Application of low-molecular-weight protein GRAB-L as PI3K signal channel activator

Abstract

The invention discloses an application of a low molecular weight protein GRAB-L as a PI3K signal channel activator. An in-vivo experiment shows that the protein GRAB-L serving as an effective component in the bitter gourd-derived extracellular vesicles can obviously reduce fasting blood glucose and postprandial blood glucose of mice, obviously reduce blood fat, relieve fatty degeneration of livers, improve the insulin secretion level and reduce the content of autoantibodies in serum of type 1 diabetes mellitus (T1DM) mice to a certain extent; as a PI3K activator, the compound can activate PI3K phosphorylation and PI3K / AKT / FOXO1 / GSK3beta signal channels, inhibit liver glycogen synthesis and gluconeogenesis, increase glycogen decomposition, glycolysis and the like to improve liver dysfunction and maintain glucose homeostasis, and has great significance in treatment of diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of a low molecular weight protein GRAB-L as a PI3K signaling pathway activator. Background Art

[0002] Diabetes mellitus (DM) is a multi-cause metabolic disease characterized by chronic hyperglycemia, accompanied by disorders of sugar, fat and protein metabolism caused by defects in insulin secretion and / or action. Long-term hyperglycemia can cause serious complications such as nephropathy, retinopathy, vasculopathy, neuropathy, cardiovascular disease, and liver disease. How to regulate sugar metabolism and maintain blood sugar homeostasis is an urgent problem to be solved in the current treatment of diabetes. Insulin is currently the most effective anti-diabetic drug, but the occurrence of insulin resistance greatly affects its therapeutic effect. Hypoglycemic drugs such as metformin, thiazolidinediones, sulfonylureas, sodium-dependent glucose transporter (SGLT2) inhibitors, dipeptidyl peptidase-4 (DPP-4) inhibitors have side effects such as long-term administration, hypoglycemia, kidney damage and pancreatitis. Therefore, it is extremely important to develop more efficient and safe anti-diabetic and hypoglycemic drugs.

[0003] Plant-derived extracellular vesicles are rich in proteins, nucleic acids, lipids, and plant-specific bioactive components. They are highly biosafe and low-cost, making them ideal candidates for hypoglycemic drugs. PI3Kα and PI3Kβ are two major subtypes of the PI3K family. In insulin signaling, the activation of PI3Kα and PI3Kβ is essential for promoting glucose uptake and utilization, glycogen synthesis, and lowering blood sugar levels. So far, there has been no report on finding an effective PI3K activator from plant extracellular vesicles that can improve glucose metabolism by regulating the PI3K signaling pathway and exert a hypoglycemic effect. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of the low molecular weight protein GRAB-L, which can be used as a PI3K activator to play a hypoglycemic effect.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] Application of low molecular weight protein GRAB-L as activator of PI3K signaling pathway.

[0007] Application of low molecular weight protein GRAB-L in preparing medicine for preventing and / or treating diabetes.

[0008] Application of low molecular weight protein GRAB-L in the preparation of drugs for preventing and / or treating hyperlipidemia

[0009] Application of low molecular weight protein GRAB-L in the preparation of blood sugar lowering and / or blood lipid lowering drugs.

[0010] Application of low molecular weight protein GRAB-L in the preparation of blood sugar and / or blood lipid lowering health products, blood sugar and / or blood lipid lowering foods or external dressings.

[0011] In the present invention, the molecular weight of the protein GRAB-L is less than 20 kDa, and its amino acid sequence is shown in SEQ ID NO.1.

[0012] The present invention adopts a mouse model of type 1 diabetes (TIDM) induced by streptozotocin (STZ) injection, and administers different concentrations of GRAB-L by intraperitoneal injection. The drug is administered every other day for two consecutive weeks. The fasting blood glucose, glucose tolerance and other data of the mice are recorded for two weeks. After administration, the contents of insulin, glucagon and insulin-related autoantibodies in the serum of the mice are detected, and the changes in the mRNA levels of liver glycogen synthesis and gluconeogenesis key enzymes are detected.

