Use of id1 inhibitors for the preparation of a medicament for the prevention and / or treatment of type 2 diabetes

Abstract

The present application relates to the application of ID1 inhibitor in the preparation of drugs for preventing and / or treating type 2 diabetes. Based on the research finding that ID1 expression is increased in the liver of high-fat diet-induced mice, db / db diabetic mice and fasting mice; inhibiting or knocking down ID1 can reduce the expression of key gluconeogenesis enzymes in the liver, reduce hepatic glucose production, reduce fasting blood glucose, improve glucose tolerance and pyruvate tolerance, and reduce liver lipid accumulation. Therefore, the present application aims to provide the application of ID1 inhibitor in the preparation of drugs for inhibiting hepatic gluconeogenesis, reducing blood glucose, improving glucose and lipid metabolism disorders and treating type 2 diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the application of ID1 inhibitors in the preparation of drugs for the prevention and / or treatment of type 2 diabetes. Background Technology

[0002] The liver is a vital organ for maintaining glucose homeostasis. In a fasting state, the liver releases glucose through glycogenolysis and gluconeogenesis to maintain blood glucose levels. Type 2 diabetes patients often exhibit hepatic insulin resistance and abnormally increased hepatic glucose production, with hyperglycemia being a significant cause of elevated fasting blood glucose. Gluconeogenesis is regulated by various hormones and transcriptional signals. The glucagon / cAMP / PKA / CREB signaling pathway promotes the expression of key gluconeogenic enzyme genes, such as PEPCK, G6Pase / G6Pc, and FBP, thereby enhancing hepatic glucose production. Therefore, inhibiting abnormally enhanced hepatic gluconeogenesis is a crucial therapeutic strategy for improving hyperglycemia in type 2 diabetes.

[0003] Currently, drug treatment for type 2 diabetes mainly includes metformin, insulin secretagogues, alpha-glucosidase inhibitors, thiazolidinediones, DPP-4 inhibitors, SGLT2 inhibitors, GLP-1 receptor agonists, GIP / GLP-1 receptor agonists, and insulin. Current clinical guidelines emphasize individualized treatment based on glycemic efficacy, risk of hypoglycemia, weight impact, cardiac and renal benefits, comorbidities, adverse reactions, and patient burden. However, existing treatments still have drawbacks, including inadequate glycemic control in some patients, insufficient long-term efficacy, adverse reactions, poor adherence, and high treatment costs. Therefore, identifying new therapeutic targets, especially novel intervention targets for abnormally enhanced hepatic gluconeogenesis, remains of great significance.

[0004] ID1, or DNA-binding repressor 1, is a member of the ID protein family. ID proteins are transcriptional regulatory proteins with a helical-loop-helical structure, but lack the basic domain required for direct DNA binding. They regulate cell differentiation, proliferation, and metabolic processes by forming complexes with basic HLH transcription factors. Previous studies have suggested a link between ID1 and abnormal glucose metabolism. For example, ID1 deficiency can enhance insulin secretion and protect mice from glucose intolerance induced by a high-fat diet. However, current research primarily focuses on the relationship between ID1 and pancreatic β-cell function. Whether ID1 participates in the regulation of hepatic gluconeogenesis and whether it can serve as a novel target for inhibiting hepatic gluconeogenesis and treating type 2 diabetes remains unclear.

[0005] Curcumin, a natural polyphenolic active ingredient in turmeric, has been reported to improve glucose and lipid metabolism, inhibit hepatic glucose production, and regulate cAMP / PKA-related signaling. Curcumin has also been reported to inhibit p300 / CBP histone acetyltransferase activity. CBP / p300, as a CREB-related coactivator, participates in the transcriptional regulation of gluconeogenesis-related genes; the selective CBP / p300 HAT inhibitor A-485 has also been reported to inhibit lipid production and hepatic gluconeogenesis. However, the specific downstream targets of curcumin in inhibiting hepatic gluconeogenesis remain unclear, and no existing technology discloses a method to inhibit hepatic gluconeogenesis and treat type 2 diabetes by inhibiting ID1 expression or function.

[0006] Therefore, it is necessary to develop a novel treatment strategy that targets ID1 and improves hyperglycemia and glucose and lipid metabolism disorders by inhibiting hepatic gluconeogenesis. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a new treatment strategy for type 2 diabetes with ID1 as the intervention target, which addresses the problems of abnormally enhanced hepatic gluconeogenesis, increased endogenous glucose production, elevated fasting blood glucose and disordered glucose and lipid metabolism in type 2 diabetes.

