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Peptide IPPKKNQDKTE ameliorates insulin resistance in HepG2 cells via blocking ROS-mediated MAPK signaling
Journal of Functional Foods 31 (2017) 287–294
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Peptide IPPKKNQDKTE ameliorates insulin resistance in HepG2 cells via blocking ROS-mediated MAPK signaling Jia-jia Song a,b, Qian Wang b, Min Du c, Bin Chen d, Xue-ying Mao a,b,⇑ a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, China College of Food Science and Nutritional Engineering, Key Laboratory of Functional Dairy, Ministry of Education, China Agricultural University, Beijing, China c Department of Animal Sciences, Washington State University, Pullman, WA, USA d Key Laboratory of Space Nutrition and Food Engineering, China Astronauts Research and Training Center, Beijing, China b
a r t i c l e
i n f o
Article history: Received 17 October 2016 Received in revised form 31 January 2017 Accepted 2 February 2017
Keywords: Insulin resistance HepG2 cells Reactive oxygen species Nrf2 HO-1
a b s t r a c t Reactive oxygen species (ROS) has been found to play an important role in insulin resistance. Whether the improvement effects of the peptide IPPKKNQDKTE on hepatic insulin resistance were mediated by ROS elimination was examined in this study. Results showed that the high glucose-induced MAPK signaling activation was inhibited by IPPKKNQDKTE in insulin-resistant HepG2 cells, which contributed to the improvement of IPPKKNQDKTE on cellular glucose uptake and the phosphorylation levels of insulin receptor substrate-1 (IRS-1) Ser636/639 and Ser307. IPPKKNQDKTE dose-dependently reduced high glucose-induced ROS production, which involved in suppression of IPPKKNQDKTE on MAPK signaling in high glucose-induced insulin-resistant HepG2 cells. Moreover, IPPKKNQDKTE increased Nrf2 nucleus translocation and induced HO-1 expression. HO-1 inhibitor partly reversed IPPKKNQDKTE-mediated suppression on high glucose-induced ROS production. Taken together, IPPKKNQDKTE reduced high glucoseinduced ROS production and MAPK activation by the activation of Nrf2/HO-1, which contributed to increase the glucose uptake and decrease the Ser phosphorylation levels of IRS-1 in insulin-resistant HepG2 cells. These results indicated that IPPKKNQDKTE plays a potential role in the management of hepatic insulin resistance. Ó 2017 Published by Elsevier Ltd.
1. Introduction Diabetes mellitus is a serious chronic metabolic disorder and has become a world-wide epidemic. In 2013, 382 million people worldwide suffered from diabetes, and the total number of people with diabetes is expected to reach 592 million by the year 2035 (Guariguata et al., 2014). Among people with diabetes, approximately 90–95% of diabetic individuals have type 2 diabetes mellitus, which is characterized by decreased response of peripheral tissues to insulin action (ie, insulin resistance) and impaired pancreatic b-cell function (Castillo, Mull, Reagan, Nemr, & Mitri, 2012; Onishi, Ono, Rabøl, Endahl, & Nakamura, 2013). Type 2 diabetes may cause many severe complications, including cardiovascular disease, nephropathy, retinopathy and neuropathy (Schram et al., 2014). Thus, it is important to prevent and manage type 2 diabetes. ⇑ Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, Key Laboratory of Functional Dairy, Ministry of Education, China Agricultural University, 100083 Beijing, China. E-mail address: [email protected] (X.-y. Mao). http://dx.doi.org/10.1016/j.jff.2017.02.005 1756-4646/Ó 2017 Published by Elsevier Ltd.
Liver is one of the major target tissues of insulin, and plays a vital role in stabilizing plasma glucose levels via regulation of hepatic glucose utilization or production. Hepatic insulin resistance contributes to hyperglycaemia and glucose intolerance, and becomes a principal component in the pathogenesis of type 2 diabetes (Galbo et al., 2013; Leclercq, Morais, Schroyen, Van Hul, & Geerts, 2007). Moreover, hepatic insulin resistance is associated with hyperinsulinemia and pancreatic b cell hyperplasia (Michael et al., 2000). It is reported that amelioration of hepatic insulin resistance reduces hyperglycemia and hyperinsulinemia, and improves glucose intolerance in type 2 diabetic Zucker diabetic fatty rats (Cordero-Herrera, Martín, Goya, & Ramos, 2015). Accumulating evidence has demonstrated that increased levels of reactive oxygen species (ROS) are an important trigger for multitypes of insulin resistance, and contribute to the onset and development of type 2 diabetes (Dong et al., 2016; Houstis, Rosen, & Lander, 2006; Matsuzawa-Nagata et al., 2008). Furthermore, ROS levels are negatively correlated with insulin sensitivity (Rains & Jain, 2011). The reduction in ROS level contributes to the improvement of hepatic insulin resistance in high-fat diet-treated mice (Zhang et al., 2013). Taken together, elimination of ROS may be
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an effective approach in the management of hepatic insulin resistance and type 2 diabetes. In order to alleviate insulin resistance and manage type 2 diabetes, several antioxidant compounds have been used. N-acetyl-lcysteine (NAC), a potent scavenger of free radicals has been shown to reduce hyperglycemia and hyperinsulinemia, and improve insulin resistance in high-fat diet-induced obese mice (Ma, Gao, & Liu, 2016). Moreover, some natural bioactive compounds, such as oleanolic acid, b-cryptoxanthin and goby fish protein hydrolysates, enhance hepatic antioxidant defenses and ameliorate hyperglycemia in diabetic animal models (Nasri et al., 2015; Ni et al., 2015; Wang et al., 2013). Among these bioactive ingredients, protein hydrolysates arouse more attentions from many researchers due to their diverse bioactive properties and high safety (Udenigwe, 2014). Glycomacropeptide (GMP) derived from bovine j-casein is a hydrophilic glycopeptide containing 64 amino acid residues. It is shown to possess antihypertensive activity, and reduce cardiovascular disease risk markers and ulcerative colitis (Keogh & Clifton, 2008; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Ming, Jia, Yan, Pang, & Chen, 2015). Our previous study has demonstrated that GMP reduced the body weight of dietary obese rats, and attenuated hepatic steatosis and oxidative stress in obese rats (Xu, Mao, Cheng, & Chen, 2013). Moreover, GMP-derived peptides inhibited the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages, alleviated intracellular ROS production and restored the activities of endogenous antioxidants in hydrogen peroxide-stimulated RAW 264.7 macrophages (Cheng, Gao, Chen, & Mao, 2015; Cheng, Gao, Song, Ren, & Mao, 2015). Besides, in our previous study, the peptide IPPKKNQDKTE which was identified from GMP-derived peptides were demonstrated to decrease the phosphorylation of insulin receptor substrate-1 (IRS-1) Ser307, an important negative regulator of insulin signaling, in high glucose-induced insulin-resistant HepG2 cells. The beneficial effects of the peptide IPPKKNQDKTE on IRS-1 Ser307 phosphorylation and downstream insulin signaling were mediated by AMP-activated protein kinase (AMPK) activation (Song et al., 2017). Apart from Ser307 phosphorylation site in IRS-1, Ser636/639 is a well-recognized phosphorylation site which negatively affects insulin signaling (Bouzakri et al., 2003; Rajesh, Sathish, Srinivasan, Selvaraj, & Balasubramanian, 2013). However, whether the peptide IPPKKNQDKTE can reduce ROS production and IRS-1 Ser636/639 phosphorylation in high glucoseinduced insulin-resistant HepG2 cells remains unclear. Thus, in the current study, we hypothesized that the improvement effects of the peptide IPPKKNQDKTE on insulin resistance in high glucose-stimulated HepG2 cells were mediated by ROS reduction. In order to verify the hypothesis, the key features of insulin resistance, and the alternation of intracellular ROS levels and related signaling pathways were evaluated. 2. Materials and methods 2.1. Materials 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-Dglucose (2-NBDG) was from Life Technologies (Carlsbad, CA, USA). Zinc (II)-protoporphyrin IX (ZnPPIX), 20 ,70 -dichlorodihydro fluorescein diacetate (DCFH-DA), Hemin and NAC were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059, SB203580, SP600125, anti-b-actin, anti-Histone H3, anti-HO-1, anti-Nrf2, anti-IRS-1, anti-p-IRS-1 (Ser307), anti-p-IRS-1 (Ser636/639), antiERK, anti-phosphorylated ERK, anti-JNK, anti-phosphorylated JNK, anti-p38, anti-phosphorylated p38 rabbit antibodies and horseradish peroxidase-conjugated anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in the study were of analytical grade.
