Use of a novel polypeptide in the preparation of a medicament for diabetes

Abstract

This invention relates to the biomedical field, specifically to interventional peptides for preparing drugs to treat diabetes, comprising: (a) SEQ ID No. 1; (b) a fusion protein formed by peptide (a) and a transmembrane peptide element; and (c) a peptide derivative of peptide (a) or (b) by deletion, insertion, or substitution of one or more amino acids, and having the same biological function as the peptide molecule described in (a) or (b). This invention has undergone extensive screening and validation at the cellular level and in animal experiments, and has also been tested using serum samples from clinical patients, confirming that the peptides can specifically lower blood glucose levels, promote cellular glucose uptake, and promote glycogen synthesis, thereby improving diabetes symptoms; it only affects glucose metabolism, therefore has few side effects, a long half-life, and significant clinical implications.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to the application of a novel polypeptide in the preparation of a drug for diabetes. BACKGROUND

[0002] Diabetes mellitus (DM) is a metabolic disease caused by impaired insulin secretion and various peripheral insulin resistance, resulting in a chronic state of hyperglycemia. Long-term poor glycemic control can lead to progressive damage to the cardiovascular, ocular, renal, and nervous tissues and organs, and even death.

[0003] The diagnosis of diabetes mellitus currently relies on blood glucose testing, including fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), and oral glucose tolerance test (OGTT). Compared with FPG, OGTT is more sensitive for the diagnosis of diabetes and impaired glucose tolerance, but it is inconvenient and time-consuming. HbA1c may be falsely high or low due to the presence of a large proportion of glycosylated hemoglobin variants in the population, greatly reducing the diagnostic value of HbA1c.

[0004] Diabetes mellitus is mainly divided into two categories according to its etiology; type I diabetes mellitus, caused by insulin secretion dysfunction due to autoimmune-mediated destruction of pancreatic beta cells; type II diabetes mellitus, caused by insulin resistance. The most common clinical manifestation of diabetes mellitus is hyperglycemia. Both type I and type II diabetes mellitus patients usually have symptoms related to hyperglycemia, such as frequent urination, polydipsia, orthostatic hypotension, dehydration, weight loss, blurred vision, etc., or no symptoms, so the diagnosis may be delayed for many years, and some patients may first present symptoms of diabetic complications.

[0005] For the treatment of diabetes mellitus, the main method is to control hyperglycemia to improve symptoms and prevent complications and reduce hypoglycemic episodes, usually combined with diet and exercise therapy. For type I diabetes mellitus, insulin is supplemented; for type II diabetes mellitus, oral hypoglycemic agents, insulin, or both are used. These treatments often require daily administration, and the requirements for patient and family monitoring are complicated.

[0006] Therefore, there is an urgent need in the art to develop drugs for the treatment or prevention of diabetes mellitus with fewer side effects and longer half-lives. SUMMARY

[0007] To solve the above problems, the present application aims to provide a therapeutic agent for treating or preventing diabetes mellitus with fewer side effects and its application. The present application provides an autophagy-related factor ATG7 that regulates glucose at different levels of glucose intake and glycogen synthesis, thereby reducing blood glucose levels.

[0008] In order to achieve the above object, the present application provides the following technical solutions.

[0009] The present application provides a novel polypeptide, characterized in that the amino acid sequence of the polypeptide comprises SEQ ID No 1.

[0010] Further, the polypeptide is derived from human ATG7 protein.

[0011] The present application also provides a use of the novel polypeptide in the preparation of a medicament for treating diabetes, characterized in that the polypeptide comprises:

[0012] (a) the novel polypeptide of claim 1;

[0013] (b) a fusion protein formed by polypeptide (a) and a cell-penetrating peptide element;

[0014] (c) a polypeptide derivative of polypeptide (a) or (b) with one or more amino acids deleted, inserted or substituted, and having the same biological function as the polypeptide molecule described in (a) or (b).

[0015] Further, the polypeptide directly activates the signal transduction factor in the Insulin signaling pathway, increases glucose uptake and glycogen synthesis, reduces glucose production, and regulates glucose metabolism.

[0016] Further, the medicament comprises:

[0017] (a) a medicament for preventing and / or treating diabetes;

[0018] (b) a medicament for lowering blood glucose levels;

[0019] (c) a preparation of an agonist of glucose uptake and glycogen synthesis.

[0020] Preferably, the dosage form of the medicament is any pharmaceutically acceptable dosage form.

