Disclosure of Invention
The invention aims to provide a preparation method and application of whey protein source hypoglycemic peptide with GLP-1 receptor agonist activity.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In one aspect, the invention provides a bovine whey protein source hypoglycemic peptide with stable gastrointestinal digestion and absorption, which is characterized in that the amino acid sequence of the peptide is shown in any one of SEQ ID NO. 1-5. The obtained peptide has the advantages of gastrointestinal digestion and absorption stability, DPP-IV inhibition activity and GLP-1 receptor agonist activity in vivo, and can improve insulin resistance and lipid accumulation induced by high-fat diet and reduce weight. The hypoglycemic peptide fragment can be used for reducing DPP-IV activity in a sample, improving GLP-1 content, activating GLP-1 receptor, and the auxiliary hypoglycemic and hypolipidemic product prepared by using the peptide fragment can be used for preventing, protecting health and auxiliary treating diseases related to hyperglycemia, hyperlipidemia, obesity and the like, and has wide application prospect in the fields of health care and medical biology.
In another aspect, the invention provides a nucleic acid molecule which encodes a peptide as set forth in any one of SEQ ID NOs 1 to 5, wherein the nucleic acid molecule is DNA or RNA.
In another aspect, the invention provides an expression vector characterized in that the nucleotide sequence of the expression vector is a nucleic acid molecule as described above.
In another aspect, the present invention provides a recombinant cell, characterized in that the recombinant cell is obtained by introducing the above-described expression vector into a host cell.
In another aspect, the invention provides a pharmaceutical composition comprising a peptide as set forth in any one of SEQ ID NOs 1 to 5, or a nucleic acid molecule as set forth above, or an expression vector as set forth above, or a recombinant cell as set forth above.
In another aspect, the present invention provides a method for preparing whey protein hydrolysate having hypoglycemic activity, characterized in that the method comprises the steps of:
s1, carrying out compound enzymolysis on bovine whey protein by using alkaline protease and pepsin at a certain temperature to obtain bovine whey protein zymolyte;
s2, performing simulated gastric digestion and simulated intestinal digestion by using bovine whey protein zymolyte;
S3, performing simulated absorption by using a fully differentiated human cloned colon adenocarcinoma cell Caco-2 single cell layer in a Transwell plate, collecting a bottom layer permeate, and freeze-drying to obtain a stable zymolyte for gastrointestinal digestion and absorption of bovine whey protein.
On the other hand, the invention proves that the peptide segment shown in any one of SEQ ID NO 1-5 can improve the uptake of HepG2 liver cells with insulin resistance and synthesize glycogen by utilizing glucose, can promote the NCI-H716 of enteroendocrine cells to secrete GLP-1, and the combined use of a plurality of peptide segments can further play roles in promoting the secretion of GLP-1 and activating GLP-1 receptors. According to an embodiment of the invention, the peptide having DPP-IV inhibitory activity and GLP-1 receptor agonist activity has the amino acid sequence described above or an amino acid sequence of a conservatively modified form thereof.
On the other hand, the in vivo level of the invention proves that the peptide fragment shown in any one of SEQ ID NO. 1-3 can reduce the weight and blood lipid level of a high-fat diet mouse, activate an insulin signal path to improve the insulin resistance level, improve the GLP-1 content in serum, reduce the lipid accumulation of white adipose tissue and regulate intestinal flora. According to an embodiment of the invention, the peptide having DPP-IV inhibitory activity and GLP-1 receptor agonist activity has the amino acid sequence described above or an amino acid sequence of a conservatively modified form thereof.
In another aspect, the present invention provides the use of the above peptide, or the above nucleic acid molecule, or the above expression vector, or the above recombinant cell, or the above pharmaceutical composition, or the above method, for the preparation of a medicament for the prevention and/or treatment of diabetes or hyperlipidemia or obesity.