2.2. Peptide synthesis The peptide IPPKKNQDKTE (purity, P95%) was prepared by the conventional Fmoc solid-phase synthesis method, which was carried out by Nanjing Leon Biological Technology Co. Ltd. (Nanjing, China). 2.3. Cell culture Human HepG2 hepatocytes were purchased from American Type Culture Collection (Rockville, MD, USA). HepG2 cells were grown in Minimum Essential Medium (MEM), supplemented with 10% fetal bovine serum, 1% MEM non-essential amino acids, 100 lg/mL streptomycin and 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA). Cells were maintained in CO2 incubator (37 °C, 5% CO2, 95% humidity). 2.4. Cell treatments A cell model of insulin resistance was developed by incubating HepG2 cells with 30 mM glucose for 24 h, as described previously (Cordero-Herrera, Martín, Goya, & Ramos, 2014; Zang et al., 2004). To evaluate the protective effect of IPPKKNQDKTE against high glucose-induced insulin resistance, HepG2 cells were treated by 5.5 mM or 30 mM glucose in the presence of various concentrations of IPPKKNQDKTE (0, 125, 250 and 500 lM) for 24 h. Later, cells were incubated with 100 nM insulin for 10 min and harvested. 2.5. Glucose uptake assay Cellular glucose uptake was monitored using 2-NBDG, a fluorescent deoxyglucose analog, as described previously (Zou, Wang, & Shen, 2005). Briefly, 2-NBDG was added at a final concentration of 10 mM after washing the treated-cells two times with phosphate buffered saline (PBS, pH 7.4). After incubation at 37 °C for 1 h, the cells were washed twice with PBS buffer to remove the unabsorbed probe. Then, the cell suspensions obtained by cell trypsinization were subjected to flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) at excitation/ emission wavelengths of 485/530 nm. Results were analyzed by FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA). 2.6. Determination of intracellular ROS The production of intracellular ROS was assayed using the fluorescent probe DCFH-DA, as described previously (Hou et al., 2015). After treatments, cells were washed with PBS (pH 7.4), and then DCFH-DA (final concentration, 10 lM) was added to the cells. After 30 min incubation at 37 °C, the cells were washed two times with PBS to remove extracellular DCFH-DA. Then, cells were trypsinized and resuspended in PBS. The intracellular ROS levels were measured by flow cytometry (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data were analyzed by FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA). 2.7. Western blot analysis After treatments, cells were harvested in cell lysis buffer (Beyotime, Haimen, Jiangsu, China), supplemented with a 1/100 dilution of protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). The supernatant of whole-cell lysates was collected by centrifugation at 12000g for 15 min at 4 °C, and used for western blot. Nuclear and cytoplasmic protein samples were prepared using nuclear and cytoplasmic extraction reagent kit
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(Beyotime, Haimen, Jiangsu, China). Equal amounts of protein samples were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) on a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The PVDF membranes were blocked with 5% nonfat dry milk or bovine serum albumin in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 2 h, and then incubated with primary antibodies overnight at 4 °C. Immunoblots were washed three times (each for 5 min with TBS-T), followed by 1 h incubation in peroxidase conjugated secondary antibody at room temperature. After three 5-min washes with TBS-T, blots were developed using enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA). Images were obtained by an Amersham Imager 600 imaging system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The optical density of the band was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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followed by Duncan’s multiple-comparison test using SPSS software (version 20.0, IBM Inc., Chicago, IL, USA). 3. Results 3.1. IPPKKNQDKTE inhibited mitogen-activated protein kinases (MAPK) signaling in high glucose-induced insulin-resistant HepG2 cells To evaluate the effect of IPPKKNQDKTE on MAPK signaling in high glucose-induced insulin-resistant HepG2 cells, the phosphorylation levels and total proteins of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 were analyzed. As shown in Fig. 1, compared with control cells, the levels of phosphorylated JNK, ERK and p38 were elevated by high
2.8. Statistics All assays were carried out at least in triplicate and data were shown as means ± standard deviations (SD). Significant differences (p < 0.05) between means were assessed with one way ANOVA,
Fig. 1. Effect of IPPKKNQDKTE on phosphorylated and total levels of JNK, ERK, p38 in high glucose-induced insulin-resistant HepG2 cells. Cells were incubated with 30 mM glucose in the presence of various concentrations (0, 250 and 500 lM) of IPPKKNQDKTE for 24 h, followed by 100 nM insulin stimulation for 10 min. Panel A showed a representative Western blot for the phosphorylated and total levels of JNK, ERK and p38. Panel B showed the densitometric analysis of three different blots. Values are means ± SD from three separate determinations. Values with different letters (a–d) in the same column are significantly different from each other (p < 0.05).
Fig. 2. Effect of IPPKKNQDKTE and MAPK inhibitors on glucose uptake in insulinresistant HepG2 cells. Cells were cultured in 5.5 and 30 mM glucose culture medium in the presence of IPPKKNQDKTE (500 lM), PD98059 (50 mM), SB203580 (10 mM) and SP600125 (40 mM) for 24 h. Then, cells were stimulated by 100 nM insulin for 10 min. Cellular glucose uptake was determined using fluorescent 2NBDG. (A) Representative histograms of 2-NBDG fluorescence. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM), blue line represented cells treated with high glucose plus PD98059 (50 mM), green line represented cells treated with high glucose plus SB203580 (10 mM) and pink line represented cells treated with high glucose plus SP600125 (40 mM). (B) The results were expressed as percentage of mean fluorescence intensity relative to control cells. Values are means ± SD from three separate determinations. Values with different letters (a–c) are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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glucose treatment. However, IPPKKNQDKTE inhibited the high glucose-induced phosphorylation of JNK, ERK and p38, especially inhibited the expression levels of p-ERK and p-p38 in a concentration-dependent manner. These results suggested that the activation of MAPK signaling was inhibited by IPPKKNQDKTE in high glucose-induced insulin-resistant HepG2 cells. 3.2. IPPKKNQDKTE and MAPK inhibitors increased the cellular glucose uptake in high glucose-induced insulin-resistant HepG2 cells In order to investigate whether the improvement of IPPKKNQDKTE on insulin resistance in high glucose-stimulated HepG2 cells was mediated by MAPK signaling, the glucose uptake in IPPKKNQDKTE and MAPK inhibitors-treated cells was analyzed. As presented in Fig. 2A and B, high glucose stimulation significantly decreased cellular glucose uptake in comparison with control cells without high glucose addition (p < 0.05). IPPKKNQDKTE treatment was able to ameliorate the reduced glucose uptake caused by high glucose. Similarly, JNK inhibitor SP600125, ERK inhibitor PD098059 and p38 inhibitor SB203580 prevented the adverse effect of glucose uptake provoked by high glucose. These results suggested that the improvement of IPPKKNQDKTE on glucose uptake in high glucose-induced insulin-resistant HepG2 cells may be related with MAPK signal inhibition.
