Use of Fam3a inhibitors in the preparation of drugs for promoting GLP-1 secretion in pancreatic alpha cells and regulating systemic glucose homeostasis

Abstract

The present invention belongs to the field of biomedicine technology. Specifically, it relates to the use of Fam3a inhibitors in the preparation of drugs that promote GLP-1 secretion in pancreatic alpha cells and regulate systemic glucose homeostasis. The research results of the present invention show that Fam3a in pancreatic alpha cells changes the body's glucose homeostasis by regulating the GLP-1 paracrine signaling pathway, that is, the specific lack of Fam3a in pancreatic alpha cells can promote the production of pancreatic GLP-1, which acts on adjacent β cells in a paracrine form to promote insulin secretion and maintain systemic glucose homeostasis. The present invention clarifies for the first time the role and specific mechanism of Fam3a in pancreatic alpha cells, and provides a new targeted treatment option for promoting pancreatic GLP-1 secretion, which has extremely high application value and social benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of Fam3a inhibitors in the preparation of drugs for promoting GLP-1 secretion in pancreatic alpha cells and regulating systemic glucose homeostasis. Background Art

[0002] Glucose homeostasis refers to the state in which blood sugar levels in the body are maintained within a relatively stable range. Under normal circumstances, the human body maintains blood sugar within a certain range through complex regulatory mechanisms to meet various physiological and metabolic needs. Hormones such as insulin and glucagon play an important role in this process. Glucose homeostasis is associated with a variety of diseases and physiological states, such as diabetes, pancreatic diseases, endocrine diseases, kidney diseases, obesity, Cushing syndrome, etc.

[0003] The body's regulation of glucose homeostasis mainly relies on the synergistic effect of pancreatic α cells and β cells to maintain the steady-state balance of systemic blood glucose. This process involves a complex and sophisticated endocrine regulatory mechanism. When the body's blood glucose is lower than the normal threshold, pancreatic α cells can secrete glucagon, activate liver glycogenolysis and gluconeogenesis, and quickly correct the falling blood glucose level. On the contrary, when the body is in a state of hyperglycemia or diabetes, it relies on the remaining pancreatic β cells to secrete insulin to promote the uptake and utilization of glucose by peripheral tissues, while inhibiting liver gluconeogenesis to achieve the effect of lowering blood glucose.

[0004] In previous studies, the regulation of glucose homeostasis was mainly targeted at the targeted treatment of pancreatic β cells. With the development of the dual hormone hypothesis, domestic and foreign scholars have gained new insights into the contribution of α cells to glucose homeostasis in recent years. We and other groups have demonstrated that blocking glucagon-glucagon receptor signaling can reduce blood glucose in diabetic animals and humans [Nat Med 2022;28:2092–9; Diabetes 2023;72:599–610], but there are still side effects such as secondary tumors and α cell hyperplasia [Proc Natl Acad Sci USA 2017;114:3006–8]. Proglucagon (encoded by GCG) can be cleaved into different polypeptides in different cells by hormone convertases (PCs). For example, in α cells, proglucagon is cleaved by PC2 (encoded by PCSK2) to form glucagon, whereas in intestinal L cells and specific neurons, it is cleaved by PC1 / 3 (encoded by PCSK1) to generate peptides such as glucagon-like peptide-1 (GLP-1) [EndocrRev 1999;20:876–913]. As an incretin, GLP-1 stimulates insulin secretion in a glucose-dependent manner and therefore plays a crucial role in glucose homeostasis [Cell Metab 2018;27:740–56]. Notably, recent studies have challenged the traditional paradigm that GLP-1 is produced only in the intestine, highlighting the ability of α cells to produce GLP-1 in response to metabolic stress [Cell Metab. 2019;30(5):976-986.e3; Curr Opin Pharmacol. 2022Apr;63:102199]. Studies have shown that compared with L cell-derived GLP-1, α cell-derived GLP-1 is more important for glucose homeostasis, because nearly 90% of L cell-derived GLP-1 is degraded by dipeptidyl peptidase-4 in the circulation [Cell Metab. 2017; 25(4): 927-934.e3]. Therefore, increasing the production of pancreatic GLP-1 has become a new strategy for regulating glucose homeostasis. Fam3a protein is a recently discovered mitochondrial protein that can be expressed in a variety of cell types. Its role in liver and pancreatic β cells has received widespread attention. Previous studies have shown that Fam3a can improve glucose metabolism and reduce fat deposition in diabetic mouse models by activating the ATP-P2 receptor-Akt and AMPK signaling pathways [Metabolism. 2018Apr; 81: 71-82]. In addition, studies have shown that Fam3a in pancreatic β cells can significantly increase the expression and secretion of insulin and improve impaired pancreatic β cell function [FASEB J. 2020 Mar;34(3):3915-3931].These research advances suggest that Fam3a has the potential to become a therapeutic target for metabolic diseases.