[0008] This invention provides the use of ID1 inhibitors in the preparation of drugs for the prevention and / or treatment of type 2 diabetes. ID1, or DNA-binding inhibitory factor 1, includes human ID1 and its animal homologs or proteins, also known as Id1 in mice.

[0009] Preferably, the ID1 inhibitor refers to a substance that can reduce ID1 gene expression, reduce ID1 mRNA levels, reduce ID1 protein expression, inhibit ID1 function, or block ID1-mediated gluconeogenesis regulation.

[0010] Preferably, the ID1 inhibitor comprises small molecule compounds, natural products, nucleic acid inhibitors, siRNA, shRNA, antisense oligonucleotides, miRNA mimics, ID1 interference sequences delivered by viral vectors, gene editing reagents, peptides, antibodies, protein degrading agents, or pharmaceutically acceptable salts, prodrugs, derivatives, and pharmaceutical compositions thereof.

[0011] Preferably, the ID1 inhibitor is used to inhibit the expression or function of hepatic ID1, thereby reducing hepatic gluconeogenesis. The reduction in hepatic gluconeogenesis is manifested by a decrease in the expression of key gluconeogenesis enzyme genes; these key gluconeogenesis enzymes include one or more of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pc), and fructose-1,6-bisphosphatase (FBP).

[0012] Preferably, the ID1 inhibitor is curcumin or a curcumin-containing pharmaceutical composition. This invention shows that curcumin can reduce ID1 expression by inhibiting the interaction between CREB and CBP, thereby reducing CREB transcriptional activity and further downregulating the expression of key gluconeogenesis enzymes such as PEPCK, G6Pc, and FBP, ultimately inhibiting hepatic gluconeogenesis.

[0013] Preferably, the ID1 inhibitor is an ID1-targeting shRNA, siRNA, or a viral vector carrying an ID1 interference sequence. Studies in this invention show that an ID1-targeting interfering adenovirus can reduce ID1 expression in mouse liver, decrease fasting and random blood glucose levels, improve glucose and pyruvate tolerance, reduce liver glycogen content, and decrease serum and liver tissue triglyceride and total cholesterol levels.

[0014] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of type 2 diabetes, the pharmaceutical composition comprising an effective dose of an ID1 inhibitor and pharmaceutically acceptable excipients.

[0015] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0016] Preferably, the dosage form of the pharmaceutical composition includes tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, lipid nanoparticle formulations, sustained-release formulations, or controlled-release formulations.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention first clearly defines the role of ID1 in the regulation of hepatic gluconeogenesis and proposes that ID1 can be used as a novel drug target for the treatment of type 2 diabetes and glucose and lipid metabolism disorders.

[0019] (2) This invention demonstrates that inhibiting ID1 can reduce hepatic glucose production. By knocking down ID1, the expression of key gluconeogenesis enzymes such as hepatic PEPCK, G6Pc and FBP can be downregulated, reducing the level of blood glucose production in the pyruvate tolerance test, suggesting that ID1 inhibitors have the effect of inhibiting hepatic gluconeogenesis.

[0020] (3) This invention demonstrates that inhibiting ID1 can improve type 2 diabetes-related hyperglycemia. Animal experiments show that ID1 knockdown can reduce fasting blood glucose in C57BL / 6 mice and db / db diabetic mice and improve glucose tolerance in db / db mice, while no significant change was observed in blood insulin levels, suggesting that this hypoglycemic effect is mainly related to the inhibition of hepatic gluconeogenesis.

[0021] (4) This invention demonstrates that inhibiting ID1 can improve glucose and lipid metabolism disorders. ID1 knockdown can reduce liver glycogen content, reduce serum and liver tissue triglyceride and total cholesterol levels, and reduce liver lipid deposition.