3.3. IPPKKNQDKTE and MAPK inhibitors decreased the phosphorylation levels of IRS-1 in high glucose-induced insulin-resistant HepG2 cells To further investigate the mechanism of the improvement of IPPKKNQDKTE on insulin resistance in HepG2 cells under high glucose conditions, the phosphorylation levels of IRS-1 in IPPKKNQDKTE and MAPK inhibitors-treated HepG2 cells were evaluated. As shown in Fig. 3A and B, high glucose promoted the phosphorylation of IRS-1 on Ser636/639 and Ser307, which was prevented by IPPKKNQDKTE treatment. In addition, blockage of ERK and p38 prevented the increase of p-IRS-1 Ser636/639 induced by high glucose, whereas JNK inhibition suppressed the enhancement in high glucose-induced phosphorylation level of IRS-1 Ser307. These results indicated that suppression of MAPK signaling may participate in the regulation of IPPKKNQDKTE on IRS-1 phosphorylation in high glucose-induced insulin-resistant HepG2 cells. 3.4. Reduction of high glucose-induced ROS production by IPPKKNQDKTE contributed to the suppression of MAPK signaling in insulin-resistant HepG2 cells To investigate whether the inhibition of IPPKKNQDKTE on MAPK signaling activation was mediated by ROS elimination, the
Fig. 3. Effect of IPPKKNQDKTE and MAPK inhibitors on the expression of p-IRS Ser636/639 and Ser307 in insulin-resistant HepG2 cells. Cells were cultured in 5.5 and 30 mM glucose culture medium in the presence of IPPKKNQDKTE (500 lM), ERK inhibitor PD98059 (50 mM), p38 inhibitor SB203580 (10 mM) and JNK inhibitor SP600125 (40 mM) for 24 h, followed by 100 nM insulin treatment for 10 min. The protein expression levels of phosphorylated IRS-1 on Ser636/639 and Ser307 were assessed by western blot. Panel A and B showed a representative blot for p-IRS-1 Ser636/639 and densitometric results from three different assays, respectively. Panel C and D showed a representative blot for p-IRS-1 Ser307 and densitometric results from three different assays, respectively. Values are means ± SD from three separate determinations. Values with different letters (a–c) are significantly different from each other (p < 0.05).
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levels of intracellular ROS in high glucose-treated HepG2 cells were monitored. Results in Fig. 4A and B clearly demonstrated that high glucose markedly increased the production of ROS, compared with control cells without glucose addition (p < 0.05). Treatment with IPPKKNQDKTE reduced the high glucose-induced ROS production in a concentration-dependent manner. Moreover, NAC, a potent ROS scavenger inhibited MAPK activation induced by high glucose in HepG2 cells (Fig. 4C). These results suggested that the blockage of IPPKKNQDKTE on high glucose-induced MAPK signaling activation may be associated with suppression of ROS production.
comparison with the control cells. To evaluate the potential role of HO-1 in the inhibition of high glucose-induced ROS production, HepG2 cells were pretreated with ZnPPIX, a HO-1 inhibitor and Hemin, an inducer of HO-1. As expected, ZnPPIX treatment partly reversed the inhibitory effect of IPPKKNQDKTE on high glucoseinduced production of ROS (Fig. 5B and C), whereas Hemin increased the suppression of IPPKKNQDKTE on high glucoseinduced ROS production. These results indicated that reduction of high glucose-induced ROS by IPPKKNQDKTE was at least partially mediated by Nrf2/HO-1 signaling activation.
3.5. Nrf2/HO-1 signaling activation involved in IPPKKNQDKTEmediated ROS elimination
4. Discussion
With the objective of evaluating the role of Nrf2/HO-1 signaling in IPPKKNQDKTE-mediated ROS elimination, the changes in Nrf2 nuclear translocation and HO-1 protein expression in HepG2 cells were assessed. As shown in Fig. 5A, IPPKKNQDKTE dosedependently increased the protein expression level of Nrf2 in nuclear fraction, whereas the protein expression level of Nrf2 in cytosol fraction was remarkably decreased by IPPKKNQDKTE dose-dependently. Moreover, the protein expression of HO-1 was up-regulated by IPPKKNQDKTE in a dose-dependent manner in
Liver plays an important regulatory role in the maintenance of blood glucose homeostasis. One third of the glucose is taken up by the liver after an oral glucose load (Moore, Coate, Winnick, An, & Cherrington, 2012). Insulin-resistant hepatocytes exhibit impaired insulin-stimulated glucose uptake and defective insulin signal transduction (Cordero-Herrera et al., 2014; Huang, Shen, & Wu, 2009). The Ser/Thr phosphorylation of IRS proteins is considered to be a molecular basis for insulin resistance (Zick, 2005). The serine phosphorylation, particularly on Ser636/639 and Ser307 of IRS-1 inhibits its tyrosine phosphorylation by inhibiting
Fig. 4. Inhibitory effect of IPPKKNQDKTE on high glucose-induced production of ROS in HepG2 cells. HepG2 cells were incubated with NAC (0 and 2 mM) for 1 h prior to the treatment of 24-h glucose (5.5 and 30 mM) and various concentration of IPPKKNQDKTE (0, 250 and 500 lM), and further exposed to 100 nM insulin for 10 min. The production of intracellular ROS was assayed using the fluorescent probe DCFH-DA. The phosphorylated and total levels of JNK, ERK, p38 were analyzed by western blot. (A) Representative flow cytometry histogram data. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, green line represented cells treated with high glucose plus IPPKKNQDKTE (250 lM), red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM) and blue line represented cells treated with high glucose plus NAC (2 mM). (B) The results were expressed as mean fluorescence intensity of each sample group. (C) A representative Western blot of p-JNK, p-ERK, p-p38, JNK, ERK and p38. (D) The densitometric analysis of three different blots. Values are means ± SD from three separate determinations. For (B), values with different letters (a–d) are significantly different from each other (p < 0.05). For (D), values with different letters (a–d) in the same column are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Effects of IPPKKNQDKTE treatment on Nrf2 nuclear translocation and HO-1 protein expression in HepG2 cells. Cells were pretreated with ZnPPIX or Hemin (0 and 10 lM) for 1 h in the absence or presence of 30 mM glucose and various concentrations of IPPKKNQDKTE (0, 125, 250 and 500 lM) for 24 h, and then stimulated by 100 nM Insulin for 10 min. The expression levels of nucleus Nrf2, cytosol Nrf2 and HO-1 were determined by western blot. Intracellular ROS was monitored by DCFH-DA. (A) and (B) showed a representative blot for nucleus Nrf2, cytosol Nrf2 and HO-1, and densitometric results from three different assays, respectively. (C) Representative histograms of DCFH-DA fluorescence. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM), green line represented cells treated with high glucose, IPPKKNQDKTE (500 lM) and ZnPPIX (10 lM) and blue line represented cells treated with high glucose, IPPKKNQDKTE (500 lM) and Hemin (10 lM). (D) The results were expressed as mean fluorescence intensity of each sample group. Values are means ± SD from three separate determinations. For (B), values with different letters (a–d) in the same column are significantly different from each other (p < 0.05). For (D), values with different letters (a–d) are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the activity of the insulin receptor kinase, which blocks the downstream effector pathways and impairs insulin signaling (Bouzakri et al., 2003; Klover & Mooney, 2004). In the present study, we demonstrated that the peptide IPPKKNQDKTE increased insulinstimulated glucose uptake in high glucose-induced insulinresistant HepG2 cells. The increased phosphorylation levels of IRS-1 on Ser636/639 and Ser307 by high glucose were reversed by peptide IPPKKNQDKTE treatment. These results indicated that the peptide IPPKKNQDKTE modulated insulin signaling transduction and increased glucose uptake in high glucose-induced insulin-resistant HepG2 cells via preventing IRS-1 Ser636/639 and Ser307 phosphorylation. It has been proposed that the activation of several serine/threonine kinases, such as AMPK and MAPK, may influence the Ser phosphorylation level of IRS-1. In our previous study, we demonstrated that AMPK signaling activation decreased Ser307 phosphorylation of IRS-1 and thereby reduced the insulin resistance in high glucose-stimulated HepG2 cells (Song et al., 2017). By contrast, the activation of MAPK is considered to involve in insulin resistance via phosphorylating IRS-1 on serine residues. Serine 307 is a major site
