3,3′,4,5′-Tetramethoxy-trans-stilbene Improves Insulin Resistance by Activating the IRS/PI3K/Akt Pathway and Inhibiting Oxidative Stress

The potential anti-diabetic effect of resveratrol derivative, 3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and its underlying mechanism in high glucose (HG) and dexamethasone (DXMS)-stimulated insulin-resistant HepG2 cells (IR-HepG2) were investigated. 3,3′,4,5′-TMS did not reduce the cell viability of IR-HepG2 cells at the concentrations of 0.5–10 µM. 3,3′,4,5′-TMS increased the potential of glucose consumption and glycogen synthesis in a concentration-dependent manner in IR-HepG2 cells. 3,3′,4,5′-TMS ameliorated insulin resistance by enhancing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), inhibiting phosphorylation of insulin receptor substrate-1 (IRS-1), and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells. Furthermore, 3,3′,4,5′-TMS significantly suppressed levels of reactive oxygen species (ROS) with up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. To conclude, the beneficial effect of 3,3′,4,5′-TMS against insulin resistance to increase glucose consumption and glycogen synthesis was mediated through activation of IRS/PI3K/Akt signaling pathways in the IR-HepG2 cells, accomplished with anti-oxidative activity through up-regulation of Nrf2.


Introduction
Diabetes is a chronic metabolic disease characterized by hyperglycemia. Over time, different complications, such as vasculopathy and retinopathy, disability or even death, occur in patients through the exacerbation of diabetes. In the past 30 years, the prevalence of diabetes has kept increasing worldwide [1]. Type 2 diabetes mellitus (T2DM) accounts for about 90% of diabetes cases and is characterized by deteriorated glucose metabolism with hyperglycemia and insulin resistance (IR) [2]. Abnormal insulin signaling may result in a reduction in glucose transport and glucose phosphorylation, and it also suppresses the activity of glycogen synthase (GS) [3]. In addition, there are close associations between IR and oxidative stress [4]. A high level of reactive oxygen species (ROS) inhibits the insulin response and exacerbates the pathologic condition of diabetic patients [5]. ROS-triggered damage varies depending on the species, location of ROS and strength of feedback [6]. Defining the tipping point of ROS causing damage remains a challenging issue. At the same time, acute or chronic hyperglycemia in diabetes increases ROS production [7].
The liver plays an extremely important role in glucose metabolism, maintaining a constant blood glucose level through glycogenesis and glycogenolysis. Insulin promotes the production of liver glycogen and inhibits gluconeogenesis to reduce glucose output in the liver [8,9]. The HepG2 cell line has the functional and physiological characteristics of normal hepatocytes, and thus, it has been used as an important model for exploring insulin resistance in vitro [10]. According to previous studies, glucocorticoids have been noted to exacerbate hyperglycemia in T2DM patients by destroying glucose homeostasis [11]. At the same time, high glucose (HG) and dexamethasone (DXMS) can induce IR, which is associated with oxidative stress [12][13][14], and the combined utilization of DXMS and HG has been used to induce insulin resistance in previous studies [15,16]. In this study, the in vitro insulin-resistant HepG2 (IR-HepG2) cell was established by treating high glucose and DXMS. It is well known that the insulin receptor substrate (IRS) protein family plays an important role in the process of insulin signal transduction, secretion and metabolism [14,17]. IRS1 and IRS2 are very important in insulin-mediated glucose and lipid metabolism [18], and the abundance status of IRS1 and IRS2 is related to the activation of the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pathway [19]. The stimulation of the PI3K/Akt signaling pathway promotes glucose uptake and activates glycogen synthase kinase 3 beta (GSK3β) to facilitate glycogen synthesis [20]. In addition, the PI3K/Akt pathway is a critical regulator in other normal cellular processes, such as growth, reproduction and apoptosis. Importantly, oxidative stress is considered a contributing factor to the onset and progression of diabetes [21]. The nuclear factor erythroid 2-related factor 2 (Nrf2), which is a key transcription factor regulating antioxidant stress, has been shown to protect cells from the damage of oxidative stress in diabetes [22]. Extensive evidence has demonstrated the anti-diabetic properties of resveratrol, trans-3,5,4-trihydroxystilbene, in animal and human studies [23,24]. 3,3 ,4,5 -tetramethoxy-trans-stilbene (3,3 ,4,5 -TMS) is a methoxy derivative of resveratrol. 3,3 ,4,5 -TMS shows superior pharmacokinetic characteristics to remedy the shortcomings of resveratrol, including low bioavailability and a short half-life. Previous results have revealed the anti-tumor, anti-inflammatory and anti-allergic activities of 3,3 ,4,5 -TMS [25][26][27]. However, the effect of 3,3 ,4,5 -TMS on high glucose-induced IR and its molecular mechanism remains to be explored. Consequently, our present study will investigate the anti-diabetic effect and the underlying mechanism of 3,3 ,4,5 -TMS in IR-HepG2 cells triggered by high glucose and DXMS.