[0005] However, the role of Fam3a in maintaining systemic glucose homeostasis has not been reported. Summary of the invention

[0006] In order to overcome the problems existing in the prior art, the object of the present invention is to provide the use of Fam3a inhibitors in the preparation of drugs for promoting GLP-1 secretion in pancreatic α cells and regulating systemic glucose homeostasis.

[0007] In order to achieve the above-mentioned purpose and other related purposes, the present invention adopts the following technical solutions:

[0008] The present application provides the use of Fam3a inhibitors in the preparation of drugs for promoting GLP-1 secretion in pancreatic α cells and regulating systemic glucose homeostasis.

[0009] As an optional mode, in the above use, the Fam3a inhibitor is the only active ingredient or one of the active ingredients of the drug.

[0010] As an optional mode, in the above use, the drug is in the form of solid, liquid, gel, semi-fluid, or aerosol.

[0011] As an optional mode, in the above-mentioned use, the subject of the drug is a mammal or a human.

[0012] As an alternative, in the above use, the mammal is selected from rodents, artiodactyls, perissodactyls, lagomorphs or primates.

[0013] As an option, in the above use, the primate is a monkey or an ape.

[0014] As an optional mode, in the above-mentioned use, the Fam3a inhibitor is a nucleic acid molecule, a carbohydrate, a lipid, a small molecule chemical drug, an antibody drug, a polypeptide, a protein or an interfering lentivirus.

[0015] As an alternative, in the above use, the nucleic acid molecule is selected from antisense oligonucleotide, double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).

[0016] As an option, in the above use, the nucleic acid molecule is selected from double-stranded RNA (dsRNA) or short hairpin RNA (shRNA).

[0017] As an option, in the above use, the double-stranded RNA (dsRNA) is small interfering RNA (siRNA).

[0018] As an optional mode, in the above use, the Fam3a inhibitor is to knock out or knock down the expression of Fam3a gene in the object targeted by the drug.

[0019] As an optional mode, in the above use, the Fam3a inhibitor is to knock out or knock down the expression of Fam3a gene in the pancreatic α cells targeted by the drug.

[0020] As an optional mode, in the above-mentioned use, the amount of the Fam3a inhibitor administered is a dose sufficient to reduce the transcription or translation of the Fam3a gene targeted by the drug, or a dose sufficient to reduce the expression or activity of the Fam3a protein targeted by the drug, so that the transcription or translation of the Fam3a gene or the expression or activity of the Fam3a protein is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] After extensive and in-depth research, the present invention found that Fam3a of pancreatic α cells changes the body's glucose homeostasis by regulating the GLP-1 paracrine signaling pathway, that is, the specific deletion of Fam3a in pancreatic α cells can promote the production of pancreatic GLP-1, which acts on adjacent β cells in a paracrine form, promotes insulin secretion, and maintains systemic glucose homeostasis.

[0023] The present invention clarifies for the first time the role and specific mechanism of Fam3a in pancreatic α cells, and provides a new targeted treatment option for promoting pancreatic GLP-1 secretion, which has extremely high application value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 : Whole body Fam3a knockout (Fam3a - / - ) Hormone expression levels in mice.

[0026] Figure 2 :The expression level of Pcsk1 was detected by knocking down or overexpressing Fam3a in α cell line (αTC1.9).

[0027] Figure 3 :α cell line transcriptomics identified Nr4a2 as a downstream molecule of Fam3a and positively regulated by Fam3a.

[0028] Figure 4: Fam3a regulates the nuclear translocation of Foxa2 in α cells through the Nr4a−Foxa2 complex.

[0029] Figure 5 :Fam3a regulates PC1 / 3 expression in α cells through Foxa2.

[0030] Figure 6 : Fam3a regulates Pcsk1 transcriptional activity in α cells through the Nr4a2−Foxa2 complex.

