CN118108790B - Preparation and application of whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity - Google Patents

Abstract

The present invention provides a hypoglycemic peptide with stable gastrointestinal digestion and absorption prepared by using bovine whey protein as a raw material. In an in vitro cell experiment, the peptide segment can improve the uptake of insulin-resistant HepG2 hepatocytes and utilize glucose to synthesize glycogen, can promote the secretion of GLP-1 by enteroendocrine cells NCI-H716, and has a certain synergistic effect. The combined use can further play the effect of promoting GLP-1 secretion and activating GLP-1 receptors. An insulin-resistant mouse model was constructed using a high-fat diet, and the peptide segment was used for intervention, which further proved that the hypoglycemic peptide has obvious effects on weight loss, reducing lipid accumulation and assisting in lowering blood lipids, and can also improve symptoms related to insulin resistance. The milk peptide provided by the present invention has gastrointestinal digestion stability and hypoglycemic activity, and can be used as a functional milk-based material for the development of foods, medicines or health products with blood sugar regulating effects, and has broad application prospects.

Description

Preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity

Technical Field

The present invention relates to the field of food and medicine, and in particular to the preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity.

Background Art

Diabetes is a chronic metabolic disease characterized by high blood sugar concentration caused by impaired insulin metabolism or insufficient secretion, with elevated blood sugar as its main feature. According to data released by the International Diabetes Federation (IDF), the number of diabetes patients worldwide reached 537 million in 2021, and is expected to reach 643 million and 783 million in 2030 and 2045, respectively. Type 2 diabetes is the most common type of diabetes, accounting for about 90% of all diabetes patients. Factors such as obesity, genetics, lifestyle and aging have led to an increase in the incidence and prevalence of T2DM.

Glucagon-like peptide-1 (GLP-1) is an intestinal hormone produced and released by enteroendocrine L cells. GLP-1 stimulates insulin biosynthesis and secretion in a glucose-dependent manner, thereby increasing sensitivity to glucose. In addition, GLP-1 can enhance insulin secretion from pancreatic β cells, inhibit glucagon secretion, attenuate gastric emptying, reduce appetite, and improve insulin resistance. These physiological effects make GLP-1 a target for the treatment of type 2 diabetes. However, the endogenous enzyme dipeptidyl peptidase-4 (DPP-IV) rapidly metabolizes GLP-1 by cleaving the N-terminal dipeptide residue, resulting in the loss of GLP-1 insulinotropic activity. DPP-IV is a novel adipokine that is highly secreted during adipogenesis/obesity and is associated with the low-grade inflammation observed. It plays a crucial role in glucose and insulin metabolism and immune regulation. Therefore, DPP-IV inhibitors are a new type of drug for the treatment of type 2 diabetes.

In addition, GLP-1 receptor agonists are a type of method for treating type 2 diabetes by activating GLP-1 receptors and exerting incretin effects. GLP-1 receptor agonists have similar effects to GLP-1, and due to structural differences from natural GLP-1, their duration of action can be prolonged. GLP-1 receptor agonists promote insulin release mainly by activating GLP-1 receptors. After binding to the GLP-1 receptor, GLP-1 receptor agonists increase calcium influx and endoplasmic reticulum calcium release through the cAMP/PKA pathway to activate calmodulin, ultimately enhancing insulin exocytosis. The use of GLP-1 receptor agonists is beneficial for patients with type 2 diabetes to achieve stable blood sugar reduction.

Summary of the invention

The purpose of the present invention is to provide a preparation and application of a whey protein-derived hypoglycemic peptide with GLP-1 receptor agonist activity.

In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

On the one hand, the present invention provides a glucose-lowering peptide with stable gastrointestinal digestion and absorption from bovine whey protein, characterized in that the amino acid sequence of the peptide is shown in any one of SEQ ID NOs: 1-5. It has been verified that the obtained peptide segment has gastrointestinal digestion and absorption stability, DPP-IV inhibitory activity and GLP-1 receptor agonist activity in vivo, and can improve insulin resistance and lipid accumulation induced by a high-fat diet, and reduce body weight. The glucose-lowering peptide segment can be used to reduce DPP-IV activity in samples, increase GLP-1 content, and activate GLP-1 receptors. The auxiliary glucose-lowering and lipid-lowering products prepared using the peptide segment can be used for the prevention, health care and auxiliary treatment of hyperglycemia, hyperlipidemia, obesity and other related diseases, and have broad application prospects in the fields of health care and medical biology.

On the other hand, the present invention provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes a peptide as shown in any one of SEQ ID NOs: 1-5, and the nucleic acid molecule is DNA or RNA.

