Study design and subjects
The current investigation was experimental in vitro study. A total of eight subjects including four obese women with body mass index (BMI) more than 30 kg/m2 and four normal-weight women with BMI between 20 and 24.9 kg/m2 were recruited for the current study. All subjects were selected among individuals who referred to Vesal blood transfusion center, Tehran province, Tehran, Iran during September 2018–December 2018 were selected. Individuals with a disease such as malignancies, coronary artery disease, osteoporosis, liver and renal dysfunctions, type 1 and 2 diabetes mellitus, myocardial infarction, systemic or local infections, autoimmune diseases, chronic or acute inflammatory disease, severe heart failure, and asthma as well as evidence of alcohol abuse and cigarette smoking were excluded. None of the subjects received medications such as anti-inflammatory drugs, immunosuppressive drugs, insulin therapy, vitamins as well as even antioxidant and micronutrient supplements during the previous 3 months.
Sample and data collection
Initially, the characteristics of precipitants such as age, blood type, blood pressure, weight, and height were carefully obtained from subjects and then, BMI was calculated. The blood samples were collected from subjects. Different laboratory tests were applied to distinguish infections such as human immunodeficiency virus (HIV), hepatitis B, hepatitis C, and syphilis. After separating plasma, the samples were transferred to the Department of Clinical Biochemistry, Tehran University of Medical Sciences. The samples were stored at − 80 °C until the isolation of exosomes.
Isolation of plasma exosomes
Plasma exosomes were isolated by ultracentrifugation as described previously [20]. Briefly, the plasma samples were diluted 1:3 volumes in sterile phosphate-buffered saline (PBS) and were centrifuged at 17,000×g for 30 min at 4 °C to remove cell debris. The supernatant was centrifuged at 100,000×g for 75 min at 4 °C using a Beckman L5-65 ultracentrifuge (Beckman Instruments, Palo Alto, CA, USA). The pellet was resuspended in PBS, filtered (0.22 μm) and then, centrifuged at 100,000×g for 75 min at 4 °C. The pellet (containing exosomes) was resuspended in PBS, aliquoted and kept at − 80 °C. The concentration of exosomes (according to their protein concentration) was measured by the Bradford method to co-incubation with HepG2 cells [23]. The hydrodynamic size and zeta potential of exosomes were determined using a Zetasizer Nano-ZS dynamic light scattering (DLS) measurement system (Malvern Zetasizer, ZEN3600, UK).
Electron microscopic imaging of exosomes
Morphology and size of exosomes were determined using Transmission Electron Microscopy (TEM). Briefly, a drop of isolated exosomes (20 μL) was placed on 300 mesh carbon-coated TEM grid for 2 min, negatively stained with 2% aqueous uranyl acetate for 1 min. Then, the grid was examined on a Zeiss EM10C TEM operating at an accelerating voltage of 100 kV [24].
Cell culture and stimulation with exosome and insulin
HepG2 cell lines were purchased from the Iranian Biological Resource Center (IBRC). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 1% penicillin–streptomycin solution, 10% fetal bovine serum (FBS) and then, incubated at 37 °C and 5% CO2. For treatments, the cells were washed with PBS and the cells were serum-starved for 12 h. Then, the cells were incubated inserum-free-DMEM supplemented with 4 µg/mL plasma exosomes derived from obese and normal-weight women, or vehicle (PBS). After 24 h incubation, HepG2 cells were induced with insulin (100 nM) for 15 min [2]. After washing again with PBS, the cells were collected for future examinations. All experiments were done on the same passage of cells and repeated three times for removing technical variables. The stimulation with insulin was only used for measuring glycogen levels, the phosphorylation of Glycogen synthase kinase 3 beta (GSK3β) and the mRNA expression of glucose 6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) genes.
Cell viability assay
The cytotoxicity of plasma exosomes against HepG2 cells was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method (Sigma). Briefly, the cells were seeded in 96-well plates and treated with the doses of 0.25–64 µg/mL the pooled exosomes obtained from obese women (PO-Exo). After 24 h of incubation at 37 °C, the media were removed and 100 μL MTT solution (0.5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. After removing the MTT solution, 100 μL dimethyl sulfoxide (DMSO) was added to each well and the plate was shaken in dark for 10 min. Finally, the absorbance was determined at 570 nm using the microplate reader (BioTek, USA). After the determination of the dose, the MTT assay was again used to evaluating the effect of plasma exosomes derived from normal-weight and obese females on cell viability.