[0013] A high-glucose and high-fat model was established by culturing AML12 cells with high glucose (100 mM) and palmitic acid (200 μM) for 48 h. After co-culture with GRAB-L for 2 days, the changes in proteins in the PI3K / AKT / FOXO1 / GSK3β signaling pathway were detected.

[0014] The results show:

[0015] 1. In the T1DM model,

[0016] ① The protein GRAB-L can significantly reduce the fasting blood glucose and postprandial blood glucose of mice.

[0017] ② Protein GRAB-L can significantly lower blood lipids, alleviate liver fatty degeneration, and improve insulin secretion levels.

[0018] ③The protein GRAB-L reduces the level of autoantibodies in the serum of T1DM mice to a certain extent.

[0019] 2. In AML12 cells, GRAB-L can activate the PI3K / AKT / FOXO1 / GSK3β signaling pathway to inhibit hepatic gluconeogenesis, indicating that GRAB-L activates PI3Kβ and its downstream signaling pathways and is a new PI3Kβ activator.

[0020] Preferably, the drug is a pharmaceutical composition consisting of the active pharmaceutical ingredient protein GRAB-L and pharmaceutically acceptable excipients.

[0021] The pharmaceutically acceptable excipients are conventional pharmaceutical excipients in the pharmaceutical field, including diluents, excipients such as water, binders such as cellulose derivatives, gelatin or polyvinyl pyrrolidone, fillers such as starch, disintegrants such as calcium carbonate or sodium bicarbonate, and other adjuvants such as flavoring agents and / or sweeteners may also be added to the composition.

[0022] Preferably, the effective concentration of the protein GRAB-L is 40 μg / kg.

[0023] Preferably, the blood sugar and / or blood lipid lowering health care products, blood sugar and / or blood lipid lowering foods, and external dressings include the active ingredient protein GRAB-L and other auxiliary materials. The blood sugar and / or blood lipid lowering health care products can be powders, granules, capsules, tablets, pills, oral liquids, etc. The blood sugar and / or blood lipid lowering foods can be processed into noodles, biscuits, beverages, and alcoholic products, etc.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention provides the use of protein GRAB-L in the preparation of drugs for preventing and / or treating diabetes, blood sugar lowering drugs or blood sugar lowering health foods. Protein GRAB-L can significantly reduce fasting blood sugar and postprandial blood sugar in mice, significantly reduce blood lipids, alleviate liver fatty degeneration, improve insulin secretion levels, and can also reduce the content of autoantibodies in the serum of T1DM mice to a certain extent, which is of great significance for the treatment of diabetes.

[0026] 2. As a PI3K activator, the low molecular weight protein GRAB-L can phosphorylate PI3K to activate the PI3K / AKT / FOXO1 / GSK3β signaling pathway, increase sugar production, reduce gluconeogenesis, and improve sugar metabolism disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart for screening GRAB-L in the present invention and a diagram of the molecular docking model of HDOCK.

[0028] Figure 2 This is a graph showing changes in fasting blood sugar in diabetic mice of the present invention after intraperitoneal injection of different concentrations of GRAB-L for two weeks.

[0029] FIG3 is a graph and a statistical graph showing changes in glucose tolerance levels in diabetic mice after intraperitoneal injection of different concentrations of GRAB-L for two weeks in the present invention; Figure 3A is the glucose tolerance level of mice in the second week, Figure 3B It is a statistical graph of the area under the glucose tolerance curve.

[0030] FIG4 is a graph showing the changes in serum total cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein levels in mice after intraperitoneal injection of different concentrations of GRAB-L in the present invention; Figure 4A This is a statistical chart of total cholesterol content in mice. Figure 4B This is a statistical chart of triglyceride content in mice. Figure 4C This is a statistical chart of the high-density lipoprotein content in mice. Figure 4D It is a statistical chart of the low-density lipoprotein content in mice.