In another aspect, the present invention provides the use of the above peptide, or the above nucleic acid molecule, or the above expression vector, or the above recombinant cell, or the above pharmaceutical composition, or the above method, for the preparation of a health product or functional food for aiding in improving insulin resistance or reducing blood lipid levels or weight loss.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention obtains the whey protein hypoglycemic peptide with stable gastrointestinal digestion and absorption through enzymolysis, simulated gastrointestinal digestion and Caco-2 cell absorption, and utilizes LC-MS/MS to identify the whey protein peptide sequence, and the obtained peptide fragments have gastrointestinal digestion stability, DPP-IV inhibitory activity and glucagon-like peptide-1 (GLP-1) receptor agonist activity.
2. The hypoglycemic peptide provided by the invention has good effect in vitro cell experiments, peptide fragments can improve the uptake of HepG2 liver cells with insulin resistance and synthesize glycogen by utilizing glucose, can promote enteroendocrine cells NCI-H716 to secrete GLP-1, and can further play the roles of promoting GLP-1 secretion and activating GLP-1 receptors by combining a plurality of peptide fragments.
3. The hypoglycemic peptide provided by the invention has excellent effects in constructing an insulin resistance mouse model by high-fat diet, and the peptide segment has obvious effects of losing weight, reducing lipid accumulation and assisting in reducing blood fat, and can improve insulin resistance related symptoms.
4. The dairy-based hypoglycemic peptide provided by the invention has gastrointestinal digestion stability and hypoglycemic activity, can be used as a functional dairy base material for developing foods, medicines or health-care products with the function of regulating blood sugar, and has wide application prospect.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will now be described in more detail by way of examples with reference to the accompanying drawings, which are not intended to limit the invention thereto, but are illustrative only.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
The quality detection index data in the examples are all average values.
In the present context, the term "conservatively modified form of an amino acid sequence" refers to an amino acid modification which does not significantly affect or alter the biological activity of a 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 invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
EXAMPLE 1 preparation of a Pestically stable hypoglycemic peptide
In this example, bovine whey protein hydrolysate having DPP-IV inhibitory activity was prepared by the following method:
(1) The enzymolysis comprises dissolving bovine whey protein in distilled water at a ratio of 10%, performing compound enzymolysis on bovine whey protein by alkaline protease and pepsin (alkaline protease: pepsin=1:1) at 37deg.C according to an enzyme-substrate ratio of 5% (w/w), performing enzymolysis for 0h,0.25h,0.5h,1h,2h,3h,4h,5h, sampling, heating at 85deg.C for 20min to inactivate enzyme, adjusting pH value of enzymolysis solution to 7.0, and freeze drying to obtain compound zymolyte (WPCH).
(2) And measuring the hydrolysis degree and DPP-IV inhibition rate of the obtained zymolyte under different hydrolysis times. The research shows that the bovine whey protein zymolyte under different hydrolysis time conditions has DPP-IV inhibition activity, and the DPP-IV inhibition rate is gradually increased while the hydrolysis degree is increased along with the extension of the hydrolysis time. When the hydrolysis time reaches 4 hours, the hydrolysis degree and the DPP-IV inhibition rate have no obvious difference with the enzymolysis product obtained in the hydrolysis time of 5 hours. In another aspect, the camel milk casein hydrolysate which has been enzymatically digested for 4 hours has the greatest DPP-IV inhibitory activity and further experiments can be performed.
