Quercetin-3-O-α-L-arabinopyranosyl-(1→2)-β-D-glucopyranoside Isolated from Eucommia ulmoides Leaf Relieves Insulin Resistance in HepG2 Cells via the IRS-1/PI3K/Akt/GSK-3β Pathway

Abstract

For nearly 2000 years, Eucommia ulmoides Oliver (EUO) has been utilized in traditional Chinese medicine (TCM) throughout China. Flavonoids present in bark and leaves of EUO are responsible for their antioxidant, anti-inflammatory, antitumor, anti-osteoporosis, hypoglycemic, hypolipidemic, antibacterial, and antiviral properties, but the main bioactive compound has not been established yet. In this study, we isolated and identified quercetin glycoside (QAG) from EUO leaves (EUOL) and preliminarily explored its molecular mechanism in improving insulin resistance (IR). The results showed that QAG increased uptake of glucose as well as glycogen production in the palmitic acid (PA)-induced HepG2 cells in a dose-dependent way. Further, we observed that QAG increases glucose transporters 2 and 4 (GLUT2 and GLUT4) expression and suppresses the phosphorylation of insulin receptor substrate (IRS)-1 at serine⁶¹², thus promoting the expression of phosphatidylinositol-3-kinase (PI3K) at tyrosine⁴⁵⁸ and tyrosine¹⁹⁹, as well as protein kinase B (Akt) and glycogen synthase kinase (GSK)-3β at serine⁴⁷³ and serine⁹, respectively. The influence posed by QAG on the improvement of uptake of glucose was significantly inhibited by LY294002, a PI3K inhibitor. In addition, the molecular docking result showed that QAG could bind to insulin receptors. In summary, our data established that QAG improved IR as demonstrated by the increased uptake of glucose and glycogen production through a signaling pathway called IRS-1/PI3K/Akt/GSK-3β.

INTRODUCTION

Diabetes mellitus (DM) is a highly prevalent metabolic disease and is hallmarked by elevated levels of blood glucose.^1,2) Diabetes prevalence has risen dramatically in recent years. Diabetes is known to affect 415 million people worldwide in 2015, and the total number of patients is expected to surge to 642 million by the year 2040, with grave social, financial, and healthcare consequences.³⁾ Type 2 diabetes mellitus (T2DM) accounts for approximately 90% of all DM cases.³⁾ Insulin resistance (IR) is attributed to inadequate insulin concentration in target cells, including hepatocytes, skeletal muscle cells, and adipocytes, all of which play important roles in developing metabolic disorders such as T2DM, hypertension, and hyperlipidemia.^1,4,5) IR is also considered a risk factor for kidney failure, retinopathy, neuropathy, vascular morbidities, and myocardial infarction.⁴⁾ As a result, it is critical to design drugs that effectively improve IR and bring benefits to IR-related diseases.

The liver is a primary site for insulin action, regulating blood glucose homeostasis by maintaining an equilibrium between glycogen storage (glycogenesis) and glucose generation via degradation of glycogen (glycogenolysis) or by de novo glucose synthesis (gluconeogenesis).⁶⁾ The core of T2DM pathophysiology, as demonstrated by several studies, incorporates the deposition of lipid metabolites in the hepatic system and skeletal muscles because of raised plasma free fatty acids (FFAs), which are closely related to IR.^4,7–11) Moreover, circulating FFAs and their metabolites can directly enter the liver, and inhibit insulin-stimulated uptake of glucose and glycogen production via interfering with the insulin signaling system.^12,13) Palmitic acid (PA) is one of the most distributed FFAs in human plasma, accounting for around 30% of total FFAs, and it has been demonstrated to mediate IR in cultured HepG2 cells.¹¹⁾ The insulin receptor substrates (IRS)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) biochemical pathway plays a pivotal role in mediating insulin metabolism.^14–17) Under conditions, a signaling biochemical pathway (IRS/PI3K/Akt/GSK-3β) is activated by insulin binding to insulin receptors, which then initiates the uptake of glucose and glycogen synthesis. Additionally, glucose transporters (GLUTs) also play a key role in blood-tissue glucose homeostasis, and GLUT2 and GLUT4, particularly GLUT2, are the primary GLUT isoforms in the liver.