[0031] FIG5 is a graph showing the changes in serum insulin and glucagon levels in mice intraperitoneally injected with different concentrations of GRAB-L in the present invention; Figure 5A This is a statistical chart of insulin levels. Figure 5B It is a statistical graph of glucagon levels.

[0032] FIG6 is a graph showing changes in the levels of glutamic acid decarboxylase (GAD) autoantibodies, insulin autoantibodies (IAA), and islet cell antibodies (ICA) in the serum of mice intraperitoneally injected with different concentrations of GRAB-L in the present invention; Fig. 6A This is a statistical chart of the content of glutamic acid decarboxylase autoantibodies. Figure 6B This is a statistical chart of insulin autoantibody levels. Figure 6C It is a statistical chart of the antibody content in pancreatic islet cells.

[0033] FIG. 7 is a graph showing changes in mRNA levels of key enzymes for glycogen synthesis and decomposition and key enzymes for gluconeogenesis in the liver of mice injected with different concentrations of GRAB-L in the present invention; wherein Fig. 7A is the relative mRNA expression level of glycogen synthase, where Figure 7B is the relative mRNA expression level of glycogen phosphorylase, where Figure 7C is the relative mRNA expression level of phosphoenolpyruvate carboxykinase, where Fig.7D is the relative mRNA expression level of fructose-1,6-bisphosphate, where Fig. 7E is the relative mRNA expression level of pyruvate carboxylase, where Figure 7F is the relative mRNA expression level of glucose-6-phosphatase.

[0034] FIG8 shows the protein changes in the PI3K / AKT / FOXO1 / GSK3β signaling pathway of AML12 cells in the present invention after co-culture with 1 μg / mL GRAB-L for 48 hours under a high-sugar and high-fat model; Fig. 8A is a protein blot, where Figure 8B It is a statistical graph of relative protein expression. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] 1. Experimental Materials and Instruments

[0037] 1.1 Experimental materials: total cholesterol assay kit, triglyceride assay kit, high-density lipoprotein assay kit, low-density lipoprotein assay kit, mouse glutamic acid decarboxylase (GAD) autoantibody (IgG) ELISA kit, mouse insulin autoantibody (IAA) ELISA kit, mouse islet cell antibody (ICA) ELISA kit, primers (Gys1, PygL, G6Pase, PC, PCK1, FBP1, RPL13A), reverse transcription kit, qPCR kit, p-PI3K (BIOSS, bs6417R), PI3K (Proteintech, 20584-1-AP), p-AKT (Proteintech, 6 6444-1-AP), AKT (Proteintech, 10176-1-AP), p-GSK-3β (CST, 9336), GSK-3β (CST, 9315), PGC-1α (HUABIO, JF09-71), p-FOXO1 (HUABIO, HA72249), FOXO1 (HUABIO, ET1608-25), β-actin (Proteintech, HRP-60008), Trizol, BCA kit, SDS-PAGE gel rapid preparation kit, streptozotocin (STZ), citric acid, sodium citrate, 0.9% saline, glucose, palmitic acid, blood glucose test strips.

[0038] Purified protein GRAB-L: 98 proteins were identified using the GCF 001995035 database (ver202001, 28666+ entries). In addition to 57 ribosomal proteins, a total of 41 proteins were screened. Consistent with previous MC-EVLP proteomics sequencing, 39 proteins were identified after IP / MS, including 9 low molecular weight proteins, such as Figure 1 GRBAIPL (glycine-rich RNA-binding, abscisic acid-inducible protein-like, 167 amino acids (its amino acid sequence is shown in SEQ ID NO.1), LOC111015433, named GRAB-L) was selected as one of the candidates for PI3Kβ activator, and GRAB-L was recombinantly purified.

[0039] 1.2 Experimental animals: Male C57BL / 6J mice were purchased from the Experimental Animal Center of Xuzhou Medical University, license number: 202404T004. All experimental animals were housed in the barrier facility of the Experimental Animal Center of Xuzhou Medical University in a clean environment with a temperature of (21±2)°C, humidity of (35±2)%, and 12h:12h day and night lighting. They had free access to food and water, and the drinking water was sterile water prepared by the Experimental Animal Center.