[0022] (5) This invention clarifies that curcumin can be used as a representative drug for downregulating ID1 and inhibiting hepatic gluconeogenesis. Curcumin can reduce the expression of ID1 in the liver of high-fat diet mice and db / db mice, and improve blood glucose and lipid metabolism, providing a new mechanistic basis for the development of curcumin or its derivatives as anti-type 2 diabetes drugs. Attached Figure Description

[0023] Figure 1 The results of experiments on the inhibition of gluconeogenesis in mouse primary liver cells by curcumin are as follows: (A) The effect of different concentrations of curcumin on the viability of mouse primary liver cells was detected by CCK-8 assay; (B) After treating mouse primary liver cells with different concentrations of curcumin and 100 μM cAMP for 24 hours, the glucose content in the culture supernatant was detected; (CE) After treating mouse primary liver cells with 50 μM curcumin and 100 μM cAMP for 8 hours, the mRNA expression of PEPCK, G6Pc, and FBP was detected; (F) After transfecting HepG2 cells with the PEPCK promoter reporter plasmid, the activity of the PEPCK promoter was detected; (G) After treating mouse primary liver cells with 100 μM cAMP and 50 μM curcumin for 16 hours, the expression of PEPCK protein was detected by Western Blot. Sub represents the gluconeogenesis substrate control, and Cur represents curcumin treatment.

[0024] Figure 2 The results show that curcumin reduces CREB transcriptional activity by inhibiting the binding of CBP to CREB. Among them, (A) HepG2 cells transfected with CRE cis-acting element reporter plasmid were treated with 100 μM cAMP and 50 μM curcumin and the CRE reporter gene activity was detected; (B) Mouse primary liver cells were treated with 100 μM cAMP and 50 μM curcumin for 12 hours and the mRNA expression of PGC1α was detected; (C) Mouse primary liver cells were treated with 100 μM cAMP and 50 μM curcumin for 6 hours and histones were extracted and the H3K27Ac level was detected; (D) HepG2 cells were treated with 50 μM curcumin and 100 μM cAMP for 6 hours and the interaction between CREB and CBP was detected by immunoprecipitation.

[0025] Figure 3Curcumin was used to inhibit the expression of liver Id1 in HFD mice and db / db mice. The mRNA levels of liver Id1 in the control group, HFD group, db / db group, and curcumin group were shown in (AB). Immunohistochemical staining of liver Id1 was shown in (CD). Data were presented in mean ± SEM format (n=7~10).

[0026] Figure 4 shows the regulation of glucose production in mouse primary liver cells by Id1; (A) Id1 expression level was detected; (B) Endogenous glucose production was detected after treatment with 100 μM cAMP and 50 μM curcumin for 24 hours. Compared with the shCtrl-Sub group, ##P<0.01; compared with the shCtrl-cAMP group, *P<0.05; (CD) treated with 100μM cAMP and 50μM curcumin for 8 hours, and the mRNA expression levels of PEPCK and G6pc were detected by qRT-PCR; compared with the shCtrl-Sub group, ##P<0.01, ###P<0.001; compared with the shCtrl-cAMP group, **P<0.01; (E) overexpression efficiency was detected; (F) treated with 100μM cAMP and 50μM curcumin for 24 hours, and the glucose content in the culture medium was measured; (GH) treated with 100μM cAMP and 50μM curcumin for 8 hours, and the mRNA expression levels of PEPCK and G6pc were detected by qRT-PCR.

[0027] Figure 5 shows the effect of Id1 overexpression on glucose metabolism in C57BL / 6 mice; (A) Id1 mRNA expression in liver, skeletal muscle, and VAT of mice injected with Ad-GFP or Ad-Id1 adenovirus; (BC) serum AST and serum ALT; (DE) fasting and random blood glucose levels; (F) IPGTT results; (G) IPITT results; (H) IPPTT results; (I) fasting and random insulin; (J) liver weight; (K) liver glycogen content; (L) expression of hepatic gluconeogenesis gene mRNA in mice. Mice were sacrificed on day 9 after adenovirus injection.

[0028] Figure 6 The effect of Id1 overexpression on lipid metabolism in C57BL / 6 mice was investigated. (AB) Serum TC and TG levels in mice injected with Ad-Id1 or Ad-GFP adenovirus vector; (CD) TC and TG content in liver tissue; (E) random body weight; (F) 24-hour food intake.

[0029] Figure 7 shows the effect of Id1 knockdown on glucose metabolism in C57BL / 6 mice; (A) expression of Id1 mRNA in the liver, skeletal muscle, and visceral adipose tissue (VAT) of mice injected with shId1 or shCtrl adenovirus vector; (BC) serum AST and ALT levels; (DE) fasting and random blood glucose levels on the 4th day after adenovirus injection; (F) IPPTT assay on the 7th day after adenovirus injection; (G) fasting and random serum insulin; (H) liver weight; (I) liver glycogen content; (J) expression of gluconeogenesis gene mRNA in mouse liver; mice were sacrificed on the 9th day after adenovirus injection.