of JNK phosphorylation in IRS-1, whereas ERK and p38 contribute to the phosphorylation of IRS-1 on Ser636/639 (Aguirre, Uchida, Yenush, Davis, & White, 2000; Cordero-Herrera, Martín, Goya, et al., 2015). The activation of ERK, JNK, or p38 MAPK and the suppression of insulin response are associated with the production of excess ROS induced by nutrient overload, inflammation and endoplasmic reticulum stress in obesity and type 2 diabetes mellitus (Son et al., 2011; Tiganis, 2011). Suppressing ROS-mediated MAPK signaling pathways alleviates insulin resistance (Cang et al., 2016). Some dietary functional ingredients, such as cocoa phenolic extracts and tartary buckwheat flavonoids, exert beneficial effects on insulin resistance via targeting MAPK signaling (CorderoHerrera, Martín, Goya, et al., 2015; Hu, Hou, Liu, & Yang, 2016). In the current study, the peptide IPPKKNQDKTE reduced high glucose-induced activation of MAPK signaling, which contributed to reduce the Ser307 and Ser636/639 phosphorylation of IRS-1. Besides, it was found that the high glucose-induced ROS production was decreased by the peptide IPPKKNQDKTE. Moreover, NAC, a potent ROS scavenger blocked the high glucose-induced activation of MAPK signaling. These results indicated that the
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suppression of the peptide IPPKKNQDKTE on high glucose-induced MAPK signaling activation was related to ROS reduction, which may contribute to the improvement of the peptide IPPKKNQDKTE on IRS-1 Ser307 and Ser636/639 phosphorylation. Taken together, the activation of AMPK pathway and the inhibition of ROS-mediated MAPK signaling by the peptide IPPKKNQDKTE synergistically inhibited the IRS-1 Ser phosphorylation and reduced the insulin resistance in HepG2 cells. The Nrf2 pathway plays an important role in counteracting oxidative stress and insulin resistance. During oxidative stress, Nrf2 can translocate into the nucleus and stimulate transcription of antioxidant proteins through binding to antioxidant response elements. It is reported that the increase in Nrf2 nuclear translocation elevated HO-1 expression and attenuated insulin resistance (Cheng, Cheng, Chiou, & Chang, 2012). HO-1 is a cytoprotective enzyme, which regulates the levels of ROS by increasing antioxidant, such as glutathione and bilirubin (Ahmad et al., 2005). Moreover, up-regulating HO-1 improves insulin sensitivity and regulates glucose metabolism in rats with type 2 diabetes (Ndisang & Jadhav, 2009). In our study, the Nrf2 nuclear translocation and HO-1 protein expression were significantly up-regulated by the peptide IPPKKNQDKTE. Moreover, HO-1 inhibitor partly reversed the inhibition of the peptide IPPKKNQDKTE on high glucose-induced ROS production, and HO-1 inducer increased the suppression of the peptide IPPKKNQDKTE on high glucoseinduced production of ROS. Altogether, the activation of Nrf2/ HO-1 signaling by the peptide IPPKKNQDKTE contributed to the inhibition of the peptide IPPKKNQDKTE on the high glucoseinduced ROS production and insulin resistance. Bioactive peptides (usually contain 2–30 amino acid residues) have exhibited many potent biological activities, including antihypertensive, antioxidative, immunomodulatory, anticancinogenic, antimicrobial, and lipid-lowering activities (Lafarga & Hayes, 2014; Udenigwe & Aluko, 2012). The bioactivity of peptides depends on their amino acid compositions and sequences. The acidic amino acids, basic amino acids and hydrophobic amino acids have been reported to contribute to the antioxidant activity of food peptides (Sarmadi & Ismail, 2010). In our study, the peptide IPPKKNQDKTE contained abundant antioxidative amino acids,
Fig. 6. The peptide IPPKKNQDKTE prevented high glucose-induced insulin resistance in HepG2 cells via blocking ROS-mediated MAPK signaling by Nrf2/HO-1 activation. Red solid line arrows represented changes in response to high glucose; Blue dotted line arrows represented changes in high glucose-stimulated cells receiving peptide intervention.
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which may contribute to enhance cellular antioxidant activity and protect against high glucose-induced insulin resistance by scavenging ROS. 5. Conclusion Our results showed that the peptide IPPKKNQDKTE protected HepG2 cells against high glucose-induced insulin resistance by blocking ROS-mediated MAPK signaling activation (Fig. 6). The peptide IPPKKNQDKTE-induced Nrf2/HO-1 activation was found to be responsible for ROS reduction. These results suggested that the peptide IPPKKNQDKTE may exert a therapeutic effect on hepatic insulin resistance via blocking ROS-mediated MAPK signaling by inducing Nrf2/HO-1 activation. Disclosure statement The authors declare no conflict of interest. Acknowledgments We are grateful for the financial support for this work by the National Natural Science Foundation of China (31371753), Open Funding Project of Key Laboratory of Space Nutrition and Food Engineering (SNFE-KF-15-01), Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2013BAD18B12-05), and National Dairy Industry Technology System-Beijing Innovation Team (NDITS-BIT). References Aguirre, V., Uchida, T., Yenush, L., Davis, R., & White, M. F. (2000). The c-Jun NH2terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser307. Journal of Biological Chemistry, 275(12), 9047–9054. Ahmad, M., Turkseven, S., Mingone, C. J., Gupte, S. A., Wolin, M. S., & Abraham, N. G. (2005). Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats. Cellular and Molecular Biology (Noisy-le-Grand, France), 51(4), 371–376. Bouzakri, K., Roques, M., Gual, P., Espinosa, S., Guebre-Egziabher, F., Riou, J., ... Vidal, H. (2003). Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes, 52(6), 1319–1325. Cang, X., Wang, X., Liu, P., Wu, X., Yan, J., Chen, J., ... Su, J. (2016). PINK1 alleviates palmitate induced insulin resistance in HepG2 cells by suppressing ROS mediated MAPK pathways. Biochemical and Biophysical Research Communications, 478(1), 431–438. Castillo, J. J., Mull, N., Reagan, J. L., Nemr, S., & Mitri, J. (2012). Increased incidence of non-Hodgkin lymphoma, leukemia, and myeloma in patients with diabetes mellitus type 2: A meta-analysis of observational studies. Blood, 119(21), 4845–4850. Cheng, A., Cheng, Y., Chiou, C., & Chang, T. (2012). Resveratrol upregulates Nrf2 expression to attenuate methylglyoxal-induced insulin resistance in HepG2 cells. Journal of Agricultural and Food Chemistry, 60(36), 9180–9187. Cheng, X., Gao, D., Chen, B., & Mao, X. (2015). Endotoxin-binding peptides derived from casein glycomacropeptide inhibit lipopolysaccharide-stimulated inflammatory responses via blockade of NF-jB activation in macrophages. Nutrients, 7(5), 3119–3137. Cheng, X., Gao, D., Song, J., Ren, F., & Mao, X. (2015). Casein glycomacropeptide hydrolysate exerts cytoprotection against H2O2-induced oxidative stress in RAW 264.7 macrophages via ROS-dependent heme oxygenase-1 expression. RSC. Advances, 5(6), 4511–4523. Cordero-Herrera, I., Martín, M. Á., Escrivá, F., Álvarez, C., Goya, L., & Ramos, S. (2015). Cocoa-rich diet ameliorates hepatic insulin resistance by modulating insulin signaling and glucose homeostasis in Zucker diabetic fatty rats. The Journal of Nutritional Biochemistry, 26(7), 704–712. Cordero-Herrera, I., Martín, M. Á., Goya, L., & Ramos, S. (2014). Cocoa flavonoids attenuate high glucose-induced insulin signalling blockade and modulate glucose uptake and production in human HepG2 cells. Food and Chemical Toxicology, 64, 10–19. Cordero-Herrera, I., Martín, M. Á., Goya, L., & Ramos, S. (2015). Cocoa flavonoids protect hepatic cells function against high glucose-induced oxidative stress. Relevance of MAPKs. Molecular Nutrition & Food Research, 59(4), 597–609.
