Application of arginine vasopressin in improving glucose tolerance of type 2 diabetic mice

Abstract

This invention provides the application of arginine vasopressin in improving glucose tolerance in type 2 diabetic mice. This invention is the first to discover that arginine vasopressin can improve glucose tolerance in individuals with impaired glucose tolerance, while having no significant effect on normal individuals. This invention utilizes AVP to improve glucose tolerance and lipid metabolism disorders in diabetes, providing a new drug option for target research, adjunctive therapy, or complication management of diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of arginine vasopressin in improving glucose tolerance in type 2 diabetic mice. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by disordered glucose and lipid metabolism. The International Diabetes Federation (IDF) classifies diabetes into two main types: Type 1 diabetes, caused by autoimmune attack leading to the destruction of pancreatic beta cells, resulting in insulin deficiency; and Type 2 diabetes mellitus (T2D), the most common type, accounting for over 95% of all diabetes cases worldwide. One of the pathological features of T2D is insulin resistance, meaning glucose cannot enter cells, primarily due to defects in glucose transporter 4 (GLUT-4) and other insulin signaling pathways. Nearly 90% of T2D patients experience elevated blood glucose levels due to this characteristic. Current T2D therapies mainly focus on symptom management but cannot repair pancreatic beta cell function or reverse insulin resistance. To date, diabetes treatment still faces many unmet clinical needs, urgently requiring the discovery of new targets and the development of new drugs to overcome the current challenges.

[0003] Arginine vasopressin (AVP) is a peptide hormone composed of nine amino acids from the hypothalamus. Existing studies have reported elevated AVP levels in various metabolic disorders, such as insulin resistance, metabolic syndrome, type 2 diabetes (T2D), and obesity. Those skilled in the art generally agree that AVP is one of the causes of impaired glucose tolerance. Furthermore, some studies have shown that reducing AVP levels in the body has a protective effect on glucose tolerance, suggesting a negative correlation between AVP levels and glucose metabolism. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention is the first to discover that, under diabetic pathological conditions, exogenous AVP supplementation not only does not impair glucose tolerance but can also significantly improve it. Furthermore, long-term AVP treatment can significantly improve glucose and lipid metabolism disorders. Therefore, this invention provides the application of arginine vasopressin in improving glucose tolerance in individuals with type 2 diabetes.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A first aspect of the present invention provides the use of arginine vasopressin or a pharmaceutically acceptable salt thereof in the preparation of a medicament comprising any of the following: 1) Medications used to improve glucose tolerance in individuals with diabetes; 2) Medications used to improve glucose and lipid metabolism disorders in individuals with diabetes.

[0006] In this invention, "pharmaceutical-grade salt" and "pharmaceutically acceptable salt" are used interchangeably, referring to any acid addition salt or base addition salt whose counterion is non-toxic to the patient at the pharmaceutical dose of the salt. Such salts and their preparation methods are well known in the art. Acids that can be used to form pharmaceutically acceptable salts include inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) and organic acids (such as acetic acid, propionic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, lactic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.). Correspondingly, the salts include, but are not limited to, acetates, adipic acid salts, hydrochlorides, hydrobromic acid salts, sulfates, phosphates, nitrates, citrates, tartrates, succinates, fumarates, maleates, lactates, methanesulfonates, benzenesulfonates, etc.

[0007] In some embodiments, the improvement in glucose tolerance includes improvement in glucose tolerance after a single dose or multiple long-term doses.

[0008] In some embodiments, the improvement in glucose tolerance is manifested by at least one of the following: a decrease in the area under the curve after the glucose tolerance test, a decrease in the peak blood glucose level at a specific time point, and a faster rate at which blood glucose returns to baseline levels.

[0009] In some embodiments, each dose of the single or multiple long-term administration is a dose that effectively improves glucose tolerance in diabetic individuals.

[0010] In this invention, the "effective dose for improving glucose tolerance in diabetic individuals," or simply "effective dose," in one specific embodiment refers to the following: This invention, verified using a diabetic mouse model, shows that a dose of 25 nmol / kg significantly improves glucose tolerance in mice. Based on this verified effective dose, those skilled in the art can reasonably expect that doses within a reasonable range around this value (e.g., 18–36 nmol / kg, preferably 23–32 nmol / kg) may also be equally effective, but this invention is not limited thereto. In a specific embodiment of this invention, the effective dose for improving glucose tolerance in diabetic individuals is 25 nmol / kg.

[0011] In this invention, based on the aforementioned effective dose in mice, those skilled in the art can convert the corresponding human equivalent dose to approximately 2.75 nmol / kg using a commonly accepted inter-animal dose conversion method. This conversion method is widely accepted and used to guide the determination of the initial human dose. In this invention, the human dose of the drug can be a dose determined based on the above conversion result, such as 2.75 nmol / kg. Those skilled in the art will understand that due to individual differences, route of administration, formulation factors, etc., the actual human dose can be appropriately adjusted, for example, any value within the range of 2–4 nmol / kg, preferably 2.5–3.5 nmol / kg, as long as the effect of improving glucose tolerance is achieved. However, it should be understood that these human dose ranges are only expected based on theoretical conversion, and the specific effective human dose needs further verification through clinical trials.

[0012] In some embodiments, the glucose and lipid metabolism disorder includes abnormalities in one or more of the following indicators: total cholesterol, triglycerides, low-density lipoprotein cholesterol, alanine aminotransferase, gamma-glutamyl transferase, glucagon, and insulin.

[0013] In some embodiments, the diabetes is type 2 diabetes or prediabetes.

[0014] Furthermore, the prediabetes is characterized by impaired glucose tolerance.

[0015] In some embodiments, the diabetic individual is a mammal, including mice or humans.

[0016] Furthermore, the mouse is db / db Any of the following: mice and DIO model mice.

