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The mixture of corn and wheat peptide prevent diabetes in NOD mice
Journal of Functional Foods 56 (2019) 163–170
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
The mixture of corn and wheat peptide prevent diabetes in NOD mice a,b,c
Suling Sun
e
f
b,c
, Guowei Zhang , Hongyan Mu , Hao Zhang , Yong Q. Chen
a,b,c,d,⁎
T
a
School of Medicine, Jiangnan University, Wuxi 214122, PR China State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China d Departments of Cancer Biology and Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA e Yichuan Maternal and Child Health Hospital, Yichuan 471300, Henan, PR China f College of Food Science and Engineering, Agricultural University, Qingdao 266109, Shandong, PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Amino acid Anti-inflammation Corn peptide Wheat peptide Type 1 diabetes
Corn peptide promotes glucagon-like peptide-1 release in type 2 diabetic animals, while wheat peptide increases pro-inflammatory cytokine response in type 1 diabetic patients. However, the effect of a mixture of corn and wheat peptide on the initiation and the development of type 1 diabtes remains unclear. Corn peptide reduced the blood glucose in wheat peptide-evoked diabetic NOD mice. A mixture of corn and wheat peptide significantly delayed the initiation and decreased the incidence of diabetes in NOD mice. In addition to the improved oral glucose tolerance, level of interleukin (IL)-6 and the insulitis score were decreased, while β-cell areas and IL-10 gene expression were increased via treatment with this peptide mixture. Meanwhile, serine and histidine levels in the serum were significantly increased in this peptide mixture treated NOD mice. Our data suggested that a mixture of corn and wheat peptide could prevent diabetes in NOD mice.
1. Introduction Type 1 diabetes is an autoimmune disease that is characterized by the destruction of insular β-cells resulting in elevated blood glucose levels due to a deficiency of insulin. There are several causes for the pathogenesis of type 1 diabetes. Notably, both genetic and environmental factors are recognized as triggers for the development of this disease (Bluestone, Herold, & Eisenbarth, 2010; Knip et al., 2005). Human diet has been proven to be an environmental factor for the initiation and progression of type 1 diabetes (Akerblom & Knip, 1998). It has been reported that cereal proteins play an important role in influencing the susceptibility to type 1 diabetes (Antvorskov, Josefsen, Engkilde, Funda, & Buschard, 2014). A higher incidence of type 1 diabetes is found in animals on a gluten-based diet (Hoorfar, Scott, & Cloutier, 1991). On the other hand, a gluten-free diet can regulate the extent of autoimmunity and reduce the incidence of diabetes in animals and the progression to type 1 diabetic patients (Elliott & Martin, 1984; Funda, Kaas, Bock, Tlaskalova, & Buschard, 1999; Pastore et al., 2003). Moreover, wheat proteins are degraded into peptides by intestinal
microbial enzymes (Helmerhorst, Zamakhchari, Schuppan, & Oppenheim, 2010). Wheat peptide derived from wheat α-gliadin exhibited a mixed pro-inflammatory cytokine response with large amounts of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in peripheral blood mononuclear cells collected from type 1 diabetic patients (Mojibian et al., 2009). It has also been shown that wheat peptide could evoke CD8+ T cell responses (Barbeau et al., 2014), which promoted the destruction of the insulin-producing beta cells in the pancreatic islets (Lohmann et al., 1997; Pinkse et al., 2005). However, the hydrolysate derived from corn protein might have a positive function in combatting type 1 diabetes. It has been reported that zein hydrolysate could promote the release of hormone glucagonlike peptide-1 (GLP-1) in mice (Hira, Mochida, Miyashita, & Hara, 2009). Corn zein hydrolysate was further associated with glucoregulatory activity and enhanced release of GLP-1 in rats (Higuchi, Hira, Yamada, & Hara, 2013; Mochida, Hira, & Hara, 2010). GLP-1 is an incretin hormone that is secreted by intestinal L-cells in response to diet (Brubaker & Anini, 2003), which causes increased β-cell mass in diabetic animals by stimulation of β-cell neogenesis, as well as the
Abbreviations: CP- plus WP-mice, a mixture of corn and wheat peptide treated mice; GLP-1, glucagon-like peptide-1; H&E, hematoxylin and eosin; IL, interleukin; NOD, non-obese diabetic; OGTT, oral glucose tolerance test; TNF, tumor necrosis factor ⁎ Corresponding author at: School of Medicine, State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, PR China and Departments of Cancer Biology and Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. E-mail address: [email protected] (Y.Q. Chen). https://doi.org/10.1016/j.jff.2019.03.020 Received 22 October 2018; Received in revised form 11 March 2019; Accepted 12 March 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Specific primers for RT-qPCR. Target gene
Forward
Reverse
IL-6 IL-10 β-actin
5′-CTCTGCAAGAGACTTCCATCCAGT-3′ 5′-GCTCTTACTGACTGGCATGAG-3′ 5′-GGCTGTATTCCCCTCCATCG-3′
5′-GAAGTAGGGAAGGCCGTGG-3′ 5′-CGCAGCTCTAGGAGCATGTG-3′ 5′-CCAGTTGGTAACAATGCCATGT-3′
Fig. 1. Corn peptide treatment reduced blood glucose levels in wheat peptide-evoked diabetic NOD mice. Wheat peptide-treated diabetic NOD mice were randomly allocated into three groups (n = 6/group) to receive 1000 mg/kg/day distilled water, 1000 mg/kg/day wheat peptide and 1000 mg/kg/day corn peptide by gavage each day for 6 weeks. Blood glucose concentration in control diabetic NOD mice (A), wheat peptidetreated diabetic NOD mice (B) and corn peptidetreated diabetic NOD mice (C). # Mice with blood glucose levels ≥25.0 mmol/L and polyuria were killed.
proliferation and inhibition of β-cell apoptosis (Brubaker & Drucker, 2004; Tourrel, Bailbe, Meile, Kergoat, & Portha, 2001). Also, it has been shown that GLP-1 reduced pancreatic insulitis in non-obese diabetic (NOD) mice (Zhang et al., 2007). Meanwhile, endogenous GLP-1 enhanced the regeneration and survival of islets (De Leon et al., 2003; Wideman et al., 2006), and was found to be deficient in type 1 diabetic patients (Zibar, Cuca, Blaslov, Bulum, & Smircic-Duvnjak, 2015). In our previous study, we found that corn peptide treatment significantly reduced the onset of type 1 diabetes and serum IL-6 level in NOD mice. Wheat- and corn-based products are the staple food for the global population due to their high calorie intensity, enriched proteins, carbohydrates, lipids and other micronutrients. However, the effects of a mixture of corn and wheat peptide on the initiation and the development of type 1 diabetic animals has not been investigated. The objective of the current study was to investigate the effects of treatment with a mixture of corn and wheat peptide on the incidence of type 1 diabetes, the area of pancreatic beta cells, serum amino acids, as well as on serum level and gene expression of cytokines in NOD mice.
serum levels of insulin were determined using a rat/mouse insulin ELISA kit purchased from EMD Millipore Inc. (Boston, USA). The IL-4, IL-6, IL-10 and TNF-α concentrations were analysed using ELISA kits from Multi Sciences (Hangzhou, China). cDNA was synthesised using the PrimeScriptTM reagent kit (Takara, Tokyo, Japan). The anti-insulin antibodies were purchased from Abcam (Cambridge, UK). 2.2. Animals Three-week-old female NOD/LtJ mice were purchased from Su Pu Si Biotechnology Co., Ltd (Suzhou, Jiangsu, China). All of the mice were maintained in the animal facility at Jiangnan University (Jiangsu, China) with free access to water and food. All experimental protocols used in this study were in compliance with the local animal ethics committee of Jiangnan University (JN. No 20150403-20170106). 2.3. Corn peptide therapy and experimental design Three-week-old NOD mice were treated with wheat peptide (500 mg/kg/day) until the onset of diabetes. Diabetic wheat peptidetreated NOD mice of similar ages were randomly allocated into three groups (n = 6/group). These groups included: (1) control diabetic NOD mice that were treated with 1000 mg/kg distilled water by gavage every day; (2) wheat peptide-treated NOD mice that were treated with 1000 mg/kg of wheat peptide by gavage every day for 6 weeks; and (3) corn peptide-treated NOD mice that were treated with 1000 mg/kg corn peptide by gavage every day for 6 weeks. Mice with blood glucose levels ≥25.0 mmol/L and polyuria were killed before 6 weeks. All other mice were killed at 6 weeks (study end).
2. Materials and methods 2.1. Materials Both the corn peptide and wheat peptide are peptide mixtures, which were obtained from Shandong Tianjiu Biological Technology Co., Ltd. (Heze, Shandong, China) and produced via enzymatic hydrolysis. Following hydrolysis, the solutions were isolated and then spray-dried. The free amino acid content and molecular weight distribution profiles of the corn and wheat peptide can be found in Tables S1 and S2. The 164
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Fig. 2. Early treatment with a mixture of corn and wheat peptide delayed the onset and reduced the incidence of diabetes in NOD mice. Three-week-old female NOD mice, 14 mice per group, were treated by gavage once a day with distilled water, 500 mg/kg wheat peptide plus 500 mg/kg corn peptide, respectively. (A) Body weights in female NOD mice were measured weekly. (B) Delayed onset and reduced incidence of diabetes in CP- and WP-treated NOD mice. Oral glucose tolerance (C), total the area under the blood glucose response curve (AUC) (D) and serum insulin (E) in female NOD mice were measured. All data are expressed as mean ± SD and differences were analysed by a t-test. * P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
25 weeks of treatment with either the mixture of corn and wheat peptide or distilled water and fixed in 4% paraformaldehyde overnight, washed with doubly distilled H2O, rehydrated with gradient ethanol solutions and embedded in paraffin. The paraffin-embedded pancreas specimens were sectioned into a 5.0 μm thickness, and subsequently stained with hematoxylin and eosin (H&E) to evaluate insulitis. To further study the morphology of islets in the pancreas, pancreatic sections were stained for insulin with insulin antibodies. The percentage of the β-cell area was quantified by calculating the ratio of the area of insulin-positive cells with respect to the total pancreatic area.
