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6J mice challenged with high-fat diet
Journal of Functional Foods 45 (2018) 190–198
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Casein glycomacropeptide hydrolysates ameliorate hepatic insulin resistance of C57BL/6J mice challenged with high-fat diet Jia-jia Songa,b, Jing Gaoa,b, Min Duc, Xue-ying Maoa,b,
T
⁎
a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, China b 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
A R T I C LE I N FO
A B S T R A C T
Keywords: Casein glycomacropeptide Insulin resistance High-fat diet AMPK MAPK
Hepatic insulin resistance plays a vital role in the development of type 2 diabetes. In the current study, the reduction effects of casein glycomacropeptide hydrolysates obtained with papain (GHP) on hepatic insulin resistance were investigated in high-fat diet (HFD)-fed C57BL/6J mice. Mice were fed with HFD for 8 weeks and then gavaged with GHP at doses of 100, 200 and 400 mg/kg body weight daily for another 8 weeks while continuing with HFD feeding. Results showed that GHP significantly decreased the levels of fasting blood glucose and serum insulin, and homeostasis model of insulin resistance index in HFD mice. The glucose tolerance and hepatic glycogen content were increased by GHP treatment in HFD mice. Besides, the hepatic steatosis and macrophages infiltration were ameliorated by GHP in HFD mice. Furthermore, GHP reduced the serine phosphorylation of IRS-1 and elevated Akt phosphorylation, which increased GSK3β phosphorylation in liver tissue of HFD mice. The decreased hepatic AMPK phosphorylation and increased hepatic MAPK phosphorylation induced by HFD were reversed by GHP, which contributed to the restoration of hepatic insulin sensitivity, reduction of hepatic steatosis and macrophage infiltration. Thus, GHP supplementation may be an alternative therapeutic approach against hepatic insulin resistance.
1. Introduction Diabetes is a serious chronic metabolic disease, which becomes increasingly common. In 2015, an estimated 415 million people worldwide had diabetes, and the number of patients with diabetes is expected to rise to 642 million by 2040 (Han Cho, 2015). More than 90% of diabetic patients have type 2 diabetes mellitus, which is characterized by decreased insulin sensitivity of peripheral tissues (i.e., peripheral insulin resistance) and relative deficiency of insulin secretion from pancreatic β cells (Chatterjee, Khunti, & Davies, 2017; Martin et al., 1992). Type 2 diabetes leads to chronic complications, such as cardiovascular diseases, end-stage renal disease and loss of visual acuity. Hence, it is vital to prevent and treat type 2 diabetes. Liver is one of insulin target tissues, and plays a major role in the control of glucose homeostasis by storing or releasing glucose according to metabolic demands. Hepatic insulin resistance is a primary feature of type 2 diabetes, and contributes to the development of metabolic complications and cardiovascular diseases (Meshkani & Adeli, 2009; Samuel et al., 2007). Hepatic insulin resistance contributes to fasting
and postprandial hyperglycemia (Leung, 2016; Samuel et al., 2007), which in turn worsens insulin sensitivity and aggravates insulin resistance in peripheral tissues (Leung, 2016; Nawano et al., 2000). Thus, ameliorating hepatic insulin resistance could be an important strategy in the regulation of glucose homeostasis and type 2 diabetes. To alleviate hepatic insulin resistance and manage type 2 diabetes, synthetic drugs such as rosiglitazone and metformin are widely used (Garabadu & Krishnamurthy, 2017; Zhou & You, 2014). Although these drugs are effective in the prevention of hepatic insulin resistance, they are usually accompanied by undesirable adverse effects (Hoffmann, Roa, Torrico, & Cubeddu, 2003; Malinowski & Bolesta, 2000). Therefore, there is an increasing interest to explore natural bioactive compounds without side effects for the reduction of hepatic insulin resistance. It is reported that salmon protein peptides, curcumins and oleanolic acids improve hepatic insulin resistance and prevent glucose intolerance in animal models (Chevrier et al., 2015; Wang et al., 2013; Wang, Zhang, Huang, Liu, & Xie, 2016). More recently, food proteinderived peptides have gained more and more attention because of their bioactive activities and high safety (Udenigwe, 2014).
⁎ 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).
https://doi.org/10.1016/j.jff.2018.03.044 Received 9 January 2018; Received in revised form 27 March 2018; Accepted 31 March 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of casein glycomacropeptide hydrolysates (GHP) on the levels of fasting blood glucose, serum insulin, homeostasis model assessment for insulin resistance (HOMA-IR) and hepatic glycogen. (A) Fasting blood glucose level; (B) Serum insulin level; (C) HOMA-IR; (D) Hepatic glycogen content. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: highfat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: highfat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
Fig. 2. Effects of casein glycomacropeptide hydrolysates (GHP) on oral glucose tolerance test (OGTT). (A) and (B) showed the blood glucose curve for the OGTT and corresponding area under the curve (AUC). ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
in high-fat diet (HFD)-fed Sprague-Dawley rats (Xu, Mao, Cheng, & Chen, 2013). Although some bioactivities of GMP are considered to be associated with its carbohydrate moieties, the polypeptide portions of GMP also play an important role in biological activity of GMP (Li & Mine, 2004; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Noh, Koh, Kim, Cho, & Lee, 2017). Compared with GMP, GMP hydrolysates obtained with papain (GHP) possessed a higher inhibition on
Glycomacropeptide (GMP), composed of 64 amino acid residues, is a sialylated phosphorylated C-terminal peptide (residues 106–169) released from κ-casein. It has been demonstrated that GMP reduces blood pressure, cardiovascular disease risk markers and ulcerative colitis (Keogh, & Clifton, 2008; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Ming, Jia, Yan, Pang, & Chen, 2015). We found that GMP attenuated hepatic steatosis and enhanced antioxidant capability 191
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Fig. 3. Effects of casein glycomacropeptide hydrolysates (GHP) on the levels of liver lipids. (A) Hepatic TG content; (B) Hepatic TC content; (C) Hepatic FFA content. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
anti-IRS-1, anti-p-IRS-1 (Ser307), anti-p-IRS-1 (Ser636/639), antiAMPKα, anti-p-AMPKα (Thr172), anti-ERK, anti-p-ERK (Thr202/ Tyr204), anti-JNK, anti-p-JNK (Thr183/Tyr185), anti-p38, anti-p-p38 (Thr180/Tyr182) antibodies, and horseradish peroxidase-conjugated anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in current study were of analytical grade.
oxidative stress and inflammatory response in RAW264.7 macrophages (Cheng, Gao, Chen, & Mao, 2015; Cheng, Gao, Song, Ren, & Mao, 2015). In addition, the peptide IPPKKNQDKTE, identified from GHP, reduced insulin resistance by increasing AMP-activated protein kinase (AMPK) phosphorylation and decreasing mitogen-activated protein kinase (MAPK) phosphorylation in high glucose-induced insulin-resistant HepG2 cells (Song et al., 2017; Song, Wang, Du, Chen, & Mao, 2017). However, whether GHP improves hepatic insulin resistance in mice remains unclear. In the current study, the effects of GHP on the levels of fasting blood glucose and serum insulin, and glucose tolerance were investigated using HFD-fed C57BL/6J mice. The levels of hepatic lipid and glycogen, and pathologic changes in liver tissue were also assessed. Moreover, the phosphorylation levels of insulin signaling-related proteins in liver were further explored.