For nearly 2000 years, Eucommia ulmoides Oliver (EUO), a single member of the Eucommia genus belonging to the Eucommiaceae family, has been utilized in traditional Chinese medicine (TCM). The bark, cortex, and leaves of EUO, are used as medicinal ingredients and have been listed in the Chinese Pharmacopoeia (2015 Edition).^18,19) One study has shown that EUO is rich in phytoconstituents, such as lignans, flavonoids, phenol, phenylpropanoids, steroids, terpenoids, and gutta-percha.¹⁸⁾ EUO possesses anti-hypertensive, anti-oxidant, anti-inflammatory, hypoglycemic, hepatoprotective, and neuroprotective activities.^18,20,21) Many studies have shown that EUO leaves (EUOL) and bark have similar compositions, and the hypoglycemic effect and IR amelioration of EUOL have been increasingly explained in recent years.^22–26) In the present work, a quercetin glycoside named quercetin-3-O-α-L-arabinopyranosyl-(1→2)-β-D-glucopyranoside (QAG), has been obtained from EUOL by applying high-speed countercurrent chromatography (HSCCC) as well as preparative HPLC, whereas identification was based on ultra HPLC equipped with quadrupole and time of flight mass spectrometry (UPLC-Q/TOF-MS) and NMR instruments analysis. The impact of QAG on IR as well as its probable molecular mechanism was investigated by utilizing a HepG2 cell model induced by PA. The observation derived from this work provides basic experimental proof for developing QAG as a drug to improve IR.

MATERIALS AND METHODS

Reagents, Primer and Antibodies

Metformin hydrochloride, bovine serum albumin (BSA), and PA were commercially obtained from the Dalian Meilun Biotechnology Co., Ltd. (Liaoning, China). LY294002 (LY, T2008) was obtained from Topscience Co., Ltd. (Shanghai, China). GLUT2 and GLUT4 (Invitrogen, Carlsbad, CA, U.S.A.) were used to synthesize glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. Antibodies used in this work have been acquired from Cell Signaling Technology, Inc. (Danvers, MA, U.S.A.): p-IRS-1 (#3203), IRS-1 (#2382), p-PI3K (#4228), PI3K (#4292), p-Akt (#4056), Akt (#9272), p-GSK-3β (#9322), GSK-3β (#9315), and GLUT4 (#2213). However, GLUT2 (#ab192599) antibody was bought from Abcam (Cambridge, MA, U.S.A.). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody and HRP-conjugated goat anti-mouse antibody are being made available from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Collection, Isolation, Purification and Identification of QAG

The EUOL was purchased from Anguo Chinese Medicinal Materials Market Co., Ltd. (Baoding, Hebei, China). Professor Pixian Shui of the Department of Pharmacognosy verified the EUOL crude materials. The powdered EUOL was extracted twice with eight times the volume of 70% ethanol at 70 °C for 3 h. Subsequently, the extract was evaporated until it got dried completely so as to obtain the total ethanol extract. The total extract was further purified using HSCCC (Tauto Biotech Co., Ltd., Shanghai, China) and preparative HPLC (BUCHI Labortechnik AG, Switzerland). The purity and molecular weight of QAG were determined by HPLC and UPLC-Q/TOF-MS, respectively. An LC2030 HPLC (Shimadzu Corporation, Kyoto, Japan) containing two pumps (LC-20AD), an autosampler (SIL-20AC), a column oven (CTO-20AC), and an SPD-20AV detector system was used for analyzing QAG. QAG was dissolved in methanol and separated on a Thermo Hypersil BDS C₁₈ column (250 × 4.6 mm, 5 µm) (Thermo Fisher Scientific, Waltham, MA, U.S.A.) with a flow rate (FR) of (1 mL min⁻¹). An elution system with gradient mode having phosphoric acid (H₃PO₄) (0.1%) in H₂O as solvent A and acetonitrile (CH₃CN) as solvent B has been set as follows: 0–50 min, 92–84% (A). We maintained a column temperature of 30 °C while the UV absorption spectra were recorded at 360 nm. QAG was identified by the Acquity UPLC system coupled with quadrupole and time of flight mass spectrometry (Q-TOF-MS), ¹³C-NMR, and ¹H-NMR. UPLC with a component of the binary solvent delivery system, an autosampler as well as a Kinetex C₁₈ column (100 × 2.1 mm, 2.6 µm) was used while maintaining the FR at 0.3 mL min⁻¹. A gradient elution system consisting of 0.1% formic acid-water (A) and acetonitrile (B) was set as follows: 0–10 min, 95–0% (A); 10–12 min, 0–0% (A); 12–16 min, 0–95% (A). 5 µL volume of sample injection was used and temperature of column was kept at 30 °C. The MS measurements have been carried out using Waters VION LC Q-TOF instrument equipped with a Turbo V source in positive ion mode. The following were the optimum parameters for mass spectrometric analysis: ion source temperature, 500 °C; atomized gas, 50 psi; auxiliary gas, 50 psi; air curtain gas, 35 psi; spray voltage, 5.5 kV; collision energies, 10 V.

