Dietary Lycium barbarum Polysaccharide Induces Nrf2/ARE Pathway and Ameliorates Insulin Resistance Induced by High-Fat via Activation of PI3K/AKT Signaling

Lycium barbarum polysaccharide (LBP), an antioxidant from wolfberry, displays the antioxidative and anti-inflammatory effects on experimental models of insulin resistance in vivo. However, the effective mechanism of LBP on high-fat diet-induced insulin resistance is still unknown. The objective of the study was to investigate the mechanism involved in LBP-mediated phosphatidylinositol 3-kinase (PI3K)/AKT/Nrf2 axis against high-fat-induced insulin resistance. HepG2 cells were incubated with LBP for 12 hrs in the presence of palmitate. C57BL/6J mice were fed a high-fat diet supplemented with LBP for 24 weeks. We analyzed the expression of nuclear factor-E2-related factor 2 (Nrf2), Jun N-terminal kinases (JNK), and glycogen synthase kinase 3β (GSK3β) involved in insulin signaling pathway in vivo and in vitro. First, LBP significantly induced phosphorylation of Nrf2 through PI3K/AKT signaling. Second, LBP obviously increased detoxification and antioxidant enzymes expression and reduced reactive oxygen species (ROS) levels via PI3K/AKT/Nrf2 axis. Third, LBP also regulated phosphorylation levels of GSK3β and JNK through PI3K/AKT signaling. Finally, LBP significantly reversed glycolytic and gluconeogenic genes expression via the activation of Nrf2-mediated cytoprotective effects. In summary, LBP is novel antioxidant against insulin resistance induced by high-fat diet via activation of PI3K/AKT/Nrf2 pathway.


Introduction
Chronic oxidative stress, characterized by the overproduction of reactive oxygen species (ROS) [1][2][3], is associated with glucose and lipid metabolic abnormalities [4][5][6]. High glucose concentrations and fatty acid levels stimulate excessive accumulation of ROS, which can cause the deleterious effects [7][8][9] and insulin resistance in peripheral metabolic tissues [10,11] in the absence or presence of antioxidants circumstance. Overall, oxidative stress represents an imbalance between production of ROS and the antioxidant defense system. The mechanistic link between increased ROS and insulin resistance is activation of several signaling pathways. Nuclear factor E2-related factor 2 (Nrf2) is important to explore the role of the endogenous antioxidant system in the prevention of insulin insensitivity in vivo, which regulates the expression of detoxifying and antioxidant genes, such as heme oxygenase 1 (HO-1), superoxide dismutase (SOD), and catalase (CAT) [12,13]. In response to oxidative stress or pharmacological activation, Nrf2 is translocated into the nucleus and induces the expression of antioxidant enzymes by binding antioxidant response element (ARE) [14][15][16]. Jun N-terminal 2 Oxidative Medicine and Cellular Longevity kinases (JNK) activation is a crucial mediator of ROSinduced insulin resistance [17]. Suppression of JNK activation prevents insulin receptor substrate-1 (IRS-1) degradation and promotes insulin signaling and insulin-dependent glucose uptake [18].
Long-term high-fat diet (HFD) aggravates the burden of antioxidative and anti-inflammatory system in the liver [6]. Impairment of endogenous redox system of liver is important in the development of insulin resistance in chronic HFD feeding [19]. In our study, we detected Nrf2 signaling pathway in vivo and in vitro. Administration of Lycium barbarum polysaccharide (LBP), a new PI3K/AKT/Nrf2 axis activator, prevented the development not only of oxidative stress but also of insulin resistance, as well as of glucose metabolic abnormalities [20][21][22]. Activated Nrf2 of LBP represents a potential novel approach in the treatment and prevention of insulin resistance induced by HFD.