[0021] Preferably, the dosage of the medicament is any pharmaceutically acceptable dosage.

[0022] The present application also provides a polynucleotide sequence, characterized in that the polynucleotide sequence can encode the polypeptide of claim 3.

[0023] The present application also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide of claim 3 and a pharmaceutically acceptable carrier or excipient.

[0024] The present application has the following advantages compared with the prior art.

[0025] The present application first discovers that autophagy-related factor ATG7 is involved in the metabolic regulation of glucose. ATG7 regulates glucose at different levels of glucose intake and glycogen synthesis, thereby reducing blood glucose levels.

[0026] In addition, the present application also first proves that the ATG7 fragment derived from the amino acid sequence at positions 626-673 of human ATG7 protein or containing the above-mentioned amino acid sequence can specifically reduce blood glucose levels, promote glucose uptake by cells, and promote glycogen synthesis, thereby improving the symptoms of diabetes; it only affects the metabolism of glucose, so the side effects are small, and the half-life is long. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The protein level of ATG7 in the serum of peripheral blood of different populations is shown. Among them, A is that the protein level of ATG7 in the serum of peripheral blood of diabetic patients is significantly higher than that of non-diabetic control group; B is the ROC curve analysis shows that AUC=0.8446.

[0028] Figure 2 The expression level of Atg7 in the serum of peripheral blood and liver tissue of type I diabetes model (STZ) mice is shown. Among them, A is that the Atg7 protein level in the serum of STZ mice is significantly higher than that of control group mice; B is that the Atg7 protein level in liver tissue is reduced.

[0029] Figure 3 ATG7 is involved in the metabolic regulation of glucose. Among them, a is that ATG7 deletion leads to a decrease in the ability of primary hepatocytes to uptake glucose; b is that ATG7 deletion leads to a lack of glycogen in primary hepatocytes; c is that ATG7 deletion leads to an increase in glucose production in primary hepatocytes; d is that ATG7 regulates glycogen synthesis in hepatocytes; e is that insulin-mediated glycogen synthesis depends on the expression of ATG7; f is that the insulin (Insulin) mediated signaling pathway depends on the expression of ATG7; g is that the binding of insulin receptor IR / IGF-1R and substrate IRS1 depends on ATG7.

[0030] Figure 4 Aap2 regulates the Insulin signaling pathway to affect the metabolism of glucose. Among them, a is that Aap2 regulates the insulin-mediated signaling pathway in primary MEF cells; b is that Aap2 regulates the insulin-mediated signaling pathway in HepG2 hepatocytes; c is that Aap2 regulates the insulin-mediated signaling pathway in Huh7 hepatocytes; d is that Aap2 increases the glucose uptake in primary hepatocytes; e is that Aap2 increases glycogen synthesis in primary hepatocytes; f is that Aap2 reduces glucose production.

[0031] Figure 5Aap2 regulates the blood glucose level of STZ mice. Among them, a is that Aap2 reduces the blood glucose level of STZ mice, n=9 per group; b is that Aap2 increases the synthesis of glycogen in the liver, n=9 per group; c is that Aap2 reduces glucose tolerance, n=8 per group; d is the AUC result. DETAILED DESCRIPTION

[0032] The application will be further described in the following experiments combined with the accompanying drawings. The following description is only a preferred embodiment of the application, which should not be used to explain or limit the scope of the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application should be included in the protection scope of the application. It should be understood that the experimental methods not specified in the following examples are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laborary Press, 1989), or the conditions recommended by the manufacturer.

[0033] I. General methods involved in the examples.

[0034] 1. Elisa detection of ATG7 protein level in serum: After collecting whole blood, stand at room temperature for 15-30 min, centrifuge at 1000-2000 g at 4°C for 10 min, and the upper layer is serum. Elisa detection of ATG7 protein level in serum, for specific use steps, see the kit instruction.

[0035] 2. Construction of STZ (Streptozotocin) type I diabetes mouse model: 6-8 week old C57 male mice, marking, weighing, recording the basic blood glucose value. Intraperitoneal injection of STZ (50 mg / kg) daily for 5 consecutive days. After 14-16 days, weigh, determine the blood glucose value, and determine the establishment of the STZ diabetes mouse model.

[0036] 3. In vivo blood glucose level detection: cut the tail tip of the mouse, use Roche blood glucose meter and blood glucose test paper to determine the blood glucose level in the mouse.