Cell Culture

The HepG2 cells, which are human hepatic carcinoma cell lines that have the basic characteristics of a normal liver cell, were obtained from the China Typical Culture Preservation Center (Wuhan, Hubei, China). The HepG2 cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Rockville, MD, U.S.A.) supplemented with a fetal bovine serum of 10% concentration (FBS, Gibco) as well as antibiotics penicillin (100 U/mL) and streptomycin (100 µg/mL) (Beyotime, Shanghai, China). The cells were incubated at 37 °C, with 5% CO₂ and 70% humidity conditions. In addition, the medium was changed every two days.

Preparation of Fatty Acid (FA)

FFAs were produced according to the previous method.^7,27) A solvent containing PA (100 mM) in NaOH (0.1 nM) was made and subjected to heating at 70 °C. The solution was then mixed in a 1 : 1 ratio with 10% endotoxin/fatty acid-free BSA to produce a 50 mM stock concentration. Prior to use, the PA/BSA solution was sterile, filtered, and stored at −20 °C.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

HepG2 cells in a volume (100 µL) were seeded in a 96-well plate with a cell density of 1 × 10⁴ cells/well and cultured overnight. The HepG2 cells were thereafter treated with QAG and/or PA for 36 h, and then the viability of cells was measured using MTT reagent (Sigma-Aldrich, St. Louis, MO, U.S.A.).²⁸⁾ For this, following the treatment, the media was replaced by a new DMEM containing 0.5 mg/mL MTT (Sigma-Aldrich), and the incubation of the cells was carried out at 37 °C for 3 h. After gently aspirating the solution, the formazan crystals were dissolved in 100 L of dimethyl sulfoxide (DMSO). Using a Cytation 3 image reader, this solution’s absorbance is assessed at a wavelength of 570 nm (BioTek, VT Lab, U.S.A.). Cell viability in terms of percentage was calculated by applying the formula below: CV (%) = (CN_treated/CN_control) × 100%.

  

Assay Investigating the Uptake of Glucose

A volume of 100 µL of HepG2 cells was seeded in wells of 96-well plate using a cell density of 1 × 10⁴ cells/well followed by culturing overnight. Afterward, the PA (0.5 mM)-induced HepG2 cells were subjected to treatment with QAG (2, 4, 8 µM) or metformin (5 mM) for 36 h. Following treatments, the medium replacement was done using a phenol red-free medium, and cells were incubated continuously for 24 h. At the same time, the same volume of phenol red-free medium was added to the wells without cells as negative control. Then, the content of glucose in the phenol red-free medium was then determined by hexokinase method using the glucose content detection kit (Maccura Biotechnology Co., Ltd., Chengdu, Sichuan, China) as per the manufacturer’s protocols. Glucose consumption rate in terms of percentage was calculated by applying the formula below: Glucose consumption rate (%) = (GC_negative – GC_treated)/(GC_negative – GC_control) × 100%. GC = Glucose content.

Glycogen Staining

A volume of 1 mL of HepG2 cells was seeded in the wells of a 6-well plate at a cell density of 2 × 10⁵ cells/well and subjected to incubation for achieving overnight growth. Cells were subsequently co-treated for 36 h with PA (0.5 mM) and QAG (2, 4, 8 µM) or metformin (5 mM). Following treatment, the medium was changed with a phenol red-free mixture, and the incubation period was extended to 24 h. Following treatment, the cells were made fixed by using 4% paraformaldehyde (PFA) and stained using the periodic acid-Schiff (PAS) glycogen staining kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) as per the manufacturer protocols. The representative images were taken with the aid of a microscope (Nikon, Tokyo, Japan). The mean optical density of the representative image was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.). The relative optical density of each group was calculated and statistically analyzed based on the control group (100%).