2.2. Preparation of casein glycomacropeptide hydrolysates GMP was hydrolyzed using papain, as described previously (Cheng, et al., 2015). Briefly, GMP was dissolved in deionized water at a concentration of 5% (w/v). The pH and temperature of solutions were adjusted to 6.0 and 55 °C, respectively. Then, papain was added at an enzyme-to-substrate ratio of 0.05. Hydrolysis was performed for 1 h at constant temperature and pH, and terminated by heating in a water bath at 85 °C for 20 min. The mixture was centrifuged at 4000g at 4 °C for 20 min with TGL-20 M high-speed centrifuge (Pingfan Instrument Co. Ltd., Changsha, China). The supernatants obtained by centrifugation were freeze-dried, and the dried hydrolysates were designated as GHP. The amino acid composition of GHP can be found in our previous study (Cheng et al., 2015).
2. Materials and methods 2.1. Materials Casein glycomacropeptide (Lactoprodan® CGMP-20, purity > 95%) was supplied by Arla Foods Ingredients (Viby, Denmark). Papain (EC 3.4.22.21, from papaya latex, 0.5–2.0 units per milligram) and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies to GSK3β, F4/80 and β-actin were obtained from Abcam (Cambridge, UK). The p-GSK3β (Ser9), anti-Akt, anti-p-Akt (Ser473),
2.3. Animals and experimental design The 5-week-old male C57BL/6J mice were obtained from Beijing 192
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Fig. 4. Effects of casein glycomacropeptide hydrolysates (GHP) on liver morphology. (A) HE staining; (B) F4/80 expression. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/ kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Arrows in panel A and B represented lipid droplets and macrophages, respectively (n = 10 in each group).
Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Mice were housed three per cage with free access to food and water at an ambient temperature of 22 ± 1 °C under 12:12 h light–dark
conditions. After acclimatization for 1 week, ten mice were kept on a normal diet (25.75% of calories from protein, 13.40% of calories from fat, 60.85% of calories from carbohydrate, Beijing KeAo Feed Co. Ltd.,
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Fig. 5. Effects of casein glycomacropeptide hydrolysates (GHP) on the phosphorylation levels of GSK3β, IRS-1 Ser and Akt. (A) A representative blot for p-GSK3β; (B) A representative blot for p-IRS-1 Ser307, p-IRS-1 Ser636/639 and p-Akt; (C) Densitometric quantification of p-GSK3β/GSK3β; (D) Densitometric quantification of p-IRS-1 Ser307, p-IRS-1 Ser636/639 and p-Akt. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
immunosorbent assay according to the manufacturer's instructions (Mercodia, Uppsala, Sweden). The homeostatic model assessment values for insulin resistance (HOMA-IR) index was calculated as previously described (Vogeser et al., 2007).
Beijing, China), and the other forty mice were fed a HFD (20.05% of calories from protein, 60.08% of calories from fat, 20.04% of calories from carbohydrate, Beijing KeAo Feed Co. Ltd., Beijing, China) for 8 weeks. Afterwards, the forty HFD-fed mice were randomly allocated into four groups (10 mice per group). These four groups of mice were continuously fed daily for another 8 weeks with HFD, HFD plus 100 mg/kg body weight of GHP (HFD + G100), HFD plus 200 mg/kg body weight of GHP (HFD + G200) and HFD plus 400 mg/kg body weight of GHP (HFD + G400), respectively. The ten mice fed a normal diet remained on a normal diet for additional 8 weeks. All mice were orally gavaged with sterile physiological saline or GHP dissolved in sterile physiological saline once daily. All of the animal protocols were approved by the Animal Experimentation Committee of China Agricultural University (Beijing, China).
2.5. Oral glucose tolerance test For oral glucose tolerance test, mice were fasted overnight and then orally gavaged with D-glucose (2 g/kg body weight). Blood was collected from tail vein at 0, 30, 60, 90 and 120 min after oral glucose. Blood glucose level was assayed with an Accu-CHEK Active glucometer (Roche Diagnostics, Mannheim, Germany). The area under the blood glucose curve (AUC) was calculated as previously described (Li et al., 2013).
2.4. Blood glucose and serum insulin analysis 2.6. Hepatic lipids and glycogen assay At the end of the animal experiments, all mice were fasted overnight. The fasting glucose level was assessed using an Accu-CHEK Active glucometer (Roche Diagnostics, Mannheim, Germany) by collecting blood samples from the tail vein. The blood samples were clotted at 4 °C overnight, and then centrifuged at 1000g for 20 min to obtain serum samples. Serum insulin was determined by enzyme-linked
The liver tissues were collected and stored at -80 °C at the end of the animal experiments. Triglycerides (TG), total cholesterols (TC), free fatty acids (FFA) and glycogens in the liver were determined using kits from BioVision (Mountain View, CA, USA) according to the manufacturer's instructions. 194
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Fig. 6. Effects of casein glycomacropeptide hydrolysates (GHP) on phosphorylation of AMPK and MAPK in liver. (A) A representative blot for p-AMPK, p-JNK, p-ERK and p-p38; (B) Densitometric quantification of p-AMPK/AMPK, p-JNK/JNK, p-ERK/ERK and p-p38/p38. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
2.7. Histological analysis of liver tissue
2.9. Statistics
Fresh liver tissues were fixed in 4% paraformaldehyde. The fixed liver tissues were paraffin-embedded, and 5 µm tissue sections were stained with Hematoxylin and Eosin (HE). For analyzing macrophages in liver tissue, paraffin sections of liver tissue were incubated with mouse anti-F4/80 antibody, followed by incubation with the corresponding secondary antibody. The F4/80-positive cells were visualized using a diaminobenzidine kit (Dako, Ely, Cambridgeshire, UK). The stained tissue sections were viewed using a light microscope (Olympus, Tokyo, Japan).
Data were expressed as means ± standard deviations (SD). Differences among groups were assessed with one way ANOVA, followed by Duncan's multiple-comparison test using SPSS software (version 20.0, IBM Inc., Chicago, IL, USA). Values of p < 0.05 were considered statistically significant.
2.8. Western blot analysis
In order to investigate the reduction effects of GHP on hepatic insulin resistance in HFD mice, the levels of fasting blood glucose and serum insulin were assayed. As shown in Fig. 1A and 1B, fasting blood glucose and serum insulin levels were significantly higher in HFD mice than those of ND mice by 1.81- and 3.21-folds, respectively (p < 0.05). However, compared with HFD mice, the mice treated with GHP showed reduced levels of fasting glucose and insulin (p < 0.05). Moreover, the HFD mice had higher HOMA-IR, an index of insulin resistance, in comparison with the ND mice (p < 0.05), while GHP-treated mice showed lower HOMA-IR than that of HFD mice (p < 0.05) (Fig. 1C). In addition, HFD mice exhibited lower levels of hepatic glycogen than that of ND mice (p < 0.05), while GHP-supplemented mice had higher glycogen content in liver tissue, in comparison to HFD mice (p < 0.05) (Fig. 1D). Collectively, these results suggested that GHP alleviated hepatic insulin resistance in HFD mice by decreasing fasting glucose and serum insulin levels and increasing hepatic glycogen content.