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Peptide IPPKKNQDKTE ameliorates insulin resistance in HepG2 cells via blocking ROS-mediated MAPK signaling Jia-jia Song a,b, Qian Wang b, Min Du c, Bin Chen d, Xue-ying Mao a,b,⇑ a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, China College of Food Science and Nutritional Engineering, Key Laboratory of Functional Dairy, Ministry of Education, China Agricultural University, Beijing, China c Department of Animal Sciences, Washington State University, Pullman, WA, USA d Key Laboratory of Space Nutrition and Food Engineering, China Astronauts Research and Training Center, Beijing, China b
a r t i c l e
i n f o
Article history: Received 17 October 2016 Received in revised form 31 January 2017 Accepted 2 February 2017
Keywords: Insulin resistance HepG2 cells Reactive oxygen species Nrf2 HO-1
a b s t r a c t Reactive oxygen species (ROS) has been found to play an important role in insulin resistance. Whether the improvement effects of the peptide IPPKKNQDKTE on hepatic insulin resistance were mediated by ROS elimination was examined in this study. Results showed that the high glucose-induced MAPK signaling activation was inhibited by IPPKKNQDKTE in insulin-resistant HepG2 cells, which contributed to the improvement of IPPKKNQDKTE on cellular glucose uptake and the phosphorylation levels of insulin receptor substrate-1 (IRS-1) Ser636/639 and Ser307. IPPKKNQDKTE dose-dependently reduced high glucose-induced ROS production, which involved in suppression of IPPKKNQDKTE on MAPK signaling in high glucose-induced insulin-resistant HepG2 cells. Moreover, IPPKKNQDKTE increased Nrf2 nucleus translocation and induced HO-1 expression. HO-1 inhibitor partly reversed IPPKKNQDKTE-mediated suppression on high glucose-induced ROS production. Taken together, IPPKKNQDKTE reduced high glucoseinduced ROS production and MAPK activation by the activation of Nrf2/HO-1, which contributed to increase the glucose uptake and decrease the Ser phosphorylation levels of IRS-1 in insulin-resistant HepG2 cells. These results indicated that IPPKKNQDKTE plays a potential role in the management of hepatic insulin resistance. Ó 2017 Published by Elsevier Ltd.
1. Introduction Diabetes mellitus is a serious chronic metabolic disorder and has become a world-wide epidemic. In 2013, 382 million people worldwide suffered from diabetes, and the total number of people with diabetes is expected to reach 592 million by the year 2035 (Guariguata et al., 2014). Among people with diabetes, approximately 90–95% of diabetic individuals have type 2 diabetes mellitus, which is characterized by decreased response of peripheral tissues to insulin action (ie, insulin resistance) and impaired pancreatic b-cell function (Castillo, Mull, Reagan, Nemr, & Mitri, 2012; Onishi, Ono, Rabøl, Endahl, & Nakamura, 2013). Type 2 diabetes may cause many severe complications, including cardiovascular disease, nephropathy, retinopathy and neuropathy (Schram et al., 2014). Thus, it is important to prevent and manage type 2 diabetes. ⇑ Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, Key Laboratory of Functional Dairy, Ministry of Education, China Agricultural University, 100083 Beijing, China. E-mail address: [email protected] (X.-y. Mao). http://dx.doi.org/10.1016/j.jff.2017.02.005 1756-4646/Ó 2017 Published by Elsevier Ltd.
Liver is one of the major target tissues of insulin, and plays a vital role in stabilizing plasma glucose levels via regulation of hepatic glucose utilization or production. Hepatic insulin resistance contributes to hyperglycaemia and glucose intolerance, and becomes a principal component in the pathogenesis of type 2 diabetes (Galbo et al., 2013; Leclercq, Morais, Schroyen, Van Hul, & Geerts, 2007). Moreover, hepatic insulin resistance is associated with hyperinsulinemia and pancreatic b cell hyperplasia (Michael et al., 2000). It is reported that amelioration of hepatic insulin resistance reduces hyperglycemia and hyperinsulinemia, and improves glucose intolerance in type 2 diabetic Zucker diabetic fatty rats (Cordero-Herrera, Martín, Goya, & Ramos, 2015). Accumulating evidence has demonstrated that increased levels of reactive oxygen species (ROS) are an important trigger for multitypes of insulin resistance, and contribute to the onset and development of type 2 diabetes (Dong et al., 2016; Houstis, Rosen, & Lander, 2006; Matsuzawa-Nagata et al., 2008). Furthermore, ROS levels are negatively correlated with insulin sensitivity (Rains & Jain, 2011). The reduction in ROS level contributes to the improvement of hepatic insulin resistance in high-fat diet-treated mice (Zhang et al., 2013). Taken together, elimination of ROS may be
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an effective approach in the management of hepatic insulin resistance and type 2 diabetes. In order to alleviate insulin resistance and manage type 2 diabetes, several antioxidant compounds have been used. N-acetyl-lcysteine (NAC), a potent scavenger of free radicals has been shown to reduce hyperglycemia and hyperinsulinemia, and improve insulin resistance in high-fat diet-induced obese mice (Ma, Gao, & Liu, 2016). Moreover, some natural bioactive compounds, such as oleanolic acid, b-cryptoxanthin and goby fish protein hydrolysates, enhance hepatic antioxidant defenses and ameliorate hyperglycemia in diabetic animal models (Nasri et al., 2015; Ni et al., 2015; Wang et al., 2013). Among these bioactive ingredients, protein hydrolysates arouse more attentions from many researchers due to their diverse bioactive properties and high safety (Udenigwe, 2014). Glycomacropeptide (GMP) derived from bovine j-casein is a hydrophilic glycopeptide containing 64 amino acid residues. It is shown to possess antihypertensive activity, and reduce cardiovascular disease risk markers and ulcerative colitis (Keogh & Clifton, 2008; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Ming, Jia, Yan, Pang, & Chen, 2015). Our previous study has demonstrated that GMP reduced the body weight of dietary obese rats, and attenuated hepatic steatosis and oxidative stress in obese rats (Xu, Mao, Cheng, & Chen, 2013). Moreover, GMP-derived peptides inhibited the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages, alleviated intracellular ROS production and restored the activities of endogenous antioxidants in hydrogen peroxide-stimulated RAW 264.7 macrophages (Cheng, Gao, Chen, & Mao, 2015; Cheng, Gao, Song, Ren, & Mao, 2015). Besides, in our previous study, the peptide IPPKKNQDKTE which was identified from GMP-derived peptides were demonstrated to decrease the phosphorylation of insulin receptor substrate-1 (IRS-1) Ser307, an important negative regulator of insulin signaling, in high glucose-induced insulin-resistant HepG2 cells. The beneficial effects of the peptide IPPKKNQDKTE on IRS-1 Ser307 phosphorylation and downstream insulin signaling were mediated by AMP-activated protein kinase (AMPK) activation (Song et al., 2017). Apart from Ser307 phosphorylation site in IRS-1, Ser636/639 is a well-recognized phosphorylation site which negatively affects insulin signaling (Bouzakri et al., 2003; Rajesh, Sathish, Srinivasan, Selvaraj, & Balasubramanian, 2013). However, whether the peptide IPPKKNQDKTE can reduce ROS production and IRS-1 Ser636/639 phosphorylation in high glucoseinduced insulin-resistant HepG2 cells remains unclear. Thus, in the current study, we hypothesized that the improvement effects of the peptide IPPKKNQDKTE on insulin resistance in high glucose-stimulated HepG2 cells were mediated by ROS reduction. In order to verify the hypothesis, the key features of insulin resistance, and the alternation of intracellular ROS levels and related signaling pathways were evaluated. 2. Materials and methods 2.1. Materials 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-Dglucose (2-NBDG) was from Life Technologies (Carlsbad, CA, USA). Zinc (II)-protoporphyrin IX (ZnPPIX), 20 ,70 -dichlorodihydro fluorescein diacetate (DCFH-DA), Hemin and NAC were obtained from Sigma-Aldrich (St. Louis, MO, USA). PD98059, SB203580, SP600125, anti-b-actin, anti-Histone H3, anti-HO-1, anti-Nrf2, anti-IRS-1, anti-p-IRS-1 (Ser307), anti-p-IRS-1 (Ser636/639), antiERK, anti-phosphorylated ERK, anti-JNK, anti-phosphorylated JNK, anti-p38, anti-phosphorylated p38 rabbit antibodies and horseradish peroxidase-conjugated anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in the study were of analytical grade.
