Antidiabetic effects of protein hydrolysates from Trachinotus ovatus and identification and screening of peptides with α-amylase and DPP-IV inhibitory activities

Abstract

In the present study, the antidiabetic properties of Trachinotus ovatus protein hydrolysates (TOH) in streptozotocin-induced diabetic mice were investigated, and peptides with α-amylase (AAM) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities were identified and screened. The results showed that TOH alleviated body weight loss, polyphagia, blood glucose elevation and insulin secretion decline in diabetic mice. After 4 weeks of TOH administration, random blood glucose (RBG) decreased significantly. The TOH groups showed a dose-dependent reduction in fasting blood glucose (FBG), especially in the high-dose TOH group, which reduced FBG by 58% versus the effect of metformin. Moreover, TOH exerted a remarkable protective effect on hepatorenal function, as evidenced by increased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and decreased serum urea levels. Histopathological studies confirmed that TOH can significantly protect the kidney and pancreas from histological changes, which was of great benefit for ensuring the normal secretion of insulin and preventing the occurrence of complications such as diabetic nephropathy. Two fractions with higher inhibitory activity against AAM and DPP-IV, F4 and F6, were obtained from the ultrafiltration of TOH-2 (≤3 kDa). A total of 19 potentially active peptides from F4 and 3 potentially active peptides from F6 were screened by LC‒MS/MS combined with bioinformatic analysis. These peptides are small molecular peptides composed of 2-6 amino acids, rich in characteristic amino acids such as proline, arginine, phenylalanine and asparagine, and contain high proportions of peptides (68% for F4, 67% for F6) with hydrophobicity ≥50%. They offer potent antidiabetic potential and could potentially bind to the active sites in the internal cavities of the target enzymes AAM and DPP-IV. In summary, this study revealed for the first time the antidiabetic effects of protein hydrolysates of Trachinotus ovatus and their derived peptides, which are promising natural ingredients with the potential to be used for the treatment or prevention of diabetes.

Keywords: Antidiabetic effects; DPP-IV; Peptide identification and screening; Protein hydrolysates; Trachinotus ovatus; α-amylase.

Graphical abstract

Fig. 1

Effects of TOH and Met on (A) weight change, (B) food intake, (C) fasting blood glucose (FBG) and (D) serum insulin (INS) in STZ-induced diabetic mice over four weeks. Values are expressed as the mean ± SD (n = 10). The lowercase letter a indicates p < 0.05 vs. mice in the DC group, the lowercase letter b indicates p < 0.05 vs. mice in the NC group, and the lowercase letter c indicates p < 0.05 vs. mice in the Met group. DC: diabetic control; NC: normal control; Met: metformin for the positive control (250 mg/kg); TOH-L: low-dose T. ovatus hydrolysates (100 mg/kg); TOH-M: medium-dose T. ovatus hydrolysates (500 mg/kg); TOH-H: high-dose T. ovatus hydrolysates (1000 mg/kg).

Fig. 2

Effects of TOH and Met on liver and kidney markers in STZ-induced diabetic mice. (A) Liver SOD level; (B) Liver CAT level; (C) Liver GPX level; (D) organ indices for liver and kidney (organ index was defined as the weight of the organ (g)/body weight (g)); (E) serum creatinine level and (F) serum urea level. Data are expressed as the mean ± SD (n = 10). The lowercase letter a indicates p < 0.05 vs. mice in the DC group, the lowercase letter b indicates p < 0.05 vs. mice in the NC group, and the lowercase letter c indicates p < 0.05 vs. mice in the Met group. DC: diabetic control; NC: normal control; Met: metformin for the positive control (250 mg/kg); TOH-L: low-dose T. ovatus hydrolysates (100 mg/kg); TOH-M: medium-dose T. ovatus hydrolysates (500 mg/kg); TOH-H: high-dose T. ovatus hydrolysates (1000 mg/kg).

Fig. 3

Effects of TOH on the histological changes in kidney tissues in STZ-induced diabetic mice. (1) Histopathological micrograph of the kidney in normal and STZ-induced diabetic mice with H&E staining (magnification, 400 × ); (2) histopathological micrograph of the kidney in normal and STZ-induced diabetic mice by Masson's trichrome staining (magnification, 400 × ). (A)–(F) represent the NC group, DC group, Met group, TOH-L group, TOH-M group and TOH-H group, respectively. The red arrows represent inflammatory cell infiltration in the renal interstitium. The green arrowheads represent vacuolar degeneration of renal tubular epithelial cells. The yellow arrowheads represent renal interstitial hemorrhage. The black arrowheads represent interstitial fibrosis. The red circles represent glomerular atrophy. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Effects of TOH on the histological changes in pancreatic tissues in STZ-induced diabetic mice. (1) Histopathological micrograph of the pancreas in normal and STZ-induced diabetic mice with H&E staining (magnification, 400 × ); (2) histopathological micrograph of the pancreas in normal and STZ-induced diabetic mice with Elastic van Gieson (EVG) staining (magnification, 400 × ). (A)–(F) represent the NC group, DC group, Met group, TOH-L group, TOH-M group and TOH-H group, respectively. The red arrows represent decreased islet volume and islet number. The red circles represent necrosis or swelling of pancreatic acini. The black boxes represent the cytoplasmic degenerative changes in the center of islets of mice in the diabetic model group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Isolation and purification of TOH and its inhibitory effects on AAM and DPP-IV. (A) AAM and DPP-Ⅳ inhibitory effects of TOH and ultrafiltrations of TOH-1 and TOH-2, where *p < 0.05 represents the comparison between TOH-2 and TOH-1; (B) gel filtration chromatographic separation of TOH-2; (C) the inhibitory effects of the fractions isolated from TOH-2 against AAM and (D) the inhibitory effects of the fractions isolated from TOH-2 against DPP- IV. The concentrations of TOH, TOH-1 and TOH-2 as well as the fractions were 10 mg/mL. The concentrations of the positive controls of acarbose and diprotin A were 5 mg/mL and 1.2 mg/mL, respectively.

Fig. 6

Molecular docking of FNFSR to the target receptors of AAM and DPP-IV. (A) Binding mode between AAM (PDB code: 3BAJ) and FNFSR. (B) Binding mode between DPP-IV (PDB code: 4A5S) and FNFSR. The 3D structure represents the interaction in docking of the peptide FNFSR at AAM or DPP- IV active sites, and the 2D structure represents the way FNFSR binds to the amino acid residues of AAM or DPP- IV active sites.