[0040] 1.3 Experimental instruments: 37°C constant temperature box, carbon dioxide incubator, clean bench, microplate reader, electronic scale, blood glucose meter, pipette, insulin needle, culture dish, Pasteur pipette, centrifuge tube, pipette gun, 6-well plate, AML12 cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences), electrophoresis instrument, electrophoresis tank, qPCR instrument, semi-dry transfer instrument.

[0041] 2. Experimental Procedure

[0042] 2.1 Establishment of experimental type 1 diabetes mouse model

[0043] The purchased mice were adaptively raised for 1 week before modeling, and were randomly divided into a model group (20 mice) and a normal control group (6 mice). The mice were fasted for 12-14 hours, and a certain amount of STZ solution (100 mg / kg) was intraperitoneally injected into the model group mice. During the injection, STZ was dissolved in 0.1 mol / L sodium citrate buffer (pH 4.5), prepared on ice in a dark place, and injected within 30 minutes after dissolution. The normal control group was not treated. The mice were fasted for 8 hours 5 days after the injection, and 20 μL of blood was taken from the tail vein of the modeled mice to measure their fasting serum glucose content. The fasting blood glucose value of the mouse was ≥11.1 mmol / L as the standard for the successful experimental type Ⅰ diabetic mouse model.

[0044] 2.2 Experimental Animal Grouping and Dosing

[0045] In this experiment, 18 mice were successfully modeled and randomly divided into the model group, 20μg / kg administration group, and 40μg / kg administration group. After the model was successfully established, the mice were injected intraperitoneally. The drugs were administered every other day for two consecutive weeks, and the blood sugar and glucose tolerance index of the mice were measured every week.

[0046] 2.3 Glucose tolerance test

[0047] 2.3.1 At 5 pm on the day before the experiment, the mice were placed in clean cages and fasted for 16 hours until 9 am the next day;

[0048] 2.3.2 At 9:00 a.m. the next day, the glucose tolerance test was started. Each mouse was weighed and fasting blood glucose was measured and recorded.

[0049] 2.3.3 The normal group and the diabetic model group were given the same volume of normal saline. After the mice were allowed to adapt for 30 minutes, preparations for oral glucose OGTT (20% glucose solution, 0.01 ml / g) were started and the timing was started;

[0050] 2.3.4 At 15min, 30min, 60min, 90min, and 120min, measure the blood glucose level of each mouse at each time point;

[0051] 2.3.5 After the experiment, each cage of mice was supplemented with feed.

[0052] 2.4 Serum total cholesterol determination

[0053] 2.4.1 Place the collected whole blood sample at room temperature for 2 hours, then centrifuge at 3000g for 10 minutes to obtain the supernatant. 2.4.2 Operation table

[0054]

[0055] 2.4.3 Calculation formula

[0056] Cholesterol content (mmol / L) = Α sample - Α blank / Α standard - Α blank * C standard 2.5 Serum triglyceride determination

[0057] 2.5.1 Place the collected whole blood sample at room temperature for 2 hours, then centrifuge at 3000g for 10 minutes to collect the supernatant. 2.5.2 Operation table

[0058]

[0059] 2.5.3 Calculation formula

[0060] Triglyceride content (mmol / L) = Α sample - Α blank / Α standard - Α blank * C standard 2.6 Serum high-density lipoprotein determination

[0061] 2.6.1 Place the collected whole blood sample at room temperature for 2 hours, then centrifuge at 3000g for 10 minutes to collect the supernatant. 2.6.2 Operation table

[0062]

[0063]

[0064] 2.6.3 Calculation formula

[0065] High-density lipoprotein content (mmol / L) = △Α sample-△Α blank / △Α standard-△Α blank*C standard

[0066] 2.7 Serum low-density lipoprotein measurement

[0067] 2.7.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant;

[0068] 2.7.2 Operation Table

[0069]

[0070] 2.7.3 Calculation formula

[0071] Low-density lipoprotein content (mmol / L) = △Α sample-△Α blank / △Α standard-△Α blank*C standard

[0072] 2.8 Mouse glutamic acid decarboxylase (GAD) autoantibody (IgG) assay

[0073] 2.8.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant. The serum and sample diluent were diluted at 1:200 for later use.