[0030] Figure 8 The effect of Id1 knockdown on lipid metabolism in C57BL / 6 mice; (AB) serum TC and TG levels in mice injected with shId1 or shCtrl adenovirus vector; (CD) TC and TG content in liver tissue; (E) random body weight; (F) 24-hour food intake.

[0031] Figure 9 The effects of Id1 knockdown on glucose metabolism in db / db mice were investigated. The results included: (AB) serum AST and ALT levels in db / db mice injected with shId1 or shCtrl adenovirus; (CD) fasting and random blood glucose levels on day 4 after adenovirus injection; (EF) IPGTT and IPPTT results; (G) fasting and random serum insulin; (H) liver weight; (I) liver glycogen content; (J) PAS staining of liver glycogen; and (K) mRNA expression of mouse liver gluconeogenesis genes. Mice were sacrificed on day 9 after adenovirus injection.

[0032] Figure 10 The effect of Id1 knockdown on lipid metabolism in db / db mice; (AB) serum TC and TG levels in db / db mice injected with shId1 or shCtrl adenovirus; (CD) TC and TG content in liver tissue; (E) Oil Red staining of liver sections; (F) random body weight; (G) body fat percentage; (H) lean body weight; (I) 24-hour food intake.

[0033] Figure 11 The results of the association analysis between ID1 gene SNP loci and type 2 diabetes and glucose metabolism indicators are presented. Among them, T2DM represents type 2 diabetes, FPG represents fasting blood glucose, 2h-PG represents blood glucose 2 hours after oral glucose tolerance test, BETA represents the effect estimate, L95 and U95 represent the lower and upper limits of the 95% confidence interval, respectively, and P represents the p-value of the significance test. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0035] Example 1

[0036] ID1 inhibitors are used in the development of drugs for treating type 2 diabetes.

[0037] In one embodiment, an ID1 inhibitor is used to prepare a medicament for the prevention and / or treatment of type 2 diabetes. The medicament reduces hepatic gluconeogenesis by decreasing hepatic ID1 expression or inhibiting ID1 function, thereby reducing endogenous glucose production.

[0038] The therapeutic effects of the drug include one or more of the following: reducing fasting blood glucose, reducing random blood glucose, improving glucose tolerance, improving pyruvate tolerance, reducing hepatic glucose production, reducing abnormal accumulation of hepatic glycogen, reducing serum triglycerides and total cholesterol, reducing liver tissue triglycerides and total cholesterol, and reducing hepatic lipid deposition.

[0039] In a preferred embodiment, the ID1 inhibitor is used to treat type 2 diabetes characterized by enhanced hepatic gluconeogenesis. This enhanced gluconeogenesis is manifested by elevated expression of key gluconeogenic enzymes such as PEPCK, G6Pc, and / or FBP. Administration of the ID1 inhibitor reduces the expression of these key gluconeogenic enzymes, decreasing hepatic glucose production and thereby improving the hyperglycemic state.

[0040] Example 2

[0041] Curcumin inhibits hepatic gluconeogenesis through the CREB-CBP-ID1 pathway:

[0042] In one implementation, curcumin reduces CREB transcriptional activity by inhibiting the interaction between CREB and CBP, thereby downregulating ID1 expression and further inhibiting hepatic gluconeogenesis.

[0043] Specifically, mouse primary liver cells were isolated, and an in vitro gluconeogenesis model was established using a sugar-free culture medium containing sodium lactate and sodium pyruvate. Gluconeogenesis was stimulated using cAMP and treated with curcumin. Figure 1 A showed that cell viability did not decrease significantly after treatment with 1-50 μM curcumin, but cell viability decreased significantly at 80 μM and 100 μM. Therefore, 50 μM was selected for subsequent experiments. Figure 1 B showed that cAMP significantly increased the glucose concentration in the culture supernatant, and 10-50 μM curcumin could reduce cAMP-induced glucose production in a concentration-dependent manner. Figure 1 CE showed that cAMP upregulated the expression of PEPCK, G6Pc and FBP mRNA, while treatment with 50 μM curcumin decreased the expression of all of these genes. Figure 1 F showed that cAMP enhanced the activity of the PEPCK promoter reporter gene, while curcumin treatment reduced this activity. Figure 1 G showed that cAMP induced an increase in PEPCK protein expression, while curcumin treatment decreased PEPCK protein levels. These results demonstrate, from multiple perspectives including cell viability, glucose production, gluconeogenesis gene mRNA, promoter activity, and protein expression, that curcumin can inhibit hepatocyte gluconeogenesis.

