Fusion protein containing improved GLP-1 polypeptide and use

Abstract

Provided is a fusion protein, the fusion protein containing a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, the GLP-1 polypeptide is selected from human GLP-1(9-37) and human GLP-1(9-36) amides, and the GLP-1 polypeptide contains G22E and / or R36G substitutions relative to a natural human GLP-1 polypeptide. The human GLP-1(9-37) and human GLP-1(9-36) amides are products respectively resulting from losing two amino acids at the N-terminus of natural human GLP-1(7-37) and human GLP-1(7-36, by means of DPP4 enzyme degradation, and were previously considered as biologically inactive fragments. Provided are a polynucleotide encoding the fusion protein, a vector and cell containing the polynucleotide, and the use thereof, for example, in the preparation of drugs for treating metabolic diseases related to lipid metabolism disorders, complications of metabolic diseases, neurodegenerative diseases and other related diseases.

Description

Improved GLP-1 polypeptide and fusion protein and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, the present application relates to an improved GLP-1 polypeptide, a fusion protein comprising the improved GLP-1 polypeptide, a polynucleotide encoding the fusion protein, a vector comprising the polynucleotide, a cell and application thereof. BACKGROUND

[0002] The liver is an important organ in vivo involved in glucose, fat and cholesterol metabolism, which maintains the metabolic balance of the body. Abnormal glucose and lipid metabolism in vivo leads to ectopic excessive deposition of lipids in hepatocytes, inducing liver lesions, which is called non-alcoholic fatty liver disease (NAFLD), commonly known as fatty liver. NAFLD includes a variety of different pathological states, simple hepatic steatosis (liver cell lipid accumulation) is the early stage of NAFLD, further development into non-alcoholic steatohepatitis (NASH) characterized by progressive inflammation and fibrosis of the liver, eventually leading to late fibrosis, cirrhosis, liver failure, liver cancer, and seriously endangering human health.

[0003] Currently, there is no effective treatment for NAFLD and NASH except lifestyle intervention. Due to the huge unmet clinical needs of NAFLD / NASH, many candidate drugs targeting the signaling molecules related to metabolic, inflammatory, oxidative stress, immune regulation, fibrosis and apoptosis have entered clinical research. Due to the complexity of glucose and lipid metabolism in vivo and the lack of recognized disease treatment targets, most drugs have not reached the primary efficacy endpoint or have been discontinued due to strong side effects. Recently, more than ten clinical 2 and 3 phase NASH clinical studies, including PPAR receptor agonists (PATIKORN C, VEETTIL S K, PHISALPRAPA P, et al. Horizon scanning of therapeutic modalities for nonalcoholic steatohepatitis [J]. Annals of hepatology, 2021, 24: 100315), farnesol X receptor agonists (YOUNOSSI Z M, RATZIU V, LOOMBA R, et al. Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial [J]. Lancet, 2019, 394 (10215): 2184-96) and the like have announced withdrawal, reflecting the current cognitive defects of the disease treatment targets.

[0004] Therefore, it is urgent to find an effective and safe treatment strategy for the clinical treatment of NAFLD and NASH. SUMMARY

[0005] One of the purposes of the present application is to provide a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from human GLP-1 (9-37) and human GLP-1 (9-36) amide, and the GLP-1 polypeptide comprises G22E and / or R36G substitutions relative to the native human GLP-1 polypeptide. The fusion protein can be obtained by various routes or means. The fusion protein provided by the present application comprises amino acid substitutions at specific positions, which prolongs the half-life of the GLP-1 polypeptide while maintaining the activity of the GLP-1 polypeptide, and has good prevention and treatment effect in human diseases and animal disease models.

[0006] Another object of the present application is to provide new biological functions of GLP-1(9-37) and GLP-1(9-36) amide and mechanisms thereof, which clarify that GLP-1(9-37) and GLP-1(9-36) amide have an effect of improving liver lipid metabolism disorder, and provide a new prevention and treatment method for NASH and liver-related metabolic diseases.

[0007] In one aspect, the present application provides a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from the group consisting of human GLP-1(9-37) and human GLP-1(9-36) amide, and the GLP-1 polypeptide comprises G22E and / or R36G substitution relative to native human GLP-1 polypeptide.

[0008] In some embodiments, the GLP-1 polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises one or more amino acid substitutions relative to native human GLP-1 polypeptide selected from the group consisting of G22E and R36G. In some embodiments, the GLP-1 polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises G22E and R36G substitution relative to native human GLP-1 polypeptide. In some embodiments, the GLP-1 polypeptide is human GLP-1(9-37), and comprises G22E and R36G substitution relative to native human GLP-1 polypeptide. In some embodiments, the GLP-1 polypeptide has an amino acid sequence set forth as SEQ ID NO: 3.

[0009] In some embodiments, the immunoglobulin Fc domain comprises or is an IgG2-Fc domain. In some embodiments, the IgG2-Fc domain is an Fc domain from human IgG2. In some embodiments, the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0010] In some embodiments, the IgG2-Fc domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. In some embodiments, the IgG2-Fc domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises A330S and P331S substitutions. In some embodiments, the IgG2-Fc domain has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises C222S, A330S, and P331S substitutions. In some embodiments, the amino acid sequence of the IgG2-Fc domain is set forth in SEQ ID NO: 6.

[0011] In some embodiments, the fusion protein provided herein comprises a GLP-1 polypeptide set forth in SEQ ID NO: 3, and comprises an immunoglobulin Fc domain set forth in SEQ ID NO: 6.

[0012] In some embodiments, the GLP-1 polypeptide is located at the N-terminus or the C-terminus of the immunoglobulin Fc domain. In some embodiments, the fusion protein provided herein comprises, from N-terminus to C-terminus, a GLP-1 polypeptide set forth in SEQ ID NO: 3 and an immunoglobulin Fc domain set forth in SEQ ID NO: 6.

[0013] In some embodiments, the GLP-1 polypeptide is directly covalently linked to the immunoglobulin Fc domain. In some embodiments, the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain via a linker. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker. In some embodiments, the linker comprises a connecting peptide that connects the GLP-1 polypeptide and the immunoglobulin Fc domain. In some embodiments, the connecting peptide comprises a glycine and serine containing linker. In some embodiments, the glycine and serine containing linker comprises one, two, three, four or more repeats of SEQ ID NO: 8 (GGGS), SEQ ID NO: 9 (GGGGS), SEQ ID NO: 10 (GGGGGS), or SEQ ID NO: 11 (GGGGGGGS). In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, in the fusion protein provided herein, the GLP-1 polypeptide has an amino acid sequence of SEQ ID NO: 3, the immunoglobulin Fc domain has an amino acid sequence of SEQ ID NO: 6, and the linker has an amino acid sequence of SEQ ID NO: 7. In some embodiments, the fusion protein provided herein comprises, from N-terminus to C-terminus, a GLP-1 polypeptide having an amino acid sequence of SEQ ID NO: 3, a linker having an amino acid sequence of SEQ ID NO: 7, and an immunoglobulin Fc domain having an amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein provided herein has an amino acid sequence of SEQ ID NO: 24 or comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0015] In some embodiments, the fusion protein provided by the present application further comprises a signal peptide.

[0016] In some embodiments, the fusion protein provided by the present application has a half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days in a human subject.

[0017] In another aspect, the present application also provides a dimer comprising two identical peptide chains connected by a disulfide bond, wherein each of the peptide chains comprises the fusion protein provided by the present application.

[0018] In another aspect, the present application also provides a nucleic acid molecule comprising a polynucleotide encoding the fusion protein provided by the present application. In some embodiments, the nucleic acid molecule provided by the present application comprises a polynucleotide sequence as set forth in SEQ ID NO: 26 or a polynucleotide sequence having at least 70% sequence identity to the polynucleotide as set forth in SEQ ID NO: 26.

[0019] In another aspect, the present application also provides a vector comprising the nucleic acid molecule provided by the present application.

[0020] In another aspect, the present application also provides a cell comprising the nucleic acid molecule or the vector provided by the present application. In some embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cell derived from a human or a Chinese hamster ovary (CHO) cell. In some embodiments, the mammalian cell is a human embryonic kidney 293 (HEK293 cell), or a CHO-K1 cell, or a CHO-S cell, or a CHO-DG44 cell.

[0021] In another aspect, the present application also provides a composition comprising the fusion protein, the dimer, the nucleic acid molecule, the vector, or the cell provided by the present application.

[0022] In another aspect, the present application provides a method for constructing the cell provided by the present application, comprising:

[0023] a) introducing a nucleic acid molecule encoding the fusion protein provided by the present application into a vector to construct an expression vector (preferably, the vector is a pKN012 vector);

[0024] b) introducing the expression vector into a cell to obtain a recombinant cell (preferably, the cell is a CHO cell, in particular a CHO-K1 cell).

[0025] In some embodiments, the present application provides a method of constructing a cell, comprising:

[0026] a) inserting the polynucleotide sequence shown in SEQ ID NO: 26 into the Ncol and Hindlll sites of the pKN012 vector to generate a pKN012-GLP1-IgG2 / Fc expression vector;

[0027] b) introducing the pKN012-GLP1-IgG2 / Fc expression vector into CHO-K1 cells to obtain a recombinant cell.

[0028] In another aspect, the present application also provides a method of producing a fusion protein, comprising the step of obtaining the fusion protein using the cell prepared by the method of constructing provided by the present application.

[0029] In another aspect, the present application provides use of the fusion protein, or the dimer, or the nucleic acid molecule, or the vector, or the cell, or the composition provided by the present application in the manufacture of a medicament for treating or preventing a disease.

[0030] In some embodiments, the disease is selected from the group consisting of a metabolic disease associated with glucose and / or lipid metabolism disorder, a complication of metabolic disease, a central metabolic disease (e.g., a neurological disease), and other related metabolic diseases. In some embodiments, the metabolic disease associated with glucose and / or lipid metabolism disorder is a liver metabolic disease associated with glucose and / or lipid metabolism disorder. In some embodiments, the metabolic disease associated with glucose and / or lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity, and metabolic syndrome. In some embodiments, the complication of metabolic disease includes a cardiovascular complication (e.g., heart failure), a kidney complication (e.g., diabetic nephropathy, chronic kidney disease), or a liver complication (e.g., fatty liver, including NAFLD and NASH) caused by metabolic disease. In some embodiments, the neurological disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, motor neuron disease, Huntington's disease, and Parkinson's disease.

[0031] In another aspect, the present application also provides a method of binding to and activating a signal cascade of long-chain-fatty-acid-CoA-ligase 1 (ACSL1) on a cell membrane of a target cell to improve lipid metabolism. In some embodiments, the method comprises exposing a GLP-1 polypeptide or a fusion protein comprising a GLP-1 polypeptide (the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide), a fusion protein, a dimer, a nucleic acid molecule, a vector, a cell or a composition provided by the present application to the target cell. In some embodiments, the target cell is an adipocyte or a hepatocyte.

[0032] In another aspect, the present application also provides a method of treating or preventing a disease, the method comprising administering to a subject a GLP-1 polypeptide or a fusion protein comprising a GLP-1 polypeptide, wherein the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide; and the disease is selected from the group consisting of a metabolic disease associated with lipid metabolism disorder, a complication of metabolic disease, a central metabolic disease (e.g., a neurological disease), and other related metabolic diseases. In some embodiments, the GLP-1 polypeptide is a GLP-1 polypeptide or a fusion protein comprising the same provided by the present application.

[0033] In some embodiments, the metabolic disease associated with lipid metabolism disorder is a liver disease associated with lipid metabolism disorder.

[0034] In some embodiments, the subject has or is at risk of having one or more symptoms selected from the group consisting of lipid deposition, elevated triglyceride level, elevated cholesterol, elevated low-density lipoprotein level, elevated free fatty acid level, increased accumulation of fatty acids, insulin resistance, elevated glutamic-pyruvic transaminase level, elevated glutamic-oxaloacetic transaminase level, elevated malondialdehyde content, decreased glutathione peroxidase (GSH-Px) activity, decreased superoxide dismutase (SOD) activity, and elevated inflammatory factor level.

[0035] In some embodiments, the metabolic disease associated with lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity, and metabolic syndrome. In some embodiments, the NAFLD is non-obese NAFLD.

[0036] In some embodiments, the complication of the metabolic disease is selected from the group consisting of cardiovascular disease (CVD) caused by metabolic disease, diabetic kidney disease (DKD), chronic kidney disease (CKD), or liver disease, e.g., fatty liver, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH). In certain embodiments, the cardiovascular disease comprises heart failure.

[0037] In some embodiments, the nervous system disease is a neurodegenerative disease (e.g., Alzheimer’s disease, motor neuron disease, Huntington’s disease, Parkinson’s disease).

[0038] Other features and advantages of the present application will be apparent from the following detailed description, taken in conjunction with the accompanying drawings in the following detailed description and specific examples. The detailed description and specific examples are given only as examples in order to more fully describe the preferred embodiments of the application, and are not intended to limit the scope of the application. Various modifications and changes will be readily suggested to those skilled in the art, which modifications and changes are to be included within the spirit and scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 shows that mouse liver does not express GLP-1R. The results of (A) RT-PCR (B) Western blot (GLP-1R antibody Mab 3F52) or (C) immunohistochemistry using mRNA or protein samples extracted from mouse liver, liver tissue sections show that mouse liver does not express GLP-1R. Islet / pancreas and brain tissues are used as positive controls. The magnification of the pictures is 400X, and the scale bar is 40 mM.

[0040] Figure 2 shows the construction and verification of GLP-1 / Fc fusion proteins. 7-37 / Fc is a fusion molecule of GLP-1 (7-37) and human immunoglobulin IgG2 Fc fragment; 9-37 / Fc is a fusion molecule of GLP-1 (9-37) and human immunoglobulin IgG2 Fc fragment. (A) GLP-1 fusion proteins are formed by GLP-1 and human IgG2-Fc via covalent disulfide bonds to form dimers; the second amino acid at the N-terminus of 7-37 / Fc is mutated to G to resist hydrolysis by DPP-4; 9-37 / Fc has two fewer amino acids at the N-terminus than 7-37 / Fc to mimic GLP-1 degraded by DPP-4 in vivo. (B) Coomassie blue-stained electrophoresis gel (C) Western blot with GLP-1 antibody (top) or IgG2 antibody (bottom). The results show that GLP-1 fusion proteins form stable dimers (~60 kDa) under non-reducing conditions. DTT: dithiothreitol.

[0041] Figure 3 shows the distribution of 7-37 / Fc and 9-37 / Fc in zebrafish. The red fluorescent labeled 7-37 / Fc and green fluorescent labeled 9-37 / Fc were mixed 1 : 1 and microinjected into zebrafish in vivo for visualization. 9-37 / Fc was mainly distributed in the liver, while no obvious liver distribution was observed for 7-37 / Fc.

[0042] Figure 4 shows that 9-37 / Fc binding to hepatocytes is independent of GLP-1R. Double (Double) or single (Single) staining of isolated hepatocytes from (A) wild type mice (B) GLP-1 receptor knockdown mice (GLP-1R KO) with Alexa Fluor fluorescent labeled 7-37 / Fc (AF647) and 9-37 / Fc (AF448) was performed and analyzed by flow cytometry. (C) INS-1 β cell line as control.

[0043] Figure 5 shows the binding affinity of 9-37 / Fc and 7-37 / Fc to HepG2 cells. (A) 9-37 / Fc binds to HepG2 cells in a concentration dependent manner (B) The binding affinity of 9-37 / Fc to HepG2 cells is nearly 50 times higher than that of 7-37 / Fc (after Fc standardization), data are expressed as mean ± standard deviation, n = 3.

[0044] Figure 6 shows the technical roadmap of animal experiments.

[0045] Figure 7 shows the preparation and concentration of SOD standard (picture from Dojindo Laboratories SOD Assay Kit Instruction Manual).

[0046] Figure 8 shows the preparation and concentration of IL-6 standard (picture from Anogen IL-6 ELISA Assay Kit Instruction Manual)

[0047] Figure 9 shows GLP-1 (9-37) reduces key gluconeogenic enzymes in liver. (A) Liver PEPCK (green) promoter transgenic zebrafish microinjected with GFP-tagged glucagon, 7-37 / Fc or 9-37 / Fc 24 h later, liver fluorescence images suggest that 9-37 / Fc reduces PEPCK expression. In comparison, glucagon increases, 7-37 / Fc does not significantly change PEPCK (quantitative results see column chart below). (B) In wild-type zebrafish, 9-37 / Fc reduces PEPCK or G6PC gene expression, glucagon increases, 7-37 / Fc does not significantly change PEPCK or G6PC expression. (C) In mice, 9-37 / Fc significantly reduces PEPCK and G6PC gene expression more than 7-37 / Fc. Data are expressed as mean ± standard deviation, n = 8, *P < 0.05, **P < 0.01, ***P < 0.001 compared with PBS; *P < 0.05 compared with 7-37 / Fc. & P < 0.05.

[0048] Figure 10 shows PA induces lipid accumulation in HepG2 cells. Oil red O staining and quantitative analysis after HepG2 cells were incubated with different concentrations of PA for 24 h. The magnification of the picture is 200X, and the scale bar is 50 μΜ. Data are expressed as mean ± standard deviation, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001 compared with control. IOD: integrated optical density; Area: area.

[0049] Figure 11 shows 9-37 / Fc improves PA-induced lipid accumulation in HepG2 cells. (A) Oil red O staining after HepG2 cells were incubated with PA alone, or in the presence of 9-37 / Fc or 7-37 / Fc for 24 h. (B) Quantitative analysis of oil red O staining images. The magnification of the picture is 200X, and the scale bar is 50 μΜ. Data are expressed as mean ± standard deviation, n = 4, *P < 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001 compared with PA, # P < 0.05, ## P < 0.01 compared with PA + 7-37, & P < 0.05. IOD: integrated optical density; Area: area. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0050] Figure 12 shows that 9-37 / Fc reduces TG content in PA-induced HepG2 cells. TG content of HepG2 cells under the above experimental conditions was detected by ELISA kit. Data are expressed as mean ± standard deviation, n = 5, compared with control, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with PA, # P < 0.05. NS represents no statistical significance.

[0051] Figure 13 shows the determination of plasma concentration of 9-37 / Fc, 7-37 / Fc fusion proteins and calculation of half-life. (A) Schematic diagram of intraperitoneal injection of fusion proteins 9-37 / Fc, 7-37 / Fc and blood sampling time points (B) Plasma drug concentration-time curve after single intraperitoneal injection of fusion proteins 9-37 / Fc, 7-37 / Fc (C) Plot of natural logarithm (InC) of plasma drug concentration of 9-37 / Fc, 7-37 / Fc against time (h), and calculate the drug half-life according to the slope after linear regression. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0052] Figure 14 shows that the effect of 9-37 / Fc on reducing high-fat diet-induced weight gain in mice is significantly weaker than that of 7-37 / Fc. After 12 weeks of intervention, (A) body weight of mice in each group (B) food intake of mice in each group. Data are expressed as mean ± standard deviation, n = 8 (n = 6 for NCD group), compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, compared with HFD + 9-37, & P < 0.05. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0053] Figure 15 shows that 9-37 / Fc improves insulin resistance in high-fat diet-induced mice, but has no significant effect on glucose tolerance. After 12 weeks of intervention, (A) intraperitoneal glucose tolerance test (IPGTT) curve of mice in each group (B) Area under the curve of IPGTT (C) Intraperitoneal insulin tolerance test (IPITT) curve of mice in each group (D) Area under the curve of IPITT. Data are expressed as mean ± standard deviation, n = 8 (n = 6 for NCD group), compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001; compared with HFD+9-37, & P < 0.05. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0054] Figure 16 shows that 9-37 / Fc reduces blood lipid and transaminase levels in high-fat diet-induced mice. Serum (A) triglyceride (B) cholesterol (C) low-density lipoprotein (D) free fatty acid (E) ALT and (F) AST levels of mice in each group after 12 weeks of intervention. Data are expressed as mean ± standard deviation, n = 8 (NCD group n = 6), compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0055] Figure 17 shows that 9-37 / Fc reduces liver lipid accumulation in high-fat diet-induced mice. After 12 weeks of intervention, (A) liver, epididymal fat (eWAT), inguinal subcutaneous fat (iWAT), pancreas and muscle tissue weight (B) liver weight / body weight percentage (C) representative pictures of liver gross morphology (D) representative pictures of liver tissue HE staining (E) liver triglyceride content (F) liver cholesterol content (G) liver tissue oil red O staining representative picture (H) NAFLD activity score calculated from liver tissue steatosis, hepatocyte ballooning and lobular inflammation according to HE staining (I) oil red O staining quantitative statistics. The magnification of the picture is 200X, the scale bar is 50 μM. Data are expressed as mean ± standard deviation, n = 8 (NCD group n = 6), compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001,#### P < 0.001. “7-37” and “9-37” in the figures represent “7-37 / Fc” and “9-37 / Fc”, respectively.

