Antioxidant Effect of Tyr-Ala Extracted from Zein on INS-1 Cells and Type 2 Diabetes High-Fat-Diet-Induced Mice

Abstract

Type 2 diabetes mellitus (T2DM) is associated with an oxidative milieu that often leads to adverse health problems. Bioactive peptides of zein possess outstanding antioxidant activity; however, their effects on hyperglycemia-related oxidative stress remain elusive. In the present study, the dipeptide Tyr-Ala (YA), a functional peptide with typical health benefits, was applied to alleviate oxidative stress in pancreatic islets under hyperglycemic conditions. By detecting viability, antioxidant ability, and insulin secretion in INS-1 cells, YA showed excellent protection of INS-1 cells from H₂O₂ oxidative stress, erasing reactive oxygen species (ROS) and promoting insulin secretion. Moreover, by Western blotting, we found that YA can regulate the PI3K/Akt signaling pathway associated with glycometabolism. After establishing a T2DM mice model, we treated mice with YA and measured glucose, insulin, hemoglobin A1C (HbA1c), total cholesterol (TC), triglyceride (TG), and malonaldehyde (MDA) levels and activities of superoxide dismutase (SOD) and glutathione (GSH) from blood samples. We observed that YA could reduce the production of glucose, insulin, HbA1c, TC, TG, and MDA, in addition to enhancing the activities of SOD and GSH. YA could also repair the function of the kidneys and pancreas of T2DM mice. Along with the decline in fasting blood glucose, the oxidative stress in islets was alleviated in T2DM mice after YA administration. This may improve the health situation of diabetic patients in the future.

Keywords: INS-1 cells; peptide; reactive oxygen species; type 2 diabetes; tyr-ala.

Figure 1

Schematic experiments of the YA effect on diabetes in vitro and in vivo. In cell assays, YA treatment increased cell viability and decreased intracellular ROS accumulation and increased insulin secretion in INS-1 cells exposed to H₂O₂, human amylin, or high glucose stimulation, which was achieved by activating the PI3K/AKT signaling pathway. In in vivo experiments, T2DM model was established by feeding a high-fat diet daily for 10 weeks and injecting STZ for 3 days in the fourth week. In T2DM mice, YA administration for 6 weeks improved the mice’s health, increased insulin secretion levels, and decreased blood sugar levels. It also increased the expression of antioxidant enzymes and decreased the levels of TG and TC. (ROS: reactive oxygen species; InsR: insulin receptor; PIP2: phosphatidylinositol 4, 5-bisphosphate; PIP3: phosphatidylinositol-3, 4, 5-triphosphate; AKT: protein kinase A; PI3K: Phosphatidylinositol 3-kinase; YA: Tyr-Ala; STZ: streptozocin).

Figure 2

Schematic flow-chart of T2DM model mice establishment and experimental drug administration. T2DM mice were established by feeding HFD daily for 10 weeks and injecting STZ intraperitoneally at the fourth week. Starting from week 5, mice were administered DL, DM, DH, DMBG for 6 weeks. Each group contained 10 mice. (ND: control feed; HFD: high fat feed; STZ: streptozocin; NS: normal saline; DL: YA 5 mg/kg; DM: YA 10 mg/kg; DH: YA 20 mg/kg; DMBG: metformin).