Real-Time PCR

A volume of 1 mL of HepG2 cells was seeded in the wells of a 6-well plate (2 × 10⁵ cells/well) and incubated overnight for culturing. Subsequently, cells were co-treated for 24 h with PA (0.5 mM) and QAG (2, 4, 8 µM) or metformin (5 mM). A total RNA from HepG2 cells was isolated by using a Total RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). For synthesizing cDNA using mRNA as a template, qPCR RT Master Mix (a ReverTra Ace kit, Toyobo Co., Ltd., Osaka, Japan) was employed, whereas Prime Script RT reagent kit (Qiagen, Valencia, CA, U.S.A.) was then utilized to perform real-time PCR as per instructions from the manufacturer. The following primers were used in this study: forward primer: 5′-ATGTCAGTGGGACTTGTGCTGC-3′ and reverse primer: 5′-AACTCAGCCACCATGAACCAGG-3′ for GLUT2, forward primer: 5′-CCATCCTGATGACTGTGGCTCT-3′ and reverse primer: 5′-GCCACGATGAACCAAGGAATGG-3′ for GLUT4, forward primer: 5′-AGCCTCAAGATCATCAGCAA-3′ and reverse primer: 5′-GTCATGAGTCCTTCCACGATAC-3′ for GAPDH. The mRNA levels for GLUT2 and GLUT4 were adjusted to those of GAPDH. The data was reported as the fold change of each sample group in comparison to the control group.

Western Blot

After treatment, on ice, HepG2 cells were lysed using 1× radioimmunoprecipitation assay (RIPA) lysis buffer solution (Cell Signaling Technology) containing protease inhibitors. We then collected the cell lysates and centrifuged them at 12000 rpm, 10 min at 4 °C, after which supernatant was transferred into new 1.5 mL tubes. The concentration of protein was measured utilizing Quick Start TM Bradford 1X Dye Reagent (Bio-Rad, CA, U.S.A.). The sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized for loading protein (30 µg) from all the samples. The proteins on the gel were transferred to a polyvinylidene fluoride (PVDF) membrane after electrophoresis. After blocking with 10% nonfat milk at room temperature for 1 h, the PVDF membrane was incubated with the primary antibodies (1 : 1000) overnight at 4 °C. After washing thrice with PBST (PBS plus 0.1% tween 20, v/v), the PVDF membrane was incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. The bands on the membrane were then detected by the UltraSignalTM ECL Western blot detection reagent (4A Biotech Co., Ltd., Beijing, China), while imaging was performed on the ChemiDoc MP Imaging System (Bio-Rad). ImageJ software (NIH) was applied to calculate the band intensities.

Molecular Docking Analysis

A molecular docking analysis was carried out as per the protocol described previously.^29,30) A three-dimensional structure of the human insulin receptor was retrieved from RCSB Protein Data Bank (PDB ID: 5HHW). 2D structure of QAG was drawn in the MOL file format and then exported using ChemDraw (PerkinElmer, Inc., MD, U.S.A.) software. The molecular docking analysis was then carried out using Autodock Version 4.2.6 to identify the combining mode. The molecular configuration of the monomers was optimized using molecular mechanics. Grid energy was calculated using the Autogrid and the Lamarca genetic algorithm (LGA) was utilized for performing docking analysis. We kept the allowed run number set at 100 while the rest of all parameters were maintained at default settings. A semi-empirical method of free energy evaluation was used to select the best combination between QAG and insulin receptors.

Statistical Analysis

GraphPad Prism (version: 8.0) was employed for performing statistical analysis (San Diego, CA, U.S.A.). All data were presented in the form of mean ± standard deviation (S.D.). A one-way ANOVA was applied to compare statistical variations among groups followed by Tukey’s multiple-comparison post hoc test or Dunnett’s test. p < 0.05 was regarded as statistically significant.