[0017] A second aspect of the present invention provides a pharmaceutical composition for improving glucose tolerance or glucose and lipid metabolism disorders in individuals with diabetes, the pharmaceutical composition comprising arginine vasopressin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0018] In this invention, the term "pharmaceutically acceptable carrier" refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to an individual receiving the drug or a combination thereof and can be administered without excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in pharmaceutical compositions may comprise fluids such as water, saline, glycerol, and ethanol. Such carriers may also contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc.

[0019] In some embodiments, the pharmaceutically acceptable carrier includes one or more of the following: diluent, excipient, flow aid, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and emulsifier.

[0020] In this invention, the term "medicine" refers to a drug containing at least one bioactive compound (e.g., spermidine). The medicines or compositions thereof described in this invention may be administered via intraperitoneal injection, subcutaneous injection, oral administration, non-gastrointestinal administration, inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or via an implanted drug storage device.

[0021] The medicaments or compositions thereof of the present invention may contain any commonly used non-toxic pharmaceutically acceptable carriers, excipients, or excipients. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrasheath, intra-injury site, and intracranial injection or infusion techniques. The medicaments or compositions thereof of the present invention may be administered to the receptor via any route, provided that the target tissue can be reached.

[0022] In some embodiments, the pharmaceutical composition further comprises at least one antidiabetic drug.

[0023] In this invention, arginine vasopressin or its pharmaceutically acceptable salt can be used in combination with one or more known antidiabetic drugs. The combined use does not produce antagonistic effects, meaning that neither drug affects the other's efficacy; the combined effect can be either simple additive (additive effect) or synergistic (synergistic effect). Those skilled in the art can determine the specific type of effect of the combined use using conventional pharmacodynamic experimental methods. The antidiabetic drug can be selected from metformin, sulfonylureas, meglitinides, thiazolidinediones, α-glucosidase inhibitors, DPP-4 inhibitors, SGLT2 inhibitors, GLP-1 receptor agonists, or insulin.

[0024] A third aspect of this invention provides a data processing method for evaluating the effect of arginine vasopressin on improving glucose tolerance in diabetic individuals, comprising the following steps: (1) Obtain glucose tolerance test data of diabetic individuals before and after administration of arginine vasopressin; (2) Calculate the area under the curve of the glucose tolerance test data; (3) Compare the changes in the area under the curve before and after administration. If the area under the curve decreases after administration, then arginine vasopressin is evaluated as having an improving effect on glucose tolerance in the diabetic individuals.

[0025] In one embodiment of the invention, "improvement" means reducing or alleviating a disease or one or more symptoms related to a disease. In some embodiments, the term refers to minimizing the spread or worsening of a disease due to the administration of one or more preventative or therapeutic agents to a patient suffering from the disease. For the purposes of the various aspects and embodiments provided by the invention, "improvement" includes, but is not limited to: reducing, alleviating, or improving one or more clinical manifestations or side effects of a treated disease or condition; improving one or more clinical outcomes; reducing the severity of a disease; delaying or slowing disease progression; improving, alleviating, or stabilizing a disease state; and other beneficial results described in the invention.

[0026] Advantages and beneficial effects of this invention: This invention is the first to discover that the effect of AVP on glucose tolerance is pathology-dependent, having no significant effect on healthy individuals, but exhibiting a clear improving effect on individuals with diabetes. This selectivity is not obvious. This invention utilizes AVP to improve glucose tolerance and glucose-lipid metabolism disorders in diabetes, providing a new drug option for target research, adjunctive therapy, or complication management of diabetes. Attached Figure Description

[0027] Figure 1 For AVP db / db The effect of glucose tolerance on mice is shown in the figure. A represents... db / db Mice were intraperitoneally injected with AVP / Saline seven days prior to GTT; B was... db / db Comparison of AUC of GTT in mice seven days after intraperitoneal injection of AVP / Saline; C represents db / db GTT was performed on mice after seven days of continuous intraperitoneal injection of AVP / Saline; D was db / db Changes in GTT levels in mice after seven days of continuous intraperitoneal injection of AVP / Saline; E represents... db / db Comparison of AUC values ​​of GTT changes after seven days of continuous intraperitoneal injection of AVP / Saline in mice.

[0028] Figure 2 For AVP db / db The effect of insulin tolerance on mice is shown in the figure. A represents... db / db Mice were intraperitoneally injected with AVP / Saline for seven days prior to ITT; B was... db / db Comparison of AUC of ITT in mice seven days after continuous intraperitoneal injection of AVP / Saline; C is db / db ITT in mice after seven days of continuous intraperitoneal injection of AVP / Saline; D was db / db Comparison of AUC of ITT in mice after seven days of continuous intraperitoneal injection of AVP / Saline.

[0029] Figure 3 For AVP db / db The effect of serum biochemical parameters and glucose metabolism hormones on mice is shown in the figure. A represents... db / db ALT and B levels in mice after seven days of continuous intraperitoneal injection of AVP / Saline were as follows: db / db AST levels in mice after seven days of continuous intraperitoneal injection of AVP / Saline; C is db / db γ-GT in mice after seven days of continuous intraperitoneal injection of AVP / Saline; D is db / db TP and E in mice after seven days of continuous intraperitoneal injection of AVP / Saline were as follows: db / db ALB in mice after seven days of continuous intraperitoneal injection of AVP / Saline; F is db / db ALP and G levels in mice after seven days of continuous intraperitoneal injection of AVP / Saline were as follows: db / db LDH in mice after seven days of continuous intraperitoneal injection of AVP / Saline; H is db / db TG in mice after seven days of continuous intraperitoneal injection of AVP / Saline; I is db / db LDL levels in mice after seven days of continuous intraperitoneal injection of AVP / Saline; J is db / db HDL and K in mice after seven days of continuous intraperitoneal injection of AVP / Saline db / db TC in mice after seven days of continuous intraperitoneal injection of AVP / Saline; L is db / db Insulin in mice after seven days of continuous intraperitoneal injection of AVP / Saline; M is db / db Glucagon levels in mice after seven consecutive days of intraperitoneal injection of AVP / Saline.