(3) And simulating gastrointestinal digestion, namely dissolving the composite zymolyte in distilled water according to the proportion of 5%, and carrying out water bath for 15min at the temperature of 85 ℃. The simulated gastric fluid was preheated at 37 ℃ and added to the sample solution in a final ratio of 1:1 (vol/vol). The pH was adjusted to 3.0 by the addition of 1M HCl. Pepsin was added to bring its activity in the final digestion mixture to 2000U/mL. The pH was maintained at 3.0 and stirred in a 37℃water bath for 2h. The simulated intestinal fluid was preheated in a 37 ℃ water bath. Simulated intestinal fluid was added to the simulated gastric digest product to a final 1:1 ratio (vol/vol). The pH was adjusted to 7.0 by the addition of 1M NaOH. Bile salts were added to give a final concentration of 10mM. The solution was placed in a 37 ℃ water bath and stirred for at least 30min to completely dissolve the bile salts. CaCl2(H2O)2 solution was added to give a final concentration of 0.6mM in simulated intestinal fluid. Pancreatin was added to give a final mixture of pancreatin activity of 100U/mL. The pH was maintained at 7.0 and stirred for 2h in a 37℃water bath. Inactivating enzyme in boiling water bath for 5min, and lyophilizing to obtain bovine whey protein analog gastrointestinal digest (GD-WPCH).
(4) Caco-2 cell uptake fully differentiated Caco-2 single cell layers were washed 2 times with pre-warmed Hank's Balanced Salt Solution (HBSS) (pH 7.2). 1.5mL of HBSS was added to the upper chamber of the Transwell plate, 2mL of HBSS was added to the bottom layer, and the mixture was equilibrated at 37℃for 30min. Subsequently, the HBSS in the upper chamber was replaced with bovine whey protein-mimicking gastrointestinal digests (25 mg/mL concentration in HBSS buffer. After incubation for 2h at 37 ℃, the bottom permeate was collected and lyophilized to obtain stable enzymatic hydrolysate (CA-WPCH) for gastrointestinal digestion and absorption of bovine whey protein.
(5) And (3) peptide segment identification, namely, niu Ruqing protein gastrointestinal digestion and absorption stable zymolyte (CA-WPCH) is dissolved in 0.1 percent formic acid solution, a high performance liquid chromatography system is connected with a mass spectrometer provided with an electrospray ionization source, the first-order spectrum detection scanning range is 50-1500 m/z, the scanning resolution is 60000, the secondary spectrum scanning range is 50-1400 m/z, and the scanning resolution is 15000. Fluidity A was 0.1% (v/v) formic acid, and mobile phase B was 80% acetonitrile (containing 0.1% formic acid). During elution, solvent B increased from 8% to 50% (v/v) at a flow rate of 200 nL/min.
(6) Peptide fragment screening, namely molecular docking is carried out on peptide fragments and DPP-IV enzyme and GLP-1 receptor. The crystal structures of DPP-IV (PDB code: 4 PNZ) and GLP-1 receptor (PDB code: 6X 18) were obtained from the RCSB protein database, the peptide fragment structure was generated in chemDraw 20.0 and converted to a steric structure in Chem3D 20.0, the DPP-IV and GLP-1 receptor and ligand and water molecules were removed in the Discovery Studio, hydrogenated and their active sites were determined, and the interactions between DPP-IV, GLP-1 receptor and peptide fragment were determined using LibDock module.
Five polypeptides were selected from the identified peptide fragments (Table 1), each of LPMHIR(SEQ ID NO:1)、KFDK(SEQ ID NO:2)、IPAVFKID(SEQ ID NO:3)、EVFR(SEQ ID NO:4)、ILDKVGINY(SEQ ID NO:5).
TABLE1 amino acid sequences identified by bovine whey protein enzymatic hydrolysate in example 1
* Alpha-La, bovine alpha-lactalbumin, beta-Lg, bovine beta-lactoglobulin, beta-lactoglobulin.
The active peptide provided by the invention can be derived from bovine whey protein composite zymolyte, and can also be obtained by solid phase synthesis. The peptide LPMHIR(SEQ ID NO:1)、KFDK(SEQ ID NO:2)、IPAVFKID(SEQ ID NO:3)、EVFR(SEQ ID NO:4)、ILDKVGINY(SEQ ID NO:5) used in the examples below was obtained by solid phase synthesis. The above-mentioned hypoglycemic peptide fragment was solid-phase synthesized by Shanghai North Biotechnology Co., ltd, and the subsequent experiments were carried out.