Cell Culture
The HepG2 cells were acquired from the China Academy of Sciences (Shanghai, China). The cells were cultured with Low-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin and non-essential amino acids (NEAA), which were obtained from Gibco (Carlsbad, CA, USA). The cells were maintained at 37 • C in a 5% CO 2 humidified incubator. In order to establish the IR cell model, the HepG2 cells were pretreated with DXMS (30 µM, dissolved in ethanol) plus D-glucose (50 mM final concentration) for 48 h. Thereafter, the experimental groups were treated with different concentrations (1 µM and 2.5 µM) of 3,3 ,4,5 -TMS for 16 h. The group not treated with DXMS, D-glucose or 3,3 ,4,5 -TMS served as the control.

MTT Assay
The cytotoxic effects of 3,3 ,4,5 -TMS and DXMS on the HepG2 cells were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). According to the protocol provided by the MTT supplier, HepG2 cells (7 × 10 3 cells/well) were seeded on 96-well culture plates and cultured overnight. Then, the cells were incubated with different concentrations of 3,3 ,4,5 -TMS and DXMS for 48 h. At the same time, ethanol and DMSO were used as vehicle to treat the cells for 48 h at the maximum administration volume of 3,3 ,4,5 -TMS and DXMS. A group with no treatment served as control. The time of incubation for 48 h in the MTT assay was selected as HepG2 cells were exposed to DXMS plus D-glucose to induce insulin resistance in latter experiments. The number of cells seeded was selected after screening the different cell densities according to the linearity range at which no cell death was observed. After incubation, the cells were refreshed with 10% MTT-containing medium at 37 • C for 3 h. At last, the supernatants were discarded, and 150 µL of DMSO was added to each well to dissolve the formazan crystals by shaking the culture plate for 30 min. The absorbance was read at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices, Silicon Valley, CA, USA). The relative cell viability was calculated as the percentage of control group.

Glucose Consumption Assay
The effects of 3,3 ,4,5 -TMS on glucose consumption potency were determined by a glucose assay kit, which was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China, A154-1-1). HepG2 cells (7 × 10 5 cells/well) were seeded on 6-well culture plates and cultured overnight. The cells were treated with DXMS plus D-glucose for 48 h and different concentrations of 3,3 ,4,5 -TMS for 16 h. According to the manufacturer's instructions, the supernatants were collected, and their glucose concentrations were measured by the kit. The contents of cell glucose were determined by the glucose oxidase method, which causes an amaranth tint in the sample. The absorbance at 505 nm was detected with a microplate spectrophotometer. The result was calculated as below: glucose consumption = glucose concentration of blank-glucose concentration of each treatment group.

Glycogen Synthesis Assay
The effect of 3,3 ,4,5 -TMS on glycogen synthesis ability was determined by a glycogen assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China, A043-1-1). HepG2 cells (7 × 10 5 cells/well) were seeded on 6-well culture plates and cultured overnight. Then, the cells were treated with DXMS, D-glucose and different concentrations of 3,3 ,4,5 -TMS as previously described in Section 2.2. According to the manufacturer's instructions, the cells were collected in phosphate-buffered saline solutions with cell disruption by ultrasonication. After further hydrolysis in alkali solution, the contents of cell glycogen were determined by sulfuric acid-anthrone method, which causes a blue tint in the sample. The absorbance at 620 nm was detected with a microplate spectrophotometer. The cell glycogen levels were calculated by comparing with the control group.

