Use of glutamate dehydrogenase inhibitor r162

Abstract

The application relates to the application technical field of glutamate enzyme inhibitors, in particular to the application of glutamate dehydrogenase inhibitor R162, and the glutamate dehydrogenase inhibitor R162 as a preparation for inducing amino acid metabolism disorder of type 2 diabetes mellitus. The application discloses the application of the glutamate dehydrogenase inhibitor R162, after intervention of R162, characteristic amino acid metabolism disorder of T2DM can be intensified, R162 can inhibit GDH expression in a specific mode, regulate the reaction between glutamic acid and alpha-KG, significantly hinder the glutamic acid oxidation and deamination process, cause abnormal accumulation of glutamic acid in cells, the pathological glutamic acid metabolism disorder further passes through multiple ways such as transamination reaction and urea cycle activation, widely influences the synthesis and conversion process of other amino acids, and the characteristic metabolism network imbalance of T2DM is preferably simulated, so that reliable experimental tools are provided for in-depth exploration of the molecular mechanism and intervention strategy of T2DM amino acid metabolism disorder.

Description

Technical Field

[0001] This invention relates to the field of glutamate inhibitor application technology, specifically to the application of glutamate dehydrogenase inhibitor R162. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by insulin resistance and progressive decline in pancreatic β-cell function. Recent research indicates that T2DM is not only a disorder of glucose and lipid metabolism but also involves complex amino acid metabolic abnormalities. Multiple clinical studies have confirmed significant amino acid metabolic imbalances in T2DM patients, such as significantly elevated urinary excretions of phenylalanine, arginine, tryptophan, tyrosine, and cysteine; and significantly decreased blood concentrations of γ-aminobutyric acid, arginine, glutamine, and phosphoethanolamine. Although studies have revealed amino acid metabolic imbalances in T2DM patients, the key regulatory nodes and intervention methods remain unclear.

[0003] The pathophysiological significance of amino acid metabolism disorder in type 2 diabetes mellitus (T2DM) has been widely recognized. However, traditional T2DM models induced by a high-fat, high-sugar diet combined with streptozotocin (STZ) injection mainly focus on the phenotype of glucose and lipid metabolism disorder, and are insufficient in reproducing the key pathological feature of abnormal amino acid metabolism (Zhou ZY, Song K, Liu ZY, et al. Branched-chain amino acids deficiency promotes diabetic cardiomyopathy by activating autophagy of cardiac fibroblasts. Theranostics. 2024;14(19):7333-7348. Published 2024 Oct 28. doi:10.7150 / thno.102708). There is still a lack of standardized animal models that can systematically simulate the characteristics of amino acid metabolism disorder in T2DM.

[0004] R162 is a highly efficient and specific inhibitor of glutamate dehydrogenase (GDH). This study aims to construct an animal model of amino acid metabolism disorder in type 2 diabetes mellitus (T2DM) using R162, providing a reliable experimental tool for further investigation into the molecular mechanisms and intervention strategies of T2DM amino acid metabolism disorder. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the glutamate dehydrogenase inhibitor R162.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Application of glutamate dehydrogenase inhibitor R162: R162 is used as a preparation to induce amino acid metabolism abnormalities in type 2 diabetes.

[0007] Furthermore, the dosage of the glutamate dehydrogenase inhibitor R162 is 3.33 mg / kg.

[0008] Furthermore, the glutamate dehydrogenase inhibitor R162 was used to construct an animal model of T2DM amino acid metabolism disorder.

[0009] Furthermore, the animal model was constructed by intraperitoneal injection of the glutamate dehydrogenase inhibitor R162 into rats after modeling.

[0010] Compared with existing technologies, this invention discloses the application of the glutamate dehydrogenase inhibitor R162. After intervention with R162, the characteristic amino acid metabolism disorder of T2DM can be aggravated. R162 regulates the reaction between glutamate and α-KG by specifically inhibiting GDH expression, significantly hindering the oxidative deamination process of glutamate, leading to abnormal accumulation of glutamate in cells. This pathological glutamate metabolism disorder then widely affects the synthesis and transformation of other amino acids through multiple pathways such as transamination reaction and urea cycle activation. It better simulates the characteristic metabolic network imbalance of T2DM, providing a reliable experimental tool for in-depth exploration of the molecular mechanism and intervention strategy of amino acid metabolism disorder in T2DM. Attached Figure Description

[0011] Figure 1 The levels of GDH and liver function indicators of rats in each group were compared (n=6); AD were the comparisons of serum GDH, liver GDH, serum ALT, and serum AST in each group of rats.