[0030] Figure 4 The graph shows the effect of a single AVP injection on glucose tolerance and insulin tolerance in different mice. A represents the change in GTT after a single intraperitoneal injection of AVP in DIO mice; B represents the comparison of AUC values ​​of the GTT changes after a single intraperitoneal injection of AVP in DIO mice; C represents the ITT after a single intraperitoneal injection of AVP in DIO mice; D represents the change in ITT after a single intraperitoneal injection of AVP in DIO mice; E represents the comparison of AUC values ​​of the ITT changes after a single intraperitoneal injection of AVP in DIO mice; F represents... db / db GTT in mice after a single intraperitoneal injection of AVP; G is db / db Comparison of AUC of GTT in mice after a single intraperitoneal injection of AVP; H represents db / db ITT of mice after a single intraperitoneal injection of AVP; I is db / dbComparison of AUCs for ITT after a single intraperitoneal injection of AVP in mice; J represents the GTT after a single intraperitoneal injection of AVP in C57 mice; K represents the AUCs for the GTT after a single intraperitoneal injection of AVP in C57 mice; L represents the ITT after a single intraperitoneal injection of AVP in C57 mice; M represents the AUCs for the ITT after a single intraperitoneal injection of AVP in C57 mice.

[0031] Figure 5 The graph shows the effect of AVP on the temperature of brown adipose tissue. Where A represents... db / db Thermographic images of mice seven days before and after continuous intraperitoneal injection of AVP / Saline; B represents... db / db Brown fat temperature in mice seven days after continuous intraperitoneal injection of AVP / Saline; C is db / db Brown fat temperature in mice after seven days of continuous intraperitoneal injection of AVP / Saline.

[0032] Figure 6 The graph shows the effects of AVP on brown adipose tissue and muscle metabolism. A represents... db / db Western blotting of UCP1 and GLUT4 in brown adipose tissue of mice after seven days of continuous intraperitoneal injection of AVP / Saline; B is db / db Western-Blot statistical analysis of UCP1 in brown adipose tissue of mice after seven days of continuous intraperitoneal injection of AVP / Saline; C represents... db / db Western-Blot plot of GLUT4 in brown adipose tissue of mice after seven days of continuous intraperitoneal injection of AVP / Saline; D represents db / db Western-Blot analysis of GLUT4 in muscle of mice after seven days of continuous intraperitoneal injection of AVP / Saline; E was... db / db Western-Blot statistical graph of GLUT4 in muscle of mice after seven days of continuous intraperitoneal injection of AVP / Saline.

[0033] Figure 7 This figure shows the effect of AVP on lipids in the liver. A represents... db / db Oil Red O staining image of mouse liver seven days after continuous intraperitoneal injection of AVP / Saline; B is... db / db A statistical chart of the area of ​​Oil Red O staining positive in the liver of mice after seven days of continuous intraperitoneal injection of AVP / Saline.

[0034] Figure 8 This is a graph showing the effect of AVP on hepatic glucose metabolism. Where A represents... db / db Mice after intraperitoneal injection of AVP / Saline for seven days Insr-B mRNA expression level; B is... db / db Mice after intraperitoneal injection of AVP / Saline for seven days Pklr mRNA expression level; C is db / db Mice after intraperitoneal injection of AVP / Saline for seven days Gys2 mRNA expression level; D is db / db Mice after intraperitoneal injection of AVP / Saline for seven days Pck1 mRNA expression level. Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Example 1: Effect of arginine vasopressin on improving glucose tolerance in type 2 diabetic mice I. Materials and Methods 1. Male db / db Twelve mice, weighing ≥40g, were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. (SCXK (Su) 2022-0016). db / db Mice were randomly divided into two groups of 6 each. The experimental group received intraperitoneal injections of AVP (25 nmol / kg) for 5 consecutive days, while the control group received intraperitoneal injections of an equal volume of physiological saline for 7 consecutive days. The experimental procedures were strictly in accordance with the regulations of the Animal Welfare and Ethics Committee of Chengdu Medical College (Chengdu Medical College Animal Ethics

[2022] No. 012).

[0037] 2. Instruments and reagents are listed in Tables 1 and 2.

[0038] Table 1. Experimental Apparatus

[0039] Table 2. Experimental Reagents

[0040] 3. Reagent preparation 1) 4% sodium pentobarbital: Weigh 1g of sodium pentobarbital solid and dissolve it in 25mL of sterile physiological saline.

[0041] 2) Glucose injection: Weigh 1.2g of glucose, add 6mL of sterile physiological saline, and prepare a 20% glucose solution. Prepare and use immediately.

[0042] 3) Insulin injection solution: Add 6 μL of insulin (100 units / mL) to 6 mL of sterile saline solution and prepare immediately before use.

[0043] 4) Arginine vasopressin injection: Take 25 mg of arginine vasopressin, add 46.11 mL of pure water to prepare a 500 µM stock solution, store at -40℃ for later use, and dilute to a 2.5 μM solution before use.

[0044] 4. Experimental Methods 1) Glucose tolerance test: After fasting for 16 hours, mice were intraperitoneally injected with 20% glucose solution at a dose of 1.0 g / kg to perform a glucose tolerance test (GTT). Tail clipping was performed at a specified time, and blood samples were collected using a blood glucose meter.

[0045] 2) Insulin tolerance test: Mice that have been fasted for 16 hours are subjected to an insulin tolerance test (ITT) via intraperitoneal injection of insulin at a dose of 1.0 units / kg. Tail clipping is performed at a specified time, and blood samples are collected using a blood glucose meter.