2.4. Treatment with a mixture of corn and wheat peptide and experimental design Three-week-old NOD mice were randomized into two groups, including a control group and mice treated with a mixture of corn and wheat peptide (CP- plus WP-mice). These two groups were treated with 1000 mg/kg/day distilled water (in the case of the control group) or a mixture of 500 mg/kg/day wheat peptide and 500 mg/kg/day corn peptide (in the case of the peptide mixture-treated group) via daily oral gavage for 25 weeks (n = 14/group). The blood glucose levels of the NOD mice were measured every week with an Accu-Check Active meter (Roche, Germany). The mice were characterized to be diabetic by the presence of blood glucose ≥11.1 mmol/L on 2 consecutive times.
2.7. ELISA for insulin and cytokine assays Blood samples were collected, centrifuged at 4000g for 10 min at 4 °C and then stored at −20 °C for ELISA analysis. The insulin levels in the sera were assayed using a rat/mouse insulin ELISA kit. The content of cytokines IL-4, IL-6, IL-10 and TNF-α in the collected sera was measured with ELISA kits purchased from Multi Sciences following the manufacturer’s instructions.
2.5. Glucose tolerance assays Two days before the mice were sacrificed, oral glucose tolerance tests (OGTTs) were performed. Animals received glucose by oral gavage (2 g/kg. BW) after 16 h fast. Blood glucose was measured at 0, 15, 30, 60 and 120 min.
2.8. Quantitative Real-Time PCR for cytokine assays
2.6. Histological and immunohistochemical examination
Total pancreatic mRNA was isolated using Trizol reagent. cDNA synthesis was prepared by the PrimeScript™ RT reagent kit. The realtime PCRs were carried out on the Bio-Rad CFX Connect Real-time
Pancreatic tissues were harvested from female NOD mice after 165
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Fig. 3. Treatment with a mixture of corn and wheat peptide reduced β-cell damage in NOD mice. (A) Representative hematoxylin and eosin (H&E)-stained sections showing pancreatic islet infiltration of control NOD mice and CP-plus WP-mice at 28 weeks of age. (B) Insulitis score of control NOD mice and CP- plus WP-mice at 28 weeks of age. The percentage of insulitis was quantified by evaluating the degree of infiltration and categorized as follows: 0-no insulitis, 1-periinsulitis or with few minimal infiltration in the islets, 2-insulitis with < 50% infiltration of the islets, 3-invasive insulitis with > 50% infiltration of the islets was quantified. (C) Representative immunohistochemistry for insulin in the pancreatic islets of control NOD mice and CP- plus WP-mice at 28 weeks of age. Red line marks the insulin area. (D) Relative beta cell areas of control NOD mice and CP- plus WP-mice at 28 weeks of age. All data are expressed as mean ± SD and differences were analysed by a t-test. *P < 0.05. CP + WP = a mixture of corn and wheat peptide treated mice.
3. Results
System (Bio-Rad, Hercules, CA). The specific primers for IL-6, IL-10 and β-actin could be found in Table 1. β-actin reaction product served as a loading control.
3.1. Corn peptide reduced the blood glucose in diabetic NOD mice The effect of corn peptide on the blood glucose levels of wheat peptide-evoked diabetic NOD mice was examined. Corn peptide treatment restored normoglycemia in three of six mice at 1–6 weeks when the initial blood glucose levels were between 11.1 and 15.0 mmol/L (Fig. 1). In contrast, none of the six mice that received wheat peptide alone and none of the control mice were normoglycemic at six weeks (Fig. 1). Thus we speculated that corn peptide treatment may neutralize the adverse impact of wheat peptide on type 1 diabetes.
2.9. Amino acid analysis Corn peptide or wheat peptide (100 mg) was respectively hydrolysed with 8 mL of 6 M HCl for 24 h at 120 °C to analyse the acidic amino acid contents. The basic amino acid contents were measured with the use of NaOH instead of HCl. Serum was thoroughly mixed with 5% (w/v) trichloroacetic acid (1:10, v/v) to precipitate the proteins, and the supernatant was then collected for analysis. The free amino acids in the serum and total amino acids contents in the corn peptide and wheat peptide were analysed with an Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) at 40 °C, as described by Shazly et al. (2017). An absorbance signal corresponding to the amino acids was detected at 338 nm in addition to a signal exhibited by proline at 262 nm.
3.2. Dietary mixture of corn and wheat peptide prevented the development of diabetes in NOD mice Gain of body weights was slightly higher in CP- plus WP-mice as compared with the control NOD mice from 13 to 20 weeks of age (Fig. 2A). CP- plus WP-mice showed delayed occurrence in diabetic development (17 weeks old) compared with control NOD mice (13 weeks old, Fig. 2B). Meanwhile, the diabetes incidence in CP- plus WP-mice was 29% at 28 weeks of age, in comparison with 71% of the control NOD mice (P < 0.01 by log rank test, Fig. 2B). During the OGTTs, CP- plus WP-mice showed significantly lower glucose levels at 30 (P < 0.05), 60 (P < 0.05) and 120 min (P < 0.01) and the area under the blood glucose response curve (P < 0.05) in comparison with the control NOD mice (Fig. 2C and D). Additionally, treatment with the mixture of corn and wheat peptide enhanced serum insulin
2.10. Statistical analysis All of the results were analysed by GraphPad Prism 5 software (San Diego, CA, USA). All results were shown as the mean ± SD. Statistical analyses were carried out by t-tests for two independent groups. The cumulative diabetes incidence was calculated with the Kaplan–Meier estimation and the statistical significance was evaluated by the log rank test. P < 0.05 was considered statistically significant. 166
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Fig. 4. Treatment with a mixture of corn and wheat peptide decreased IL-6 and increased IL-10 levels in NOD mice. Serum levels of IL-6 (A), IL-10 (B), IL-4 (C) and TNF-α (D) in the control group and in NOD mice treated with the mixture of corn and wheat peptide were measured by ELISA. The mRNA expression of IL-6 (E) and IL-10 (E) were analysed using quantitative real-time PCR. All data are expressed as mean ± SD and differences were analysed by a t-test. *P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
3.4. Dietary mixture of corn and wheat peptide reduced serum level and gene expression of IL-6 in NOD mice
concentration by two-fold over the control level (P < 0.05, Fig. 2E). These results imply that early treatment with the mixture of corn and wheat peptide prevented type 1 diabetes development, and that β-cell function was improved by this peptide mixture.
In type 1 diabetes, β-cells are destroyed by an autoimmune mechanism in which cytokine-induced apoptosis is thought to play an important role (Eizirik & Mandrup-Poulsen, 2001). Thus, we quantified the serum levels of pro-inflammatory cytokines (IL-6 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) in control NOD mice and CP- plus WP-mice at the endpoint. The serum IL-6 level was significantly decreased in CP- plus WP-mice versus the control NOD mice (P < 0.05, Fig. 4A). In addition, the mixture of corn and wheat peptide significantly reduced serum levels of anti-inflammatory IL-10 when compared to the control NOD mice (P < 0.05, Fig. 4B). However, there was no significant difference in the serum IL-4 and TNF-α levels in these two groups (Fig. 4C and D). Quantitative Real-time PCR analyses further confirmed that the mixture of corn and wheat peptide inhibited the mRNA expression of IL-6 (P < 0.01, Fig. 4E) and activated the gene expression of IL-10 (P < 0.05, Fig. 4F). The prevention of hyperglycemia in CP- plus WP-mice was accompanied by reduction the serum level and mRNA expression of IL-6, suggesting that the mixture of corn and wheat peptide was an anti-inflammatory mediator.
3.3. Dietary mixture of corn and wheat peptide inhibited pancreatic insulitis in NOD mice Histological analysis of pancreatic islets obtained from mice at the end of the treatment showed that the mixture of corn and wheat peptide ameliorated the destruction of islets. The islets in pancreata of the control NOD mice were heavily infiltrated by leukocytes and few islets remained β-cell (Fig. 3A). The average insulitis score exhibited by the control NOD mice was significantly higher than that observed in CPplus WP-mice (P < 0.01, Fig. 3B), indicating that treatment with the mixture of corn and wheat peptide reduced the severity of lymphocytic infiltration. We also measured the beta cell areas in both groups. There was a significant increase in the β-cell area and most islets were not infiltrated for CP- plus WP-mice (P < 0.05, Fig. 3C and D). All of these data indicated that the mixture of corn and wheat peptide attenuated the inflammatory autoimmune process.
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peptides, we then analysed the total amino acid contents in the corn and wheat peptide. As shown in Table 3, the glutamine, proline and leucine contents were very high in the corn and wheat peptide, whereas the serine and histidine contents were low. Our results indicated that the histidine and serine contents in the corn and wheat peptide were not correlated with their concentrations in the sera collected from CPplus WP-mice.