[0012] Figure 2 The comparison of glucose and lipid metabolism levels among the rat groups (n=6) was conducted as follows: A) Comparison of blood glucose levels among the rat groups; B) Comparison of blood lipid levels among the rat groups.

[0013] Figure 3 The following are the pathological morphological results of the livers of rats in each group: A. Results of HE, Oil Red O, and Masson staining of the livers of rats in each group; B. Relative area of ​​Oil Red O stained positive areas; C. Relative area of ​​Masson stained positive areas.

[0014] Figure 4 The expression levels of GDH mRNA and protein in the liver of rats in each group were compared. A. GDH protein expression level in the liver of rats in each group; B. GDH mRNA expression level in the liver of rats in each group; C. Relative expression level of GDH protein in the liver of rats in each group.

[0015] Figure 5 Comparison of α-KG metabolic levels in the livers of rats in each group (n = 6).

[0016] Figure 6 Comparison of amino acid metabolic levels in the livers of rats in each group (n = 6).

[0017] Note: In the figure, compared with the normal group, aP < 0.05, aaP < 0.01; compared with the model group, bP < 0.05, bbP < 0.01; compared with the high-dose R162 group, cP < 0.05, ccP < 0.01. Specific implementation manner

[0018] The technical solution of the present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only illustrative explanations of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0019] The animals, main reagents, and statistical methods used in the following embodiments are specifically as follows: 1. Experimental animals: 36 SPF-grade female healthy SD rats, purchased from Shanghai Slack Experimental Animal Co., Ltd., with the license number SCXK (Shanghai) 2022-0004, 8 weeks old, body weight (200 ± 20); housed in the Experimental Animal Center of Fujian University of Traditional Chinese Medicine, with the license number: SYXK (Fujian) 2023-0004; housing conditions: environmental temperature (22 ± 1) °C, humidity (50 ± 5)%; freely fed and watered, drinking water and bedding wood chips are changed daily, light is alternated between light and dark every 12 hours, and the animal experiment has been approved by the Medical Ethics Committee of Fujian University of Traditional Chinese Medicine (FJTCM IACUC 2024072), abiding by ethical principles and conforming to the relevant national and departmental guidelines for the protection and use of experimental animals.

[0020] 2. Reagents: High-fat, high-sugar feed containing 70.7% ordinary feed, 7% refined lard, 2% cholesterol, 0.3% pig bile salts, and 20% sucrose (Fuzhou Nordens Biotechnology Co., Ltd., batch number H1022); STZ and glutamate dehydrogenase inhibitor R162 (Shandong Cisco Biotechnology Co., Ltd., batch numbers SJ-MA0060 and 64302-87-0 respectively); GDH enzyme-linked immunosorbent assay kit (Wuhan Yilairuit Biotechnology Co., Ltd., catalog number E-BC-K759-M); rat C-peptide enzyme-linked immunosorbent assay kit (Wuhan Huamei Bioengineering Co., Ltd., catalog number CSB-E05067r); total cholesterol (TC), triglyceride (TG), non-esterified fatty acid (NEFA), and alanine aminotransferase (ALT). Aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits (Nanjing Jiancheng Technology Co., Ltd., catalog numbers A111-2-1, A110-1-1, A042-2-1, C009-2-1, C010-2-1, respectively); Hematoxylin-eosin (HE) staining kit (Beijing Lanjieke Technology Co., Ltd., batch number BL700B); Oil Red O staining solution (Beijing Solarbio Science & Technology Co., Ltd., catalog numbers G1261, G1340, respectively); GDH antibody, β-actin antibody, rabbit secondary antibody (Wuhan Sanying Biotechnology Co., Ltd., catalog numbers 14299-1-AP, 66009-1-Ig, SA00001-2, respectively); SPARKeasy tissue RNA rapid extraction kit, SPARKscript II All-in-one RT Supermix for qPCR, 2×SYBR Green qPCR Mix (Shandong Sikejie Biotechnology Co., Ltd., product numbers are AC0205-A, AG0305-B, and AH0104-B respectively).

[0021] 3. Statistical Methods: SPSS 27.0 software was used for data analysis, and Graphpad Prism 10.0 and Adobe Illustrator 2022 software were used for plotting. For intergroup comparisons, one-way ANOVA was used if the distribution was normal; LSD test was used for pairwise comparisons with homogeneous variances; Tamhance's T2 test was used for unequal variances; and Kruskal-Wallis H rank-sum test was used if the distribution was not normal. P < 0.05 was considered statistically significant.