EXAMPLE 2 determination of the DPP-IV inhibitory Activity of peptide fragments
To a 96-well plate, 100. Mu.L of Gly-Pro-PNA HCl solution (1 mM), 50. Mu.L of LTris-HCl buffer (100 mM, pH 8.0), 20. Mu.L of sample (10 mg/mL), incubation at 37℃for 10min, 30. Mu.L of DPP-IV Tris-HCl solution (0.02. Mu.g/. Mu.L) were added for enzyme reaction for 30min, absorbance at 405nm was measured, 3 replicates were performed for each sample, and DPP-IV inhibition was calculated according to the formula. The sample control group replaced DPP-IV with the same volume of Tris-HCl buffer (100 mM, pH 8.0), the negative control group replaced the sample with the same volume of Tris-HCl buffer (100 mM, pH 8.0), and the blank control group replaced the sample and DPP-IV with the same volume of Tris-HCl buffer (100 mM, pH 8.0). DPP-IV inhibition is calculated using the following formula.
DPP-IV is also called CD26, is a transmembrane serine protease, belongs to one member of prolyl oligopeptidase family, can specifically cleave N-terminal dipeptide residue of GLP-1, namely AA-Pro or AA-ala (AA is any amino acid), and is one of key enzymes for promoting degradation and inactivation of GLP-1 in vitro and in vivo. DPP-IV is widely distributed in vivo, and is present not only in plasma but also in tissue organs such as epithelial cells of kidney, small intestine, bile duct and pancreas, endothelial cells of blood vessels, and the like. Therefore, the selective inhibition of DPP-4 can improve the concentration of GLP-1 in vivo, prolong the action time of the GLP-1, inhibit the generation of glucagon and prolong the duration of insulin secretion stimulated by the GLP-1. Thus, DPP-IV enzyme plays a vital role in glucose and insulin metabolism. The DPP-IV inhibition rate of the peptide fragment is shown in fig. 1 and table 2, and the peptide fragments LPMHIR (LR), KFK (KK) and IPAVFKID (ID) all show larger DPP-IV inhibition activity (> 70%) by using sitagliptin drug (sitagliptin) as a positive control group. Wherein, at the same concentration, the peptide KFDK shows the maximum DPP-IV inhibition rate which is as high as 78.28+/-4.29 percent. Furthermore, the in vitro DPP-IV inhibition of peptide fragment EVFR (ER), ILDKVGINY (IY) was <50%.
TABLE 2 DPP-IV inhibition by peptide fragment
Semi-inhibitory concentration (half maximal inhibitory concentration, IC50) refers to the concentration at which one substance inhibits another substance, such as an enzyme, by 50% to measure sensitivity. Much research on milk-derived DPP-IV inhibitory peptides, IC50 =580.0 μm of peptide VLGP derived from bovine beta-casein, IC50 = 424.0 μm derived from bovine beta-lactoglobulin VLVLDTDYK, and smaller IC50＝286.0μM.IC50 values of peptide WLAHKAL derived from bovine alpha-lactalbumin, indicate stronger inhibition of DPP-IV by peptide. The results of the measurement of the IC50 value of the hypoglycemic peptide fragment in this example are shown in Table 3.
TABLE 3 semi-inhibitory concentration of hypoglycemic peptide fragment
Example 3 improving Effect of peptide fragments on glucose utilization in insulin resistant HepG2 cells
The effect of peptide fragments on glucose utilization and glycogen synthesis by insulin resistant liver cells was evaluated by constructing insulin resistant HepG2 cells.