2.2. Peptide synthesis The peptide IPPKKNQDKTE (purity, P95%) was prepared by the conventional Fmoc solid-phase synthesis method, which was carried out by Nanjing Leon Biological Technology Co. Ltd. (Nanjing, China). 2.3. Cell culture Human HepG2 hepatocytes were purchased from American Type Culture Collection (Rockville, MD, USA). HepG2 cells were grown in Minimum Essential Medium (MEM), supplemented with 10% fetal bovine serum, 1% MEM non-essential amino acids, 100 lg/mL streptomycin and 100 U/mL penicillin (Invitrogen, Carlsbad, CA, USA). Cells were maintained in CO2 incubator (37 °C, 5% CO2, 95% humidity). 2.4. Cell treatments A cell model of insulin resistance was developed by incubating HepG2 cells with 30 mM glucose for 24 h, as described previously (Cordero-Herrera, Martín, Goya, & Ramos, 2014; Zang et al., 2004). To evaluate the protective effect of IPPKKNQDKTE against high glucose-induced insulin resistance, HepG2 cells were treated by 5.5 mM or 30 mM glucose in the presence of various concentrations of IPPKKNQDKTE (0, 125, 250 and 500 lM) for 24 h. Later, cells were incubated with 100 nM insulin for 10 min and harvested. 2.5. Glucose uptake assay Cellular glucose uptake was monitored using 2-NBDG, a fluorescent deoxyglucose analog, as described previously (Zou, Wang, & Shen, 2005). Briefly, 2-NBDG was added at a final concentration of 10 mM after washing the treated-cells two times with phosphate buffered saline (PBS, pH 7.4). After incubation at 37 °C for 1 h, the cells were washed twice with PBS buffer to remove the unabsorbed probe. Then, the cell suspensions obtained by cell trypsinization were subjected to flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) at excitation/ emission wavelengths of 485/530 nm. Results were analyzed by FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA). 2.6. Determination of intracellular ROS The production of intracellular ROS was assayed using the fluorescent probe DCFH-DA, as described previously (Hou et al., 2015). After treatments, cells were washed with PBS (pH 7.4), and then DCFH-DA (final concentration, 10 lM) was added to the cells. After 30 min incubation at 37 °C, the cells were washed two times with PBS to remove extracellular DCFH-DA. Then, cells were trypsinized and resuspended in PBS. The intracellular ROS levels were measured by flow cytometry (BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data were analyzed by FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA). 2.7. Western blot analysis After treatments, cells were harvested in cell lysis buffer (Beyotime, Haimen, Jiangsu, China), supplemented with a 1/100 dilution of protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). The supernatant of whole-cell lysates was collected by centrifugation at 12000g for 15 min at 4 °C, and used for western blot. Nuclear and cytoplasmic protein samples were prepared using nuclear and cytoplasmic extraction reagent kit
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(Beyotime, Haimen, Jiangsu, China). Equal amounts of protein samples were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) on a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The PVDF membranes were blocked with 5% nonfat dry milk or bovine serum albumin in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 2 h, and then incubated with primary antibodies overnight at 4 °C. Immunoblots were washed three times (each for 5 min with TBS-T), followed by 1 h incubation in peroxidase conjugated secondary antibody at room temperature. After three 5-min washes with TBS-T, blots were developed using enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA). Images were obtained by an Amersham Imager 600 imaging system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The optical density of the band was measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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followed by Duncan’s multiple-comparison test using SPSS software (version 20.0, IBM Inc., Chicago, IL, USA). 3. Results 3.1. IPPKKNQDKTE inhibited mitogen-activated protein kinases (MAPK) signaling in high glucose-induced insulin-resistant HepG2 cells To evaluate the effect of IPPKKNQDKTE on MAPK signaling in high glucose-induced insulin-resistant HepG2 cells, the phosphorylation levels and total proteins of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 were analyzed. As shown in Fig. 1, compared with control cells, the levels of phosphorylated JNK, ERK and p38 were elevated by high
2.8. Statistics All assays were carried out at least in triplicate and data were shown as means ± standard deviations (SD). Significant differences (p < 0.05) between means were assessed with one way ANOVA,
Fig. 1. Effect of IPPKKNQDKTE on phosphorylated and total levels of JNK, ERK, p38 in high glucose-induced insulin-resistant HepG2 cells. Cells were incubated with 30 mM glucose in the presence of various concentrations (0, 250 and 500 lM) of IPPKKNQDKTE for 24 h, followed by 100 nM insulin stimulation for 10 min. Panel A showed a representative Western blot for the phosphorylated and total levels of JNK, ERK and p38. Panel B showed the densitometric analysis of three different blots. Values are means ± SD from three separate determinations. Values with different letters (a–d) in the same column are significantly different from each other (p < 0.05).
Fig. 2. Effect of IPPKKNQDKTE and MAPK inhibitors on glucose uptake in insulinresistant HepG2 cells. Cells were cultured in 5.5 and 30 mM glucose culture medium in the presence of IPPKKNQDKTE (500 lM), PD98059 (50 mM), SB203580 (10 mM) and SP600125 (40 mM) for 24 h. Then, cells were stimulated by 100 nM insulin for 10 min. Cellular glucose uptake was determined using fluorescent 2NBDG. (A) Representative histograms of 2-NBDG fluorescence. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM), blue line represented cells treated with high glucose plus PD98059 (50 mM), green line represented cells treated with high glucose plus SB203580 (10 mM) and pink line represented cells treated with high glucose plus SP600125 (40 mM). (B) The results were expressed as percentage of mean fluorescence intensity relative to control cells. Values are means ± SD from three separate determinations. Values with different letters (a–c) are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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glucose treatment. However, IPPKKNQDKTE inhibited the high glucose-induced phosphorylation of JNK, ERK and p38, especially inhibited the expression levels of p-ERK and p-p38 in a concentration-dependent manner. These results suggested that the activation of MAPK signaling was inhibited by IPPKKNQDKTE in high glucose-induced insulin-resistant HepG2 cells. 3.2. IPPKKNQDKTE and MAPK inhibitors increased the cellular glucose uptake in high glucose-induced insulin-resistant HepG2 cells In order to investigate whether the improvement of IPPKKNQDKTE on insulin resistance in high glucose-stimulated HepG2 cells was mediated by MAPK signaling, the glucose uptake in IPPKKNQDKTE and MAPK inhibitors-treated cells was analyzed. As presented in Fig. 2A and B, high glucose stimulation significantly decreased cellular glucose uptake in comparison with control cells without high glucose addition (p < 0.05). IPPKKNQDKTE treatment was able to ameliorate the reduced glucose uptake caused by high glucose. Similarly, JNK inhibitor SP600125, ERK inhibitor PD098059 and p38 inhibitor SB203580 prevented the adverse effect of glucose uptake provoked by high glucose. These results suggested that the improvement of IPPKKNQDKTE on glucose uptake in high glucose-induced insulin-resistant HepG2 cells may be related with MAPK signal inhibition.