Table 2 The mixture of corn and wheat peptide increased serum serine and histidine levels in NOD mice. Free amino acid
Controla (ng/mL)
CP + WPb (ng/mL)
Asp Glu Asn Ser Gln His Gly Thr Ala Arg Tyr Cys-s Val Met Trp Phe Ile Leu Lys Pro Total amino acids
13.42 ± 1.33 26.97 ± 2.15 5.68 ± 2.23 4.07 ± 1.31 73.03 ± 9.33 6.72 ± 0.39 21.81 ± 5.31 13.22 ± 1.41 20.27 ± 5.37 144.24 ± 77.73 14.26 ± 3.82 3.25 ± 3.65 31.42 ± 3.91 8.20 ± 0.48 18.52 ± 5.47 11.47 ± 1.13 11.06 ± 1.08 16.57 ± 1.86 37.16 ± 5.41 19.76 ± 3.97 501.72 ± 72.31
12.58 ± 0.36 26.10 ± 4.06 6.49 ± 0.67 6.41 ± 0.33* 77.49 ± 1.85 10.32 ± 0.67** 21. 61 ± 2.15 12.13 ± 1.34 20.84 ± 3.57 195.92 ± 33.18 14.98 ± 2.79 0.68 ± 0.32 30.22 ± 0.05 8.71 ± 0.15 13.63 ± 3.23 12.66 ± 1.26 11.68 ± 0.89 19.52 ± 1.29 40.98 ± 1.76 17.24 ± 1.81 560.19 ± 36.08
4. Discussion In this study, we reported on the effects of treatment with a mixture of corn and wheat peptide on the development of type 1 diabetes in NOD mice. Previous studies have aimed to develop antigen-based immunotherapy for the treatment of type 1 diabetes. Dietary proteinbased peptides have been found to be effective compounds for exerting anti-inflammatory effects (Majumder, Mine, & Wu, 2016) and reducing the incidence of type 1 diabetes (Jiang et al., 2015). Corn peptide derived from corn-based by-products have been shown to promote GLP-1 release in type 2 diabetic animals (Hira et al., 2009), and thus corn peptide treatment may counteract the pro-inflammatory effect of wheat peptide in type 1 diabetic animals Notably, in our study we have shown for the first time that early administration with a mixture of corn and wheat peptide prevented the development of type 1 diabetes in NOD mice (Fig. 2B), indicating that corn peptide treatment could neutralize the pro-inflammatory effect of a wheat peptide diet on type 1 diabetes. However, the partial diabetes reversal (50%) among corn peptide-treated recent-onset NOD mice indicted that the normoglycemic restoration capabilities of the corn peptide is limited and early intervention might be a more efficient treatment strategy. Type 1 diabetes is characterized by the infiltration of immune cells into the pancreas. This inflammation selectively leads to dysfunction and finally death of β-cells. However, dampening the inflammatory response can mitigate the damage to the β-cells (Cohen, 2002). The significantly increased beta cell area and reduced average insulitis score achieved via treatment with the mixture of corn and wheat peptide (Fig. 3A–D) was sufficient to reduce the blood glucose levels in NOD mice, and suggested that this peptide mixture regulated the inflammation among NOD mice. Subsequently, we measured the cytokine levels in sera collected from both groups, and we found that the serum level and pancreatic mRNA expression of IL-6 in CP- plus WP-mice were significantly lower compared to those found in the control NOD mice (Fig. 4A and E). Systemic inflammation was enhanced in youth with type 1 diabetes (Snell-Bergeon et al., 2010). The pro-inflammatory cytokines cause dysfunction to the beta cells and apoptosis. Several pro-inflammatory markers were elevated in subjects with type 1 diabetes. Elevated levels of pro-inflammatory cytokine IL-6 have previously been reported in children and adolescents with type 1 diabetes (Rosa et al., 2008; SnellBergeon et al., 2010). Liese et al. reported that the biomarker IL-6 levels in youth with type 1 diabetes was not influenced by dietary intake (Liese et al., 2018). Consistent with our results, curcumin administration reduced the concentration of serum IL-6 and blood glucose from STZ-induced rats (Jain, Rains, Croad, Larson, & Jones, 2009), indicating that immune suppression in type 1 diabetes could be achieved via a proinflammatory cytokine blockade (Ryden et al., 2017). IL-10, an immunosuppressive and anti-inflammatory cytokine secreted by monocytes/macrophages lineages and immune cells, was first identified as cytokine synthesis inhibitory factor (Fiorentino, Bond, & Mosmann, 1989), and has a beneficial effect on autoimmune diabetes. Exogenous IL-10 provided protection against destructive insulitis of pancreatic beta cells, and delayed the occurrence and limited the incidence of type 1 diabetes among NOD mice (Pennline, Roquegaffney, & Monahan, 1994). In addition, IL-10 was expressed in the pancreas of type 1 diabetic mice (Teros et al., 2000), whereas IL-10 was deficient in subjects with type 1 diabetes (Rabinovitch & Suarez-Pinzon, 2003).
a The concentration of free amino acids in the sera of control NOD mice was analysed by HPLC. b The concentration of free amino acids in the sera of NOD mice treated with the mixture of corn and wheat peptide was analysed by HPLC. All data are expressed as mean ± SD and significance was analysed by the t-test. * P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
Table 3 Total amino acid content in corn and wheat peptide. Total amino acid
Wheat peptide (g/100 g)
Corn peptide (g/100 g)
Asp Glu Ser His Gly Thr Ala Arg Tyr Cys-s Val Met Trp Phe Ile Leu Lys Pro
2.78 ± 0.04 37.36 ± 0.04 3.16 ± 0.03 1.37 ± 0.04 2.69 ± 0.04 1.77 ± 0.01 1.83 ± 0.05 2.12 ± 0.03 1.66 ± 0.03 0.61 ± 0.01 3.68 ± 0.02 1.09 ± 0.02 0.45 ± 0.01 4.67 ± 0.04 3.41 ± 0.01 4.98 ± 0.04 1.04 ± 0.03 12.94 ± 0.01
5.72 ± 0.05 23.45 ± 0.02 3.49 ± 0.01 1.35 ± 0.01 1.98 ± 0.01 2.09 ± 0.01 6.17 ± 0.01 1.54 ± 0.01 3.38 ± 0.01 0.18 ± 0.01 4.22 ± 0.01 1.96 ± 0.02 0.34 ± 0.04 4.52 ± 0.03 3.84 ± 0.05 12.68 ± 0.04 0.95 ± 0.03 11.87 ± 0.01
The content of total amino acids in corn and wheat peptide were analysed by HPLC. All data are expressed as mean ± SD.
3.5. Dietary mixture of corn and wheat peptide increased serum serine and histidine levels in NOD mice Serum amino acid concentrations are shown in Table 2. The glucogenic amino acids of serine (∼6.41 vs. ∼4.07 ng/mL, P < 0.05) and histidine (∼10.32 vs. ∼6.72 ng/mL, P < 0.01) were apparently reduced to lower concentrations in control NOD mice compared to CPplus WP- mice (Table 2). However, other free amino acids (including leucine, ∼19.52 vs. ∼16.57 ng/mL) in sera were similar between the two groups (Table 2). Differences in metabolomic profiles have been shown to be associated with dietary proteins (Holmes et al., 2008). To investigate the correlation of serum amino acids and the amino acids of indicated 168
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Conflict of interest
Peptide IA-2-P2 (insulinoma antigen-2 (IA-2), which is used to create the fused peptide not only reduced the incidence of diabetes but also enhanced the production of IL-10 by splenocytes (Shen et al., 2017). In present study, the pancreatic gene expression of IL-10 was higher in CPplus WP-mice when compared to the control NOD mice (Fig. 4F). In contrast, we found that the IL-10 levels were higher in sera collected from control NOD mice than in those taken from CP- plus WP-mice (Fig. 4B), which may be due to its role in suppressing the cytotoxic effects of pro-inflammatory IL-6 in NOD mice (Souza, Gurgul-Convey, Elsner, & Lenzen, 2008). Dietary plant proteins influence the incidence of type 1 diabetes, and in turn diabetes can potentially alter the metabolism of proteins (Jensen-Waern et al., 2009). Protein catabolism increases in subjects with type 1 diabetes (Hamadeh & Hoffer, 2003). However, higher levels of plasma amino acids could stimulate protein synthesis. In addition, insulin release was enhanced in response to elevated concentrations of amino acids found in subjects treated with protein diets (Schmid, Schusdziarra, Schulte-Frohlinde, Maier, & Classen, 1989). A previous study has reported that the amino acid concentrations were lower in patients with human preclinical type 1 diabetes compared to controls (la Marca et al., 2013). In combination with glucose, amino acids derived from dietary proteins and released from intestinal epithelial cells can activate insulin secretion. Arginine and branched chain amino acids (isoleucine, alanine and leucine) have been found to be essential for insulin secretion, which inhibits protein degradation in children and adult humans with type 1 diabetes (Pacy, Nair, Ford, & Halliday, 1989). In addition, plasma-branched chain amino acids levels, especially those of leucine, can serve as effective biomarkers for the detection of diabetes. It has been shown that an elevated plasma leucine level is associated with a greater risk for the development type 1 diabetes (JensenWaern et al., 2009). However, treatment with the mixture of corn and wheat peptide apparently increased the levels of leucine, isoleucine and alanine in sera but this impact was not statistically significant (Table 2). Interestingly, the serum concentrations of only serine and histidine among the glucogenic amino acids were significantly increased in CPplus WP-mice (Table 2). This is consistent with observations that plasma serine and histidine were significantly decreased in Zucker diabetic fatty rats (Wijekoon, Skinner, Brosnan, & Brosnan, 2004). It has been shown that the capacities of histidine- and serine-pyruvate aminotransferase were enhanced when blood glucose levels were elevated (Noguchi, Okuno, & Kido, 1976). Serine and histidine could be converted to pyruvate. Thus, the observed reduction in serine and histidine from control NOD mice may be due to the increased activity of their degradative enzyme. Furthermore, L-serine treatment has been shown to decrease the incidence of diabetes in NOD mice (Holm et al., 2018), suggesting that increased serine may provide protection against the development of type 1 diabetes. However, the elevated serine and histidine levels in sera from CP- plus WP-mice were not related to their content in corn and wheat peptide (Table 3), proving that levels of the two amino acids in sera may be influenced not only by supplementation but also by metabolism.