Measurement of triglyceride and glycogen
Glycogen levels were determined using the Glycogen Assay kit (Abcam Company, Cambridge, UK) following the manufacturer’s instructions. To the measurement of triglyceride (TG), the HepG2 cells were carefully washed four times with PBS and then, RIPA buffer was added to the cells on ice for 30 min. After ultrasonication, supernatant (50 µL) was applied for the measurement of protein using BCA assay. Next, a mixture of methanol and chloroform (1:2) were added to the rest of the supernatant on ice for 30 min and samples were centrifuged at 12,000×g for 5 min at 4 °C. After removing the upper solution, the lower solution was dried at 60 °C. Finally, 20 µL PBS was added to samples and vortexed for about one min [25]. TG levels were measured by kit based on the manufacturer’s instructions (Pars Azmoon Co., Tehran, Iran). The absorbance was measured at 570 and 546 nm for glycogen and TG assays using the ELISA reader (BioTek, USA), respectively.
Oil red O staining
For oil red O staining, 5 × 105 HepG2 cells were stained by the Oil Red O method to determine cellular lipid droplet accumulation. At first, HepG2 cells were washed several times with PBS and after fixation with 10% formalin, cells were stained with Oil Red O solution for 30 min at room temperature. Finally, the cells were washed four times with PBS and observed under a light microscope [26].
Measurement of hepatokines
The enzyme-linked immunosorbent assay (ELISA) method was carried out to detect the protein levels of hepatokines in the supernatant of HepG2 cells. The levels of hepatokines [fibroblast growth factor 21 (FGF21) and fetuin-A] were measured using the ELISA kit for FGF21 (R&D Systems, Inc., Minneapolis, MN), and for fetuin-A (Abcam Company, Cambridge, UK) under the supplier’s instructions. The absorbance was measured at 450 nm using the ELISA reader (BioTek, USA), respectively.
RNA extraction and quantitative real-time PCR
For evaluation of gluconeogenesis, gene expression of G6pase and PEPCK enzymes was determined using the quantitative real-time PCR (qRT-PCR). At first, RNA extraction was carried out using total RNA purification kit (Qiagen, GmbH, Hilden, Germany) and cDNA synthesis was performed using cDNA synthesis kit (TaKaRa Bio, Tokyo, Japan). Additional file 1: Table S1 showed the sequences of all used primers [27]. Gene expression of G6pase, PEPCK, and β-actin was determined by the qRT-PCR method using SYBR Green RealQ Plus Master Mix Green (Ampliqon, Skovlunde, Denmark) on the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, USA). The mRNA expression of G6pase and PEPCK genes was normalized to β-actin expression. The delta–delta CT method was used to calculate the relative gene expression [28].
Western blot analysis
The isolation of exosomes was approved by western blot analysis using the antibody of CD63 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Also, the phosphorylation of GSK3β at Ser-9 position was determined in the cells treated with the doses of 2 and 4 µg/mL the pooled exosomes obtained from normal-weight (PN-Exo) females and PO-Exo as well as in the cells treated with the dose of 4 µg/mL obese exosome (O-Exo) and normal-weight exosome (N-Exo) using the western blot analysis. Briefly, the cells were lysed by RIPA buffer (50 mM Tris–HCl, 1% Triton X-100, pH 7.4, 0.2% SDS, 0.2% sodium deoxycholate, 1 mM PMSF and 1 mM Na-EDTA) containing protease inhibitor cocktail and then, the cell lysate was fractionated using SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Five percent non-fat dry milk was applied to blocking. Immunoblots were incubated with antibodies against GAPDH, p-GSK3β, and GSK3β (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 18 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min. The bound proteins were observed by chemiluminescence using enhanced electrochemiluminescence (ECL) reagents and subsequent autoradiography. Eventually, the protein bands were quantitated using ImageJ software.
Statistical analysis
Descriptive statistics were applied to describe the participants’ characteristics. Continuous variables were evaluated for normality using the Shapiro–Wilk test. Not normally distributed data were logarithmically transformed. Normally distributed variables were considered as mean ± standard error of the mean (SEM) and differences between three and two groups were examined by one way ANOVA with Tukey HSD post hoc test and Student t-test, respectively. Not normally distributed variables were presented as the median and interquartile range (IQR) and differences between three and two groups were examined using the Kruskal–Wallis H and the Mann–Whitney U tests, respectively. The correlation between variables was measured by spearman correlation tests. P-value < 0.05 was considered significant. All analyses were carried out using IBM SPSS AMOS 21.0 (SPSS, Inc., Chicago, IL) and GraphPad Prism software, version 8 (GraphPad Software, La Jolla, CA).