Phenolic compounds ameliorate the glucose uptake in HepG2 cells' insulin resistance via activating AMPK

Journal of Functional Foods 19 (2015) 487–494

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Phenolic compounds ameliorate the glucose uptake in HepG2 cells’ insulin resistance via activating AMPK Anti-diabetic effect of phenolic compounds in HepG2 cells Qun Huang, Lei Chen *, Hui Teng *, Hongbo Song *, Xiaoqi Wu, Meiyu Xu College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

The preventive effects of phenolic compounds on insulin signalling and on both glucose

Received 26 June 2015

production and uptake were studied in insulin-responsive human HepG2 cells treated with

Received in revised form 31 August

high glucose. The influence of insulin in different-dose insulin and at different action times

2015

on the IR of HepG2 cells, and changes of glucose uptake and glycogen level were detected

Accepted 8 September 2015

by using cell culture glucose uptake analysis. Seven compounds, agrimonolide (1),

Available online

desmethylagrimonolide (2), quercetin (3), luteolin (4), luteolin-7-O-glucoside (5), kaempferol (6), and apigenin (7), were used. The IR model was successfully established in HepG2 cells

Keywords:

after the action of 5 × 10−7 M insulin for 24 h. The analysis of glucose uptake showed that

Phenolics

1–3 samples had significant glucose lowering activity and exhibited no difference with

HepG2

metformin (P > 0.05). All compounds had low cytotoxicity in HepG2 cells within the test con-

insulin resistance

centration. Compounds 1 and 2 pre-treatment also prevented the inactivation of the AKT

AKT pathway

pathway and AMPK, which play an important role in glycometabolism. © 2015 Published by Elsevier Ltd.

AMPK

1.

Introduction

Insulin is an important hormone in nutrient metabolism. Due to the remarkable therapeutic effect of purified and synthetic insulin, natural products were gradually abandoned in many nations and areas. However, complications in macrovascular or microvascular functions are still associated in patients receiving insulin injection. Insulin resistance is a hormone that facilitates the transport of blood sugar

(glucose) from the bloodstream into cells throughout the body for use as fuel and is the clinical characteristic of type 2 diabetes. In response to the normal increase in blood sugar after a meal, the pancreas secretes insulin into the bloodstream. With insulin resistance, the normal amount of insulin secreted is not sufficient to move glucose into the cells – thus the cells are said to be “resistant” to the action of insulin. Insulin resistance is a fundamental aspect of the aetiology of type 2 diabetes and is also linked to a wide array of other pathophysiologic sequelae including hypertension and

* Corresponding authors. College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China. Tel.: +86 591 83756316; fax: +86 591 83756316. E-mail addresses: [email protected] (L. Chen); [email protected] (H. Teng); [email protected] (H. Song). http://dx.doi.org/10.1016/j.jff.2015.09.020 1756-4646/© 2015 Published by Elsevier Ltd.

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Journal of Functional Foods 19 (2015) 487–494

hyperlipidaemia (Kahn & Flier, 2000). These impairments in insulin action play an important role not only in the development of hyperglycaemia of non-insulin dependent diabetes but also in the pathogenesis of long term complications. Insulinsensitive tissues, such as liver, fat, and muscle, are typically involved in regulating whole body fuel metabolism (LeRoith & Gavrilova, 2006). The liver plays many essential roles in maintaining normal physiology. It is also a vulnerable target of many drugs or other chemicals because it is involved in complex metabolism (Chen & Kang, 2013; Chen, Kang, & Suh, 2014). Thus, this organ is not able to control glucose homeostasis and there is a mis-regulation of the insulin pathway (Klover & Mooney, 2004). HepG2 cells were used in this study due to their common physiologic function to lipid or glucose metabolism with normal hepatic cells (Xu, Ma, & Purcell, 2003). At the cellular level, insulin binds and activates the insulin receptor (IR) by phosphorylating key tyrosine residues. This is followed by tyrosine phosphorylation of insulin receptor substrates (IRS) and subsequent activation of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (PKB/AKT) pathway (Klover & Mooney, 2004). AMP-activated protein kinase (AMPK) is a ubiquitous serine/ threonine protein kinase, proposed as a “fuel gage”. Recent discoveries have exhibited that AMPK activation plays a significant role in the regulation of body weight, systemic glucose homeostasis, lipid metabolism and mitochondrial biogenesis, making it an attractive therapeutic target for the treatment of diabetes (Wang, Zhao, Liu, Diao, & Kong, 2015). In the liver, AMPK is a key master switch in regulating glucose and lipid metabolism (Lin, Huang, & Lin, 2007; Meng et al., 2015). Pharmacologic studies have reported that Xian Hecao (AP) has broad biological properties, such as antioxidant, nitric oxide scavenging (Tsai, Tsai, Yu, & Ho, 2007), acetylcholinesterase (Jung & Park, 2007) and α-glucosidase inhibitory (Asano, Ishibe, & Kurachi, 2006) activities. Phenolic compounds such as quercetin,