The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by the National Key Research and Development Program of China (2017YFD0400200), the National Natural Science Foundation of China Grants No. 3141128 (YQ.C) and No. 31771539 (YQ.C), and the National First-Class Discipline Program of Food Science and Technology (JUFSTR20180101). We wish to thank Hong-yang Pan and Qin Yang for providing technical assistance with the amino acid analysis, Xiao-hong Gu and Shang-wei Chen for their technical assistance with molecular weight analysis. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.03.020. References Akerblom, H. K., & Knip, M. (1998). Putative environmental factors in type 1 diabetes. Diabetes-Metabolism Reviews, 14(1), 31–67. Antvorskov, J. C., Josefsen, K., Engkilde, K., Funda, D. P., & Buschard, K. (2014). Dietary gluten and the development of type 1 diabetes. Diabetologia, 57(9), 1770–1780. Barbeau, W. E., Hontecillas, R., Horne, W., Carbo, A., Koch, M. H., & Bassaganya-Riera, J. (2014). Elevated CD8 T cell responses in type 1 diabetes patients to a 13 amino acid coeliac-active peptide from alpha-gliadin. Clinical and Experimental Immunology, 175(2), 167–171. Bluestone, J. A., Herold, K., & Eisenbarth, G. (2010). Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature, 464(7293), 1293–1300. Brubaker, P. L., & Anini, Y. (2003). Direct and indirect mechanisms regulating secretion of glucagon-like peptide-1 and glucagon-like peptide-2. Canadian Journal of Physiology and Pharmacology, 81(11), 1005–1012. Brubaker, P. L., & Drucker, D. J. (2004). Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology, 145(6), 2653–2659. Cohen, I. R. (2002). Peptide therapy for type I diabetes: The immunological homunculus and the rationale for vaccination. Diabetologia, 45(10), 1468–1474. De Leon, D. D., Deng, S. P., Madani, R., Ahima, R. S., Drucker, D. J., & Stoffers, D. A. (2003). Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes, 52(2), 365–371. Eizirik, D. L., & Mandrup-Poulsen, T. (2001). A choice of death – The signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia, 44(12), 2115–2133. Elliott, R. B., & Martin, J. M. (1984). Dietary protein: A trigger of insulin-dependent diabetes in the BB rat? Diabetologia, 26(4), 297–299. Fiorentino, D. F., Bond, M. W., & Mosmann, T. R. (1989). Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. The Journal of Experimental Medicine, 170(6), 2081–2095. Funda, D. P., Kaas, A., Bock, T., Tlaskalova, H., & Buschard, K. (1999). Gluten-free diet prevents diabetes in NOD mice. Diabetes, 48 A215–A215. Hamadeh, M. J., & Hoffer, L. J. (2003). Effect of protein restriction on sulfur amino acid catabolism in insulin-dependent diabetes mellitus. American Journal of PhysiologyEndocrinology and Metabolism, 284(2), E382–E389. Helmerhorst, E. J., Zamakhchari, M., Schuppan, D., & Oppenheim, F. G. (2010). Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity. Plos One, 5(10). Higuchi, N., Hira, T., Yamada, N., & Hara, H. (2013). Oral administration of corn zein hydrolysate stimulates GLP-1 and GIP secretion and improves glucose tolerance in male normal rats and Goto-Kakizaki rats. Endocrinology, 154(9), 3089–3098. Hira, T., Mochida, T., Miyashita, K., & Hara, H. (2009). GLP-1 secretion is enhanced directly in the ileum but indirectly in the duodenum by a newly identified potent stimulator, zein hydrolysate, in rats. American Journal of Physiology-Gastrointestinal and Liver Physiology, 297(4), G663–G671. Holm, L. J., Haupt-Jorgensen, M., Larsen, J., Giacobini, J. D., Bilgin, M., & Buschard, K. (2018). L-serine supplementation lowers diabetes incidence and improves blood glucose homeostasis in NOD mice. Plos one, 13(3) e0194414–e0194414. Holmes, E., Loo, R. L., Stamler, J., Bictash, M., Yap, I. K. S., Chan, Q., ... Elliott, P. (2008). Human metabolic phenotype diversity and its association with diet and blood pressure. Nature, 453(7193) 396–U350. Hoorfar, J., Scott, F. W., & Cloutier, H. E. (1991). Dietary plant materials and development of diabetes in the BB rat. Journal of Nutrition, 121(6), 908–916. Jain, S. K., Rains, J., Croad, J., Larson, B., & Jones, K. (2009). Curcumin supplementation lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose, and glycosylated hemoglobin in diabetic rats. Antioxidants & Redox Signaling, 11(2), 241–249. Jensen-Waern, M., Andersson, M., Kruse, R., Nilsson, B., Larsson, R., Korsgren, O., & Essen-Gustavsson, B. (2009). Effects of streptozotocin-induced diabetes in domestic
5. Conclusions An oral mixture of corn and wheat peptide delayed the onset and lowered the incidence of type 1 diabetes. Furthermore, treatment with this mixture of corn and wheat peptide decreased serum level and pancreatic gene expression of IL-6 and insulitis, and increased pancreatic β-cell areas, pancreatic gene expression of IL-10 and serum levels of serine and histidine in the treated subjects. These findings demonstrate that treatment with a mixture of corn and wheat peptide can provide protection against the progression of type 1 diabetes through attenuating the inflammation in NOD mice. Our results suggest that a mixture of corn and wheat peptide treatment could protect against diabetes in NOD mice. 169
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291–299. Rosa, J. S., Flores, R. L., Oliver, S. R., Pontello, A. M., Zaldivar, F. P., & Galassetti, P. R. (2008). Sustained IL-1 alpha, IL-4, and IL-6 elevations following correction of hyperglycemia in children with type 1 diabetes mellitus. Pediatric Diabetes, 9(1), 9–16. Ryden, A. K., Perdue, N. R., Pagni, P. P., Gibson, C. B., Ratliff, S. S., Kirk, R. K., ... Boursalian, T. E. (2017). Anti-IL-21 monoclonal antibody combined with liraglutide effectively reverses established hyperglycemia in mouse models of type 1 diabetes. Journal of Autoimmunity, 84, 65–74. Schmid, R., Schusdziarra, V., Schulte-Frohlinde, E., Maier, V., & Classen, M. (1989). Role of amino acids in stimulation of postprandial insulin, glucagon, and pancreatic polypeptide in humans. Pancreas, 4(3), 305–314. Shazly, A. B., He, Z., El-Aziz, M. A., Zeng, M., Zhang, S., Qin, F., & Chen, J. (2017). Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates. Food Chemistry, 232, 753–762. Shen, L., Lu, S., Huang, D., Li, G., Liu, K., Cao, R., ... Wu, J. (2017). A rationally designed peptide IA-2-P2 against type 1 diabetes in streptozotocin-induced diabetic mice. Diabetes & Vascular Disease Research, 14(3), 184–190. Snell-Bergeon, J. K., West, N. A., Mayer-Davis, E. J., Liese, A. D., Marcovina, S. M., D'Agostino, R. B., Jr., ... Dabelea, D. (2010). Inflammatory markers are increased in youth with type 1 diabetes: The search case-control study. Journal of Clinical Endocrinology & Metabolism, 95(6), 2868–2876. Souza, K. L. A., Gurgul-Convey, E., Elsner, M., & Lenzen, S. (2008). Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells. Journal of Endocrinology, 197(1), 139–150. Teros, T., Hakala, R., Ylinen, L., Liukas, A., Arvilommi, P., Sainio-Pollanen, S., ... Simell, O. (2000). Cytokine balance and lipid antigen presentation in the NOD mouse pancreas during development of insulitis. Pancreas, 20(2), 191–196. Tourrel, C., Bailbe, D., Meile, M. J., Kergoat, M., & Portha, B. (2001). Glucagon-like peptide-1 and exendin-4 stimulate beta-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes, 50(7), 1562–1570. Wideman, R. D., Yu, I. L. Y., Webber, T. D., Verchere, C. B., Johnson, J. D., Cheung, A. T., & Kieffer, T. J. (2006). Improving function and survival of pancreatic islets by endogenous production of glucagon-like peptide 1 (GLP-1). Proceedings of the National Academy of Sciences of the United States of America, 103(36), 13468–13473. Wijekoon, E. P., Skinner, C., Brosnan, M. E., & Brosnan, J. T. (2004). Amino acid metabolism in the Zucker diabetic fatty rat: Effects of insulin resistance and of type 2 diabetes. Canadian Journal of Physiology and Pharmacology, 82(7), 506–514. Zhang, J., Tokui, Y., Yamagata, K., Kozawa, J., Sayama, K., Iwahashi, H., ... Miyagawa, J. I. (2007). Continuous stimulation of human glucagon-like peptide-1 (7–36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes. Diabetologia, 50(9), 1900–1909. Zibar, K., Cuca, J. K., Blaslov, K., Bulum, T., & Smircic-Duvnjak, L. (2015). Difference in glucagon-like peptide-1 concentrations between C-peptide negative type 1 diabetes mellitus patients and healthy controls. Annals of Clinical Biochemistry, 52(2), 220–225.