RESULTS

Isolation, Purification, and Identification of QAG

The EUOL were extracted with 70% ethanol. The total ethanol extract was then purified using HCSCC and preparative HPLC. The purity of the obtained compound was determined using HPLC-UV at 360 nm and its purity was 98.32% (Fig. 1A). It was then identified using the UPLC-Q/TOF-MS and NMR instruments. The MS peak indicated that the measured accurate mass of this compound was 597.1377 [M + H]⁺ (Fig. 1B), and its molecular formula was speculated to be C₂₆H₂₈O₁₆. The ¹³C-NMR data (Fig. 1C) were as follow: ¹³C-NMR (101 MHz, methanol-d4) δ179.56 (C-4), 165.79 (C-7), 163.12 (C-5), 158.35 (C-2), 158.19 (C-9), 149.70 (C-4′), 146.03 (C-3′), 135.10 (C-3), 123.40 (C-6′), 123.13 (C-1′), 117.26 (C-5′), 116.01 (C-2′), 105.74 (C-1‴), 105.40 (C-10), 100.77 (C-6), 99.70 (C-1″), 94.51 (C-8), 82.31 (C-2″), 78.35 (C-5″), 78.21 (C-3″), 77.08 (C-3‴), 74.93 (C-2‴), 70.97 (C-4″), 70.97 (C-4‴), 66.69 (C-5‴), 62.33 (C-6″). The ¹H-NMR data (Fig. 1D) were as follow: ¹H-NMR (400 MHz, methanol-d₄) δ: 7.70–7.58 (m, 2H), 6.86 (d, J = 8.5 Hz, 1H), 6.37 (s, 1H), 6.18 (s, 1H), 5.50 (d, J = 7.5 Hz, 1H), 4.75 (d, J = 3.1 Hz, 1H), 3.92 (dd, J = 11.4, 4.4 Hz, 1H), 3.78–3.66 (m, 2H), 3.64–3.44 (m, 3H), 3.43–3.34 (m, 3H), 3.27–3.17 (m, 2H). The above data were consistent with those of QAG in literature.³¹⁾

Fig. 1. The HPLC, MS, NMR and Structure of QAG

(A) HPLC analysis of total ethanol extract of EUOL and QAG. S1: total ethanol extract of EUOL, S2: QAG. (B) The mass spectrum of QAG in positive mode. (C) ¹³C-NMR of QAG. (D) ¹H-NMR of QAG.

QAG Improved Cell Viability in the PA-Induced HepG2 Cells

The cytotoxicity of QAG or PA alone on HepG2 cells was first investigated to determine whether QAG can increase the cell viability of PA-induced HepG2 cells. No considerable decrease in cell viability was observed after 36 h for QAG (20 µM or less), whereas PA (0.5 mM or more) remarkably decreased the viability of the HepG2 cells as illustrated by Fig. 2A. QAG’s effect on 0.5 mM PA-induced HepG2 cells was investigated. QAG improved cell viability in PA-induced HepG2 cells dose-dependently as shown in Fig. 2C.

Fig. 2. The Impacts of QAG and PA on the Cell Viability in the HepG2 Cells

(A) The HepG2 cells were incubated with QAG (0–1280 µM) for 36 h. (B) The HepG2 cells were incubated with PA (0–1 mM) for 36 h. (C) The HepG2 cells were incubated with QAG (2, 4, 8 µM), 0.5 mM PA, and metformin for 36 h, and cell viability was measured using the MTT assay. Statistical data are presented as mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

QAG Led to an Increase in Uptake of Glucose in the PA-Induced HepG2 Cells

Once IR is increased, uptake of glucose and consumption of cells were greatly reduced.^32,33) To examine the impact of QAG on the uptake of glucose in the PA-induced HepG2 cells, the cellular uptake of glucose was determined using the hexokinase method. PA significantly reduced cellular glucose consumption in a dose-dependent manner, according to the findings (Fig. 3A), whereas QAG reversed the decrease in glucose consumption in the PA-induced HepG2 cells dose-dependently (Fig. 3B). In summary, QAG improved IR in the HepG2 cells induced by PA.