[0046] 3) ELISA assay: After 7 consecutive days of intraperitoneal injection of AVP / Saline, [the following was administered]... db / db Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (4 mL / kg), and blood was collected from the heart. After standing at room temperature for 30 min, the blood was centrifuged at 4℃ and 3000 rpm for 15 min, and the supernatant was collected as serum samples. The serum was aliquoted and placed in... Store at 80℃, avoiding repeated freeze-thaw cycles. Prepare standard solutions of different concentrations by serially diluting the standards according to the ELISA kit instructions for plotting a standard curve. Set up blank wells, standard wells, and sample wells. Add different concentrations of standard to the standard wells; add 50 μL of mouse serum sample to the sample wells. Each sample and standard should be in replicates. After adding samples, gently vortex to mix, and incubate at room temperature for 1.5 hours as per the instructions. After incubation, discard the liquid in the wells, wash the plate three times with washing buffer, allowing it to stand for 1 minute each time, and pat dry any remaining liquid. Add 1:500 enzyme-labeled secondary antibody to each well, gently mix, and incubate again for 1 hour. Repeat the washing steps to thoroughly remove unbound enzyme-labeled material. Add TMB chromogenic solution to each well, and react in the dark for 10 minutes until a clear blue color appears in the wells. Add stop solution to each well; the color will change from blue to yellow. Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm. A standard curve was plotted with the concentration of the standard on the x-axis and the OD value on the y-axis. The corresponding concentration was calculated based on the sample OD value for subsequent statistical analysis.

[0047] 4) Serum biochemical marker detection: After 7 consecutive days of intraperitoneal injection of AVP / Saline, [the following was administered]... db / db Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (4 mL / kg), and blood was collected from the heart. After standing at room temperature for 30 min, the blood was centrifuged at 3000 rpm for 15 min at 4℃, and the supernatant was collected as serum samples. The serum was aliquoted and placed in... Store at 80℃, avoiding repeated freeze-thaw cycles. Use a veterinary biochemical analyzer to test the following: Lipid profile (four items): Total cholesterol (TC), Triglycerides (TG), High-density lipoprotein cholesterol (HDL-C), Low-density lipoprotein cholesterol (LDL-C). Liver function tests (seven items): Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transferase (GGT), Albumin (ALB), Total protein (TP), Bilirubin (BIL). All procedures must be performed strictly according to the instrument's standard operating procedures and the reagent kit instructions.

[0048] 5) Statistical methods: GraphPad Prism 10 software was used for statistical analysis. Experimental data are expressed as mean ± standard error (±SE). This indicates that the comparison between the means of the two groups of samples uses... Unpaired t test The test results are as follows: P A value <0.05 is considered statistically significant for the difference between groups.

[0049] II. Experimental Results 1. After one week of continuous administration of arginine vasopressin db / db Changes in glucose tolerance in mice: To clarify the regulatory effect of AVP on blood glucose levels in T2D model mice, this study investigated... db / db Mice underwent a glucose tolerance test. GTT results before and after one week of intraperitoneal injection of AVP showed no difference in the area under the glucose tolerance curve (AUC) between the control group and the AVP group before injection (control group n=6, 2075.00±227.30 vs AVP group 2282.00±101.00, n=6). P >0.05, Figure 1 (B in the middle). db / db After 7 days of AVP intervention, the area under the curve (AUC) of the GTT change value in the AVP group was significantly reduced (control group n=6, 752.60±126.00 vs AVP group 215.40±74.08, n=6). P <0.01, Figure 1 (E in the text) demonstrates that continuous AVP intervention can improve glucose tolerance in T2D mice.

[0050] 2. After one week of continuous administration of arginine vasopressin db / db Changes in insulin tolerance in mice: To investigate the effect of AVP on insulin sensitivity in T2D mice, insulin levels were measured before AVP injection and one week after continuous injection. db / db Mice underwent an insulin tolerance test. The results showed that the area under the insulin tolerance curve (AUC) did not change significantly before and after AVP intervention (before injection, control group n=6, 98.12±9.25 vs AVP group 100.50±7.52, n=6).P >0.05, Figure 2 In the B group; after injection, the control group (n=6, 132.90±10.02) vs. the AVP group (122.40±4.98, n=6) P >0.05, Figure 2 The D in the figure indicates that under the experimental conditions, continuous AVP intervention did not have a significant effect on insulin sensitivity in diabetic mice.

[0051] 3. After one week of continuous administration of arginine vasopressin db / db Changes in serum biochemical parameters and glucose metabolism hormones in mice: To assess the effects of AVP on... db / db To investigate the effects on systemic metabolic function in mice, we examined serum biochemical parameters and levels of hormones related to glucose metabolism. Serum biochemical analysis revealed that alanine aminotransferase (ALT), a marker of liver injury, was significantly elevated (103.60±7.34 U / L in the control group (n=4) vs. 82.00±3.88 U / L in the AVP group (n=5)). P <0.05, Figure 3 In the A group, gamma-glutamyl transferase (γ-GT) was compared with that of the control group (n=5, 0.32±0.08 U / L) and the AVP group (0.1±0.00 U / L, n=5). P <0.05, Figure 3 The levels of C in the blood were significantly reduced, suggesting reduced liver damage and inflammation. Furthermore, triglycerides (TG) (control group n=5, 1.05±0.06 mmol / L vs AVP group 1.34±0.06 mmol / L, n=6) were significantly lower. P <0.01, Figure 3 H), low-density lipoprotein cholesterol (LDL-C) (control group n=6, 0.43±0.04mmol / L vs AVP group 0.70±0.09mmol / L, n=4, P <0.05, Figure 3 The levels of I) and serum total cholesterol (TC) (control group n=6, 3.62±0.16mmol / L vs AVP group 4.38±0.30mmol / L, n=5) were compared. P <0.05, Figure 3 The levels of potassium (K) in the control group were significantly elevated, while high-density lipoprotein cholesterol (HDL-C) did not change significantly (control group n=6, 2.88±0.11mmol / L vs AVP group 3.22±0.17mmol / L, n=6). P >0.05, Figure 3 (J in the text) indicates that AVP has been significantly altered. db / db Lipid transport and metabolism in mice. ELISA results showed that AVP intervention significantly reduced... db / dbSerum glucagon levels in mice (control group n=6, 243.80±11.28 mU / L vs AVP group 154.60±18.58 mU / L, n=5) P <0.01, Figure 3 The M in the control group increased insulin concentration (control group n=6, 39.18±3.35 mIU / L vs AVP group 52.56±3.98 mIU / L, n=5). P <0.05, Figure 3 (L in the text), this trend can explain the experimental results of AVP improving glucose tolerance.