Western Blot Assay
HepG2 cells (7 × 10 5 cells/well) were seeded on 6-well culture plates and cultured overnight. Treatments of the cells were the same as above in Section 2.2. After treatment, the medium was discarded, and the residual medium was washed away by phosphatebuffered saline (PBS). The cells were harvested and lysed with RIPA solution containing 1% Protease Inhibitor Cocktail and 1% phenylmethanesulfonyl fluoride (PMSF), both of which were obtained from Beyotime Biotechnology. The above operations were all performed on ice. Subsequently, the cell lysates were centrifuged at 15,000 rpm for 30 min at 4 • C to collect supernatants. The BCA Protein assay kit (Beyotime Biotechnology, Shanghai, China) was used to measure the total protein contents. Protein samples were separated by 8-10% SDS/PAGE gels, and all materials for SDS-PAGE were obtained from Bio-Rad (Hercules, CA, USA). Afterwards, the proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat milk (Bio-Rad) for 2 h, followed by incubation with the appropriate primary antibodies overnight at 4 • C. After washing with Tris Buffered Saline with Tween-20 (TBST) buffer, the membranes were incubated with the secondary antibodies for 2 h at room temperature. Finally, the protein bands were detected by enhanced chemiluminescence (ECL) reagent purchased from Thermo Fisher Scientific (Waltham, MA, USA) and ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis
In this study, all the values were shown as mean ± S.E.M from at least three independent experiments. One-way analysis of variance (ANOVA) was used to analyze the differences among groups by GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). The Newman-Keuls multiple comparison tests were applied for post hoc pairwise comparisons. A p < 0.05 indicated statistically significant difference.

The Effect of 3,3 ,4,5 -TMS and DXMS on the HepG2 Cell Viability
MTT assay was used to determine the effects of 3,3 ,4,5 -TMS and DXMS on cell viability. The results showed that 3,3 ,4,5 -TMS did not show cytotoxic effect at low concentrations (0.5-10 µM) but significantly decreased the cell viability of HepG2 cells at high concentrations (25 and 50 µM) (Figure 1a). Moreover, DXMS, as well as the solvents (0.25% v/v DMSO or 0.3% v/v ethanol), had no negative effect on the cell viability of HepG2 cells (Figure 1b).
Curr. Issues Mol. Biol. 2022, 2, FOR PEER REVIEW 4 which were obtained from Beyotime Biotechnology. The above operations were all performed on ice. Subsequently, the cell lysates were centrifuged at 15,000 rpm for 30 min at 4 °C to collect supernatants. The BCA Protein assay kit (Beyotime Biotechnology, Shanghai, China) was used to measure the total protein contents. Protein samples were separated by 8-10% SDS/PAGE gels, and all materials for SDS-PAGE were obtained from Bio-Rad (Hercules, CA, USA). Afterwards, the proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat milk (Bio-Rad) for 2 h, followed by incubation with the appropriate primary antibodies overnight at 4 °C. After washing with Tris Buffered Saline with Tween-20 (TBST) buffer, the membranes were incubated with the secondary antibodies for 2 h at room temperature. Finally, the protein bands were detected by enhanced chemiluminescence (ECL) reagent purchased from Thermo Fisher Scientific (Waltham, MA, USA) and ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis
In this study, all the values were shown as mean ± S.E.M from at least three independent experiments. One-way analysis of variance (ANOVA) was used to analyze the differences among groups by GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). The Newman-Keuls multiple comparison tests were applied for post hoc pairwise comparisons. A p < 0.05 indicated statistically significant difference.

Effect of 3,3 ,4,5 -TMS on Glucose Consumption
To investigate the potential effect of 3,3 ,4,5 -TMS on glucose metabolism in IR-HepG2 cells, the glucose concentration of cell culture supernatants was detected by the glucose assay kit. Compared with the control group (mean = 1.735 mM), glucose consumption of the high glucose-DXMS-treated group was significantly reduced with a mean value of 0.745 mM. Conversely, the IR-HepG2 cells significantly consumed more glucose to a level comparable with control after the treatment of 3,3 ,4,5 -TMS at 2.5 µM (Figure 2a). The mean values of 1 and 2.5 µM 3,3 ,4,5 -TMS were 1.018 and 1.564 mM, respectively.