Construction of an insulin-resistant HepG2 cell model, namely inoculating HepG2 liver cells into a 96-well plate and attaching for 24 hours. A600 mM glucose PBS solution was prepared, and 10. Mu.L of the solution was aspirated through a 0.22 μm aqueous filter to give a 30mM glucose concentration in the wells, and 10. Mu.L of PBS was added to the blank. Culturing in a 37 ℃ and 5% CO2 incubator for 24 hours, and measuring the glucose consumption rate and the relative glycogen content. As shown in FIG. 2, the glucose consumption rate and the intracellular relative glycogen content of the high-sugar-stimulated HepG2 cells were significantly lower than those of the normal HepG2 cells, and the insulin-resistant HepG2 cell model was successfully constructed.
Effect on glucose consumption rate and relative glycogen content HepG2 hepatocytes were seeded in 96-well plates, after 24h of adherence, 10 μl of glucose (30 mM final concentration) was added, together with 10 μl of 100 μm final concentration of glycopeptide PBS solution, the blank wells replaced glucose solution and sample solution with the same volume of PBS, and the standard wells replaced sample solution with the same volume of PBS. After 24h of culture, the culture medium was aspirated and a glucose oxidase test kit (Nanjing institute of bioengineering, A154-1-1) was used. Cells were inoculated into 6cm diameter dishes and after 12h of adherence, 30mM glucose was added to stimulate for 24h. Cells were digested with pancreatin to form a cell suspension, centrifuged at 1000rpm for 10min, and the supernatant was discarded to leave a cell pellet. 1mL of PBS buffer was added to the cell pellet, gently mixed, centrifuged at 1000rpm for 10min, and the supernatant was discarded to leave the cell pellet. 0.2mL of PBS buffer solution is added, the mixture is crushed by ultrasonic waves under the ice-water bath condition, and the mixture is directly measured without centrifugation. And determining the glycogen content of the cells by using a glycogen kit.
As shown in fig. 2 and 3, the intervention of peptide fragments LPMHIR (LR), KFDK (KK), ipafkid (ID) significantly increased the glucose consumption rate and intracellular glycogen content of insulin resistant HepG2 cells compared to the control group, indicating that the peptide fragments increased the glucose uptake utilization capacity of insulin resistant liver cells, and had the effects of reducing blood glucose and improving insulin resistance to some extent. The peptide EVFR (ER) and ILDKVGINY (IY) have no obvious effect on improving the glucose uptake utilization capacity of insulin resistant liver cells.
Example 4 Effect of peptide fragments on secretion of GLP-1 by enteroendocrine cells
Taking an NCI-H716 cell of the enteroendocrine cell as a model, researching the promotion effect of peptide fragments on the secretion of GLP-1 by the enteroendocrine cell and whether the synergistic effect of promoting the secretion of GLP-1 exists between the peptide fragments.
The NCI-H716 cells with stable growth are inoculated into a 12-hole culture plate coated with matrigel according to the density of 1.5X106 for culturing for 48 hours, the NCI-H716 cells are respectively incubated with five peptide fragments for 24 hours, the positive control group uses sitagliptin, and the GLP-1 content in the cell supernatant is detected by using an enzyme-linked immunosorbent assay kit. NCI-H716 is a human colorectal adenocarcinoma cell, can secrete GLP-1 and other hormones, and is used for researching the influence of active peptide intervention on secretion of GLP-1 by intestinal epithelial cells. As shown in FIG. 4, with reference to the control group, peptide fragments LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) all promote secretion of GLP-1 by NCI-H716, indicating that the hypoglycemic peptide can promote secretion of GLP-1 at the cellular level, and exert effects of reducing blood glucose, improving insulin resistance, and the like through GLP-1. Synthesis of secreted GLP-1 exerts beneficial effects by binding to the cell surface GLP-1 receptor. The GLP-1 receptor agonist can activate calmodulin through increasing the calcium ion inflow and the endoplasmic reticulum calcium ion release through the cAMP/PKA pathway after being combined with the GLP-1 receptor, and promote the release of insulin. To further determine whether the peptide fragment possesses activity as a GLP-1 receptor agonist, cAMP levels in the culture supernatant are determined. As shown in FIG. 5, peptide stretches LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3), EVFR (SEQ ID NO: 4) all increased cAMP levels to some extent, indicating that hypoglycemic peptide stretches can activate GLP-1 receptor by increasing cAMP levels.