On the other hand, the present invention provides an expression vector, characterized in that the nucleotide sequence of the expression vector is the above-mentioned nucleic acid molecule.

On the other hand, the present invention provides a recombinant cell, characterized in that the recombinant cell is obtained by introducing the above expression vector into a host cell.

On the other hand, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a peptide as shown in any one of SEQ ID NOs: 1-5, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell.

On the other hand, the present invention provides a method for preparing a whey protein hydrolysate having hypoglycemic activity, characterized in that the method comprises the following steps:

S1. Performing a composite enzymatic hydrolysis of bovine whey protein using alkaline protease and pepsin at a certain temperature to obtain a bovine whey protein hydrolysate;

S2. Simulated gastric digestion and simulated intestinal digestion using bovine whey protein hydrolysate;

S3. The fully differentiated human cloned colon adenocarcinoma Caco-2 monolayer in the Transwell plate was used for simulated absorption, and the bottom permeate was collected and freeze-dried to obtain the stable enzymatic hydrolysate of bovine whey protein for gastrointestinal digestion and absorption.

On the other hand, the present invention proves at the cellular level that the peptides shown in any one of SEQ ID NOs: 1-5 can increase the uptake of insulin-resistant HepG2 hepatocytes and use glucose to synthesize glycogen, and can promote the secretion of GLP-1 by enteroendocrine cells NCI-H716, and the combined use of multiple peptides can further promote the secretion of GLP-1 and activate the GLP-1 receptor. According to an embodiment of the present invention, the peptide with DPP-IV inhibitory activity and GLP-1 receptor agonist activity has the above-mentioned amino acid sequence or its conservatively modified amino acid sequence.

On the other hand, the present invention has demonstrated at the in vivo level that the peptide segment shown in any one of SEQ ID NO: 1-3 can reduce the body weight and blood lipid level of mice fed a high-fat diet, activate the insulin signaling pathway to improve insulin resistance, increase the GLP-1 content in serum, reduce lipid accumulation in white adipose tissue, and regulate intestinal flora. According to an embodiment of the present invention, the peptide with DPP-IV inhibitory activity and GLP-1 receptor agonist activity has the above-mentioned amino acid sequence or its conservatively modified amino acid sequence.

On the other hand, the present invention provides the use of the above-mentioned peptide, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition, or the above-mentioned method in the preparation of drugs for preventing and/or treating diabetes, hyperlipidemia or obesity.

On the other hand, the present invention provides the use of the above-mentioned peptide, or the above-mentioned nucleic acid molecule, or the above-mentioned expression vector, or the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition, or the above-mentioned method in preparing a health care product or functional food that assists in improving insulin resistance or lowering blood lipid levels or losing weight.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention obtains whey protein hypoglycemic peptides with stable gastrointestinal digestion and absorption through enzymatic hydrolysis, simulated gastrointestinal digestion, and Caco-2 cell absorption. The whey protein peptide sequence is identified by LC-MS/MS, and the obtained multiple peptide segments have gastrointestinal digestion stability, DPP-IV inhibitory activity, and glucagon-like peptide-1 (GLP-1) receptor agonist activity.

2. The glucose-lowering peptide provided by the present invention has good effects in in vitro cell experiments. The peptide segment can increase the uptake of insulin-resistant HepG2 hepatocytes and utilize glucose to synthesize glycogen, and can promote the secretion of GLP-1 by enteroendocrine cells NCI-H716. The combined use of multiple peptide segments can further promote the secretion of GLP-1 and activate the GLP-1 receptor.

3. The glucose-lowering peptide provided by the present invention also has excellent effects in the construction of an insulin-resistant mouse model on a high-fat diet. The peptide segment has obvious effects on weight loss, reducing lipid accumulation and assisting in lowering blood lipids, and can also improve symptoms related to insulin resistance.

4. The milk-based glucose-lowering peptide provided by the present invention has gastrointestinal digestion stability and blood sugar-lowering activity, and can be used as a functional milk-based material for developing foods, medicines or health products with blood sugar-regulating effects, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the in vitro DPP-IV inhibition rate of the peptide fragments.

Figure 2 shows the effect of the peptides on glucose utilization in insulin-resistant HepG2 cells.

FIG3 shows the effect of the peptides on the relative glycogen content in insulin-resistant HepG2 cells.

FIG4 shows the effect of the peptides on GLP-1 secretion in NCI-H716 cells.

FIG5 shows the effect of the peptides on cAMP in NCI-H716 cells.

FIG6 shows the study of the synergistic effect of peptides on GLP-1 secretion in NCI-H716 cells.

