CN109078187B - Application of pancreatic GnIH receptor function regulator in preparation of medicine for treating type 2 diabetes - Google Patents
Application of pancreatic GnIH receptor function regulator in preparation of medicine for treating type 2 diabetes Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention relates to application of a pancreatic GnIH receptor function regulator in preparing a medicament for treating type 2 diabetes, and also relates to application of a pancreatic GnIH secretion regulator in preparing a medicament for treating type 2 diabetes. The inventors found that the expression of GnIH receptor exists in mouse pancreatic tissues and pancreatic alpha cell lines, and also found that RFamide-related peptide-3, which is a homolog of GnIH, can promote the activity and proliferation of pancreatic alpha-TC 1 clone 6 cells by binding to GPR147, which is a receptor thereof.
Description
Technical Field
The invention relates to a medicament for treating type 2 diabetes, in particular to application of a pancreatic GnIH receptor function regulator in preparing a medicament for treating type 2 diabetes and application of a pancreatic GnIH secretion regulator in preparing a medicament for treating type 2 diabetes.
Background
The pathogenesis of type 2 diabetes is not completely clear, however, abnormal islet hormone secretion is one of the important mechanisms in the pathogenesis of type 2 diabetes.
In pancreas, insulin secreted by beta cells of pancreatic islets mainly plays a role in reducing blood sugar, while glucagon secreted by alpha cells mainly plays a role in increasing blood sugar, and the stability of the blood sugar of the body is ensured by maintaining a certain balance between the two. Current research has found that patients with type 2 diabetes have both insulin resistance and insulin hyposecretion, and treatment is also focused primarily on the number and function of islet beta cells. However, it has also been found that glucagon levels are abnormally elevated in type 2 diabetic patients and, moreover, the number of alpha cells is also increased. Thus, the cellular number and function of α is closely linked to type 2 diabetes.
Disclosure of Invention
The research of the invention finds that the expression of GnIH receptors (G protein coupled receptor 147, GPR147, also called NPFFR) exists in mouse pancreatic tissues and a pancreatic alpha cell line (alpha-TC 1 clone 6), and also finds that the homologue RFamide-related peptide-3(RFRP-3) of GnIH can promote the activity and proliferation of pancreatic alpha-TC 1 clone 6 cells by combining with the receptor GPR 147.
Based on the above findings, the present invention provides, in one aspect, the use of a modulator of pancreatic GnIH receptor function for the preparation of a modulator of islet hormone secretion.
The pancreatic GnIH receptor function regulator is a pancreatic GnIH receptor function agonist and a pancreatic GnIH receptor function blocker.
In another aspect, the invention provides the use of a modulator of pancreatic GnIH receptor function in the manufacture of a formulation for modulating the number of pancreatic alpha cells.
In another aspect, the invention provides the use of a modulator of pancreatic GnIH receptor function for the preparation of a formulation for modulating glucagon secretion.
In yet another aspect, the present invention provides the use of a modulator of pancreatic GnIH receptor function for the manufacture of a medicament for the treatment of type 2 diabetes.
In a further aspect, the present invention provides the use of a pancreatic GnIH secretion modulator for the manufacture of an islet hormone secretion modulator.
Meanwhile, the invention provides application of the pancreatic GnIH secretion regulator in preparing a preparation for regulating the quantity of pancreatic alpha cells.
The invention also provides application of the pancreatic GnIH secretion regulator in preparing a preparation for regulating glucagon secretion.
In another aspect, the invention provides the use of a pancreatic GnIH secretion modulator for the manufacture of a medicament for the treatment of type 2 diabetes.
Dosage forms of the drug developed based on the present invention include both oral and injectable dosage forms.
Drawings
FIGS. 1A and 1B demonstrate the presence of mRNA expression of GnIH receptor (GPR147) in mouse pancreatic alpha and beta cell lines using RT-PCR (agarose gel electrophoresis); FIG. 1C demonstrates the presence of GPR147 protein expression in mouse pancreatic islets and pancreatic α cells using protein electrophoresis; FIG. 1D demonstrates GPR147 mRNA expression in pancreatic alpha cells using fluorescence in situ hybridization; figures 1E and 1F demonstrate GPR147 protein expression in pancreatic alpha cells and islet tissue using immunohistochemical techniques.