3.3. IPPKKNQDKTE and MAPK inhibitors decreased the phosphorylation levels of IRS-1 in high glucose-induced insulin-resistant HepG2 cells To further investigate the mechanism of the improvement of IPPKKNQDKTE on insulin resistance in HepG2 cells under high glucose conditions, the phosphorylation levels of IRS-1 in IPPKKNQDKTE and MAPK inhibitors-treated HepG2 cells were evaluated. As shown in Fig. 3A and B, high glucose promoted the phosphorylation of IRS-1 on Ser636/639 and Ser307, which was prevented by IPPKKNQDKTE treatment. In addition, blockage of ERK and p38 prevented the increase of p-IRS-1 Ser636/639 induced by high glucose, whereas JNK inhibition suppressed the enhancement in high glucose-induced phosphorylation level of IRS-1 Ser307. These results indicated that suppression of MAPK signaling may participate in the regulation of IPPKKNQDKTE on IRS-1 phosphorylation in high glucose-induced insulin-resistant HepG2 cells. 3.4. Reduction of high glucose-induced ROS production by IPPKKNQDKTE contributed to the suppression of MAPK signaling in insulin-resistant HepG2 cells To investigate whether the inhibition of IPPKKNQDKTE on MAPK signaling activation was mediated by ROS elimination, the
Fig. 3. Effect of IPPKKNQDKTE and MAPK inhibitors on the expression of p-IRS Ser636/639 and Ser307 in insulin-resistant HepG2 cells. Cells were cultured in 5.5 and 30 mM glucose culture medium in the presence of IPPKKNQDKTE (500 lM), ERK inhibitor PD98059 (50 mM), p38 inhibitor SB203580 (10 mM) and JNK inhibitor SP600125 (40 mM) for 24 h, followed by 100 nM insulin treatment for 10 min. The protein expression levels of phosphorylated IRS-1 on Ser636/639 and Ser307 were assessed by western blot. Panel A and B showed a representative blot for p-IRS-1 Ser636/639 and densitometric results from three different assays, respectively. Panel C and D showed a representative blot for p-IRS-1 Ser307 and densitometric results from three different assays, respectively. Values are means ± SD from three separate determinations. Values with different letters (a–c) are significantly different from each other (p < 0.05).
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levels of intracellular ROS in high glucose-treated HepG2 cells were monitored. Results in Fig. 4A and B clearly demonstrated that high glucose markedly increased the production of ROS, compared with control cells without glucose addition (p < 0.05). Treatment with IPPKKNQDKTE reduced the high glucose-induced ROS production in a concentration-dependent manner. Moreover, NAC, a potent ROS scavenger inhibited MAPK activation induced by high glucose in HepG2 cells (Fig. 4C). These results suggested that the blockage of IPPKKNQDKTE on high glucose-induced MAPK signaling activation may be associated with suppression of ROS production.
comparison with the control cells. To evaluate the potential role of HO-1 in the inhibition of high glucose-induced ROS production, HepG2 cells were pretreated with ZnPPIX, a HO-1 inhibitor and Hemin, an inducer of HO-1. As expected, ZnPPIX treatment partly reversed the inhibitory effect of IPPKKNQDKTE on high glucoseinduced production of ROS (Fig. 5B and C), whereas Hemin increased the suppression of IPPKKNQDKTE on high glucoseinduced ROS production. These results indicated that reduction of high glucose-induced ROS by IPPKKNQDKTE was at least partially mediated by Nrf2/HO-1 signaling activation.
3.5. Nrf2/HO-1 signaling activation involved in IPPKKNQDKTEmediated ROS elimination
4. Discussion
With the objective of evaluating the role of Nrf2/HO-1 signaling in IPPKKNQDKTE-mediated ROS elimination, the changes in Nrf2 nuclear translocation and HO-1 protein expression in HepG2 cells were assessed. As shown in Fig. 5A, IPPKKNQDKTE dosedependently increased the protein expression level of Nrf2 in nuclear fraction, whereas the protein expression level of Nrf2 in cytosol fraction was remarkably decreased by IPPKKNQDKTE dose-dependently. Moreover, the protein expression of HO-1 was up-regulated by IPPKKNQDKTE in a dose-dependent manner in
Liver plays an important regulatory role in the maintenance of blood glucose homeostasis. One third of the glucose is taken up by the liver after an oral glucose load (Moore, Coate, Winnick, An, & Cherrington, 2012). Insulin-resistant hepatocytes exhibit impaired insulin-stimulated glucose uptake and defective insulin signal transduction (Cordero-Herrera et al., 2014; Huang, Shen, & Wu, 2009). The Ser/Thr phosphorylation of IRS proteins is considered to be a molecular basis for insulin resistance (Zick, 2005). The serine phosphorylation, particularly on Ser636/639 and Ser307 of IRS-1 inhibits its tyrosine phosphorylation by inhibiting
Fig. 4. Inhibitory effect of IPPKKNQDKTE on high glucose-induced production of ROS in HepG2 cells. HepG2 cells were incubated with NAC (0 and 2 mM) for 1 h prior to the treatment of 24-h glucose (5.5 and 30 mM) and various concentration of IPPKKNQDKTE (0, 250 and 500 lM), and further exposed to 100 nM insulin for 10 min. The production of intracellular ROS was assayed using the fluorescent probe DCFH-DA. The phosphorylated and total levels of JNK, ERK, p38 were analyzed by western blot. (A) Representative flow cytometry histogram data. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, green line represented cells treated with high glucose plus IPPKKNQDKTE (250 lM), red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM) and blue line represented cells treated with high glucose plus NAC (2 mM). (B) The results were expressed as mean fluorescence intensity of each sample group. (C) A representative Western blot of p-JNK, p-ERK, p-p38, JNK, ERK and p38. (D) The densitometric analysis of three different blots. Values are means ± SD from three separate determinations. For (B), values with different letters (a–d) are significantly different from each other (p < 0.05). For (D), values with different letters (a–d) in the same column are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Effects of IPPKKNQDKTE treatment on Nrf2 nuclear translocation and HO-1 protein expression in HepG2 cells. Cells were pretreated with ZnPPIX or Hemin (0 and 10 lM) for 1 h in the absence or presence of 30 mM glucose and various concentrations of IPPKKNQDKTE (0, 125, 250 and 500 lM) for 24 h, and then stimulated by 100 nM Insulin for 10 min. The expression levels of nucleus Nrf2, cytosol Nrf2 and HO-1 were determined by western blot. Intracellular ROS was monitored by DCFH-DA. (A) and (B) showed a representative blot for nucleus Nrf2, cytosol Nrf2 and HO-1, and densitometric results from three different assays, respectively. (C) Representative histograms of DCFH-DA fluorescence. Gray background represented control (untreated) cells, black line represented high glucose-treated cells, red line represented cells treated with high glucose plus IPPKKNQDKTE (500 lM), green line represented cells treated with high glucose, IPPKKNQDKTE (500 lM) and ZnPPIX (10 lM) and blue line represented cells treated with high glucose, IPPKKNQDKTE (500 lM) and Hemin (10 lM). (D) The results were expressed as mean fluorescence intensity of each sample group. Values are means ± SD from three separate determinations. For (B), values with different letters (a–d) in the same column are significantly different from each other (p < 0.05). For (D), values with different letters (a–d) are significantly different from each other (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the activity of the insulin receptor kinase, which blocks the downstream effector pathways and impairs insulin signaling (Bouzakri et al., 2003; Klover & Mooney, 2004). In the present study, we demonstrated that the peptide IPPKKNQDKTE increased insulinstimulated glucose uptake in high glucose-induced insulinresistant HepG2 cells. The increased phosphorylation levels of IRS-1 on Ser636/639 and Ser307 by high glucose were reversed by peptide IPPKKNQDKTE treatment. These results indicated that the peptide IPPKKNQDKTE modulated insulin signaling transduction and increased glucose uptake in high glucose-induced insulin-resistant HepG2 cells via preventing IRS-1 Ser636/639 and Ser307 phosphorylation. It has been proposed that the activation of several serine/threonine kinases, such as AMPK and MAPK, may influence the Ser phosphorylation level of IRS-1. In our previous study, we demonstrated that AMPK signaling activation decreased Ser307 phosphorylation of IRS-1 and thereby reduced the insulin resistance in high glucose-stimulated HepG2 cells (Song et al., 2017). By contrast, the activation of MAPK is considered to involve in insulin resistance via phosphorylating IRS-1 on serine residues. Serine 307 is a major site