[0056] Figure 18 shows that 9-37 / Fc increases the antioxidant enzymes GSH-Px and SOD activities in the liver of high-fat diet mice, and improves oxidative stress. (A-C) The content of lipid peroxidation product MDA (A), the activity of antioxidant enzyme GSH-Px (B), and the activity of antioxidant enzyme SOD (C) in the liver tissue of mice in each group were detected by chemical colorimetry. (D) Representative pictures of DCFA-DA fluorescent staining of frozen sections of liver tissue of mice in each group and (E) statistical analysis of average fluorescence intensity. The magnification of the pictures is 200X, and the scale bar is 50 mM. Data are expressed as mean ± standard deviation, n = 8 (n = 6 for NCD group), compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001. “7-37” and “9-37” in the figures represent “7-37 / Fc” and “9-37 / Fc”, respectively.

[0057] Figure 19 shows that 9-37 / Fc reduces the levels of inflammatory factors such as IL-6 and TNF-a in the serum and liver of high-fat diet mice. (A) Western blot detection of IL-6 and TNF-a protein expression in the liver tissue of mice in each group (B-C) Quantitative statistics of IL-6 and TNF-a protein expression content, and the data of each group are normalized to the protein expression amount of the NCD group (D) RT-qPCR detection of IL-6 and TNF-a gene expression in the liver tissue of mice in each group, and the data of each group are normalized to the gene expression amount of the NCD group (E-F) ELISA detection of IL-6 and TNF-a content in the serum of mice in each group. Data are expressed as mean ± standard deviation, n = 6-8, compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001; compared with HFD+7-37, &P<0.05. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0058] Figure 20 shows that 9-37 / Fc does not affect the body weight of high-fat diet-induced mice. (A) Body weight (B) food intake of mice in each group fed with normal diet or high-fat diet for 16 weeks. Data are expressed as mean ± standard deviation, n=8 (n=6 for NCD group), compared with NCD, * P<0.05, ** P<0.01; compared with HFD, # P<0.05, ## P<0.01, ### P<0.001, compared with HFD+9-37, & P<0.05, && P<0.01, &&& P<0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0059] Figure 21 shows that 9-37 / Fc improves insulin resistance and has no significant change in impaired glucose tolerance in high-fat diet-induced mice. (A) Intraperitoneal glucose tolerance curve (B) Area under the curve of IPGTT (C) Intraperitoneal insulin tolerance curve (D) Area under the curve of IPITT of mice in each group after 16 weeks of intervention. Data are expressed as mean ± standard deviation, n= (8 NCD group n=6), compared with NCD, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.001; compared with HFD, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.001; compared with HFD+9-37, & P<0.05, && P<0.01, &&& P<0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0060] Figure 22 shows that 9-37 / Fc reduces blood lipid and transaminase levels in high-fat diet-induced mice. (A) Triglyceride (B) Cholesterol (C) Low-density lipoprotein (D) Free fatty acid (E) ALT (F) AST levels in serum of mice in each group after 16 weeks of intervention. Data are expressed as mean ± standard deviation, n=8 (n=6 for NCD group), compared with NCD, *P<0.05, ** P<0.01, *** P<0.001, **** P<0.001; compared with HFD, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0061] Figure 23 shows that 9-37 / Fc reduces liver lipid accumulation in high-fat diet-induced mice. After 16 weeks of intervention, the (A) liver, epididymal white adipose tissue (eWAT), inguinal subcutaneous adipose tissue (iWAT), pancreas and muscle tissue weights of mice in each group (B) liver weight / body weight percentage (C) representative pictures of liver gross morphology (D) representative pictures of liver tissue HE staining (E) liver triglyceride content (F) liver cholesterol content (G) representative pictures of liver tissue oil red O staining (H) NAFLD activity score was calculated from three aspects of liver steatosis, hepatocyte ballooning and lobular inflammation according to HE staining (J) oil red O staining quantification. The magnification of the pictures is 200X, and the scale bar is 50 μΜ. Data are expressed as mean ± standard deviation, n = 8 (NCD group n = 6), compared with NCD, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.001; compared with HFD, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0062] Figure 24 shows that 9-37 / Fc increases the antioxidant enzyme GSH-Px and SOD activity in the liver of high-fat diet mice, and improves oxidative stress. After 16 weeks of intervention, (A-C) colorimetric detection of (A) lipid peroxidation product MDA content (B) antioxidant enzyme GSH-Px activity (C) antioxidant enzyme SOD activity in liver tissue of mice in each group (D) representative pictures of DCFA-DA fluorescent staining of liver tissue frozen sections of mice in each group and (E) average fluorescence intensity statistics. The magnification of the pictures is 200X, and the scale bar is 50 μΜ. Data are expressed as mean ± standard deviation, n = 8 (NCD group n = 6), compared with NCD, * P<0.05, ** P<0.01, ***P < 0.001, **** P < 0.001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0063] Figure 25 shows that 9-37 / Fc reduces the levels of inflammatory factors such as IL-6 and TNF-a in the serum and liver of high-fat diet mice. (A) Western blot detection of IL-6 and TNF-a protein expression in liver tissue of mice in each group (B-C) Quantitative statistics of IL-6 and TNF-a protein expression content, the data of each group is normalized to the protein expression amount of the NCD group (D) RT-qPCR detection of IL-6 and TNF-a gene expression in liver tissue of mice in each group, the data of each group is normalized to the gene expression amount of the NCD group (E-F) ELISA detection of IL-6 and TNF-a content in serum of mice in each group. Data represent mean ± standard deviation, n = 6-8, compared with NCD, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001; compared with HFD+7-37, & P < 0.05. "7-37" and "9-37" in the figure represent "7-37 / Fc" and "9-37 / Fc", respectively.

[0064] Figure 26 shows that 9-37 / Fc reduces the expression of genes related to hepatic lipid synthesis and promotes the expression of genes related to lipid catabolism. HepG2 cells were incubated with PA alone, or in the presence of 9-37 / Fc or 7-37 / Fc for 24 hours, and proteins and mRNA were extracted. (A) Western blot detected the expression level of SREBP-1 protein (B) qRT-PCR detected the expression level of lipid synthesis related genes SREBP-1, FAS, and ACC (C) qRT-PCR detected the expression level of lipid β-oxidation related genes ACOX1, PPARa, and CPT1. Western blot detected the expression of SREBP-1, PPARa, and CPT1 proteins in liver tissues of mice fed with high-fat diet and administered with 7-37 / Fc or 9-37 / Fc (D) prophylactic intervention for 12 weeks or (E) therapeutic intervention for 4 weeks (F-G) qRT-PCR detected the expression of lipid synthesis and catabolism related genes in the liver of mice in the above prophylactic and therapeutic studies. (H) Flow cytometry detected the DCFH-DA fluorescence intensity of HepG2 cells in each group (I) Elisa kit detected the ATP content in HepG2 cells. Data are expressed as mean ± standard deviation, n = 3-8, (A-C, H, I) compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with PA, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001 (D-G) compared with the NCD group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001; compared with the HFD group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.001. “7-37” and “9-37” in the figure represent “7-37 / Fc” and “9-37 / Fc”, respectively.

[0065] Figure 27 shows 9-37 / Fc inhibits PA and high-fat diet-induced p-ERK expression. HepG2 cells were incubated with PA alone, or in the presence of 9-37 / Fc or 7-37 / Fc for 24 hours (A) Western blot detected the expression of p-ERK and p-PKA in each treatment group and (A’) quantification statistics. Mice were given 9-37 / Fc or 7-37 / Fc (B) prophylactic intervention for 12 weeks Western blot detected the expression of p-ERK protein in liver tissue of each group of mice and (B’) quantification statistics (C) treatment intervention for 4 weeks Western blot detected the expression of p-ERK protein in liver tissue of each group of mice and (C’) quantification statistics. Data are expressed as mean ± standard deviation, n = 3-8, (A’) compared with control, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001; compared with PA, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with PA + 7-37, & P < 0.05 (B’-C’) compared with NCD group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001; compared with HFD, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with HFD + 7-37, & P < 0.05. “7-37” and “9-37” in the figure represent “7-37 / Fc” and “9-37 / Fc”, respectively.

[0066] Figure 28 shows extraction and identification of liver cell membrane proteins. (A) Silver staining and Coomassie blue staining of electrophoresis gel of total protein (Lysate) and extracted liver cell membrane (PM). (B) Western blot with Na + / K + ATPase) and GAPDH antibodies. Abbreviation: PM, plasma membrane.

[0067] Figure 29 shows mass spectrometry identifies potential receptors for GLP-1(9-37). Using the 9-37 / Fc fusion protein tool, based on its high affinity for binding to hepatocytes and its Fc region binding to protein A, rat hepatocytes were isolated with sucrose gradient centrifugation to extract the plasma membrane (PM), 300 μg of 9-37 / Fc (or 7-3 / Fc, IgG-Fc as a control) was mixed with 30 μΐ of agarose beads and coupled to form protein pre-coated agarose beads at 4 °C overnight, then 300 μg of PM protein was incubated in 1 mL reaction volume at 4 °C overnight for pull-down, unbound proteins were washed away, bound PM proteins were eluted with acidic buffer, separated by SDS-PAGE, stained with silver, protein bands were cut, trypsin digested and subjected to mass spectrometry analysis. (A) Silver-stained SDS-PAGE, protein bands in white boxes were subjected to mass spectrometry analysis. (B) Proteins bound to 9-37 / Fc in silver-stained gels were identified by mass spectrometry in descending order of protein Sum PEP score. (C) Chimera software simulated the interaction mode diagram of ACSL1 and 9-37 / Fc, where ACSL1 is meat color, GLP-1(9-37) is rainbow color, and amino acid residues that form hydrogen bonds are dark blue. Abbreviations: Exp. Q-value: the experimental; Sum PEP score: Sum posterior error probability score; PSMs: peptide spectrum matches; AAs: amino acids; MW [kDa]: molecular weight (kDa); calc. pi: calculated isoelectric point.

[0068] Figure 30 shows ACSL1 is located on the membrane of HepG2 cells. HepG2 cells were subjected to immunofluorescence staining of ACSL1 (red) and DAPI (blue), and typical pictures were selected for display. (A-B) ACSL1 showed the distribution characteristics of immunofluorescence surface staining of antigens. (C) Pictures were merged with bright field. (D) Control experiments were performed using IgG antibody and (E) fluorescent secondary antibody alone. (F) HepG2 cells were permeabilized with 0.1% Triton X-100 before staining with anti-ACSL antibody. Pictures were taken at 400X magnification, and the scale bar is 10 μΜ.

[0069] Figure 31 shows 9-37 / Fc but not 7-37 / Fc binds to ACSL1 on hepatocyte membrane and undergoes endocytosis. HepG2 cells were incubated with 100 nM 9-37 / Fc or 7-37 / Fc at 4°C for 4 h, then switched to 37°C for 30 min, cells were stained with fluorescent labeled antibody (A) Confocal images show 9-37 / Fc (green) presents obvious binding and endocytosis at 37°C (B) Cells were double-stained (ACSL1: red; hlgG: green) show 9-37 / Fc co-localizes with ACSL1 on cell membrane and intracellular, suggesting ACSL1-mediated 9-37 / Fc endocytosis (C) Western blot verified the efficiency of ACSL1 siRNA knockdown (D) Western blot verified the efficiency of ACSL1 -OE overexpression (E) Binding curve of 9-37 / Fc to HepG2 cells after knockdown or overexpression of ACSL1, and affinity constant (Kd) and maximum specific binding coefficient (Bmax). Data represent mean ± s.d., n = 3. Picture magnification is 400X, scale bar 40 mM. Compared with control, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0070] Figure 32 shows knockdown of ACSL1 attenuates the inhibitory effect of 9-37 / Fc on lipid accumulation in HepG2 cells. Knockdown or non-knockdown of ACSL1 with PA alone, or in the presence of 9-37 / Fc, each treatment group (A) Representative pictures of oil red O staining and quantitative statistical analysis. Picture magnification is 200X, scale bar 50 mM. (B) Triglyceride content. Data represent mean ± s.d., n = 3, * P < 0.05, ** P < 0.01. IOD, integrated optical density; Area, area; TG, triglyceride.

[0071] Figure 33 shows overexpression of ACSL1 enhances the effect of 9-37 / Fc on reducing PA-induced lipid accumulation. Overexpression or non-overexpression of ACSL1 with PA alone, or in the presence of 9-37 / Fc, each treatment group (A) Representative pictures of oil red O staining and quantitative statistical analysis. Picture magnification is 200X, scale bar 50 mM (B) Triglyceride content. Data represent mean ± s.d., n = 3, compared with control, * P < 0.05, ** P < 0.01, *** P < 0.001. IOD, integrated optical density; Area, area; TG, triglyceride.

[0072] Figure 34 shows 9-37 / Fc inhibits ERK phosphorylation and lipid synthesis, promotes beta oxidation in HepG2 cells through ACSL1. (A) Western blotting detected the protein expression levels of ACSL1, p-ERK, SREBP-1 and PPARa in each group after ACSL1 siRNA treatment and statistical quantification. (B) Western blotting detected the protein expression levels of ACSL1, p-ERK, SREBP-1 and PPARa in each group after ACSL1-OE treatment and statistical quantification. Data are expressed as mean ± standard deviation, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0073] Figure 35 shows the vector information of ACSL1 overexpression plasmid.

[0074] Figure 36 is a schematic diagram of the vector pKN012-GLP1-IgG2 / Fc. DETAILED DESCRIPTION

[0075] The following is a detailed description that helps a person of ordinary skill in the art to practice the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the present application are only used to describe the specific embodiments and are not intended to limit the present application. All publications, patent applications, patents, drawings and other references mentioned herein are incorporated by reference in their entirety into the present application.

[0076] I. Definitions or Terms

[0077] All features disclosed in the specification can be combined in any way. Each feature disclosed in the specification can be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed in the present application is only an example of a series of equivalent or similar features.

[0078] The terms "peptide," "polypeptide," and "protein," as used herein, refer to a chain of two or more amino acid residues, whether natural or non-natural, connected, whether or not post-translational modifications (e.g., glycosylation or phosphorylation) are present. A polypeptide in the present application can include, for example, 3 to 3500 natural or non-natural amino acid residues. The protein can be a single polypeptide chain or a multi-subunit protein (e.g., can consist of 2 or more polypeptides). "Peptide," "polypeptide," and "protein" as described herein can be used interchangeably, can contain natural amino acids, can contain non-natural amino acids, can contain modified amino acids, or can contain unmodified amino acids, or analogs, mimetics of amino acids. The peptides, polypeptides, or proteins described in the present application can be obtained by any method known in the art, for example, but not limited to, by natural isolation, recombinant expression, chemical synthesis, and the like.

[0079] The term "amino acid," as used herein, refers to an organic compound containing an amino (-NH2) and carboxyl (-COOH) functional groups, and a side chain specific to each amino acid. The amino acid names are also represented in the standard one-letter or three-letter code in the present application, which is summarized as follows:

[0080] The term "GLP-1 polypeptide," as used herein, includes a GLP-1 polypeptide having lysine at amino acid residue position 34 or corresponding to amino acid residue position 34, for example, a polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 2. Specifically, "GLP-1 polypeptide" as used herein includes, but is not limited to, GLP-1 (7-37), GLP-1 (7-36-NH2) ("GLP-1 (7-36-NH2) can also be interchangeably referred to herein as "GLP-1 (7-36)" or "GLP-1 (7-36) amide"), GLP-1 (9-37), GLP-1 (9-36-NH2) ("GLP-1 (9-36-NH2) can also be interchangeably referred to herein as "GLP-1 (9-36)" or "GLP-1 (9-36) amide"), or other GLP-1 analogs having lysine at the amino acid residue position 34 or corresponding to the amino acid residue position 34. For example, the GLP-1 polypeptide can comprise a sequence from Liraglutide (Liraglutide, from Novo Nordisk ), Semaglutide (Semaglutide, from Novo Nordisk ), Albiglutide (Albiglutide, from GlaxoSmithKline ), Taspoglutide (Roche), Dulaglutide (Dulaglutide, from Eli Lilly ) or GLP-1 polypeptides of LY2428757 (Eli Lilly and Company), can also include GLP-1 polypeptides disclosed in WO2021163972A1, CN111217915A, WO2011056713A2, and WO2000034332A1, each incorporated herein by reference. For example, the above polypeptides can be GLP-1 analogs comprising a “KG” amino acid motif sequence.

[0081] The term “polynucleotide” or “nucleic acid” as used herein refers to two or more nucleotides covalently linked. Unless the context clearly indicates otherwise, this term generally includes, but is not limited to, deoxyribonucleotides (DNA) and ribonucleotides (RNA), which can be single-stranded (ss) or double-stranded (ds). For example, the polynucleotide or nucleic acid molecules of the present application can consist of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions. Mixtures of single- and double-stranded regions, hybrid molecules comprising RNA and DNA, which can be single-stranded or, more typically, double-stranded or mixtures of single- and double-stranded regions. In addition, the polynucleotide molecules can consist of triple-stranded regions comprising RNA or DNA, or both RNA and DNA.

[0082] The term “oligonucleotide” as used herein generally refers to a polynucleotide of up to 200 base pairs in length, and can be single- or double-stranded. The sequences provided herein can be DNA sequences or RNA sequences, however it is understood that the sequences provided include both DNA and RNA, as well as complementary RNA and DNA sequences, unless the context clearly indicates otherwise. For example, the sequence 5’-GAATCC-3’ is understood to include 5’-GAAUCC-3’, 5’-GGATTC-3’, and 5’-GGAUUC-3’.

[0083] The term "sequence identity" or "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or between two polynucleotide sequences. To determine the percent identity of two amino acid sequences or of two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or polynucleotide sequence for optimal alignment with a second amino acid or polynucleotide sequence), and the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. In other words, the percent (%) sequence identity of the amino acid sequences (or of a nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or of bases) that are the same in both the candidate and reference sequences (or the shorter of the two) by the total number of amino acid residues (or of bases) in the candidate sequence or in the reference sequence, using the same amino acid residues (or bases) of the reference sequence with which the candidate sequence is compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In some embodiments, the two sequences are of the same length. The percent identity between two sequences can also be determined using a mathematical algorithm. One preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (see Karlin, S. and S. F. Altschul, Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci U S A, 1990. 87(6): p. 2264-8). Such an algorithm is incorporated in the NBLAST and XBLAST programs (Altschul, S. F., et al., Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10) which can be used to perform BLAST nucleotide searches using the NBLAST nucleotide program set, for example, score = 100, word length = 12, to obtain nucleotide sequences homologous to a particular polynucleotide molecule. BLAST protein searches can be performed using the XBLAST program set, for example, score = 50, word length = 3, to obtain amino acid sequences homologous to a protein molecule described herein.For purposes of comparison, Gapped BLAST (see Altschul, S.F., et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997. 25(17): p. 3389-402) can be used. Alternatively, PSI-BLAST can be used to perform an iterated search to detect distant relationships between molecules. When using the BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm that is suitable for sequence comparison is the algorithm of Myers and Miller (see Myers, E.W. and W. Miller, Optimal alignments in linear space. Comput Appl Biosci, 1988. 4(1): p. 11-7), incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing for gaps.

[0084] In the present application, a "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue that does not abrogate the properties of the protein. Suitable conservative amino acid substitutions can be made, for example, by replacing one of the following groups of amino acids with another: hydrophobic (e.g., between an alanine, isoleucine, valine, leucine, or methionine); polar (e.g., between an arginine and a lysine, between a glutamine and an asparagine); basic (e.g., between a lysine, an arginine, or a histidine); or acidic (e.g., between an aspartic acid and a glutamic acid). The phrase "conservative amino acid substitution" also includes the use of a chemically derivatized residue or a non-natural amino acid in place of a non-derivatized residue or a natural amino acid, provided that the polypeptide exhibits the necessary activity.