[0052] In summary, the results indicate that AVP significantly increased serum insulin levels in individuals with type 2 diabetes (T2D) while decreasing glucagon levels, suggesting that the effect of AVP in alleviating glucose tolerance is related to glucose metabolism hormones.

[0053] Example 2: Effect of a single dose of arginine vasopressin on improving glucose tolerance in type 2 diabetic mice. I. Materials and Methods 1. Twenty-four male C57 mice, weighing 18-25 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. They were randomly divided into two groups of 12 mice each. One group was fed a high-fat diet (DIO) for 12 weeks and then injected with streptozotocin (STZ) at a dose of 100 mg / kg. Five days after STZ injection, mice with a 3-hour fasting blood glucose level ≥13.9 mmol / L were defined as T2D model mice (DIO mice). db / db Twelve mice, weighing ≥40 g, were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. C57 mice, DIO mice, and... db / db Mice were randomly divided into two groups of 6 each. The experimental group received a single intraperitoneal injection of AVP, while the control group received a single intraperitoneal injection of an equal volume of physiological saline. The experimental procedures were strictly in accordance with the regulations of the Animal Welfare and Ethics Committee of Chengdu Medical College (Chengdu Medical College Animal Ethics

[2022] No. 3).

[0054] 2. The instruments and reagents are the same as in Example 1, and other items are shown in Table 3.

[0055] Table 3. Experimental Reagents

[0056] 3. Streptozotocin (STZ) injection: Dissolve 0.12 g of streptozotocin in 12 mL of 0.1 M sodium citrate buffer (pH 4.5) to prepare a 1% injection solution. Prepare and use immediately. Inject 100 mg / kg into mice on a high-fat diet. Other reagents are prepared in the same way as in Example 1.

[0057] 4. Experimental Methods: Mice were fasted for 16 hours, and then intraperitoneally injected with insulin to perform an insulin tolerance test (ITT). The dose was 0.75 units / kg (C57 wild-type mice and DIO) or 1.0 units / kg ( db / db Mice). Tails were clipped at a specified time, and blood samples were collected using a blood glucose meter. Other experimental methods were the same as in Example 1.

[0058] II. Experimental Results 1. A single dose of arginine vasopressin has an effect on C57, db / db Effects of AVP on glucose tolerance in DIO mice: A single intraperitoneal injection of AVP significantly reduced glucose tolerance in DIO mice (control group n=6, 46.89±4.90 vs AVP group 25.01±3.34, n=6). P <0.01, Figure 4 B) AUC of GTT change value and db / db Mice (control group n=7, 3515.00±110.70 vs AVP group 2820.00±103.20, n=6) P <0.001, Figure 4 The AUC of GTT in G57 mice was measured. Notably, a single AVP injection did not affect the AUC of GTT in C57 mice (control group n=6, 913.70±100.70 vs AVP group 965.30±118.80, n=6). P >0.05, Figure 4 These results suggest that short-term effects of AVP can specifically improve glucose tolerance in T2D mice without affecting glucose tolerance in normal mice.

[0059] 2. A single dose of arginine vasopressin does not affect C57. db / db Insulin tolerance in DIO mice: A single injection of AVP does not change db / db Mice (control group n=6, 86.59±7.25 vs AVP group 85.81±13.24, n=6) P >0.05, Figure 4 The AUC of ITT in IQT mice and DIO mice (control group n=6, 25.37±4.41 vs AVP group 30.19±4.48, n=6) were compared. P >0.05, Figure 4 The AUC of the change in ITT in the E group also did not change the AUC of ITT in C57 mice (control group n=5, 400.70±81.79 vs AVP group 389.50±40.22, n=6). P >0.05, Figure 4 The M in the figure indicates that a single injection of AVP did not directly affect C57. db / db Insulin sensitivity in DIO mice.

[0060] The results in summary show that, under the same experimental conditions, AVP had no significant effect on glucose tolerance in wild-type mice, suggesting that its improving effect may be T2D specific, exerting its effect only in the pathological state of hyperglycemia, rather than in the physiological condition of normal blood glucose. Meanwhile, a single AVP administration had an effect on the glucose tolerance of three types of mice (wild-type, medium-type, and low-type). db / db Insulin tolerance in both the DIO model and long-term AVP treatment did not change significantly, a result consistent with long-term AVP treatment. db / db The results of the ITT experiment in mice were consistent, indicating that AVP does not directly affect peripheral insulin sensitivity, and this effect is independent of whether the mice are in a pathological state of hyperglycemia.

[0061] Therefore, AVP can reproduce its long-term regulatory effect in the acute intervention phase, specifically improving glucose metabolism disorders in T2D state, and this effect does not depend on the immediate change in insulin sensitivity.

[0062] Example 3: Effects of arginine vasopressin on thermogenesis in brown adipose tissue and metabolism in the liver and muscles of type 2 diabetic mice. Influence I. Materials and Methods 1. The experimental animals are the same as in Example 1.