pigs with focus on the amino acid metabolism. Laboratory Animals, 43(3), 249–254. Jiang, H., Tong, Y., Yan, D., Jia, S., Ostenson, C. G., & Chen, Z. (2015). The soybean peptide vglycin preserves the diabetic beta-cells through improvement of proliferation and inhibition of apoptosis. Scientific Reports, 5, 15599. Knip, M., Veijola, W., Virtanen, S. M., Hyoty, H., Vaarala, O., & Akerblom, H. K. (2005). Environmental triggers and determinants of type 1 diabetes. Diabetes, 54, S125–S136. la Marca, G., Malvagia, S., Toni, S., Piccini, B., Di Ciommo, V., & Bottazzo, G. F. (2013). Children who develop type 1 diabetes early in life show low levels of carnitine and amino acids at birth: does this finding shed light on the etiopathogenesis of the disease? Nutrition & Diabetes, 3. Liese, A. D., Ma, X., Ma, X., Mittleman, M. A., The, N. S., Standiford, D. A., ... Puett, R. C. (2018). Dietary quality and markers of inflammation: No association in youth with type 1 diabetes. Journal of Diabetes and Its Complications, 32(2), 179–184. Lohmann, T., Halder, T., Morgenthaler, N. G., KhooMorgenthaler, U. Y., Seissler, J., Engler, J., ... Kalbacher, H. (1997). T cell responses to the novel autoantigen IA2 in IDDM patients and healthy controls. Diabetologia, 40 327-327. Majumder, K., Mine, Y., & Wu, J. (2016). The potential of food protein-derived antiinflammatory peptides against various chronic inflammatory diseases. Journal of the Science of Food and Agriculture, 96(7), 2303–2311. Mochida, T., Hira, T., & Hara, H. (2010). The corn protein, zein hydrolysate, administered into the ileum attenuates hyperglycemia via its dual action on glucagon-like peptide1 secretion and dipeptidyl peptidase-IV activity in rats. Endocrinology, 151(7), 3095–3104. Mojibian, M., Chakir, H., Lefebvre, D. E., Crookshank, J. A., Sonier, B., Keely, E., & Scott, F. W. (2009). Diabetes-specific HLA-DR-restricted proinflammatory T-Cell response to wheat polypeptides in tissue transglutaminase antibody-negative patients with type 1 diabetes. Diabetes, 58(8), 1789–1796. Noguchi, T., Okuno, E., & Kido, R. (1976). Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase. The Biochemical Journal, 159(3), 607–613. Pacy, P. J., Nair, K. S., Ford, C., & Halliday, D. (1989). Failure of insulin infusion to stimulate fractional muscle protein synthesis in type I diabetic patients. Anabolic effect of insulin and decreased proteolysis. Diabetes, 38(5), 618–624. Pastore, M. R., Bazzigaluppi, E., Belloni, C., Arcovio, C., Bonifacio, E., & Bosi, E. (2003). Six months of gluten-free diet do not influence autoantibody titers, but improve insulin secretion in subjects at high risk for type 1 diabetes. Journal of Clinical Endocrinology & Metabolism, 88(1), 162–165. Pennline, K. J., Roquegaffney, E., & Monahan, M. (1994). Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse. Clinical Immunology and Immunopathology, 71(2), 169–175. Pinkse, G. G. M., Tysma, O. H. M., Bergen, C. A. M., Kester, M. G. D., Ossendorp, F., van Veelen, P. A., ... Roep, B. O. (2005). Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes. Proceedings of the National Academy of Sciences of the United States of America, 102(51), 18425–18430. Rabinovitch, A., & Suarez-Pinzon, W. L. (2003). Role of cytokines in the pathogenesis of autoimmune diabetes mellitus. Reviews in Endocrine & Metabolic Disorders, 4(3),
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
The mixture of corn and wheat peptide prevent diabetes in NOD mice a,b,c
Suling Sun
e
f
b,c
, Guowei Zhang , Hongyan Mu , Hao Zhang , Yong Q. Chen
a,b,c,d,⁎
T
a
School of Medicine, Jiangnan University, Wuxi 214122, PR China State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China d Departments of Cancer Biology and Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA e Yichuan Maternal and Child Health Hospital, Yichuan 471300, Henan, PR China f College of Food Science and Engineering, Agricultural University, Qingdao 266109, Shandong, PR China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Amino acid Anti-inflammation Corn peptide Wheat peptide Type 1 diabetes
Corn peptide promotes glucagon-like peptide-1 release in type 2 diabetic animals, while wheat peptide increases pro-inflammatory cytokine response in type 1 diabetic patients. However, the effect of a mixture of corn and wheat peptide on the initiation and the development of type 1 diabtes remains unclear. Corn peptide reduced the blood glucose in wheat peptide-evoked diabetic NOD mice. A mixture of corn and wheat peptide significantly delayed the initiation and decreased the incidence of diabetes in NOD mice. In addition to the improved oral glucose tolerance, level of interleukin (IL)-6 and the insulitis score were decreased, while β-cell areas and IL-10 gene expression were increased via treatment with this peptide mixture. Meanwhile, serine and histidine levels in the serum were significantly increased in this peptide mixture treated NOD mice. Our data suggested that a mixture of corn and wheat peptide could prevent diabetes in NOD mice.
1. Introduction Type 1 diabetes is an autoimmune disease that is characterized by the destruction of insular β-cells resulting in elevated blood glucose levels due to a deficiency of insulin. There are several causes for the pathogenesis of type 1 diabetes. Notably, both genetic and environmental factors are recognized as triggers for the development of this disease (Bluestone, Herold, & Eisenbarth, 2010; Knip et al., 2005). Human diet has been proven to be an environmental factor for the initiation and progression of type 1 diabetes (Akerblom & Knip, 1998). It has been reported that cereal proteins play an important role in influencing the susceptibility to type 1 diabetes (Antvorskov, Josefsen, Engkilde, Funda, & Buschard, 2014). A higher incidence of type 1 diabetes is found in animals on a gluten-based diet (Hoorfar, Scott, & Cloutier, 1991). On the other hand, a gluten-free diet can regulate the extent of autoimmunity and reduce the incidence of diabetes in animals and the progression to type 1 diabetic patients (Elliott & Martin, 1984; Funda, Kaas, Bock, Tlaskalova, & Buschard, 1999; Pastore et al., 2003). Moreover, wheat proteins are degraded into peptides by intestinal
microbial enzymes (Helmerhorst, Zamakhchari, Schuppan, & Oppenheim, 2010). Wheat peptide derived from wheat α-gliadin exhibited a mixed pro-inflammatory cytokine response with large amounts of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in peripheral blood mononuclear cells collected from type 1 diabetic patients (Mojibian et al., 2009). It has also been shown that wheat peptide could evoke CD8+ T cell responses (Barbeau et al., 2014), which promoted the destruction of the insulin-producing beta cells in the pancreatic islets (Lohmann et al., 1997; Pinkse et al., 2005). However, the hydrolysate derived from corn protein might have a positive function in combatting type 1 diabetes. It has been reported that zein hydrolysate could promote the release of hormone glucagonlike peptide-1 (GLP-1) in mice (Hira, Mochida, Miyashita, & Hara, 2009). Corn zein hydrolysate was further associated with glucoregulatory activity and enhanced release of GLP-1 in rats (Higuchi, Hira, Yamada, & Hara, 2013; Mochida, Hira, & Hara, 2010). GLP-1 is an incretin hormone that is secreted by intestinal L-cells in response to diet (Brubaker & Anini, 2003), which causes increased β-cell mass in diabetic animals by stimulation of β-cell neogenesis, as well as the
Abbreviations: CP- plus WP-mice, a mixture of corn and wheat peptide treated mice; GLP-1, glucagon-like peptide-1; H&E, hematoxylin and eosin; IL, interleukin; NOD, non-obese diabetic; OGTT, oral glucose tolerance test; TNF, tumor necrosis factor ⁎ Corresponding author at: School of Medicine, State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, PR China and Departments of Cancer Biology and Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA. E-mail address: [email protected] (Y.Q. Chen). https://doi.org/10.1016/j.jff.2019.03.020 Received 22 October 2018; Received in revised form 11 March 2019; Accepted 12 March 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 Specific primers for RT-qPCR. Target gene
Forward
Reverse
IL-6 IL-10 β-actin
5′-CTCTGCAAGAGACTTCCATCCAGT-3′ 5′-GCTCTTACTGACTGGCATGAG-3′ 5′-GGCTGTATTCCCCTCCATCG-3′
5′-GAAGTAGGGAAGGCCGTGG-3′ 5′-CGCAGCTCTAGGAGCATGTG-3′ 5′-CCAGTTGGTAACAATGCCATGT-3′
Fig. 1. Corn peptide treatment reduced blood glucose levels in wheat peptide-evoked diabetic NOD mice. Wheat peptide-treated diabetic NOD mice were randomly allocated into three groups (n = 6/group) to receive 1000 mg/kg/day distilled water, 1000 mg/kg/day wheat peptide and 1000 mg/kg/day corn peptide by gavage each day for 6 weeks. Blood glucose concentration in control diabetic NOD mice (A), wheat peptidetreated diabetic NOD mice (B) and corn peptidetreated diabetic NOD mice (C). # Mice with blood glucose levels ≥25.0 mmol/L and polyuria were killed.