[0022] Example 1 1. Experimental Grouping and Treatment: Thirty-six healthy female SPF-grade SD rats were acclimatized for one week and then subjected to an oral glucose tolerance test (OGTT). After 12 hours of fasting with unlimited water, blood was collected from the tail tip, and fasting blood glucose was measured using a micro-glucose meter. Immediately afterwards, the rats were administered 2 g / kg of 50% glucose injection via gavage, and the gavage time was recorded. Blood glucose was measured again 2 hours after gavage. Rats with fasting blood glucose >6.1 mmol / L or 2-hour postprandial blood glucose >7.8 mmol / L were excluded. After stratification by fasting body weight, 6 rats were randomly selected and fed a normal diet as the control group; the remaining 30 rats were fed a high-fat, high-sugar diet for modeling. After 4 weeks of feeding, the modeling rats were intraperitoneally injected with STZ (25 mg / kg) for 2 consecutive days. 72 hours after the injection, an OGTT was performed again. Rats with fasting blood glucose >11.1 mmol / L and postprandial blood glucose >16.7 mmol / L were considered to have successfully developed a diabetic model. During the 4-week modeling and subsequent screening process, two rats died (one died from acute STZ toxicity and the other died due to accidental death during gavage). Two other rats were excluded because their blood glucose levels did not meet the established modeling criteria. Ultimately, 24 rats were selected from the remaining 26 successful modeling rats for subsequent experiments. The fasting weight of the modeling rats was measured the following day, and they were stratified by weight and randomly divided into four groups using a random number table: the model group (intraperitoneal injection of an equal volume of physiological saline), the high-dose R162 group (intraperitoneal injection of 3.33 mg / kg R162), the medium-dose R162 group (intraperitoneal injection of 2.50 mg / kg R162), and the low-dose R162 group (intraperitoneal injection of 2.00 mg / kg R162). Injections were administered once daily for 14 consecutive days. During the injection period, the normal control group continued to be fed a normal diet, while the other groups continued to be fed a high-fat, high-sugar diet. The rats' condition was observed and recorded. After the injection, the rats were fasted for 12 hours but allowed free access to water before being harvested.

[0023] 2. Sample Processing: Rats were anesthetized by intraperitoneal injection of 20% urethane solution (5 mL / kg). After the corneal reflex disappeared and there was no response to painful stimuli, the rats were fixed in a supine position. The abdominal cavity was opened to fully expose the abdominal aorta, and the required blood volume was drawn using a disposable blood collection needle. After blood collection, euthanasia was performed by cervical dislocation, and the blood sample was immediately placed on ice to prevent blood clotting and degradation. Subsequently, the sample was centrifuged at 2500 rpm for 15 minutes at 4°C to separate the serum for later use. After the liver was removed, it was washed twice in pre-cooled pure water, and excess water was absorbed with filter paper. Then, the outer 1 / 3 to outer 2 / 3 of the right lobe of the liver was excised. Part of the liver was fixed in 4% paraformaldehyde, and the remaining liver was homogenized. The homogenate was centrifuged at 2500 rpm for 15 minutes at 4°C, and the supernatant was stored at -80°C for later analysis.

[0024] The serum, liver, liver homogenate, and other materials obtained in Example 1 were used in Examples 2-6 below.

[0025] In Example 1, blood glucose was measured again 2 hours after gavage, which is the same as the postprandial blood glucose in Example 3.

[0026] Example 2 Enzyme-linked immunosorbent assay (ELISA) was used to detect serum GDH, ALT, AST and liver GDH levels, mainly to show key targets and liver function levels. The procedure was strictly performed in accordance with the ELISA kit instructions.

[0027] See results Figure 1 Compared with the normal group, the serum and liver GDH levels in the model group and the high, medium, and low dose R162 groups were significantly reduced (P<0.01). Compared with the model group, the serum GDH levels in the high-dose and medium-dose R162 groups were significantly reduced (P<0.01), and the liver GDH level in the high-dose R162 group was also significantly lower than that in the model group (P<0.01). The high-dose R162 showed the most significant inhibitory effect on GDH, with its serum GDH level significantly lower than that in the low-dose group, and the liver GDH level in the high-dose R162 group significantly lower than that in the medium and low-dose groups (P<0.01).