[0031] Figure 7 : Pancreatic α-cell-specific knockout of Fam3a (Fam3a α- / - ) Glucose metabolism phenotype of mice.

[0032] Figure 8 : Pancreatic α-cell-specific knockout of Fam3a (Fam3a α- / - ) improves pancreatic islet function in diabetic mice. DETAILED DESCRIPTION

[0033] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0034] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0037] All experimental data below were analyzed and plotted using GraphPad Prism 9.0 or R language package. Experimental data are expressed as mean ± standard error (mean ± SEM). Data analysis methods were performed using analysis of variance (ANOVA) with Tukey-Kramer post hoc test, Student's T test, or Mann-Whitney test according to data distribution and grouping, and P < 0.05 indicated statistically significant differences.

[0038] Example:

[0039] 1. Experimental methods:

[0040] Animal experiments

[0041] Transgenic mouse construct: Fam3a flox / flox The mice (gifted by Professor Yang Jichun's research group at Peking University Health Science Center) were crossed with Gcg-Cre transgenic mice (gifted by Professor Xu Chengran's research group at Peking University Health Science Center). Their offspring were self-fertilized to achieve multiplication, and finally the pancreatic α-cell-specific Fam3a gene knockout mice were obtained, which were named Fam3a α- / - In this study, the control mice were littermates of Flox or Cre mice (Flox / cre mice). In addition, another transgenic mouse in this study was a systemic knockout of Fam3a (Fam3a - / - ) mice (donated by Professor Yang Jichun's research group at the School of Basic Medical Sciences, Peking University Health Science Center), and wild-type C57BL / 6J mice (WT mice) were used as control mice. When the mice were 12 weeks old (n=5-6 per group), their metabolic indicators, including body weight, random blood glucose, fasting blood glucose, and intraperitoneal glucose tolerance test (GTT), were tested. In addition, to detect whether the active GLP-1 secreted by pancreatic α cells acts on adjacent pancreatic β cells in a paracrine form. After an overnight fast, Fam3a α- / - Mice and their control Flox / cre mice were injected with Ex9-39 (blocking GLP-1 signaling pathway) or vehicle, and ipGTT was performed 30 minutes later.

[0042] The diabetic mouse model was established using the streptozotocin (STZ) method. Male Fam3a mice aged 12 weeks α- / - After overnight fasting, mice and their control Flox / cre mice (n = 8 in each group) were given a single large dose of 125 mg / kg STZ intraperitoneal injection. Blood glucose was tested on the 7th day after injection, and the model was considered successful when fasting blood glucose ≥ 11.1 mmol / L.

[0043] The animal experiments strictly followed the Peking University Animal Experiment Management Regulations and were reviewed by the Peking University Animal Experiment Ethics Committee.

[0044] After the in vivo experiments on mice were completed, samples were collected and subsequent experiments were performed.

[0045] (1) Collection of plasma and pancreatic tissue: Mouse plasma was collected at the end of the experiment and frozen at -80°C for subsequent experiments. A small amount of fresh pancreatic tissue was collected near the tail of the pancreas and frozen in liquid nitrogen. The remaining pancreatic tissue was fixed with 10% paraformaldehyde, dehydrated routinely, and embedded in paraffin for sectioning.

[0046] (2) Detection of the area of ​​pancreatic α and β cells in pancreatic tissue: Paraffin sections of pancreatic tissue were subjected to routine immunofluorescence staining (insulin, glucagon, and DAPI) to calculate the proportion of pancreatic α and β cell areas in pancreatic tissue.

[0047] (3) Hormone determination: Mouse plasma or pancreatic tissue protein lysate was used to detect insulin (Alpco, USA), glucagon (R&D, USA) and active GLP-1 (Alpco, USA) using ELISA kits.

[0048] (4) Extraction and intervention of primary mouse pancreatic islet cells: extraction of Fam3a α- / - Primary islet cells from mice and their control Flox / cre mice (n = 8 per group). To detect basal hormone secretion, 25 islets were selected from each group, and the islet culture supernatant was collected after 24 hours of incubation in high glucose (25mmol / L) medium to detect the expression levels of glucagon, insulin and active GLP-1. To detect the paracrine effect of GLP-1 secretion by pancreatic α cells, 25 islets were selected from each group, and the islet culture supernatant was collected 24 hours after intervention with Ex9-39 or vehicle control to determine the insulin level. All hormone test results were corrected using the total protein content of the islets.