Figure 7 shows the mass spectrum of the peptide LPMHIR.

FIG8 shows the mass spectrum of the peptide KFDK.

FIG9 shows the mass spectrum of the peptide IPAVFKID.

Figure 10 shows the effect of peptides on glucose tolerance in insulin-resistant mice induced by a high-fat diet. A is the blood glucose monitoring results at different times after oral gavage; B is the effect of different peptides on the area under the curve during the OGTT test.

Figure 11 shows the effect of peptides on insulin resistance in mice induced by a high-fat diet. A is the blood glucose monitoring results at different times after intraperitoneal injection of insulin; B is the effect of different peptides on the area under the blood glucose curve of mice.

FIG12 shows the effect of the peptide fragments on the DPP-IV enzyme activity in the serum of mice with insulin resistance induced by a high-fat diet.

FIG13 shows the effect of the peptide fragments on the serum GLP-1 content in mice with insulin resistance induced by a high-fat diet.

FIG. 14 shows the effect of the peptides on the expression of GLP-1R mRNA in the liver of high-fat diet-induced insulin-resistant mice.

FIG. 15 shows the effect of the peptides on body weight in mice fed a high-fat diet.

FIG. 16 shows the effect of the peptides on lipid accumulation in the liver of insulin-resistant mice induced by a high-fat diet.

Figure 17 shows the effect of peptides on the diversity of intestinal flora in insulin-resistant mice induced by a high-fat diet. A is the effect of different peptides on the Ace index of intestinal flora; B is the effect of different peptides on the Chao index of intestinal flora; C is the effect of different peptides on the Shannon index of intestinal flora.

FIG18 shows the effect of the peptides on the composition of intestinal flora in high-fat diet-induced insulin-resistant mice.

DETAILED DESCRIPTION

The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The present invention is described in detail below through embodiments in conjunction with the accompanying drawings, but the present invention is not limited thereto and is only described as an example.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

The quality inspection index data in the embodiments are all average values.

In the text, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or change the biological activity of the polypeptide comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the xanthine oxidase inhibitory peptides of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains.

Example 1. Preparation of hypoglycemic peptides that are stable to gastrointestinal digestion

In this example, a bovine whey protein hydrolysate having DPP-IV inhibitory activity was prepared by the following method:

(1) Enzymatic hydrolysis: bovine whey protein was dissolved in distilled water at a ratio of 10%, and the bovine whey protein was subjected to composite enzymatic hydrolysis at 37°C using alkaline protease and pepsin (alkaline protease: pepsin = 1:1) at a ratio of 5% (w/w) of enzyme to substrate; samples were taken after enzymatic hydrolysis for 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, and the enzymes were inactivated by heating at 85°C for 20 min; the pH value of the enzymatic hydrolyzate was adjusted to 7.0, and the composite enzymatic hydrolyzate (WPCH) was obtained by freeze-drying.

(2) Determine the degree of hydrolysis and DPP-IV inhibition rate of the hydrolysates obtained under different hydrolysis times. The study found that the bovine whey protein hydrolysates under different hydrolysis time conditions all have DPP-IV inhibition activity. As the hydrolysis time increases, the degree of hydrolysis increases and the DPP-IV inhibition rate gradually increases. When the hydrolysis time reaches 4h, there is no significant difference in the degree of hydrolysis and DPP-IV inhibition rate compared with the hydrolysate obtained after 5h of hydrolysis. On the other hand, the camel casein protein hydrolysate hydrolyzed for 4h has the greatest DPP-IV inhibition activity, which can be further experimented.

(3) Simulated gastrointestinal digestion: The composite enzymatic hydrolysate was dissolved in distilled water at a ratio of 5% and placed in a water bath at 85°C for 15 min. Preheat simulated gastric juice at 37°C and add the simulated gastric juice to the sample solution at a final ratio of 1:1 (vol/vol). Add 1M HCl to adjust the pH to 3.0. Add pepsin to a final activity of 2000U/mL in the digestion mixture. Keep the pH at 3.0 and stir in a water bath at 37°C for 2 h. Preheat simulated intestinal juice in a water bath at 37°C. Add simulated intestinal juice to the simulated gastric digestion product to a final ratio of 1:1 (vol/vol). Add 1M NaOH to adjust the pH to 7.0. Add bile salts to a final concentration of 10mM. Place the solution in a water bath at 37°C and stir for at least 30 min to completely dissolve the bile salts. Add CaCl ₂ (H ₂ O) ₂ solution to a final concentration of 0.6mM in the simulated intestinal juice. Pancreatin was added to make the pancreatin activity in the final mixture 100 U/mL. The pH value was maintained at 7.0 and stirred in a 37°C water bath for 2 h. The enzyme was inactivated in a boiling water bath for 5 min and freeze-dried to obtain bovine whey protein simulated gastrointestinal digest (GD-WPCH).