LBP Ameliorates Insulin Resistance Induced by HFD in C57BL/6J Mice.
To determine whether LBP reduces HFDinduced insulin resistance, glucose, insulin, and pyruvate tolerance tests were measured. Intraperitoneal glucose tolerance test (IPGTT) showed that LBP (100 mg/kg) significantly reduced blood glucose 30 min after injection; area under the curve (AUC) reflected the point (Figure 1(a), < 0.05). As shown in Figure 1(b), intraperitoneal insulin tolerance test (IPITT) showed that LBP improved insulin-mediated glucose-lowering 60 min after injection; AUC also reflected the point ( < 0.05). In Figure 1(c), intraperitoneal pyruvate tolerance test (IPPTT) showed that LBP lowered blood glucose 60 min after injection; AUC also indicated the point ( < 0.05). On 24 weeks, we, respectively, measured blood glucose, insulin, and pyruvate concentrations in serum of different groups. As shown in Table 1, LBP reduced blood glucose and insulin concentrations ( < 0.05, < 0.01) and increased pyruvate concentration compared to HFD-fed mice ( < 0.05).

LBP Modulates Glycolytic and Gluconeogenic Genes
Expression in Liver of HFD-Fed Mice. In order to get a first insight into a potential interdependence between LBP and glucose metabolism, we analyzed glycolytic and gluconeogenesis genes expression levels with qRT-PCR. As shown in Figure 1(d), LBP increased GCK and PK mRNA levels and also decreased PEPCK and G6Pase mRNA levels ( < 0.05). LBP obviously elevated GCK and PK activities (Figures 1(e) and 1(f), < 0.05, < 0.01).

LBP Activates PI3K/AKT Signaling Pathway In Vitro and
In Vivo. We examined the effects of LBP on PI3K/AKT signaling pathway in vivo and in vitro. Treatment of LBP significantly enhanced the phosphorylation expression of IRS-1, PI3K, and AKT in liver (Figure 2(a)). HepG2 cells were incubated for 12 hrs with LBP in the presence of 200 M palmitate. From 100 g/mL to 600 g/mL concentration, LBP significantly promoted an increase of phospho-IRS-1, -PI3K, and -AKT levels (Figure 4(a)).

LBP Regulates Phosphorylation Levels of GSK3 and JNK via PI3K/AKT Signaling Pathway.
In in vivo experiment, treatment of LBP effectively inhibited phospho-JNK level and increased phospho-GSK3 level in liver of HFD-fed mice (Figure 2(a)). The mRNA levels of inflammatory genes were analyzed by qRT-PCR in liver. As shown in Figure 2(b), LBP lowered expressions of MCP-1, IL-6, and TNF-( < 0.01). In Figures 2(c) and 2(e), LBP significantly promoted glycogen synthesis and glucose utilization ( < 0.01). In addition, LBP significantly reduced hepatic glucose production (HGP) in liver ( Figure 2(d), < 0.05). In in vitro experiment, cells were pretreated with 100-600 g/mL LBP for 12 hrs in the presence of palmitate. LBP significantly increased phosphorylation level of GSK3 and reduced phosphorylation level of JNK depending on dose-concentration ( Figure 4(a)). In Figures 4(c) and 4(d), LBP also increased glycogen contents and decreased glucose production of palmitate-stimulated cells ( < 0.05, < 0.01). When cells were pretreated for 2 hrs with 10 M LY294002 and 2 M Wortmannin of PI3K/AKT inhibitor, respectively, and then treated for 12 hrs with 300 g/mL LBP, we found that inhibitor-induced phospho-JNK level was suppressed by LBP, and inhibitor-suppressed phospho-GSK3 level was reversed by LBP ( Figure 4(b)). Based on these findings, we conclude that LBP-supplement boosts cellular glycogen metabolism through activation of PI3K/AKT and JNK signaling pathway.

LBP Induces Nrf2 Phosphorylation Level In Vivo and In
Vitro. To test whether LBP activates Nrf2 expression in vivo, we delivered LBP at 100 mg/kg by intragastric administration to C57BL/6J mice for one day, respectively. We found that LBP could activate phospho-Nrf2 level, without any change in total Nrf2 protein expression (Figures 3(a) and 3(b), < 0.05). We also examined whether LBP directly induced Nrf2 expression in vitro. Palmitate-stimulated cells were incubated in LBP for 12 hrs. As shown in Figure 5(a), LBP significantly increased phospho-Nrf2 expression in a dose-dependent manner, without any change in total Nrf2 expression. To investigate the detailed mechanisms underlying the induction of Nrf2 expression by LBP, we explored the nuclear translocation of Nrf2 with immunofluorescence experiments. 200 M palmitate obviously resulted in phospho-Nrf2 migrating from nucleus to cytosol, while 300 g/mL LBP caused the nuclear translocation of phospho-Nrf2 ( Figure 5(b)).