of JNK phosphorylation in IRS-1, whereas ERK and p38 contribute to the phosphorylation of IRS-1 on Ser636/639 (Aguirre, Uchida, Yenush, Davis, & White, 2000; Cordero-Herrera, Martín, Goya, et al., 2015). The activation of ERK, JNK, or p38 MAPK and the suppression of insulin response are associated with the production of excess ROS induced by nutrient overload, inflammation and endoplasmic reticulum stress in obesity and type 2 diabetes mellitus (Son et al., 2011; Tiganis, 2011). Suppressing ROS-mediated MAPK signaling pathways alleviates insulin resistance (Cang et al., 2016). Some dietary functional ingredients, such as cocoa phenolic extracts and tartary buckwheat flavonoids, exert beneficial effects on insulin resistance via targeting MAPK signaling (CorderoHerrera, Martín, Goya, et al., 2015; Hu, Hou, Liu, & Yang, 2016). In the current study, the peptide IPPKKNQDKTE reduced high glucose-induced activation of MAPK signaling, which contributed to reduce the Ser307 and Ser636/639 phosphorylation of IRS-1. Besides, it was found that the high glucose-induced ROS production was decreased by the peptide IPPKKNQDKTE. Moreover, NAC, a potent ROS scavenger blocked the high glucose-induced activation of MAPK signaling. These results indicated that the
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suppression of the peptide IPPKKNQDKTE on high glucose-induced MAPK signaling activation was related to ROS reduction, which may contribute to the improvement of the peptide IPPKKNQDKTE on IRS-1 Ser307 and Ser636/639 phosphorylation. Taken together, the activation of AMPK pathway and the inhibition of ROS-mediated MAPK signaling by the peptide IPPKKNQDKTE synergistically inhibited the IRS-1 Ser phosphorylation and reduced the insulin resistance in HepG2 cells. The Nrf2 pathway plays an important role in counteracting oxidative stress and insulin resistance. During oxidative stress, Nrf2 can translocate into the nucleus and stimulate transcription of antioxidant proteins through binding to antioxidant response elements. It is reported that the increase in Nrf2 nuclear translocation elevated HO-1 expression and attenuated insulin resistance (Cheng, Cheng, Chiou, & Chang, 2012). HO-1 is a cytoprotective enzyme, which regulates the levels of ROS by increasing antioxidant, such as glutathione and bilirubin (Ahmad et al., 2005). Moreover, up-regulating HO-1 improves insulin sensitivity and regulates glucose metabolism in rats with type 2 diabetes (Ndisang & Jadhav, 2009). In our study, the Nrf2 nuclear translocation and HO-1 protein expression were significantly up-regulated by the peptide IPPKKNQDKTE. Moreover, HO-1 inhibitor partly reversed the inhibition of the peptide IPPKKNQDKTE on high glucose-induced ROS production, and HO-1 inducer increased the suppression of the peptide IPPKKNQDKTE on high glucoseinduced production of ROS. Altogether, the activation of Nrf2/ HO-1 signaling by the peptide IPPKKNQDKTE contributed to the inhibition of the peptide IPPKKNQDKTE on the high glucoseinduced ROS production and insulin resistance. Bioactive peptides (usually contain 2–30 amino acid residues) have exhibited many potent biological activities, including antihypertensive, antioxidative, immunomodulatory, anticancinogenic, antimicrobial, and lipid-lowering activities (Lafarga & Hayes, 2014; Udenigwe & Aluko, 2012). The bioactivity of peptides depends on their amino acid compositions and sequences. The acidic amino acids, basic amino acids and hydrophobic amino acids have been reported to contribute to the antioxidant activity of food peptides (Sarmadi & Ismail, 2010). In our study, the peptide IPPKKNQDKTE contained abundant antioxidative amino acids,
Fig. 6. The peptide IPPKKNQDKTE prevented high glucose-induced insulin resistance in HepG2 cells via blocking ROS-mediated MAPK signaling by Nrf2/HO-1 activation. Red solid line arrows represented changes in response to high glucose; Blue dotted line arrows represented changes in high glucose-stimulated cells receiving peptide intervention.
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which may contribute to enhance cellular antioxidant activity and protect against high glucose-induced insulin resistance by scavenging ROS. 5. Conclusion Our results showed that the peptide IPPKKNQDKTE protected HepG2 cells against high glucose-induced insulin resistance by blocking ROS-mediated MAPK signaling activation (Fig. 6). The peptide IPPKKNQDKTE-induced Nrf2/HO-1 activation was found to be responsible for ROS reduction. These results suggested that the peptide IPPKKNQDKTE may exert a therapeutic effect on hepatic insulin resistance via blocking ROS-mediated MAPK signaling by inducing Nrf2/HO-1 activation. Disclosure statement The authors declare no conflict of interest. Acknowledgments We are grateful for the financial support for this work by the National Natural Science Foundation of China (31371753), Open Funding Project of Key Laboratory of Space Nutrition and Food Engineering (SNFE-KF-15-01), Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2013BAD18B12-05), and National Dairy Industry Technology System-Beijing Innovation Team (NDITS-BIT). References Aguirre, V., Uchida, T., Yenush, L., Davis, R., & White, M. F. (2000). The c-Jun NH2terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser307. Journal of Biological Chemistry, 275(12), 9047–9054. Ahmad, M., Turkseven, S., Mingone, C. J., Gupte, S. A., Wolin, M. S., & Abraham, N. G. (2005). Heme oxygenase-1 gene expression increases vascular relaxation and decreases inducible nitric oxide synthase in diabetic rats. Cellular and Molecular Biology (Noisy-le-Grand, France), 51(4), 371–376. Bouzakri, K., Roques, M., Gual, P., Espinosa, S., Guebre-Egziabher, F., Riou, J., ... Vidal, H. (2003). Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes, 52(6), 1319–1325. Cang, X., Wang, X., Liu, P., Wu, X., Yan, J., Chen, J., ... Su, J. (2016). PINK1 alleviates palmitate induced insulin resistance in HepG2 cells by suppressing ROS mediated MAPK pathways. Biochemical and Biophysical Research Communications, 478(1), 431–438. Castillo, J. J., Mull, N., Reagan, J. L., Nemr, S., & Mitri, J. (2012). Increased incidence of non-Hodgkin lymphoma, leukemia, and myeloma in patients with diabetes mellitus type 2: A meta-analysis of observational studies. Blood, 119(21), 4845–4850. Cheng, A., Cheng, Y., Chiou, C., & Chang, T. (2012). Resveratrol upregulates Nrf2 expression to attenuate methylglyoxal-induced insulin resistance in HepG2 cells. Journal of Agricultural and Food Chemistry, 60(36), 9180–9187. Cheng, X., Gao, D., Chen, B., & Mao, X. (2015). Endotoxin-binding peptides derived from casein glycomacropeptide inhibit lipopolysaccharide-stimulated inflammatory responses via blockade of NF-jB activation in macrophages. Nutrients, 7(5), 3119–3137. Cheng, X., Gao, D., Song, J., Ren, F., & Mao, X. (2015). Casein glycomacropeptide hydrolysate exerts cytoprotection against H2O2-induced oxidative stress in RAW 264.7 macrophages via ROS-dependent heme oxygenase-1 expression. RSC. Advances, 5(6), 4511–4523. Cordero-Herrera, I., Martín, M. Á., Escrivá, F., Álvarez, C., Goya, L., & Ramos, S. (2015). Cocoa-rich diet ameliorates hepatic insulin resistance by modulating insulin signaling and glucose homeostasis in Zucker diabetic fatty rats. The Journal of Nutritional Biochemistry, 26(7), 704–712. Cordero-Herrera, I., Martín, M. Á., Goya, L., & Ramos, S. (2014). Cocoa flavonoids attenuate high glucose-induced insulin signalling blockade and modulate glucose uptake and production in human HepG2 cells. Food and Chemical Toxicology, 64, 10–19. Cordero-Herrera, I., Martín, M. Á., Goya, L., & Ramos, S. (2015). Cocoa flavonoids protect hepatic cells function against high glucose-induced oxidative stress. Relevance of MAPKs. Molecular Nutrition & Food Research, 59(4), 597–609.
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