In combination with examples 2,3, peptide stretches LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) have good GLP-1 receptor agonist activity. On this basis, it was further investigated whether there is a synergistic effect in promoting GLP-1 secretion between peptide stretches LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3). As shown in fig. 6, the combined intervention with simultaneous peptide administration resulted in higher GLP-1 bleeding levels in the cell culture medium than the intervention alone. The combined intervention of the peptide LPMHIR and KFDK and the combined intervention of the peptide KFDK and IPAVFKID has the GLP-1 secretion effect higher than the sum of the effects of the independent interventions, which shows that KFDK and LPMHIR, IPAVFKID respectively have synergistic effects, and the combined use of the peptide LPMHIR and the peptide KFDK and IPAVFKID can improve the action of promoting GLP-1 secretion and activating GLP-1 receptor. Peptides LPMHI and IPAVFKID do not have significant synergy. The results show that the combined use of KFDK and LPMHIR or KFDK and IPAVFKID can further play the role of the hypoglycemic peptide in promoting GLP-1 secretion and activating GLP-1 receptor.
Example 5 improving Effect of peptide fragments on insulin resistance in high fat diet mice
According to examples 2, 3 and 4, peptide stretches LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) have better hypoglycemic effect, and can simultaneously play the dual roles of the DPP-IV inhibitor and the GLP-1 agonist in vitro. Thus, animal experiments were performed using whey protein-derived hypoglycemic peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) to investigate their hypoglycemic effects in vivo. The mass spectra of the three peptide fragments are shown in FIGS. 7-9.
Male C57/6J mice of 3-4 weeks of age were selected and purchased from Peking Vitrellis laboratory techniques Co., ltd.) and raised according to SPF-grade animal raising standards. Animal experiments were approved by the animal experiment committee of the Chinese university of agriculture. Mice were free to drink water and were acclimatized to a week in a 22+ -1deg.C, 12:12 day-night cycle environment. After 1 week of acclimation, mice were randomly divided into Normal Diet (ND) groups (n=6) and High-fat diet (HFD) groups (n=30). The ND group mice were fed with normal feed, and the HFD group mice were fed with high fat feed (energy ratio of 20% protein, 20% carbohydrate, 60% fat) for 10 weeks, to construct high fat diet-induced insulin resistant mice. Fasting blood glucose and fasting insulin were measured after 10 weeks, and insulin resistance index (HOMA-IR) was calculated. The insulin resistance index of the HFD group mice is more than 2.69, which indicates that the high-fat diet induced insulin resistance model is successfully constructed. High-fat diet mice were randomly assigned to HFD group, LPMHIR group (LR), KFDK group (KK group), IPAVFKID group (ID group), sitagliptin group (SITAGLIPTIN group), 6 each. The mice were monitored weekly for food intake and body weight during ten weeks of intervention with the corresponding intervention substances.
Nine weeks after intervention, mice were fasted without water withdrawal for 8h, were subjected to glucose lavage at 2g/kg BW dose, and tail tip blood was collected at the 0 th, 30 th, 60 th, 90 th and 120 th min of the lavage, blood glucose levels were measured with a glucometer, and the Area under the curve (AUC) was calculated.
As shown in fig. 10, the mice had an increase in blood glucose after gastric lavage, followed by a decrease, and became substantially flat after 90 min. The blood glucose levels of mice in the HFD group were consistently maintained at higher levels than those in the ND group, with the Area under the curve (AUC) during the OGTT test being 1.39 times that of the ND group, significantly higher than that of the control group, indicating impaired glucose tolerance and reduced glucose regulation in mice induced by a high fat diet. The intervention of the hypoglycemic peptide and the intervention of the sitagliptin improve the regulating capability of HFD mice on blood sugar, and have better improving effect on glucose tolerance damage induced by high-fat diet. Sitagliptin intervention reduced AUC in HFD mice by 27.2%. The three hypoglycemic peptide fragments LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) respectively reduce the AUC of the HFD mice by 18.8%,23.0% and 21.3%. The results show that the hypoglycemic peptide can improve the glucose tolerance damage induced by the high-fat diet and improve the glucose regulating capacity of the high-fat diet mice.
