Hypoglycemic Effect of Vitexin in C57BL/6J Mice and HepG2 Models

Apigenin-8-C-glucoside (vitexin), a natural phytochemical contained in hawthorn, has been reported to have versatile beneficial bioactivities, such as antioxidation, anticancer property, and adipogenesis inhibition.+e present research aimed to determine the influence of vitexin on insulin resistance elicited by HFD in mice and HepG2 cells. Vitexin markedly alleviated body weight gain and improved glucose and insulin intolerance induced by HFD. Vitexin partially normalized blood glucose, cholesterol, TNF-α, and hepatic lipid content. Moreover, vitexin recovered the reduced glucose uptake induced by glucosamine. +e present results indicate that vitexin prevents HFD-induced insulin resistance.


Introduction
In recent years, incidence of type 2 diabetes has increased dramatically all over the world, and diabetes is usually accompanied by many systemic complications, such as heart, eye, and blood vessel diseases, which poses a great threat to patients [1]. e liver is an insulin-sensitive organ and plays an important role in maintaining glucose and lipid metabolism. Insulin resistance in the liver is closely linked with the occurrence and progression of type 2 diabetes [2]. In the liver, insulin resistance has been found to reduce glucose uptake and glycogen synthesis and to inhibit insulin signaling activation [3,4]. HepG2 cells share the same lipid or glucose metabolism with normal hepatic cells [5]. In this metabolism, insulin activates an insulin receptor, and insulin receptor substrates are phosphorylated, which eventually leads to the activation of the PI3K/AKT pathway [6]. ER stress plays a critical role in the progression of liver insulin resistance [3]. ER stress promotes adipokine production and induces adipose tissue inflammation and dysfunction [7]. In the liver, ER stress interferes with hepatic glucose metabolism and impairs insulin sensitivity [8].
Vitexin is a natural flavonoid presenting in several consumable plants, such as hawthorn (Crataegus pinnatifida) and chayote (Sechium edule) [9,10]. e plants have been historically used as Chinese medicine for curing toxification, intestinal inflammation, and digestive disorders three thousand years ago [11,12]. Recently, a few studies reported that vitexin exhibits various bioactivities, including anti-inflammatory, anticancer, and antihypertension effects [13,14]. Vitexin was shown to reduce the pro-hyperalgesic cytokine level, while enhancing the anti-hyperalgesic level [15]. Vitexin was also found to ameliorate streptozotocin-or lipopolysaccharide-induced damage of the pancreas and islet tissue via the upregulation of antioxidative factors, including Nrf2 and GPx [16]. Moreover, vitexin obtained from Cynometra cauliflora Linn. increased the translocation of GLUT4 from the cell cytoplasm to membrane, indicating its beneficial potential on glucose uptake [17]. Although the protection of the pancreas and the promotion of glucose uptake by vitexin may suggest its beneficial effects on glucose homeostasis, the information of vitexin on insulin resistance and hepatic ER stress response is scarce. Animal studies reported that HFD-induced obesity is closely associated with insulin resistance [18,19]. Also, glucosamine has been widely used to induce ER stress in vitro [4]. us, the present research aimed to determine vitexin's influence on HFD and glucosamine-elicited insulin resistance in C57BL/6J mice and in HepG2 cells.  All animal experimental procedures were followed according to the Guide for the Care and Use of Laboratory Animals: Eighth Edition, ISBN-10: 0-309-15396-4, and the animal protocol was approved by the Jiangsu University Animal Ethics Committee. After a two-week adaptation, mice were separated into 3 groups (9 mice per group): LFD, HFD, and HFD + Vitexin (5 mg vitexin/kg BW/day) for 8 weeks. e HFD (60% of energy from fat, TP23400) and LFD (10% kcal from fat, TP23402) were from Trophic Animal Feed High-tech Co., Ltd. (Nantong, Jiangsu Province, China). e vitexin dose was based on a previous study [20].

Materials and Methods
At the end of the study, mice were fasted overnight before being sacrificed by CO 2 asphyxiation. Blood was collected by cardiac puncture. Serum was obtained from blood by centrifugation for 20 min at 2000 rpm. Part of the liver was fixed in buffered formalin (10%). e ITT and GTT were performed as previously described [21].

Serum Parameters and Liver Triglycerides Quantitation.
e TG from the liver and serum and serum glucose levels were quantified using commercial kits according to manufacturers' instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China). e level of serum TNF-α was detected using the ELISA kits (FcMACS Co., Ltd, Nanjing, Jiangsu Province, China).