proliferation and inhibition of β-cell apoptosis (Brubaker & Drucker, 2004; Tourrel, Bailbe, Meile, Kergoat, & Portha, 2001). Also, it has been shown that GLP-1 reduced pancreatic insulitis in non-obese diabetic (NOD) mice (Zhang et al., 2007). Meanwhile, endogenous GLP-1 enhanced the regeneration and survival of islets (De Leon et al., 2003; Wideman et al., 2006), and was found to be deficient in type 1 diabetic patients (Zibar, Cuca, Blaslov, Bulum, & Smircic-Duvnjak, 2015). In our previous study, we found that corn peptide treatment significantly reduced the onset of type 1 diabetes and serum IL-6 level in NOD mice. Wheat- and corn-based products are the staple food for the global population due to their high calorie intensity, enriched proteins, carbohydrates, lipids and other micronutrients. However, the effects of a mixture of corn and wheat peptide on the initiation and the development of type 1 diabetic animals has not been investigated. The objective of the current study was to investigate the effects of treatment with a mixture of corn and wheat peptide on the incidence of type 1 diabetes, the area of pancreatic beta cells, serum amino acids, as well as on serum level and gene expression of cytokines in NOD mice.
serum levels of insulin were determined using a rat/mouse insulin ELISA kit purchased from EMD Millipore Inc. (Boston, USA). The IL-4, IL-6, IL-10 and TNF-α concentrations were analysed using ELISA kits from Multi Sciences (Hangzhou, China). cDNA was synthesised using the PrimeScriptTM reagent kit (Takara, Tokyo, Japan). The anti-insulin antibodies were purchased from Abcam (Cambridge, UK). 2.2. Animals Three-week-old female NOD/LtJ mice were purchased from Su Pu Si Biotechnology Co., Ltd (Suzhou, Jiangsu, China). All of the mice were maintained in the animal facility at Jiangnan University (Jiangsu, China) with free access to water and food. All experimental protocols used in this study were in compliance with the local animal ethics committee of Jiangnan University (JN. No 20150403-20170106). 2.3. Corn peptide therapy and experimental design Three-week-old NOD mice were treated with wheat peptide (500 mg/kg/day) until the onset of diabetes. Diabetic wheat peptidetreated NOD mice of similar ages were randomly allocated into three groups (n = 6/group). These groups included: (1) control diabetic NOD mice that were treated with 1000 mg/kg distilled water by gavage every day; (2) wheat peptide-treated NOD mice that were treated with 1000 mg/kg of wheat peptide by gavage every day for 6 weeks; and (3) corn peptide-treated NOD mice that were treated with 1000 mg/kg corn peptide by gavage every day for 6 weeks. Mice with blood glucose levels ≥25.0 mmol/L and polyuria were killed before 6 weeks. All other mice were killed at 6 weeks (study end).
2. Materials and methods 2.1. Materials Both the corn peptide and wheat peptide are peptide mixtures, which were obtained from Shandong Tianjiu Biological Technology Co., Ltd. (Heze, Shandong, China) and produced via enzymatic hydrolysis. Following hydrolysis, the solutions were isolated and then spray-dried. The free amino acid content and molecular weight distribution profiles of the corn and wheat peptide can be found in Tables S1 and S2. The 164
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Fig. 2. Early treatment with a mixture of corn and wheat peptide delayed the onset and reduced the incidence of diabetes in NOD mice. Three-week-old female NOD mice, 14 mice per group, were treated by gavage once a day with distilled water, 500 mg/kg wheat peptide plus 500 mg/kg corn peptide, respectively. (A) Body weights in female NOD mice were measured weekly. (B) Delayed onset and reduced incidence of diabetes in CP- and WP-treated NOD mice. Oral glucose tolerance (C), total the area under the blood glucose response curve (AUC) (D) and serum insulin (E) in female NOD mice were measured. All data are expressed as mean ± SD and differences were analysed by a t-test. * P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
25 weeks of treatment with either the mixture of corn and wheat peptide or distilled water and fixed in 4% paraformaldehyde overnight, washed with doubly distilled H2O, rehydrated with gradient ethanol solutions and embedded in paraffin. The paraffin-embedded pancreas specimens were sectioned into a 5.0 μm thickness, and subsequently stained with hematoxylin and eosin (H&E) to evaluate insulitis. To further study the morphology of islets in the pancreas, pancreatic sections were stained for insulin with insulin antibodies. The percentage of the β-cell area was quantified by calculating the ratio of the area of insulin-positive cells with respect to the total pancreatic area.
2.4. Treatment with a mixture of corn and wheat peptide and experimental design Three-week-old NOD mice were randomized into two groups, including a control group and mice treated with a mixture of corn and wheat peptide (CP- plus WP-mice). These two groups were treated with 1000 mg/kg/day distilled water (in the case of the control group) or a mixture of 500 mg/kg/day wheat peptide and 500 mg/kg/day corn peptide (in the case of the peptide mixture-treated group) via daily oral gavage for 25 weeks (n = 14/group). The blood glucose levels of the NOD mice were measured every week with an Accu-Check Active meter (Roche, Germany). The mice were characterized to be diabetic by the presence of blood glucose ≥11.1 mmol/L on 2 consecutive times.
2.7. ELISA for insulin and cytokine assays Blood samples were collected, centrifuged at 4000g for 10 min at 4 °C and then stored at −20 °C for ELISA analysis. The insulin levels in the sera were assayed using a rat/mouse insulin ELISA kit. The content of cytokines IL-4, IL-6, IL-10 and TNF-α in the collected sera was measured with ELISA kits purchased from Multi Sciences following the manufacturer’s instructions.
2.5. Glucose tolerance assays Two days before the mice were sacrificed, oral glucose tolerance tests (OGTTs) were performed. Animals received glucose by oral gavage (2 g/kg. BW) after 16 h fast. Blood glucose was measured at 0, 15, 30, 60 and 120 min.
2.8. Quantitative Real-Time PCR for cytokine assays
2.6. Histological and immunohistochemical examination
Total pancreatic mRNA was isolated using Trizol reagent. cDNA synthesis was prepared by the PrimeScript™ RT reagent kit. The realtime PCRs were carried out on the Bio-Rad CFX Connect Real-time
Pancreatic tissues were harvested from female NOD mice after 165
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Fig. 3. Treatment with a mixture of corn and wheat peptide reduced β-cell damage in NOD mice. (A) Representative hematoxylin and eosin (H&E)-stained sections showing pancreatic islet infiltration of control NOD mice and CP-plus WP-mice at 28 weeks of age. (B) Insulitis score of control NOD mice and CP- plus WP-mice at 28 weeks of age. The percentage of insulitis was quantified by evaluating the degree of infiltration and categorized as follows: 0-no insulitis, 1-periinsulitis or with few minimal infiltration in the islets, 2-insulitis with < 50% infiltration of the islets, 3-invasive insulitis with > 50% infiltration of the islets was quantified. (C) Representative immunohistochemistry for insulin in the pancreatic islets of control NOD mice and CP- plus WP-mice at 28 weeks of age. Red line marks the insulin area. (D) Relative beta cell areas of control NOD mice and CP- plus WP-mice at 28 weeks of age. All data are expressed as mean ± SD and differences were analysed by a t-test. *P < 0.05. CP + WP = a mixture of corn and wheat peptide treated mice.
3. Results
System (Bio-Rad, Hercules, CA). The specific primers for IL-6, IL-10 and β-actin could be found in Table 1. β-actin reaction product served as a loading control.
3.1. Corn peptide reduced the blood glucose in diabetic NOD mice The effect of corn peptide on the blood glucose levels of wheat peptide-evoked diabetic NOD mice was examined. Corn peptide treatment restored normoglycemia in three of six mice at 1–6 weeks when the initial blood glucose levels were between 11.1 and 15.0 mmol/L (Fig. 1). In contrast, none of the six mice that received wheat peptide alone and none of the control mice were normoglycemic at six weeks (Fig. 1). Thus we speculated that corn peptide treatment may neutralize the adverse impact of wheat peptide on type 1 diabetes.
2.9. Amino acid analysis Corn peptide or wheat peptide (100 mg) was respectively hydrolysed with 8 mL of 6 M HCl for 24 h at 120 °C to analyse the acidic amino acid contents. The basic amino acid contents were measured with the use of NaOH instead of HCl. Serum was thoroughly mixed with 5% (w/v) trichloroacetic acid (1:10, v/v) to precipitate the proteins, and the supernatant was then collected for analysis. The free amino acids in the serum and total amino acids contents in the corn peptide and wheat peptide were analysed with an Agilent 1100 HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) at 40 °C, as described by Shazly et al. (2017). An absorbance signal corresponding to the amino acids was detected at 338 nm in addition to a signal exhibited by proline at 262 nm.