3. Results 3.1. GHP reduced the levels of fasting blood glucose and serum insulin, and increased hepatic glycogen content in HFD mice
Frozen liver tissue samples were homogenized in ice-cold RIPA lysis buffer (Beyotime, Haimen, Jiangsu, China), supplemented with protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). The whole lysates were centrifuged at 12,000g for 15 min at 4 °C, and supernatants were collected for western blot. Protein concentrations were quantitated using a bicinchoninic acid protein assay. Proteins were separated on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) in a transfer buffer. After 2 h blocking at room temperature using 5% bovine serum albumin in Trisbuffered saline (TBS) containing 0.1% Tween 20 (TBS-T), the membranes were incubated with primary antibodies overnight at 4 °C. After the incubation, the membranes were washed three times, and then followed by incubation with peroxidase conjugated secondary antibody at room temperature for 1 h. Then, the membranes were washed three times with TBS-T, followed by detection using enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA). Images were visualised by an Amersham Imager 600 imaging system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band intensities were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3.2. GHP improved glucose tolerance in HFD mice To further investigate insulin resistance-reducing effects of GHP, the oral glucose tolerance test (OGTT) was carried out. At 30 min after glucose intake, the levels of blood glucose in HFD mice were higher 195
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hyperglycemia and glucose intolerance, and long-term hyperglycemia exacerbates insulin resistance (Cefalu, 2014; Meshkani & Adeli, 2009). Hepatic insulin resistance plays a substantial role in the development of type 2 diabetes mellitus, thus improving hepatic insulin resistance may be a therapeutic approach for the treatment of type 2 diabetes. Some functional ingredients, such as anthocyanins, flavonoids and protein hydrolysates attenuate hepatic insulin resistance, which effectively reduce the levels of fasting blood glucose, and restore insulin sensitivity in HFD mice (Boonloh et al., 2015; Naowaboot, Wannasiri, & Pannangpetch, 2016; Zhang et al., 2013). Similarly, our results showed that oral administration of GHP reduced the levels of fasting blood glucose and serum insulin, and improved glucose tolerance. Moreover, GHP alleviated the reduction of hepatic glycogen induced by HFD in mice. Collectively, GHP reduced hepatic insulin resistance of mice challenged with HFD. Hepatic insulin resistance is associated with impaired insulin signaling. Serine/threonine phosphorylation of IRS-1 is a molecular basis for insulin resistance (Zick, 2005). IRS phosphorylation in serine residues, especially Ser307 and Ser636/639 is enhanced in insulin-resistant state, which inhibits tyrosine phosphorylation of IRS-1, and further inactivates the transmission of insulin signaling (CorderoHerrera et al., 2015; Hotamisligil, 2003; Zhang et al., 2013). Reducing serine phosphorylation of IRS-1 is considered as a target for the reversal of insulin resistance (Sykiotis & Papavassiliou, 2001). GSK3β, a critical substrate of IRS-1/Akt signaling, can reduce glycogen synthesis. Inhibition of GSK3β activity (i.e., increased phosphorylation at Ser9) leads to activation of glycogen synthesis (Klover & Mooney, 2004). In the current study, GHP supplementation alleviated the phosphorylation levels of IRS-1 at Ser636/639 and Ser307, and elevated the expression levels of p-Akt in liver tissue of HFD mice. Moreover, HFD decreased GSK3β phosphorylation, which was reversed by GHP treatment. These results showed that GHP restored the impaired hepatic insulin signaling in HFD mice, which contributed to the increase in hepatic glycogen synthesis and reduction of hepatic insulin resistance. Hepatic lipid accumulation was associated with insulin resistance and type 2 diabetes mellitus (Birkenfeld & Shulman, 2014; Perry, Samuel, Petersen, & Shulman, 2014). The levels of circulating FFA were elevated under obese condition, which plays a pathogenic role in the development of hepatic insulin resistance (Gao et al., 2010). FFA activates protein kinase (PK) Cδ and nicotinamide adenine dinucleotide phosphate oxidase, which lead to serine phosphorylation of IRS and subsequent impairment of hepatic insulin signaling (Pereira et al., 2014). Moreover, FFA can be esterified into diacylglycerols (DG), TG and ceramides in the liver. Hepatic DG leads to PKCε activation, which decreases insulin-stimulated tyrosine phosphorylation of IRS and hepatic glycogen synthesis (Birkenfeld & Shulman, 2014; Jornayvaz et al., 2011). Increased ceramides also contribute to the development of hepatic insulin resistance (Konstantynowicz-Nowicka, Harasim, Baranowski, & Chabowski, 2015). In addition, macrophages contribute directly to hepatic steatosis and insulin resistance in diet-induced obesity via producing inflammatory cytokines (De Taeye et al., 2007; Huang et al., 2010). Our results proved that GHP significantly reduced the levels of hepatic FFA and TG in HFD mice, and GHP alleviated HFDinduced hepatic steatosis. Moreover, GHP reduced the infiltration of macrophages into liver tissue of HFD mice. These results indicated that GHP restored impaired insulin signaling and alleviating hepatic insulin resistance by reducing hepatic lipids accumulation and macrophages infiltration. The hepatic insulin sensitivity and the levels of hepatic lipids can be regulated by protein kinases, such as AMPK and MAPK. AMPK is a heterotrimer composed of a catalytic subunit (α) and two regulatory subunits (β and γ). Once AMPK is phosphorylated on α-Thr172, AMPK is activated (Oakhill, Scott, & Kemp, 2012). The activation of AMPK increases the phosphorylation of Akt and GSK3β, and ameliorates insulin resistance in db/db mice (Zheng et al., 2015). Moreover, HFDinduced hepatic steatosis and macrophage infiltration are inhibited by