Cell
Culture. HepG2 cells were maintained as previously reported [22]. After incubation with 18 mm glucosamine for 18 h to induce insulin resistance [4], the HepG2 cells were serum-starved for 2 h and then incubated with vitexin (20 μM) for 24 h, with 100 nM insulin added in the last 15 min. e vitexin and glucosamine concentrations were in accordance with previous publications [4,20], and the concentrations had no influence on cell viability as determined by the MTT assay, as previously reported [23,24]. Glucosamine was dissolved in PBS, and vitexin was dissolved in DMSO. All groups had the same amount of DMSO.
ere were three treatment groups, i.e., Control, Glucosamine, and Glucosamine + Vitexin groups. Briefly, HepG2 cells were treated with or without 18 mM glucosamine for 18 h and starved in F-12K (5.5 mM glucose) serum-free media for 2 h. en, cells were treated with or without 20 μM vitexin for 24 h following the treatment of 100 nM insulin for 15 min (serum-free medium). After washing, the fluorescent signal was quantified with a plate reader at excitation/emission of 465/ 540 nm (SpectraMax i3, Shanghai, China) and normalized by protein content (BCA protein assay kit, Beyotime Biotechnology Inc. Shanghai, China).

Statistical Analyses.
Data were analyzed with one-way ANOVA (Tukey's multiple-range test) with SAS software (SAS Institute Inc., Cary, NC, USA). A P < 0.05 was considered statistically significant.

Vitexin Decreased Body Weight in HFD-Treated Mice.
As shown in Figure 1(a), the HFD group possessed higher body weight than the LFD and HFD + Vitexin groups during week 8.
e HFD + Vitexin group had no significantly different body weight compared to the LFD group. All three groups had similar total food intake (Figure 1(b)).

Vitexin Improved Glucose Homeostasis. ITT and GTT
were performed to determine the influence of vitexin on glucose metabolism, and results are shown in Figure 2. For the ITT, HFD-fed mice had a higher blood glucose level than mice fed with LFD at all points in time (Figure 2(a)). Compared to the HFD-fed mice, vitexin significantly reduced the HFD-induced blood glucose increase after 0, 15, 30, 60, and 120 minutes (Figure 2(a)). Similarly, HFD significantly increased the AUC of the ITT, and the AUC was significantly reduced by vitexin treatment (Figure 2(b)). For the GTT results, mice fed with a HFD had a significantly higher blood glucose level at 15, 30, and 60 minutes, as well as the AUC, than in the LFD group and the HFD + Vitexin group (Figures 2(c) and 2(d)). e results indicate that vitexin significantly alleviated insulin and glucose intolerance induced by the HFD. Table 1, in HFD-fed mice, the glucose and TG levels were significantly increased compared with the LFD-fed mice and were markedly decreased in the HFD + Vitexin group. In addition, vitexin reduced the HFD-induced TNF-α level. No differences were observed for the three parameters between the LFD and HFD + Vitexin groups.

Vitexin Reduced Liver Fat Content.
e H&E staining results (Figure 3(a)) directly showed the difference of lipid content in the liver tissue in each group of mice. Mice fed with HFD had higher liver fat content than mice fed with the    LFD, and the liver fat content was significantly reduced by vitexin treatment (Figure 3(b)).

Influence of Vitexin and Glucosamine on Cell Viability and
Glucose Uptake in HepG2 Cells. Glucosamine (18 mM) and vitexin (20 μM) did not affect cell viability (Figure 4(a)). Reduced glucose uptake is an important marker of insulin resistance [26]. Glucosamine reduced glucose uptake by 56% compared to the control, while vitexin recovered glucose uptake by 50% compared to the glucosamine group (Figure 4(b)). ese results suggested that vitexin alleviated glucose uptake reduction induced by glucosamine.

Discussion
Our present research demonstrated that vitexin improved HFD-induced insulin resistance in C57BL/6J male mice and recovered glucosamine-induced glucose uptake reduction.
In the present study, vitexin significantly recovered insulin tolerance and glucose tolerance impaired by HFD, and it increased glucose uptake reduced by glucosamine treatment, which are consistent with a previous study showing that oral administration of 1 to 200 mg vitexin/kg body weight effectively reduced the blood glucose level in sucrose-loaded mice [27,28]. Excessive accumulation of fat and insulin resistance is closely linked with inflammation, as with excess TNF-α release [29]. Vitexin contained in mung bean extracts reduced the intramuscular TNF-α in KK-Ay diabetic mice [30]. Vitexin also inhibited TNF-α production in LPS-treated rats and pancreatic β-cells [31]. Consistent with the studies, the current research demonstrated that vitexin potently reduced the serum TNF-α level induced by a HFD in C57BL/6J mice.
Knowledge of serum vitexin concentration in human metabolism is limited, while the bioavailability of vitexin is relatively low [32]. e absolute bioavailability of vitexin is only around 4.9% when rats are orally administered with 30 mg/kg vitexin [33]. e vitexin concentration we used in the current in vitro study might be relatively high. Studies to increase vitexin's bioavailability are therefore necessary to better utilize vitexin as a functional food ingredient. In summary, vitexin improved HFD-triggered insulin resistance and reduced hepatic fat accumulation in C57 BL/ 6J mice. erefore, vitexin can be considered as a potential functional food ingredient which may be combined with preventive strategies to alleviate insulin resistance and type 2 diabetes.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.