[0063] 2. The instruments and reagents are the same as in Example 1, and other items are shown in Tables 4 and 5.

[0064] Table 4. Experimental Apparatus

[0065] Table 5. Experimental Reagents

[0066] 3. Reagent preparation 1) Electrophoresis buffer: 24 g Tris base, 150 g g glycine and 8 g sodium dodecyl sulfonate, 800 mL double-distilled water are placed in a 1 L beaker, stirred to dissolve and then brought to a final volume of 1000 mL. Store at room temperature for later use.

[0067] 2) Transfer buffer: 3.03 g Tris base, 14.41 g glycine, and 300 mL double-distilled water are placed together in a 1 L beaker, stirred to dissolve, and then 200 mL methanol is added to bring the volume to 1000 mL. Store at room temperature for later use.

[0068] 3) TBST: Weigh 8.8 g NaCl and 2.4 g Tris-base into a 1 L beaker, add 1000 mL of double-distilled water, stir to dissolve, adjust the pH to around 7.4 using HCl titration solution, and finally add 0.5 mL Tween 20, stir to dissolve, and store at room temperature for later use.

[0069] 4) Blocking solution: Add 10 g of skim milk powder or BSA to 150 mL of TBST and stir thoroughly to dissolve. Make up to 200 mL with TBST and store at 4°C.

[0070] 4. Thermal imaging to observe brown adipose tissue temperature: During the active period of mice (22:00), the surface temperature of the brown adipose tissue between the scapulae was continuously monitored using a thermal imager before and after continuous intraperitoneal injection of AVP. Each mouse was monitored for 1 minute each time to dynamically evaluate the effect of AVP on the heat production function of brown adipose tissue.

[0071] 5. Western Blot experiment: Western Blot was used to detect the expression of UCP1, GLUT4 in brown adipose tissue and GLUT4 in muscle tissue of AVP group and control group (Saline) mice.

[0072] Experimental Materials and Sample Collection (Muscle / Brown Fat GLUT4, Brown Fat UCP1 Content): This experiment used... db / db Gastrocnemius muscle and BAT from mice were used as samples. The expression level of GLUT4 protein was analyzed using Western blot, with β-actin as an internal control protein. Mice were quickly sacrificed, and BAT and gastrocnemius muscle tissues were removed, rinsed with cold physiological saline, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0073] Protein extraction and quantification: Tissue lysis: Approximately 0.01 g of tissue sample was placed in pre-chilled 1×RIPA lysis buffer (a mixture of 10×RIPA lysis buffer and deionized water at a ratio of 1:9, with 100× protease inhibitor added to achieve a final concentration of 1×). The tissue was thoroughly homogenized using a homogenizer under ice bath conditions, and then incubated at 4°C for 30 min for lysis. After lysis, the sample was centrifuged at 12000 rpm for 10 min, and the supernatant was collected as the total protein extract.

[0074] Protein quantification: The BCA protein concentration assay kit was used, following the instructions. The absorbance of the sample was measured at 562 nm, and the protein concentration was calculated based on the bovine serum albumin (BSA) standard curve.

[0075] SDS-PAGE Electrophoresis and Protein Transfer: Gel Preparation and Electrophoresis: Prepare 10%–12% gradient acrylamide gels (separating gels) using Tris–glycine electrophoresis buffer containing SDS. Denature the protein samples in the loading buffer and load them into the gels. Perform constant-voltage electrophoresis: The stacking gel is electrophoresed at 80 V, then the voltage is increased to 100 V after the sample enters the separating gel, until the protein bands are fully separated. Protein Transfer: After electrophoresis, transfer the proteins from the gel to a PVDF membrane (0.45 μm pore size). The PVDF membrane is pre-activated with methanol and placed in a wet transfer apparatus, where it is transferred at a constant current of 100 mA for 1 h at 4°C.

[0076] Protein blocking and antibody incubation: Blocking: Prepare a 5% skim milk blocking solution using TBS buffer (TBST) containing 0.1% Tween-20 and block the PVDF membrane for 1 h at room temperature to block non-specific binding sites. Primary antibody incubation: Add mouse anti-mouse anti-GLUT4 (proteintech, dilution 1:1000) and anti-β-actin primary antibody (Beyotime, dilution 1:5000) separately or in combination to the membrane and incubate overnight at 4°C. Add anti-UCP1 primary antibody (Abcam, dilution 1:1000) and anti-β-actin primary antibody (Beyotime, dilution 1:5000) separately or in combination to the membrane and incubate overnight at 4°C. Washing: Wash the membrane 3 times with TBST buffer for 5 min each time after each antibody incubation. Secondary antibody incubation: Add goat anti-rabbit IgG-HRP secondary antibody (Biosharp, dilution 1:5000) and incubate for 1 h at room temperature. Wash again: Wash the membrane three times with TBST buffer for 5 min each time to remove unbound secondary antibody.

[0077] Color development and imaging: Color development was performed using the SignalFire ECL chemiluminescence kit from Cell Signaling, and protein band signals were recorded using the QTouch imaging system from Rayward.