Effect of 3,3′,4,5′-TMS on Glucose Consumption
To investigate the potential effect of 3,3′,4,5′-TMS on glucose metabolism in IR-HepG2 cells, the glucose concentration of cell culture supernatants was detected by the glucose assay kit. Compared with the control group (mean = 1.735 mM), glucose consumption of the high glucose-DXMS-treated group was significantly reduced with a mean value of 0.745 mM. Conversely, the IR-HepG2 cells significantly consumed more glucose to a level comparable with control after the treatment of 3,3′,4,5′-TMS at 2.5 µM (Figure 2a). The mean values of 1 and 2.5 µM 3,3′,4,5′-TMS were 1.018 and 1.564 mM, respectively.
With the advances in knowledge on the pathophysiology of diabetes, IR has captured more and more attention in T2DM. Normal signal transduction of insulin promotes glucose transportation from blood to different cells, stabilizing blood glucose concentration at a normal level. In the liver, insulin activates glycogen synthesis and glycolysis and inhibits gluconeogenesis [29]. Glucose and DXMS were used together to induce IR in HepG2 cells. DXMS not only can induce IR but also can exacerbate IR induced by high glucose [15]. Combination treatment with high glucose and DXMS has been proven to be an effective method for establishing insulin resistance models [16]. DXMS causes damage to mitochondrial function, leading to exaggerated levels of ROS in mitochondria [30]. Insulin resistance involves complex mechanisms and processes, including oxidative stress [31,32]. The current study showed that 3,3 ,4,5 -TMS at high concentrations (25 and 50 µM) significantly lowered the cell viability of HepG2 cells. Low concentrations (0.5-10 µM) of 3,3 ,4,5 -TMS had no effect on cell viability. This result was consistent with the previous findings, which revealed the anti-tumor potency of this resveratrol derivative [33].
In T2DM, IR results in a reduction in glucose consumption in hepatocytes [34], modulating glucose transport activity and glucose metabolism [35]. Specific transporter proteins are required for facilitated diffusion of glucose into cells, such as glucose transporters (GLUTs) and sodium-glucose cotransporters (SGLTs) [36]. GLUT2 has been shown to be down-regulated in IR models to reduce glucose consumption in the IR-HepG2 cells [37][38][39]. After insulin stimulates the transfer of glucose to cells in the liver and muscle, glucose is converted into pyruvate through glycolysis, which supplies energy to mitochondria through the tricarboxylic acid cycle (TCA) [40]. However, hepatic glycolytic activity and glucose oxidation are impaired in T2DM [41]. In this study, we found that 3,3 ,4,5 -TMS (2.5 µM) was effective to increase glucose consumption and glycogen synthesis with enhanced serine phosphorylation of GSK3β in IR-HepG2 cells. Lower concentration at 1 µM showed minor but non-significant improvement. Several studies have reported that insulin induces the inactivation of GSK3β. GSK3β becomes inactivated by phosphorylation at Ser9; this inactivation subsequently reduces the inhibitory phosphorylation of glycogen synthase (GS), activating GS and thereby promoting glycogen synthesis [42].
The PI3K/Akt pathway is involved in the mechanism of insulin transduction [43]. In general, insulin is secreted by the pancreas and then binds with the insulin receptor on the cell surface, triggering an intracellular cascade. After insulin binding to a receptor, IRS proteins are recruited to stimulate the PI3K/Akt pathway downstream [44], regulating glucose uptake, glucose synthesis, gluconeogenesis, protein synthesis, cell growth and differentiation [43]. IR is associated with decreased IRS1 and IRS2 activities, which, in turn, inactivate PI3K/Akt signaling [45]. Notably, deficiency or knockout of IRS2 can lead to IR and activate gluconeogenesis in HepG2 cells [46]. Our results showed that 3,3 ,4,5 -TMS modulated the IRS/PI3K/Akt pathway to ameliorate IR and T2DM potentially.

Conclusions
In conclusion, this study is the first to show that 3,3 ,4,5 -TMS could improve IR in HepG2 cells by inhibiting oxidative stress and activating the IRS/PI3K/Akt pathway, indicating the potential of 3,3 ,4,5 -TMS for the treatment of diabetes ( Figure 5). The effect of 3,3 ,4,5 -TMS in diabetes still needs to be further studied in vivo. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.