Fig. 3. The Impact of QAG on Uptake of Glucose Rate in the PA-Induced HepG2 Cells

(A) Influence of PA on the rate of glucose consumption among HepG2 cells. (B) Impact of QAG and metformin on the rate of glucose consumption in the PA-induced HepG2 cells. The data are represented as the mean ± S.D. (n = 3). * p < 0.05, *** p < 0.001.

QAG Increased Synthesis of Glycogen in the PA-Induced HepG2 Cells

A decrease in uptake of glucose as well as glycogen synthesis can be found in cells with IR.^16,34,35) In this experiment, we investigated the impact exerted by QAG on the synthesis of glycogen in the HepG2 (PA-induced) cells using the PAS glycogen staining method, and glycogen was dyed purple-red with the PAS staining reagent. The results in Fig. 4 show that the cells stained purple-red in the PA-induced HepG2 cells were considerably reduced in comparison to the cells present in the control group, demonstrating that PA has reduced glycogen synthesis. However, after treatment with concentrations gradient of QAG or metformin, the cells stained purple-red were significantly increased in the PA-induced HepG2 cells. These findings represent that QAG promoted the synthesis of glycogen in PA-induced HepG2 cells.

Fig. 4. QAG Increased Glycogen Synthesis in the PA-Induced HepG2 Cells

(A) Representative images of cells stained purple-red using the PAS glycogen staining method. Magnification: 10×, bar scale: 200 µm; Magnification: 20×, bar scale: 100 µm. (B) A Bar chart indicating a relative optical density of positive aera based on control group. Data are reported as mean ± S.D. (n = 3). ** p < 0.01, *** p < 0.001.

QAG Improved GLUT2 and GLUT4 Expression in PA-Induced HepG2 Cells

IR is usually associated with reduced or dysfunctional glucose transporters. GLUT2 and GLUT4, acting as important glucose transporters in the liver, are essential for cell glucose homeostasis.^36–39) In this study, the influence of QAG on the expression of GLUT2 and GLUT4 was investigated by detecting their mRNA and protein levels by performing RT-PCR and Western blot analyses, respectively. As shown in Fig. 5, QAG significantly increased the mRNA level of GLUT2 and GLUT4 in the PA-induced HepG2 cells. Data obtained from Western blot analysis illustrated that QAG improved the expression of proteins GLUT2 and GLUT4 in PA-induced HepG2 cells dose-dependently (Fig. 5B). In summary, QAG improved the expression of GLUT2 and GLUT4.

Fig. 5. QAG Improved GLUT2 and GLUT4 Expression in the PA-Induced HepG2 Cells

The PA-induced HepG2 cells were treated with QAG or metformin for 24 h, then the mRNA level (A) and protein expression (B) of GLUT2 and GLUT4 were quantified by RT-PCR and Western blot analyses, respectively. Data are represented as mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

QAG Upregulated the IRS-1/PI3K/Akt/GSK-3β Pathway in HepG2 Cells

The IRS-1/PI3K/Akt/GSK-3β pathway plays a crucial role in regulating the uptake of glucose and glycogen synthesis.^34,35,40) In this study, the effect of QAG on the modulation of the IRS-1/PI3K/Akt/GSK-3β signaling pathway in the HepG2 cells was investigated. Figure 6 clearly demonstrates that QAG could significantly reduce the phosphorylation of IRS-1 at Ser612, implying activation of insulin signaling. The reduction of p-IRS-1(S612)/IRS-1 ratio leading to a significant elevation of p-PI3K(Y458/Y199)/PI3K, p-Akt(S473)/Akt, and p-GSK-3β(S9)/GSK-3β, which indicating the upregulation of IRS-1/PI3K/Akt/GSK-3β pathway. Furthermore, we also observed the effect of QAG on pathway in PA-induced HepG2 cells, and the results showed that QAG could upregulate this pathway (Fig. 7). In summary, QAG upregulated the IRS-1/PI3K/Akt/GSK-3β signaling pathway in HepG2 cells.

Fig. 6. QAG Upregulated the IRS-1/PI3K/Akt/GSK-3β Pathway in HepG2 Cells

(A) After treating HepG2 cells with QAG at the specified concentrations for 24 h, the protein was collected for Western blot analysis. A bar chart indicates the relative protein expression of p-IRS-1/IRS-1 (B), p-PI3K/PI3K (C), p-Akt/Akt (D), and p-GSK-3β/GSK-3β (E). Data were expressed as mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the controls.