FIG. 2, ortholog of GnIH, RFRP-3, promotes pancreatic alpha cell viability in comparison to control (SF) under conditions of high glucose (25mM) and Serum starvation (Serum conservation).
FIG. 3 shows that RFRP-3 can increase the expression of Proliferating Cell Nuclear Antigen (PCNA) of pancreatic alpha cells by using protein electrophoresis technique.
FIGS. 4A and 4B, using protein electrophoresis techniques, RFRP-3 was found to function via the PI3K/AKT and ERK1/2 pathways; FIGS. 4C and 4D, the effect of RFRP-3 in promoting pancreatic alpha cell viability was abolished after addition of the antagonist of GPR147 (RF 9).
Fig. 5 shows that after GnIH binds to GPR147, which is its receptor, it exerts its activity and proliferation promoting effect through PI3K/AKT and ERK1/2 pathways, and after adding RF9, the binding of GnIH to its receptor is blocked, and this pathway is blocked and the effect disappears.
Detailed Description
The terms of the invention are explained:
GnIH (gonadotropin-inhibiting hormone) is a hormone secreted from the hypothalamus which negatively regulates the reproductive function of the body, is a neuropeptide secreted from the hypothalamus and plays a role as a "commander" in the hypothalamic-pituitary-gonadal axis. Recent studies have found that GnIH is involved in regulating food intake in animals and presumably in energy metabolism.
The pancreatic GnIH receptor function regulator is a substance influencing the functions of the pancreatic GnIH receptor (gonadotropin-inhibiting hormone receptor distributed in pancreas), and comprises the following components: pancreatic GnIH receptor blockers, pancreatic GnIH receptor agonists, and the like.
The pancreatic GnIH receptor function agonist is a substance which can be combined with a GnIH receptor and promotes corresponding physiological effect and drug effect, and comprises RFamide-related peptide-1; RFamide-related peptide-3; prolactin-releasing peptide; KGFF03, and the like.
The pancreatic GnIH receptor function blocker refers to a substance which can be combined with GnIH receptor, inhibit and block corresponding physiological effect and drug effect, and comprises 1-Adamantanecarbonyl-Arg-Phe-NH2trifluoroacetate salt (RF9 trifluoroacetate salt); GJ 14; BIBP 3226; KGFF09, and the like.
The pancreatic GnIH secretion regulator of the invention refers to a substance which affects the secretion of pancreatic GnIH.
The inventor discovers that: the receptor GPR147 of GnIH is present in the pancreas, and binding of GnIH to the receptor GPR147 promotes the viability and proliferation of pancreatic alpha cells.
Animal model: male C57BL/6 mouse pancreas and mouse pancreatic alpha cell line (alpha-TC 1 clone 6)
Experimental materials: GPR147 antibody (cat # orb157323, Biorbyt, UK); glucagon antibody (cat # ab10988, Abcam, UK); RFRP-3 (Cat: H-5846.0001, Bachem, Switzerland); a GPR147 upstream primer ATG AGC GGC TTG GTA CAG G, GPR147 downstream primer CGG AAA GGG TGT ACG ATG CA of RT-PCR, Servicobio, Wuhan; RevertAId First Strand cDNA Synthesis Kit (cat # K1622, Thermo Fisher Scientific, USA); FastStart Universal SYBR Green Master (Rox) kit (cat # 04913914001, Roche Life Science, USA); fluorescent in situ hybridization GPR147 probe sequence: 5'-FAM-GGGTA GGAGC GGTTG CGAGC ATCCA G-FAM-3', Wuhan Serviceibio.