[0049] 1.2. Cell experiments

[0050] Cell line culture and experiments: In this study, the α cell line (αTC1.9) was purchased from the ATCC cell bank in the United States (https: / / www.atcc.org / ). The human embryonic kidney 293T cell line (293T) was purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (https: / / www.cellbank.org.cn / ).

[0051] (1) Construction of a cell line that knocks down or overexpresses a specific gene: αTC1.9 cell line, using Lipofectamine™ RNAiMAX transfection reagent to transfect small interfering RNA (siRNA) targeting specific genes (e.g., Fam3a, Foxa2, and Nr4a2) (exemplary siRNA sequences are shown in Table 1 below) into the αTC1.9 cell line to construct an α cell line with specific gene knockdown. The sequence of the Fam3a gene is shown in SEQ ID NO.1.

[0052] Table 1: Small interfering RNA (siRNA) sequences targeting Fam3a gene

[0053]

[0054] Instead, overexpression plasmids targeting specific genes (e.g., Fam3a, Foxa2, and Nr4a2) were transfected into the αTC1.9 cell line using Lipofectamine™ 2000 transfection reagent to construct an α cell line overexpressing a specific gene. RNA or protein was extracted from the cells 24 hours after transfection, and the efficiency of specific gene knockdown or overexpression was verified by RT-PCR or Western blot.

[0055] (2) Detection of the effect of Fam3a on α cell phenotype: RNA and protein were routinely extracted from αTC1.9 cell lines and control cells with knockdown or overexpression of Fam3a, and RT-PCR was used to detect the mRNA expression levels of α cell phenotype-related markers (e.g., Gcg, Pcsk2, and mafB, etc.) and Pcsk1. Western blot was used to detect the protein expression level of Pcsk1. ELISA kits were used to detect the secretion level of active GLP-1.

[0056] (3) Screening of key molecules that Fam3a regulates PC1 / 3 expression by transcriptomics in α cells: αTC1.9 cells were transfected with Fam3a small interfering RNA (siFam3a) or control siRNA (n = 3 per group) and then subjected to transcriptomic sequencing. The DESeq2R software package was used to identify differentially expressed genes, and P value < 0.05 and |log2foldchange| ≥ 1 were set as the thresholds for significant differential expression. The volcano plot and principal component analysis plot of differential gene expression were visualized using the R software package. Subsequently, the Search Tool for the Retrieval of Interacting Genes (STRING) database (http: / / string-db.org / ) was used to construct a protein-protein interaction (PPI) network of transcription factors, and the cytoHubba plug-in in the Cytoscape 3.10.1 software was used to screen the top 24 transcription factor key genes. In addition, the GOSemSim package in the R software was used for Friends analysis to identify the top 10 key genes with the highest biological function semantic similarity.

[0057] (4) Detection of the interaction between Foxa2 and Nr4a2 proteins: GRAMM-X (http: / / gramm.x3dna.org / ) was used to perform rigid protein-protein docking between Nr4a2 and Foxa2. The protein domains of Nr4a2 and Foxa2 were obtained from the Protein Data Bank (PDB, http: / / www.rcsb.org / ). AlphaFold was used to predict the structures of Nr4a2 (AF-Q06219-F1) and Foxa2 (AF-P36683-F1). Pymol (version 2.5) and PDBePISA (https: / / www.ebi.ac.uk / pdbe / pisa / ) were used for protein interaction analysis and visualization analysis. Subsequently, protein co-immunoprecipitation (Co-IP) technology was used to verify the interaction between Foxa2 and Nr4a2 proteins.

[0058] (5) Detection of Fam3a regulating Pcsk1 transcriptional activity through the Foxa2−Nr4a2 complex: The Pcsk1 promoter region (-2000 bp − +100 bp) was cloned into the pGL3-based luciferase reporter vector to establish the Pcsk1-luciferase reporter gene. αTC1.9 cells were co-transfected with the RL-TK Renilla luciferase gene, the Pcsk1 promoter-luciferase gene, and the Foxa2 plasmid, the Nr4a2 plasmid, or the empty vector using Lipofectamine™ 2000. After 24 hours of transfection, a dual-luciferase reporter gene assay was performed (Promega, USA). To further identify the specific binding site of Foxa2 on the Pcsk1 promoter, the full-length Pcsk1 promoter was gradually truncated into three sequences: truncated sequence 1 (-1420 bp − +50 bp), truncated sequence 2 (-810 bp − +50 bp), and truncated sequence 3 (-530 bp − +50 bp). Subsequently, mutants of Foxa2 binding sites (sites 2, 6, and 10) were introduced into truncated sequence 3, and dual-luciferase reporter gene assays were performed.