Mice were fasted without water for 6h, insulin was intraperitoneally injected at a BW dose of 0.75U/kg, tail tip blood was collected from the mice at 0, 30, 60, 90 and 120min of injection, blood glucose levels were measured with a glucometer, a curve was drawn, and the Area under the curve (AUC) was calculated.
As shown in fig. 11, the mice in the HFD group had significantly higher fasting blood glucose levels than the ND group, and the blood glucose was reduced more slowly and with a lower amplitude after insulin injection, 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 resulted in elevated fasting blood glucose and impaired insulin resistance in the mice. The blood sugar reducing peptide and sitagliptin intervene to increase the blood sugar reducing speed of HFD mice, and the blood sugar reducing amplitude is increased, so that the area under the curve is obviously reduced. Sitagliptin intervention reduced AUC in HFD mice by 42.3%. Three kinds of hypoglycemic peptides LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) improve glucose tolerance induced by high-fat diet, and decrease AUC of HFD mice by 26.1%, 33.3% and 30.8%, respectively, which indicates that the hypoglycemic peptides improve insulin resistance of the HFD mice and reduce insulin resistance degree.
Ten weeks after intervention, mice were sacrificed, serum was collected, and DPP-IV enzyme activity in serum was determined, and the effect of the application example of the present invention on DPP-IV enzyme activity in serum of insulin resistant mice induced by high-fat diet was investigated. As shown in fig. 12, DPP-IV enzyme activity was significantly increased in serum of HFD mice compared to ND group. The intervention of different glycopeptides reduces DPP-IV activity in serum of HFD mice, which indicates that the intervention of the glycopeptides can inhibit DPP-IV activity in high-fat diet mice, thereby playing a role in inhibiting degradation and inactivation of GLP-1.
GLP-1 activity in serum is measured, and the influence of the application example of the invention on GLP-1 content in serum of insulin resistant mice induced by high-fat diet is explored. GLP-1 not only can stimulate insulin secretion, but also has protective effects on pancreatic beta cells and cardiovascular systems, can inhibit beta cell apoptosis, promote beta cell growth and proliferation, up regulate insulin secretion, reduce blood sugar, reduce food intake, promote gastric emptying and weight loss, and plays an important role in relieving diabetes. As shown in figure 13, GLP-1 content in serum of HFD mice was significantly reduced compared to ND group, indicating that GLP-1 exerted less effect in improving insulin resistance in high fat diet mice. The intervention of different hypoglycemic peptides obviously improves the GLP-1 content in serum of a high-fat diet mouse, LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) respectively increase the GLP-1 content in serum of the HFD mouse by 46.4%, 57.8% and 61.8%, which shows that the hypoglycemic peptides in the application example are beneficial to increasing the GLP-1 content in serum and play a role in improving insulin resistance by GLP-1. On the other hand, GLP-1 exerts its beneficial effects by binding to GLP-1 receptors on the surface of different tissue cells. To investigate the effect of the hypoglycemic peptide fragment on GLP-1R expression in the liver of high-fat diet-induced insulin resistant mice, GLP-1R expression in the liver of mice was examined by PCR, as shown in FIG. 14. Compared with ND group, HFD diet remarkably reduces the expression of GLP-1R mRNA, which shows that high-fat diet reduces the expression of GLP-1R while inducing the reduction of the content of serum GLP-1, and is unfavorable for the combination of GLP-1 and histiocyte receptor so as to play a role in improving blood sugar. Intervention of the hypoglycemic peptide fragments LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) can improve the expression of GLP-1R mRNA in liver tissues of HFD mice and activate GLP-1 receptors.