(4) Caco-2 cell absorption: The fully differentiated Caco-2 monolayer was washed twice with preheated Hank's balanced salt solution (HBSS) (pH 7.2). 1.5 mL HBSS was added to the upper chamber of the Transwell plate, and 2 mL HBSS was added to the bottom chamber, and the plates were equilibrated at 37°C for 30 min. Subsequently, the HBSS in the upper chamber was replaced with bovine whey protein simulated gastrointestinal digest (concentration of 25 mg/mL, dissolved in HBSS buffer). After incubation at 37°C for 2 h, the bottom permeate was collected and freeze-dried to obtain bovine whey protein gastrointestinal digestion and absorption stable enzymatic hydrolysate (CA-WPCH).

(5) Peptide identification: The stable enzymatic hydrolysate of bovine whey protein digested and absorbed by the gastrointestinal tract (CA-WPCH) was dissolved in 0.1% formic acid solution, and the high performance liquid chromatography system was connected to a mass spectrometer equipped with an electrospray ionization source. The first order spectrum detection scanning range was 50-1500 m/z, the scanning resolution was 60000, and the second order spectrum scanning range was 50-1400 m/z, the scanning resolution was 15000. The flowability A was 0.1% (v/v) formic acid, and the mobile phase B was 80% acetonitrile (containing 0.1% formic acid). During the elution process, the solvent B was increased from 8% to 50% (v/v) at a flow rate of 200 nL/min.

(6) Peptide screening: Molecular docking was performed with peptides and DPP-IV enzyme and GLP-1 receptor. The crystal structures of DPP-IV (PDB code: 4PNZ) and GLP-1 receptor (PDB code: 6X18) were obtained from the RCSB protein database; the peptide structures were generated in ChemDraw 20.0 and converted into stereostructures in Chem3D 20.0; the DPP-IV and GLP-1 receptors were freed of ligands and water molecules, hydrogenated, and their active sites were determined in Discovery Studio; the interaction between DPP-IV, GLP-1 receptor, and peptides was determined using the LibDock module.

Five polypeptides were screened out from the identified peptides (Table 1), namely LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), EVFR (SEQ ID NO: 4), and ILDKVGINY (SEQ ID NO: 5).

Table 1 Amino acid sequence obtained by identifying the bovine whey protein hydrolysate in Example 1

*α-La: bovine α-lactalbumin; β-Lg: bovine β-lactoglobulin.

The active peptides provided by the present invention can be derived from a composite enzymatic hydrolysate of bovine whey protein, or can be obtained by solid phase synthesis. The peptide segments LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), EVFR (SEQ ID NO: 4), and ILDKVGINY (SEQ ID NO: 5) used in the following examples are obtained by solid phase synthesis. Shanghai Nuoyou Biotechnology Co., Ltd. was commissioned to solid phase synthesize the above-mentioned hypoglycemic peptide segments for subsequent experiments.

Example 2. Determination of the DPP-IV inhibitory activity of peptide fragments

100 μL of Gly-Pro-PNA HCl solution (1 mM), 50 μL of Tris-HCl buffer (100 mM, pH 8.0), and 20 μL of sample (10 mg/mL) were added to a 96-well plate, incubated at 37°C for 10 min, and 30 μL of DPP-Ⅳ Tris-HCl solution (0.02 μg/μL) was added for 30 min enzyme reaction. The absorbance value at 405 nm was measured, and each sample was analyzed three times. The DPP-Ⅳ inhibition rate was calculated according to the formula. The sample control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of DPP-Ⅳ, the negative control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of the sample, and the blank control group used the same volume of Tris-HCl buffer (100 mM, pH 8.0) instead of the sample and DPP-Ⅳ. The DPP-Ⅳ inhibition rate was calculated using the following formula.