than those of NC mice (p < 0.05), and kept at a high level after 120 min (Fig. 2A). However, the rise in blood glucose was significantly inhibited by GHP supplement (p < 0.05). Compared with ND mice, the AUC of glucose response in HFD mice was markedly increased (p < 0.05). The deteriorated glucose tolerance was improved by GHP treatment (Fig. 2B). These results indicated that GHP improved glucose tolerance in HFD mice, which contributed to the reduction of hepatic insulin resistance in HFD mice. 3.3. GHP reduced hepatic steatosis and macrophage infiltration in HFD mice To determine the effects of GHP on liver profiles, the hepatic TG, TC and FFA contents were analyzed. As shown in Fig. 3, compared with ND mice, the levels of TG, TC and FFA in liver tissue were significantly increased in HFD mice (p < 0.05). High levels of TG, TC and FFA in liver induced by HFD were decreased by GHP supplementation (p < 0.05). Consistently, HE staining showed that the liver of HFD mice had more serious fatty degeneration compared with that of ND mice, while administration of GHP dramatically reduced the levels of HFD-induced hepatic steatosis (Fig. 4A). Additionally, HFD consumption increased liver macrophage infiltration, demonstrated by F4/80 staining (Fig. 4B). However, the elevation effects of HFD on hepatic macrophage infiltration were blocked by GHP treatment. Taken together, these results showed that GHP decreased hepatic lipid accumulation and macrophage infiltration in HFD-fed mice. 3.4. GHP improved hepatic insulin signaling in HFD mice To investigate the effects of GHP on insulin signaling in liver tissue, the phosphorylation levels of key glycogenic proteins, IRS-1 Ser636/ 639, IRS-1 Ser307 and Akt were evaluated. Fig. 5A and 5C showed that feeding a HFD suppressed the phosphorylation levels of glycogen synthase kinase (GSK) 3β in liver tissue (p < 0.05). However, GHP treatment markedly increased GSK3β phosphorylation in liver tissue of HFD mice (p < 0.05). Moreover, as shown in Fig. 5B and 5D, HFD treatment markedly increased the phosphorylation levels of insulin receptor substrate-1 (IRS-1) on serine 636/639 and 307 and reduced Akt phosphorylation in liver (p < 0.05). However, GHP reduced HFDinduced phosphorylation of IRS-1 Ser636/639 and Ser307 in liver, and increased the level of p-Akt in liver of HFD mice (p < 0.05). Together, these results demonstrated that chronic GHP supplementation significantly restored the impairment of hepatic insulin signaling in mice fed HFD. 3.5. GHP increased the phosphorylation levels of AMPK and decreased MAPK phosphorylation in liver tissue of HFD mice To elucidate the potential mechanism underlying GHP-mediated improvement in hepatic insulin signaling, the phosphorylation levels of AMPK and MAPK in liver tissue were assessed. As shown in Fig. 6, compared with ND mice, the liver of HFD mice had lower AMPK phosphorylation and higher phosphorylation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 (p < 0.05). Supplementation of GHP, especially a dose of 400 mg/kg body weight, increased AMPK phosphorylation and inhibited the phosphorylation levels of JNK, ERK and p38 in liver of HFD mice (p < 0.05). Altogether, these results demonstrated that GHP reduced hepatic insulin resistance in HFD mice which was associated with increased phosphorylation levels of AMPK and decreased MAPK phosphorylation. 4. Discussion Liver plays a crucial role in the regulation of glucose homeostasis. Hepatic insulin resistance contributes to the development of 196
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AMPK activation (Carvalho et al., 2012; Han et al., 2011). Administration of AMPK activator enhances liver and muscle insulin action, improves glucose tolerance and decreases fasting concentrations of glucose and insulin in insulin-resistant rat (Buhl et al., 2002; Iglesias et al., 2002). Furthermore, activation of ERK and p38 has been characterized as a key event in insulin resistance and type 2 diabetes (Liu & Cao, 2009; Nandipati, Subramanian, & Agrawal, 2017). It is reported that inhibition of p38/ERK MAPK pathways improves hepatic insulin sensitivity (Geng et al., 2018). In current study, GHP treatment at a dose of 100 mg/kg body weight did not show inhibition effects on MAPK phosphorylation. However, GHP (100 mg/kg body weight) increased the phosphorylation levels of AMPK, which played an important role in the improvement of hepatic insulin resistance. By contrast, we found that the phosphorylation levels of ERK and p38 were markedly decreased by GHP (200 and 400 mg/kg body weight), while GHP (200 and 400 mg/kg body weight) treatment increased AMPK phosphorylation levels. Although JNK activation is also associated with hepatic insulin resistance, our results showed that GHP (only 400 mg/ kg body weight) displayed significant inhibition on the phosphorylation of JNK. Similarly, the different inhibition extent of bioactive compound on JNK, p38 and ERK was also reported in other researches (Geng et al., 2017; Wu et al., 2017). These results indicated that the improvement of hepatic insulin resistance by GHP was mainly mediated by inhibition on the phosphorylation of ERK and p38 and activation of AMPK.
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A., Ye, J., Frangioudakis, G., Saha, A. K., Tomas, E., Ruderman, N. B., ... Kraegen, E. W. (2002). AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats. Diabetes, 51(10), 2886–2894. Jornayvaz, F. R., Birkenfeld, A. L., Jurczak, M. J., Kanda, S., Guigni, B. A., Jiang, D. C., ... Shulman, G. I. (2011). Hepatic insulin resistance in mice with hepatic overexpression of diacylglycerol acyltransferase 2. Proceedings of the National Academy of Sciences, 108(14), 5748–5752. Keogh, J. B., & Clifton, P. (2008). The effect of meal replacements high in glycomacropeptide on weight loss and markers of cardiovascular disease risk. The American Journal of Clinical Nutrition, 87(6), 1602–1605. Klover, P. J., & Mooney, R. A. (2004). Hepatocytes: Critical for glucose homeostasis. The International Journal of Biochemistry & Cell Biology, 36(5), 753–758. Konstantynowicz-Nowicka, K., Harasim, E., Baranowski, M., & Chabowski, A. (2015). New evidence for the role of ceramide in the development of hepatic insulin resistance. PLoS ONE, 10(1), e0116858. Leung, P. S. (2016). The potential protective action of vitamin D in hepatic insulin resistance and pancreatic islet dysfunction in type 2 diabetes mellitus. Nutrients, 8(3), 147. Li, E. W., & Mine, Y. (2004). Immunoenhancing effects of bovine glycomacropeptide and its derivatives on the proliferative response and phagocytic activities of human macrophagelike cells, U937. Journal of Agricultural and Food Chemistry, 52(9), 2704–2708. Li, Y., Xiao, J., Tian, H., Pei, Y., Lu, Y., Han, X., ... Fang, F. (2013). The DPP-4 inhibitor MK0626 and exercise protect islet function in early pre-diabetic kkay mice. Peptides, 49, 91–99. Liu, Z., & Cao, W. (2009). p38 mitogen-activated protein kinase: a critical node linking insulin resistance and cardiovascular diseases in type 2 diabetes mellitus. Endocrine, Metabolic & Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune, Endocrine & Metabolic Disorders), 9(1), 38–46. Malinowski, J. M., & Bolesta, S. (2000). Rosiglitazone in the treatment of type 2 diabetes mellitus: A critical review. Clinical Therapeutics, 22(10), 1151–1168. Martin, B. C., Warram, J. H., Krolewski, A. S., Soeldner, J. S., Kahn, C. R., & Bergman, R. N. (1992). Role of glucose and insulin resistance in development of type 2 diabetes mellitus: Results of a 25-year follow-up study. The Lancet, 340(8825), 925–929. Meshkani, R., & Adeli, K. (2009). Hepatic insulin resistance, metabolic syndrome and cardiovascular disease. Clinical Biochemistry, 42(13), 1331–1346. Miguel, M., Manso, M. A., López-Fandiño, R., Alonso, M. J., & Salaices, M. (2007). Vascular effects and antihypertensive properties of κ-casein macropeptide. International Dairy Journal, 17(12), 1473–1477. Ming, Z., Jia, Y., Yan, Y., Pang, G., & Chen, Q. (2015). Amelioration effect of bovine casein glycomacropeptide on ulcerative colitis in mice. Food and Agricultural Immunology, 26(5), 717–728. Nandipati, K. C., Subramanian, S., & Agrawal, D. K. (2017). Protein kinases: Mechanisms