[0074] 2.8.2 Set up blank wells without any solution. Set up 3 negative control wells and 2 positive control wells; add 100 μL of negative control and positive control respectively. Add 100 μL of diluted sample to the static wells; cover with adhesive strips and incubate at 37°C for 60 minutes;

[0075] 2.8.3 Discard the liquid, add washing solution, let stand for 2 minutes, then discard the liquid and pat dry; aspirate and wash each well, and repeat this process four times;

[0076] 2.8.4 Add 100 μL of enzyme label (except blank wells), cover the membrane and incubate at 37℃ for 30 minutes;

[0077] 2.8.5 Repeat 2.8.3 and wash the plate four times;

[0078] 2.8.6 Add 50 μL of substrate A and substrate B to each well; incubate at 37°C in the dark for 15 minutes;

[0079] 2.8.7 Add 50 μL of stop solution and mix gently; use the blank well as zero and the wavelength as 450 nm to read the absorbance using a microplate reader.

[0080] 2.9 Determination of insulin autoantibodies (IAA) in mice

[0081] 2.9.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant. The serum and sample diluent were diluted at 1:800 for later use.

[0082] 2.9.2 Set up blank wells without any solution; add 100 μL of negative control, positive control or diluted sample to each well, cover with adhesive strips, and incubate at 37°C for 30 minutes;

[0083] 2.9.3 Discard the liquid, add washing solution, let stand for 2 minutes, then discard the liquid and pat dry; aspirate and wash each well, and repeat this process four times;

[0084] 2.9.4 Add 100 μL of enzyme label (except blank wells), cover the membrane and incubate at 37℃ for 30 minutes;

[0085] 2.9.5 Repeat 2.9.3 and wash the plate four times;

[0086] 2.9.6 Add 90 μL of TMB substrate to each well and incubate at 37°C in the dark for 20 minutes;

[0087] 2.9.7 Add 50 μL of stop solution and mix gently; use the blank well as zero and the wavelength as 450 nm to read the absorbance using a microplate reader.

[0088] 2.10 Mouse islet cell antibody (ICA) assay

[0089] 2.10.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant. The serum and sample diluent were diluted at 1:200 for later use.

[0090] 2.10.2 Set up blank wells without any solution; add 100 μL of negative control, positive control or diluted sample to each well, cover with adhesive strips, and incubate at 37°C for 60 minutes;

[0091] 2.10.3 Discard the liquid, add washing solution, let stand for 2 minutes, then discard the liquid and pat dry; aspirate and wash each well, and repeat this process four times;

[0092] 2.10.4 Add 100 μL of enzyme label (except blank wells), cover the membrane and incubate at 37°C for 60 minutes;

[0093] 2.10.5 Repeat 2.10.3 and wash the plate four times;

[0094] 2.10.6 Add 50 μL of substrate A and substrate B to each well; incubate at 37°C in the dark for 15 minutes;

[0095] 2.10.7 Add 50 μL of stop solution and mix gently. Set the blank well as zero and the wavelength as 450 nm with a microplate reader to read the absorbance.

[0096] 2.11 Mouse insulin assay

[0097] 2.11.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant. The serum and sample diluent were diluted at a ratio of 1:128 for later use.

[0098] 2.11.2 Add samples. Set up zero well, standard well, and sample well. Add 100 μL of sample diluent to the zero well, and add 100 μL / well of graded diluted standard or sample to be tested to the remaining wells. Be careful not to generate bubbles.

