CN114903994A - Application of BCKDK as target point in preparation of type 2 diabetes mellitus medicines - Google Patents

Abstract

The invention relates to application of BCKDK as a target spot in preparation of a type 2 diabetes mellitus medicine. The invention firstly proves the regulation and control effect of BCKDK on hepatic gluconeogenesis, and the specific inhibition of BCKDK is expected to become a new target point for treating type 2 diabetes.

Description

Application of BCKDK as target point in preparation of type 2 diabetes mellitus medicines

Technical Field

The invention belongs to the field of type 2 diabetes mellitus medicines, and particularly relates to application of BCKDK as a target point in preparation of a type 2 diabetes mellitus medicine.

Background

Glucose is an energy source and a metabolic intermediate product of living cells of mammals, and provides power for normal operation of tissues and organs such as brains, blood cells and the like. The normal body maintains blood glucose at a relatively stable level through the synergistic action of various metabolic organs, and in this metabolic network, the liver participates in maintaining blood glucose levels constant through gluconeogenesis and glycogenolysis. When the body is in a state of satiety, glycogen production is inhibited and glucose is stored in the form of glycogen; when the organism is in a short-term hunger state, glycogen decomposition is the main factor for hepatic glucose production; when the body is in a long-term hungry state, glycogen is depleted, the main mode of hepatic glycogenesis is converted into gluconeogenesis, and the body's blood glucose homeostasis is maintained by converting various non-sugar substances into glucose.

Diabetes mellitus is a group of metabolic diseases characterized by chronic hyperglycemia caused by multiple causes, and has become a worldwide public health problem seriously threatening human health. According to the international diabetes alliance (IDF) statistics: the number of diabetes patients worldwide has reached 4.15 billion in 2015, and this figure is expected to reach 6.42 billion by 2040 years. Although 14 different classes of hypoglycemic agents are currently in clinical use, continuous control of blood glucose in diabetic patients remains a significant challenge. Therefore, the research, development and supplement of the novel hypoglycemic drug can provide further guarantee for the prevention and treatment of diabetes. Diabetes, a complex disease of carbohydrate metabolism, involves the dysfunction of various tissues or organs. The research finds that the hepatic gluconeogenesis of the type 2 diabetes (T2DM) patient is increased by 40 percent compared with the normal people, so that the body generates excessive endogenous glucose, which is not only one of the reasons for fasting hyperglycemia of the patient, but also participates in the occurrence of postprandial hyperglycemia of the patient, and the research proves that the hepatic gluconeogenesis for maintaining normal level has important significance on the blood sugar control of the T2DM patient. Therefore, the research on the regulation mechanism of the liver gluconeogenesis pathophysiological process has great significance for the prevention and treatment of T2 DM.

Branched Chain Amino Acids (BCAAs) belong to the essential amino acids, including leucine, isoleucine and valine, accounting for 20% of the total protein intake in the human diet. Previous studies have suggested that BCAAs contribute to muscle protein synthesis and maintenance of blood glucose homeostasis, but recently, studies have reported that an increase in plasma BCAA levels is closely associated with the onset of insulin resistance, T2 DM. Cross-sectional studies of various people of different ethnic groups at home and abroad show that the blood plasma BCAA level of patients with insulin resistance, T2DM and obesity is higher than that of normal people. Later, researches show that the early increase of BCAA is closely related to the generation and development of T2DM and insulin resistance, and is expected to become a prediction factor of diabetes. Therefore, the influence of changing BCAA intake on the metabolism of the organism is attracted by researchers, and a plurality of reports find that when the intake of three BCAA is increased simultaneously, the insulin sensitivity of mice is weakened; however, when only leucine was supplemented, the insulin sensitivity of mice could be significantly improved. In these animal experiment-related studies, although there is no direct study on BCAA and hepatic gluconeogenesis, BCAA intake was positively correlated with hepatic gluconeogenesis levels in view of the results of the pyruvate tolerance test and the expression of hepatic gluconeogenesis-critical genes.

Plasma levels of BCAA are also associated with oxidative metabolism of BCAA, in addition to being dependent on exogenous intake. Branched-chain aminotransferase (BCAT) mediates the transamination reaction of the first step of BCAA metabolism, branched-chain alpha-ketoacid dehydrogenase (BCKDH) mediates the oxidative decarboxylation of the second step into an irreversible reaction, BCKDHA is the catalytic subunit of BCKDH, the activity of which depends on its phosphorylation level. Branched-chain ketoacid dehydrogenase kinase (BCKDK) inhibits the activity of BCKDH by phosphorylating BCKDHA, and BT2 is a specific inhibitor of BCKDK; protein phosphatase 2C (PP2Cm, mitochondrial type) activates BCKDH activity by dephosphorylating BCKDHA. So far, no research report is available on the relationship between BCAA oxidative metabolism key enzyme and hepatic gluconeogenesis. Therefore, the research on the regulation and control effect of BCKDK on hepatic gluconeogenesis is of great significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of BCKDK as a target point in preparing the type 2 diabetes mellitus medicaments, and the invention firstly proves the regulation and control effect of the BCKDK on hepatic gluconeogenesis, and the specific inhibition of the BCKDK is expected to become a new target point for treating type 2 diabetes mellitus.