[0078] 6. RT-qPCR experiment 1) Total RNA extraction from the liver: Before first use, add 48 mL of anhydrous ethanol to each bottle of Buffer RW2 and mix thoroughly. Take 10-20 mg of fresh sample into an EP tube and add 1 mL of lysis buffer LB. Homogenize using a high-speed, low-temperature tissue homogenizer. Incubate the lysate at room temperature for 5 min to allow complete separation of the nucleic acid-protein complex. Add 0.2 mL of RNA extraction aid, tighten the cap, shake vigorously for 15 s, incubate at room temperature for 2-3 min, and centrifuge at 12000 rpm for 15 min. The sample will separate into three layers: an orange-yellow lower organic phase, an intermediate layer, and a colorless upper aqueous phase. Transfer 500 μL of the upper aqueous phase containing total RNA to a new centrifuge tube, add 250 μL of anhydrous ethanol, and mix by inverting several times (a precipitate will form). Transfer the resulting solution and precipitate together to a Spin Columns & Collection Tube (place the adsorption column in the collection tube). Centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 500 μL of Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 500 μL of Buffer RW2 to the adsorption column, centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube (add 48 mL of anhydrous ethanol to Buffer RW2 before use). Repeat step 7 once. Return the adsorption column to the collection tube and centrifuge at 12000 rpm for 2 min. Place the adsorption column into a new 1.5 mL centrifuge tube, add 50-100 μL of Buffer TB, incubate at room temperature for 1-2 min, centrifuge at 12000 rpm for 1 min to obtain the RNA solution.

[0079] 2) Primer Information: Full gene sequences were searched from the National Center for Biotechnology Information (NCBI) database, and gene-specific primers were designed and screened using Primer Premier primer design software. All primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by UltraPAGE.

[0080] Table 6. Primer Information

[0081] 3) Detection Results: The Threshold cycle (CT) values ​​of each sample tested during the PCR process were analyzed using QuantStudio™ Design & Analysis SE Software (Thermo). Our laboratory calculated the relative mRNA expression level of the gene using 2-ΔΔCT, and the fold change in gene mRNA expression was expressed as 2-ΔΔCT.

[0082] 7. Oil Red O Staining: Liver tissue is embedded in OCT embedding medium and sectioned using a cryostat, typically to a thickness of 18 μm. The sections are mounted on glass slides and air-dried at room temperature for 60 minutes. The dried sections are then rinsed with ultrapure water for 1 minute, followed by immersion in 60% isopropanol for 60 seconds to remove some non-specific background. Immediately after immersion, the sections are transferred to freshly filtered Oil Red O staining solution and stained at room temperature in the dark for 15 minutes. The sections are then transferred back to 60% isopropanol and quickly rinsed for 15 seconds to remove excess dye until the background is clean. The sections are then gently rinsed with ultrapure water for 1 minute to thoroughly remove the isopropanol. Next, the sections are immersed in hematoxylin staining solution for 30 seconds, followed by rinsing with running water or weakly alkaline water for 5 minutes to allow the blue color to return. Finally, neutral resin is added directly for mounting, and a coverslip is placed on top. The sections are then observed under an OLYMPUS X63 microscope.

[0083] 8. Statistical methods: Image J was used to perform quantitative statistical analysis on the Oil Red O staining results. The images were standardized, the area of ​​the positive region was measured, and the data were exported for statistical analysis to compare the differences in lipid content among different groups.

[0084] All experimental results were statistically analyzed using GraphPad Prism 10 software. Experimental data are expressed as mean ± standard error (±SE). This indicates that the comparison between the means of the two groups of samples uses... Unpaired t test The test results are as follows: P A value <0.05 is considered statistically significant for the difference between groups.

[0085] II. Experimental Results 1. Increased arginine vasopressin levels after one week of continuous administration. db / db Thermodynamics of Brown Adipose Tissue in Mice: To investigate whether AVP affects brown adipose tissue function in T2D mice, this study used infrared thermography to monitor temperature changes in brown adipose tissue in the scapular region of mice before and after AVP intervention. Results showed that during the active nocturnal period (22:00), there was no significant difference in BAT temperature between the control group and the AVP group before injection (control group n=6, 35.66±0.26℃ vs AVP group 35.93±0.28℃, n=6). P >0.05, Figure 5 (B in the text); but after a week of continuous AVP injections, db / db The temperature of the brown adipose tissue region in mice was significantly higher than that in the saline control group (control group n=6, 35.76±0.15℃ vs AVP group 36.62±0.10℃, n=6). P <0.001, Figure 5 (C in the text) suggests that AVP can promote db / db Thermogenesis occurs in brown fat in mice.

[0086] 2. Continuous administration of arginine vasopressin for 1 week has an effect on... db / db Effects of UCP1 and GLUT4 expression in mouse brown adipose and muscle tissue: To investigate whether changes occurred in brown adipose tissue thermogenesis and metabolism, Western blotting was used to detect the effects of AVP intervention for one week. db / db Expression levels of UCP1 and GLUT4 in brown adipose tissue of mice. Results showed that UCP1 expression in brown adipose tissue of mice in the AVP intervention group was significantly upregulated (control group n=6, 1.07±0.11 vs AVP group 2.01±0.18, n=6). P <0.01, Figure 6 In the B group, GLUT4 expression showed no significant difference compared to the control group (control group n=3, 1.00±0.10 vs AVP group 0.90±0.12, n=3). P >0.05, Figure 6 (C in the text). Notably, in skeletal muscle tissue, AVP significantly increased GLUT4 expression levels (control group n=4, 1.00±0.14 vs AVP group 1.62±0.08, n=6). P <0.01, Figure 6 (E in the text). The above results suggest that AVP enhancement... db / db The thermogenesis of brown adipose tissue in mice is directly related to the upregulation of UCP1 expression in the tissue, but it does not change glucose uptake. Therefore, the improvement of glucose metabolism by AVP not only stems from increasing thermogenesis of brown adipose tissue and enhancing energy expenditure, but may also improve systemic glucose metabolism in type 2 diabetes mellitus (T2D) by promoting the ability of skeletal muscle to take up glucose.