5. Conclusion In summary, GHP reduced the levels of fasting blood glucose and serum insulin, improved glucose tolerance, and increased glycogen content in HFD mice, which was associated with the improvement of insulin signaling in liver tissue. Moreover, GHP reduced HFD-induced hepatic lipid accumulation and macrophage infiltration, which contributed to alleviate hepatic insulin resistance. These beneficial effects of GHP in HFD mice were mediated by the activation of AMPK and the inhibition of MAPK signaling. Our results suggest that GHP may possess potential of alleviating hepatic insulin resistance and type 2 diabetes. 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 (Grant No. 31371753), and Beijing Dairy Industry Innovation Team (BAIC06-2018). References Birkenfeld, A. L., & Shulman, G. I. (2014). Nonalcoholic fatty liver disease, hepatic insulin resistance, and type 2 diabetes. Hepatology, 59(2), 713–723. Boonloh, K., Kukongviriyapan, V., Kongyingyoes, B., Kukongviriyapan, U., Thawornchinsombut, S., & Pannangpetch, P. (2015). Rice bran protein hydrolysates improve insulin resistance and decrease pro-inflammatory cytokine gene expression in rats fed a high carbohydrate-high fat diet. Nutrients, 7(8), 6313–6329. Buhl, E. S., Jessen, N., Pold, R., Ledet, T., Flyvbjerg, A., Pedersen, S. B., ... Lund, S. (2002). Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome. Diabetes, 51(7), 2199–2206. Carvalho, B. M., Guadagnini, D., Tsukumo, D., Schenka, A. A., Latuf-Filho, P., Vassallo, J., ... Saad, M. (2012). Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice. Diabetologia, 55(10), 2823–2834. Cefalu, W. T. (2014). Paradoxical insights into whole body metabolic adaptations following SGLT2 inhibition. The Journal of Clinical Investigation, 124(2), 485–487. Chatterjee, S., Khunti, K., & Davies, M. J. (2017). Type 2 diabetes. The Lancet, 389(10085), 2239–2251. 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-κB 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
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Casein glycomacropeptide hydrolysates ameliorate hepatic insulin resistance of C57BL/6J mice challenged with high-fat diet Jia-jia Songa,b, Jing Gaoa,b, Min Duc, Xue-ying Maoa,b,
T
⁎
a
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, China b 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
A R T I C LE I N FO
A B S T R A C T
Keywords: Casein glycomacropeptide Insulin resistance High-fat diet AMPK MAPK
Hepatic insulin resistance plays a vital role in the development of type 2 diabetes. In the current study, the reduction effects of casein glycomacropeptide hydrolysates obtained with papain (GHP) on hepatic insulin resistance were investigated in high-fat diet (HFD)-fed C57BL/6J mice. Mice were fed with HFD for 8 weeks and then gavaged with GHP at doses of 100, 200 and 400 mg/kg body weight daily for another 8 weeks while continuing with HFD feeding. Results showed that GHP significantly decreased the levels of fasting blood glucose and serum insulin, and homeostasis model of insulin resistance index in HFD mice. The glucose tolerance and hepatic glycogen content were increased by GHP treatment in HFD mice. Besides, the hepatic steatosis and macrophages infiltration were ameliorated by GHP in HFD mice. Furthermore, GHP reduced the serine phosphorylation of IRS-1 and elevated Akt phosphorylation, which increased GSK3β phosphorylation in liver tissue of HFD mice. The decreased hepatic AMPK phosphorylation and increased hepatic MAPK phosphorylation induced by HFD were reversed by GHP, which contributed to the restoration of hepatic insulin sensitivity, reduction of hepatic steatosis and macrophage infiltration. Thus, GHP supplementation may be an alternative therapeutic approach against hepatic insulin resistance.
1. Introduction Diabetes is a serious chronic metabolic disease, which becomes increasingly common. In 2015, an estimated 415 million people worldwide had diabetes, and the number of patients with diabetes is expected to rise to 642 million by 2040 (Han Cho, 2015). More than 90% of diabetic patients have type 2 diabetes mellitus, which is characterized by decreased insulin sensitivity of peripheral tissues (i.e., peripheral insulin resistance) and relative deficiency of insulin secretion from pancreatic β cells (Chatterjee, Khunti, & Davies, 2017; Martin et al., 1992). Type 2 diabetes leads to chronic complications, such as cardiovascular diseases, end-stage renal disease and loss of visual acuity. Hence, it is vital to prevent and treat type 2 diabetes. Liver is one of insulin target tissues, and plays a major role in the control of glucose homeostasis by storing or releasing glucose according to metabolic demands. Hepatic insulin resistance is a primary feature of type 2 diabetes, and contributes to the development of metabolic complications and cardiovascular diseases (Meshkani & Adeli, 2009; Samuel et al., 2007). Hepatic insulin resistance contributes to fasting
and postprandial hyperglycemia (Leung, 2016; Samuel et al., 2007), which in turn worsens insulin sensitivity and aggravates insulin resistance in peripheral tissues (Leung, 2016; Nawano et al., 2000). Thus, ameliorating hepatic insulin resistance could be an important strategy in the regulation of glucose homeostasis and type 2 diabetes. To alleviate hepatic insulin resistance and manage type 2 diabetes, synthetic drugs such as rosiglitazone and metformin are widely used (Garabadu & Krishnamurthy, 2017; Zhou & You, 2014). Although these drugs are effective in the prevention of hepatic insulin resistance, they are usually accompanied by undesirable adverse effects (Hoffmann, Roa, Torrico, & Cubeddu, 2003; Malinowski & Bolesta, 2000). Therefore, there is an increasing interest to explore natural bioactive compounds without side effects for the reduction of hepatic insulin resistance. It is reported that salmon protein peptides, curcumins and oleanolic acids improve hepatic insulin resistance and prevent glucose intolerance in animal models (Chevrier et al., 2015; Wang et al., 2013; Wang, Zhang, Huang, Liu, & Xie, 2016). More recently, food proteinderived peptides have gained more and more attention because of their bioactive activities and high safety (Udenigwe, 2014).