[0099] 2.11.3 Cover the ELISA plate with film and incubate at 37℃ for 2h;

[0100] 2.11.4 Washing 1) Remove the sealing film, discard the liquid, and pat dry; 2) Wash the strips with washing solution (1×), 350-400μL per well. After washing, shake off the liquid and pat dry the strips. Repeat this step 4 times;

[0101] 2.11.5 Add 100 μL of detection antibody (1×) to each well, cover with sealing film, and incubate at 37℃ for 1 hour;

[0102] 2.11.6 Repeat step 2.11.4; add 100 μL HRP-labeled secondary antibody (1×) to each well, cover with sealing film, and incubate at 37°C for 40 min;

[0103] 2.11.7 Repeat step 2.11.4;

[0104] 2.11.8 Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 15-20 minutes;

[0105] 2.11.9 Add 100 μL of stop solution to each well. The blue color will turn yellow. Use a microplate reader to measure the optical density (OD value) of each well at a wavelength of 450 nm. Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Use professional software (such as Origin, ELISACalc, etc.) to perform four-parameter fitting (4-PL). Calculate the fitted concentration from the standard curve based on the OD value of the sample, and multiply it by the dilution factor to get the actual concentration of the sample.

[0106] 2.12 Mouse glucagon determination

[0107] 2.12.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant. The serum and sample diluent were diluted at a ratio of 1:10 for later use.

[0108] 2.12.2 Set up zero well, standard well, and sample well. Add 100μL of sample diluent to zero well, and add 100μL / well of graded diluted standard or sample to be tested to the remaining wells, cover with film and incubate at 37℃ for 2h;

[0109] 2.12.3 Take out the plate without washing, add 100 μL (1x) biotin antibody to each well, cover with film and incubate at 37℃ for 1 hour;

[0110] 2.12.4 Discard the liquid, add washing solution, let stand for 2 minutes, then discard the liquid and pat dry. Aspirate and wash each well, and repeat this process four times;

[0111] 2.12.5 Add 100 μL enzyme-labeled avidin, cover the membrane and incubate at 37°C for 60 minutes;

[0112] 2.12.6 Repeat 2.12.4 and wash the plate four times;

[0113] 2.12.7 Add 90 μL of TMB substrate to each well; incubate at 37°C in the dark for 15-30 minutes;

[0114] 2.12.8 Add 50 μL of stop solution and mix gently; use the blank well as zero and the wavelength as 450 nm to read the absorbance using a microplate reader.

[0115] 2.13 Real-time fluorescence quantitative nucleic acid amplification monitoring

[0116] 2.13.1 RNA extraction:

[0117] 1. Tissue homogenization: Take 50 mg of mouse liver tissue, add 1 mL of Trizol, and homogenize with a homogenizer;

[0118] 2. Place the homogenized sample at room temperature (15-30°C) for 5 minutes to completely separate the nucleic acid-protein complex;

[0119] 3. Centrifuge at 10,000 × g for 10 minutes at 2-8°C and collect the supernatant;

[0120] 4. Add 0.2 mL of chloroform for every 1 mL of Trizol used, shake vigorously for 15 seconds, and leave at room temperature for 3 minutes;

[0121] 5. Centrifuge at 10,000 × g for 15 minutes at 2-8°C. The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase, and an intermediate layer. The RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 60% of the Trizol reagent used;

[0122] 6. Transfer the aqueous phase to a new tube. Add 0.5 ml of isopropanol for every 1 mL of Trizol used and leave at room temperature for 10 minutes;

[0123] Centrifuge at 7.2-8℃, 10,000×g for 10 minutes. After centrifugation, a gelatinous precipitate will appear on the sides and bottom of the tube. Remove the supernatant;

[0124] 8. Wash the RNA precipitate with 75% ethanol. For every 1 mL of Trizol used, add at least 1 mL of 75% ethanol; centrifuge at no more than 7500 × g at 2-8°C for 5 minutes and discard the supernatant;

[0125] 9. Leave to dry at room temperature or vacuum dry the RNA precipitate for about 5-10 minutes; add 200 μL of nuclease-free water;