quercitrin, hyperoside, taxifoliol, luteolin-7-O-beta-Dglucopyranoside and rutin constitute an important part of the human diet, and it can be found in AP. The antidiabetic potential and our ongoing interest in bioactive natural compounds prompted us to investigate this plant. The aim of the study was to test the potential chemoprotective effect of phenolic compounds from AP against insulin signalling restraint induced by a high glucose challenge in HepG2 cultured cells. Thus, key proteins in the signalling transduction pathway of the insulin, as well as glucose production, glucose uptake and glycogen content, were evaluated.

2.

Materials and methods

2.1.

Plant materials and chemicals

Seven phenolic compounds, agrimonolide (AM), desmethylagrimonolide (DA), quercetin (PubChem: 5280343), luteolin (PubChem: 5280445), luteolin-7-O-glucoside (PubChem: 5291488), kaempferol (PubChem: 5280863), and apigenin (PubChem: 5280443), were previously isolated from A. pilosa Ledeb and used in this study (Fig. 1). Human recombinant insulin was obtained from Korean Cell Line Bank (Seoul, Korea). Foetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Hyclone (Logan, UT, USA); human insulin, PD98059, palmitate, anthrone reagent, anthrone, and Bradford reagent were purchased from SigmaAldrich (St. Louis, MO, USA).

2.2.

Insulin-resistant HepG2 cell model (IRM)

Insulin-resistant cell model was induced according to the previous method [5]. Human HepG2 cells were grown in DMEM

Fig. 1 – Molecular structure of phenolic compounds used in this study.

Journal of Functional Foods 19 (2015) 487–494

medium supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 1% streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. After plating in 96-well for 24 h, the medium was changed to DMEM containing 1000 mg/mL D-glucose, 4 mM glutamine and 1% FBS. Subsequently, the medium was exchanged with FBS-free medium containing 5 × 10−5, 5 × 10−6, 5 × 10−7, 5 × 10−8, and 5 × 10−9 mol/L insulin; incubation was conducted in this medium for 12–48 h. At the end of the treatment, the optimum insulin concentration and incubation time were obtained to induct the insulin-resistant model (IR).

2.3.

2.6.

Preparation of cell lysates

Cells were lysed at 4 °C in a buffer containing 25 mM HEPES (pH 7.5), 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol, 0.1% Trition X-100, 200 mM β-glycerolphosphate, 0.1 mM Na3VO4, 2 µg/mL leupeptin and 1 mM phenylmethylsulphonyl fluoride. The supernatants were collected, assayed for protein concentration by using the Bio-Rad (Richmond, CA, USA) protein assay kit according to the manufacture’s specifications, aliquoted and stored at 80 °C until used for Western blot analyses.

Cytotoxicity assay 2.7.

To assess cell viability, culture media from cells exposed to the different compounds were tested by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, essentially as described by a previous study (Chen & Kang, 2013) The HepG2 cells were seeded on 96 multi-well plates at 1 × 105 cells/well and cultured for 24 h. After incubation for 48 h, 25 µL of MTT solution (5 mg/mL in phosphate-buffered saline, pH 7.4) was added to each well and incubated further for 4 h at 37 °C. Upon termination, the supernatant was aspirated and the MTT formazan, formed by metabolically viable cells, was dissolved in dimethyl sulphoxide (DMSO) 150 µL by mixing for 30 min on a gyratory shaker, and the plates were scanned at 540 nm.

