| Type 2 diabetes mellitus is an endocrine and metabolic system disease characterized by insulin resistance or persistent hyperglycemia,affecting more than 400million people worldwide.Food-derived bioactive peptides to have substantial potential in maintaining blood glucose homeostasis.As one of the four traditional fermented bean products,douchi is not only rich in protein,amino acid,organic acids,minerals,soluble sugars,carbohydrates,vitamins and other nutrients,but also has a variety of biological activities,such as antioxidant activity,hypoglycemic activity,hypotensive activity,etc.Therefore,douchi was used as the study material,and the hypoglycemic effect and related mechanism of douchi-derived peptides in the process of digestion-absorption/transport-metabolism were systematically investigated using in vitro simulated digestion,Caco-2 monolayer cell model,high-fat diet-induced insulin resistance model,and L6 cell model.The main research contents and results are as follows:（1）Study on the extraction and hypoglycemic activity of douchi peptide water extract.Douchi peptides were extracted by alcohol,water and Tris-HCl buffer,respectively.It was found that the peptide content of douchi peptides extracted by water was the highest,which was 23.99±0.84 g/100g,showed a better glucose uptake promoting activity in L6 cells,and had an IC50 value of 0.39±0.12 mg/m L againstα-glucosidase.Furthermore,the peptide content（23.99±0.84 g/100g）and the total amino acids content（22.63±1.42 g/100g）in the Mucor type Yongchuan douchi peptide extracts were higher than other fermented douchi extracts（Aspergillus type,bacterial type）.Moreover,the inhibitory percentage of Yongchuan douchi peptide extracts onα-glucosidase activity reached 76.39%at a concentration of 4 mg/m L.Compared with high-fat diet-fed mice,4%douchi peptide extracts supplementation not only reduced the homeostasis model assessment insulin resistance（HOMA-IR）index,but also improved glucose intolerance,indicating that Yongchuan douchi peptide extract has hypoglycemic activity.（2）Isolation,extraction and identification of Yongchuan douchi water extract.The Yongchuan douchi peptide extract was fractionated and purified successively by solid-phase microextraction,cation-exchange chromatography and reversed-phase liquid chromatography,respectively,and the fractions F4-1-6,F4-1-9 and F4-3-12 with the stronger glucose uptake promoting activity in L6 cells were obtained.Furthermore,western blot analysis revealed that the fractions F4-1-6,F4-1-9 and F4-3-12 increased the translocation of glucose transporter 4（GLUT4）from vesicles to cell membrane by activating adenosine monophosphate（AMP）-activated protein kinase（AMPK）,p38mitogen activated protein kinase（MAPK）and p44/42 MAPK signaling pathways,thus promoting glucose uptake in L6 cells.UPLC-Q-TOF-MS/MS was used to identify 5,11 and 5 soybean-derived protein peptide sequences with peptide lengths of 2-6 amino acids from the three components,respectively.（3）The molecular mechanism of hypoglycemic activity of douchi peptide.Based on mass spectrometry,7 peptides with relative intensities higher than 40%were selected for glucose uptake experiment in L6 cells.Peptides with the stronger glucose uptake promoting activity were found to be Val-Tyr（VY）and Ser-Phe-Leu-Leu-Arg（SFLLR）.Western blot was further used to study the effects of peptides VY and SFLLR on the activation of protein kinase B（AKT）,AMPK,p38 MAPK and p44/42 MAPK signaling pathways and GLUT4 translocation;pathways inhibitors were used to clarify the key signaling pathways.The results showed that VY and SFLLR promoted the translocation of GLUT4 by activating the AMPK and MAPK pathways rather than the AKT pathway,thereby promoting glucose uptake in L6 cells.Molecular docking experiments showed that SFLLR had stronger inhibitory activity againstα-glucosidase than VY.Multiple-reaction monitoring liquid chromatography mass spectrometry showed that the content of VY and SFLLR in Yongchuan douchi extracts were 13.53±0.15 mg/100g dry base（VY）and 128.40±0.04 mg/100g dry base（SFLLR）,respectively.（4）The digestion,absorption and transport properties of douchi-derived peptides VY and SFLLR.Computer simulation analysis showed that VY had better gastrointestinal stability than that of SFLLR.VY was resistant to gastrointestinal tract digestion and could cross Caco-2 cell monolayers intact via both tight junctions（TJs）-mediated passive paracellular pathway and Peptide-transporters 1（Pep T1）-mediated active route.In comparison,SFLLR was partially degraded into small fragments of SFLL,SFL,and SF by the digestive system,leading to increased glucose uptake activity.Although19.68%of SFLLR,22.95%of SFLL,and 27.8%of SFL were hydrolyzed by aminopeptidase N or dipeptidyl peptidase IV during transport,most of them could still be transported intact.SFL was transported via both paracellular diffusion and Pep T1-mediated routes,while SFLLR and SFLL were via paracellular route only.（5）Effects of douchi-derived peptides VY and SFLLR on glucose homeostasis and gut dysbacteriosis in high-fat diet-induced insulin resistant mice.Feeding C57BL/6 mice with a high-fat diet containing 60%fat for 8 weeks successfully induced insulin resistance mice model.After supplementation of douchi-derived peptides（doses:10 and 50 mg/kg body weight/day）for 8 weeks,especially SFLLR,the body weight gain,serum glucose and insulin content were significantly reduced.The insulin resistance,liver damage and lipid accumulation were improved in high-fat diet-induced insulin resistant mice.In addition to reducing the pro-inflammatory factors（TNF-α,IL-6 and MCP-1）in the serum of high-fat diet mice,peptide supplementation down-regulated the expression of pro-inflammatory factors in adipose tissue and liver tissue,increased the anti-inflammatory factor IL-10 in serum,and up-regulated the expression of anti-inflammatory factor in adipose tissue and liver tissue.In both liver and muscle,VY and SFLLR not only activated glycogen synthesis by regulating insulin receptor substrate-1（IRS-1）/AKT/glycogen synthase kinase 3β（GSK3β）and AMPK/GSK3β-mediated pathways,but also inhibited gluconeogenesis by regulating IRS-1/AKT/Fox O1,AMPK/Fox O1 and MAPK/Fox O1-mediated pathways,thereby ameliorating glucose metabolism disorders in high-fat diet-fed mice.Furthermore,VY and SFLLR supplementation could reversed HFD-induced gut dysbacteriosis by decreasing the abundance of Enterococcus,Oscillibacter,and Deferribacteraceae,which were positively associated with glycolipid metabolic-associated markers including body weight gain,serum FBG,serum insulin,serum GHb,serum TC,serum TG,serum LDL-C,serum pro-inflammatory factors（TNF-α,IL-6,and MCP-1）,and negatively associated with serum IL-10 and SCFAs,but also increased probiotic abundances of Alistipes,Lactobacillus,Faecalibaculum,Akkermansia,and Bifidobacterium,which were negatively related to glycolipid metabolic disorders makers,and positively related to serum IL-10 and SCFAs.In summary,this study identified hypoglycemic peptides from douchi,revealed the effects of simulated gastrointestinal digestion and absorption on the stability,transport,and the hypoglycemic activity of douchi-derived peptides;and elucidated the hypoglycemic mechanisms of douchi-derived peptides in vivo and in vitro,thus laying a theoretical foundation for the future study and development of natural drugs or functional foods towards lowering blood glucose,and promoting the sustainable growth of the soybean industry,especially the douchi industry. |