[0037] 4. Culture of Atg7 knockout mouse primary embryonic liver (Mouse Embryonic Hepatocyte, MEH) cells: After mating of Atg7 heterozygous mice, the pregnant female mouse (E14) takes the embryonic mouse, takes the liver, collagenase digestion, and subcultures inoculation after 48 hours of culture according to the experimental requirements.

[0038] 5. Culture of primary hepatocytes: C57 mice are killed by cervical dislocation, the liver is taken, cut and ground, centrifuged, trypsin digested, centrifuged, and inoculated on rat tail collagen.

[0039] 6. Hepatic glycogen detection: Take the liver tissue of mice, or primary cultured embryonic hepatocytes, and measure the hepatic glycogen. Refer to the kit instruction for specific experimental procedures.

[0040] 7. Glucose uptake experiment detection: After primary cultured embryonic hepatocytes are given Aap for 24 hours, measure the glucose uptake. Refer to the kit instruction for specific experimental procedures.

[0041] 8. Glucose production experiment detection: Incubate primary cultured embryonic hepatocytes or hepatocytes in glucose production medium (sugar-free DMEM without phenol red, 20 mM sodium lactate, and 2 mM sodium pyruvate) for 4 hours, take the supernatant, and measure the glucose concentration. Refer to the kit instruction for specific experimental procedures. Measure the sample protein concentration by BCA method as sample calibration.

[0042] 9. In vitro glucose level detection: Lyse the cells in RIPA lysis buffer at 4°C for 30 min, centrifuge the lysis buffer at 13000 rpm at 4°C for 20 min, separate, and take the supernatant to detect the glucose level. Refer to the kit instruction for specific experimental procedures. Measure the sample protein concentration by BCA method as sample calibration.

[0043] 10. Glucose tolerance test experiment: Inject glucose intraperitoneally into mice that have been fasted for 16 hours, with 1 g of glucose per kilogram of body weight. Measure the blood glucose level at 0, 15, 30, 50, 120 min. Use the blood glucose levels at 0 (BG0), 30 (BG30), 60 (BG60), 90 (BG90), and 120 (BG120) min to calculate the area under the curve (AUC) of the IPGTT result by the following formula: AUC (min·mg / dL) = 30min × [1 / 2 × (BG0 + BG120) + 1 × (BG30 + BG60 + BG90)].

[0044] 11. Western blotting: Lyse the cells in RIPA lysis buffer at 4°C for 30 min, centrifuge at 13000 rpm at 4°C for 20 min, and take the supernatant. Measure the sample protein concentration by BCA method and adjust to the same concentration. Add loading buffer, mix, and then perform protein SDS polyacrylamide gel electrophoresis at 95°C for 10 min. Electrotransfer to a PVDF membrane, block with 5% skim milk at room temperature for 1 hour, dilute the primary antibody in the antibody diluent to the appropriate concentration, and incubate at 4°C overnight. Wash 3 times with PBST, incubate the secondary antibody at room temperature for 1-4 hours, wash 3 times with PBST, and develop by ECL.

[0045] 12. Cell culture and transfection: Human hepatocarcinoma cell line HepG2 was cultured in MEM medium with 10% FBS, 1% NEAA, human hepatocarcinoma cell line Huh7 and human endometrial carcinoma KLE cell line were cultured in DMEM medium with 10% FBS, primary fetal liver cells and primary hepatocytes were cultured in DMEM medium with 15% FBS. All transfection was done by Lipofectamin 3000 (Invitrogen).

[0046] 13. Co-immunoprecipitation experiment: Cells were lysed in RIPA lysis buffer at 4°C for 30 min, then centrifuged at 13000 rpm, 4°C for 20 min, the supernatant was taken, and the protein concentration of the sample was determined by BCA method. The supernatant was quantitatively taken and added with the corresponding antibody, and incubated at 4°C overnight. Protein A / G-beads were added and incubated at 4°C for 4-6 hours. After the immunoprecipitation reaction, centrifugation was performed at 4°C, 3000g for 5 min, the supernatant was discarded, the protein A / G-beads were washed for 3 times, 2x SDS loading buffer 40 μL was added, 95°C, 10 min, and Western blotting detection and analysis.

[0047] II. Antibodies, kits and drugs used in the experiments in the examples.

[0048] 1. Elisa detection kit of human ATG7, Wuhan Huamei; Elisa detection kit of mouse ATG7, LsBio.

[0049] 2. Glycogen detection and glucose uptake detection kit of BIOVISION.

[0050] 3. Glucose detection kit of Sigma.