[0085] In the present application, the term "fusion protein" refers to a protein comprising two or more polypeptides that form distinct functional domains. For example, a GLP-1 fusion protein as described herein comprises a GLP-1 polypeptide and an immunoglobulin Fc domain.

[0086] In the present application, the term "linker" refers to any chemical moiety that is capable of covalently linking one moiety to another moiety. For example, a "linker" can be an artificial polypeptide having 1, 2, 3, 4, or 5 amino acid residues, or a length between 5 and 15, 20, 30, 50, or more amino acid residues, linked by peptide bonds, and used to link one or more polypeptides. A linker can or can not have a secondary structure. Linker sequences are known in the art, for example, those disclosed in Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., Structure 2:1121-1123 (1994).

[0087] In the present application, the term "CH2" refers to constant heavy chain 2, which is one of the domains of an immunoglobulin heavy chain. Similarly, the term "CH3" refers to constant heavy chain 3, which is another domain of an immunoglobulin heavy chain.

[0088] In the present application, the term "hinge" when used in the context of an immunoglobulin (e.g., IgG), refers to the flexible region between the antigen-binding fragment (Fab) and the crystallizable fragment (Fc).

[0089] The term "vector" as used herein refers to a vehicle into which a genetic element can be operatively inserted and which provides for expression of the genetic element, e.g., production of a protein, RNA or DNA encoded by the genetic element, or replication of the genetic element. A vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed within the host cell. By way of example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses, among others. Vectors can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors can contain a replication origin. Vectors can also include components that assist in their entry into cells, including but not limited to, viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors.

[0090] In the present application, the term "pharmaceutical grade" refers to the chemical purity or proportion of a drug, a biological macromolecule, or a reagent that meets the requirements for drug production.

[0091] In the present application, the term "treatment" refers to administration of an effective amount of a compound or composition or formulation to a subject, which can consist of a single administration, or alternatively include a series of procedures. As is well known in the art, "treatment" is the method used to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, partial or complete reversal of the disease, amelioration or palliation of the disease and its conditions, whether partial or total, or alleviation of one or more of the signs or symptoms of the disease. Beneficial or desired clinical results include improved fasting blood glucose and / or HbAlc levels, weight loss, improved liver lipid content, and improved cognitive function, motor coordination, etc.

[0092] In the present application, the term "subject" also referred to as "patient" or "object" refers to all animals including mammals, and preferably refers to humans. The "subject" can also be a domestic animal, such as a cow, pig, sheep, poultry, and horse; or a rodent, such as a rat, mouse; or a primate, such as an ape, monkey, chimpanzee, gorilla, orangutan, baboon; or a domesticated animal, such as a dog and cat.

[0093] In the present application, the term "pharmaceutically acceptable carrier" refers to any biologically or otherwise acceptable carrier, agent, excipient. The use of such a pharmaceutically acceptable carrier in treating formulations is well known in the art, for example, various ingredients that can be included in pharmaceutical formulations are described in Allen, L.V., Jr., Remington: The Science and Practice of Pharmacy: from the past into the future. Int J Pharm Compd, 2012. 16(5): p. 358-62.

[0094] In the present application, the term "therapeutically effective amount" refers to any dose that elicits the desired effect in a subject, which refers to alleviating symptoms, slowing disease progression, preventing disease onset, etc. The amount can be effective for multiple administrations and / or over a period of time to achieve the intended effect. As used herein, the term can refer to an amount that elicits an improvement in liver lipid deposition in a subject, or can also refer to an amount that elicits a reduction in triglyceride content in a subject.

[0095] In the present application, when referring to the "combined use" of two or more substances (e.g., two or more compounds, two or more compositions, etc.), it is meant that the two or more substances are administered to a subject, both of which are biologically active at the same time. The exact circumstances of the administration will depend on the pharmacokinetics of the two or more substances in the presence of each other. However, the "combined use" of the two or more substances does not necessarily have to be administered to the subject at the same time, the sequential administration of the two or more substances to the subject is also considered to be a "combined use". Moreover, the "combined use" of the two or more substances does not necessarily have to be administered to the subject by the same route of administration, for example, the first substance is administered to the subject by intravenous injection, the second substance is administered to the subject by oral administration, which is also considered to be a "combined use".

[0096] In understanding the scope of the present application, the term "comprising" and its derivatives, as used herein, are open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to like terms, such as "comprising," "having," "including," and their derivatives, and their equivalents.

[0097] As used herein, the terms "consisting of and "consisting essentially of are closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also preclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0098] Numerical ranges as used herein are inclusive of the numbers recited and endpoints (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5, etc.). It is also to be understood that all numbers and / or fractions thereof are presumed to be modified by the term "about."

[0099] Further, as used herein, terms such as "substantially," "about," and "approximately" mean an acceptable quantity of deviation from the recited quantity, such that the end result is not significantly changed. These terms should be interpreted as including a deviation of up to ±10% of the modified term. For example, the term "about" can mean ±1-10% of the reference number, such as up to 10%, up to 8%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% of the reference number.

[0100] As used in this specification and the appended claims, the singular forms“a,”“an,” and“the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing“a compound” includes mixtures of two or more compounds. It should also be noted that the term“or” is generally employed in its sense of

[0101] Furthermore, those skilled in the art will recognize that the definitions and embodiments described in particular sections are intended to apply to other embodiments described herein. For example, in the following paragraphs, different aspects of the application are described in more detail. Each of these aspects thus described can be combined with any other aspect or aspects unless explicitly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0102] II. Proteins and fusion proteins

[0103] In one aspect, the present application provides a fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, and the GLP-1 polypeptide comprises a G22E and / or R36G substitution relative to the native human GLP-1 polypeptide.

[0104] GLP-1 polypeptide

[0105] The unprocessed native human GLP-1 polypeptide has 37 amino acids, the amino acid sequence of which is set forth in SEQ ID NO:4, and is also commonly referred to as "GLP-1 (1-37)." Native human GLP-1 (1-37) is processed in the small intestinal epithelial cells to secrete GLP-1 (7-37) polypeptide or GLP-1 (7-36-NH2) ("GLP-1 (7-36-NH2)" is also referred to herein as "GLP-1 (7-36)," "GLP-1 (7-36)amide," or "GLP-1 (7-36)amide") into the blood circulation, where the N-terminal first two amino acids (histidine, alanine) are degraded by dipeptidyl peptidase 4 (DPP-4) to form GLP-1 (9-37) and GLP-1 (9-36-NH2) ("GLP-1 (9-36-NH2)" is also referred to herein as "GLP-1 (9-36)," "GLP-1 (9-36)amide," or "GLP-1 (9-36)amide"). Exemplary amino acid sequences of GLP-1 (7-37) and GLP-1 (7-36-NH2) are set forth in SEQ ID NO:23 and SEQ ID NO:25, respectively. Exemplary amino acid sequences of GLP-1 (9-37) and GLP-1 (9-36-NH2) are set forth in SEQ ID NO:2 and SEQ ID NO:1, respectively.

[0106] Unless otherwise specified, the amino acid positions of the GLP-1 polypeptides referred to in this application correspond to the amino acid positions of SEQ ID NO:4. For example, "GLP-1 (9-36)amide" refers to a GLP-1 polypeptide fragment formed by the amino acids between positions 9 and 36 of SEQ ID NO:4; "GLP-1 (9-37)" refers to a GLP-1 polypeptide fragment formed by the amino acids between positions 9 and 37 of SEQ ID NO:4. In some embodiments, the amino acid sequence of GLP-1 (9-36)amide is set forth in SEQ ID NO:1. In some embodiments, the amino acid sequence of GLP-1 (9-37) is set forth in SEQ ID NO:2.

[0107] Unless otherwise specified, the naming convention for the amino acid mutations referred to in this application is: the name of the amino acid before the mutation - the position of the amino acid where the mutation occurs - the name of the amino acid after the mutation. For example, a G22E mutation in a GLP-1 polypeptide refers to a mutation of glycine (G) at position 22 of SEQ ID NO:4 to glutamic acid (E).

[0108] In some embodiments, the GLP-1 polypeptide in the fusion protein provided herein can be a human-derived GLP-1 polypeptide. In some embodiments, the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide. In some embodiments, the GLP-1 polypeptide is human GLP-1 (9-37). In some embodiments, the GLP-1 polypeptide is GLP-1 (9-36) amide.

[0109] In some embodiments, the GLP-1 polypeptide can comprise an amino acid substitution, such as one or both of G22E, R36G mutations relative to a native human GLP-1 polypeptide, but still retains the function of the GLP-1 polypeptide (e.g., the function of ameliorating a hepatic glycolipid metabolic disorder).

[0110] In some embodiments, the GLP-1 polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, but still retains the function of the GLP-1 polypeptide (e.g., the function of ameliorating a hepatic glycolipid metabolic disorder).

[0111] In some embodiments, the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, and comprises a G22E substitution relative to a native human GLP-1 polypeptide. In some embodiments, the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, and comprises a R36G substitution relative to a native human GLP-1 polypeptide. In some embodiments, the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, and comprises both G22E and R36G substitutions relative to a native human GLP-1 polypeptide.

[0112] In some embodiments, the GLP-1 polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and comprises one or more amino acid substitutions relative to a native human GLP-1 polypeptide selected from the group consisting of G22E and R36G.

[0113] In some embodiments, the GLP-1 polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises G22E and R36G substitutions relative to native human GLP-1 polypeptide.

[0114] In some embodiments, the GLP-1 polypeptide is human GLP-1 (9-37) having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises G22E and R36G substitutions relative to native human GLP-1 polypeptide.

[0115] In some embodiments, the GLP-1 polypeptide is human GLP-1 (9-36) amide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises G22E and R36G substitutions relative to native human GLP-1 polypeptide.

[0116] In some embodiments, the GLP-1 polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, comprises one or more amino acid substitutions relative to native human GLP-1 polypeptide selected from the group consisting of G22E and R36G, and still retains the function of GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolism disorder).

[0117] In some embodiments, the GLP-1 polypeptide has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, includes G22E and R36G substitutions relative to native human GLP-1 polypeptide, and still retains the function of GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolic disorder).

[0118] In some embodiments, the GLP-1 polypeptide is human GLP-1 (9-37) having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, includes G22E and R36G substitutions relative to native human GLP-1 polypeptide, and still retains the function of GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolic disorder).

[0119] In some embodiments, the GLP-1 polypeptide is human GLP-1 (9-36) amide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, includes G22E and R36G substitutions relative to native human GLP-1 polypeptide, and still retains the function of GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolic disorder).

[0120] In some embodiments, the GLP-1 polypeptide has an amino acid sequence comprising the amino acid sequence set forth as SEQ ID NO: 3. In some embodiments, the GLP-1 polypeptide has an amino acid sequence set forth as SEQ ID NO: 3.

[0121] Fc domain

[0122] In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is the Fc domain of immunoglobulin IgG. In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is the Fc domain of immunoglobulin IgG1. In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is the Fc domain of immunoglobulin IgG2 (also referred to herein as "IgG2-Fc" or "IgG2 / Fc"). In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is the Fc domain of immunoglobulin IgG3. In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is the Fc domain of immunoglobulin IgG4.

[0123] In some embodiments, the immunoglobulin Fc domain in the fusion protein provided in this application is an Fc domain derived from human immunoglobulin IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0124] In some embodiments, the immunoglobulin Fc domain of the fusion protein provided in this application includes an IgG2-Fc domain. In some embodiments, the immunoglobulin Fc domain of the fusion protein provided in this application is an IgG2-Fc domain. In some embodiments, the IgG2-Fc domain described in this application is an Fc domain derived from human IgG2.

[0125] In some embodiments, the IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0126] In some embodiments, the IgG2-Fc domain includes a C222S substitution. While not bound by any particular theory, it is thought that the C222S substitution of the IgG2-Fc domain can increase the flexibility of the N-terminal hinge region by removing the disulfide bond between the two monomers of the homodimer. This increased flexibility of the N-terminal hinge region can reduce the binding affinity of the Fcγ receptor, thereby reducing antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0127] In some embodiments, the IgG2-Fc domain comprises A330S and P331S substitutions. While not bound by any particular theory, it is believed that the A330S and P331S substitutions in the IgG2-Fc domain reduce affinity for the Fcγ receptor and C1q complement protein.

[0128] In some embodiments, the IgG2-Fc domain includes C222S, A330S, and P331S substitutions.

[0129] Other amino acid residue positions of the IgG2-Fc domain described herein, including, for example, C222, A330, and P331, correspond to positions of human IgG2 as set forth in Genbank Accession No. QRG33935.1. The IgG2-Fc portion can be 227 amino acids as set forth in SEQ ID NO: 5, corresponding to, for example, amino acids 219-445 of human IgG2 as set forth in Genbank Accession No. QRG33935.1. One of skill in the art will readily recognize the residue positions of the amino acids, for example, C222, A330, and P331, in Fc fragments shorter or longer than those set forth in reference sequences SEQ ID NO: 5 or SEQ ID NO: 6.

[0130] In some embodiments, the IgG2-Fc domain described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. Wherein the fusion protein has improved half-life compared to a GLP-1 polypeptide that is not fused to an IgG / Fc domain or that is fused to an IgG4 / Fc domain.

[0131] In some embodiments, the IgG2-Fc domain described herein has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S. In some embodiments, the IgG2-Fc domain described herein has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

[0132] In some embodiments, the IgG2-Fc domain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity compared to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises A330S and P331S substitutions. In some embodiments, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises A330S and P331S substitutions.

[0133] In some embodiments, the IgG2-Fc domain further comprises a C222S substitution. In some embodiments, the IgG2-Fc domain has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity compared to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises C222S, A330S, and P331S substitutions. In some embodiments, the IgG2-Fc domain has at least 90% sequence identity compared to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and comprises C222S, A330S, and P331S substitutions.

[0134] In some embodiments, the IgG2-Fc domain has the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG2-Fc domain has the amino acid sequence of SEQ ID NO: 6.

[0135] In some embodiments, in the fusion proteins described herein, the GLP-1 polypeptide has the amino acid sequence of SEQ ID NO: 3, and the immunoglobulin Fc domain has the amino acid sequence of SEQ ID NO: 6.

[0136] The positions in SEQ ID NO: 3 corresponding to the positions in SEQ ID NO: 24 at which the G22E, R36G substitutions of the GLP-1 polypeptide referred to in this application occur are shown in the following table. In addition, the positions in QRG33935.1 corresponding to the positions in SEQ ID NO: 24 at which the C222S, A330S, P331S substitutions on the IgG2-Fc domain referred to in this application occur are also shown in the following table.

[0137] In some embodiments, in the fusion protein provided in this application, the GLP-1 polypeptide is located at the N-terminus of the immunoglobulin Fc domain. In some embodiments, in the fusion protein provided in this application, the GLP-1 polypeptide is located at the C-terminus of the immunoglobulin Fc domain.

[0138] In some embodiments, the fusion protein provided in this application comprises, from N-terminus to C-terminus, a GLP-1 polypeptide and an immunoglobulin Fc domain, the amino acid sequence of the GLP-1 polypeptide is set forth in SEQ ID NO: 3, and the amino acid sequence of the immunoglobulin Fc domain is set forth in SEQ ID NO: 6.

[0139] Linker

[0140] In some embodiments, the GLP-1 polypeptide in the fusion protein provided in this application is covalently linked (e.g., directly linked or linked through a linker) to the immunoglobulin Fc domain.

[0141] In some embodiments, the GLP-1 polypeptide is directly covalently linked to the immunoglobulin Fc domain.

[0142] In some embodiments, the GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain through a linker.

[0143] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

[0144] In some embodiments, the linker comprises a connecting peptide. In some embodiments, the linker comprises a connecting peptide connecting the GLP-1 polypeptide and the immunoglobulin Fc domain.

[0145] The GLP-1 polypeptide and the immunoglobulin Fc domain (e.g., an IgG Fc domain, particularly an IgG2-Fc domain) in the fusion protein provided in this application can be connected through a connecting peptide.

[0146] In the present invention, the "linker peptide" refers to any moiety that links different functional domains of a polypeptide together. The linker peptide can have any suitable length and structure.

[0147] In the present invention, the term "cleavable linker" refers to a linker that is sensitive to proteases, pH, or chemicals, etc. in vivo and is easily cleaved in the presence of the above factors.

[0148] In the present invention, the term "non-cleavable linker" refers to a linker that is stable to proteases, pH, or chemicals, etc. in vivo and is not easily cleaved.

[0149] In the present invention, the term "flexible linker" refers to a linker that, when linking different protein components, can increase the spatial extensibility so that the spatial folding, conformation of the protein components are as little affected by each other as possible.

[0150] In the present invention, the term "rigid linker" refers to a linker that, when linking different protein components, can maintain a fixed distance between the protein components.

[0151] In the present invention, the term "helical linker" refers to a linker in which the rigid unit can form a helix (e.g. an α-helix) within itself or between the same adjacent sequences, so that the resulting fusion protein has a relatively stable spatial conformation.

[0152] In the present invention, the term "non-helical linker" refers to a linker that cannot form a helix structure.

[0153] In some embodiments, the linker peptide comprises a glycine and serine containing linker. In some embodiments, the glycine and serine containing linker comprises one, two, three, four or more repeats as set forth in SEQ ID NO: 8 (GGGS), SEQ ID NO: 9 (GGGGS), SEQ ID NO: 10 (GGGGGS), or SEQ ID NO: 11 (GGGGGGGS).

[0154] In some embodiments, the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 7 or comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the amino acid sequence of the linker is as set forth in SEQ ID NO: 7.

[0155] In some embodiments, in the fusion protein provided herein, the amino acid sequence of the GLP-1 polypeptide is as set forth in SEQ ID NO: 3, the amino acid sequence of the immunoglobulin Fc domain is as set forth in SEQ ID NO: 6, and the amino acid sequence of the linker is as set forth in SEQ ID NO: 7.

[0156] In some embodiments, the fusion protein provided herein comprises, from N-terminus to C-terminus, a GLP-1 polypeptide, a linker, and an immunoglobulin Fc domain, wherein the amino acid sequence of the GLP-1 polypeptide is as set forth in SEQ ID NO: 3, the amino acid sequence of the linker is as set forth in SEQ ID NO: 7, and the amino acid sequence of the immunoglobulin Fc domain is as set forth in SEQ ID NO: 6.

[0157] Fusion protein

[0158] In some embodiments, the fusion protein provided herein increases the in vivo half-life and / or yield of a GLP-1 polypeptide (e.g., GLP-1 (9-37), GLP-1 (9-36) amide, etc.). Thus, the fusion protein, as well as reagents for preparing the fusion protein, etc., can be used in the preparation of a medicament.

[0159] In some embodiments, the fusion protein provided herein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24.

[0160] In some embodiments, the fusion protein provided herein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein provided herein comprises an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein provided herein has the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion protein provided herein has the amino acid sequence of SEQ ID NO: 22.

[0161] In some embodiments, the fusion protein provided herein comprises an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein provided herein comprises an amino acid sequence with at least about 80% (e.g., at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein provided herein comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein provided herein has the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein provided herein has the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion protein provided herein has the amino acid sequence of SEQ ID NO: 24.

[0162] In some embodiments, the fusion protein provided herein comprises an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the GLP-1 polypeptide comprises G22E and R36G substitutions.

[0163] In some embodiments, the fusion protein provided herein comprises an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the immunoglobulin Fc domain comprises C222S, A330S, and P331S substitutions.

[0164] In some embodiments, the fusion protein provided herein comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the GLP-1 polypeptide comprises G22E and R36G substitutions, and the immunoglobulin Fc domain comprises C222S, A330S, and P331S substitutions.

[0165] In some embodiments, the fusion protein provided herein comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the GLP-1 polypeptide comprises G22E and R36G substitutions, and the fusion protein still retains the function of the GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolism disorder).

[0166] In some embodiments, the fusion protein provided herein comprises an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the immunoglobulin Fc domain comprises C222S, A330S, and P331S substitutions, and the fusion protein still retains the function of the GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolism disorder).

[0167] In some embodiments, the fusion protein provided herein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 24, and the GLP-1 polypeptide comprises G22E and R36G substitutions, and the immunoglobulin Fc domain comprises C222S, A330S, and P331S substitutions, and the fusion protein still retains the function of the GLP-1 polypeptide (e.g., the function of improving hepatic glycolipid metabolism disorder).

