CN115006536A - Application of Sgk1 as target in preparation of medicine for inhibiting gluconeogenesis - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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 Sgk1 as a target in preparation of a medicine for inhibiting gluconeogenesis. The invention proves the regulation and control effect of Sgk1 on gluconeogenesis in vitro and in vivo, and Sgk1 is likely to become a target for inhibiting gluconeogenesis in liver by medicine, thereby being expected to become a new target for treating type 2 diabetes.
Description
Technical Field
The invention belongs to the field of type 2 diabetes mellitus medicines, and particularly relates to application of Sgk1 as a target point in preparation of a medicine for inhibiting gluconeogenesis.
Background
Type 2 diabetes is an endocrine-metabolic disease characterized by chronic hyperglycemia, is mainly caused by insufficient insulin secretion or reduced peripheral insulin sensitivity, and is one of ten chronic diseases in the world. The international diabetes consortium (IDF) recent data showed that the population suffering from diabetes worldwide in 2019 has reached 4.63 hundred million.
The hyperglycemia existing in the diabetic patients for a long time can cause various tissues, particularly cardiovascular, nerve, eye, kidney, limbs and the like to suffer chronic damage, induce dysfunction, cause various complications such as cardiovascular diseases, visual loss, renal failure and the like, seriously affect the health of the patients and bring huge burden to the society and the country. Therefore, elucidation of the pathogenesis of diabetes and prevention and treatment of diabetes by effective means have been the focus of public health concern worldwide.
Glucose is an important energy source of organisms, and the maintenance of blood glucose homeostasis is a prerequisite for ensuring the normal function of organ tissues. Throughout the feeding-fasting cycle, the liver plays a key role in maintaining glucose homeostasis by regulating glycogenesis, glycogenolysis, glycolysis, and gluconeogenesis. More and more researches show that abnormally increased hepatic gluconeogenesis is one of the factors which have the greatest contribution to fasting hyperglycemia of type 2 diabetes patients and is also an important pathophysiological change in the occurrence and development processes of type 2 diabetes. Therefore, the regulation mechanism of hepatic gluconeogenesis is deeply explored, abnormal increase of liver gluconeogenesis is inhibited, and the method has great significance for preventing and treating type 2 diabetes.
Sgk1 is a serine/threonine protein kinase, also known as serum/glucocorticoid-regulated kinase 1, because its mRNA levels are rapidly elevated by stimulation with serum and/or glucocorticoids. Sgk1 has been reported to have a broad role, involved in the regulation of various ion channels, membrane transporters, cell proliferation and apoptosis. There is some evidence supporting the involvement of Sgk1 in the regulation of glucose homeostasis, and studies have shown that over-activation of Sgk1 can impair insulin secretion by islet beta cells, but appears to also enhance insulin sensitivity in peripheral organs. There are also reports in the literature that Sgk1 is also associated with the development of diseases such as hypertension, obesity, diabetes, etc. It is therefore of great importance to study the role of Sgk1 in type 2 diabetes.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of Sgk1 as a target point in preparing a medicine for inhibiting gluconeogenesis, and the invention proves that Sgk1 has a regulation and control effect on gluconeogenesis, and Sgk1 is likely to become a target point for inhibiting gluconeogenesis by the medicine, so that the Sgk1 is expected to become a new target point for treating type 2 diabetes.
The invention provides application of Sgk1 as a target point in preparation of a medicament for inhibiting gluconeogenesis.
Preferably, the medicament reduces hepatic gluconeogenesis by interfering with or inhibiting Sgk 1.
Preferably, the medicine takes Sgk1 as a target spot, and is prepared into a preparation by matching with pharmaceutically acceptable auxiliary materials or auxiliary components.
Preferably, the preparation is selected from one of injection, subcutaneous implant, tablet, powder, granule, capsule, oral liquid and sustained release preparation.
Advantageous effects
The invention proves the regulation and control effect of Sgk1 on gluconeogenesis in vitro and in vivo, and Sgk1 is likely to become a target for inhibiting gluconeogenesis in liver by medicine, thereby being expected to become a new target for treating type 2 diabetes.