The experimental method comprises the following steps:
RT-PCR. Total RNA was extracted by TRIzol method, RNA concentration was measured (D260/D280 was between 1.8-2.0), 2. mu.g of RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit, and 2. mu.L of cDNA was subjected to fluorescent quantitative PCR using the FastStart Universal SYBR Green Master kit. The amplification conditions were: pre-denaturation at 95 deg.C for 10 min; the cycle was 40 times, 95 ℃, 15s → 60 ℃, 60 s. The amplification products were separated by electrophoresis on a 1.5% agarose gel and detected.
2. Fluorescence in situ hybridization and immunofluorescence. alpha-TC 1 clone 6 cells were fixed with 4% paraformaldehyde (DEPC) for 20min, washed three times with PBS, gene-circled and digested with protease for 25min, followed by glucagon antibody (1:200 dilution) and incubated overnight at 4 ℃. After PBS washing, CY 3-labeled rabbit anti-mouse secondary antibody is added dropwise, the mixture is incubated at room temperature for 1h, after PBS washing is continued, the prehybridization solution is added dropwise, the mixture is incubated at room temperature for 1h, the hybridization solution containing GPR147 probe (8ng/mL) is added dropwise, and the mixture is incubated at 37 ℃ overnight. The SSC eluate is washed free of hybridization and the nuclei are counterstained with DAPI. And (4) dripping an anti-fluorescence quenching agent, sealing and then placing under a fluorescence microscope for shooting.
3. Western blot experiment. alpha-TC 1 clone 6 cells and mouse islets are washed three times by precooled PBS, RIPA lysate containing phosphatase inhibitor, 1% PMSF and 1% protease inhibitor is dripped, the mixture is placed on ice for cracking for 10min, and then the supernatant is collected by centrifugation, and the protein concentration is determined by BCA method. Mu.g of protein samples were electrophoresed using 10% SDS-PAGE, blocked with 5% skimmed milk powder for 2h at room temperature after the transfer, washed with TBST and incubated overnight at 4 ℃ with the corresponding primary antibody. And (3) washing the TBST three times, adding corresponding secondary antibody, incubating at room temperature for 1h, washing the TBST three times, and placing the TBST in a chemiluminescence imaging system for detection. The grey values of the respective bands were analyzed with ImageJ.
4. Immunohistochemistry. Mouse pancreas was fixed with 4% paraformaldehyde, and sections were paraffin embedded and deparaffinized. After the cell slide of alpha-TC 1 clone 6 was fixed with 4% paraformaldehyde at room temperature for 20 min. Antigen retrieval and inactivation of endogenous peroxidase were then performed and blocked with 3% BSA for 30 min. Then, the GPR147 antibody (1:200) was added dropwise and incubated overnight at 4 ℃, and after washing off the primary antibody, the HRP-labeled rabbit secondary antibody (1:200) was added dropwise and incubated at room temperature for 1 hour. DAB solution was incubated, hematoxylin was added to counterstain nuclei and photographed under a microscope.
The experimental results are as follows: FIGS. 1A and 1B demonstrate the presence of mRNA expression of GnIH receptor (GPR147) in mouse pancreatic alpha and beta cell lines using RT-PCR (agarose gel electrophoresis); FIG. 1C demonstrates the presence of GPR147 protein expression in mouse pancreatic islets and pancreatic α cells using protein electrophoresis; FIG. 1D demonstrates GPR147 mRNA expression in pancreatic alpha cells using fluorescence in situ hybridization; figures 1E and 1F demonstrate GPR147 protein expression in pancreatic alpha cells and islet tissue using immunohistochemical techniques.
FIG. 2, RFRP-3, an ortholog of GnIH, promoted the viability and proliferation of pancreatic alpha cells in comparison to the control (SF) under conditions of high glucose (25mM) and Serum starvation (Serum conservation).
FIG. 3 shows that RFRP-3 can increase the expression of Proliferating Cell Nuclear Antigen (PCNA) of pancreatic alpha cells by applying protein electrophoresis technology, and further proves that RFRP-3 can promote the activity and proliferation of pancreatic alpha cells.