3.2. Dietary mixture of corn and wheat peptide prevented the development of diabetes in NOD mice Gain of body weights was slightly higher in CP- plus WP-mice as compared with the control NOD mice from 13 to 20 weeks of age (Fig. 2A). CP- plus WP-mice showed delayed occurrence in diabetic development (17 weeks old) compared with control NOD mice (13 weeks old, Fig. 2B). Meanwhile, the diabetes incidence in CP- plus WP-mice was 29% at 28 weeks of age, in comparison with 71% of the control NOD mice (P < 0.01 by log rank test, Fig. 2B). During the OGTTs, CP- plus WP-mice showed significantly lower glucose levels at 30 (P < 0.05), 60 (P < 0.05) and 120 min (P < 0.01) and the area under the blood glucose response curve (P < 0.05) in comparison with the control NOD mice (Fig. 2C and D). Additionally, treatment with the mixture of corn and wheat peptide enhanced serum insulin
2.10. Statistical analysis All of the results were analysed by GraphPad Prism 5 software (San Diego, CA, USA). All results were shown as the mean ± SD. Statistical analyses were carried out by t-tests for two independent groups. The cumulative diabetes incidence was calculated with the Kaplan–Meier estimation and the statistical significance was evaluated by the log rank test. P < 0.05 was considered statistically significant. 166
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Fig. 4. Treatment with a mixture of corn and wheat peptide decreased IL-6 and increased IL-10 levels in NOD mice. Serum levels of IL-6 (A), IL-10 (B), IL-4 (C) and TNF-α (D) in the control group and in NOD mice treated with the mixture of corn and wheat peptide were measured by ELISA. The mRNA expression of IL-6 (E) and IL-10 (E) were analysed using quantitative real-time PCR. All data are expressed as mean ± SD and differences were analysed by a t-test. *P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
3.4. Dietary mixture of corn and wheat peptide reduced serum level and gene expression of IL-6 in NOD mice
concentration by two-fold over the control level (P < 0.05, Fig. 2E). These results imply that early treatment with the mixture of corn and wheat peptide prevented type 1 diabetes development, and that β-cell function was improved by this peptide mixture.
In type 1 diabetes, β-cells are destroyed by an autoimmune mechanism in which cytokine-induced apoptosis is thought to play an important role (Eizirik & Mandrup-Poulsen, 2001). Thus, we quantified the serum levels of pro-inflammatory cytokines (IL-6 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) in control NOD mice and CP- plus WP-mice at the endpoint. The serum IL-6 level was significantly decreased in CP- plus WP-mice versus the control NOD mice (P < 0.05, Fig. 4A). In addition, the mixture of corn and wheat peptide significantly reduced serum levels of anti-inflammatory IL-10 when compared to the control NOD mice (P < 0.05, Fig. 4B). However, there was no significant difference in the serum IL-4 and TNF-α levels in these two groups (Fig. 4C and D). Quantitative Real-time PCR analyses further confirmed that the mixture of corn and wheat peptide inhibited the mRNA expression of IL-6 (P < 0.01, Fig. 4E) and activated the gene expression of IL-10 (P < 0.05, Fig. 4F). The prevention of hyperglycemia in CP- plus WP-mice was accompanied by reduction the serum level and mRNA expression of IL-6, suggesting that the mixture of corn and wheat peptide was an anti-inflammatory mediator.
3.3. Dietary mixture of corn and wheat peptide inhibited pancreatic insulitis in NOD mice Histological analysis of pancreatic islets obtained from mice at the end of the treatment showed that the mixture of corn and wheat peptide ameliorated the destruction of islets. The islets in pancreata of the control NOD mice were heavily infiltrated by leukocytes and few islets remained β-cell (Fig. 3A). The average insulitis score exhibited by the control NOD mice was significantly higher than that observed in CPplus WP-mice (P < 0.01, Fig. 3B), indicating that treatment with the mixture of corn and wheat peptide reduced the severity of lymphocytic infiltration. We also measured the beta cell areas in both groups. There was a significant increase in the β-cell area and most islets were not infiltrated for CP- plus WP-mice (P < 0.05, Fig. 3C and D). All of these data indicated that the mixture of corn and wheat peptide attenuated the inflammatory autoimmune process.
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peptides, we then analysed the total amino acid contents in the corn and wheat peptide. As shown in Table 3, the glutamine, proline and leucine contents were very high in the corn and wheat peptide, whereas the serine and histidine contents were low. Our results indicated that the histidine and serine contents in the corn and wheat peptide were not correlated with their concentrations in the sera collected from CPplus WP-mice.
Table 2 The mixture of corn and wheat peptide increased serum serine and histidine levels in NOD mice. Free amino acid
Controla (ng/mL)
CP + WPb (ng/mL)
Asp Glu Asn Ser Gln His Gly Thr Ala Arg Tyr Cys-s Val Met Trp Phe Ile Leu Lys Pro Total amino acids
13.42 ± 1.33 26.97 ± 2.15 5.68 ± 2.23 4.07 ± 1.31 73.03 ± 9.33 6.72 ± 0.39 21.81 ± 5.31 13.22 ± 1.41 20.27 ± 5.37 144.24 ± 77.73 14.26 ± 3.82 3.25 ± 3.65 31.42 ± 3.91 8.20 ± 0.48 18.52 ± 5.47 11.47 ± 1.13 11.06 ± 1.08 16.57 ± 1.86 37.16 ± 5.41 19.76 ± 3.97 501.72 ± 72.31
12.58 ± 0.36 26.10 ± 4.06 6.49 ± 0.67 6.41 ± 0.33* 77.49 ± 1.85 10.32 ± 0.67** 21. 61 ± 2.15 12.13 ± 1.34 20.84 ± 3.57 195.92 ± 33.18 14.98 ± 2.79 0.68 ± 0.32 30.22 ± 0.05 8.71 ± 0.15 13.63 ± 3.23 12.66 ± 1.26 11.68 ± 0.89 19.52 ± 1.29 40.98 ± 1.76 17.24 ± 1.81 560.19 ± 36.08
4. Discussion In this study, we reported on the effects of treatment with a mixture of corn and wheat peptide on the development of type 1 diabetes in NOD mice. Previous studies have aimed to develop antigen-based immunotherapy for the treatment of type 1 diabetes. Dietary proteinbased peptides have been found to be effective compounds for exerting anti-inflammatory effects (Majumder, Mine, & Wu, 2016) and reducing the incidence of type 1 diabetes (Jiang et al., 2015). Corn peptide derived from corn-based by-products have been shown to promote GLP-1 release in type 2 diabetic animals (Hira et al., 2009), and thus corn peptide treatment may counteract the pro-inflammatory effect of wheat peptide in type 1 diabetic animals Notably, in our study we have shown for the first time that early administration with a mixture of corn and wheat peptide prevented the development of type 1 diabetes in NOD mice (Fig. 2B), indicating that corn peptide treatment could neutralize the pro-inflammatory effect of a wheat peptide diet on type 1 diabetes. However, the partial diabetes reversal (50%) among corn peptide-treated recent-onset NOD mice indicted that the normoglycemic restoration capabilities of the corn peptide is limited and early intervention might be a more efficient treatment strategy. Type 1 diabetes is characterized by the infiltration of immune cells into the pancreas. This inflammation selectively leads to dysfunction and finally death of β-cells. However, dampening the inflammatory response can mitigate the damage to the β-cells (Cohen, 2002). The significantly increased beta cell area and reduced average insulitis score achieved via treatment with the mixture of corn and wheat peptide (Fig. 3A–D) was sufficient to reduce the blood glucose levels in NOD mice, and suggested that this peptide mixture regulated the inflammation among NOD mice. Subsequently, we measured the cytokine levels in sera collected from both groups, and we found that the serum level and pancreatic mRNA expression of IL-6 in CP- plus WP-mice were significantly lower compared to those found in the control NOD mice (Fig. 4A and E). Systemic inflammation was enhanced in youth with type 1 diabetes (Snell-Bergeon et al., 2010). The pro-inflammatory cytokines cause dysfunction to the beta cells and apoptosis. Several pro-inflammatory markers were elevated in subjects with type 1 diabetes. Elevated levels of pro-inflammatory cytokine IL-6 have previously been reported in children and adolescents with type 1 diabetes (Rosa et al., 2008; SnellBergeon et al., 2010). Liese et al. reported that the biomarker IL-6 levels in youth with type 1 diabetes was not influenced by dietary intake (Liese et al., 2018). Consistent with our results, curcumin administration reduced the concentration of serum IL-6 and blood glucose from STZ-induced rats (Jain, Rains, Croad, Larson, & Jones, 2009), indicating that immune suppression in type 1 diabetes could be achieved via a proinflammatory cytokine blockade (Ryden et al., 2017). IL-10, an immunosuppressive and anti-inflammatory cytokine secreted by monocytes/macrophages lineages and immune cells, was first identified as cytokine synthesis inhibitory factor (Fiorentino, Bond, & Mosmann, 1989), and has a beneficial effect on autoimmune diabetes. Exogenous IL-10 provided protection against destructive insulitis of pancreatic beta cells, and delayed the occurrence and limited the incidence of type 1 diabetes among NOD mice (Pennline, Roquegaffney, & Monahan, 1994). In addition, IL-10 was expressed in the pancreas of type 1 diabetic mice (Teros et al., 2000), whereas IL-10 was deficient in subjects with type 1 diabetes (Rabinovitch & Suarez-Pinzon, 2003).
a The concentration of free amino acids in the sera of control NOD mice was analysed by HPLC. b The concentration of free amino acids in the sera of NOD mice treated with the mixture of corn and wheat peptide was analysed by HPLC. All data are expressed as mean ± SD and significance was analysed by the t-test. * P < 0.05; **P < 0.01. CP + WP = a mixture of corn and wheat peptide treated mice.