Drawings
FIG. 1 shows the results of the Sgk1 knockdown in example 1 without affecting the cell viability of mouse liver primary cells, wherein sh-Ctrl is control unloaded virus and sh-Sgk1 is Sgk1 interfering adenovirus.
FIG. 2 is the result of suppressing gluconeogenesis of mouse liver primary cells by knockdown of Sgk1 in example 1, in which (A-D) was treated with or without 100. mu.M 8-Br-cAMP for 8 hours and mRNA expression levels of Sgk1 and PEPCK, G6Pase and FBPase were measured by qRT-PCR; treatment with or without 100. mu.M 8-Br-cAMP for 24 hours, (E) detection of PCK protein expression level by Western Blot (internal reference GAPDH), (F) detection of glucose production in cell culture medium by glucose oxidase method.
FIG. 3 shows the results that GSK650394 did not affect the cell viability of mouse liver primary cells in example 1, wherein (A) the chemical structure of GSK 650394; (B) the CCK-8 assay detects cell viability of mouse liver primary cells.
FIG. 4 is the mRNA expression levels of GSK650394 in example 1 for inhibiting gluconeogenic key enzymes PEPCK (A), G6Pase (B) and FBPase (C).
FIG. 5 shows the results of GSK650394 inhibiting glucose production by mouse liver primary cells in example 1.
FIG. 6 shows the results of testing the expression levels of Sgk1 gene of Liver (Liver) (A), Skeletal muscle (Skeletal muscle) (B) and Adipose tissue (Adipose tissue) (C) by qRT-PCR after Sgk1 interfering adenovirus is injected into C57BL/6 mice via tail vein in example 2.
Fig. 7 shows the mRNA expression levels of liver fbpase (a), G6Pase (B) and pepck (c) (n ═ 6) detected by qRT-PCR using specific knockdown liver Sgk1 in example 2.
FIG. 8 shows the results of random body weight monitoring (A) after one week of tail vein injection of control or Sgk1 interfering adenovirus, respectively, in example 2; (B) body weight fasted for 16 hours; (C) food intake in 24 hours; (D) random blood glucose levels.
FIG. 9 shows the results of the liver-specific knockdown of Sgk1 in example 2 to inhibit hepatic gluconeogenesis in mice.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the claims appended to the present application.
Male C57BL/6 mice at 8 weeks of age were purchased from Experimental animals technology, Inc. of Weitongli, Beijing. Sodium pyruvate, dexamethasone, 8-bromo-3 '-5' -Cyclic adenosine monophosphate (8-Br-3 '-5' -Cyclic adenosine monophosphate, cAMP) was purchased from Sigma. The Cell Counting Kit-8(CCK-8) Kit was purchased from Beyotime.
Example 1
Transfection of Sgk1 interferes with adenovirus to suppress gluconeogenesis of mouse liver primary cells:
inoculating mouse liver primary cells in a 96-well plate, transfecting Sgk1 to interfere adenovirus, and performing a CCK-8 test 24 hours later, wherein the test specifically comprises the following steps: 1) inoculating mouse liver primary cells into 96-well plate, culturing in incubator (37 deg.C, 5% CO) with at least 4 multiple wells 2 ). 2) After the cells adhere to the wall, Sgk1 is transfected to interfere adenovirus for 24h, and then culture solution is changed100. mu.l of low-sugar DMEM culture solution containing 0.25% BSA and 10% CCK-8 solution was prepared and cultured for 1 hour. 3) And (4) oscillating for 10 seconds in a microplate reader, measuring absorbance at the wavelength of 450nm, and finally calculating to obtain the cell activity. Results as shown in figure 1, the knockdown of Sgk1 did not affect the cell viability of mouse liver primary cells compared to the control virus (sh-Ctrl) (figure 1).