The GnIH can promote the activity and proliferation of pancreatic alpha cells, so that the pancreatic GnIH receptor function regulator has the function of regulating the quantity of the pancreatic alpha cells and further regulating the glucagon level, and the pancreatic GnIH secretion regulator also has the function of regulating the quantity of the pancreatic alpha cells and further regulating the glucagon level.
To further verify whether the above-mentioned effects of GnIH act by binding to its receptor and thereby initiating intracellular signal transduction, the inventors selected antagonists of GnIH receptors for relevant studies.
The inventor researches and discovers that: the GnIH antagonist blocks the combination of GnIH and receptor GPR147, further blocks PI3K/AKT and ERK1/2 signal channel, inhibits the activity and proliferation of pancreatic alpha cells, further regulates the level of glucagon in the body, and the agonist and/or antagonist of GnIH receptor can be used for the development and application of medicaments related to the treatment of type 2 diabetes.
Animal model: mouse pancreatic alpha cell line (alpha-TC 1 clone 6)
Experimental biological materials: antagonist RF9 of GPR147 (cat # G-4800.0005, Bachem, Switzerland); PCNA antibody (cat # 2586T, Cell Signaling Technology, USA).
The experimental method comprises the following steps: the CCK8 method measures cell viability. Cells were digested and plated in 96-well plates (5X 10)3Per well), the next day, the medium was changed to DMEM medium containing 25mM glucose for 72 h. Then adding serum-free high-sugar DMEM medium for culturing for 12h, and then continuing culturing for 24 h by using serum-free medium containing RFRP-3 with corresponding concentration. Then, Cell Counting Kit-8 solution (10. mu.L/well) was added thereto, and after culturing for 1 to 4 hours, the cells were placed in a microplate reader to measure the optical density at a wavelength of 450 nm.
The experimental results are as follows: FIGS. 4A and 4B, using protein electrophoresis techniques, RFRP-3 was found to function via the PI3K/AKT and ERK1/2 pathways. FIGS. 4C and 4D, addition of GPR147 antagonist (RF9), the PI3K/AKT and ERK1/2 pathways were blocked, and the effect of RFRP-3 in promoting pancreatic alpha cell viability and proliferation was abolished, reducing glucagon levels, as shown in FIG. 5.
Claims (2)
1. Use of a modulator of pancreatic GnIH receptor function RF9 for the preparation of a formulation for modulating the number of pancreatic alpha cells.
2. Use of a pancreatic GnIH receptor function modulator RF9 for the manufacture of a medicament for the treatment of type 2 diabetes.
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| WO2004089399A1 (en) * | 2003-04-01 | 2004-10-21 | Takeda Pharmaceutical Company Limited | Novel uses of rfrp and ot7t022 |
| CN102316735A (en) * | 2008-12-23 | 2012-01-11 | 哈佛大学校长及研究员协会 | The micromolecular inhibitor of gangrenosum acne apoptosis |
| CN107412769A (en) * | 2017-06-30 | 2017-12-01 | 中国人民解放军第四军医大学 | Pancreas GnRH receptor function modulators prepare the application for the treatment of diabetes B medicine |
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| WO2004089399A1 (en) * | 2003-04-01 | 2004-10-21 | Takeda Pharmaceutical Company Limited | Novel uses of rfrp and ot7t022 |
| CN102316735A (en) * | 2008-12-23 | 2012-01-11 | 哈佛大学校长及研究员协会 | The micromolecular inhibitor of gangrenosum acne apoptosis |
| CN107412769A (en) * | 2017-06-30 | 2017-12-01 | 中国人民解放军第四军医大学 | Pancreas GnRH receptor function modulators prepare the application for the treatment of diabetes B medicine |
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| Title |
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| Editorial: The Roles of GnIH in Reproductive Function and Behavior;Takayoshi Ubuka等;《FRONTIERS IN ENDOCRINOLOGY》;20180131;第9卷(第19期);第1-2页 * |
| Takayoshi Ubuka等.Editorial: The Roles of GnIH in Reproductive Function and Behavior.《FRONTIERS IN ENDOCRINOLOGY》.2018,第9卷(第19期),第1-3页. * |
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