[0028] Elevated ALT and AST levels indicate liver dysfunction. Serum ALT and AST levels were measured to assess the effect of R162 on liver function. Compared with the normal group, serum ALT levels in the model group and the high- and low-dose R162 groups were significantly increased (P<0.05 or P<0.01), and serum AST levels in the model group and the high-, medium-, and low-dose R162 groups were also significantly increased (P<0.01). However, there were no significant differences in ALT and AST levels among the different R162 dose groups compared with the model group. This indicates that liver damage was present in both the model group and each injection group, and the level of damage was similar among the model group and each injection group, suggesting that R162 injection did not cause additional liver function damage.

[0029] Example 3 Serum TC, TG, NEFA, and liver NEFA levels were detected using biochemical methods, with procedures strictly following the instructions of the test kits. Rat serum C-peptide levels were detected using an enzyme-linked immunosorbent assay (ELISA), with procedures strictly following the instructions of the test kits.

[0030] See Figure 2Compared with the normal group, the fasting and postprandial blood glucose levels of rats in the model group and the high-dose R162 group were significantly increased (P<0.01), and the blood glucose level in the high-dose R162 group was further higher than that in the model group (P<0.01). The serum C-peptide level of rats in the model group and the high-dose R162 group was significantly decreased (P<0.01), and the C-peptide level in the high-dose R162 group was lower than that in the model group (P<0.05). Compared with the normal group, the serum TC, TG, NEFA and liver NEFA levels of rats in the model group and the high-dose R162 group were significantly increased (P<0.01), and the TC, TG and liver NEFA levels in the high-dose R162 group were higher than those in the model group (P<0.05 or P<0.01).

[0031] Example 4 Liver tissue staining: HE staining: After washing, the tissue was embedded in paraffin, sectioned, and then subjected to dewaxing, hematoxylin staining, 1% hydrochloric acid ethanol differentiation, tap water immersion for blue return, eosin staining, washing, and resin mounting. Oil Red O staining: Frozen sections were fixed with 70% ethanol, washed with distilled water, stained with diluted Oil Red solution, protected from light and sealed, then washed with 60% ethanol and distilled water, finally counterstained with hematoxylin, washed with distilled water, and mounted with glycerol gelatin. Masson staining: After mordant staining and washing, the tissue was subjected to hematoxylin staining, 1% hydrochloric acid ethanol differentiation, ammonia water for blue return, then immersed in Masson's solution, washed with 1% acetic acid, 2% phosphomolybdic acid, and 2% orange G, finally dehydrated with ethanol, cleared with xylene, and mounted with neutral resin.

[0032] Quantitative analysis of liver pathology: Image J (Version 2.16.0) image analysis software was used for quantitative analysis. For Oil Red O stained sections, five non-overlapping regions were randomly captured at 200x magnification for each section. Orange-red lipid droplets were uniformly identified using a color threshold setting, and the percentage of lipid droplet area to the total liver tissue area within the field of view was calculated. For Masson stained sections, five regions were also randomly captured at 200x magnification. Blue-green collagen fibers were identified using a threshold setting, and the percentage of collagen fiber area to the total area was calculated.

[0033] See Figure 3HE staining of the liver showed that in the normal group, rat liver cells were arranged radially and uniformly around the central vein, with regular cell morphology and no obvious fatty degeneration in the cytoplasm, and the liver lobule structure was intact. In contrast, the liver cells in the model group and the high-dose R162 group were disordered, with irregular cell morphology, round lipid droplets of varying sizes visible in the cytoplasm, nucleus translocation, and damaged liver lobule structure, indicating significant fatty degeneration and hepatocyte damage. Masson staining showed that the liver lobule structure of the normal group was clear, and no obvious collagen fiber proliferation was observed around the liver tissue. However, proliferating collagen fibers were observed in the liver tissue of the model group and the high-dose R162 group, indicating liver fibrosis. Quantitative analysis showed that compared with the normal group, the relative area of ​​collagen fibers in the model group and the high-dose R162 group was increased (P<0.01). Oil Red O staining showed that in the normal group, rat hepatocytes showed blue nuclei and no obvious orange-red lipid droplets, indicating no significant lipid deposition. However, in the model group and the high-dose R162 group, large areas of deep orange-red lipid droplets were observed in the hepatocytes, confirming severe lipid deposition. Quantitative analysis showed that compared with the normal group, the relative area of ​​Oil Red O staining in the model group and the high-dose R162 group was increased (P<0.01), and the relative area of ​​Oil Red O staining in the high-dose R162 group was greater than that in the model group (P<0.01).