2.4.

489

Glucose uptake in IR

Western blotting

HepG2 cells were cultured in 6-well plates with compounds in different concentrations for 24 h and lysed on ice in 120 µL RIPA buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 1% NP40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) from Cell Signaling Technology (Beverly, MA, USA). The samples were electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, blocked for 1 h in 5% (w/v) bovine serum albumin, and then incubated with primary antibodies overnight at 4 °C, followed by incubation with appropriate secondary antibodies for 1 h at room temperature. The bands were detected using chemiluminescent reagent and autoradiographic film.

Cellular glucose uptake was assayed in 10 µL of medium by enzymatic methods with a glucose assay kit. Cells were plated in 96-well plates at a rate of 1 × 105 cells per well, and after incubation for 24 h, the serum-free medium containing 5 × 10−7 mol/L insulin was replaced with different concentrations of the samples. At the end of the incubation periods, the medium was removed, and glucose concentrations were determined as described previously (Sigma-Aldrich). The glucose concentration of the wells with cells was subtracted from the glucose of the blank wells to obtain the amount of glucose consumption.

2.8.

2.5.

HepG2 cells are widely used for biochemical and nutritional studies as a cell culture model of human hepatocytes since they retain their morphology and most of their function in culture (Brandon, Bosch, Deenen, & Levink, 2006; Lin & Lin, 2008). Thus, this cell line has been extensively used to study the hepatic glucose production and the modulation of the insulin pathway in vitro (Lin et al., 2007). Insulin resistance in insulin sensitive organs results in metabolic disorder such as hyperglycaemia, hyperinsulinaemia and hyper triglyceridaemia, which are common features of type 2 diabetes. Insulin resistance in liver cells mainly causes impaired glycogen synthesis, failed to suppress glucose production, which is the major contribution to hyperglycaemia. In order to establish an in vitro insulin resistant model of liver cells and evaluate the effects of different insulin concentrations on glucose metabolism in the cell model, HepG2 cells were incubated with 5 × 10−5, 5 × 10−6, 5 × 10−7, 5 × 10−8, and 5 × 10−9 mol/L insulin to build an insulin-resistant cell

Determination of glycogen content

To measure the content of glycogen, HepG2 cells were seeded on 96-well cell culture plates at a density of 1 × 105 cells/well. Following 24 h of stabilization, the cells were pretreated with or without samples at concentrations of 0.1, 1, and 10 µM PD98059 (ERK inhibitor), 30 µM of sodium salicylate (5 mM) for 30 min, followed by stimulation with palmitate (PA) (0.5 mM) for another 24 h. Then, cells were washed twice with phosphatebuffered saline (PBS) and cultured in DMEM containing 100 nM insulin for 3 h. Incubation media were discarded and cells were collected and washed to remove extracellular glucose. The glycogen content of the cells was determined by the anthrone reagent. The amount of blue compound generated by the reaction was assayed at 620 nm. The protein content of the collected HepG2 cells was quantified with Bradford reagent. Values were presented in the ratio of mg glycogen/mg protein.

Statistical analysis

Statistical analyses were performed by Statistical Analysis System (SAS, Cary, NC, USA). Data were subjected to analysis of variance (ANOVA), followed by mean comparisons by Duncan’s multiple range test at p < 0.05.

3.

Result and discussion

3.1.

Insulin dose-dependent effect of HepG2 cells

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Table 1 – Influence of insulin concentrations on glucose consumption in HepG2 cells.