LBP Activates Nrf2/ARE Pathway via PI3K/AKT Signaling.
To further elucidate, the effects of LBP on Nrf2/ARE pathway were examined. LBP induced HO-1, SOD2, and CAT protein expression in liver of HFD-fed mice (Figure 3(a)). LBP also increased SOD and CAT activities in liver (Table 1, < 0.01). As shown in Figure 5(a), LBP significantly increased the protein expression of HO-1, SOD2, and CAT in a dosedependent manner in the palmitate-stimulated environment. In Table 1 and Figure 3   liver GSH and GSH/GSSG levels, which was significantly higher than HFD-fed mice by 24 weeks. However, GSSG levels were higher in the HFD-fed group compared with LBP-supplementation group ( < 0.05, < 0.01). LBP significantly decreased intracellular ROS level (Figure 3(d)). Cells were pretreated with inhibitor and then treated with LBP. These results showed that LBP significantly activated Nrf2/ARE pathway in the presence of inhibitor ( Figure 5(c)). It was an important point that inhibitor-induced intracellular ROS levels were reversed by 300 g/mL LBP ( Figure 5(d)).
Taken together, our data suggests that LBP induces phosphorylation level of Nrf2 and activates Nrf2/ARE pathway via PI3K/AKT signaling.

Discussion
Oxidative stress is a major factor in the development of various liver diseases, affects liver function and induces hepatic insulin resistance, and is ultimately attributed to liver injury [23,24]. It is essential to control the progressive Oxidative Medicine and Cellular Longevity oxidative stress to upregulate cellular redox system [25]. The previous study has already reported that plant drugs reduce oxidative stress to maintain peripheral insulin sensitivity through increasing antioxidant or anti-inflammation effects [26][27][28]. Lycium barbarum (L. barbarum) (Gouqizi, Wolfberry), whose bioactive components are Lycium barbarum polysaccharide (LBP), is well known in traditional Chinese herbal medicine. Previous studies have shown that LBP can protect liver function and reduce blood glucose levels [29][30][31]. But the effect of LBP on Nrf2-mediated insulin resistance is still not clear. We made a hypothesis in our study that investigated molecular mechanism underlying the activation of PI3K/AKT/Nrf2 signaling pathway by LBP treatment.
The discovery of Nrf2 as main factor in the expression of endogenous antioxidant genes favored study exploiting Nrf2 as target of drugs or nutritional supplements [27,32,33]. In the present study, we found that LBP supplementation can affect status of Nrf2 expression in vivo and in vitro. First, our data suggested that treatment of LBP can increase the phosphorylation level of Nrf2 in liver of HFD-fed mice. Second, our results showed that LBP effectively increased the phospho-Nrf2 expression in dosedependent manner in palmitate-induced HepG2 cells. Third, inhibitor-suppressed Nrf2 expression was activated by LBP. Consistent with previous studies, it was shown that drugs had a positively antioxidative effect on animal models via activation of PI3K/AKT/Nrf2 pathway [34,35]. In vitro and in vivo results finally indicated that LBP as a new Nrf2 activator directly induced Nrf2 expression. Nrf2 is essential for cellular protective mechanisms against oxidative stress or inflammation through the transcriptional activation of ARE-dependent expressions of genes encoding detoxification enzymes and antioxidant enzymes such as HO-1, SOD2, CAT, and GSH [12,[36][37][38][39]. A previous study revealed that Nrf2deficiency reduced the expression of detoxification enzymes in liver, resulting in oxidative stress [40,41]. Antioxidant enzymes can improve insulin resistance in in vivo study [42]. Our results suggested that LBP effectively upregulated HO-1, SOD2, and CAT expression and reduced intracellular ROS levels through Nrf2 activity in vivo and in vitro. These results indicated that activation of Nrf2 is central to the induction of potent cellular antioxidant and detoxification systems.
JNK is a crucial mediator of insulin resistance, activated by the accumulation of ROS [43,44]. Our results suggested that HFD-induced phospho-JNK was increased in liver. In contrast, treatment of LBP significantly reduced JNK phosphorylation level and expression of inflammatory genes, such as MCP-1, IL-6, and TNF-. We further confirmed that LBP prevented expression of inflammatory factor by HFDinduced via PI3K/AKT/JNK pathway. Taken together, LBP, as an inhibitor of JNK, results in marked improvement of insulin sensitivity in mouse models of HFD-induced insulin resistance.
Activation of transcription factor Nrf2 is one of the major cellular defense lines against oxidative stress but also influences genes involved in glucose metabolism [32]. We investigated the effects of LBP on glucose homeostasis in HFD-fed mice for 24 weeks. We found that treatment with LBP increased phospho-GSK3 level and glycolytic enzymes expression but decreased gluconeogenic enzymes expression.
Oxidative Medicine and Cellular Longevity In in vitro experiment, LBP also significantly increased hepatic glucose uptake and export through Nrf2 and JNK signaling. In summary, these studies demonstrate a critical role for Nrf2 in protecting the liver from stress likely by coordinately regulating expression of genes in cytoprotective and metabolic pathways.
In conclusion, our results provide a link between Nrf2 activity, oxidative stress, and insulin resistance and demonstrate in vivo and in vitro that high-fat induced-insulin resistance could be ameliorated by LBP through upregulating PI3K/AKT/Nrf2 signaling pathway. The current study suggests that LBP may be a promising role for managing insulin 8 Oxidative Medicine and Cellular Longevity resistance-associated oxidative stress in acute or chronic liver damage.