DPP-IV, also known as CD26, is a transmembrane serine protease and a member of the prolyl oligopeptidase family. It can specifically cleave the N-terminal dipeptide residue of GLP-1: AA-Pro or AA-Ala (AA is any amino acid), and is one of the key enzymes that promote the degradation and inactivation of GLP-1 in vivo and in vitro. DPP-IV is widely distributed in the body, not only in plasma, but also in tissues and organs such as kidneys, small intestines, bile ducts and pancreatic epithelial cells, and vascular endothelial cells. Therefore, selective inhibition of DPP-4 can increase the concentration of GLP-1 in the body and prolong its duration of action. It can also inhibit the production of glucagon and prolong the duration of insulin secretion stimulated by GLP-1. Therefore, the DPP-IV enzyme plays a vital role in glucose and insulin metabolism. The DPP-IV inhibition rate of the peptides is shown in Figure 1 and Table 2. The positive control group used sitagliptin, and the peptides LPMHIR (LR), KFDK (KK), and IPAVFKID (ID) all showed a large DPP-IV inhibitory activity (>70%). Among them, at the same concentration, the peptide KFDK showed the greatest DPP-Ⅳ inhibition rate, up to 78.28±4.29%. In addition, the in vitro DPP-Ⅳ inhibition rate of the peptides EVFR (ER) and ILDKVGINY (IY) was <50%.

Table 2 DPP-Ⅳ inhibition rate of peptides

Half maximal inhibitory concentration (IC ₅₀ ) refers to the concentration at which a substance has an inhibitory effect of 50% on another substance, such as an enzyme, and is used to measure sensitivity. There are many studies on milk-derived DPP-Ⅳ inhibitory peptides. The IC ₅₀ of the peptide segment VLGP derived from bovine β-casein is 580.0 μM, the IC ₅₀ of the peptide segment VLVLDTDYK derived from bovine β-lactoglobulin is 424.0 μM, and the IC ₅₀ of the peptide segment WLAHKAL derived from bovine α-lactalbumin is 286.0 μM. The smaller the IC ₅₀ value, the stronger the inhibitory effect of the peptide segment on DPP-Ⅳ. The results of the determination of the IC ₅₀ value of the glucose-lowering peptide segment in this example are shown in Table 3.

Table 3 Half inhibitory concentration of glucose-lowering peptide segments

Example 3. Effect of peptides on improving glucose utilization in insulin-resistant HepG2 cells

By constructing insulin-resistant HepG2 cells, the effects of the peptides on glucose utilization and glycogen synthesis in insulin-resistant liver cells were evaluated.

Construction of HepG2 cell model of insulin resistance: HepG2 hepatocytes were inoculated in 96-well plates and adhered for 24 hours. A 600mM glucose PBS solution was prepared, filtered through a 0.22μm aqueous filter, and 10μL was aspirated to make the glucose concentration in the well 30mM. 10μL of PBS was added to the blank control group. The cells were cultured in a 37°C, 5% _CO2 incubator for 24 hours, and the glucose consumption rate and relative glycogen content were measured. The results are shown in Figure 2. The glucose consumption rate and relative glycogen content of HepG2 cells stimulated by high sugar were significantly lower than those of normal HepG2 cells, and the HepG2 cell model of insulin resistance was successfully constructed.

Effect on glucose consumption rate and relative glycogen content: HepG2 hepatocytes were inoculated in 96-well plates. After 24 hours of attachment, 10 μL of glucose (final concentration of 30 mM) was added, and 10 μL of PBS solution of glucose-lowering peptide with a final concentration of 100 μM was added at the same time. The blank wells were replaced with the same volume of PBS instead of glucose solution and sample solution, and the standard wells were replaced with the same volume of PBS instead of sample solution. After 24 hours of culture, the culture medium was aspirated and the glucose oxidase detection kit (Nanjing Jiancheng Bioengineering Institute, A154-1-1) was used. The cells were inoculated into a culture dish with a diameter of 6 cm. After 12 hours of attachment, 30 mM glucose was added for stimulation for 24 hours. The cells were digested with trypsin to make a cell suspension, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded to keep the cell pellet. 1 mL of PBS buffer was added to the cell pellet, gently mixed, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded to keep the cell pellet. 0.2 mL of PBS buffer was added, ultrasonically disrupted under ice water bath conditions, and directly measured without centrifugation. The glycogen content of cells was measured using a glycogen assay kit.

The glucose consumption rate and relative intracellular glycogen content of each group are shown in Figures 2 and 3. Compared with the control group, the intervention of peptides LPMHIR (LR), KFDK (KK), and IPAVFKID (ID) significantly increased the glucose consumption rate and intracellular glycogen content of insulin-resistant HepG2 cells, indicating that the peptides improved the glucose uptake and utilization ability of insulin-resistant liver cells, and to a certain extent played a role in lowering blood sugar and improving insulin resistance. The peptides EVFR (ER) and ILDKVGINY (IY) had no obvious effect on improving the glucose uptake and utilization ability of insulin-resistant liver cells.