Control Insulin

Concentration of insulin (mol/L)

Glucose consumption (mmol/L)

Glucose consumption ratio (%)

0 5 × 10−5 5 × 10−6 5 × 10−7 5 × 10−8 5 × 10−9

3.97 ± 0.04c 2.94 ± 0.08b 3.04 ± 0.30b 2.29 ± 0.09a 2.85 ± 0.02b 3.13 ± 0.07b

25.95 23.43 42.32 28.21 21.16

Note. Values represent mean ± SE from three independent repeats. Different letters indicate a significant difference by Duncan’s multiple range test at p < 0.05.

model. The cells were stimulated with fresh insulin for 24 h and the glucose uptake by these cells was carried out. The glucose uptake by the cells was detected by the method of glucose oxidizes/peroxides (GOD-POD). As shown in Table 1, following 5 × 10−7 mol/L insulin incubation of HepG2 cells, there was the most significant decrease in the consumption of extracellular glucose (P < 0.05) compared with blank control without insulin pretreatment. As depicted in Table 2, the addition of insulin to HepG2 cells grown in DMEM containing different amounts of glucose induced a statistically significant decrease (P < 0.05) in the glucose consumption after incubation for 24 h.

3.2.

Cell viability and proliferation

To determine the potential effects on cell viability and proliferation of compounds in a human hepatic cell line (HepG2),

Table 2 – Influence of insulin on glucose consumption in HepG2 cells at different action times. Time

Insulin concentration (M)

Glucose consumption (mM)

12 h

5 × 10−5 5 × 10−6 5 × 10−7 5 × 10−8 5 × 10−9 5 × 10−5 5 × 10−6 5 × 10−7 5 × 10−8 5 × 10−9 5 × 10−5 5 × 10−6 5 × 10−7 5 × 10−8 5 × 10−9 5 × 10−5 5 × 10−6 5 × 10−7 5 × 10−8 5 × 10−9

3.92 ± 0.26h 3.92 ± 0.27h 3.96 ± 0.37 h 3.38 ± 0.22d,e,f 3.42 ± 0.31d,e,f 3.10 ± 0.03b,c,d 3.19 ± 0.02b,c,d 2.68 ± 0.14a 3.00 ± 0.05b,c 2.90 ± 0.18a,b 3.25 ± 0.12c,d,e 3.69 ± 0.18f,g 3.98 ± 0.05h 3.57 ± 0.05e,f,g 3.85 ± 0.26g,h 3.44 ± 0.05d,e,f 3.33 ± 0.22c,d,e 3.92 ± 0.15h 3.99 ± 0.12h 3.34 ± 0.09d,e

24 h

36 h

48 h

Note. Values represent mean ± SE from three independent repeats. Different letters indicate a significant difference by Duncan’s multiple range test at p < 0.05.

cells were exposed to a high concentration of 20 µM for 24 h. Treatment of HepG2 cells for 24 h with tested samples did not evoke changes in cell viability, as determined by the MTT assay, indicating that the concentrations selected for the study did not damage cell integrity during the period of incubation (Fig. 2B). Similarly, treatment with compounds did not affect cell growth, indicating no impairment of cell proliferative machinery and preservation of a regular cell cycle (Fig. 2A).

3.3. Effect of isolated compounds on sensitivity to exogenous insulin in insulin-resistant HepG2 cells The effect of compounds on insulin resistance was comparable to that of metformin, a biguanide agent that reduces hyperinsulinaemia and improves hepatic insulin resistance. The glucose consumption of the insulin-resistant model was significantly improved by EA fraction treatment. As shown in Fig. 2C, compounds 1 and 3 had the strongest activity with lowering values of 51.2 and 55.0%, respectively, at 20 µM concentration. Compound 1 consisting of 2 and 3 showed the best activity in all tested compounds with values of 62.3, 59.6, and 66.9%, respectively, and had no significant differences with metformin (70.5%). Shisheva and Shechter (1992) reported that quercetin treatment of intact rat adipocytes blocked insulinstimulated effects on glucose metabolism, including glucose transport, oxidation, and its incorporation into lipids, suggesting that an active receptor tyrosine kinase is necessary for mediating these bioeffects. Insulin-resistant glucose uptake is a prominent feature of both type I (DeFronzo, Hendler, & Simonson, 1982) and type II DM (Scarlett, Kolterman, Ciaraldi, Kao, & Olefsky, 1983) in humans and of experimental models of diabetes in rats. This defect is evident in vivo as reduced glucose uptake in response to insulin measured by euglycaemic clamp (Rossetti, Smith, Shulman, Papachristou, & DeFronzo, 1987) and in isolated adipose cells in vitro as reduced glucose transport response to insulin. Therefore, the modulation of the mentioned targets could be beneficial for the prevention and control of diabetes. An earlier study of Nomura et al. (2008) also demonstrated that certain flavonoids such as luteolin, quercetin, apigenin and kaempferol showed inhibitory effects on the insulin signalling pathway in mouse adipose cells by suppressing insulin receptor phosphorylation and subsequent inhibition of Akt activation.