Preparation of Lycium barbarum
Polysaccharide. LBP was extracted from L. barbarum as previously described [45]. Briefly, the dried fruit of L. barbarum was put in boiling deionized water. The water extract was filtered through a filter paper to remove impurities. The crude extract was concentrated to the volume under vacuum at 40 ∘ C and diluted to deionized water. Then, the extract was precipitated with 95% ethanol, followed by centrifugation to remove the supernatant. Then, the precipitate was collected and ground into powder. The powder of LBP was dissolved in normal saline for mice experiment, filtered through a 0.22 m filter, and stored at −20 ∘ C.

Animal Care and Treatment.
Male C57BL/6J mice from Beijing Vital River Biological Co., Ltd., were housed in standard cages placed in a room at 22 ± 2 ∘ C temperature, 55 ± 1% relative humidity, and a 12/12 light/dark cycle. All mice consumed a commercial diet and tap water for 2 weeks prior to their division into three groups ( = 10 per group): ND (10% energy from fat, D12450B, USA), HFD (60% energy from fat, D12492, USA), and 100 mg/kg LBP-supplemented diet. At the end of the animal experiments in 24 weeks, liver tissue was isolated, one sample was prepared for RNA isolation and analysis of gene expression, and another sample was frozen in liquid nitrogen and stored at −80 ∘ C. The animal experiments were approved by the Animal Research Committee of Ningxia Medical University, China.

Cell Culture and Treatment.
HepG2 cells were generously provided from Peking University and cultured in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco, USA) in 5% CO 2 at 37 ∘ C. The cells were incubated in 200 M palmitate (Sigma) for 12 hrs and then treated with 100-600 g/mL LBP for 12 hrs. In addition, the cells were pretreated with 10 M LY294002 for 2 hrs (Cell Signaling Technology) and 2 M Wortmannin (Sigma) for 2 hrs and treated with 300 g/mL LBP for 12 hrs.

Immunofluorescence Staining.
HepG2 cells were planted on coverslips and incubated overnight. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and washed with cold PBS. And cells were treated with 0.1% Triton X-100 for 10 min. Cells were incubated for 1 hr with anti-pNrf2 (1 : 100) at room temperature and washed with cold PBS. Cells were incubated for 1 hr with FITC-conjugated goat anti-rabbit IgG (1 : 200) in the dark at room temperature and washed twice with cold PBS. The nuclei were stained with DAPI (5 mg/mL) (Beyotime, China) for 5 min in the dark. Finally, coverslips were observed under a fluorescence microscope (Olympus IX71).

Detection of Intracellular ROS Generation.
For the detection of hepatic superoxide production, an oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate the in situ production of superoxides. The 10 m thick liver frozen sections were incubated for 1 hr with 50 M DHE (Beyotime, China). Fluorescent signals were scanned using a fluorescence microscopy.