Example 4. Effect of peptide fragments on GLP-1 secretion by enteroendocrine cells

Using enteroendocrine cells NCI-H716 cells as a model, the effects of peptides on promoting the secretion of GLP-1 by enteroendocrine cells and whether there is a synergistic effect between peptides in promoting the secretion of GLP-1 were studied.

Stably growing NCI-H716 cells were inoculated at a density of 1.5×10 ⁶ in a 12-well culture plate coated with matrix gel and cultured for 48 hours. NCI-H716 cells were incubated with five peptides for 24 hours, and sitagliptin was used as the positive control group. The GLP-1 content in the cell supernatant was detected using an enzyme-linked immunosorbent assay kit. NCI-H716 is a human colorectal adenocarcinoma cell that can secrete hormones such as GLP-1. It is used to study the effect of active peptide intervention on the secretion of GLP-1 by intestinal epithelial cells. As shown in Figure 4, with the control group as the benchmark, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) all promoted the secretion of GLP-1 by NCI-H716, indicating that hypoglycemic peptides can promote the secretion of GLP-1 at the cellular level, and exert the effects of lowering blood sugar and improving insulin resistance through GLP-1. The synthetically secreted GLP-1 exerts a beneficial effect by binding to the GLP-1 receptor on the cell surface. After binding to the GLP-1 receptor, the GLP-1 receptor agonist increases calcium influx and endoplasmic reticulum calcium release through the cAMP/PKA pathway to activate calmodulin and promote insulin release. In order to further determine whether the peptide has the activity as a GLP-1 receptor agonist, the cAMP level in the culture supernatant was measured. As shown in Figure 5, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), and EVFR (SEQ ID NO: 4) all increased the cAMP level to a certain extent, indicating that the glucose-lowering peptide can activate the GLP-1 receptor by increasing the cAMP level.

Combined with Examples 2 and 3, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQID NO: 3) have good GLP-1 receptor agonist activity. On this basis, it was further studied whether there was a synergistic effect between the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) in promoting GLP-1 secretion. As shown in Figure 6, when the combined intervention of the peptides was given at the same time, the GLP-1 secretion level in the cell culture medium was higher than that of the single intervention. The GLP-1 secretion effect of the combination of peptides LPMHIR+KFDK and the combination of peptides KFDK+IPAVFKID was higher than the sum of the effects of the single interventions, indicating that KFDK has a synergistic effect with LPMHIR and IPAVFKID, respectively, and the combined use can improve the effects of both in promoting GLP-1 secretion and activating the GLP-1 receptor. Peptides LPMHI and IPAVFKID do not have obvious synergistic effects. The above results indicate that the combined use of KFDK and LPMHIR or KFDK and IPAVFKID can further exert the role of glucose-lowering peptides in promoting GLP-1 secretion and activating GLP-1 receptors.

Example 5. Effect of peptide fragments on improving insulin resistance in mice fed a high-fat diet

According to Examples 2, 3, and 4, the peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) have good hypoglycemic effects and can simultaneously play the dual roles of DPP-IV inhibitor and GLP-1 agonist in vitro. Therefore, whey protein-derived hypoglycemic peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) were used for animal experiments to study their hypoglycemic effects in vivo. The mass spectra of the three peptides are shown in Figures 7-9.

Male C57/6J mice aged 3-4 weeks were selected and purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd. They were raised in accordance with the SPF animal husbandry standards. The animal experiments were reviewed and approved by the Animal Experiment Committee of China Agricultural University. The mice were free to eat and drink water and adapted for one week in an environment with a 22±1℃ and 12:12 day and night cycle. After 1 week of adaptation, the mice were randomly divided into a normal diet (Normal diet, ND) group (n=6) and a high-fat diet (High-fat diet, HFD) group (n=30). The mice in the ND group were fed with ordinary feed, and the mice in the HFD group were fed with a high-fat diet (energy supply ratio 20% protein, 20% carbohydrates, 60% fat) for 10 weeks to construct high-fat diet-induced insulin resistance mice. After 10 weeks, fasting blood glucose and fasting insulin were measured, and the insulin resistance index (HOMA-IR) was calculated. The insulin resistance index of mice in the HFD group was greater than 2.69, indicating that the high-fat diet-induced insulin resistance model was successfully constructed. The high-fat diet mice were randomly divided into HFD group, LPMHIR group (LR), KFDK group (KK group), IPAVFKID (ID group), and sitagliptin group (Sitagliptin group), with 6 mice in each group. The corresponding intervention substances were given for ten weeks, during which the food intake and body weight of the mice were monitored every week.