[0126] 2.13.2 Reverse transcription was performed using the ABScript II cDNA First Strand Synthesis Kit (RK20400). Reverse transcription conditions were: 65°C for 5 min, 42°C for 1 h, and 80°C for 5 min;

[0127] 2.13.3 Real-time fluorescence quantitative PCR was performed using Vazyme's ChamQ SYBR qPCR Master Mix. All reactions were repeated three times. Reverse transcription conditions were: 65°C for 5 min, 42°C for 1 h, 80°C for 5 min; PCR conditions were: 95°C for 5 min, 95°C for 10 s, 60°C for 30 s for 45 cycles; primers were purchased from Shanghai Biotechnology Co., Ltd. (sequences are shown in Table 1). The comparative threshold period (2 -△△ct ) method to analyze the data.

[0128] Table 1 Primer sequence list

[0129]

[0130]

[0131] 2.14 Cell culture

[0132] Mouse liver cell line AML12 (FH0338) was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. AML12 cells were cultured in DMEM / F12 medium (containing 10% fetal bovine blood, 1% penicillin-streptomycin, 1% iTS and 40ng / mL dexamethasone) at 37°C, 5% CO2 and 95% air in a humidified environment. Cells were stimulated with palm (PA, 200μM) and high glucose (100mM) for 48h, and treated with GRAB-L (1μg / mL).

[0133] 2.15 Protein immunoblotting

[0134] 2.15.1 Cell protein extraction: Cell lysis buffer (RIPA + 1% PMSF + 1% phosphatase inhibitor), the whole process was performed on ice, the cells were washed three times with pre-cooled PBS, the PBS in the dish was aspirated, 200 μL of the prepared cell lysis buffer was added to each large dish, the cells were scraped into a pre-cooled 1.5 mL EP tube, vortexed for 60 seconds, let stand on ice for 10 minutes, and then vortexed for 30 seconds, repeated three times, and finally centrifuged at 14000 rpm, 4°C for 25 minutes, and the supernatant was collected. The supernatant was the extracted total cell protein.

[0135] 2.15.2 Protein concentration determination: The BCA method was used with 0.5 mg / mL bovine serum albumin (BSA) as the standard protein. The determination was performed according to the steps in the kit instructions. Three replicate wells were measured for each sample. The average concentration of each sample was calculated based on the standard curve and adjusted to the same concentration using RIPA lysis buffer.

[0136] 2.15.3Western Blot:

[0137] 1. Sample treatment: Add 5× protein loading buffer to samples of the same volume and protein amount, and heat in a metal bath for 5 minutes to denature the protein;

[0138] 2. Prepare gel: Prepare separation gel and concentrated gel of different concentrations according to molecular weight. For proteins with large molecular weight, use separation gel of small concentration, and for proteins with small molecular weight, use separation gel of high concentration.

[0139] 3 Electrophoresis: After gelation, install the electrophoresis tank, load an equal amount of protein sample, and perform constant voltage electrophoresis. Run at 80V for 30 minutes, then switch to 120V and continue running until bromophenol blue reaches the bottom of the glass plate, ending the electrophoresis.

[0140] 4. Transfer: After electrophoresis, transfer the protein to a methanol-activated PVDF membrane using a wet transfer method (250 mA, 120 min) or a semi-dry transfer method (100 mA, 35 min).

[0141] 5 Blocking: Prepare 5% skim milk as blocking solution. After transfer, place the PVDF membrane in the blocking solution and shake slowly at room temperature for 2 hours.

[0142] 6 Incubate with primary antibody: After blocking, recover the blocking solution, wash the membrane with 1×Washing Buffer (10 min×3 times), add the diluted primary antibody, and incubate overnight at 4°C

[0143] 7 Incubate with secondary antibody: Recover the primary antibody, wash the membrane with 1× Washing Buffer (10 min×3 times), add the diluted secondary antibody, and incubate at room temperature for 1 hour;

[0144] 8. Development: Recover the secondary antibody and wash the membrane with 1× Washing Buffer (10 min×3 times); prepare the exposure solution according to the instructions of the ECL kit for development, and use ImageJ software to process and analyze the gray value of the exposed bands.