[0044] Further investigation was conducted to examine the effect of curcumin on CREB-CBP signal. Figure 2 A showed that cAMP stimulation enhanced CRE reporter gene activity, while curcumin treatment significantly reduced cAMP-induced CRE transcriptional activity. Figure 2 B showed that cAMP upregulated the mRNA expression of PGC1α, a co-activation factor of CREB, while curcumin treatment decreased the expression of PGC1α. Figure 2 C showed that cAMP treatment increased H3K27 acetylation levels, while curcumin decreased H3K27Ac levels. Figure 2 The results of co-precipitation of D showed that CBP was detectable in CREB immunoprecipitate. Treatment with curcumin significantly reduced the amount of CBP co-precipitated with CREB, while it was still visible in CREB immunoprecipitate and the control group. These results indicate that curcumin can inhibit the interaction between CREB and CBP and related transcriptional activation processes, thereby reducing ID1 expression and inhibiting gluconeogenesis.

[0045] Example 3

[0046] Curcumin is used as an ID1 expression inhibitor to treat type 2 diabetes.

[0047] In a preferred embodiment, the ID1 inhibitor is curcumin or a curcumin-containing pharmaceutical composition. Curcumin can reduce hepatic ID1 expression and improve type 2 diabetes-related metabolic abnormalities by inhibiting hepatic gluconeogenesis.

[0048] Specifically, mice with abnormal glucose metabolism induced by a high-fat diet were selected as a model and administered curcumin by gavage. The curcumin dose was 100 mg / kg / day, administered continuously for 8 weeks. After the administration period, fasting blood glucose, random blood glucose, glucose tolerance, insulin tolerance, pyruvate tolerance, liver glycogen content, and expression of key enzymes in liver gluconeogenesis were measured in the mice.

[0049] Figure 3 A and Figure 3C shows that, in the high-fat diet experiment, compared with the normal diet group (NCD), the high-fat diet group (HFD) mice had increased Id1 mRNA levels in the liver and enhanced immunohistochemical staining of Id1 in liver tissue; after administration of curcumin, Id1 mRNA levels decreased and immunohistochemical staining weakened. Data for each group are expressed as mean ± SEM, with sample sizes n = 7–10. These results indicate that a high-fat diet can induce Id1 expression in mouse liver, while curcumin can reduce this expression.

[0050] Figure 3 B and Figure 3 The results showed that, compared with non-diabetic littermate control db / m mice, db / db mice had increased liver Id1 mRNA levels and enhanced immunohistochemical staining of liver Id1. After curcumin treatment, db / db mice showed decreased liver Id1 mRNA levels and weakened immunohistochemical staining. Data for each group are expressed as mean ± SEM, with sample sizes n = 7–10. These results further indicate that curcumin can downregulate Id1 expression in the liver of diabetic model mice.

[0051] The above results indicate that curcumin can be used as a drug that can downregulate ID1 expression and inhibit hepatic gluconeogenesis, and can be used to prepare drugs for the treatment of type 2 diabetes and related glucose and lipid metabolism disorders.

[0052] Example 4

[0053] ID1 knockdown inhibits glucose production in hepatocytes:

[0054] In one implementation, an interference sequence targeting ID1 is used to reduce ID1 expression in hepatocytes to verify the regulatory role of ID1 in hepatic gluconeogenesis.

[0055] Specifically, primary mouse liver cells were divided into a control interference group and an ID1 knockdown group. The ID1 knockdown group was transfected with adenovirus carrying the ID1 interference sequence, while the control group was transfected with the corresponding empty vector or negative control virus. After transfection, hepatocyte gluconeogenesis was stimulated with cAMP, and the glucose content and expression of key gluconeogenesis enzymes in the culture supernatant were detected.