[0168] In some embodiments, the fusion protein provided herein has a half-life of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days in a subject (e.g., a human subject). As demonstrated in the examples herein, the GLP-1 fusion proteins disclosed herein, by sequence engineering, have a longer half-life.

[0169] In some embodiments, the fusion protein provided herein further comprises a signal peptide.

[0170] In the present application, the term "signal peptide" refers to a polypeptide that allows the fusion protein to be secreted into the extracellular medium. Such a polypeptide can also be referred to as "leader peptide", "polypeptide precursor", "prepeptide", etc. The use of signal peptides to direct the secretion of proteins is known in the art (e.g., US8658174, the contents of which are incorporated herein by reference in their entirety). Examples of signal peptides include, but are not limited to, human CD33 signal peptide, human growth hormone-releasing hormone (GHRH) signal peptide, human alpha-1-microglobulin / bikunin precursor (AMBP) signal peptide, Gaussia luciferase signal peptide, murine immunoglobulin heavy chain signal peptide, murine immunoglobulin kappa light chain signal peptide. The signal peptide is cleaved during the secretion process.

[0171] In another preferred embodiment, the fusion protein provided herein is pharmaceutical grade.

[0172] In another aspect, the present application also provides a dimer comprising two identical peptide chains connected by a disulfide bond, wherein each peptide chain comprises the fusion protein provided herein.

[0173] III. Nucleic acid

[0174] In another aspect, the present application also provides a nucleic acid molecule comprising a polynucleotide encoding a polypeptide (e.g., a GLP-1 polypeptide, an immunoglobulin Fc domain, etc.) or a fusion protein of the present application.

[0175] The term "nucleic acid" or "nucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see, Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0176] In some embodiments, the polynucleotides described herein are codon-optimized, e.g., optimized for humans. The nucleic acid molecules can be used in the methods described herein.

[0177] Polypeptides and fusion proteins can be synthesized using standard protein chemistry techniques (see, Bodanszky, M., Principles of peptide synthesis. 2 nd rev.ed. Springer laboratory. 1993, Berlin; New York: Springer-Verlag. Xii, 329 p.). Additionally, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Mode 1396; Milligen / Biosearch 9600). In addition, the peptides, polypeptides, or fragments or variants thereof described herein can be recombinantly produced using various expression systems well known in the art.

[0178] In some embodiments, the nucleic acid molecule described herein comprises a polynucleotide sequence as set forth in SEQ ID NO: 26 or a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polynucleotide sequence as set forth in SEQ ID NO: 26. In some embodiments, the nucleic acid molecule described herein comprises a polynucleotide sequence as set forth in SEQ ID NO: 26. In some embodiments, the nucleic acid molecule described herein consists of a polynucleotide sequence as set forth in SEQ ID NO: 26.

[0179] IV. Vectors and Cells

[0180] In another aspect, the present application also provides a vector comprising the nucleic acid molecule described herein.

[0181] Any vector suitable for the intended use can be used. For example, some vectors can be introduced into an expression system, such as a mammalian, insect, or bacterial expression system, for expression and purification of the expressed protein. Some vectors can be used to produce viruses. These different vectors are well known in the art.

[0182] In some embodiments, the vector is used with an in vitro expression system to produce the fusion protein. Expression and purification of the fusion protein can be performed by any suitable method known in the art.

[0183] Possible expression vectors include, but are not limited to, plasmids or modified viruses (e.g., replication-defective retroviruses, including lentiviral vectors, adenoviruses, and adeno-associated viruses, etc.). In some embodiments, the expression vector that can be used with the fusion protein of the present application is a pKN012 vector, which can be obtained through commercial channels (e.g., Beijing Konosys Technology Co., Ltd.).

[0184] The vector can comprise suitable regulatory sequences and components. Suitable regulatory sequences can be selected from a variety of sources, including bacteria, fungi, viruses, mammalian or insect genes. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. In addition, other sequences, such as an origin of replication, additional restriction sites, enhancers and sequences which influence transcription induction capabilities, can be incorporated into the expression vector as appropriate. In some embodiments, the regulatory sequences direct or increase expression in neural tissue and / or cells. In some embodiments, the vector is a viral vector. The recombinant expression vectors can further comprise a marker gene that facilitates the selection of host cells transformed, infected or transfected with the vector expressing the fusion proteins described herein. The recombinant expression vectors can also comprise additional expression cassettes encoding, for example, fusion moieties that can aid in detection, including, for example, tags and markers described herein.

[0185] In some embodiments, the vector comprises one or more, optionally the components shown in FIG. 36. For example, in some embodiments, the vector comprising a nucleic acid molecule encoding a GLP-1 fusion protein is pKN012-GLP1-IgG2.

[0186] A variety of methods for transducing cells can be used, including viral vectors, “naked” DNA, DNA in liposomes or other nanoparticles, adjuvant-assisted DNA, gene guns, etc. For example, retroviral vectors, such as lentiviral vectors, can also be used to transduce cells in vivo. Other vector systems that can be used to practice the present application include adenoviral and adeno-associated viral based vectors.

[0187] In another aspect, the present application also provides a recombinant cell comprising a nucleic acid molecule encoding a fusion protein described herein, or comprising a vector described herein.

[0188] Stably-expressing recombinant cells can be made by, for example, transforming, transfecting or transducing a recombinant cell with a vector comprising a polynucleotide, preferably any of the polynucleotides described herein.

[0189] In some embodiments, the cell described herein is a prokaryotic cell. In some embodiments, the cell described herein is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cell derived from a human. For example, the mammalian cell is a human embryonic kidney 293 (HEK293 cell), e.g., a HEK293T cell, a HEK293S cell, or a HEK293F cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell, e.g., a CHO-K1 cell, a CHO-S cell, or a CHO-DG44 cell.

[0190] In some embodiments, the recombinant cell of the present application is obtained by suspension adaptation of a Chinese hamster ovary cell, exemplified by a CHO-K1 cell.

[0191] V. Methods of constructing a cell, and methods of producing a fusion protein

[0192] In another aspect, the present application also provides a method of constructing a recombinant cell, comprising:

[0193] a) introducing a nucleic acid molecule encoding the fusion protein described herein into a vector to construct an expression vector;

[0194] b) introducing the expression vector into a cell to obtain a recombinant cell.

[0195] In some embodiments, the cell is a CHO cell, e.g., a CHO-K1 cell, a CHO-S cell, or a CHO-DG44 cell.

[0196] In some embodiments, the vector can be a pKN012 vector.

[0197] In some embodiments, the method of constructing a recombinant cell comprises the following steps:

[0198] a) inserting the polynucleotide sequence set forth in SEQ ID NO: 26 into the Ncol and HindIII sites of a pKN012 vector to generate a pKN012-GLP1-IgG2 / Fc expression vector;

[0199] b) introducing the pKN012-GLP1-IgG2 / Fc expression vector into a CHO-K1 cell to obtain a recombinant cell.

[0200] In another aspect, the present application also provides a method of producing a fusion protein, comprising the step of obtaining a fusion protein using a recombinant cell described herein or a cell prepared by the method of constructing described herein.

[0201] As described herein, fusion proteins comprising GLP-1 polypeptides can be synthesized. As shown herein, the fusion proteins can also be produced using recombinant cells, including, for example, recombinant Chinese hamster ovary cells that express the GLP-1 fusion proteins. Accordingly, the present application also provides a method of producing a GLP-1 fusion protein, the method comprising culturing a recombinant cell that expresses the GLP-1 fusion protein, wherein the culturing comprises one or more process steps or materials described in the Examples. For example, the method can comprise one or more process steps or materials described in Example 1 of the present application.

[0202] In some embodiments, the recombinant cell that expresses the GLP-1 fusion protein is produced using HEK293T, HEK293S, HEK293F, and / or CHO cells (e.g., CHO-K1 cells), which can be used to produce the recombinant polypeptides and / or fusion proteins, e.g., under conditions suitable for in vivo use.

[0203] VI. Compositions

[0204] In another aspect, the present application also provides a composition comprising a fusion protein, dimer, nucleic acid molecule, vector, or recombinant cell described herein.

[0205] In some embodiments, the composition can further comprise a suitable diluent or carrier. In another preferred embodiment, the carrier is a pharmaceutically acceptable carrier.

[0206] In some embodiments, the composition is a pharmaceutical composition.

[0207] In some embodiments, the composition is a pharmaceutical composition comprising a GLP-1 fusion protein and a pharmaceutically acceptable carrier.

[0208] In some embodiments, the composition further comprises buffered saline.

[0209] In some embodiments, the composition further comprises a sugar.

[0210] In some embodiments, the composition further comprises a surfactant. The pharmaceutical compositions of the present application can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of unit doses. The compositions can be prepared in various forms.

[0211] In some embodiments, the composition comprises a GLP-1 fusion protein formulated in a formulation, administered at a dose of about 0.25 mg to about 20 mg, e.g., about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg of the GLP-1 fusion protein.

[0212] The GLP-1 fusion protein, nucleic acid molecule, vector, recombinant cell, or composition can be used in the preparation of a medicament and / or for use, e.g., by parenteral, intravenous, subcutaneous, or intramuscular administration.

[0213] In some embodiments, the GLP-1 fusion protein or composition thereof can be administered parenterally or formulated for parenteral administration.

[0214] In some embodiments, the GLP-1 fusion protein or composition thereof can be administered subcutaneously or formulated for subcutaneous administration.

[0215] In some embodiments, the GLP-1 fusion protein or composition thereof can be administered intravenously or formulated for intravenous administration.

[0216] In some embodiments, the GLP-1 fusion protein or composition thereof can be administered intramuscularly or formulated for intramuscular administration.

[0217] Diluents suitable for the GLP-1 fusion protein and / or cells include, but are not limited to, saline solutions, pH buffered solutions, and diluents described herein, as well as glycerol solutions or other solutions suitable for freezing polypeptides and / or cells.

[0218] Diluents suitable for the nucleic acid and / or vector include, but are not limited to, saline solutions, pH buffered solutions, and diluents described herein, as well as water, etc.

[0219] In another preferred embodiment, the diluent is sterile.

[0220] VII. Methods and Uses of Fusion Proteins for Treating and Preventing Disease

[0221] As shown herein, the GLP-1 polypeptide G22E and / or R36G substitution, and / or one or more of the C222S, A330S, and P331S substitutions in the IgG2 / Fc portion increase the yield, activity, and / or half-life of the GLP-1 fusion protein. The GLP-1 fusion proteins, nucleic acids, vectors, and recombinant cells described herein are suitable for the manufacture of medicaments and compositions thereof, and for the corresponding therapeutic uses.

[0222] In another aspect, the present application also provides a method of treating or preventing a disease, comprising administering to a subject having the disease a fusion protein, a dimer, a nucleic acid molecule, a vector, a cell, or a composition provided by the present application.

[0223] In another aspect, the present application also provides the use of a fusion protein, a dimer, a nucleic acid molecule, a vector, a cell, or a composition described herein in the manufacture of a medicament for treating or preventing a disease.

[0224] In another aspect, the present application also provides the use of a fusion protein, a dimer, a nucleic acid molecule, a vector, a cell, or a composition described herein for treating or preventing a disease.

[0225] In some embodiments, the medicament comprising the fusion protein is administered in an amount of about 0.2 mg to about 20 mg of the fusion protein per person per time. In some embodiments, the amount of the fusion protein is about 1 mg to about 10 mg. In some embodiments, the amount of the fusion protein is about 1 mg to about 5 mg. In some embodiments, the amount of the fusion protein is about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg.

[0226] Different dosage forms can be used, suitable dosage forms can include but are not limited to solutions, suspensions, pills, tablets.

[0227] In some embodiments, the treatment regimen can include multiple administrations.

[0228] In some embodiments, the fusion protein or composition thereof is administered once every 3 days, or once a week, or once every two weeks.

[0229] In some embodiments, the fusion protein, dimer, nucleic acid molecule, vector, cell, or composition thereof is administered in a regimen of once a week followed by once a week for 4 consecutive weeks. In another preferred embodiment, the treatment further comprises an initial dose of 1 mg one week prior to the start of the administration regimen.

[0230] In some embodiments, the fusion protein, dimer, nucleic acid molecule, vector, cell, or composition thereof is administered in a regimen of once a week followed by once a week for 4 consecutive weeks. In another preferred embodiment, the treatment further comprises an initial dose of 1 mg one week prior to the start of the administration regimen.

[0231] In some embodiments, the fusion protein, dimer, nucleic acid molecule, vector, cell, or composition thereof is administered via parenteral, intravenous, subcutaneous, intramuscular, or the like.

[0232] In some embodiments, the disease is selected from the group consisting of a metabolic disease associated with a glucose and / or lipid metabolism disorder, a complication of a metabolic disease, a central metabolic disease (e.g., a neurological disease), and other related metabolic diseases.

[0233] In some embodiments, the disease or condition is a metabolic disease associated with a glucose and / or lipid metabolism disorder.

[0234] In some embodiments, the metabolic disease associated with a glucose and / or lipid metabolism disorder is a liver metabolic disease associated with a glucose and / or lipid metabolism disorder. In some embodiments, the metabolic disease associated with a glucose and / or lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, and metabolic syndrome.

[0235] In some embodiments, the metabolic disease associated with a glucose and / or lipid metabolism disorder is or comprises diabetes. In some embodiments, the metabolic disease associated with a glucose and / or lipid metabolism disorder is type 2 diabetes. In another embodiment, the metabolic disease associated with a glucose and / or lipid metabolism disorder is or comprises obesity. In another embodiment, the metabolic disease associated with a glucose and / or lipid metabolism disorder is or comprises nonalcoholic fatty liver disease (NAFLD). In another embodiment, the metabolic disease associated with a glucose and / or lipid metabolism disorder is nonalcoholic liver fibrosis (NASH). In another embodiment, the metabolic disease associated with a glucose and / or lipid metabolism disorder is metabolic syndrome.

[0236] In some embodiments, the subject is newly diagnosed or has been previously diagnosed with diabetes (e.g., type 2 diabetes). Diabetes can be diagnosed in a variety of ways, such as fasting plasma glucose (FPG). According to the American Diabetes Association, diabetes is diagnosed when fasting plasma glucose is greater than or equal to 126 mg / dl.

[0237] In some embodiments, the subject has an increased likelihood of developing diabetes (e.g., type 2 diabetes). For example, the subject can be predisposed to developing diabetes, such as due to the subject being obese or the subject having a genetic predisposition, such as when the subject has a family history of diabetes.

[0238] In some embodiments, the subject is obese. Obesity can be defined by reference to body mass index (BMI). For example, the World Health Organization (WHO) defines obesity as having a BMI equal to or greater than 30. In another embodiment, the subject has a BMI of at least about 20 kg / m 2 In another embodiment, the subject can have a blood glucose that is higher than the average for a person of comparable body weight, but not high enough to be diagnosed with diabetes. In another embodiment, the subject can also be an individual with a family history of diabetes.

[0239] In some embodiments, the subject is newly diagnosed or has been previously diagnosed as having NAFLD or NASH. In another embodiment, the subject has an increased likelihood of developing NAFLD or NASH. For example, the subject can have a genetic predisposition to developing NAFLD or NASH.

[0240] In some embodiments, the disease is a complication of a metabolic disease. In some embodiments, the complication of a metabolic disease includes a cardiovascular complication (e.g., coronary heart disease, sudden cardiac death, heart failure, etc.), a renal complication (e.g., acute kidney injury, diabetic nephropathy), or a liver complication (e.g., fatty liver, including NAFLD and NASH) arising from a metabolic disease.

[0241] In some embodiments, the disease or disorder is a neurological disease. In some embodiments, the neurological disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), motor neuron disease, Huntington’s disease, and Parkinson’s disease (PD).

[0242] In some embodiments, the neurodegenerative disease is Alzheimer’s disease. In other embodiments, the neurodegenerative disease is motor neuron disease. In other embodiments, the neurodegenerative disease is Huntington’s disease. In other embodiments, the neurodegenerative disease is Parkinson’s disease.

[0243] In some embodiments, the subject is newly diagnosed or has been previously diagnosed with Alzheimer’s disease. In other embodiments, the subject has an increased likelihood of developing Alzheimer’s disease. For example, the subject can be a subject predisposed to developing Alzheimer’s disease due to the subject’s genetics, such as when the subject has a family history of Alzheimer’s disease or a Tau or APP mutation associated with Alzheimer’s disease.

[0244] In some embodiments, the subject is newly diagnosed or has been previously diagnosed with motor neuron disease. In other embodiments, the subject has an increased likelihood of developing motor neuron disease. For example, the subject can be a subject predisposed to developing motor neuron disease due to the subject’s genetics, such as when the subject has a family history of motor neuron disease.

[0245] In some embodiments, the subject is newly diagnosed or has been previously diagnosed with Huntington’s disease. In other embodiments, the subject has an increased likelihood of developing Huntington’s disease. For example, the subject can be a subject predisposed to developing Huntington’s disease due to the subject’s genetics, such as when the subject has a family history of Huntington’s disease.

[0246] In some embodiments, the subject is newly diagnosed or has been previously diagnosed with Parkinson’s disease. In other embodiments, the subject has an increased likelihood of developing Parkinson’s disease. For example, the subject can be a subject predisposed to developing Parkinson’s disease due to the subject’s genetics, such as when the subject has a family history of Parkinson’s disease.

[0247] In another aspect, the present application also provides a method of binding to and activating a signal cascade of long-chain-fatty-acid-CoA-ligase 1 (ACSL1) on the cell membrane of a target cell to improve lipid metabolism, the method comprising exposing the target cell to a GLP-1 polypeptide or a fusion protein comprising a GLP-1 polypeptide selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, a fusion protein, a dimer, a nucleic acid molecule, a vector, a cell or a composition thereof of the present application. In some embodiments, the target cell is an adipocyte. In some embodiments, the target cell is a hepatocyte. The method can be performed in vitro or in vivo.

[0248] The term "ACSL1", also known as Long-chain-fatty-acid-CoA ligase 1, long-chain-fatty-acid-CoA synthetase 1, is an enzyme responsible for activating long-chain fatty acids, playing an important role in the synthesis and distribution of triglycerides, as used in the present application. The ACSL1 referred to in the present application includes any variant, conformation, isoform, and species homolog of ACSL1 expressed by a cell naturally or by a cell transfected with an ACSL1 gene. For example, the ACSL1 described herein can refer to ACSL1 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). An exemplary ACSL1 amino acid sequence is shown in NCBI Reference Sequence: NP_001273637.1 (SEQ ID NO: 29). Other exemplary amino acid sequences of ACSL1 can also be obtained by the skilled person through public databases (e.g., GenBank, UniProt, OMIM, etc.).

[0249] The term "ACSL1" used in the present application is intended to encompass any form of ACSL1, for example 1) naturally unprocessed ACSL1 molecule, "full-length" ACSL1 chain or naturally occurring variants of ACSL1, including, for example, splice variants or allelic variants; 2) any form of ACSL1 produced by processing in a cell; or 3) full-length, fragments (e.g., truncated forms, extracellular / transmembrane domains) or modified forms (e.g., mutant forms, glycosylated / PEGylated, His-tag / immunofluorescent fusion forms) of ACSL1 subunits produced by recombinant methods.

[0250] In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide described in the present application binds to amino acids corresponding to any one, two, three, four, or five positions selected from the group consisting of position 316, 320, 324, 409, and 488 of SEQ ID NO: 29 of ACSL1 on the cell membrane of a target cell. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide described in the present application binds to position 316, 320, 324, 409, and 488 of SEQ ID NO: 29 of ACSL1 on the cell membrane of a target cell.

[0251] In another aspect, the present invention also provides a method for binding to and activating the signaling cascade of multiple proteins (e.g., selected from one or more proteins in the table below) on the cell membrane of a target cell to improve lipid metabolism, the method comprising exposing the target cell to a GLP-1 peptide or a fusion protein containing a GLP-1 peptide (the GLP-1 peptide being selected from human GLP-1(9-37) and human GLP-1(9-36) amide), the fusion protein of the present invention, a dimer, a nucleic acid molecule, a carrier, a cell or a combination thereof.

[0252] The fusion proteins, dimers, nucleic acid molecules, vectors, cells, or compositions provided by this invention can be used in combination with any other known drugs or therapies for the treatment or prevention of diseases.

[0253] VIII. Methods of treating or preventing disease using GLP-1(9-37) and GLP-1(9-36) amides or their fusion proteins.