After nine weeks of intervention, the mice were fasted but not watered for 8 h, and were gavaged with glucose at a dose of 2 g/kg BW. Blood was collected from the tail tip of the mice at 0, 30, 60, 90 and 120 min after gavage. Blood glucose levels were measured with a blood glucose meter, and curves were drawn to calculate the area under the curve (Area under cure, AUC).

As shown in Figure 10, the blood sugar of mice rose after oral administration of glucose, then fell, and basically flattened after 90 minutes. Compared with the ND group, the blood sugar of mice in the HFD group was always maintained at a higher level. The area under the curve (AUC) during the OGTT test was 1.39 times that of the ND group, which was significantly higher than that of the control group, indicating that under the induction of a high-fat diet, the glucose tolerance of mice was impaired and the ability to regulate blood sugar decreased. The intervention of hypoglycemic peptides and sitagliptin intervention improved the ability of HFD mice to regulate blood sugar, and had a good improvement effect on the impairment of glucose tolerance induced by a high-fat diet. Sitagliptin intervention reduced the AUC of HFD mice by 27.2%. The three hypoglycemic peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQID NO: 2), and IPAVFKID (SEQ ID NO: 3) reduced the AUC of HFD mice by 18.8%, 23.0%, and 21.3%, respectively. The above results indicate that glucose-lowering peptides can improve impaired glucose tolerance induced by a high-fat diet and enhance the blood glucose regulation ability of mice fed a high-fat diet.

The mice were fasted but not watered for 6 h, and insulin was injected intraperitoneally at a dose of 0.75 U/kg BW. Blood was collected from the tail tip of the mice at 0, 30, 60, 90 and 120 min after injection. Blood glucose levels were measured with a blood glucose meter, and curves were drawn to calculate the area under the curve (Area under cure, AUC).

As shown in Figure 11, compared with the ND group, the fasting blood glucose level of mice in the HFD group was significantly increased, and the blood glucose decreased more slowly after insulin injection, with a lower decrease. The area under the curve (AUC) was 1.63 times that of the ND group, which was significantly higher than that of the control group, indicating that high-fat diet feeding caused the mice to have increased fasting blood glucose and impaired insulin tolerance. The intervention of hypoglycemic peptides and sitagliptin increased the rate of blood glucose decrease in HFD mice, while the decrease in blood glucose increased, and the area under the curve was significantly reduced. Sitagliptin intervention reduced the AUC of HFD mice by 42.3%. The three hypoglycemic peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) improved the impaired glucose tolerance induced by high-fat diet, and reduced the AUC of HFD mice by 26.1%, 33.3%, and 30.8%, respectively, indicating that the hypoglycemic peptides improved the insulin tolerance of HFD mice and reduced the degree of insulin resistance.

After ten weeks of intervention, the mice were killed, the serum was collected, and the DPP-IV enzyme activity in the serum was measured to explore the effect of the application example of the present invention on the DPP-IV enzyme activity in the serum of mice with insulin resistance induced by a high-fat diet. As shown in Figure 12, compared with the ND group, the DPP-IV enzyme activity in the serum of HFD mice increased significantly. The intervention of different glucose-lowering peptides reduced the DPP-IV activity in the serum of HFD mice, indicating that the glucose-lowering peptide intervention can inhibit the DPP-IV activity in mice fed a high-fat diet, thereby inhibiting the degradation and inactivation of GLP-1.

The activity of GLP-1 in serum was determined to explore the effect of the application example of the present invention on the GLP-1 content in the serum of mice with insulin resistance induced by a high-fat diet. GLP-1 can not only stimulate the secretion of insulin, but also has a protective effect on pancreatic β cells and the cardiovascular system, inhibits the apoptosis of β cells, promotes β cell growth and proliferation, upregulates insulin secretion, lowers blood sugar, and is related to reducing food intake, promoting gastric emptying and weight loss, and plays an important role in alleviating diabetes. As shown in Figure 13, compared with the ND group, the GLP-1 content in the serum of HFD mice was significantly reduced, indicating that in mice on a high-fat diet, GLP-1 has a lower effect on improving insulin resistance. The intervention of different glucose-lowering peptides significantly increased the GLP-1 content in the serum of mice fed a high-fat diet. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) increased the GLP-1 content in the serum of HFD mice by 46.4%, 57.8%, and 61.8%, respectively, indicating that the glucose-lowering peptides in this application example are beneficial to increase the GLP-1 content in serum and play the role of GLP-1 in improving insulin resistance. On the other hand, GLP-1 exerts its beneficial effects by binding to GLP-1 receptors on the surface of different tissue cells. In order to study the effect of glucose-lowering peptide segments on the expression of GLP-1R in the liver of insulin-resistant mice induced by a high-fat diet, the expression of GLP-1R in the liver of mice was detected by PCR, as shown in Figure 14. Compared with the ND group, the HFD diet significantly reduced the expression of GLP-1R mRNA, indicating that the high-fat diet reduced the expression of GLP-1R while inducing a decrease in serum GLP-1 content, which is not conducive to the binding of GLP-1 to tissue cell receptors to improve blood sugar. Intervention with the glucose-lowering peptide segments LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) can increase the expression of GLP-1R mRNA in the liver tissue of HFD mice and activate the GLP-1 receptor.