[0034] Example 5 Western blot analysis of GDH protein expression in liver tissue: Total protein was extracted from liver tissue, and the total protein concentration was determined using the BCA method. After sample loading, proteins were separated by gel electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 2 hours, and then incubated overnight at 4°C with primary antibody GDH and β-actin (both 1:1000). Secondary antibody (1:5000) was added, and the membrane was incubated at room temperature for 2 hours. Developed using ECL solution in the dark, the band gray values ​​were analyzed using ImageLab software after exposure and normalization.

[0035] RT-qPCR detection of GDH mRNA expression in liver tissue: Total RNA was extracted from liver tissue, and its concentration and quality were detected by spectrophotometer. cDNA was synthesized, and then GDH and β-actin were amplified using an RT-qPCR instrument. The primer sequences are as follows: GDH upstream primer (F): 5'-TCAACATACTTGGTCAAGCCAGC-3' (23 bp), downstream primer (R): 5'-GACCATCTCAACCAGGCCCAG-3' (21 bp); β-actin upstream primer (F): 5'-CTGGGACGACATGGAGAAAA-3' (20 bp), downstream primer (R): 5'-AAGGAAGGCTGGAAGAGTGC-3' (20 bp). The total reaction volume was 20 μL. The reaction conditions were: 94℃ for 75 seconds; 95℃ for 40 seconds, followed by annealing and extension according to different targets, for a total of 40 cycles. Using β-actin as an internal reference, 2 - The relative expression level of GDH mRNA was calculated using the ΔΔCt method, and the data were normalized.

[0036] See Figure 4 There was no statistically significant difference in GDH mRNA expression in the liver of rats among the groups (P>0.05). Compared with the normal group, the protein expression level of GDH in the liver of rats in the model group and the high-dose R162 group was significantly reduced (P<0.01), and the protein expression level of GDH in the liver of the high-dose R162 group was even lower than that in the model group (P<0.05).

[0037] Example 6 Targeted metabolomics detection of alpha-ketoglutarate (α-KG) and amino acid metabolites in the liver: A 600MRM high-throughput targeted metabolomics detection technique was used to quantitatively analyze amino acid metabolites in rat liver samples via liquid chromatography-tandem mass spectrometry (LC-MS / MS). 25 mg of liver tissue was weighed and added to 1500 μL of pre-chilled extraction buffer (methanol:acetonitrile:water = 2:2:1, containing an isotope internal standard). After homogenization, sonication, incubation at -40℃, and centrifugation, the supernatant was concentrated, reconstituted, and finally transferred to vials for LC-MS / MS analysis. An Agilent 1290 UHPLC ultra-high performance liquid chromatograph and a SCIEX 6500 QTRAP+ mass spectrometer were used in multiple reaction monitoring (MRM) mode for detection. Target metabolites were quantified using a standard curve method. Data processing was performed using SCIEX Analyst Workstation Software and BIOTREE BioBud software.

[0038] See Figure 5 Compared with the normal group, the α-KG metabolic level in the liver of rats in the model group and the high-dose R162 group was significantly reduced (P<0.01), while the high-dose R162 group showed a more significant decrease in α-KG level than the model group (P<0.05).

[0039] See Figure 6 Compared with the normal group, the metabolic levels of glutamate, L-aspartate, threonine, lysine, L-citrulline, and arginine in the liver of rats in the model group and the high-dose R162 group were significantly increased (P<0.05 or P<0.01). Specifically, the metabolic levels of glutamate, L-aspartate, lysine, and L-citrulline in the high-dose R162 group were further increased compared with the model group (P<0.05 or P<0.01), and the metabolic level of ornithine in this group was significantly increased compared with the normal group (P<0.05). Compared with the normal group, the metabolic levels of alanine, valine, serine, L-tryptophan, L-phenylalanine, L-tyrosine, L-leucine, and L-proline in rats in the model group and the high-dose R162 group were significantly decreased (P<0.01).

[0040] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of the glutamate dehydrogenase inhibitor R162, characterized by: R162, an inhibitor of glutamate dehydrogenase, is used as an agent that induces amino acid metabolism abnormalities in type 2 diabetes.

2. The application of the glutamate dehydrogenase inhibitor R162 according to claim 1, characterized in that: The dosage of the glutamate dehydrogenase inhibitor R162 is 3.33 mg / kg.

3. The application of the glutamate dehydrogenase inhibitor R162 according to claim 1, characterized in that: The glutamate dehydrogenase inhibitor R162 was used to construct an animal model of T2DM amino acid metabolism disorder.

4. The application of the glutamate dehydrogenase inhibitor R162 according to claim 3, characterized in that: The animal model was constructed by intraperitoneal injection of the glutamate dehydrogenase inhibitor R162 into rats after modeling.

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