Control unloaded viruses (sh-Ctrl) and Sgk1 interfering adenoviruses (sh-Sgk1) were transfected into mouse liver primary cells for 24 hours, respectively, pretreated with 100nM dexamethasone for 16 hours, and then the culture was changed to a sugar-free culture containing gluconeogenic substrates (10mM sodium lactate and 1mM sodium pyruvate), and divided into sub-groups containing only gluconeogenic substrates and 100. mu.M 8-Br-cAMP-treated groups. Cell total RNA was extracted by Trizol with or without 100. mu.M 8-Br-cAMP for 8 hours, mRNA expression levels of Sgk1 and PEPCK, G6Pase and FBPase were detected by real-time fluorescent quantitative PCR after reverse transcription, and as a result, as shown in FIGS. 2A-D, cAMP stimulated gene expression of Sgk1, and Sgk1 interfering adenovirus both significantly down-regulated mRNA expression levels of Sgk1 in mouse primary hepatocytes (FIG. 2A). Meanwhile, knock-down of Sgk1 in mouse liver primary cells significantly down-regulated mRNA expression levels of three gluconeogenic key enzymes PEPCK, G6Pase and FBPase stimulated by cAMP (fig. 2B-2D). In addition, the protein expression level of PCK (internal reference GAPDH) was measured by Western Blot with or without 100. mu.M 8-Br-cAMP for 24 hours, and as a result, as shown in FIG. 2E, the cAMP-induced PCK protein expression was also decreased; the amount of glucose produced in the cell culture medium was measured by the glucose oxidase method, and the results are shown in fig. 2F, where the knock-down of Sgk1 significantly inhibited cAMP-stimulated endogenous glucose production in mouse liver primary cells. Taken together, the knockdown of Sgk1 inhibited gluconeogenesis in mouse liver primary cells.
Sgk1 inhibitor GSK650394 inhibits mouse liver primary cell gluconeogenesis:
mouse liver primary cells were inoculated into a 96-well plate, and after 24-hour treatment with 0-10 μ M of an inhibitor GSK650394 (chemical structure shown in fig. 3A) of Sgk1 at different concentrations, the CCK-8 assay (same above) was performed, and as a result, as shown in fig. 3B, GSK650394 did not affect the cell viability of the mouse liver primary cells.
Separating the primary mouse liver cells, pretreating the cells with 100nM dexamethasone for 16 hours after the cells adhere to the walls, and then changing the culture solution into a sugar-free culture solution containing gluconeogenesis substrates, wherein the four groups are sequentially as follows: a sub-group containing only gluconeogenic substrates; 100 μ McAMP treated group; 5 μ M GSK650394 treatment group; the group was co-treated with 100. mu.M cAMP and 5. mu.M GSK650394, and mRNA expression levels of gluconeogenesis key enzymes PEPCK, G6Pase and FBPase were detected by qRT-PCR 8 hours after the treatment of mouse liver primary cells. Real-time fluorescent quantitative PCR results showed that GSK650394 significantly reduced cAMP-stimulated PEPCK, G6Pase and FBPase mRNA expression levels (fig. 4A-4C). In addition, the amount of glucose produced in the cell culture medium was measured by the glucose oxidase method 24 hours after treating primary mouse liver cells with 100. mu.M cAMP and 5. mu.M GSK 650394. Gluconeogenesis assay results showed that GSK650394 also significantly inhibited cAMP-stimulated endogenous glucose production (fig. 5).
In conclusion, both the Sgk1 interfering adenovirus and the Sgk1 inhibitor GSK650394 can inhibit the gluconeogenesis level of mouse liver primary cells, which indicates that the expression level of Sgk1 in liver is positively correlated with the gluconeogenesis in liver.