The invention provides application of BCKDK as a target point in preparation of a type 2 diabetes mellitus medicine.

Preferably, the medicament reduces hepatic gluconeogenesis by interfering with or inhibiting BCKDK.

Preferably, the medicine takes BCKDK as a target point, and is matched with pharmaceutically acceptable auxiliary materials or auxiliary components to prepare a preparation for use.

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 firstly proves the regulation and control effect of BCKDK on hepatic gluconeogenesis, and the specific inhibition of BCKDK is expected to become a new target point for treating type 2 diabetes.

Drawings

FIG. 1 (A) the mRNA expression level of BCKDK was measured 12 hours after the mouse liver primary cells were treated with 100. mu.M cAMP; (B) after the primary mouse liver cells are treated by 100 mu M cAMP for 16 hours, detecting the protein expression levels of BCKDK, p-BCKDHA and BCKDHA; c57BL/6 mice empty stomach overnight, (C) detecting the mRNA expression level of liver BCKDK, (D) detecting the protein expression levels of liver BCKDK, p-BCKDHA and BCKDHA; detecting the mRNA expression level of liver BCKDK after the db/db mouse fasts overnight (E), and detecting the protein expression levels of liver BCKDK, p-BCKDHA and BCKDHA.

FIG. 2 (A) protein expression levels of BCKDK after treatment of mouse liver primary cells with 100. mu.M cAMP and 10. mu.g/ml Cycloheximide (CHX) for the corresponding time; (B) quantitative statistics were performed on WB band signal intensities in a by Image J software.

In fig. 3 (a), after BT2 treatment, the phosphorylation level of BCKDHA, a target protein of BCKDK, was detected; after the primary mouse liver cells are treated by BT2 and cAMP (B), detecting the generation amount of endogenous glucose; (C-E) detecting the mRNA expression level of a gluconeogenic key enzyme; (G) transfecting a PEPCK promoter plasmid by using a HepG2 cell, and detecting the activity of the PEPCK promoter through a reporter gene; (H) detecting the protein expression level of PEPCK; mRNA expression of PEPCK was detected after treating mouse liver primary cells with different concentrations of BT2 and 100. mu.M cAMP (F).

FIG. 4 is a graph showing body weight (A), random blood glucose (B), fasting 16-hour blood glucose (C), and intraperitoneal injection of glucose tolerance test (D) in mice in the db/m-Vehicle, db/db-Vehicle, and db/db-BT2 groups after one week of continuous administration.

FIG. 5 (A) detection of overexpression efficiency by Western blotting after transfection of mouse liver primary cells with control adenovirus or BCKDK overexpression adenovirus; (B) after mouse liver primary cells transfect control adenovirus or BCKDK overexpression adenovirus, treating for 24 hours by 100 mu McAMP, and collecting culture medium supernatant to measure endogenous glucose production; (C-E) after the mouse liver primary cells are transfected with control adenovirus or BCKDK overexpression adenovirus, treating the cells with 100 mu M cAMP for 12 hours, and detecting the mRNA expression level of gluconeogenesis key enzyme; (F-G) mRNA expression levels of PEPCK and FBP were measured 12 hours after transfection of mouse liver primary cells with control adenovirus or BCKDK overexpressing adenovirus and treatment with 200. mu.M BT2 and 100. mu.M cAMP.

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 may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

After the mouse liver primary cells were treated with 100 μ McAMP for 16 hours, the mRNA expression level of BCKDK of the liver primary cells did not change significantly (fig. 1A), but WB results showed a significant increase in BCKDK protein expression level, and in response, the phosphorylation level of BCKDHA, the target protein, was also significantly increased (fig. 1B). In C57BL/6 mice, the mRNA levels of liver BCK-DK were not different in the fasted state compared to the fed state (fig. 1C), while the protein expression level of BCKDK and the phosphorylation level of BCKDHA were significantly increased on average (fig. 1D). Compared with normal mice, the protein expression level of the liver BCKDK of db/db mice is higher, the phosphorylation level of target protein BCKDHA is also increased, and the mRNA expression level of the BCKDK is not changed correspondingly (FIGS. 1E-1F). Protein expression levels of BCKDK were measured 3 hours, 6 hours after treatment of mouse liver primary cells with 100. mu.M cAMP and 10. mu.g/ml Cycloheximide (CHX) and found to be significantly enhanced in BCKDK protein stability in the presence of cAMP stimulation (FIGS. 2A-2B).