[0145] In order to identify the specific protein components that regulate PI3Kβ, the inventors performed immunoprecipitation-mass spectrometry analysis on bitter melon vesicles to find proteins that interact with PI3Kβ. The low molecular weight protein GRAB-L was selected as one of the candidates for PI3Kβ activators and GRAB-L was recombinantly purified. Figure 1 The molecular docking results of HDOCK showed that PI3Kβ (green) and GRAB-L can be stably combined together through hydrogen bonds formed by amino acids at different sites and multiple hydrophobic interactions.

[0146] Figure 2 The results showed that the injection of 20μg / kg and 40μg / kg GRAB-L for two weeks can significantly reduce the fasting blood glucose of diabetic mice. The results in Figure 3 show that the injection of 20μg / kg and 40μg / kg GRAB-L can improve the glucose tolerance level of diabetic mice, and the treatment of 40μg / kg GRAB-L is better than the treatment of 20μg / kg GRAB-L. The results in Figure 4 show that the injection of 20μg / kg and 40μg / kg GRAB-L treatment leads to a decrease in the total cholesterol and triglyceride levels and an increase in the high-density lipoprotein level in diabetic mice, thus having a good lipid-lowering effect. Figure 5A The results showed that insulin levels in diabetic mice increased after injection of 20μg / kg and 40μg / kg GRAB-L. Glucagon levels were significantly increased in T1D mice, but significantly decreased under 40μg / kg GRAB-L treatment ( Figure 5B ). The results of Figure 6 show that 40 μg / kg GRAB-L treatment significantly reduced the levels of IAA, ICA and GAD. The results of Figure 7 show that GRAB-L inhibits gluconeogenesis and promotes glycogen synthesis. The results of Figure 8 show that GRAB-L treatment significantly increased the phosphorylation levels of PI3K, AKT, Fox01 and GSK-3β, and increased the protein level of PGC-1α in AML12 cells treated with high glucose and high fat.

[0147] The above results show that PI3Kβ and GRAB-L can be stably combined together through hydrogen bonds and multiple hydrophobic interactions formed by amino acids at different sites. GRAB-L can reduce fasting blood glucose and glucose tolerance in mice, improve insulin secretion levels in mice, reduce blood lipids in mice, and improve the titer of insulin autoantibodies in diabetic mice to a certain extent. At the same time, GRAB-L can improve the changes in key enzymes of glycogen synthesis and gluconeogenesis in mice. At the cellular level, GRAB-L can phosphorylate PI3K to activate the PI3K / AKT / FOXO1 / GSK3β signaling pathway, increase sugar production, reduce gluconeogenesis, and improve sugar metabolism disorders.

[0148] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. Application of low molecular weight protein GRAB-L as activator of PI3K signaling pathway.

2. Application of the low molecular weight protein GRAB-L in the preparation of drugs for preventing and / or treating diabetes.

3. Application of the low molecular weight protein GRAB-L in the preparation of drugs for preventing and / or treating hyperlipidemia.

4. Application of low molecular weight protein GRAB-L in the preparation of hypoglycemic and / or hypolipidemic drugs.

5. Application of the low molecular weight protein GRAB-L in the preparation of blood sugar and / or blood lipid lowering health products, blood sugar and / or blood lipid lowering foods, and external dressings.

6. The use according to any one of claims 1 to 5, characterized in that: The protein GRAB-L has a molecular weight of less than 20 kDa, and its amino acid sequence is shown in SEQ ID NO.

1.

7. The use according to any one of claims 2 to 4, characterized in that: The medicine is a medicine composition consisting of the medicine active ingredient protein GRAB-L and pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that: The effective concentration of the protein GRAB-L is 40 μg / kg.

9. The use according to claim 5, characterized in that: The blood sugar and / or blood lipid lowering health care product, blood sugar and / or blood lipid lowering food, and external dressing comprise the active ingredient protein GRAB-L and other auxiliary materials.

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