[0254] In another aspect, the present invention provides a method for treating or preventing a disease, the method comprising administering a GLP-1 polypeptide or a fusion protein containing a GLP-1 polypeptide to a subject suffering from the disease, wherein the GLP-1 polypeptide is selected from human GLP-1(9-37) and human GLP-1(9-36) amide; the disease is selected from the group consisting of: metabolic diseases related to lipid metabolism disorders, complications of metabolic diseases, and neurological diseases, as well as other related diseases.

[0255] In some embodiments, the GLP-1 polypeptide is selected from one or more of the GLP-1 polypeptides described in Part II (Proteins and Fusion Proteins) herein. In some embodiments, the GLP-1 polypeptide is selected from human GLP-1 (9-37) and human GLP-1 (9-36) amides, and contains G22E and / or R36G substitutions relative to the native human GLP-1 polypeptide.

[0256] In some embodiments, the GLP-1 polypeptide has at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises one or more amino acid substitutions relative to native human GLP-1 polypeptide selected from the group consisting of G22E and R36G. In some embodiments, the GLP-1 polypeptide comprises G22E and R36G substitutions relative to native human GLP-1 polypeptide.

[0257] In some embodiments, the GLP-1 polypeptide has an amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the GLP-1 polypeptide has an amino acid sequence set forth as SEQ ID NO: 3.

[0258] The inventors of the present application have found that the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide provided by the present application has an important regulatory effect on liver lipid metabolism. In some embodiments, the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits hepatic gluconeogenesis, improves liver steatosis, oxidative stress, and inflammatory response, and / or reduces liver enzyme and blood lipid levels, thereby playing a liver-protecting role in both prevention and treatment. Therefore, in some embodiments, the metabolic disease associated with lipid metabolism disorder is a liver lipid metabolism disorder-related disease.

[0259] In some embodiments, after administration of the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide, the liver steatosis, liver oxidative stress, and inflammatory response of the subject are improved, and the body weight and glucose tolerance of the subject do not change significantly.

[0260] In some embodiments, the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of a lipid synthesis-related gene. In some embodiments, the lipid synthesis-related gene is selected from the group consisting of SREBP-1, ACC, FAS, and SCD-1. In some embodiments, the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of the lipid synthesis-related genes SREBP-1, ACC, and FAS. In some embodiments, the GLP-1 polypeptide or fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of the lipid synthesis-related gene SCD-1.

[0261] In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application promotes the expression of a beta-oxidation related gene. In some embodiments, the beta-oxidation related gene is selected from the group consisting of ACOX1, PPARa, CPT1 and P4a14. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application promotes the expression of the beta-oxidation related genes ACOX1, PPARa and CPT1. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application promotes the expression of the beta-oxidation related gene P4a14.

[0262] In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of a lipid synthesis related gene SREBP-1, ACC and FAS, and promotes the expression of a beta-oxidation related gene ACOX1, PPARa and CPT1. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of a lipid synthesis related gene SCD-1, and promotes the expression of a beta-oxidation related gene P4a14.

[0263] In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the expression of p-ERK. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application dose-dependently inhibits the expression of p-ERK.

[0264] In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application inhibits the production of reactive oxygen species (ROS) in HepG2 cells. In some embodiments, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application also improves the reduction of adenosine triphosphate (ATP) content.

[0265] Without being bound by any theory, the GLP-1 polypeptide or the fusion protein comprising the GLP-1 polypeptide provided by the present application is endocytosed by binding to the hepatocyte membrane ACSL1 and co-localizes in the cell, inhibits the downstream p-ERK signaling pathway, thereby inhibiting the expression of lipid synthesis genes (e.g., SREBP-1, ACC, FAS and SCD-1), promoting the expression of beta-oxidation related genes (e.g., ACOX1, PPARa, CPT1 and P4a14), improving liver lipid accumulation, and directly regulating liver lipid metabolism.

[0266] In some embodiments, the subject has or is at risk of having one or more symptoms selected from the group consisting of lipid deposition, elevated triglyceride levels, elevated cholesterol, elevated low-density lipoprotein levels, elevated free fatty acid levels, increased accumulation of fatty acids, insulin resistance, elevated glutamic-pyruvic transaminase levels, elevated glutamic-oxaloacetic transaminase levels, elevated malondialdehyde content, decreased glutathione peroxidase (GSH-Px) activity, decreased superoxide dismutase (SOD) activity, elevated levels of inflammatory factors.

[0267] In some embodiments, the metabolic disease associated with lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, and metabolic syndrome. In some embodiments, the subject is a newly diagnosed or previously diagnosed patient of NAFLD or NASH. In another embodiment, the subject has an increased likelihood of developing NAFLD or NASH. For example, the subject can have a genetic predisposition for NAFLD or NASH.

[0268] In some embodiments, the NAFLD is non-obese NAFLD.

[0269] In some embodiments, the complication of the metabolic disease is selected from the group consisting of cardiovascular disease (CVD) caused by the metabolic disease, diabetic kidney disease (DKD), chronic kidney disease (CKD), or liver disease, e.g., nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH).

[0270] In some embodiments, the disease or disorder is a nervous system disease. In some embodiments, the nervous system disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease (AD), motor neuron disease, Huntington’s disease, and Parkinson’s disease (PD).

[0271] IX. Sequence Information

[0272] The SEQ ID NOs referred to in this application and their corresponding amino acid sequences or nucleotide sequences are shown in the following table.

[0273] The foregoing disclosure generally describes the present application. The present application can be more completely understood in reference to the following detailed description. The following examples are described for illustration only and are not intended to limit the scope of the application. Changes and alterations in the embodiments will become apparent to those of ordinary skill in the art which are made without departing from the scope of the application. Although specific terms are employed herein, they are used in a descriptive sense only and should not be construed as limiting.

[0274] The following non-limiting examples are provided to describe the present application.

[0275] Examples

[0276] The experimental methods used in the following examples are conventional unless otherwise specified.

[0277] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0278] Example 1. Construction and verification of 9-37 / Fc and 7-37 / Fc fusion proteins (YN-101 / YN-011)

[0279] In this example, the inventors constructed GLP-1(9-37 / Fc) (hereinafter abbreviated as "9-37 / Fc" or "GLP-1(9-37 / Fc)") and GLP-1(7-37 / Fc) (hereinafter abbreviated as "7-37 / Fc" or "GLP-1(7-37 / Fc)") using genetic engineering recombinant protein technology.

[0280] 1.1 Plasmid construction and expression of 7-37 / Fc

[0281] A vector encoding 7-37 / Fc was constructed, which fusion protein comprises human GLP-1(7-37) and human IgG2 / Fc (containing hinge, CH2 and CH3 regions in human IgG2 heavy chain, i.e. Hinge-CH2-CH3). The cDNA fragment encoding the fusion protein 7-37 / Fc (as shown in SEQ ID NO: 28) was chemically synthesized and inserted into the Ncol and HindIII sites of the pKN012 vector to generate pKN012-GLP-1-IgG2 / Fc.

[0282] The pKN012-GLP-1-IgG2 / Fc stable expression vector was transformed into E. coli DH5a competent cells. The plasmid containing pKN012-GLP-1-IgG2 / Fc was extracted and digested with PvuI. After electrophoresis, there was a target band with the correct molecular weight. The linearized plasmid was quantified after ethanol precipitation and used for stable transfection.

[0283] To establish CHO-K1 cells (Lot# 58995535 / ATCC) stably expressing GLP-1-IgG2 / Fc, 2 μg of linearized pKN012-GLP-1-IgG2 / Fc was used to transfect CHO-K1 cells growing in 6-well plates (2.5 x 10 5 Twenty-four hours after transfection, cells were dispersed and cultured in CD-CHO medium containing MSX (methionine sulfoximine, 100 μM / L) and those cells that had stably integrated the recombinant plasmid into the genome were selected. The medium was changed every 3 days until colonies were formed. Individual colonies were isolated and expanded into stable cell lines, and the tissue culture supernatant of the cell lines grown in 24-well plates was tested for 7-37 / Fc using a rat GLP-1 RIA kit. Cells capable of secreting the fusion protein were selected for further identification. The amino acid sequence of the resulting 7-37 / Fc is shown in SEQ ID NO: 27 (also referred to as “YN-011” in this application).

[0284] 1.2. Plasmid construction and expression of 9-37 / Fc

[0285] Using similar procedures as in Example 1.1, a vector encoding 9-37 / Fc was constructed, except that the cDNA fragment encoding 9-37 / Fc is shown in SEQ ID NO: 26, and the amino acid sequence of the resulting 9-37 / Fc is shown in SEQ ID NO: 24 (also referred to as “YN-101” in this application).

[0286] The fusion protein monomers expressed above are linked to form homodimers through the naturally occurring intermolecular disulfide bond of the IgG2-Fc molecule (Figure 2A). DTT (Dithiothreitol) is a strong reducing agent that can reduce the disulfide bond in the fusion protein, causing it to break and cleaving the dimer into monomers. Coomassie blue staining and Western blotting with GLP-1 antibody or human IgG2 antibody showed that the GLP-1 fusion proteins (7-37 / Fc and 9-37 / Fc) formed stable dimers with a molecular weight of about 60 kDa and monomers with a molecular weight of about 35 kDa under non-reducing and reducing conditions, respectively (Figure 2B, C). The above results indicate that the GLP-1 / Fc fusion protein was successfully constructed.

[0287] Example 2. Distribution of GLP-1 (9-37) in vivo and its binding to the liver

[0288] In vivo, the half-life of native GLP-1 is short (1-2 min). Once released into the blood circulation, the N-terminal two amino acids of GLP-1 are rapidly degraded by DPP-4 to form the metabolic derivative GLP-1(9-37). The half-life of GLP-1(9-37) is relatively long (8-10 min), and it is the main form of GLP-1 in the postprandial plasma, accounting for 80-90% of the total circulating GLP-1. GLP-1 can also be degraded by endogenous neutral endopeptidase (NEP) into smaller GLP-1 polypeptide fragments GLP-1(28-37) and GLP-1(32-37). The exact mechanism of GLP-1 metabolic degradation in vivo and its physiological significance are not fully understood. Early studies have shown that GLP-1(9-37) has very low affinity for GLP-1R binding to pancreatic beta cells, about 1 / 100 of GLP-1(7-37), and has no effect on stimulating insulin secretion.

[0289] At present, the biological role of GLP-1(9-37) has not been determined, and it has been considered as an inactive GLP-1 metabolic derivative or an antagonist of GLP-1R. Studies using GLP-1R antagonists or GLP-1R knockout (KO) mice have shown that the cell protective effect of GLP-1(9-37) is independent of the classic GLP-1R. Since the liver does not express GLP-1R, the inventors hypothesized that the direct biological effect of GLP-1 on the liver is mainly exerted by its metabolic derivative GLP-1(9-37).

[0290] In order to further explore whether GLP-1(9-37) has a direct biological regulatory effect on the liver, the distribution of GLP-1(9-37) in vivo and its binding ability to hepatocytes were determined. In this embodiment, the fusion protein GLP-1(9-37 / Fc) prepared in Example 1 (the amino acid sequence of which is shown in SEQ ID NO: 24) was used to explore the distribution of GLP-1(9-37 / Fc) in zebrafish, and its binding ability to wild-type (WT) and GLP-1R KO mouse primary hepatocytes and human hepatoma cell line HepG2.

[0291] 2.1. Experimental method

[0292] 2.1.1 Agarose gel electrophoresis

[0293] Mouse tissue sampling

[0294] (1) The mice were fasted overnight before sampling, and free water was provided. Tissue cryopreservation tubes were prepared in advance and labeled.

[0295] (2) Intraperitoneal injection of 10% chloral hydrate (dose: 150 μL / 20 g).

[0296] (3) After the mouse righting reflex disappears, fix the mouse on the operation board, disinfect the surface with alcohol, and cut a cross-shaped incision at the lower end of the abdominal cavity. Cut open the abdominal and thoracic cavities, expose the thoracic and abdominal cavities, and cut small openings on each lobe of the liver for easy perfusion.

[0297] (4) Insert the scalp needle into the left ventricle and perfuse physiological saline at a uniform speed for 30-40 mL until the liver turns white.

[0298] (5) Quickly separate the mouse liver, pancreas, and brain tissues. Prioritize the separation of the mouse pancreas to prevent rapid degradation of the pancreas.

[0299] (6) Place a portion of the above tissues in the corresponding labeled tissue cryogenic tube, then immediately place it in liquid nitrogen for quick freezing treatment, and finally place it in a -80°C refrigerator for long-term storage for subsequent RNA and protein extraction.

[0300] (7) Take another portion of liver and pancreas tissues and place them in 4% paraformaldehyde fixative solution. After 24 hours of room temperature fixation, send them to Wuhan Saivier Biological Technology Co., Ltd. for paraffin embedding and sectioning for immunohistochemical staining.

[0301] Extraction of mouse liver RNA

[0302] (1) Cut 10 mg of liver, pancreas, and brain tissues and place them in pre-cooled tissue homogenization tubes, then add 1 mL of 4°C pre-cooled TRIzol. Perform the entire process quickly on ice.

[0303] (2) Add one large and two small steel balls, homogenize with a homogenizer (70 HZ, 60 s, stop for 10 s, repeat 3 times) until there is no obvious large sediment, then place it on ice for 30 min.

[0304] (3) 4°C centrifuge, 12000 rpm for 15 min.

[0305] (4) Absorb the supernatant into a new RNA and DNA enzyme-free 1.5 mL centrifuge tube (with labels).

[0306] (5) Room temperature for 5 min, add 200 μL of chloroform to each tube, shake vigorously until pink, and then room temperature for about 10 min.

[0307] (6) 4°C centrifuge, 12000 rpm for 15 min.

[0308] (7) Move the uppermost 300 μL of colorless transparent liquid to a new RNA and DNA enzyme-free 1.5 mL centrifuge tube.

[0309] (8) Add 300 μL of isopropanol, mix gently, and then ice for 30 min.

[0310] (9) 4°C centrifuge at 12000 rpm for 15 min. White precipitate can be observed at the bottom of the EP tube. Discard the supernatant.

[0311] (10) Add 1 mL of 75% ethanol prepared with DEPC water and mix well. 4°C centrifuge at 12000 rpm for 10 min.

[0312] (11) Discard the supernatant and dry the EP tube at room temperature (about 10 min) until the precipitate becomes transparent. Add 15-30 μL of DEPC water to dissolve the RNA completely.

[0313] (12) Take 1 μL of the RNA sample and measure the concentration and purity using a NanoDrop 2000 spectrophotometer. Before measurement, adjust the zero of the spectrophotometer with DEPC water. Wipe the measurement point with paper before and after each sample measurement. Read the OD 260 / 280 and OD 260 / 230 ratio. The OD 260 / 280 ratio is 1.8-2.0, and the OD 260 / 230 ratio is greater than 2.0.

[0314] cDNA synthesis

[0315] (1) Prepare the following reaction mixture in an eight-tube:

[0316] (2) After 65°C incubation for 5 min, quickly cool on ice. This step can denature the template RNA and improve the efficiency of reverse transcription.

[0317] (3) Prepare the following 20 μL reverse transcription system in the above eight-tube:

[0318] (4) Cover the tube cap, mix gently and slowly, and then place it in a PCR amplifier. Set the following reverse transcription program:

[0319] (5) Store the reverse transcription synthesized cDNA at -20°C for subsequent PCR reaction.

[0320] PCR reaction

[0321] (1) Dissolve the primers: centrifuge the upper and lower primer lyophilized powder at 12,000 rpm for 5 min at 4°C; prepare a 100 μM primer stock solution with DEPC water; dilute 10 times (10 μM) and store at -20°C.

[0322] (2) Prepare the following 50 μL PCR reaction system in an eight-tube on ice:

[0323] (3) After the PCR reaction system is prepared, mix gently, centrifuge, and place in a PCR amplifier. Set the program as follows:

[0324] (4) After the reaction is completed, place on ice for subsequent DNA gel electrophoresis.

[0325] DNA gel electrophoresis

[0326] (1) Prepare the gel: Dissolve 1.5 g of agarose powder in 100 mL of 1x TAE solution in a microwave oven at high heat for 4-5 min until the solution becomes clear and there is no solid precipitate.

[0327] (2) After the solution is cooled to 50-60°C, add 10 μL of 10,000x Gelgreen dye and shake well. After inserting the comb vertically in the gel preparation plate, slowly pour the solution in the bottle into the gel preparation plate, taking care not to have air bubbles. Let it stand for about 30 min to solidify the gel.

[0328] (3) Remove the comb and place the gel in the electrophoresis tank. Pour 1x TAE solution to immerse the gel. Use a pipette to add 5-10 μL of PCR reaction product to each gel well, and add 100 bp DNA maker 5 μL to the other well. Electrophorese under constant voltage (U = 120 V).

[0329] (4) After the bands are separated at a suitable distance (about 30 min), remove the gel and place it in an imaging analyzer for development and imaging. Analyze the GLP-1R expression according to the electrophoretic bands in the desired position of the image.

[0330] 2.1.2 Tissue protein extraction and concentration determination (BCA method)

[0331] Extract tissue protein

[0332] (1) Prepare protease and phosphatase inhibitors: Dissolve each piece of phosphatase inhibitor (Roche, lot#04906845001) and protease inhibitor (Roche, lot#04693132001) in 500 μL of double distilled water to prepare 10x phosphatase inhibitor stock solution and 20x protease inhibitor stock solution, and store at 4°C.

[0333] (2) Prepare protein lysis solution: Dilute the protease and phosphatase inhibitors to 1x with RIPA protein lysis solution. Operate on ice throughout, and prepare fresh.

[0334] (3) Cut 10 mg of liver, pancreas and brain tissue respectively, and place in 2 mL homogenization tube, and add 500 μL of protein lysis solution.

[0335] (4) Add a large and small steel ball, and put the homogenate tube into the automatic tissue grinder (set conditions: 70HZ, 60s, stop for 10s, repeat 3 times)

[0336] (5) After homogenization, put the homogenate tube into a 4°C shaker and lyse for 40 min. At this time, pre-cool the high-speed centrifuge at 4°C. Note that the tube cap should be tightly covered to prevent liquid leakage, and the grinder iron should be pre-cooled at -20°C.

[0337] (6) 4°C centrifugation, 14,000 rpm, 30 min;

[0338] (7) Take the supernatant to another EP tube, and note that it should not be sucked into the precipitate. The protein concentration can be determined by BCA kit or stored in -80°C refrigerator for short-term preservation.

[0339] BCA method for determining protein concentration

[0340] (1) Prepare the protein standard: dilute the BSA (2 mg / mL) stock solution in the BCA concentration detection kit with double distilled water to 1 mg / mL BSA working stock solution, and place it at room temperature for standby.

[0341] (2) Prepare different BSA standard concentrations according to the following table:

[0342] (3) After vortexing and mixing, the BSA standard is ready for use.

[0343] (4) Prepare the working solution: prepare the working solution according to the volume ratio of A liquid: B liquid = 50:1, and use it immediately. Each sample sets 3 replicate wells.

[0344] (5) Add samples in 96-well plates according to the following system: add 20 μL of different concentrations of BSA standard or sample to each well. Due to the high concentration of tissue samples, dilute the sample 20 times before determination of the concentration, i.e. add 1 μL of protein sample to each well, and add 19 μL of double distilled water.

[0345] (6) Add 160 μL of working solution to each well, and note that there should be no bubbles when adding samples to affect the absorbance reading.

[0346] (7) Place the 96-well plate in a 37°C shaker for 30 min.

[0347] (8) Detect the absorbance value (OD value) of the standard and sample at 562 nm. According to the linear fitting of the OD value and concentration of the standard, calculate the protein concentration of the sample to be tested.

[0348] (9)According to the instructions, add 5X protein loading buffer to the protein sample, dilute the protein loading buffer to 1X, and mix well. Set the metal bath program to a temperature of 100°C and a speed of 350 rpm. When the temperature reaches 100°C, place the protein sample in the bath and heat for 10 minutes. After heating, quickly place the sample on ice to cool, and then proceed with Western blotting.

[0349] 2.1.3 Western blot

[0350] (1) Electrophoresis solution: Weigh 3.0 g of Tris powder, 14.4 g of glycine powder, and 1 g of SDS powder into a glass bottle. Add double-distilled water to make up to 1 L, mix well, and store at room temperature.

[0351] (2) Transfer solution: Weigh 3.0 g of Tris powder and 14.4 g of glycine powder into a glass bottle. Add 100 mL of anhydrous methanol, and make up to 1 L with double-distilled water. Mix well and store at room temperature. The transfer solution should be pre-cooled at 4°C before use.

