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Research Article Higd1a Protects Cells from Lipotoxicity Under High-Fat Exposure

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Research Article Higd1a Protects Cells from Lipotoxicity Under High-Fat Exposure Hindawi Oxidative Medicine and Cellular Longevity Volume 2019, Article ID 6051262, 15 pages https://doi.org/10.1155/2019/6051262 Research Article Higd1a Protects Cells from Lipotoxicity under High-Fat Exposure Tong Li ,1 Wen-jing Xian ,2 Yang Gao ,1 Shuai Jiang ,1 Qi-hong Yu ,1 Qi-chang Zheng ,1 and Yong Zhang 1 1Department of Hepatobiliary Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China Correspondence should be addressed to Yong Zhang; [email protected] Received 3 November 2018; Revised 7 January 2019; Accepted 16 January 2019; Published 8 April 2019 Academic Editor: Sharad S. Singhal Copyright © 2019 Tong Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hypoxia-inducible gene domain family member 1A (Higd1a) has recently been reported to protect cells from hypoxia by helping to maintain normal mitochondrial function. The potential induction of Higd1a under high-fat exposure and whether it could protect cells from oxidative stress attracted our attention. Initially, 0.4 mM oleic acid and 0.2 mM palmitate were added to the growth media of HepG2 and LO2 cells for 72 hours. We discovered increased Higd1a expression, and knocking down Higd1a impaired mitochondrial transmembrane potential and induced cell apoptosis. We then identified that elevated reactive oxygen species (ROS) is responsible for increased Higd1a expression. Furthermore, we found that ROS promoted Higd1a expression by upregulating HIF-1a and PGC-1a expressions, and these two proteins could exert synergistic effects in inducing Higd1a expression. Taken together, these data suggest that Higd1a plays positive roles in protecting cells from oxidative stress, and ROS could induce Higd1a expression by upregulating PGC-1a and HIF-1a expressions. 1. Introduction [6, 7]. Impaired mitochondrial β-oxidation and defective mitochondrial respiratory chain subsequently contribute to Nonalcoholic fatty liver disease (NAFLD) is one of the most hepatic steatosis [8, 9]. Thus, protecting mitochondria from prevalent liver diseases and is characterized by a wide range oxidative stress is one of the key aspects in treating NAFLD. of alterations, including simple steatosis at early stages and Hypoxia-induced gene domain protein-1a (Higd1a) (also steatohepatitis in advanced stages, in which fatty liver is known as HIG1 or HIMP1-a) is a 10.4 kDa mitochondrial accompanied by inflammation, hepatocyte ballooning, liver inner membrane protein and is predicted to have two trans- fibrosis, and disrupted glucose homeostasis and insulin resis- membrane domains oriented in an “N-terminal outside-C- tance [1–3]. Due to the increasing prevalence of obesity and terminal outside and loop inside” conformation [10, 11]. type II diabetes, the incidence of NAFLD is increasing dra- Higd1a is induced by hypoxia and ischemia and may aid in matically, and NAFLD constitutes a global health concern, cell survival in the context of severe stress by protecting affecting not only adults but also children [4, 5]. Mitochon- mitochondria [12–16]. Higd1a could also be induced dur- dria play a central role in nutrient metabolism and provide ing central nervous maturation. Under this circumstance, energy required for a myriad of cell functions. In NAFLD Higd1a may facilitate cell apoptosis and promote tissue patients, the rates of fatty acid oxidation (FAO) exceed the remodeling [17]. Thus, the exact functions of Higd1a depend tricarboxylic acid cycle (TCA) capacity, resulting in mito- on the developmental stage and cellular microenvironment. chondrial fatty acid overload and leading to incomplete In the present study, we found that Higd1a expression is FAO and accumulation of reactive oxygen species (ROS) increased under high-fat exposure and protects cells from that contribute to mitochondria dysfunction and cell damage oxidative stress. Moreover, we found that ROS promotes 2 Oxidative Medicine and Cellular Longevity the expression of Higd1a by upregulating HIF-1a and PCG- 2.5. Oil Red O Staining. Treated cells were washed three times 1a. These results may help us to better understand the protec- with iced PBS and fixed with 4% paraformaldehyde for 30 tive response of cells under oxidative stress. minutes. After fixation, cells were washed three times and stained with Oil Red O solution (Shenggong, Shanghai, 2. Materials and Methods China) for 15 minutes at room temperature. Cells were then washed with PBS three times to remove redundant staining 2.1. Cells and Transfections. HepG2 and LO2 cells were and observed under a microscope. purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Medical Sciences (Shanghai, ° 2.6. Inhibitor Studies. The antioxidant N-acetyl-L-cysteine China). These cells were maintained at 37 C in DMEM was purchased from Sigma and was added to