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The Role of Overexpressed Apolipoprotein AV in Insulin-Resistant Hepatocytes

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The Role of Overexpressed Apolipoprotein AV in Insulin-Resistant Hepatocytes Hindawi BioMed Research International Volume 2020, Article ID 3268505, 13 pages https://doi.org/10.1155/2020/3268505 Research Article The Role of Overexpressed Apolipoprotein AV in Insulin-Resistant Hepatocytes Wang Zhao,1 Yaqiong Liu,1 Xiaobo Liao,2 and Shuiping Zhao 1 1Department of Cardiovascular Medicine, e Second Xiangya Hospital, Central South University, No. 139 Middle Renmin Road, Changsha, Hunan 410011, China 2Department of Cardiovascular Surgery, e Second Xiangya Hospital, Central South University, No. 139 Middle Renmin Road, Changsha, Hunan 410011, China Correspondence should be addressed to Shuiping Zhao; [email protected] Received 31 January 2019; Revised 16 July 2019; Accepted 16 August 2019; Published 21 April 2020 Academic Editor: Kui Li Copyright © 2020 Wang Zhao et al. *is 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. In this paper, we sought to explore the relationship between apolipoprotein AV (APOAV) overexpression and insulin resistance in hepatocytes. *e insulin-resistant HepG2 cell model was constructed, and then, APOAV-overexpressed HepG2 cells (B-M) were induced by infecting with a recombinant adenovirus vector. Microarray data were developed from B-M samples compared with negative controls (A-con), and the microarray data were analyzed by bioinformatic methods. APOAV-overexpression induced 313 upregulated genes and 563 downregulated ones in B-M sample. *e differentially expressed genes (DEGs) were significantly classified in fat digestion and absorption pathway. Protein-protein interaction network was constructed, and AGTR1 (angiotensin II receptor type 1) and P2RY2 (purinergic receptor P2Y, G-protein coupled 2) were found to be the significant nodes closely related with G-protein related signaling. Additionally, overexpression of APOAV could change the expression of Glut4 and release the insulin resistance of hepatic cells. *us, APOAV overexpression may prevent the insulin resistance in liver cells by mediating the genes such as AGTR1 and P2RY2. 1. Introduction lipid metabolism [6, 7]. APOAV is mainly expressed in liver and closely associated with high-density lipoprotein [8]. Insulin resistance has become an increasingly common Moreover, APOAV is also found to participate in the metabolic syndrome in people around the word. *e main metabolism of triglycerides by stimulating hydrolysis of understanding of the mechanism for insulin resistance is the triglycerides, reducing the lipoprotein production rate, and glycometabolism disorder in liver [1]. Insulin resistance facilitating clearance of triglyceride-rich lipoprotein [9]. A results in excessive glucose consumption in liver, which previous study suggests that APOAV is downregulated in contributes to the development of hyperglycemia [2]. In insulin-treated mice and the expression of APOAV a con- addition, hyperglycemia is frequently associated with met- tributor for hypertriglyceridemia [10]. *e decreased ex- abolic syndromes such as hypertension, coronary heart pression APOAV is involved in insulin resistance-related disease, and diabetes [3, 4]. Type 2 diabetes (T2D) is a hypertriglyceridemia [6]. APOAV seems to be closely related common metabolic disorder that characterizes high blood with insulin resistance in diabetics, but whether the over- sugar, insulin deficiency, and insulin resistance. T2D (non- expression of APOAV could prevent insulin resistance needs insulin-dependent) is the risk for cardiovascular disease that to be further determined. is the main cause for death of diabetes patients [5]. Microarray technology has been widely used to generate Recently, apolipoprotein AV (APOAV), also served as the gene expression data on a genomic scale [11]. In this the APOA5, has been found to be a novel apolipoprotein and paper, we performed the microarray analysis for the human to play a key role in the processes of insulin resistance and hepatoma (HepG2) cells to discover the differentially 2 BioMed Research International expressed genes (DEGs) induced by APOAV overexpression. then transferred to a polyvinylidene difluoride (PVDF) HepG2 cells, which are characterized by the common membrane. After blocked with 5% skimmed milk for 1 h, the physiological function in glucose metabolism with normal membrane was incubated with primary antibodies hepatic cells [12], were used in this study. *erefore, in the (APOAV5, 1 :1000; beta actin, 1 :1000) at 4°C overnight. present study, we sought to explore the potential mechanism Following washing with TBST for 3 times, the membrane of relationship between APOAV expression and insulin was incubated with secondary antibody (antirabbit IgG- resistance in hepatocytes. HRP, 1 : 5000; antimouse IgG-HRP, 1 : 5000) for 2 h at room temperature. Finally, the proteins were visualized using the 2. Materials and Methods ECL chemiluminescence reagent. 