[0352] (3) Membrane washing solution (TBST): Measure 50 mL of 20x TBS, add 950 mL of double-distilled water, and then add 1 mL of Tween-20. Mix well and store at room temperature.

[0353] (4) Blocking solution: Dissolve 2.5 g of skimmed milk powder in 50 mL of TBST solution. Mix well to prepare a 5% milk blocking solution, and store for later use.

[0354] (5) Prepare SDS-PAGE gels according to the following table:

[0355] (6) Place a 50 mL centrifuge tube on ice. First, prepare the lower gel according to the table. Add each component to the centrifuge tube and mix well by gently inverting the tube. Be careful not to introduce air bubbles. Slowly pour the prepared lower gel solution into the double-layered gel glass plate. Stop when the liquid surface is about 1 cm from the upper edge of the short glass plate. Gently add 1 mL of double-distilled water to the top to prevent air bubbles and tilting of the liquid surface. Place the glass plate at room temperature for 30 minutes. After the gel solidifies, a clear boundary line will appear.

[0356] (7) Pour out the upper double-distilled water. Prepare the upper gel according to the table. Mix well and add about 2 mL to the double-layered gel glass plate. Gently insert the comb. After about 30 minutes, when the upper gel has solidified, proceed with the following gel electrophoresis or store at 4°C for a short period (within one week).

[0357] (8) Electrophoresis

[0358] 1) Insert the gel plate into the electrophoresis slot holder. Pour the electrophoresis solution between the two gel plates, making sure that the solution covers the inner slot of the gel plate.

[0359] 2) Carefully remove the combs, making sure there are no air bubbles. Load the appropriate amount of protein sample (10-30 μg) and 5 μL of protein marker into the wells using a micro-loading pipette tip. Be careful not to overflow the wells. Fill the empty lanes with the same volume of 1 x protein loading buffer.

[0360] 3) Connect the power supply and set the voltage to 60 V to start the electrophoresis. When the samples have entered the resolving gel, adjust the voltage to 120 V (at this point, the protein marker will separate into multiple blue and red bands). After about 1 h, when the bromophenol blue indicator has reached the bottom of the resolving gel, end the electrophoresis.

[0361] (9) Transfer

[0362] 1) Prepare a 5.5 cm x 8.5 cm PVDF membrane and activate it by soaking in methanol for 30 s - 1 min. Place the membrane in the transfer buffer and prepare pre-chilled transfer buffer and ice box.

[0363] 2) After the electrophoresis is complete, remove the gel plate and gently rinse the surface with water to remove any foam. Pour the transfer buffer into the tray, pry open the gel plate, remove the concentrated gel on the top, and transfer it to the transfer buffer. Assemble the transfer sandwich in the following order: the cathode (black) and sponge of the transfer sandwich, filter paper soaked in transfer buffer, gel, PVDF membrane, filter paper, sponge, and anode (white) of the transfer sandwich. Make sure there are no air bubbles between the layers.

[0364] 3) Insert the transfer sandwich into the transfer slot with the corresponding electrodes, pour the transfer buffer, and place it in the ice box. Transfer at 200 mA for 2 h.

[0365] (10) Blocking

[0366] Remove the transferred PVDF membrane and place it in TBST. Pour off the TBST, add 5% milk blocking solution, cover the PVDF membrane, and block it slowly on a shaker at room temperature for 1 h (rotation speed 40 rpm).

[0367] (11) Primary antibody incubation

[0368] Remove the blocking solution and wash the membrane with TBST for 3 times, 5 min each time. Cut the membrane according to the molecular weight, add the primary antibody at the appropriate dilution, and incubate it on a shaker at 4°C overnight.

[0369] (12) Membrane washing

[0370] Remove the PVDF membrane after overnight incubation and store the antibody at -20°C. Place the removed PVDF membrane in TBST and rinse it quickly for 3 times, 15 min each time.

[0371] (13) Secondary antibody incubation

[0372] Add the corresponding species of secondary antibody proportionally prepared in blocking solution, slowly block at room temperature for 1 hour.

[0373] (14) Wash the membrane

[0374] Remove the secondary antibody, place the PVDF membrane in TBST solution, quickly rinse 3 times, each time for 15 min.

[0375] (15) ECL chemiluminescence

[0376] Prepare fresh developing working solution (A solution and B solution = 1:1) according to the instructions in the ECL luminescence kit, evenly drop on the membrane, and develop and collect images in the imager under light-proof conditions.

[0377] (16) Protein expression analysis

[0378] Use Image J software to statistically analyze the gray value of the western blot band, correct with the gray value of the reference protein, and calculate the relative expression amount of the target protein.

[0379] 2.1.4 Coomassie blue staining

[0380] (1) Dilute 9-37 / Fc (8.6 mg / mL) and 7-37 / Fc (10 mg / mL) fusion proteins to 0.1 mg / mL with pre-cooled PBS.

[0381] (2) Label 4 ep tubes as reduced 9-37 / Fc, non-reduced 9-37 / Fc, reduced 7-37 / Fc, and non-reduced 7-37 / Fc. Take 20 μL of diluted 9-37 / Fc and 7-37 / Fc fusion proteins (0.1 mg / mL) into each tube, and place on ice. Add 5 μL of 5X reduced protein loading buffer (containing DTT) to the tubes labeled as reduced 9-37 / Fc and reduced 7-37 / Fc, and add 5 μL of 5X non-reduced protein loading buffer (without DTT) to the other two tubes. Mix well.

[0382] (3) Set the program of the metal bath to temperature 100℃ and rotation speed 350 rpm. After the temperature rises to 100℃, place the protein sample in it and heat for 10 min. After heating, quickly place on ice to cool, and then proceed to Coomassie blue staining or Western blotting.

[0383] (4) SDS-PAGE gel electrophoresis same as Western blotting section.

[0384] (5) After electrophoresis, take out the gel and put it into the appropriate amount of Coomassie brilliant blue staining solution, make sure that the staining solution can fully cover the gel. Place it on a horizontal shaker and slowly shake it for 2 hours at room temperature.

[0385] (6) Pour out the staining solution. The staining solution can be recycled and reused at least 2-3 times.

[0386] (7) Add the appropriate amount of Coomassie brilliant blue staining and decolorizing solution, make sure that the decolorizing solution can fully cover the gel. Place it on a horizontal shaker and slowly shake it for 4-24 hours at room temperature. Change the decolorizing solution 2-4 times during this period until the blue background is basically all removed and the protein band staining effect reaches the expected.

[0387] 2.1.5 Immunohistochemical staining

[0388] (1) Fix the specimen: Put the liver tissue and pancreas tissue into 4% paraformaldehyde and fix it in a 4°C shaker overnight, then send it to Wuhan Saivier Biological Company for paraffin embedding and sectioning.

[0389] (2) Bake the slice: Put the slice into a slice holder and bake it in a 65°C oven for 2 hours.

[0390] (3) De-waxing and hydration: Put the slice into the following solutions in sequence, xylene (I) 60 min→ xylene (II) 5 min→ 100% alcohol (I) 5 min→ 90% alcohol (II) 5 min→ 80% alcohol (III) 5 min→ 70% alcohol (IV) 5 min→ slightly drain and quickly put it into 1x PBS buffer (PH 7.4)→ slowly wash it on a shaker for 3 times, 5 min each time.

[0391] (4) Antigen repair: Immerse the slice holder in 200 mL EDTA antigen repair solution (PH 8.0), wrap the repair box with transparent tape and place it in a microwave oven. Follow the steps below to repair: high fire for 3 min→ stop fire for 2.5 min for insulation→ high fire for another 30 s→ stop fire for 2.5 min for insulation→ high fire for 30 s→ stop fire for 1 min for insulation. Pay attention to observe whether the repair solution evaporates during microwave heating, causing dry slices.

[0392] (5) After the slice holder cools naturally at room temperature for about 1 hour, pour out the original EDTA antigen repair solution, add 1x PBS buffer and slowly wash it on a shaker at room temperature for 3 times, 5 min each time.

[0393] (6) Pour out the PBS buffer, put the slice holder into a box containing 3% hydrogen peroxide solution and place it in a dark place at room temperature for about 20 min to block endogenous peroxidase. Remove the 3% hydrogen peroxide solution, add 1x PBS buffer and slowly wash it on a shaker at room temperature for 3 times, 5 min each time.

[0394] (7) Blocking: Take out the sections and let them dry a little on the blotting paper. Draw a circle around the tissue with a histological pen, making sure not to touch the tissue. Cover the entire tissue with 60 μL of 10% goat serum in lx PBS (blocking solution) and place the sections in a humid chamber at room temperature for 30 min. Add a small amount of water to the humid chamber to prevent the liquid from evaporating.

[0395] (8) Primary antibody incubation: Remove the blocking solution and place the sections horizontally in a humid chamber. Add 60 μL of primary antibody diluted in 1% goat serum (prepared in lx PBS) to cover the entire tissue. Add 1% goat serum diluted in lx PBS to the negative control group. Incubate the sections at 4°C overnight.

[0396] (9) Secondary antibody incubation: Remove the primary antibody and place the sections in a slide rack containing lx PBS buffer. Wash the sections slowly on a shaker at room temperature for 3 times, 5 min each time. Take out the sections and let them dry a little on the blotting paper. Add 60 μL of secondary antibody (HRP labeled) diluted in 1% goat serum to cover the entire tissue. Place the sections in a humid chamber at room temperature for 60 min.

[0397] (10) DAB color development: Remove the secondary antibody and place the sections in a slide rack containing lx PBS buffer. Wash the sections slowly on a shaker at room temperature for 3 times, 5 min each time. Take out the sections and let them dry a little on the blotting paper. Add the color developing solution under a microscope and observe the color development of the tissue. The positive color development is brownish yellow. The color development time should be consistent for each group. Stop the color development with running tap water when the color development is complete.

[0398] (11) Place the sections in a slide rack containing lx PBS buffer and transfer them to lx PBS buffer. Wash the sections slowly on a shaker at room temperature for 3 times, 5 min each time.

[0399] (12) Nuclei staining: Place the sections in Harris hematoxylin for 3 min or so (the staining time can be adjusted according to the observation under a microscope). Then place the sections in 1% hydrochloric acid alcohol and ammonia water in sequence. Wash the sections thoroughly with running tap water between each step.

[0400] (13) Dehydration: Place the sections in the following solutions in sequence: 70% alcohol (I) for 5 min, 80% alcohol (II) for 5 min, 90% alcohol (III) for 5 min, 100% alcohol (IV) for 5 min, xylene (I) for 5 min, and xylene (II) for 5 min until the sections are dehydrated and transparent.

[0401] (14) Mounting: Take out the sections from xylene and let them dry in a fume hood. Add 10 μL of neutral resin diluted in xylene (diluted to a flowable state on the sections) to each section. Carefully cover the sections with coverslips to prevent air bubbles from forming between the coverslips and the slides.

[0402] (15) Photography: Microscopy observation and image collection for relevant analysis.

[0403] 2.1.6 Zebrafish microinjection

[0404] Alexa Fluor 647 Microscale Protein Labeling Kit (Invitrogen, lot# A30009) and Alexa Fluor 488 Microscale Protein Labeling Kit (Invitrogen, lot# A30006) were used to label 7-37 / Fc (red fluorescence) and 9-37 / Fc (green fluorescence) according to the manufacturer’s instructions. TM 647 Microscale Protein Labeling Kit (Invitrogen, lot# A30009) and Alexa Fluor TM 488 Microscale Protein Labeling Kit (Invitrogen, lot# A30006) were used to label 7-37 / Fc (red fluorescence) and 9-37 / Fc (green fluorescence) according to the manufacturer’s instructions. Zebrafish at 3 days post-fertilization were anesthetized with piperocaine and then injected with 7-37 / Fc and 9-37 / Fc fusion proteins through the main vein according to standard microinjection methods. One hour after injection, fluorescence microscopy was used to observe the distribution of red and green fluorescence in zebrafish.

[0405] 2.1.7 Flow cytometry analysis of 7-37 / Fc and 9-37 / Fc binding to hepatocytes

[0406] The method of mouse hepatocyte isolation and primary hepatocyte culture was as reported (SHAO W, WANG D, CHIANG Y T, et al. The Wnt signaling pathway effector TCF7L2 controls gut and brain proglucagon gene expression and glucose homeostasis, Diabetes, 2013, 62(3):789-800.). The isolated organs from GLP-1R KO mice, including pancreas and liver, were provided by Dr. Daniel Drucker. INS-1 cells were cultured in RPMI 1640 medium containing 50 μΜ β-mercaptoethanol, 10% fetal bovine serum. 7-37 / Fc (red fluorescence labeling) and 9-37 / Fc (green fluorescence labeling) were labeled according to the method in 2.1.6. Primary hepatocytes and INS-1 cells were incubated with 7-37 / Fc (1 μg / mL, red), 9-37 / Fc (1 μg / mL, green), or both, and then used for flow cytometry assay.

[0407] 2.1.8 Human hepatoma cell line HepG2 cell recovery and culture

[0408] Reagent preparation

[0409] (1) Prepare 500 mL of HepG2 cell complete culture medium as follows: DMEM, high glucose: 7 mL; DMSO: 1 mL, fetal bovine serum: 2 mL.

[0410] (2) Prepare 10 mL cell freezing solution as follows: DMEM, high glucose: 445 mL; fetal bovine serum: 50 mL; penicillin / streptomycin: 5 mL.

[0411] Cell recovery

[0412] (1) Alcohol wipe the clean bench and sterilize with UV light for more than 30 min. Set the water bath to 37°C and preheat PBS and complete medium.

[0413] (2) Take a cell freezing tube from the liquid nitrogen tank and quickly place it in the 37°C water bath. Gently shake the tube to quickly thaw the freezing solution in 1-2 min.

[0414] (3) Use a pipette to aspirate the cell solution from the freezing tube and add it to the previously prepared 15 mL centrifuge tube. Then add 2 times the volume of HepG2 cell complete medium (5 mL) and gently blow to form a cell suspension.

[0415] (4) Centrifuge at 1000 rpm for 5 min. Remove the supernatant and add 10 mL of fresh HepG2 cell complete medium. Gently blow to form a cell suspension.

[0416] (5) Add 10 mL of cell suspension to a 10 cm culture dish and gently shake the dish to distribute the cells evenly. Place the cells in a 37°C cell culture incubator with 5% CO2. The next day, observe the cell adhesion under a microscope and replace the fresh medium accordingly.

[0417] Cell passage

[0418] (1) When the cell density reaches 80% confluence, it is ready for passage.

[0419] (2) Alcohol wipe the clean bench and sterilize with UV light for more than 30 min. Prepare a 37°C water bath in advance to preheat PBS buffer, 0.25% trypsin solution, and complete medium.

[0420] (3) Aspirate the original medium and add 2-3 mL of PBS buffer to gently shake the dish to remove any residual serum. Then add 1 mL of 0.25% trypsin solution and place it in a 37°C cell culture incubator for 2 min.

[0421] (4) Observe the cell digestion under a microscope to ensure complete digestion without over-digesting the cells.

[0422] (5) Aspirate the 0.25% trypsin solution and add 1 mL of PBS buffer to gently rinse the cells once. Aspirate the 1 mL of PBS buffer and add 3 mL of fresh HepG2 complete medium to gently mix and collect the cell suspension.

[0423] (6) Add the mixed cell suspension dropwise to three new cell culture dishes, and add 9 mL of complete culture medium to each dish. Gently shake the culture dishes back and forth and side to side to distribute the cells evenly. Place the cells in a cell culture incubator containing 5% CO2 at 37°C and observe the cell adhesion and growth under a microscope the next day.

[0424] Cell cryopreservation

[0425] (1) Once the cell density reaches 80% confluence, the cells can be cryopreserved. The same applies to cell passage steps (2)-(4) in 2.1.8 below.

[0426] (2) Label the cryovials and prepare the cryopreservation solution for later use.

[0427] (3) Remove the culture dish from the incubator and add 10 times the volume of culture medium containing 10% FBS to stop the digestion. Gently pipette the cells to detach them from the bottom of the dish and transfer them to a 15mL centrifuge tube.

[0428] (4) Centrifuge at 1000 rpm for 5 min at room temperature.

[0429] (5) Discard the supernatant, add 1 mL of cell cryopreservation solution, mix with a pipette and transfer into a labeled cryopreservation tube.

[0430] (6) Place the cryovials in a cell cryopreservation box containing 250 mL of isopropanol and place them vertically at -80°C overnight. The next day, remove the cryovials and quickly place them in liquid nitrogen for preservation.

[0431] 2.1.9 Ligand-receptor binding assay based on cell ELISA

[0432] (1) HepG2 cells were passaged according to the method in Example 2.1.8 and seeded into 96-well cell culture plates with black transparent bottom at a seeding density of 10,000 / well.

[0433] (2) On the second day after seeding, add 100 μL of PBS buffer to each well and gently wash the cells twice. Then add 90 μL of binding buffer (DMEM high glucose medium containing 0.1% BSA, pH=7.5) and place in a 4°C refrigerator.

[0434] (3) In a clean bench, 7-37 / Fc, 9-37 / Fc, or IgG-Fc proteins were serially diluted 10-fold using binding buffer pre-cooled at 4°C to obtain 7-37 / Fc, 9-37 / Fc, or IgG-Fc protein solutions of different concentrations (10... -11 M–10 -4 M).

[0435] (4) Take out the 96-well plate and place it on ice. Add 10 μL of 7-37 / Fc, 9-37 / Fc or IgG-Fc protein solution of different concentrations to each well, respectively. Incubate at 4°C for 4 hours.

[0436] (5) Quickly take out the 96-well plate and place it on ice. Absorb the original culture medium containing 7-37 / Fc, 9-37 / Fc or IgG-Fc protein, and gently rinse the cells with pre-cooled PBS once.

[0437] (6) Dilute the fluorescently labeled human IgG-Fc secondary antibody with the binding buffer at a ratio of 1:2000 in the dark. Add 100 μL to each well and incubate at 4°C for 1 hour.

[0438] (7) Take out the 96-well plate and place it on ice. Absorb the original culture medium, and gently rinse the cells with pre-cooled PBS twice.

[0439] (8) Absorb the PBS, and set the SpectraMax i3x enzyme marker parameters as follows: fluorescence emission detection, excitation light: 490 ± 9 nm, emission light: 530 ± 15 nm, bottom reading method. Read the fluorescence value of each well, draw the binding curve by four-parameter fitting, and calculate Bmax and EC50.

[0440] 2.1.10 Statistical analysis

[0441] Statistical analysis and plotting were performed using GraphPad Prism 9.4.1 software (GraphPad Software Inc., San Diego, CA, USA). Experimental data were shown as mean ± standard deviation (mean ± SD). Student’s test was used for comparison between two groups. One-way ANOVA analysis was used for comparison among three groups or more groups, and Dunnett-t test was used for pairwise comparison between groups. Two-way ANOVA analysis was used for comparison between two groups at different time points, and Sidak’s multiple comparison test was used for pairwise comparison between groups. P<0.05 was considered statistically significant.

[0442] 2.2. Experimental results

[0443] 2.2.1. C57BL / 6 mice do not express GLP-1R in the liver

[0444] GLP-1 exerts biological effects by binding to GLP-1 receptor, therefore, in order to study the regulatory effect of GLP-1 on the liver and its mechanism, the expression of GLP-1R in the liver was first detected. The mRNA of mouse liver tissue was extracted for RT-PCR, and Western blot and immunohistochemical staining were performed using GLP-1R antibody, with islet / pancreas and brain tissue as positive controls, to detect whether GLP-1R was expressed in the liver of mice. The results showed that the mouse liver did not express GLP-1R (Fig. 1A-C).

[0445] 2.2.2. Distribution of 7-37 / Fc and 9-37 / Fc in zebrafish in vivo

[0446] In order to study whether GLP-1 (9-37) can exert a regulatory effect on the liver, the distribution of GLP-1 (7-37) and GLP-1 (9-37) in vivo was first detected. A mixture of fluorescently labeled 7-37 / Fc (Alexa Fluor 647, red) and 9-37 / Fc (Alexa Fluor 488, green) at a ratio of 1:1 was microinjected into zebrafish, and microscopic photography was performed 1 hour later. The images showed that 9-37 / Fc (green) was mainly distributed in the liver, and partially in the pancreas; 7-37 / Fc (red) was mainly distributed in the pancreas and islet, and no obvious liver distribution was observed (Fig. 3).