⁎ 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).
https://doi.org/10.1016/j.jff.2018.03.044 Received 9 January 2018; Received in revised form 27 March 2018; Accepted 31 March 2018 1756-4646/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Effects of casein glycomacropeptide hydrolysates (GHP) on the levels of fasting blood glucose, serum insulin, homeostasis model assessment for insulin resistance (HOMA-IR) and hepatic glycogen. (A) Fasting blood glucose level; (B) Serum insulin level; (C) HOMA-IR; (D) Hepatic glycogen content. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: highfat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: highfat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
Fig. 2. Effects of casein glycomacropeptide hydrolysates (GHP) on oral glucose tolerance test (OGTT). (A) and (B) showed the blood glucose curve for the OGTT and corresponding area under the curve (AUC). ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
in high-fat diet (HFD)-fed Sprague-Dawley rats (Xu, Mao, Cheng, & Chen, 2013). Although some bioactivities of GMP are considered to be associated with its carbohydrate moieties, the polypeptide portions of GMP also play an important role in biological activity of GMP (Li & Mine, 2004; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Noh, Koh, Kim, Cho, & Lee, 2017). Compared with GMP, GMP hydrolysates obtained with papain (GHP) possessed a higher inhibition on
Glycomacropeptide (GMP), composed of 64 amino acid residues, is a sialylated phosphorylated C-terminal peptide (residues 106–169) released from κ-casein. It has been demonstrated that GMP reduces blood pressure, cardiovascular disease risk markers and ulcerative colitis (Keogh, & Clifton, 2008; Miguel, Manso, López-Fandiño, Alonso, & Salaices, 2007; Ming, Jia, Yan, Pang, & Chen, 2015). We found that GMP attenuated hepatic steatosis and enhanced antioxidant capability 191
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Fig. 3. Effects of casein glycomacropeptide hydrolysates (GHP) on the levels of liver lipids. (A) Hepatic TG content; (B) Hepatic TC content; (C) Hepatic FFA content. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
anti-IRS-1, anti-p-IRS-1 (Ser307), anti-p-IRS-1 (Ser636/639), antiAMPKα, anti-p-AMPKα (Thr172), anti-ERK, anti-p-ERK (Thr202/ Tyr204), anti-JNK, anti-p-JNK (Thr183/Tyr185), anti-p38, anti-p-p38 (Thr180/Tyr182) antibodies, and horseradish peroxidase-conjugated anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in current study were of analytical grade.
oxidative stress and inflammatory response in RAW264.7 macrophages (Cheng, Gao, Chen, & Mao, 2015; Cheng, Gao, Song, Ren, & Mao, 2015). In addition, the peptide IPPKKNQDKTE, identified from GHP, reduced insulin resistance by increasing AMP-activated protein kinase (AMPK) phosphorylation and decreasing mitogen-activated protein kinase (MAPK) phosphorylation in high glucose-induced insulin-resistant HepG2 cells (Song et al., 2017; Song, Wang, Du, Chen, & Mao, 2017). However, whether GHP improves hepatic insulin resistance in mice remains unclear. In the current study, the effects of GHP on the levels of fasting blood glucose and serum insulin, and glucose tolerance were investigated using HFD-fed C57BL/6J mice. The levels of hepatic lipid and glycogen, and pathologic changes in liver tissue were also assessed. Moreover, the phosphorylation levels of insulin signaling-related proteins in liver were further explored.
2.2. Preparation of casein glycomacropeptide hydrolysates GMP was hydrolyzed using papain, as described previously (Cheng, et al., 2015). Briefly, GMP was dissolved in deionized water at a concentration of 5% (w/v). The pH and temperature of solutions were adjusted to 6.0 and 55 °C, respectively. Then, papain was added at an enzyme-to-substrate ratio of 0.05. Hydrolysis was performed for 1 h at constant temperature and pH, and terminated by heating in a water bath at 85 °C for 20 min. The mixture was centrifuged at 4000g at 4 °C for 20 min with TGL-20 M high-speed centrifuge (Pingfan Instrument Co. Ltd., Changsha, China). The supernatants obtained by centrifugation were freeze-dried, and the dried hydrolysates were designated as GHP. The amino acid composition of GHP can be found in our previous study (Cheng et al., 2015).
2. Materials and methods 2.1. Materials Casein glycomacropeptide (Lactoprodan® CGMP-20, purity > 95%) was supplied by Arla Foods Ingredients (Viby, Denmark). Papain (EC 3.4.22.21, from papaya latex, 0.5–2.0 units per milligram) and insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies to GSK3β, F4/80 and β-actin were obtained from Abcam (Cambridge, UK). The p-GSK3β (Ser9), anti-Akt, anti-p-Akt (Ser473),
2.3. Animals and experimental design The 5-week-old male C57BL/6J mice were obtained from Beijing 192
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Fig. 4. Effects of casein glycomacropeptide hydrolysates (GHP) on liver morphology. (A) HE staining; (B) F4/80 expression. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/ kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Arrows in panel A and B represented lipid droplets and macrophages, respectively (n = 10 in each group).
Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Mice were housed three per cage with free access to food and water at an ambient temperature of 22 ± 1 °C under 12:12 h light–dark
conditions. After acclimatization for 1 week, ten mice were kept on a normal diet (25.75% of calories from protein, 13.40% of calories from fat, 60.85% of calories from carbohydrate, Beijing KeAo Feed Co. Ltd.,
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Fig. 5. Effects of casein glycomacropeptide hydrolysates (GHP) on the phosphorylation levels of GSK3β, IRS-1 Ser and Akt. (A) A representative blot for p-GSK3β; (B) A representative blot for p-IRS-1 Ser307, p-IRS-1 Ser636/639 and p-Akt; (C) Densitometric quantification of p-GSK3β/GSK3β; (D) Densitometric quantification of p-IRS-1 Ser307, p-IRS-1 Ser636/639 and p-Akt. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
immunosorbent assay according to the manufacturer's instructions (Mercodia, Uppsala, Sweden). The homeostatic model assessment values for insulin resistance (HOMA-IR) index was calculated as previously described (Vogeser et al., 2007).
Beijing, China), and the other forty mice were fed a HFD (20.05% of calories from protein, 60.08% of calories from fat, 20.04% of calories from carbohydrate, Beijing KeAo Feed Co. Ltd., Beijing, China) for 8 weeks. Afterwards, the forty HFD-fed mice were randomly allocated into four groups (10 mice per group). These four groups of mice were continuously fed daily for another 8 weeks with HFD, HFD plus 100 mg/kg body weight of GHP (HFD + G100), HFD plus 200 mg/kg body weight of GHP (HFD + G200) and HFD plus 400 mg/kg body weight of GHP (HFD + G400), respectively. The ten mice fed a normal diet remained on a normal diet for additional 8 weeks. All mice were orally gavaged with sterile physiological saline or GHP dissolved in sterile physiological saline once daily. All of the animal protocols were approved by the Animal Experimentation Committee of China Agricultural University (Beijing, China).
2.5. Oral glucose tolerance test For oral glucose tolerance test, mice were fasted overnight and then orally gavaged with D-glucose (2 g/kg body weight). Blood was collected from tail vein at 0, 30, 60, 90 and 120 min after oral glucose. Blood glucose level was assayed with an Accu-CHEK Active glucometer (Roche Diagnostics, Mannheim, Germany). The area under the blood glucose curve (AUC) was calculated as previously described (Li et al., 2013).