Mice were monitored for weight changes during the intervention period, and the effect of the application examples of the invention on the improvement of obesity induced by a high-fat diet was investigated. As shown in fig. 15, HFD group mice significantly increased in weight compared to ND group by 1.52 times. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) can significantly reduce obesity induced by high fat diet, and reduce body weight of mice.
Mice epididymal fat was subjected to H & E staining to investigate the effect of the application examples of the present invention on lipid accumulation induced by high-fat diet. As shown in fig. 16, the cell diameter of epididymal fat of HFD mice was increased and fat index was increased compared to ND group, indicating that high fat diet induced lipid accumulation in mice and increased white adipocytes. Intervention with LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) significantly reduced white fat diameter, indicating that the peptide fragments have an improved effect on reducing lipid accumulation.
The blood lipid level of the mice was characterized by measuring Total Cholesterol (TC), total Triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in the serum of the mice. As shown in table 4, high fat diet intervention significantly increased mice TC, TG, LDL-c, causing hyperlipidemia. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2) and IPAVFKID (SEQ ID NO: 3) are given for intervention, TC, TG, LDL-c level in serum of HFD mice is reduced, HDL-c level is increased, and peptide intervention is shown to remarkably improve blood lipid level of high-fat diet mice, improve hyperlipidemia and have an auxiliary blood lipid lowering effect.
TABLE 4 Effect of hypoglycemic peptide fragment intervention on HFD mouse blood lipid levels
The effects of the application examples of the invention on the index in the serum of the high-fat diet induced insulin resistant mice are explored by measuring the fasting insulin and glycosylated hemoglobin of the mice. Glycosylated hemoglobin (HbA 1 c) is the product of the combination of hemoglobin in red blood cells and glucose in blood, and is directly and effectively responsive to blood glucose levels in patients over the past 3-6 months, and is positively correlated with insulin resistance levels, directly correlated with cholesterol, triglycerides and low density lipoprotein cholesterol, and negatively correlated with high density lipoprotein cholesterol. As shown in table 5, HFD mice had significantly elevated glycosylated hemoglobin levels compared to ND group, indicating that high fat diet induced diabetes and insulin resistance in mice. The intervention of LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) significantly reduced glycosylated hemoglobin levels in high fat diet mice.
TABLE 5 influence of hypoglycemic peptide intervention on HFD mouse serum index
Alpha diversity analysis reflects the diversity and variability of microbial communities, with the most common indices including Ace index, chao index, and Shannon index. The intestinal flora of mice is measured, and the influence of the application example of the invention on the diversity of the intestinal flora of the high-fat diet induced insulin resistant mice is explored. As shown in fig. 17, both Ace index and Chao index of HFD mice were significantly reduced compared to ND group, indicating that HFD significantly reduced the intestinal microbial abundance of mice. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) increased the diversity of intestinal flora in high-fat diet mice and was recovered to some extent.
Intestinal microorganisms are thought to be a factor associated with fat storage, weight gain and insulin resistance. When weight gain is in an obese state, bacteroides abundance decreases and firmicutes abundance increases. As shown in fig. 18, the ratio of bacteroides/firmicutes relative abundance in the HFD group mice intestinal flora was significantly reduced compared to ND group, indicating that a high fat diet can cause an imbalance in the intestinal flora of the mice at the portal level. LPMHIR (SEQ ID NO: 1), KFDK (SEQ ID NO: 2), IPAVFKID (SEQ ID NO: 3) intervene to significantly increase the ratio of Bacteroides/Thick-walled bacteria, demonstrating that the three peptide fragments regulate the intestinal flora composition of HFD mice at the portal level, which is beneficial to exerting the effect of the intestinal flora in improving obesity, insulin resistance and other related symptoms.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.