[0447] 2.2.3. 9-37 / Fc binding to hepatocytes does not depend on GLP-1R

[0448] Further separation of hepatocytes from wild-type (WT) and GLP-1R - / - (KO) mice was performed for flow cytometry to detect the binding of 9-37 / Fc and 7-37 / Fc to hepatocytes. The results showed that 9-37 / Fc had strong specific binding to hepatocytes of both WT and KO mice. In contrast, 7-37 / Fc showed no obvious binding to hepatocytes of WT or GLP-1R - / - mice (Fig. 4A, B). The β cell line INS-1 was used as a positive control, and showed that both 9-37 / Fc and 7-37 / Fc could bind to islet β cells (Fig. 4C). The above results indicate that 9-37 / Fc binds to hepatocytes in a GLP-1R-independent manner, while 7-37 / Fc does not bind to hepatocytes.

[0449] 2.2.4. Comparison of the binding affinity of 9-37 / Fc and 7-37 / Fc to HepG2 cells

[0450] The binding of 9-37 / Fc and 7-37 / Fc to HepG2 cells was further tested in human hepatoma cell line HepG2. Ligand-receptor binding assay based on cell ELISA showed that 9-37 / Fc bound to HepG2 cells in a protein concentration-dependent manner, and reached the maximum binding at 10 μM of 9-37 / Fc, with an equilibrium dissociation constant (Kd) of 37.56 ± 8.2 nM. The equilibrium dissociation constant (Kd) of 7-37 / Fc to HepG2 cells was 1659 ± 35.3 nM (Fig. 5A, B). The above results showed that 9-37 / Fc has a higher affinity to bind to HepG2 cells than 7-37 / Fc, about 50 times higher than 7-37 / Fc.

[0451] 2.3. Summary

[0452] 9-37 / Fc is mainly distributed in the liver, while 7-37 / Fc is mainly distributed in the pancreas and pancreatic islets, with no obvious liver distribution. The liver does not express GLP-1R, and 9-37 / Fc binds to liver cells in a GLP-1R-independent manner, while 7-37 / Fc does not bind to liver cells.

[0453] Example 3. 9-37 / Fc improves liver glucose and lipid metabolism disorders

[0454] The liver is an important organ for glucose metabolism, fat and cholesterol metabolism, and participates in the regulation of glucose and lipid metabolism balance in the body. The liver participates in the regulation of glycogen synthesis, decomposition and gluconeogenesis to maintain normal blood glucose levels in the body. The liver also participates in the regulation of multiple important links in the process of lipid metabolism, including the uptake and synthesis of fatty acids, the processing, storage, oxidative decomposition and output of lipids. Under normal circumstances, these processes are subject to complex and precise regulation, allowing the liver to maintain a dynamic balance between lipid metabolism and glucose metabolism. Hepatic lipid accumulation and inflammation caused by glucose and lipid metabolism disorders are key factors in the development of NAFLD disease. Lipids accumulated in hepatocytes undergo lipid peroxidation in response to reactive oxygen species, activating liver macrophages to release inflammatory mediators and inducing progressive inflammation, i.e. non-alcoholic steatohepatitis (NASH).

[0455] Previous studies have shown that GLP-1 (9-37) reduces glucose production in mouse primary hepatocytes in vitro. GLP-1 (9-37) can still reduce glucose production after the use of GLP-1R antagonist Ex (9-39). Similar results were observed in vivo. GLP-1 (9-37) can still reduce liver glucose production in obese subjects after the use of Ex (9-39), and does not affect insulin and glucagon levels. In addition, GLP-1 (9-37) reduces high-fat diet-induced obesity in mice, improves insulin resistance, and accompanying hyperglycemia and lipogenesis. In the study of Example 2, it was found that GLP-1 (9-37) can be localized to the liver of zebrafish and bind to mouse primary hepatocytes and HepG2 cells, while GLP-1 (7-37) does not bind to hepatocytes. Moreover, the binding of GLP-1 (9-37) to hepatocytes is not dependent on GLP-1R. The binding of GLP-1 (9-37) to hepatocytes independent of the classic GLP-1R may have physiological effects such as regulating hepatocyte glucose and lipid metabolism. Therefore, in order to study whether this specific binding has physiological significance, in Example 3, GLP-1 (9-37) was used to intervene in a high-fat diet-induced NAFLD mouse model and a palmitic acid-induced hepatocyte lipid accumulation model to study the regulatory effects of GLP-1 (9-37) on liver glucose and lipid metabolism and inflammatory response.

[0456] 3.1. Experimental methods

[0457] 3.1.1 Preparation of reagents

[0458] 7-37 / Fc and 9-37 / Fc fusion proteins (0.3 mg / mL) for animal experiments

[0459] Take 300 μL of 7-37 / Fc fusion protein stock solution (60 kD) with a concentration of 10 mg / mL, dilute with sterile normal saline to 10 mL to prepare a working solution of 0.3 mg / mL. Similarly, take 338 μL of 9-37 / Fc fusion protein stock solution (60 kD) with a concentration of 8.86 mg / mL, dilute with sterile normal saline to 10 mL to prepare a working solution of 0.3 mg / mL. The working solution is prepared fresh every week and stored at 4°C.

[0460] 7-37 / Fc and 9-37 / Fc fusion proteins (50 μM) for cell experiments

[0461] ​Add 1.5 mL of 10 mg / mL 7-37 / Fc fusion protein stock solution to 8.5 mL of sterile PBS buffer to prepare a 50 μM 7-37 / Fc working solution. Similarly, add 1.75 mL of 8.86 mg / mL 9-37 / Fc fusion protein stock solution to 8.25 mL of sterile PBS buffer to prepare a 50 μM 9-37 / Fc working solution. The commonly used working concentration is 100 nM. Store the working solution at 4°C for short periods, and the stock solution at -80°C for long periods.

[0462] Palmitic acid (PA) solution (6mM stock solution)

[0463] (1) Weigh 1g of BSA powder and dissolve it completely in 19mL of double-distilled water. Heat in a water bath at 55℃ for more than 30min to ensure that the BSA is completely dissolved.

[0464] (2) Weigh 40 mg of NaOH (molecular weight: 40.0 g / mol) and dissolve it in 10 mL of double-distilled water to prepare a NaOH solution with a concentration of 0.1 mol / L. Prepare and use immediately.

[0465] (3) Weigh 33.4 mg palmitic acid (molecular weight: 256.42 g / mol) and dissolve it in 1 mL of 0.1 mol / L NaOH solution. Heat in a metal bath at 90 °C for at least 30 min until completely dissolved.

[0466] (4) Quickly add 1 mL of NaOH solution containing dissolved palmitic acid to BSA solution, mix well, and continue heating in a 55°C water bath for 30 min to obtain 6 mM palmitic acid stock solution.

[0467] (5) Add 1 mL of NaOH solution without palmitic acid to another tube of BSA solution to make the control group solution.

[0468] (6) Sterilize by filtration in a clean bench and dispense into individual containers. Store at -20°C. Before use, heat in a 55°C water bath until the solution is fully dissolved.

[0469] Oil Red O staining solution (0.5% stock solution)

[0470] (1) Weigh 0.2g of Oil Red powder and dissolve it in 40mL of isopropanol to prepare Oil Red stock solution. Wrap the stock solution in aluminum foil and shake it overnight on a shaker at 4℃. Store at 4℃ protected from light.

[0471] (2) When using, add 6 mL of oil red mother liquor to 4 mL of double-distilled water and let it stand at room temperature for 10 min. Filter twice through a 0.22 μM disposable sterile filter. It is ready for use when it turns dark red and there is no sediment. It is recommended to use it within 2 hours.

[0472] 20% glucose injection

[0473] Weigh 2 g of glucose and dissolve it in 10 mL of sterile water, filter it through a 0.22 μM disposable sterile filter,

[0474] Store at 4°C and use within 1 month.

[0475] Insulin injection (0.75 UI / 10 mL)

[0476] Take 18.75 μL of recombinant human insulin (400 IU / 10 mL) and dissolve it in 10 mL of normal saline to prepare an insulin injection of 0.75 UI / 10 mL. The insulin injection should be prepared fresh on the day of the experiment.

[0477] 3.1.2 Animal grouping and treatment

[0478] (1) Determination of the half-life of 7-37 / Fc and 9-37 / Fc fusion proteins

[0479] Sixteen 6-week-old male C57BL / 6J mice were randomly divided into four groups: 7-37 / Fc low dose (0.3 μg / g), 7-37 / Fc high dose (0.9 μg / g), 9-37 / Fc low dose (0.3 μg / g), and 9-37 / Fc high dose (0.9 μg / g) injection groups. The mice received a single intraperitoneal injection of the corresponding group fusion protein, and 24, 48, 72, 96, 120, 144, and 168 hours after intraperitoneal injection of 9-37 / Fc and 7-37 / Fc fusion proteins (0.3 μg / g, 0.9 μg / g), blood was collected from the retro-orbital plexus. ELISA was used to detect the plasma concentration of each group of mice at the specified time points to draw the drug concentration-time curve and calculate the half-life.

[0480] (2) Animal experiment design

[0481] After 60 six to seven-week-old C57BL / 6 male mice were acclimated for one week, the mice were numbered. The animal experiment was divided into two parts, and the protective effect of 7-37 / Fc and 9-37 / Fc on fatty liver was studied from the aspects of prevention and treatment of NAFLD. In the model of preventing fatty liver induced by high-fat diet in mice, 30 mice were divided into four groups of normal diet (n=6), high-fat diet (n=8), high-fat diet+7-37 / Fc (n=8, 0.3 μg / g, intraperitoneal injection, twice a week) and high-fat diet+9-37 / Fc (n=8, 0.3 μg / g, intraperitoneal injection, twice a week) for 12 weeks. In the model of treating fatty liver induced by high-fat diet in mice, 30 mice were divided into normal diet (n=6) and high-fat diet for 12 weeks (n=24). The mice in the high-fat diet group were divided into three groups, and two groups were treated with 7-37 / Fc (n=8, 0.3 μg / g, intraperitoneal injection, twice a week) or 9-37 / Fc (n=8, 0.3 μg / g, intraperitoneal injection, twice a week) for 4 weeks, a total of 16 weeks.

[0482] (3) Experimental procedure and animal sampling

[0483] The body weight and food intake of mice in the prevention and treatment groups were monitored every week, and the glucose tolerance and insulin tolerance tests were performed one week before the end of the intervention. After the experimental intervention was completed, the mice in each group were fasted overnight, intraperitoneally injected with 10% chloral hydrate (150 μL / 20 g), and the eyeballs were quickly removed to collect blood. The mice were then heart perfused with normal saline. After blood collection, the whole blood was placed at room temperature for more than 30 min, centrifuged at 3000 rpm for 30 min at 4°C. After perfusion, the mouse liver, bilateral inguinal subcutaneous white fat, epididymal fat, pancreas and muscle tissues were quickly separated, and the liver was weighed and photographed. A portion of the liver tissue was fixed in 4% paraformaldehyde solution for 24 hours for paraffin and frozen section embedding. A portion of the fresh liver was immediately placed in a -80°C freezer for triglyceride content, cholesterol content, active oxygen staining section, MDA content, antioxidant enzyme GPH-Px and SOD activity determination. The remaining fresh liver tissue was placed in a cryopreservation tube, transferred to a -80°C freezer after quick freezing in liquid nitrogen, and stored for subsequent RNA and protein detection. The animal technical roadmap used in the present application is shown in Figure 6.

[0484] 3.1.3 Mouse retro-orbital plexus blood collection

[0485] (1) Prepare the following reagents and consumables: capillary glass tube, cotton ball, 10 μL gun head and pipette gun, 1.5 mL EDTA anticoagulant tube, 75% alcohol sprayer, gauze.

[0486] (2) Intraperitoneally inject 10% chloral hydrate at a dose of 0.03 mL / 10 g, and the blood can be collected after the mouse's righting reflex disappears.

[0487] (3) After anesthesia, cut off the mouse whiskers, and place a piece of paper under the mouse to make it lie on its side. Disinfect the left hand with 75% alcohol, and pull the skin on both sides of the mouse's neck with the left hand to make the eyeballs protrude (be careful not to pinch the mouse to death).

[0488] (4) Break the capillary glass tube, leaving 1-2 cm, and use the flat end to pierce the inner corner of the mouse's eye. Adjust the position up, down, left, and right to bleed, but do not pierce too deeply at once to prevent scabbing from affecting the next blood collection. Collect 100 μL after bleeding. Remove the glass tube, press the cotton ball to stop bleeding, and supplement 0.1-0.2 mL of 0.9% normal saline. Place the mouse on the cotton glove for warming, and wait for recovery.

[0489] (5) After blood collection, place at room temperature for 30 min, then centrifuge at 3000 rpm at 4°C for 10 min. Absorb the upper serum, and store at -80°C.

[0490] 3.1.4 Mouse plasma 7-37 / Fc and 9-37 / Fc concentration determination

[0491] Determine the plasma 7-37 / Fc and 9-37 / Fc concentrations by ELISA, and the detection concentration range is 15.625 ng / mL-1000 ng / mL.

[0492] Prepare solutions

[0493] (1) ELISA washing solution (0.05% PBST): add 0.5 mL Tween-20 to 1 L 0.01 mol / L PBS, mix well, and store at 4°C for 3 months after preparation.

[0494] (2) Coating buffer (CBS): dissolve one CBS capsule in 100 mL double-distilled water, mix well until completely dissolved, and the pH value is 9.6±0.2. Store at 4°C for 3 months after preparation.

[0495] (3) ELISA blocking / dilution solution (I-block in PBST): dissolve 0.2 g I-block powder in 100 mL 0.01 mol / L PBS containing 50 μL Tween-20, filter with a 0.22 μM disposable sterile filter after complete dissolution, store at 4°C for 1 month after preparation.

[0496] (4) ELISA termination solution (2M sulfuric acid): first add 22.4 mL concentrated sulfuric acid to 150 mL double-distilled water, mix well, then add pure water to 200 mL, and store at room temperature for 3 months after preparation.

[0497] Experimental procedure

[0498] (1) Coating 96-well plate: Use coating buffer to dilute anti-GLP-1 antibody to a concentration of 2 pg / mL, add 100 pL per well to the 96-well plate coated enzyme-labeled plate, seal with sealing film, and incubate at 4°C overnight in the dark.

[0499] (2) Wash the plate: wash each well with 300 pL of washing solution, 3 min each time, repeat 3 times, and then pat dry.

[0500] (3) Blocking: Add 200 pL of blocking solution to each well, seal with sealing film, and place the enzyme-labeled plate in a 37°C incubator for 2 h ± 5 min.

[0501] (4) Wash the plate: wash each well with 300 pL of washing solution, 3 min each time, repeat 3 times, and then pat dry.

[0502] (5) Prepare 7-37 / Fc and 9-37 / Fc standard concentration: Take 7-37 / Fc stock solution (10 mg / mL), use 100% mouse serum as the dilution matrix, and prepare the standard curve solution as follows:

[0503] Take 9-37 / Fc stock solution (8.86 mg / mL), use 100% mouse serum as the dilution matrix, and prepare the standard curve solution as follows:

[0504] (6) Sample addition: Take the sample out in advance, melt it on ice, dilute the standard, test sample and blank sample (100% mouse serum) 10 times with sample diluent, add 100 pL per well to the corresponding well of the enzyme-labeled plate, set up a duplicate well for each sample, seal with sealing film, and incubate the enzyme-labeled plate at 37°C for 1 h ± 5 min. Note that due to the high concentration of the sample, dilute it 3 times (0.3 pg / g dose group) or 10 times (0.9 pg / g dose group) with 100% mouse serum before adding the sample.

[0505] (7) Wash the plate: wash each well with 300 pL of washing solution, 3 min each time, repeat 3 times, and then pat dry.

[0506] (8) Add HRP-labeled IgG-Fc antibody: dilute the HRP-labeled IgG-Fc antibody 20,000 times with ELISA blocking / dilution solution, 100 pL per well, seal with sealing film, and incubate the enzyme-labeled plate at 37°C for 1 h ± 5 min.

[0507] (9) Wash the plate: wash each well with 300 pL of washing solution, 3 min each time, repeat 3 times, and then pat dry.

[0508] (10) Color development: add 100 pL of TMB color developing solution to each well, react at room temperature for 15 min ± 5 min in the dark.

[0509] (11) Termination: Add 50 μL of termination solution to each well, and mix by hand gently.

[0510] (12) Read plate: After termination of the reaction, within 15 min, measure the absorbance of each well of the enzyme-labeled plate using a dual-wavelength, with a detection wavelength of 450 nm and a correction wavelength of 630 nm.

[0511] (13) Data calculation: Use the enzyme-labeled instrument software SoftMax Pro 7.0.3 to fit the standard curve with four parameters, and then calculate the concentration value of the sample to be tested.

[0512] 3.1.5 Intraperitoneal glucose tolerance test (IPGTT) in mice

[0513] (1) Prepare a 20% glucose solution according to the foregoing method.

[0514] (2) The night before the test, fast the mice overnight (> 16 hours): remove the original cage and place the mice in a clean cage without feed, but with unlimited water.

[0515] (3) Fasting blood glucose (0 min) detection: at 8 am, first weigh each group of mice and mark the number on the tail of the mouse with a marker pen. Use ophthalmic scissors to gently cut a 2 mM opening at the end of the mouse tail, and gently squeeze the blood out of the cut along the tail root. Use a blood glucose meter to detect and record the fasting blood glucose of each mouse.

[0516] (4) Inject 20% glucose solution into the mice at 10 μL / g of body weight, i.e. the injection dose is 2 g / kg, and pay attention to whether there is blood or liquid leakage after injection.

[0517] (5) Use a timer to time, and use a blood glucose meter to detect and record the blood glucose value at the end of the mouse tail at 15, 30, 60, 90 and 120 min after injection of 20% glucose.

[0518] (6) Keep quiet and gentle during the whole operation to prevent the mice from being stressed and affecting the blood glucose results.

[0519] 3.1.6 Intraperitoneal insulin tolerance test (IPITT) in mice

[0520] (1) Prepare an insulin injection solution (0.75 UI / 10 mL) according to the foregoing method. Prepare 20% glucose injection solution in advance to prevent the mice from dying of hypoglycemia during the experiment.

[0521] (2) Morning 8 o'clock mice fast, remove the original cage, put the mice into clean cages without feed, unlimited water. After 4 hours of fasting, at noon 12 o'clock, each group of mice were weighed and marked with a marker pen on the tail of the mouse.

[0522] (3) Fasting blood glucose (0 min) detection: after weighing, the tail end of the mouse was gently cut with ophthalmic scissors to form a 2 mM small opening, and the tail end was gently squeezed to bleed along the tail root. The fasting blood glucose of each mouse was detected and recorded using a blood glucose meter.

[0523] (4) The mice were injected intraperitoneally with insulin injection solution according to 10 μL / g body weight, that is, the injection dose was 0.75 UI / kg, and attention was paid to whether blood or liquid leaked out after injection.

[0524] (5) Timing with a timer, the blood glucose value at the end of the mouse tail was detected with a blood glucose meter at 15, 30, 60, 90 min after insulin injection and recorded.

[0525] (6) The whole operation should be kept quiet and gentle to prevent the stress of the mice from affecting the blood glucose results. If the mice show hypoglycemic reaction during the experiment, the experiment should be terminated immediately and 20% glucose solution should be injected intraperitoneally to prevent the mice from dying of hypoglycemia.

[0526] 3.1.7 Hematoxylin-eosin (HE) staining of liver sections

[0527] The specimen was fixed and baked as in Example 2, Section 2.1.5. After fixation and baking, the following operations were performed:

[0528] (1) De-waxing and hydration: the sections were placed in the following solutions in turn, xylene (I) 10 min→xylene (II) 10 min→xylene (III) 10 min→100% alcohol (I) 5 min→90% alcohol (II) 5 min→80% alcohol (III) 5 min→70% alcohol (IV) 5 min→ slightly drained and quickly placed in double distilled water→ slowly washed on a shaker for 3 times, 5 min each time.

[0529] (2) HE staining: the sections were moved into hematoxylin staining solution and stained for 10 min. After staining, the sections were placed in a section holder containing double distilled water and slowly washed on a shaker for 3 times, 5 min each time. Then the sections were moved into 1% hydrochloric acid ethanol differentiation solution, and the differentiation of cell nucleus and cytoplasm was observed under a microscope until it was clear. The sections were placed in a section holder containing double distilled water, and the cell nucleus showed blue color. Then the sections were moved into eosin staining solution for 1-5 min, and the excess staining solution was washed away with double distilled water.