[0087] 3. Continuous administration of arginine vasopressin for 1 week has an effect on... db / db Effects of AVP on lipid droplets in mouse liver tissue: To clarify the effects of AVP on lipid metabolism in the liver of T2D mice, two groups were compared. db / db Mouse liver sections were analyzed using Oil Red O staining, and the area of ​​hepatic lipid droplets was quantitatively assessed. Histological staining results showed that, compared with the control group, the AVP intervention group exhibited significantly reduced red lipid droplet staining and a markedly smaller lipid droplet volume in liver sections. Statistical analysis of the images indicated that AVP intervention significantly reduced... db / db The percentage of lipid droplet area in mouse liver (control group n=6, 18548.00±679.20 μm² vs AVP group 14198.00±981.80 μm², n=6) P <0.01, Figure 7 (B in the text) suggests that continuous AVP treatment can reduce lipid accumulation in the liver.

[0088] 4. Continuous administration of arginine vasopressin for 1 week has an effect on... db / dbEffects of AVP on hepatic glucose metabolism in mice: To clarify the effects of AVP on hepatic glucose metabolism in T2D mice, we compared two groups... db / db Genes related to hepatic glucose metabolism in mice were examined. RT-qPCR analysis showed that AVP intervention did not affect hepatic insulin receptors. Insr-B (Control group n=5, 1.00±0.04 vs AVP group 1.06±0.05, n=6) P >0.05, Figure 8 A) Key enzymes in glycolysis Pklr (Control group n=5, 1.03±0.13 vs AVP group 0.10±0.22, n=6) P >0.05, Figure 8 B) Key enzymes in glycogen synthesis Gys2 (Control group n=5, 1.20±0.32 vs AVP group 0.93±0.15, n=6) P >0.05, Figure 8 C in the middle) and gluconeogenesis enzyme Pck1 (Control group n=5, 1.02±0.08 vs AVP group 1.02±0.11, n=6) P >0.05, Figure 8 The expression of D) mRNA in the liver was also observed. These results suggest that the mechanism by which AVP improves glucose tolerance in T2D mice may not be through regulation of hepatic insulin receptor signaling, glycolysis, gluconeogenesis, or glycogen synthesis pathways.

[0089] In summary, these results indicate that AVP can effectively improve the thermogenesis of brown adipose tissue, muscle glucose uptake, and hepatic lipid metabolism by synergistically regulating these functions. db / db Glucose and lipid metabolism disorders in mice. AVP significantly increased the temperature of brown adipose tissue and increased the expression of UCP1. AVP may reduce the metabolic burden on the liver by transporting lipids from the liver to the circulating blood through some transport mechanism (increased TC, TG, and LDL-C), thereby increasing uptake by other peripheral organs. The metabolic capacity of brown adipose tissue and muscle tissue will be greatly improved. This may be an important potential mechanism by which AVP improves T2D metabolism.

[0090] AVP significantly enhanced db / dbThermogenesis in mice via brown adipose tissue (BAT) not only increases local tissue temperature but also upregulates the expression level of UCP1 in brown adipose tissue, suggesting that AVP may alleviate diabetes-related metabolic disorders by activating the brown adipose tissue thermogenesis pathway. Simultaneously, AVP upregulates GLUT4 expression in skeletal muscle, enhancing glucose uptake and utilization in muscle tissue and improving peripheral glucose metabolism. Further histological analysis of liver tissue showed that AVP effectively reduced the degree of hepatic steatosis and decreased lipid deposition, indicating its positive role in maintaining hepatic lipid metabolic homeostasis. In summary, AVP improves glucose and lipid metabolism disorders through multi-tissue synergistic regulation, providing experimental evidence for its application in the treatment of diabetes.

[0091] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of arginine vasopressin or a pharmaceutically acceptable salt thereof for the manufacture of a medicament, characterized in that, The drug includes any of the following: 1) Medications used to improve glucose tolerance in individuals with diabetes; 2) Medications used to improve glucose and lipid metabolism disorders in individuals with diabetes.

2. Use according to claim 1, characterized in that, The improvement in glucose tolerance includes improvement in glucose tolerance after a single dose or multiple long-term doses.

3. Use according to claim 2, characterized in that, The improvement in glucose tolerance is manifested by at least one of the following: a decrease in the area under the curve after the glucose tolerance test, a decrease in the peak blood glucose level at a specific time point, and a faster rate at which blood glucose returns to baseline.

4. Use according to claim 2, characterized in that, The dose for each single or multiple long-term administration is the dose that effectively improves glucose tolerance in diabetic individuals. Preferably, the dose that effectively improves glucose tolerance in diabetic individuals is selected from 18–36 nmol / kg; Preferably, the effective dose for improving glucose tolerance in diabetic individuals is 25 nmol / kg.

5. The application according to claim 1, characterized in that, The glucose and lipid metabolism disorder includes abnormalities in one or more of the following indicators: total cholesterol, triglycerides, low-density lipoprotein cholesterol, alanine aminotransferase, gamma-glutamyl transferase, glucagon, and insulin.

6. The application according to claim 1, characterized in that, The diabetes referred to is type 2 diabetes or prediabetes; Preferably, the prediabetes is characterized by impaired glucose tolerance.

7. The application according to claim 1, characterized in that, The diabetic individual is a mammal, including mice or humans; Preferably, the mouse is db / db Any of the following: mice and DIO model mice.

8. A pharmaceutical composition for improving glucose tolerance or glucose and lipid metabolism disorders in individuals with diabetes, characterized in that, The pharmaceutical composition comprises arginine vasopressin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also contains at least one antidiabetic drug.

10. A data processing method for evaluating the effect of arginine vasopressin on improving glucose tolerance in diabetic individuals, characterized in that, Includes the following steps: (1) Obtain glucose tolerance test data of diabetic individuals before and after administration of arginine vasopressin; (2) Calculate the area under the curve of the glucose tolerance test data; (3) Compare the changes in the area under the curve before and after administration. If the area under the curve decreases after administration, then arginine vasopressin is evaluated as having an improving effect on glucose tolerance in the diabetic individuals.

Images