Table 3 Total amino acid content in corn and wheat peptide. Total amino acid
Wheat peptide (g/100 g)
Corn peptide (g/100 g)
Asp Glu Ser His Gly Thr Ala Arg Tyr Cys-s Val Met Trp Phe Ile Leu Lys Pro
2.78 ± 0.04 37.36 ± 0.04 3.16 ± 0.03 1.37 ± 0.04 2.69 ± 0.04 1.77 ± 0.01 1.83 ± 0.05 2.12 ± 0.03 1.66 ± 0.03 0.61 ± 0.01 3.68 ± 0.02 1.09 ± 0.02 0.45 ± 0.01 4.67 ± 0.04 3.41 ± 0.01 4.98 ± 0.04 1.04 ± 0.03 12.94 ± 0.01
5.72 ± 0.05 23.45 ± 0.02 3.49 ± 0.01 1.35 ± 0.01 1.98 ± 0.01 2.09 ± 0.01 6.17 ± 0.01 1.54 ± 0.01 3.38 ± 0.01 0.18 ± 0.01 4.22 ± 0.01 1.96 ± 0.02 0.34 ± 0.04 4.52 ± 0.03 3.84 ± 0.05 12.68 ± 0.04 0.95 ± 0.03 11.87 ± 0.01
The content of total amino acids in corn and wheat peptide were analysed by HPLC. All data are expressed as mean ± SD.
3.5. Dietary mixture of corn and wheat peptide increased serum serine and histidine levels in NOD mice Serum amino acid concentrations are shown in Table 2. The glucogenic amino acids of serine (∼6.41 vs. ∼4.07 ng/mL, P < 0.05) and histidine (∼10.32 vs. ∼6.72 ng/mL, P < 0.01) were apparently reduced to lower concentrations in control NOD mice compared to CPplus WP- mice (Table 2). However, other free amino acids (including leucine, ∼19.52 vs. ∼16.57 ng/mL) in sera were similar between the two groups (Table 2). Differences in metabolomic profiles have been shown to be associated with dietary proteins (Holmes et al., 2008). To investigate the correlation of serum amino acids and the amino acids of indicated 168
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Conflict of interest
Peptide IA-2-P2 (insulinoma antigen-2 (IA-2), which is used to create the fused peptide not only reduced the incidence of diabetes but also enhanced the production of IL-10 by splenocytes (Shen et al., 2017). In present study, the pancreatic gene expression of IL-10 was higher in CPplus WP-mice when compared to the control NOD mice (Fig. 4F). In contrast, we found that the IL-10 levels were higher in sera collected from control NOD mice than in those taken from CP- plus WP-mice (Fig. 4B), which may be due to its role in suppressing the cytotoxic effects of pro-inflammatory IL-6 in NOD mice (Souza, Gurgul-Convey, Elsner, & Lenzen, 2008). Dietary plant proteins influence the incidence of type 1 diabetes, and in turn diabetes can potentially alter the metabolism of proteins (Jensen-Waern et al., 2009). Protein catabolism increases in subjects with type 1 diabetes (Hamadeh & Hoffer, 2003). However, higher levels of plasma amino acids could stimulate protein synthesis. In addition, insulin release was enhanced in response to elevated concentrations of amino acids found in subjects treated with protein diets (Schmid, Schusdziarra, Schulte-Frohlinde, Maier, & Classen, 1989). A previous study has reported that the amino acid concentrations were lower in patients with human preclinical type 1 diabetes compared to controls (la Marca et al., 2013). In combination with glucose, amino acids derived from dietary proteins and released from intestinal epithelial cells can activate insulin secretion. Arginine and branched chain amino acids (isoleucine, alanine and leucine) have been found to be essential for insulin secretion, which inhibits protein degradation in children and adult humans with type 1 diabetes (Pacy, Nair, Ford, & Halliday, 1989). In addition, plasma-branched chain amino acids levels, especially those of leucine, can serve as effective biomarkers for the detection of diabetes. It has been shown that an elevated plasma leucine level is associated with a greater risk for the development type 1 diabetes (JensenWaern et al., 2009). However, treatment with the mixture of corn and wheat peptide apparently increased the levels of leucine, isoleucine and alanine in sera but this impact was not statistically significant (Table 2). Interestingly, the serum concentrations of only serine and histidine among the glucogenic amino acids were significantly increased in CPplus WP-mice (Table 2). This is consistent with observations that plasma serine and histidine were significantly decreased in Zucker diabetic fatty rats (Wijekoon, Skinner, Brosnan, & Brosnan, 2004). It has been shown that the capacities of histidine- and serine-pyruvate aminotransferase were enhanced when blood glucose levels were elevated (Noguchi, Okuno, & Kido, 1976). Serine and histidine could be converted to pyruvate. Thus, the observed reduction in serine and histidine from control NOD mice may be due to the increased activity of their degradative enzyme. Furthermore, L-serine treatment has been shown to decrease the incidence of diabetes in NOD mice (Holm et al., 2018), suggesting that increased serine may provide protection against the development of type 1 diabetes. However, the elevated serine and histidine levels in sera from CP- plus WP-mice were not related to their content in corn and wheat peptide (Table 3), proving that levels of the two amino acids in sera may be influenced not only by supplementation but also by metabolism.
The authors declare that there are no conflicts of interest. Acknowledgments This study was supported by the National Key Research and Development Program of China (2017YFD0400200), the National Natural Science Foundation of China Grants No. 3141128 (YQ.C) and No. 31771539 (YQ.C), and the National First-Class Discipline Program of Food Science and Technology (JUFSTR20180101). We wish to thank Hong-yang Pan and Qin Yang for providing technical assistance with the amino acid analysis, Xiao-hong Gu and Shang-wei Chen for their technical assistance with molecular weight analysis. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.03.020. References Akerblom, H. K., & Knip, M. (1998). Putative environmental factors in type 1 diabetes. Diabetes-Metabolism Reviews, 14(1), 31–67. Antvorskov, J. C., Josefsen, K., Engkilde, K., Funda, D. P., & Buschard, K. (2014). Dietary gluten and the development of type 1 diabetes. Diabetologia, 57(9), 1770–1780. Barbeau, W. E., Hontecillas, R., Horne, W., Carbo, A., Koch, M. H., & Bassaganya-Riera, J. (2014). Elevated CD8 T cell responses in type 1 diabetes patients to a 13 amino acid coeliac-active peptide from alpha-gliadin. Clinical and Experimental Immunology, 175(2), 167–171. Bluestone, J. A., Herold, K., & Eisenbarth, G. (2010). Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature, 464(7293), 1293–1300. Brubaker, P. L., & Anini, Y. (2003). Direct and indirect mechanisms regulating secretion of glucagon-like peptide-1 and glucagon-like peptide-2. Canadian Journal of Physiology and Pharmacology, 81(11), 1005–1012. Brubaker, P. L., & Drucker, D. J. (2004). Minireview: Glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology, 145(6), 2653–2659. Cohen, I. R. (2002). Peptide therapy for type I diabetes: The immunological homunculus and the rationale for vaccination. Diabetologia, 45(10), 1468–1474. De Leon, D. D., Deng, S. P., Madani, R., Ahima, R. S., Drucker, D. J., & Stoffers, D. A. (2003). Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes, 52(2), 365–371. Eizirik, D. L., & Mandrup-Poulsen, T. (2001). A choice of death – The signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia, 44(12), 2115–2133. Elliott, R. B., & Martin, J. M. (1984). Dietary protein: A trigger of insulin-dependent diabetes in the BB rat? Diabetologia, 26(4), 297–299. Fiorentino, D. F., Bond, M. W., & Mosmann, T. R. (1989). Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. The Journal of Experimental Medicine, 170(6), 2081–2095. Funda, D. P., Kaas, A., Bock, T., Tlaskalova, H., & Buschard, K. (1999). Gluten-free diet prevents diabetes in NOD mice. Diabetes, 48 A215–A215. Hamadeh, M. J., & Hoffer, L. J. (2003). Effect of protein restriction on sulfur amino acid catabolism in insulin-dependent diabetes mellitus. American Journal of PhysiologyEndocrinology and Metabolism, 284(2), E382–E389. Helmerhorst, E. J., Zamakhchari, M., Schuppan, D., & Oppenheim, F. G. (2010). Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity. Plos One, 5(10). Higuchi, N., Hira, T., Yamada, N., & Hara, H. (2013). Oral administration of corn zein hydrolysate stimulates GLP-1 and GIP secretion and improves glucose tolerance in male normal rats and Goto-Kakizaki rats. Endocrinology, 154(9), 3089–3098. Hira, T., Mochida, T., Miyashita, K., & Hara, H. (2009). GLP-1 secretion is enhanced directly in the ileum but indirectly in the duodenum by a newly identified potent stimulator, zein hydrolysate, in rats. American Journal of Physiology-Gastrointestinal and Liver Physiology, 297(4), G663–G671. Holm, L. J., Haupt-Jorgensen, M., Larsen, J., Giacobini, J. D., Bilgin, M., & Buschard, K. (2018). L-serine supplementation lowers diabetes incidence and improves blood glucose homeostasis in NOD mice. Plos one, 13(3) e0194414–e0194414. Holmes, E., Loo, R. L., Stamler, J., Bictash, M., Yap, I. K. S., Chan, Q., ... Elliott, P. (2008). Human metabolic phenotype diversity and its association with diet and blood pressure. Nature, 453(7193) 396–U350. Hoorfar, J., Scott, F. W., & Cloutier, H. E. (1991). Dietary plant materials and development of diabetes in the BB rat. Journal of Nutrition, 121(6), 908–916. Jain, S. K., Rains, J., Croad, J., Larson, B., & Jones, K. (2009). Curcumin supplementation lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose, and glycosylated hemoglobin in diabetic rats. Antioxidants & Redox Signaling, 11(2), 241–249. Jensen-Waern, M., Andersson, M., Kruse, R., Nilsson, B., Larsson, R., Korsgren, O., & Essen-Gustavsson, B. (2009). Effects of streptozotocin-induced diabetes in domestic
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