3.4.

Glycogen synthesis in HepG2 Cells

As shown in Fig. 3, PA (500 µM) treatment significantly decreased glycogen content in control. In the presence of compounds and PD98059, glycogen in HepG2 cells increased significantly. Glycogen is present as a storage form of D-glucose biological sample, in animal tissues, particularly in muscles and in the liver. The main insulin action in the liver is to increase glycogen synthesis, and hepatic insulin resistance is characterized by the reduced capacity of insulin to increase glycogen synthesis. Stimulation of palmitate resulted in a decrease in glycogen content; compounds 1, 2, 3 and 5 effectively increased insulin-mediated glycogen synthesis in hepatocytes. Glycogen synthesis from different precursors and its degradation are known to be dependent on nutritional and hormonal factors, principally glucose and insulin. An increased

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Fig. 2 – Cell viability and dose-dependent effects of compounds on glucose consumption in HepG2 cells. A: Changes in morphology of cells following 24 h treatment without insulin (control) or with insulin (IR model). B: Cytotoxicity of compounds on HepG2 cell. Cells were treated with 25 µg/mL concentrations of samples for 24 h. Blank, non-treated cells. * Significant difference from blank. C: Effects of isolated compounds on glucose consumption in HepG2 cells (Mean ± SD, n = 3), values follows by different lowercase letters with in the same row are significant difference at p < 0.05.

Fig. 3 – Changes of glycogen content in cells in all groups (Mean ± SD, n = 3), values follows by different lowercase letters with in the same row are significant difference at p < 0.05. *Significant difference from Blank.

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glucokinase activity enhanced glucose utilization and glucose uptake in the liver (Jung, Yoon, Bae, Min, & Choi, 2008). The elevation of hepatic glucokinase activity could increase the utilization of blood glucose for glycogen storage in the liver (Shisheva & Shechter, 1992). Insulin decreased the hepatic glucose output by activating glycogen synthesis and glycolysis, and by inhibiting gluconeogesis (McGarry, 1992). The liver plays a critical role in maintaining blood glucose concentration both through its ability to supply glucose to the circulation via glycogenolysis and gluconeogenesis in the post absorptive state and to remove glucose from the circulation after meal ingestion. However, in diabetes, the gluconeogenic pathway is aberrantly activated and supplies a relatively larger amount of glucose into the circulation.

3.5. AM and DA prevent high-glucose induced downregulation of AKT AKT lays downstream of PI3K and facilitates glucose uptake and glycogen synthesis in the liver, and directly contributes to the activity of GS, which is the key molecular mediating the metabolic effects of insulin signalling (Whiteman, Cho, & Birnbaum, 2002). To evaluate the potential protective effect of representative compounds AM and DA against the alterations caused on AKT by a high glucose concentration, the phosphorylated and total levels of the mentioned proteins were analysed in cell lysates by Western blot analysis. Treatment of HepG2 cells with AM and DA for 24 h evoked a significant increase in the phosphorylated levels of AKT (Fig. 4), as previously reported. Likewise, pre-treatment of HepG2 cells with AM and DA prevented the diminution in the p-AKT levels caused by 30 mM glucose. There was no difference in the total levels of AKT. All these results suggest that AM and DA restrained the inhibition of the AKT pathway, which constitutes a key route in the insulin signalling cascade.