[0051] III. Commercial antibodies used in the experiments in the examples are from the following companies respectively.

[0052] 1. Rabbit anti-ATG7 antibody, mouse anti-GFP antibody, mouse anti-α-tubulin antibody of Sigma.

[0053] 2. Mouse anti-ATG7 antibody, mouse anti-IR antibody of Santa Cruz.

[0054] 3. Rabbit anti-IRS-1, IRS-1 phosphorylation, AKT, AKT phosphorylation, rabbit anti-GFP antibody of CST.

[0055] IV. Unless otherwise specified, other drugs and reagents in the examples are from Sigma Company.

[0056] V. Experimental animals: C57 mice from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0057] Six, Aap2 peptide directly synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. is applied in the present application.

[0058] Example 1 Elisa detection of ATG7 protein level in serum.

[0059] 1.1 Detection of ATG7 protein level in serum of diabetic patients and normal people.

[0060] Peripheral blood samples of diabetic patients and normal people from the First Affiliated Hospital of China Medical University were collected, serum was extracted, and Elisa detection was performed, see General Method 1.

[0061] The results showed that the ATG7 protein level in the serum of diabetic patients was significantly higher than that in the normal group, see Figure 1 A. It can be seen that the ATG7 protein level in the peripheral blood serum of diabetic patients is significantly higher than that in the normal population. ROC curve analysis showed AUC = 0.8446, see Figure 1 B. It can be seen that the ATG7 protein level has very high specificity and sensitivity for distinguishing between diabetic and non-diabetic controls.

[0062] 1.2 Detection of ATG7 protein level in peripheral blood serum of STZ diabetic model mice.

[0063] 1.2.1 Construction of STZ diabetic model mice, see General Methods 2 and 3.

[0064] 1.2.2 Collection of peripheral blood samples of STZ diabetic model mice and normal C57 mice, extraction of serum, Elisa detection, see General Method 1.

[0065] The results showed that the Atg7 protein level in the serum of STZ model mice was significantly higher than that in the normal group of mice, see Figure 2 A.

[0066] 1.3 Detection of Atg7 protein expression level in liver tissue.

[0067] Take the liver tissue of STZ model mice and normal mice, add RIPA lysis buffer, homogenate, 4℃ vortex for 45min, 13000rpm, 4℃, centrifuge for 20min, take the supernatant, measure the sample protein concentration by BCA method, and adjust to the same concentration. Western blot detection, see General Method 8.

[0068] The results showed that the Atg7 protein level in the liver tissue of STZ mice was lower than that in the normal group of mice, see Figure 2 B.

[0069] Example 2 ATG7 is involved in the regulation of insulin-mediated glucose metabolism.

[0070] 2.1 Detection of glucose metabolism in primary embryonic hepatocytes.

[0071] 2.1.1 Culture of primary embryonic hepatocytes, see General Method 4.

[0072] 2.1.2 Detection of glucose uptake, glycogen synthesis and glucose production in primary embryonic hepatocytes, see General Methods 5, 6 and 7.

[0073] The results show that the absence of Atg7 leads to a decrease in glucose uptake, a decrease in glycogen synthesis and an increase in glucose production in primary embryonic hepatocytes, see Figure 3 a, 3b and 3c.

[0074] 2.2 ATG7 regulates glycogen synthesis

[0075] 2.2.1 Detection of glycogen expression level in HepG2 cells transfected with ATG7 siRNA or myc-ATG7 plasmid, see General Methods 5 and 10.

[0076] 2.2.2 Detection of glycogen expression level in HepG2 cells transfected with ATG7 siRNA and stimulated with Insulin for 4 hours, see General Methods 5 and 10.

[0077] The results show that the absence of ATG7 leads to a decrease in glycogen synthesis; the increase in ATG7 expression leads to an increase in glycogen synthesis; ATG7 regulates glycogen synthesis, see Figure 3 d.

[0078] The increase in Insulin-mediated glycogen synthesis is dependent on the expression of ATG7, see Figure 3 e.

[0079] 2.3 Detection of the Insulin signaling pathway regulated by ATG7.

[0080] 2.3.1 Detection of the changes in the signaling pathway in WT cells and KO (Atg7- / -) cells in primary cultured MEF cells stimulated with Insulin.

[0081] The results show that Atg7 does not affect the activation of the Insulin receptor IR / IGF-1R upon Insulin stimulation; the activation of the substrate IRS1 and the downstream signal AKT are regulated by Atg7, see Figure 3 f.