Fig. 7. QAG Upregulated the IRS-1/PI3K/Akt/GSK-3β Pathway in PA-Induced HepG2 Cells

(A) The PA-induced HepG2 cells were treated with QAG (2, 4, 8 µM) or metformin (5 mM) for 24 h, the protein was collected for Western blot analysis. A bar chart indicates the relative protein expression of p-IRS-1/IRS-1 (B), p-PI3K/PI3K (C), p-Akt/Akt (D), and p-GSK-3β/GSK-3β (E). Data were expressed as mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

QAG Increased Uptake of Glucose via the PI3K/Akt/GSK-3β Signaling Pathway

LY294002, a PI3K inhibitor, was used to inhibit PI3K to investigate whether the effect of QAG on IR is related to the regulation of the signaling pathway called PI3K/Akt/GSK-3. The downstream substrates of PI3K and rate of glucose consumption in the HepG2 cells were then detected. The results revealed that LY294002 counteracted the impact of QAG on PI3K/Akt/GSK-3β signaling pathway activation (Figs. 8A–D), as well as the glucose consumption rate in the HepG2 cells (Fig. 8E). In summary, QAG improved the uptake of glucose by activating the aforementioned biochemical signaling pathway.

Fig. 8. QAG Improved Uptake of Glucose by Upregulating the Signaling Pathway Called PI3K/Akt/GSK-3β

(A–D) A QAG treatment was performed on HepG2 cells with or without LY294002 for a period of 24 h, after which the protein was collected for Western blot analysis. The bar chart indicates the relative expression of p-PI3K/PI3K, p-Akt/Akt, and p-GSK-3β/GSK-3β. (E) QAG was applied on the PA-induced HepG2 cells with and without LY294002 for 24 h. The cells were further subjected to measurement of glucose consumption rate through a glucose content detection kit. The bar chart indicates the glucose consumption rate. Data are reported as mean ± S.D. (n = 6). * p < 0.05, *** p < 0.001.

Molecular Docking of QAG with Insulin Receptors

The activation of the insulin signaling pathway is dependent on insulin receptors. The compound binding to the extracellular domain of the insulin receptor induces conformational alterations in the intracellular domain and causes activation of insulin signaling.^29,30) The current study used a molecular docking approach to investigate how QAG interacts with the insulin receptor to regulate a signaling pathway called IRS-1/PI3K/Akt/GSK-3. As shown in Fig. 9, QAG interacted with three residues, including GLU-1135, PRO-1129, and ASP-1170 in the active pocket of the insulin receptor, and the calculated binding energy (in kcal/mol) was −4.91 kcal/mol, suggesting that there was a good binding capacity of QAG with the insulin receptor.

Fig. 9. The Best-Scored Docking Pose of QAG inside the Binding Pocket of the Insulin Receptor

The yellow dotted line and the numbers next to it indicate the hydrogen bonds and their bond lengths.

DISCUSSION

IR, one of the main pathophysiological characteristics of T2DM, is closely associated with an increase in plasma FFAs.^4,8) High levels of circulating FFAs are considered to be critical in the initiation and promotion of IR.^27,41) PA, a common saturated FFA in blood, was shown to induce IR in hepatocytes by upregulating inflammatory cytokines, some microRNAs such as microRNA-33a, microRNA-33b, and microRNA-221, and serine/tyrosine hyperphosphorylation of IRS-1 and IRS-2.^5,7,42) HepG2 cells are derived from human hepatoma tissues, which retained many biological characteristics of normal liver cells and had insulin receptor and post-receptor functional defects. This makes them an ideal cell model for studying IR.^43–45) This study, which is consistent with previous studies, revealed that PA reduced uptake of a glucose molecule and glycogen synthesis in HepG2 cells to a significant extent, indicating that the cell model of IR was successfully established.^4,7) QAG, a flavonoid isolated from EUOL, increased uptake of glucose as well as glycogen synthesis in PA-induced HepG2cells, thus improving IR. In addition, PA decreased the cell activity of the HepG2 cells, which may be related to a glucose metabolism disorder, while QAG restored the cell viability of PA-induced HepG2 cells.^42,46–49)

