded protein response (UPR) through the actions of canonical sensors IRE1, PERK, and ATF6 to restore ER homeostasis. The excessive accumulation of unfolded or misfolded proteins stimulates the ERS sensors to uncouple from GRP78 [6-7]. Among these sensors, IRE1 a ribonuclease that splices uXBP1 transcripts into the active sXBP1. Then sXBP1 is translocated into the nucleus to promote the transcription of lipogenic enzymes such as acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase 1 (SCD1) [8]. In addition, PERK regulates fatty acid synthase (FAS) via sterol regulatory element binding protein-1c (SREBP1c) [9]. In the past few decades, plant-derived bioactives have been demonstrated to have the potential to prevent lipids-associated disorders. An epidemiological study involving 1938 healthy adults showed that intake of adequate phytochemicals was beneficial, lowering body weight and body fat [10]. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, especially in broccoli, was reported to regulate a number of metabolic enzymes including phase I & phase II metabolic enzymes, and lipid metabolism-related enzymes/proteins [11]. Oral administration of the sulfur-radish extract and of sulforaphane after carbon tetrachloride (CCl4)-induced liver injury both decreased the serum level of alanine aminotransferase reduced the necrotic zones, inhibited lipid peroxidation, and induced phase 2 enzymes without affecting cytochrome P450-2E1 (CYP2E1). [12]. Moreover, long-term consumption of whole broccoli counters NAFLD development enhanced by a Western diet as well as hepatic tumorigenesis induced by DEN in B6C3F1 mice. [13] Therefore, we further hypothesized that SFN presents a preventive effect on the abnormal lipid metabolism via ERS mechanisms. In the present study, high-lipid induced abnormal lipid metabolism models were established both in vitro and in vivo to explore the protective effects of SFN and focusing on the ER-dependent/independent mechanisms. Material and Method This article is protected by copyright. All rights reserved. www.mnf-journal.com Page 5 Molecular Nutrition & Food Research 2.1 Reagent and Preparation Sulforaphane (1-isothiocyanato-4-(methylsulfinyl) butane, SFN, purity≥98%), purchased from LKT laboratories (St. Paul, MN), stock solution (100 mmol/L) was prepared in dimethyl sulfoxide (DMSO) and stored -20oC. 4-phenyl butyric acid (4-PBA) was purchased from Sigma-Aldrich (Saint Louis, USA). Antibodies against SREBP-1c, XBP1, lamin B were from Proteintech Biotechnologies (USA). Antibody against PERK was from Santa Cruz Biotechnology (USA). TRIZOL reagent kit, TransScript One-Step gDNA Removel and cDNA Synthesis SuperMix kit, TransStart Top Green qPCR SuperMix kit were purchased from TransGen Biotech (China). Kits for measuring triglycerides and cholesterol were purchased from Nanjing Jiancheng Biotechnologies (China). The animal chow was purchased from Keaoxieli Feed Co., Ltd (Beijing, China). Polyene Phosphatidylcholine (PPC) was purchased from Pharmacy (China) and dissolved in soya-bean oil. Oleic acid and palmitic acid were bought from Sigma-Aldrich (Saint Louis, USA). Palmitic acid was prepared as described previously [14]. Briefly, palmitate and oleate were dissolved in 0.1 mol/L NaOH at 70oC and filtered respectively to prepare 100 mmol/L palmitate stocks and 100 mmol/L oleate stocks. Five percent (w/v) FFA-free BSA (Sigma) solution was prepared in double-distilled H2O. 100 mmol/L FA and 5% BSA solution were mixed by 1:1 (v/v) in at 60℃ and filtered. 2.2 Cell Culture and Treatment Immortalized human hepatocytes (defined as HHL-5) were kindly supplied by Professor Arvind Petal, Medical Research Council (MRC) Virology Unit, UK. HHL-5 hepatocytes were cultured in DMEM/F12 (1:1) medium with 10% fetal bovine serum, at 37oC and 5% carbon dioxide. After reaching 70% confluence, cells were pre-treated with SFN once (1, 5, 10, 20 µmol/L) for 24 h, followed by 0.25 mmol/L FAs (oleic acid: palmitic acid, 2:1) for 5 days. 2.3 Establishment of NAFLD Animal Model and Treatments All the procedures were conducted in conformity with the guidelines of the Institutional Animal Care Use Committee, Heilongjiang