2.4. Blood glucose and serum insulin analysis 2.6. Hepatic lipids and glycogen assay At the end of the animal experiments, all mice were fasted overnight. The fasting glucose level was assessed using an Accu-CHEK Active glucometer (Roche Diagnostics, Mannheim, Germany) by collecting blood samples from the tail vein. The blood samples were clotted at 4 °C overnight, and then centrifuged at 1000g for 20 min to obtain serum samples. Serum insulin was determined by enzyme-linked
The liver tissues were collected and stored at -80 °C at the end of the animal experiments. Triglycerides (TG), total cholesterols (TC), free fatty acids (FFA) and glycogens in the liver were determined using kits from BioVision (Mountain View, CA, USA) according to the manufacturer's instructions. 194
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Fig. 6. Effects of casein glycomacropeptide hydrolysates (GHP) on phosphorylation of AMPK and MAPK in liver. (A) A representative blot for p-AMPK, p-JNK, p-ERK and p-p38; (B) Densitometric quantification of p-AMPK/AMPK, p-JNK/JNK, p-ERK/ERK and p-p38/p38. ND: mice fed normal diet; HFD: mice fed high-fat diet; HFD + G100: high-fat diet mice received GHP at a dose of 100 mg/kg body weight; HFD + G200: high-fat diet mice received GHP at a dose of 200 mg/kg body weight; HFD + G400: high-fat diet mice received GHP at a dose of 400 mg/kg body weight. Results were expressed as the mean ± SD (n = 10 in each group). Different letters indicate significant differences between columns (p < 0.05).
2.7. Histological analysis of liver tissue
2.9. Statistics
Fresh liver tissues were fixed in 4% paraformaldehyde. The fixed liver tissues were paraffin-embedded, and 5 µm tissue sections were stained with Hematoxylin and Eosin (HE). For analyzing macrophages in liver tissue, paraffin sections of liver tissue were incubated with mouse anti-F4/80 antibody, followed by incubation with the corresponding secondary antibody. The F4/80-positive cells were visualized using a diaminobenzidine kit (Dako, Ely, Cambridgeshire, UK). The stained tissue sections were viewed using a light microscope (Olympus, Tokyo, Japan).
Data were expressed as means ± standard deviations (SD). Differences among groups were assessed with one way ANOVA, followed by Duncan's multiple-comparison test using SPSS software (version 20.0, IBM Inc., Chicago, IL, USA). Values of p < 0.05 were considered statistically significant.
2.8. Western blot analysis
In order to investigate the reduction effects of GHP on hepatic insulin resistance in HFD mice, the levels of fasting blood glucose and serum insulin were assayed. As shown in Fig. 1A and 1B, fasting blood glucose and serum insulin levels were significantly higher in HFD mice than those of ND mice by 1.81- and 3.21-folds, respectively (p < 0.05). However, compared with HFD mice, the mice treated with GHP showed reduced levels of fasting glucose and insulin (p < 0.05). Moreover, the HFD mice had higher HOMA-IR, an index of insulin resistance, in comparison with the ND mice (p < 0.05), while GHP-treated mice showed lower HOMA-IR than that of HFD mice (p < 0.05) (Fig. 1C). In addition, HFD mice exhibited lower levels of hepatic glycogen than that of ND mice (p < 0.05), while GHP-supplemented mice had higher glycogen content in liver tissue, in comparison to HFD mice (p < 0.05) (Fig. 1D). Collectively, these results suggested that GHP alleviated hepatic insulin resistance in HFD mice by decreasing fasting glucose and serum insulin levels and increasing hepatic glycogen content.
3. Results 3.1. GHP reduced the levels of fasting blood glucose and serum insulin, and increased hepatic glycogen content in HFD mice
Frozen liver tissue samples were homogenized in ice-cold RIPA lysis buffer (Beyotime, Haimen, Jiangsu, China), supplemented with protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). The whole lysates were centrifuged at 12,000g for 15 min at 4 °C, and supernatants were collected for western blot. Protein concentrations were quantitated using a bicinchoninic acid protein assay. Proteins were separated on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) in a transfer buffer. After 2 h blocking at room temperature using 5% bovine serum albumin in Trisbuffered saline (TBS) containing 0.1% Tween 20 (TBS-T), the membranes were incubated with primary antibodies overnight at 4 °C. After the incubation, the membranes were washed three times, and then followed by incubation with peroxidase conjugated secondary antibody at room temperature for 1 h. Then, the membranes were washed three times with TBS-T, followed by detection using enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA). Images were visualised by an Amersham Imager 600 imaging system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band intensities were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3.2. GHP improved glucose tolerance in HFD mice To further investigate insulin resistance-reducing effects of GHP, the oral glucose tolerance test (OGTT) was carried out. At 30 min after glucose intake, the levels of blood glucose in HFD mice were higher 195
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hyperglycemia and glucose intolerance, and long-term hyperglycemia exacerbates insulin resistance (Cefalu, 2014; Meshkani & Adeli, 2009). Hepatic insulin resistance plays a substantial role in the development of type 2 diabetes mellitus, thus improving hepatic insulin resistance may be a therapeutic approach for the treatment of type 2 diabetes. Some functional ingredients, such as anthocyanins, flavonoids and protein hydrolysates attenuate hepatic insulin resistance, which effectively reduce the levels of fasting blood glucose, and restore insulin sensitivity in HFD mice (Boonloh et al., 2015; Naowaboot, Wannasiri, & Pannangpetch, 2016; Zhang et al., 2013). Similarly, our results showed that oral administration of GHP reduced the levels of fasting blood glucose and serum insulin, and improved glucose tolerance. Moreover, GHP alleviated the reduction of hepatic glycogen induced by HFD in mice. Collectively, GHP reduced hepatic insulin resistance of mice challenged with HFD. Hepatic insulin resistance is associated with impaired insulin signaling. Serine/threonine phosphorylation of IRS-1 is a molecular basis for insulin resistance (Zick, 2005). IRS phosphorylation in serine residues, especially Ser307 and Ser636/639 is enhanced in insulin-resistant state, which inhibits tyrosine phosphorylation of IRS-1, and further inactivates the transmission of insulin signaling (CorderoHerrera et al., 2015; Hotamisligil, 2003; Zhang et al., 2013). Reducing serine phosphorylation of IRS-1 is considered as a target for the reversal of insulin resistance (Sykiotis & Papavassiliou, 2001). GSK3β, a critical substrate of IRS-1/Akt signaling, can reduce glycogen synthesis. Inhibition of GSK3β activity (i.e., increased phosphorylation at Ser9) leads to activation of glycogen synthesis (Klover & Mooney, 2004). In the current study, GHP supplementation alleviated the phosphorylation levels of IRS-1 at Ser636/639 and Ser307, and elevated the expression levels of p-Akt in liver tissue of HFD mice. Moreover, HFD decreased GSK3β phosphorylation, which was reversed by GHP treatment. These results showed that GHP restored the impaired hepatic insulin signaling in HFD mice, which contributed to the increase in hepatic glycogen synthesis and