The weight changes of mice were monitored during the intervention period to explore the improvement effect of the application example of the present invention on high-fat diet-induced obesity. As shown in Figure 15, compared with the ND group, the weight of mice in the HFD group increased significantly, reaching 1.52 times that of the ND group. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) can significantly reduce obesity induced by a high-fat diet and reduce the weight of mice.

Mouse epididymal fat was taken for H&E staining to explore the effect of the application example of the present invention on lipid accumulation induced by a high-fat diet. As shown in Figure 16, compared with the ND group, the cell diameter of epididymal fat in HFD mice increased and the fat index increased, indicating that the high-fat diet induced lipid accumulation in mice and enlarged white adipocytes. The intervention of LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) significantly reduced the diameter of white fat, indicating that the peptide segment has an improvement effect on reducing lipid accumulation.

Total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in mouse serum were measured to characterize the blood lipid level of mice. As shown in Table 4, high-fat diet intervention significantly increased TC, TG, and LDL-c in mice, causing hyperlipidemia. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) intervention reduced the levels of TC, TG, and LDL-c in the serum of HFD mice, and increased the level of HDL-c, indicating that peptide intervention significantly improved the blood lipid level of mice on a high-fat diet, improved hyperlipidemia, and had the effect of assisting in lowering blood lipids.

Table 4 Effects of glucose-lowering peptide intervention on blood lipid levels in HFD mice

The fasting insulin and glycated hemoglobin of mice were measured to explore the effect of the application examples of the present invention on the indicators in the serum of mice with insulin resistance induced by a high-fat diet. Glycated hemoglobin (HbA1c) is the product of the combination of hemoglobin in red blood cells and glucose in the blood. It can directly and effectively reflect the patient's blood sugar level in the past 3-6 months, and is positively correlated with the level of insulin resistance, directly correlated with cholesterol, triglycerides and low-density lipoprotein cholesterol, and negatively correlated with high-density lipoprotein cholesterol. As shown in Table 5, compared with the ND group, the glycated hemoglobin level of HFD mice was significantly increased, indicating that the high-fat diet induced diabetes and insulin resistance in mice. Giving LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) intervention significantly reduced the glycated hemoglobin level of mice on a high-fat diet.

Table 5 Effects of hypoglycemic peptide intervention on serum parameters of HFD mice

Alpha diversity analysis reflects the diversity and variability of microbial communities. The most commonly used indexes include the Ace index, Chao index, and Shannon index. The intestinal flora of mice was measured to explore the effect of the application example of the present invention on the diversity of intestinal flora in mice with insulin resistance induced by a high-fat diet. As shown in Figure 17, compared with the ND group, the Ace index and Chao index of HFD mice were significantly decreased, indicating that HFD significantly reduced the richness of intestinal microorganisms in mice. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) peptide intervention increased the diversity of intestinal flora in mice fed a high-fat diet and restored it to a certain extent.

Intestinal microorganisms are considered to be factors related to fat storage, weight gain, and insulin resistance. When weight gain is in an obese state, the abundance of Bacteroidetes decreases and the abundance of Firmicutes increases. As shown in Figure 18, compared with the ND group, the ratio of the relative abundance of Bacteroidetes/Firmicutes in the intestinal flora of mice in the HFD group decreased significantly, indicating that a high-fat diet can cause an imbalance in the intestinal flora of mice at the phylum level. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), and IPAVFKID (SEQ ID NO: 3) intervention significantly increased the ratio of Bacteroidetes/Firmicutes, indicating that the three peptides regulate the intestinal flora composition of HFD mice at the phylum level, which is beneficial to the role of intestinal flora in improving obesity, insulin resistance and other related symptoms.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (3)

1. The application of the peptide shown as SEQ ID NO. 2 in preparing a medicament for preventing and/or treating diabetes.

2. The application of the peptide shown as SEQ ID NO. 2 in preparing medicines for preventing and/or treating hyperlipidemia.

3. The application of the peptide shown as SEQ ID NO. 2 in preparing a medicament for preventing and/or treating obesity.