BCKDK inhibitor BT2 inhibits hepatic gluconeogenesis: after treatment with 200 μ M BT2, the phosphorylation level of BCKDHA, the target protein of BCKDK, was significantly reduced (fig. 3A). After the primary mouse liver cells are treated with 200 mu M BT2 and 100 mu M cAMP for 24 hours, collecting culture medium supernatant to measure the endogenous glucose production; although BT2 did not affect endogenous glucose production by hepatic primary cells in the basal state, cAMP-induced endogenous glucose production was significantly inhibited by BT2 (fig. 3B). After the primary mouse liver cells were treated with 200. mu.M BT2 and 100. mu.M cAMP for 12 hours, the mRNA expression levels of key gluconeogenesis enzymes were measured, and the gene expression levels of key gluconeogenesis enzymes G6Pc, FBP and PEPCK were similarly changed, i.e., significantly inhibited under cAMP stimulation conditions (FIGS. 3C-3E). The inhibition effect was found to be BT2 dose-dependent by detecting PEPCK mRNA expression 12 hours after treating mouse liver primary cells with 0, 25, 50, 100, 150, 200 μ M BT2 and 100 μ M cAMP, respectively, and PEPCK mRNA levels were reduced by about 50% at BT2 treatment concentration of 100 μ M (fig. 3F). PEPCK promoter activity was detected by reporter gene after transfection of PEPCK promoter plasmid in HepG2 cells followed by 100 μ M cAMP and 200 μ M BT2 treatment for 24 hours, and the reporter gene results showed that cAMP significantly increased PEPCK promoter activity, but PEPCK promoter activity was reduced after simultaneous administration of BT2 treatment (fig. 3G). In addition, cAMP induction of protein expression levels of PEPCK in liver primary cells was also blocked by BT2 (fig. 3H). These data indicate that BCKDK inhibitor BT2 can inhibit hepatic gluconeogenesis by reducing the transcriptional expression of key gluconeogenesis enzymes.

BT2 reduced hepatic glucose production in db/db mice: to further clarify the metabolic regulation effect of BT2, especially the in vivo regulation effect on hepatic gluconeogenesis, type 2 diabetes model mice, db/db mice, were administered with 1 week old intraperitoneal injections of BT 2. Experimental animals at 4 weeks of age were divided into three groups: the first group was db/m control mice without spontaneous diabetes given saline intraperitoneal injection (db/m-Vehicle); the second group is db/db mice given intraperitoneal injections of physiological saline (db/db-Vehicle); the third group was db/db mice, BT2 at 20mg kg ^-1 ·day ^-1 The dose of (c) was given intraperitoneally (db/db-BT 2). After 1 week of continuous administration, BT2 did not affect body weight and random blood glucose in db/db mice (FIGS. 4A-4B), but fasting blood glucose was significantly lower in db/db-BT2 mice than in db/db-Vehicle mice (FIG. 4C). After fasting the mice overnight for 16 hours, fasting blood glucose (i.e., 0 minute) was measured, and then sodium pyruvate was intraperitoneally injected into the mice at a dose of 2g/kg body weight, followed by measuring blood glucose 15, 30, 60 and 120 minutes after the injection of sodium pyruvate,blood glucose levels in the db/db-BT2 group mice were found to be lower than those in the db/db-Vehicle group mice at 0, 15, 30 and 60 minutes, and were particularly significant at 15 minutes with statistical differences (FIG. 4D). These in vivo data suggest that BT2 has inhibitory effect on hepatic gluconeogenesis and certain improving effect on sugar metabolism disorder of db/db mice.

Overexpression of BCKDK enhances hepatocyte gluconeogenesis: after the primary mouse liver cells are transfected with control adenovirus or BCKDK overexpression adenovirus, detecting overexpression efficiency by using Western blotting; after mouse liver primary cells were transfected with control adenovirus or BCKDK overexpression adenovirus, 100. mu. McAMP was treated for 24 hours, and the culture medium supernatant was collected to measure the endogenous glucose production. It was found that endogenous glucose production could be further increased upon cAMP induction after overexpression of BCKDK in mouse liver primary cells (fig. 5A) (fig. 5B). After the mouse liver primary cells were transfected with control adenovirus or BCKDK overexpression adenovirus, the mRNA expression level of gluconeogenesis key enzymes was detected by treating with 100 μ M cAMP for 12 hours, and the results show that cAMP can already significantly improve the mRNA expression levels of gluconeogenesis key enzymes PEPCK, FBP and G6Pc, and BCKDK overexpression further increases their expression levels (FIGS. 5C-5E). After the mouse liver primary cells were transfected with the control adenovirus or the BCKDK overexpression adenovirus, the mRNA expression levels of PEPCK and FBP were detected by treating 200 μ M BT2 and 100 μ M cAMP for 12 hours, and it is noted that the gluconeogenesis promotion effect of BCKDK overexpression can still be reversed by BCKDK inhibitor BT2, the mouse liver primary cells were treated with BT2 based on BCKDK overexpression, and the mRNA levels of PEPCK and FBP were significantly lower than those of BCKDK overexpression only (FIGS. 5F-5G). The opposite effects of BCKDK overexpression and a BCKDK inhibitor on hepatic gluconeogenesis indicate that the BCKDK not only participates in regulating the metabolism of branched chain amino acid, but also has an important regulation and control effect on hepatic gluconeogenesis.

Claims (4)

1. An application of BCKDK in preparing a medicament for treating type 2 diabetes.

2. The use of claim 1, wherein the medicament reduces hepatic gluconeogenesis by interfering with or inhibiting BCKDK.

3. The use of claim 1, wherein the medicament is prepared into a preparation by taking BCKDK 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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