[0059] 2. Experimental results:

[0060] 2.1. Whole-body knockout of Fam3a - / - ) Increased secretion of active GLP-1 in mice

[0061] First, the expression of Fam3a in different islet cell subsets was detected and the results showed that (see Figure 1 A): Fam3a is mainly expressed in pancreatic β cells, followed by pancreatic α cells. Next, Fam3a was detected - / -The results showed that the hormone levels in fasting plasma and pancreatic α cells of mice and their control mice (WT mice) were Figure 1 B-1E): Compared with WT mice, Fam3a - / - There was no significant change in fasting plasma glucagon in mice, but the level of fasting plasma active GLP-1 increased significantly; the hormone levels in pancreatic tissue also showed a similar trend. The above results suggest that Fam3a deficiency can significantly increase the production of pancreatic GLP-1.

[0062] 2.2. Fam3a negatively regulates Pcsk1 expression in α cells

[0063] In the αTC1.9 cell line, Fam3a expression was inhibited by small interfering RNA technology, or Fam3a was overexpressed by plasmid transfection to explore the effect of Fam3a on α cell maturation markers, PC1 / 3 expression, and active GLP-1 levels. The results showed (see Figure 2 ): Compared with the control group (Ctrl), knockdown of Fam3a (siFam3a) can significantly downregulate the expression of α cell maturation-related markers (such as Pcsk2, MafB, Arx and Pax6), and significantly upregulate the expression levels of Pcsk1 gene and protein, and increase the secretion of active GLP-1. In contrast, overexpression of Fam3a (oeFam3a) can significantly reduce the expression levels of Pcsk1 gene and protein, and has a trend of reducing the secretion of active GLP-1 (P = 0.094), but has no significant effect on other α cell maturation-related markers. The above results suggest that in the α cell line, Fam3a negatively regulates the expression of PC1 / 3, thereby affecting the secretion of active GLP-1.

[0064] 2.3. Nr4a2 is a downstream molecule of Fam3a and is positively regulated by Fam3a

[0065] In the αTC1.9 cell line, small interfering RNA technology was used to inhibit Fam3a expression (siFam3a) and transcriptomic sequencing analysis was performed. The results showed that (see Figure 3 AD): Principal component analysis (PCA) showed that the siFam3a and control groups (Ctrl) were clearly separated. 473 differentially expressed genes were identified in the two groups, of which 326 were upregulated and 147 were downregulated. The protein interaction network (PPI) of transcription factors in the differential genes showed that the top 10 transcription factors were: Nr4a2, Six3, Fosl2, Egr1, Lhx3, Nfia, Isl2, Hes6, Otx1 and Onecut1, among which Nr4a2 scored the highest. In addition, Nr4a2 was also included in the top 5 key genes in the Friends analysis. Therefore, Nr4a2 was screened as a key downstream molecule of Fam3a and verified. The results showed (see Figure 3 EH): Compared with the control group (Ctrl), knockdown of Fam3a significantly downregulated the expression levels of Nr4a2 mRNA and protein; conversely, overexpression of Fam3a significantly upregulated the expression levels of Nr4a2 mRNA and protein. The above results suggest that Fam3a positively regulates the expression level of Nr4a2 in α cells.

[0066] 2.4. Fam3a regulates the nuclear translocation of Foxa2 in α cells through the Nr4a2-Foxa2 complex