the growth with 10% fetal bovine serum (Gibco, Invitrogen, USA). media at a concentration of 1 mM. Higd1a siRNA (50 nM), HIF-1a siRNA (50 nM), PGC-1a siRNA (50 nM), pcDNA HIF-1a, pcDNA PGC-1a, and corre- 2.7. Western Blotting and Antibodies. Total cell proteins sponding negative controls were designed and synthesized were lysed in RIPA buffer with phosphatase inhibitors by RiboBio Co. (Guangzhou, China). Cell transfection was and a protease inhibitor cocktail (Sigma, USA). The follow- performed using Lipofectamine 3000 (Invitrogen, USA) ing antibodies for western blotting were purchased from according to the supplier’s protocol. All sequences are pro- Santa Cruz (USA): mouse anti-Higd1a (1 : 1000), rabbit vided in Supplementary Table 1. anti-HIF-1a, rabbit anti-PGC-1a (1 : 1000), and rabbit anti- GAPDH (1 : 1000). The rabbit/mouse secondary antibodies 2.2. Patients. Liver tissue specimens were obtained from (1 : 3000) were obtained from Proteintech, China. Proteins patients who underwent liver resection for benign liver were separated by 10% SDS-polyacrylamide gels and trans- disease (polycystic liver or hepatic hemangioma), and the ferred to PVDF membranes. After blocking, blots were specimens were obtained at least 2 cm away from the liver probed with specific antibodies at room temperature for 2 lesion. NAFLD was identified by pathological examination. hours and then incubated with the appropriate secondary Specimens were stored in liquid nitrogen within 30 minutes antibody. The protein bands were detected using a Bio-Rad after removal from patients during surgery. Informed con- imaging system (Bio-Rad, USA). All experiments were sent forms were signed by all patients. repeated at least three times, and the representative bands fi 2.3. Animals. C57BL/6 mice were housed in sterilized iso- are indicated in the gures. lators at 22 ± 1°C with 55-65% relative humidity and a 10 h light/14 h dark cycle. Mice were fed a standard diet 2.8. qRT-PCR. Total RNA was extracted from cells/liver (SD, composed of 20%, 70%, and 10% calories from protein, tissues using TRIzol reagent (Invitrogen) according to the ’ carbohydrate, and fat, respectively; Research Diets D12450B, manufacturer s instructions. Reverse transcription was per- Research Diets Inc., USA) or a high-fat diet (HFD, composed formed using a reverse transcription kit (Takara). Quantita- of 20%, 20%, and 60% calories from protein, carbohydrate, tive real-time reverse transcription PCR (qRT-PCR) was and fat, respectively; Research Diet D12492, Research Diets conducted using the quantitative SYBR Green PCR kit Inc., USA). Six weeks after feeding, adenovirus expressing (Takara). Targets were normalized to glyceraldehyde-3- Higd1a shRNA or control shRNA (Vigen Biosciences, phosphate dehydrogenase (GAPDH). All qRT-PCR reac- Shandong, China) (the sequences are shown in Supple- tions were performed in triplicate. The qRT-PCR primers mentary Table 1) at a titer of 4 ∗ 109 was injected to the are presented in Supplementary Table 2. caudal vein of HFD-fed mice. All mice were sacrificed and analyzed 7 days after virus injection. The Animal Care and 2.9. Measurement of Mitochondrial Transmembrane Use Committee of Union Hospital, Tongji Medical College, Potential. Mitochondrial transmembrane potential (MMP) Huazhong University of Science and Technology, approved assay kit with JC-1 (Beyotime, China) was used to assess the MMP. Briefly, cells were incubated with JC-1 working the animal protocol. The animal experiments adhered to fl ° the NIH Guide for the Care and Use of Laboratory Animals. uid at 37 C for 20 minutes. After incubation, cells were washed twice with JC-1 dyeing buffer. Fluorescence was then 2.4. Preparation of FFAs. Sodium oleate and sodium palmi- detected using a fluorescence microscope (Olympus, Japan). tate were dissolved separately in ethanol at a concentration of 100 mM. Then, BSA solution (20%, W/V) was heated to 2.10. Measurement of Reactive Oxygen Species Production. ° 45 C, and oleate and palmitate solutions were added to the After treatment of FFAs (or FFAs plus NAC) for 72 hours, BSA solution to obtain a mixed FA stock solution (5 mM; 5 μM DCFH-DA (2′,7′-dichlorodihydrofluorescein diace- oleate and palmitate at final ratio of 2 : 1). We also prepared tate, Beyotime, China) was added to the cells and incubated single fatty acid stock solutions to which only oleate or for 30 minutes. Following the incubation period, the cells palmitate solutions were added to the BSA solution. Stock were washed with PBS and analyzed using flow cytometry. solutions were then diluted in incubation medium contain- ing 5 mg/ml of fatty acid-free BSA (Boehringer Mannheim 2.11. Cell Proliferation Assay. A total of 5×103 cells per GmbH, Mannheim, Germany) to desired concentrations. well were seeded in 96-well plates. Seventy-two hours after HepG2 and LO2 cells at 80% confluence were exposed to FFA treatment, cell viability was determined using the the FFAs for 72 hours.
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