[0082] 2.3.2 Detection of the changes in the binding of the receptor IR / IGF-1R to the substrate IRS1 upon the absence of ATG7 by co-immunoprecipitation experiments, see General Method 11.

[0083] Results showed that Insulin increased the binding of IR / IGF-1R to IRS1, and the absence of ATG7 blocked the binding of IR / IGF-1R to IRS1, leading to the blockage of Insulin signaling pathway, see Figure 3 g.

[0084] Example 3 The polypeptide Aap2 of the present application regulates the Insulin signaling pathway to affect glucose metabolism.

[0085] 3.1 Aap2 regulates the Insulin signaling pathway

[0086] Insulin or Aap2 stimulates primary MEF cells, HepG2 cells and Huh7 cells, and WB detects the Insulin signaling pathway.

[0087] Results showed that Aap2 can directly activate the signal transduction factor in the Insulin signaling pathway, see Figure 4 a, 4b and 4c.

[0088] 3.2 Aap2 regulates glucose metabolism

[0089] In primary cultured hepatocytes, Aap2 was given, and 24 hours later, glucose uptake, glycogen synthesis and glucose production were detected, see General Methods 6, 7 and 8.

[0090] Results showed that Aap2 increased glucose uptake and glycogen synthesis, and reduced glucose production, and Aap2 regulated glucose metabolism, see Figure 4 d, 4e and 4f.

[0091] Example 4 STZ diabetic model mice were used to detect the effect of Aap2 on reducing blood glucose.

[0092] STZ mice were injected intraperitoneally with Aap2 every other day, and blood glucose levels were detected, and after 10 days, the mice were executed by cervical dislocation, and liver tissue was taken to detect glycogen, see General Methods 3 and 6.

[0093] Results showed that Aap2 reduced the blood glucose level of STZ mice, n = 9 mice / group, see Figure 5 a; and increased glycogen synthesis in the liver of STZ mice, n = 9 mice / group, see Figure 5 b.

[0094] STZ mice were injected intraperitoneally with Aap2, and 24 hours later, a glucose tolerance test was performed, n = 8 mice / group, see General Methods.

[0095] Results showed that after STZ mice were given Aap2, glucose tolerance was reduced, see Figure 5 c, 5d is the AUC result. SEQUENCE LISTING <110> CHINA MEDICAL UNIVERSITY <120> Use of a novel polypeptide in the preparation of a medicament for diabetes <141> 09-DEC-2021 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 48 <212> PRT <213> Homo sapiens <400> 1 Val Leu Asp Gln Tyr Glu Arg Glu Gly Phe Asn Phe Leu Ala Lys Val 1 5 10 15 Phe Asn Ser Ser His Ser Phe Leu Glu Asp Leu Thr Gly Leu Thr Leu 20 25 30 Leu His Gln Glu Thr Gln Ala Ala Glu Ile Trp Asp Met Ser Asp Asp 35 40 45

Claims

1. Use of a polypeptide in the manufacture of a medicament for the treatment of diabetes, wherein the polypeptide has an amino acid sequence as set forth in SEQ ID No 1, and wherein the polypeptide is derived from a human ATG7 protein.

2. The use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of diabetes, wherein the medicament comprises: (a) a medicament for the prevention and / or treatment of diabetes; (b) a medicament for lowering blood glucose levels.

3. Use according to claim 2, characterized in that, The medicament in the use is in any pharmaceutically therapeutically acceptable dosage form.

4. Use according to claim 2, characterized in that, The medicament in the use is in any pharmaceutically therapeutically acceptable dosage.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the polypeptide in the use according to claim 1 and a pharmaceutically acceptable carrier or excipient.

3. The use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of diabetes, wherein the polypeptide has an amino acid sequence as set forth in SEQ ID No 1, and wherein the polypeptide is derived from a human ATG7 protein.

4. The use of a polypeptide according to claim 3 in the manufacture of a medicament for the treatment of diabetes, wherein the medicament comprises: (a) a medicament for the prevention and / or treatment of diabetes; (b) a medicament for lowering blood glucose levels. The medicament in the use is in any pharmaceutically therapeutically acceptable dosage form. The medicament in the use is in any pharmaceutically therapeutically acceptable dosage. The pharmaceutical composition comprises the polypeptide in the use according to claim 3 and a pharmaceutically acceptable carrier or excipient.

5. The use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of diabetes, wherein the polypeptide

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