Example 2
12 8-week-old male C57BL/6 mice were randomly divided into a control unloaded virus group (sh-Ctrl) and an Sgk1 interfering adenovirus group (sh-Sgk1), tail vein-injected with control unloaded virus or Sgk1 interfering adenovirus, respectively, and mRNA expression levels of Sgk1 in Liver (Liver), Skeletal muscle (Skeletal muscle) and Adipose tissue (Adipose tissue) were measured by qRT-PCR on two groups of mice fasted for 16 hours on day 9 after injection, respectively. The results showed that the mRNA expression level of Sgk1 in the livers of the sh-Sgk1 group mice was significantly reduced compared to the control group mice (fig. 6A), while there was no significant change in skeletal muscle and adipose tissue (fig. 6B-6C), which was consistent with the hepatotropic properties of the viruses used, demonstrating that Sgk1 interfering adenoviruses can specifically reduce the expression of Sgk1 in the C57BL/6 mouse livers. In addition, mRNA expression levels of gluconeogenesis key enzymes PEPCK, G6Pase and FBPase in mouse liver tissues are detected by qRT-PCR, and the result shows that the mRNA expression level of Sgk1 interfering adenovirus group mouse liver FBPase is remarkably reduced (figure 7A), the mRNA expression level of G6Pase is downward-regulated (figure 7B) and the mRNA expression level of PEPCK is not remarkably changed (figure 7C) compared with a control group, so that the liver-specific knock-down of Sgk1 is proved to inhibit the expression of C57BL/6 mouse liver FBPase.
The two groups of mice were injected with control or Sgk1 interfering adenovirus in tail vein, and after one week of injection, random body weight, body weight after 16 hours of fasting, food intake at 24 hours, and random blood glucose level were monitored, and it was found that there was no significant difference in random body weight, fasting body weight, food intake, etc. (fig. 8A-8C). However, mice injected with Sgk1 interfering adenovirus had a significant drop in random blood glucose compared to control mice (fig. 8D). To confirm whether liver-specific knockdown Sgk1 affected hepatic gluconeogenesis, one week after adenovirus injection, pyruvate tolerance test (IPPTT) was performed on two groups of mice fasted for 16 hours, mouse body weight and blood glucose (i.e., 0 min) were measured and recorded after the mice were fasted for 16 hours, each mouse was intraperitoneally injected with 2g/kg body weight of sodium pyruvate, timing was started immediately after injection was completed, blood was sequentially measured by tail-cutting and blood glucose levels were recorded at 0, 15, 30, 60 and 120 min after sodium pyruvate solution injection, and it was found that the blood glucose levels of the sh-Sgk1 group mice were lower at each time point than those of the control group, and that the difference between the 30 and 60 min time points was particularly significant (fig. 9). Thus, liver-specific knockdown of Sgk1 reduced blood glucose and improved pyruvate tolerance in C57BL/6 mice. The above results indicate that Sgk1 also has a role in regulating hepatic gluconeogenesis at the in vivo level.
Claims (4)
1. An application of Sgk1 in preparing medicine for suppressing gluconeogenesis.
2. The use of claim 1, wherein the medicament reduces hepatic gluconeogenesis by interfering with or inhibiting Sgk 1.
3. The use of claim 1, wherein the medicament is prepared into a preparation by using Sgk1 as a target point and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
4. The use according to claim 3, wherein the formulation is selected from one of injection, subcutaneous implant, tablet, powder, granule, capsule, oral liquid, and sustained release formulation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317786A (en) * | 2007-04-18 | 2012-01-11 | 特提斯生物科学公司 | Diabetes correlativity biological marker and method of application thereof |
| CN110279860A (en) * | 2019-07-31 | 2019-09-27 | 上海交通大学医学院附属瑞金医院 | Nrg4 is preparing the application in diabetes medicament as target spot |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317786A (en) * | 2007-04-18 | 2012-01-11 | 特提斯生物科学公司 | Diabetes correlativity biological marker and method of application thereof |
| CN110279860A (en) * | 2019-07-31 | 2019-09-27 | 上海交通大学医学院附属瑞金医院 | Nrg4 is preparing the application in diabetes medicament as target spot |
Non-Patent Citations (3)
| Title |
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| BEN ZHOU等: "Serum- and glucocorticoid-induced kinase drives hepatic insulin resistance by directly inhibiting AMP-activated protein kinase", 《CELL REPORTS》 * |
| SILVIA VELAZQUEZ-GARCIA等: "SAT-153 Role Of Increased Serum- And Glucocorticoid-inducible Kinase 1 (SGK1) Activity In Gluconeogenesis And Liver Metabolism During Metabolic Syndrome", 《JOURNAL OF THE ENDOCRINE SOCIETY》 * |
| 刘率男 等: "糖尿病治疗新靶点糖原合成酶激酶-3抑制剂的研究进展", 药学学报 * |
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