[0067] To further explore whether Nr4a2 is involved in the expression regulation of PC1 / 3, the protein expression and transcriptional levels of PC1 / 3 were detected after knocking down Nr4a2. Figure 4 A and B), knocking down Nr4a2 can significantly upregulate the protein expression level of PC1 / 3, but cannot regulate its transcription level. Considering that Nr4a2 may interact with other proteins to exert its biological function, the Nr4a2 interacting protein (Nr4a2-PPI) and the transcription factor binding to the Pcsk1 promoter (predicted using three databases: TFBD, Jaspar, and Genecards) were used to screen the Nr4a2 interacting protein. The results showed (see Figure 4 CE), the Venn diagram shows that three intersecting proteins were screened out, namely: Foxa2, Egr1 and Atf3; the protein docking model was used to further predict the interaction between Nr4a2 and Foxa2. The enlarged image of the protein docking interface showed that Nr4a2 and Foxa2 were stably docked and bound by hydrogen bonds at amino acid residues (such as Gln404Glu265); immunoprecipitation technology further verified this interaction. Subsequently, the regulatory effect of Fam3a on Foxa2 was detected. The results showed (see Figure 4 FJ), cell immunofluorescence staining suggested that Fam3a overexpression could offset the translocation of Foxa2 out of the nucleus caused by insulin stimulation; immunoblotting experiments of cytoplasm and nucleus separation further confirmed this Foxa2 nuclear translocation effect; in addition, immunoblotting experiments showed that Nr4a2 knockdown caused Foxa2 to translocate from the nucleus to the cytoplasm, while Fam3a overexpression reversed this effect. The above results indicate that Fam3a regulates the nuclear translocation of Foxa2 in α cells through the Nr4a2-Foxa2 complex.

[0068] 2.5. Fam3a regulates Pcsk1 transcriptional activity in α cells through the Nr4a2-Foxa2 complex

[0069] In the αTC1.9 cell line, Foxa2 expression was inhibited by small interfering RNA technology, or Foxa2 was overexpressed by plasmid transfection to explore the effect of Foxa2 on PC1 / 3 expression and active GLP-1 levels. The results showed that (see Figure 5 AH), compared with the control group (Ctrl), knockdown of Foxa2 (siFoxa2) significantly upregulated Pcsk1 expression and PC1 / 3 levels, and increased the secretion of active GLP-1; on the contrary, compared with the control group (Ctrl), overexpression of Foxa2 (oeFoxa2) significantly downregulated Pcsk1 expression and PC1 / 3 levels, and reduced the secretion of active GLP-1. In addition, overexpression of Fam3a can offset the effect of increased PC1 / 3 expression induced by knockdown of Foxa2. The above results indicate that Foxa2 is involved in mediating the negative regulation of PC1 / 3 by Fam3a.

[0070] Previous studies have shown that Foxa2 can directly bind to the promoter of the target gene and regulate its transcription level. In order to explore whether the Nr4a2-Foxa2 complex can directly regulate the transcription level of Pcsk1, a dual luciferase reporter gene experiment was performed. The results showed that (see Figure 6 A and B), there are a total of 10 potential Foxa2 binding sites in the full-length Pcsk1 promoter sequence (-2000 bp − +100 bp); dual luciferase reporter gene experiments showed that Foxa2 significantly inhibited the promoter activity of the full-length and three truncated sequences of Pcsk1, indicating that the core binding region of Foxa2 is located in the truncated sequence 3, including binding sites 10, 6, and 2. To further confirm the specific binding site of Foxa2-mediated Pcsk1 transcriptional inhibition, mutation sites were introduced in sites 10, 6, and 2 of the truncated sequence 3 of the Pcsk1 promoter. The results showed (see Figure 6 CE), dual luciferase reporter gene experiments showed that mutations at sites 10 and 6 reversed the transcriptional inhibition of Pcsk1 by Foxa2, while mutations at site 2 had no such effect, indicating that Foxa2 specifically binds to sites 10 and 6 of the Pcsk1 promoter. In addition, overexpression of Nr4a2 enhanced the inhibitory effect of Foxa2 on Pcsk1 promoter activity. The above results indicate that Fam3a regulates the transcriptional activity of Pcsk1 in α cells through the Nr4a2-Foxa2 complex.