Figure 3

Antioxidant activity of YA in INS-1 cells. (A) The effect of YA on the toxicity of H₂O₂ and hA in INS-1 cells. INS-1 cells were treated with H₂O₂ (50 µM), co-incubation of H₂O₂ (50 µM) and YA (10, 20, and 40 µM), hA (20 µM), co-incubation of hA (20 µM) and YA (10, 20, and 40 µM), and YA (40 µM) alone for 24 h. Cell viability was measured using an MTT viability assay. (*** p < 0.001 vs. Control; ^^ p < 0.01 vs. H₂O₂; ^^^ p < 0.001 vs. H₂O₂; ^### p < 0.001 vs. hA); (B) Effect of YA on reactive oxygen species (ROS) accumulation in INS-1 cells. INS-1 cells were treated with H₂O₂ (50 µM) or YA (10 µM, 20 µM, 40 µM) for 24 h. The DCFH-DA method was used to assay the rate of ROS. (** p < 0.01 vs. H₂O_2, *** p < 0.001 vs. H₂O₂); (C) Effect of different concentrations of YA on GSIS/BIS levels in INS-1 cells cultured by medium with 40 mM glucose. INS-1 cells were plated in a 96-well plate overnight and then treated with either glucose (40 µM) or co-incubation of glucose (40 µM) and YA (10 µM, 20 µM, 40 µM) for 24 h. (** p < 0.01 vs. glucose, *** p < 0.001 vs. glucose). All data are expressed as mean ± SD for each group (n = 3). HG: glucose 40 mM.

Figure 4

Effects of YA treatment on PI3K/Akt signaling pathway in INS-1 cells. INS-1 cells were treated with 40 μM YA and 40 mM glucose for 24 h, the PI3K (A) and phosphorylation of Akt473/Akt308 (B) were detected using Western blot. Equal amounts of proteins from each sample were separated on SDS-PAGE. Phosphorylation of Akt473/Akt308 was probed by p-Akt (Ser 473) and p-Akt (Thr 308) antibody. PI3K was probed by PI3K-p85 antibody. Total β-actin and Akt were taken as two control groups separately. Values are means ± SD from three representative experiments (n = 3). (* p < 0.05 vs. glucose, ** p < 0.01 vs. glucose, *** p < 0.001 vs. glucose).

Figure 5

Effects of YA on body weight of T2DM mice. (A) Body weight differences between the different mouse groups. (B) Body weights of different groups mice on the 10th week. Data are expressed as mean ± SD for each group. (n = 8) (DMBG: metformin) (*** p < 0.001 vs. Control; ** p < 0.01 vs. Control; * p < 0.05 vs. Control).

Figure 6

Fasting plasma glucose (FPG) of different groups during administration. Values are expressed as mean ± SD for each group. (n = 8) (*** p < 0.001 vs. Control group, ^ p < 0.05 vs. T2DM Model group at the 10th week, ^^ p < 0.01 vs. T2DM Model group at the 10th week, ^^^ p < 0.001 vs. T2DM Model group at the 10th week).

Figure 7

Effect of YA on FPG in different groups. (A) FPG of different groups at different time points; (B) FPG of different groups at 120 min. Data are expressed as mean ± SD for each group. (n = 8; DMBG: metformin) (### p < 0.001 vs. control, * p < 0.05 vs. T2DM model, ** p < 0.01 vs. T2DM model, *** p < 0.001 vs. T2DM model).

Figure 8

Effect of YA on the levels of insulin, hemoglobin A1C (HbA1c), triglyceride (TG), total cholesterol (TC), and malonaldehyde (MDA), and on the activities of superoxide dismutase (SOD) and glutathione (GSH) in serum. (A) insulin; (B) HbA1c; (C) TG; (D) TC; (E) SOD; (F) MDA; (G) GSH. Data are expressed as mean ± SD for each group. (n = 8; DMBG: metformin) (^# p < 0.05 vs. control, ^## p < 0.01 vs. control, ^### p < 0.001 vs. control, * p < 0.05 vs. model, ** p < 0.01 vs. model, *** p < 0.001 vs. model).

Figure 9

Representative hematoxylin and eosin (H&E) staining sections of pancreases from mice in the different groups (×200). (A). control; (B). T2DM mice; (C). YA 5 mg/mL; (D). YA 10 mg/mL; (E). YA 20 mg/mL; (F). DMBG 100 mg/mL.

Figure 10

Representative H&E staining sections of kidneys from mice in the different groups (×200). (A). control; (B). T2DM mice; (C). YA 5 mg/mL; (D). YA 10 mg/mL; (E). YA 20 mg/mL; (F). DMBG 100 mg/mL.