3.6. AM and DA prevent high-glucose induced downregulation of AMPK phosphorylation AMPK has been proven to be required for antidiabetic effects of some clinical drugs in insulin-resistant human HepG2 cells (Hardie, 2011). In recent years, considerable research indicated that the activation of AMPK in the liver, skeleton muscles, and adipose tissue promotes glucose uptake, insulin sensitivity and fatty acid oxidation and mitochondrial biogenesis (O’Neill, 2013). Thus, in this study the effects of AM and DA on the phosphorylation of AMPK was evaluated. IR HepG2 cells were pretreated with increasing doses of the sample for 24 h. As a result, AM and DA significantly stimulated the phosphorylation of AMPK in a dose-dependent manner (Fig. 5). In addition, these two compounds significantly reversed the phosphorylation level of AMPK by IR HepG2 cells compared with control cells at concentrations of 10–20 µM. This result suggested that AMPK was supposed to be a key molecule in the glycometabolism signal transduction pathway targeted by AM and DA. AMPK is an intracellular energy sensor implicated in the regulation of cellular metabolism, in which phosphorylation is suppressed in insulin-resistant hepatic cells (Hardie, 2011; Zang et al., 2004). In this line, AM and DA contribute to restore the diminished p-AMPK levels in the liver of insulin-resistant

Fig. 4 – Protective effect of AM and DA on the diminished phosphorylated and total AMPK levels evoked by 30 mM glucose in HepG2 cells. Cells treated with 10 and 20 µM samples for 24 h were exposed to 30 mM glucose (Glu) for 24 h and then incubated with 100 nM (Insulin) for 10 min. (Up) Bands of representative experiments. (Down) Percent values of p-AMPK/AMPK ratio relative to the control condition. Equal loading of Western blots was ensured by β-actin. Different letters over bars indicate statistically significant differences (p < 0.05).

mice and in high-glucose-incubated adipocytes, respectively, which helped to prevent hyperglycaemia and insulin resistance (Guo, Guo, Jiang, Li, & Ling, 2012; Si et al., 2011; Yamashita, Okabe, Natsume, & Ashida, 2012). Phenolic compounds have also been shown to activate AMPK in hepatic cells and in high glucose-induced insulin resistant HepG2 cells (Lin & Lin, 2008; Zhang et al., 2010) and, consequently, to modulate cellular metabolism. The flavonol glycoside derived from Sophorae flos improves glucose uptake in 3T3-L1 cells at 20 µM (Ha et al., 2010). Similarly, naringin reversed the reduced phosphorylated levels of AMPK in primary hepatocytes cultured in high glucose as well as in the liver of mice fed with a high-fat diet (Pu et al., 2012). 7-O-methyl aromadendrin, isolated from Inula viscosa, stimulates the reactivation of phosphorylation of AMPK in HepG2 cells at 10 µM (Zhang et al., 2010). For the first time, we report the representative phenolic compounds from A. pilosa Ledeb which could enhance glucose consumption of IR HepG2 cells.

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Conflict of interest There is no a conflict of interest.

Acknowledgement This research was supported by the Construction Project of Top University at Fujian Agriculture and Forestry University of China (Grant No. 612014042) and scientific research project for young and middle-aged teachers of fujian province in 2015. REFERENCES

Fig. 5 – Preventive effect of AM and DA on the decreased phosphorylated and total levels of AKT induced by 30 mM glucose in HepG2 cells. Cells treated with 10 and 20 µM samples for 24 h were exposed to 30 mM glucose for 24 h and then incubated with 100 nM (Insulin) for 10 min. (Up) Bands of representative experiments. (Down) Percent values of p-AMPK/AMPK ratio relative to the control condition. Equal loading of Western blots was ensured by β-actin. Different letters over bars indicate statistically significant differences (p < 0.05).

4.

Conclusions

In summary, phenolic constituents from AP extract alleviate hepatic insulin resistance, as they had a glucose-lowering effect and low toxicity in HepG2 cells. In addition, AM and DA, at least in part, increase glucose utilization, which seemingly was mediated via elevating glucokinase activity and hepatic glycogen concentration. Therefore, it may play a crucial role in increasing insulin sensitivity, which is very meaningful in the treatment of type 2 diabetes. Although the understanding of high glucoseinduced insulin resistance has recently progressed, effective therapeutic strategies to prevent or delay the development of this damage remain limited. Further studies are required for mechanism and animal test confirmation.

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