[0071] 2.6. Pancreatic α-cell-specific knockout of Fam3a α- / - ) mice improve whole-body glucose homeostasis via GLP-1 paracrine signaling

[0072] Detection of Fam3a α- / -The glucose tolerance level, fasting plasma glucagon, fasting plasma and pancreatic tissue active GLP-1, and plasma insulin levels of mice and their littermate control Flox / cre mice were measured; pancreatic tissue sections were immunofluorescently stained to analyze the area of ​​pancreatic α and β cells; primary pancreatic islets were extracted from mice to detect the levels of the above hormones. The results showed (see Figure 7 AF): Compared with Flox / cre mice, Fam3a α- / - The glucose tolerance of mice was significantly improved, the fasting plasma glucagon level was significantly reduced, the fasting plasma and pancreatic tissue active GLP-1 levels were significantly increased, and the insulin secretion 15 minutes after glucose loading was significantly increased. The results of immunofluorescence staining and quantitative analysis of pancreatic tissue suggest that (see Figure 7 GI): Compared with Flox / cre mice, Fam3a α- / - The proportion of pancreatic α and β cells in the pancreatic tissue of mice did not change significantly, suggesting that Fam3a α- / - The increase in plasma insulin levels in mice was not related to the number of β cells, but may be related to the improvement of β cell function. Results from primary mouse islets showed (see Figure 7 JL), compared with Flox / cre mice, Fam3a α- / - The secretion of glucagon in primary mouse islets was significantly reduced, while the secretion of active GLP-1 and insulin was significantly increased. The above results suggest that the loss of Fam3a in pancreatic α cells can cause the main secretory product of α cells to change from glucagon to active GLP-1, while promoting insulin secretion.

[0073] Next, Fam3a α- / - Mice and Flox / cre mice were intraperitoneally injected with Ex9-39 or solvent to block the GLP-1 signaling pathway, and ipGTT experiments were performed 30 minutes later. α- / - Primary pancreatic islets from mice and Flox / cre mice were treated with Ex9-39 or vehicle for 24 hours, and the insulin level in the islet culture supernatant was measured. The results showed that (see Figure 7 MN): Compared with Flox / cre mice, Fam3a α- / - The glucose tolerance of mice was significantly improved, and compared with the vehicle control, Fam3a α- / - Treatment of mice with Ex9-39 significantly reversed this trend; meanwhile, the results of primary mouse islets suggested (see Figure 7 O): Compared with Flox / cre mice, Fam3a α- / -The insulin level of primary mouse islets increased significantly, and compared with the vehicle control, this trend was significantly reversed after treatment with Ex9-39. The above results suggest that Fam3a deficiency can promote the secretion of GLP-1 from pancreatic α cells, which acts on pancreatic β cells in a paracrine manner and promotes insulin secretion, thereby regulating systemic glucose homeostasis.

[0074] 2.7. Pancreatic α-cell-specific knockout of Fam3a (Fam3a α- / - ) Improves pancreatic islet function in diabetic mice

[0075] Detection of Fam3a in diabetic model α- / - Plasma glucagon, plasma insulin, pancreatic tissue active GLP-1 and plasma insulin levels of mice and their littermate control Flox / cre mice were measured; metabolic indices of mice were measured, including fasting body weight, fasting blood glucose and random blood glucose. The results showed that (see Figure 8 AC): In the diabetic state, compared with Flox / cre mice, Fam3a α- / - The plasma glucagon level of mice was significantly reduced, and the pancreatic tissue active GLP-1 level and plasma insulin level were significantly increased. At the same time, compared with Flox / cre mice, Fam3a α- / - There was no significant difference in fasting body weight, fasting blood glucose and random blood glucose of mice (see Figure 8 DF). The above results suggest that Fam3a can improve pancreatic β-cell function by increasing pancreatic active GLP-1 under the metabolic stress state of diabetes, thereby maintaining pancreatic metabolic balance.

[0076] 3. Conclusion

[0077] Fam3a improves the body's glucose homeostasis by regulating the GLP-1 paracrine signaling pathway derived from pancreatic α cells. In the treatment of diabetes, it can enhance the ability of the pancreas to adapt to metabolic stress, thereby maintaining normal function.

[0078] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of Fam3a inhibitor in the preparation of a drug for treating diabetes, characterized in that: The Fam3a inhibitor is a small interfering RNA that works by reducing Fam3a gene expression in the target subject; The small interfering RNA comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are selected from the sequences shown in SEQ ID NO.4-5, SEQ ID NO.6-7 or SEQ ID NO.8-9.

2. The use according to claim 1, characterized in that: The Fam3a inhibitor is the sole active ingredient or one of the active ingredients of the drug.

3. The use according to claim 1, characterized in that: The amount of the Fam3a inhibitor administered is a dose sufficient to reduce the transcription or translation of the Fam3a gene targeted by the drug, or sufficient to reduce the expression or activity of the Fam3a protein targeted by the drug.

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