2.1. Insulin-Resistant HepG2 Cell Model Construction. 2.3. Recombinant Plasmid Vector with APOAV. *e APOAV HepG2 cell lines (Cell Culture Center of Peking Union gene was amplified by PCR with the primers, such as 5′-ACA ° Medical Science, Beijing, China) were cultured at 37 C in CGGATCCATGGCAAGCATGGCTGCCGT-3′ (forward) Dulbecco’s modified eagle medium (DMEM) supplemented and 5′-ACACGAATTCTCAGGGGTCCCCCAGATG-3′ with 10% (v/v) FBS (fetal bovine serum) and 100 IU peni- (reverse). After the plasmid pHBAd-MCMV-GFP was cillin/streptomycin/amphotericin in a humidified 5% CO2 digested with BamH1 and EcoR1, the recombinant plasmid atmosphere. When entering the logarithmic phase, the cells vector was constructed by connecting with APOAV at 4°C were washed by PBS, and then digested with trypsin enzyme. overnight. *e positive recombinant clone was named as After the cells turned round, cell suspension was put into pHBAd-MCMV-APOAV-GFP. *e expressions of APOAV 15 ml centrifuge tube and centrifuged at 1000 rpm for 3 min 3 and GFP were regulated by cytomegalovirus (CMV) and inoculated in 96-well plates at the density of 5 ×10 cells/ promoter. cm2. In order obtain the insulin-resistant HepG2 cell model, the cells were treated with insulin at a series of concentration 2.4. Insulin-Resistant HepG2 Cells Infection by Recombinant gradients. *e insulin-resistant HepG2 cell model was in- Adenovirus. HEK293 was the common cell for packing the duced as described previously [13]. Briefly, HepG2 cells were recombinant adenoviruses because of its E1 genes. To subjected to 24h-culture of 5 ×10−5, 5 ×10−6, 5 ×10−7, and generate recombinant adenovirus vectors with APOAV 5 ×10−8 mol/L bovine insulin, followed by incubating in expression, the pHBAd-MCMV-APOAV-GFP and adeno- 10−9 mol/L insulin for 24 h. And to determine whether the virus skeleton plasmid vector of pHBAd-BHG were coin- insulin model was successfully constructed, glucose uptake fected into HEK293 cells with the lipofectamine 2000 kit rate and liver glycogen synthesis were measured. For glucose following the manufacturer’s instruction. After 6 h trans- uptake rate measurement, the supernatant was absorbed into formation, the recombinant adenoviruses were harvested. a 15 mL centrifuge tube at 1500 rpm/6 min, and transferred *en, the insulin-resistant HepG2 cells were cultured and to a 1.5 ml EP tube. After the supernatant was diluted 6 times infected with the recombinant adenovirus vector for 48 h. with distilled water, and the glucose uptake rate was de- *e successful infection by recombinant adenovirus was termined by the urine glucose assay kit-oxidase assay determined, when the expression of GFP was observed by according to the manufacturer’s instructions. Glucose up- fluorescence microscopy. take rate � (preinduced glucose concentration-postinduced glucose concentration)/preinduced glucose concentration. 2.5. RNA Isolation. Total RNA of insulin-resistant HepG2 For glycogen synthesis measurement, (1) the cells were cells with APOAV overexpression (B-M) and negative digested with trypsin, centrifuged (3000 g, 5 min) and controls (A-con) was isolated using TRIzol reagent (Invi- weighed; (2) hydrolysis: sample weight (mg): lye volume trogen, Carlsbad, CA) according to the manufacturer’s (μL) � 1 : 3, boiled in boiling water for 20 min; (3) liver protocol. *e expected quality of RNA was determined by glycogen detection solution (1%); (4) the standard glucose measuring the absorbance ratios (A260/A280) between 1.8 solution: the standard glucose solution was prepared into 25, and 2.0 under the spectrophotometer. 12.5, 6.25, 3.125, and 1.5625 g/ml with distilled water; (5) OD620 nm value of each tube was measured; and (6) standard curve was drawn for calculating the liver glycogen 2.6. Microarray Analysis. *e RNA library was constructed concentration. by using the NEBNext ultra directional RNA library prep kit for illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s instruction. After the library 2.2. Western Blot Analysis. To determine the native APOAV quality was assessed by Aglient 2100 Bioanalyzer (Agilent expression in the induced model cells and insulin-resistant Technologies, Palo Alto, CA), the next-generation se- HepG2 cells infected by recombinant adenovirus, western quencing was performed based on the Illumina HiSeq4000 blot analysis was performed. Briefly, the cells were lysed in a platform (Illumina Inc., San Diego, CA). Read counts were radioimmunoprecipitation assay (RIPA) buffer supple- normalized by TMM (trimmed mean of M values) in the mented with for
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