GLUT2 and GLUT4, especially GLUT2, are insulin-regulated transmembrane glucose transporters that are involved in glucose homeostasis, the control of which is strongly governed via a signaling pathway called IRS-1/PI3K/Akt/GSK-3β.^37,42,50) In insulin signaling, IRS-1 is the major and better-characterized protein that activates multiple signaling pathways that regulate the uptake of glucose and glycogen synthesis.⁵¹⁾ The binding of insulin to its receptor results in the autophosphorylation of IRS-1, leading to tyrosine phosphorylation and activation of PI3K and the translocation of GLUTs to the cell surface, which subsequently promote the transport of glucose into cells.^4,7,42,50) PA induces serine hyperphosphorylation of IRS-1 and failure to activate downstream proteins, resulting in blocked insulin signaling interruption. Interestingly, in the current study, QAG restored IRS-1 activity by inhibiting its phosphorylation at serine⁶¹². The activated IRS-1 then phosphorylated PI3K at tyrosine⁴⁵⁸ and tyrosine¹⁹⁹, and Akt at serine⁴⁷³, promoting the expression, maturation, and translocation of GLUT2 and GLUT4 to the cell membrane from the cytoplasm, and ultimately modulating glucose homeostasis.^7,42) QAG also promoted the phosphorylation of p-GKS-3β at serine⁹ thereby inhibiting the GSK-3β activity, which led to an increase in GS and initiated glycogen synthesis.^30,35) Thus, QAG improves IR by promoting the uptake of glucose and glycogen synthesis by improving the expression of GLUT2 and GLUT4. The PI3K inhibitor (LY294002) was used for estimating the improvement effect of QAG on IR, and its correlation with the PI3K/Akt/GSK-3β biochemical signaling pathway. These observations displayed that LY294002 abolished the activation of PI3K/Akt/GSK-3β pathway by QAG and partly reversed the effect of QAG on the increase in uptake of glucose, and this phenomenon suggests that QAG may also improve glucose uptake via other PI3K-independent signaling pathways. The PI3K/Akt pathway plays an important but not decisive role in the regulation of hepatic glucose uptake, and AMP activated protein kinase (AMPK), MEK/extracellular signal-regulated kinase (ERK) pathway is also involved in the regulation of glucose uptake.^52,53) Although GLUT2 and GLUT4 are the major transporters of hepatic glucose uptake, GLUT1, GLUT5, GLUT8, GLUT9 and GLUT10 have also been found to be involved in glucose transport.⁵⁴⁾ Whether the above mechanisms are involved in the improvement of glucose uptake by QAG needs further study. In summary, QAG improved IR by upregulating the IRS-1/PI3K/Akt/GSK-3β pathway, at least in part. Furthermore, the molecular docking approach was applied in the virtual screening of drugs and guides the discovery and development of new potential drugs.^29,55) The molecular docking result showed that QAG had good binding capacities to the insulin receptor, supporting our findings that QAG upregulated the signaling pathway called IRS-1/PI3K/Akt/GSK-3β.

Many health benefits have been reported for quercetin and its glycosides, including antioxidant, anti-inflammatory, anti-allergic, antitumor, and insulin resistance-improving properties.^56–58) In the present work, we made an attempt to isolate a quercetin glycoside QAG from EUOL, and for the first time, it was observed that QAG could improve IR in vitro analysis. QAG is more soluble in water than quercetin, indicating that QAG has good absorption and biodistribution profile. In addition, the terminal glucose on the attached disaccharides was difficult to hydrolyze, thus QAG may be absorbed in a non-deglycosylated form and exerts the IR improving effect.⁵⁹⁾ Although our current findings indicate that QAG improves IR in vitro, in vivo validation is required.

CONCLUSION

A quercetin glycoside QAG was isolated from EUOL and found to be able to alleviate IR through the biochemical signaling pathway called IRS-1/PI3K/Akt/GSK-3β, which provided evidence for developing QAG into a novel anti-IR drug.

Acknowledgments

We thank all the authors for their contributions to this work, and thank the financial support from the Technology Department of Sichuan Province (2010JY0126), Education Department of Sichuan Province (11ZA243), Health Department of Sichuan Province (100228).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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