[0056] Figure 4A shows that, compared with the shCtrl control group, shId1 treatment significantly reduced Id1 mRNA expression in primary hepatocytes, confirming the effectiveness of knockdown. Figure 4B shows that cAMP stimulation significantly increased glucose production in control cells, while under cAMP stimulation, the glucose concentration in the culture supernatant of the shId1 group was lower than that of the shCtrl group. Figures 4C and 4D show that cAMP significantly induced the expression of PEPCK and G6Pc mRNA, and the cAMP-induced expression of both decreased after Id1 knockdown. These results indicate that reducing ID1 expression can directly inhibit hepatocyte gluconeogenesis.

[0057] Example 5

[0058] ID1 overexpression promotes gluconeogenesis:

[0059] In one implementation, the promoting effect of ID1 on gluconeogenesis was verified by overexpressing ID1.

[0060] Specifically, mouse primary liver cells were divided into a control group and an ID1 overexpression group. The ID1 overexpression group was transfected with adenovirus carrying the ID1 coding sequence, while the control group was transfected with empty adenovirus. After transfection, gluconeogenesis was stimulated with cAMP, and the amount of glucose produced and the expression of PEPCK and G6Pc were measured.

[0061] Figure 4E shows the Western blot results, indicating that Flag-labeled Id1 protein was detectable in the Ad-Id1 group, confirming the effectiveness of Id1 overexpression. Figure 4F shows that under substrate basal state, cAMP stimulation state, and curcumin treatment conditions, the glucose concentration in the culture supernatant of the Ad-Id1 group was higher than that of the Ad-GFP control group. Figures 4G and 4H show that under the above treatment conditions, the expression of PEPCK and G6Pc mRNA in the Ad-Id1 group was higher than that in the Ad-GFP group; even with curcumin administration, the gluconeogenesis-related indicators in the Id1 overexpression group were still higher than those in the corresponding control group. These results indicate that ID1 can promote hepatocyte gluconeogenesis and can partially counteract the inhibitory effect of curcumin on gluconeogenesis.

[0062] Further, in C57BL / 6 mice, Ad-Id1 adenovirus was injected via tail vein to overexpress liver Id1, while the control group was injected with Ad-GFP adenovirus. Figure 5A shows that Ad-Id1 mainly increased liver Id1 mRNA expression, while no significant changes were observed in Id1 expression in skeletal muscle and visceral adipose tissue. Figures 5B-C show no significant difference in serum AST and ALT levels between the two groups, suggesting that this treatment did not cause significant liver damage. Figures 5D-E show that Id1 overexpression increased fasting blood glucose, while random blood glucose did not show significant changes. Figures 5F-H show that the blood glucose curve increased after glucose loading in the Ad-Id1 group, and the hypoglycemic response after insulin loading differed, with a significant increase in blood glucose after pyruvate loading, suggesting that glucose tolerance, insulin sensitivity, and hepatic gluconeogenesis were affected. Figure 5I shows no significant difference in serum insulin levels between fasting and fed states; Figures 5J-K show no significant changes in liver weight and liver glycogen content; Figure 5L shows increased expression of liver PEPCK, G6Pc, and FBP mRNA. The above results indicate that hepatic Id1 overexpression can enhance gluconeogenesis and lead to abnormal glucose metabolism.

[0063] Figure 6 Further AD studies showed that Id1 overexpression increased serum total cholesterol, serum triglycerides, liver tissue total cholesterol, and liver tissue triglycerides in C57BL / 6 mice. Figure 6EF analysis showed no significant differences in body weight and 24-hour food intake between the Ad-Id1 group and the Ad-GFP group. Therefore, the increased lipid levels induced by Id1 overexpression were not due to differences in body weight or food intake, suggesting that ID1 can directly or indirectly promote lipid metabolism disorders.

[0064] Example 6

[0065] Knocking down ID1 in vivo improves abnormal glucose metabolism:

[0066] In one implementation, the effect of ID1 inhibitors on improving glucose metabolism was verified by knocking down ID1 in vivo.

[0067] Specifically, C57BL / 6 mice were selected and injected via tail vein with adenovirus carrying an ID1 interference sequence to reduce ID1 expression in the liver. The control group was injected with the corresponding control virus. After administration, fasting blood glucose, random blood glucose, pyruvate tolerance, serum insulin, liver glycogen content, and expression of key enzymes in liver gluconeogenesis were measured in the mice.