reduction of hepatic insulin resistance. Hepatic lipid accumulation was associated with insulin resistance and type 2 diabetes mellitus (Birkenfeld & Shulman, 2014; Perry, Samuel, Petersen, & Shulman, 2014). The levels of circulating FFA were elevated under obese condition, which plays a pathogenic role in the development of hepatic insulin resistance (Gao et al., 2010). FFA activates protein kinase (PK) Cδ and nicotinamide adenine dinucleotide phosphate oxidase, which lead to serine phosphorylation of IRS and subsequent impairment of hepatic insulin signaling (Pereira et al., 2014). Moreover, FFA can be esterified into diacylglycerols (DG), TG and ceramides in the liver. Hepatic DG leads to PKCε activation, which decreases insulin-stimulated tyrosine phosphorylation of IRS and hepatic glycogen synthesis (Birkenfeld & Shulman, 2014; Jornayvaz et al., 2011). Increased ceramides also contribute to the development of hepatic insulin resistance (Konstantynowicz-Nowicka, Harasim, Baranowski, & Chabowski, 2015). In addition, macrophages contribute directly to hepatic steatosis and insulin resistance in diet-induced obesity via producing inflammatory cytokines (De Taeye et al., 2007; Huang et al., 2010). Our results proved that GHP significantly reduced the levels of hepatic FFA and TG in HFD mice, and GHP alleviated HFDinduced hepatic steatosis. Moreover, GHP reduced the infiltration of macrophages into liver tissue of HFD mice. These results indicated that GHP restored impaired insulin signaling and alleviating hepatic insulin resistance by reducing hepatic lipids accumulation and macrophages infiltration. The hepatic insulin sensitivity and the levels of hepatic lipids can be regulated by protein kinases, such as AMPK and MAPK. AMPK is a heterotrimer composed of a catalytic subunit (α) and two regulatory subunits (β and γ). Once AMPK is phosphorylated on α-Thr172, AMPK is activated (Oakhill, Scott, & Kemp, 2012). The activation of AMPK increases the phosphorylation of Akt and GSK3β, and ameliorates insulin resistance in db/db mice (Zheng et al., 2015). Moreover, HFDinduced hepatic steatosis and macrophage infiltration are inhibited by
than those of NC mice (p < 0.05), and kept at a high level after 120 min (Fig. 2A). However, the rise in blood glucose was significantly inhibited by GHP supplement (p < 0.05). Compared with ND mice, the AUC of glucose response in HFD mice was markedly increased (p < 0.05). The deteriorated glucose tolerance was improved by GHP treatment (Fig. 2B). These results indicated that GHP improved glucose tolerance in HFD mice, which contributed to the reduction of hepatic insulin resistance in HFD mice. 3.3. GHP reduced hepatic steatosis and macrophage infiltration in HFD mice To determine the effects of GHP on liver profiles, the hepatic TG, TC and FFA contents were analyzed. As shown in Fig. 3, compared with ND mice, the levels of TG, TC and FFA in liver tissue were significantly increased in HFD mice (p < 0.05). High levels of TG, TC and FFA in liver induced by HFD were decreased by GHP supplementation (p < 0.05). Consistently, HE staining showed that the liver of HFD mice had more serious fatty degeneration compared with that of ND mice, while administration of GHP dramatically reduced the levels of HFD-induced hepatic steatosis (Fig. 4A). Additionally, HFD consumption increased liver macrophage infiltration, demonstrated by F4/80 staining (Fig. 4B). However, the elevation effects of HFD on hepatic macrophage infiltration were blocked by GHP treatment. Taken together, these results showed that GHP decreased hepatic lipid accumulation and macrophage infiltration in HFD-fed mice. 3.4. GHP improved hepatic insulin signaling in HFD mice To investigate the effects of GHP on insulin signaling in liver tissue, the phosphorylation levels of key glycogenic proteins, IRS-1 Ser636/ 639, IRS-1 Ser307 and Akt were evaluated. Fig. 5A and 5C showed that feeding a HFD suppressed the phosphorylation levels of glycogen synthase kinase (GSK) 3β in liver tissue (p < 0.05). However, GHP treatment markedly increased GSK3β phosphorylation in liver tissue of HFD mice (p < 0.05). Moreover, as shown in Fig. 5B and 5D, HFD treatment markedly increased the phosphorylation levels of insulin receptor substrate-1 (IRS-1) on serine 636/639 and 307 and reduced Akt phosphorylation in liver (p < 0.05). However, GHP reduced HFDinduced phosphorylation of IRS-1 Ser636/639 and Ser307 in liver, and increased the level of p-Akt in liver of HFD mice (p < 0.05). Together, these results demonstrated that chronic GHP supplementation significantly restored the impairment of hepatic insulin signaling in mice fed HFD. 3.5. GHP increased the phosphorylation levels of AMPK and decreased MAPK phosphorylation in liver tissue of HFD mice To elucidate the potential mechanism underlying GHP-mediated improvement in hepatic insulin signaling, the phosphorylation levels of AMPK and MAPK in liver tissue were assessed. As shown in Fig. 6, compared with ND mice, the liver of HFD mice had lower AMPK phosphorylation and higher phosphorylation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 (p < 0.05). Supplementation of GHP, especially a dose of 400 mg/kg body weight, increased AMPK phosphorylation and inhibited the phosphorylation levels of JNK, ERK and p38 in liver of HFD mice (p < 0.05). Altogether, these results demonstrated that GHP reduced hepatic insulin resistance in HFD mice which was associated with increased phosphorylation levels of AMPK and decreased MAPK phosphorylation. 4. Discussion Liver plays a crucial role in the regulation of glucose homeostasis. Hepatic insulin resistance contributes to the development of 196
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AMPK activation (Carvalho et al., 2012; Han et al., 2011). Administration of AMPK activator enhances liver and muscle insulin action, improves glucose tolerance and decreases fasting concentrations of glucose and insulin in insulin-resistant rat (Buhl et al., 2002; Iglesias et al., 2002). Furthermore, activation of ERK and p38 has been characterized as a key event in insulin resistance and type 2 diabetes (Liu & Cao, 2009; Nandipati, Subramanian, & Agrawal, 2017). It is reported that inhibition of p38/ERK MAPK pathways improves hepatic insulin sensitivity (Geng et al., 2018). In current study, GHP treatment at a dose of 100 mg/kg body weight did not show inhibition effects on MAPK phosphorylation. However, GHP (100 mg/kg body weight) increased the phosphorylation levels of AMPK, which played an important role in the improvement of hepatic insulin resistance. By contrast, we found that the phosphorylation levels of ERK and p38 were markedly decreased by GHP (200 and 400 mg/kg body weight), while GHP (200 and 400 mg/kg body weight) treatment increased AMPK phosphorylation levels. Although JNK activation is also associated with hepatic insulin resistance, our results showed that GHP (only 400 mg/ kg body weight) displayed significant inhibition on the phosphorylation of JNK. Similarly, the different inhibition extent of bioactive compound on JNK, p38 and ERK was also reported in other researches (Geng et al., 2017; Wu et al., 2017). These results indicated that the improvement of hepatic insulin resistance by GHP was mainly mediated by inhibition on the phosphorylation of ERK and p38 and activation of AMPK.
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