[0068] Figure 7A shows that shId1 adenovirus significantly reduced Id1 mRNA expression in the liver of C57BL / 6 mice, while no decrease was observed in Id1 expression in skeletal muscle and visceral adipose tissue, indicating that knockdown mainly occurred in the liver. Figures 7B-C show no significant difference in serum AST and ALT levels between the two groups. Figures 7D-E show that Id1 knockdown reduced fasting blood glucose, and random blood glucose showed a decreasing trend. Figure 7F shows that after pyruvate loading, blood glucose in the shId1 group was lower than that in the shCtrl group, suggesting reduced hepatic glucose production. Figure 7G shows no significant change in serum insulin levels during fasting and feeding; Figure 7H shows no significant change in liver weight; Figure 7I shows a decrease in liver glycogen content; Figure 7J shows a decrease in hepatic PEPCK, G6Pc, and FBP mRNA expression. These results indicate that in vivo inhibition of ID1 can reduce hepatic gluconeogenesis and improve abnormal glucose metabolism, without significantly increasing hepatotoxic indicators.

[0069] Figure 8 AC showed that knocking down Id1 in C57BL / 6 mice reduced serum total cholesterol, serum triglycerides, and total cholesterol in liver tissue. Figure 8 D shows a decreasing trend in triglycerides in liver tissue, but statistical significance is not indicated in the figure. Figure 8 E showed that the shId1 group experienced a slight decrease in weight. Figure 8 F showed no significant difference in 24-hour food intake between the two groups. These results indicate that inhibiting ID1 in normal mice can improve some blood lipid and liver lipid parameters, but this effect cannot be fully explained by changes in food intake.

[0070] Further knockdown of Id1 was performed in db / db diabetic mice. Figure 9AB showed that there was no significant difference in serum AST and ALT levels between the shId1 group and the shCtrl group. Figure 9 CD showed that Id1 knockdown significantly reduced fasting blood glucose and random blood glucose. Figure 9 EF showed that the blood glucose levels in the shId1 group were lower than those in the control group after both glucose and pyruvate loading, indicating improved glucose tolerance and reduced hepatic glucose production. Figure 9 G showed no significant changes in serum insulin levels during both fasting and eating states; Figure 9 H showed a decrease in liver weight; Figure 9 I and Figure 9 PAS staining of J showed a decrease in both liver glycogen content and liver tissue glycogen staining. Figure 9 K showed decreased expression of hepatic Id1, PEPCK, G6Pc, and FBP mRNA. These results indicate that ID1 inhibitors can improve hyperglycemia in type 2 diabetic models, and their effect is related to the inhibition of hepatic gluconeogenesis.

[0071] Example 7

[0072] Knockdown of ID1 in vivo improves lipid metabolism disorders

[0073] In one implementation, ID1 inhibitors can also be used to improve type 2 diabetes-related lipid metabolism disorders.

[0074] Specifically, after knocking down hepatic ID1 expression in C57BL / 6 mice and db / db mice, the levels of total cholesterol and triglycerides in serum and liver tissue were measured, and the lipid deposition in the liver was observed.

[0075] Figure 10 AD showed that knocking down Id1 in db / db mice decreased serum total cholesterol, serum triglycerides, liver tissue total cholesterol, and liver tissue triglycerides. Figure 10 Oil Red O staining with E showed that lipid droplet staining in the liver tissue of the shId1 group was reduced compared with that of the shCtrl group, indicating that lipid deposition in the liver was reduced. Figure 10 F shows that the shId1 group experienced a decrease in weight; Figure 10 GH showed no significant changes in body fat / weight ratio and lean body mass / weight ratio; Figure 10 I showed no significant difference in food intake over 24 hours. Combined with... Figure 8 and Figure 10 The above results indicate that inhibiting ID1 can improve lipid metabolism abnormalities and liver lipid deposition in normal mice and diabetic model mice, and the related changes cannot be simply attributed to decreased food intake.

[0076] Example 8

[0077] Pharmaceutical compositions containing ID1 inhibitors:

[0078] The present invention also provides a pharmaceutical composition for treating type 2 diabetes, the pharmaceutical composition comprising an effective dose of an ID1 inhibitor and a pharmaceutically acceptable carrier, excipient or excipient.

[0079] In one embodiment, the active ingredient of the pharmaceutical composition is curcumin. The curcumin can be prepared into oral formulations with pharmaceutically acceptable excipients, including tablets, capsules, granules, pills, oral liquids, suspensions, sustained-release formulations, or controlled-release formulations.

[0080] In another embodiment, the active ingredient of the pharmaceutical composition is siRNA, shRNA, or antisense oligonucleotide targeting ID1. The nucleic acid-based ID1 inhibitor can be delivered to the liver via lipid nanoparticles, viral vectors, or non-viral delivery systems to reduce hepatic ID1 expression.

[0081] The pharmaceutical composition can be used alone or in combination with existing hypoglycemic drugs. These existing hypoglycemic drugs include metformin, SGLT2 inhibitors, GLP-1 receptor agonists, DPP-4 inhibitors, α-glucosidase inhibitors, thiazolidinediones, insulin, or analogues thereof.

[0082] Example 9

[0083] Screening for anti-type 2 diabetes drugs targeting ID1:

[0084] This invention also provides a method for screening candidate drugs for treating type 2 diabetes. The method includes the following steps:

[0085] Establish a hepatocyte gluconeogenesis model; treat with candidate drugs; detect whether the candidate drugs reduce ID1 expression or inhibit ID1 function; further detect whether the candidate drugs reduce PEPCK, G6Pc and / or FBP expression, and whether they reduce hepatocyte glucose production.

[0086] If a candidate drug can reduce ID1 expression or function, and simultaneously reduce hepatocyte glucose production and downregulate the expression of key gluconeogenesis enzymes, then the candidate drug can be used as a candidate drug for the treatment of type 2 diabetes or metabolic diseases related to abnormal hepatic gluconeogenesis.

[0087] The screening methods described above can be used to discover novel antidiabetic drugs targeting ID1, providing a basis for developing novel therapeutic strategies to inhibit hepatic gluconeogenesis.

[0088] Example 10

[0089] Association analysis between ID1 gene polymorphism and the risk of type 2 diabetes:

[0090] To further illustrate the clinical association between ID1 and type 2 diabetes, an association analysis was performed on ID1 gene SNP sites using T2DM, FPG, and 2h-PG as outcomes, and the effect estimate BETA, 95% confidence interval, and P-value were recorded. Figure 11 The results showed that for the T2DM outcome, the BETA was -0.3763312, with a 95% confidence interval of -0.6603664 to -0.0922951, and a P value of 0.00940862, indicating a significant negative association. For the FPG outcome, the BETA was -0.0071553, with a 95% confidence interval of -0.0177361 to 0.00342556, and a P value of 0.185162, which did not reach statistical significance. For the 2h-PG outcome, the BETA was -0.0370167, with a 95% confidence interval of -0.0619816 to -0.0120518, and a P value of 0.00369395, indicating a significant negative association. The above results indicate that the ID1 gene SNP site is associated with type 2 diabetes mellitus (T2DM) and 2-hour postprandial blood glucose, while the association with fasting blood glucose did not reach statistical significance in this analysis, further supporting the association between ID1 and population glucose metabolism phenotype.

Claims

1. Application of ID1 inhibitors in the preparation of drugs for the prevention and / or treatment of type 2 diabetes.

2. The application according to claim 1, characterized in that, The ID1 inhibitor refers to a substance that can reduce ID1 gene expression, reduce ID1 mRNA levels, reduce ID1 protein expression, inhibit ID1 function, or block ID1-mediated gluconeogenesis regulation.

3. The application according to claim 1, characterized in that, The ID1 inhibitors include small molecule compounds, natural products, nucleic acid inhibitors, siRNA, shRNA, antisense oligonucleotides, miRNA mimics, ID1 interference sequences delivered by viral vectors, gene editing reagents, peptides, antibodies, protein degrading agents, or pharmaceutically acceptable salts, prodrugs, derivatives, and pharmaceutical compositions thereof.

4. The application according to claim 1, characterized in that, The ID1 inhibitor is used to suppress the expression or function of hepatic ID1, thereby reducing hepatic gluconeogenesis.

5. The application according to claim 1, characterized in that, The ID1 inhibitor is curcumin or a pharmaceutical composition containing curcumin.

6. The application according to claim 1, characterized in that, The ID1 inhibitor is an shRNA, siRNA, or a viral vector carrying an ID1 interference sequence that targets ID1.

7. A pharmaceutical composition for the prevention and / or treatment of type 2 diabetes, characterized in that, The pharmaceutical composition comprises an effective dose of an ID1 inhibitor and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, injections, lyophilized powder injections, lipid nanoparticle formulations, sustained-release formulations, or controlled-release formulations.

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