[0530] Dehydration and mounting were the same as in Example 2, Section 2.1.5.

[0531] 3.1.8 Oil red O staining of liver sections

[0532] The specimen fixation section is the same as HE staining. After the fixation section is completed, the following operations are performed:

[0533] (1) Oil red O staining: The frozen section is left to stand at room temperature for 15-20 min. After the water vapor on the slide disappears, the section is placed in the oil red O staining solution for 8 min.

[0534] (2) Differentiation: After the staining is completed, the section is washed with PBS and differentiated with 75% alcohol.

[0535] (3) Hematoxylin restaining: The section is washed with PBS and moved into the hematoxylin staining solution for 2 min.

[0536] (4) Mounting and statistics: The section is washed with PBS, and after the section is slightly dried, it is mounted with glycerol gelatin. The section is observed under a microscope and photographed. The lipid droplet content in the image is quantitatively analyzed by using ImagePro-Plus 6.0 software.

[0537] 3.1.9 Detection of liver triglyceride (TG) content

[0538] 100 mg of mouse liver tissue is weighed and added to a homogenizing tube containing 1 mL of anhydrous ethanol. Three steel balls are added, and the homogenizing tube is placed in a full-automatic sample grinder. The conditions are set as follows: 60 Hz, 60 s each time, and 3 times. The sample amount is added to the blank well, the standard well and the sample well in a 96-well plate according to the following table. 250 μL of working solution is added to each well, and the plate is incubated at 37°C for 10 min. The absorbance value (OD value) of each well is measured at 510 nm by using an enzyme label instrument. The liver triglyceride content is calculated according to the formula.

[0539] Triglyceride content (mg / g) = (OD value of sample well-OD value of blank well) / (OD value of standard well-OD value of blank well) x standard concentration (mmol / L) x 639 g / mol.

[0540] 3.1.10 Detection of liver cholesterol (TC) content

[0541] 100 mg of mouse liver tissue is weighed and added to a homogenizing tube containing 1 mL of anhydrous ethanol. Three steel balls are added, and the homogenizing tube is placed in a full-automatic sample grinder. The conditions are set as follows: 60 Hz, 60 s each time, and 3 times. The sample amount is added to the blank well, the standard well and the sample well in a 96-well plate according to the following table. 250 μL of working solution is added to each well, and the plate is incubated at 37°C for 10 min. The absorbance value (OD value) of each well is measured at 510 nm by using an enzyme label instrument. The liver triglyceride content is calculated according to the formula.

[0542] Cholesterol content (mg / g) = (OD value of sample well - OD value of blank well) / (OD value of standard well - OD value of blank well) x standard concentration (mmol / L) x 386.65 g / mol.

[0543] 3.1.11 ROS staining of liver sections (DCFH-DA)

[0544] Fix the specimen and slice it as for HE staining. After fixing the slice, perform the following operations:

[0545] (1) DCFH-DA staining: rinse the slice with double-distilled water (30 s) and place it in 10 μmmol / L DCFH-DA staining solution for 10 min in the dark.

[0546] (2) After staining, wash the slice in a beaker containing double-distilled water for 1 min, and repeat the washing twice.

[0547] (3) DAPI staining: place the slice in 100 ng / mL DAPI staining solution for 5 min in the dark.

[0548] (4) After staining, wash the slice in a beaker containing double-distilled water for 1 min, and repeat the washing twice.

[0549] (5) Immediately observe the slice under a microscope and take a photograph.

[0550] 3.1.12 Determination of blood lipid and transaminase levels

[0551] After taking blood from the mice by enucleation, let the blood stand at room temperature for 30 min, and then centrifuge it at 3000 rpm and 4°C for 30 min. Carefully pipette the upper serum into a new 1.5 mL EP tube and store it at -80°C. Use a Hitachi automatic analyzer to detect the serum triglyceride, cholesterol, low-density lipoprotein, glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase, etc.

[0552] 3.1.13 Extraction and concentration determination of tissue proteins, and Western blotting

[0553] The experimental procedures are the same as those in Example 2, Section 2.1.2 and 2.1.3.

[0554] 3.1.14 Determination of MDA content in liver tissue

[0555] Weigh 50 mg of mouse liver tissue, and place the homogenization tube into a full-automatic sample grinder. Set the conditions as follows: 60 Hz, 60 s each time, 3 times. Add the sample into the 96-well plate according to the following table to set the blank well, standard well, sample well and control well. Calculate the MDA content in the liver tissue according to the formula.

[0556] MDA content (nmol / mg) = (OD value of sample well - OD value of control well) / (OD value of standard well - OD value of blank well) x standard concentration (10 mmol / L) / protein concentration to be detected (mg / mL).

[0557] 3.1.15 Reverse transcription reaction (RT-PCR) and fluorescent quantitative PCR experiment (qPCR)

[0558] Tissue RNA extraction is the same as Example 2.

[0559] 3.1.15.1 RT-PCR

[0560] (1) Prepare the following 20 μL reverse transcription system with an eight-way tube on ice:

[0561] (2) After the sample is added, cover the tube cap, mix gently and centrifuge, and put it into the PCR amplifier. Set the following reverse transcription program:

[0562] (3) The cDNA synthesized by reverse transcription can be directly used for qPCR experiment, or can be stored at -20°C.

[0563] 3.1.15.2 Real-time fluorescent quantitative PCR (qPCR)

[0564] (1) Prepare the following 10 μL PCR reaction system with a 96-well plate on ice:

[0565] (2) After the sample is added, cover the tube cap, mix gently and centrifuge, and put it into the fluorescent quantitative amplifier. Set the following program:

[0566] 3.1.16 Detection of SOD activity in liver tissue

[0567] (1) Weigh 100 mg of mouse liver tissue, slightly rinse the tissue with physiological saline, and remove as much blood as possible. Use a water-absorbing paper to dry the water on the tissue.

[0568] (2) Add 400-900 μL sucrose buffer (0.25 mol / L sucrose, 10 mmol / L HEPES, 1 mmol / L EDTA, pH 7.4) and use a Teflon homogenizer to homogenize the sample.

[0569] (3) After homogenization, centrifuge the sample at 10,000 x g for 60 min, and transfer the supernatant to a new test tube for detection.

[0570] (4) Prepare working solution: Dilute 1 mL WST Solution with 19 mL Buffer Solution. Pipette 1 mL Enzyme Solution into the cuvette and mix well. Dilute 15 μL Enzyme Solution with 2.5 mL Dilution buffer.

[0571] (5) Prepare SOD standard solution according to Figure 7: Add 200 μL Dilution Buffer to SOD standard solution to obtain 200 U / mL SOD standard solution. Dilute 200 U / mL SOD standard solution with Dilution Buffer to obtain SOD standard solutions of different concentrations.

[0572] (6) Set up sample blank control wells (blank 1), reagent blank control wells (blank 2), standard wells and sample wells in 96-well plates according to the table below. Add 200 μL working solution to each well, incubate at 37°C for 20 min, and measure the absorbance value (OD value) of each well at 450 nm with a microplate reader. Calculate the SOD activity of liver tissue according to the formula.

[0573] SOD activity (mU / mg) = (OD value of sample well - OD value of blank 1) / (OD value of standard well - OD value of blank 2) x standard concentration (mU / mL) / protein concentration to be tested (mg / mL).

[0574] 3.1.17 Liver tissue GSH-Px activity

[0575] Sample processing is the same as 3.1.16.

[0576] (1) Let the tissue homogenate supernatant undergo enzymatic reaction according to the table below (reagent is preheated at 37°C in advance):

[0577] (2) Mix well, centrifuge at 4000 rpm for 10 min, and take 1 mL supernatant for color development reaction

[0578] (3) Set up sample blank wells, standard wells, control wells and sample wells in 96-well plates according to the table below, stand at room temperature for 15 min, and measure the absorbance value (OD value) of each well at 412 nm with a microplate reader. Calculate the GSH-Px activity of liver tissue according to the formula.

[0579] GSH-Px activity is defined as: 1 μmol / L decrease in GSH concentration in the reaction system per minute per mg of protein, minus the effect of non-enzymatic reaction, as one unit of enzyme activity.

[0580] GSH-Px activity (mU / mg) = (OD value of control well - OD value of sample well) / (OD value of standard well - OD value of blank well) x standard concentration (20 μmol / L) x 5 / protein concentration (mg / mL) / reaction time.

[0581] 3.1.18 ELISA method for detecting IL-6 and TNF-a content in serum

[0582] Sample processing is as described above. The following is an example of IL-6 detection.

[0583] (1) Prepare 1X Wash Buffer: dilute 60 mL of 20X Wash Buffer with double distilled water to 1200 mL, mix well;

[0584] (2) Prepare IL-6 standard: add 1 mL of Calibrator Diluent II to the recombinant mouse IL-6 standard tube. Get 800 pg / mL IL-6 standard solution. After standing at room temperature for 15 min, add 0.5 mL of Calibrator Diluent II to the 800 pg / mL IL-6 standard solution for dilution by a factor of two, to get IL-6 standards of different concentrations (Figure 8).

[0585] (3) Add 50 μL of sample or standard to the enzyme-labeled plate coated with IL-6 antibody at the bottom, and incubate at room temperature for 60 min.

[0586] (4) Add 50 μL of anti-IL-6 Biotin conjugate to each well, and incubate at room temperature for 60 min after sealing the plate.

[0587] (5) Discard the liquid in the enzyme-labeled plate, and wash the plate 5 times with 1X Wash Buffer, 3 min each time. After the last washing, invert the enzyme-labeled plate on absorbent paper to absorb the remaining liquid.

[0588] (6) Add 100 μL of Avidin-HRP conjugate to each well, and incubate at room temperature for 60 min after sealing the plate.

[0589] (7) Repeat step (5).

[0590] (8) Add 100 μL of TMB color developing solution to each well, and incubate at room temperature for 15 min.

[0591] (9) Add 100 μL of stop solution to each well, and detect the absorbance value (OD value) at 450 nm with an enzyme-labeled instrument.

[0592] (10) Data calculation: using the software Soft Max Pro 7.0.3 of the microplate reader to fit the standard curve with four parameters, and then calculating the concentration of the sample according to the OD value of each sample.

[0593] 3.1.19 Resuscitation and culture of HepG2 cells

[0594] The same as the part 2.1.8 of Example 2.

[0595] 3.1.20 Determination of triglyceride in HepG2 cells

[0596] (1) HepG2 cells were grown to 80% density in a 6-well plate, and then 0.25% trypsin was added for 2 min to terminate the digestion, and the cell suspension was collected into a 1.5 mL EP tube by gently blowing.

[0597] (2) Centrifugation at 1500 rpm for 5 min.

[0598] (3) Try to suck the supernatant clean, add 1 mL of isopropanol to each tube and mix well, then stand at room temperature for 10 min to lyse the cells.

[0599] (4) Centrifugation at 3000 rpm for 15 min.

[0600] (5) Carefully pipette the supernatant into a new 1.5 mL ep tube, being careful not to suck the cell precipitate at the bottom, add 300 μL of 1 mmol / L NaOH to each tube, and mix well until no obvious particles are visible. Then determine the protein concentration according to the BCA method described above.

[0601] (6) Set the metal bath parameters to 90°C and the rotation speed to 350 rpm. Put the ep tube containing the supernatant into the metal bath and heat until the liquid is completely evaporated (about 30 min).

[0602] (7) After the liquid is completely evaporated, add 30 μL of isopropanol to resolubilize, mix well and centrifuge.

[0603] (8) According to the table below, set the blank wells, standard wells and sample wells in a 96-well plate, add 200 μL of working solution to each well, incubate at 37°C for 10 min, measure the absorbance value (OD value) of each well at 510 nm with a microplate reader, and calculate the intracellular triglyceride content according to the formula.

[0604] Triglyceride content (mmol / g) = (OD value of sample well - OD value of blank well) / (OD value of standard well - OD value of blank well) x standard concentration / 5.

[0605] 3.1.21 Oil red staining of HepG2 cells

[0606] (1) Discard the original culture medium, rinse the 24-well plate with PBS for 2 times.

[0607] (2) Discard the PBS, add 4% paraformaldehyde to fix the cells for 10 minutes at room temperature.

[0608] (3) Wash the cells with PBS for 3 times.

[0609] (4) Add 300 μL of 60% isopropanol (prepared with double distilled water) to each well, incubate at room temperature for 5 minutes to facilitate the subsequent oil red dye into the cells.

[0610] (5) Remove the 60% isopropanol, add 500 μL of oil red working solution to each well, incubate at room temperature for 30 minutes.

[0611] (6) Remove the oil red working solution, wash the cells with PBS for 5 times to wash away the excess oil red dye.

[0612] (7) Add 300 μL of 60% isopropanol to each well for about 10 seconds, until the intercellular matrix is clear, immediately remove the 60% isopropanol, and wash the cells with PBS for 3 times.

[0613] (8) Stain the cell nucleus with hematoxylin for 30 seconds, wash the cells with PBS for 3 times to wash away the excess hematoxylin dye.

[0614] (9) After mounting with glycerol gelatin, observe the staining under a microscope.

[0615] 3.1.22 Statistical analysis

[0616] The same as Example 2, Section 2.1.10.

[0617] 3.2. Experimental results

[0618] 3.2.1. 9-37 / Fc reduces hepatic gluconeogenesis

[0619] To investigate whether the binding of 9-37 / Fc to the liver has physiological significance, we detected two key enzymes of hepatic gluconeogenesis: PEPCK and G6PC after injecting 9-37 / Fc in zebrafish and mice. We injected GFP-labeled liver (green) promoter plasmid into zebrafish to construct transgenic zebrafish (Tg:PEPCK:eGFP). The results showed that glucagon increased the fluorescence intensity of PEPCK compared with the control group injected with PBS, and 9-37 / Fc could significantly reduce the fluorescence intensity of PEPCK, while 7-37 / Fc had no significant change on the fluorescence intensity of PEPCK (Figure 9A, A’). The results of RT-qPCR were consistent with the fluorescence results (Figure 9B). It suggested that 9-37 / Fc could reduce the expression of PEPCK and G6PC genes in zebrafish, while 7-37 / Fc had no significant effect on the expression of PEPCK and G6PC genes. Further, we injected PBS or 7-37 / Fc and 9-37 / Fc 0.3 μg / g into the abdominal cavity of mice to detect the gene expression of PEPCK and G6PC in the liver. The results showed that 7-37 / Fc and 9-37 / Fc significantly reduced the expression of PEPCK and G6PC genes in the liver of mice, and 9-37 / Fc had more significant effect than 7-37 / Fc (Figure 9C).

[0620] 3.2.2. 9-37 / Fc reduces palmitic acid (PA)-induced lipid accumulation and triglyceride content in HepG2 cells

[0621] PA induces lipid accumulation in HepG2 cells

[0622] Firstly, we used PA to treat HepG2 cells to construct a high-fat model at the cellular level. Oil red O staining results showed that compared with control, PA could promote the increase of red lipid droplet particles in HepG2 in a concentration-dependent manner, suggesting that the modeling was successful (Figure 10). We selected 300 μM PA concentration for subsequent cell experiments.

[0623] 9-37 / Fc improves PA-induced lipid accumulation in HepG2 cells

[0624] To further investigate whether GLP-1(9-37) directly mediates the regulation of GLP-1 on hepatic lipid metabolism, HepG2 cells were co-incubated with different concentrations of 7-37 / Fc and 9-37 / Fc (10, 30, 100 nM) and 300 μM PA for 24 hours, and the lipid deposition in each group of cells was detected by oil red O staining. The results showed that 9-37 / Fc could reduce PA-induced lipid deposition, and 9-37 / Fc at concentrations of 30 nM and 100 nM had statistical significance. 7-37 / Fc had no significant change on PA-induced lipid deposition. Compared with 7-37 / Fc, 9-37 / Fc at the same concentration had a stronger effect, and at a concentration of 100 nM, it had statistical significance (Figure 11).

[0625] 9-37 / Fc reduces the triglyceride (TG) content of PA-induced HepG2 cells

[0626] Consistent with the results of oil red staining, the results of TG content detection in HepG2 cells showed that 9-37 / Fc 100 nM could reduce the increase of PA-induced TG content. 7-37 / Fc 100 nM had no significant effect on the TG content in cells (Figure 12).

[0627] 3.2.3. Preventive effect of 9-37 / Fc on fatty liver induced by high-fat diet in mice

[0628] Calculation of administration frequency of 9-37 / Fc and 7-37 / Fc fusion proteins

[0629] WT wild type and high-fat diet-fed NAFLD mouse models were used to further investigate the regulatory effect of 9-37 / Fc on hepatic lipid metabolism. To determine the appropriate animal administration dose and administration frequency, the plasma concentration and half-life of 9-37 / Fc and 7-37 / Fc fusion proteins in mice were first detected. 16 8-week-old male C57BL / 6J mice were randomly divided into 4 groups, namely 7-37 / Fc low-dose group (7-37 / Fc 0.3 μg / g), 7-37 / Fc high-dose group (7-37 / Fc 0.9 μg / g), 9-37 / Fc low-dose group (9-37 / Fc 0.3 μg / g) and 9-37 / Fc high-dose group (9-37 / Fc 0.9 μg / g) injection group. The mice received intraperitoneal injection of fusion proteins once according to the corresponding group, and blood samples were collected from the retro-orbital plexus at 24, 48, 72, 96, 120, 144 and 168 hours after intraperitoneal injection of fusion proteins at 30 min before administration (Figure 13A).

[0630] ELISA method was used to detect the plasma concentration of each group of mice at the above time points and draw the plasma drug concentration-time curve after single intraperitoneal injection of fusion protein 9-37 / Fc, 7-37 / Fc (Figure 13B). According to Figure 13B, the peak concentration of the drug (Cmax, Table 1) can be directly obtained. The natural logarithm (lnC) of the plasma drug concentration of each group of mice was plotted against time (h), and the linear fitting was calculated according to the slope to calculate the drug half-life (T1 / 2, Table 1). The results showed that the plasma concentration reached the highest peak at 24 hours after injection of 9-37 / Fc, 7-37 / Fc fusion protein, and the Cmax was 328.36 ng / mL, 1321.92 ng / mL, 358.54 ng / mL and 1110.80 ng / mL (9-37 / Fc 0.3 μg / g, 9-37 / Fc 0.9 μg / g, 7-37 / Fc 0.3 μg / g and 7-37 / Fc 0.9 μg / g, respectively). The T1 / 2 was 33.16-38.23 hours, and there was no significant difference in T1 / 2 between each group. According to the literature (WAN Y, BAO X, HUANG J, et al. Novel GLP-1 Analog Supaglutide Reduces HFD-Induced Obesity Associated with Increased Ucp-1 in White Adipose Tissue in Mice. Frontiers in physiology, 2017, 8:294.) and considering the tolerance of mice, the subsequent animal experiment of 9-37 / Fc, 7-37 / Fc was administered at a dose of 0.3 μg / g, and the administration frequency was twice a week.

[0631] Table 1. Pharmacokinetic parameters after single intraperitoneal injection of fusion protein 9-37 / Fc, 7-37 / Fc

[0632] C max , peak plasma concentration; T 1 / 2 , elimination half-life, T 1 / 2 = 0.693 / λz, where λz is the terminal elimination rate constant (the negative value of the slope obtained by linear regression of the logarithmic plasma concentration against time is λz). Data are presented as mean ± standard deviation, n = 4.

[0633] 9-37 / Fc reduces the effect of high-fat diet-induced weight gain in mice significantly weaker than 7-37 / Fc

[0634] 6-week-old male C57BL / 6J mice were randomly divided into 4 groups and fed with normal diet (NCD), high-fat diet (HFD), high-fat diet plus 7-37 / Fc (HFD+7-37) and high-fat diet plus 9-37 / Fc (HFD+9-37) for 12 weeks. The mice in HFD+7-37 and HFD+9-37 groups were injected intraperitoneally with 7-37 / Fc and 9-37 / Fc fusion protein at 0.3 μg / g twice a week, respectively, until the end of feeding. The body weight and food intake of mice in each group were monitored every week. It was found that the body weight of mice in HFD and HFD+9-37 groups increased significantly compared with that of mice in NCD group, although the body weight of mice in HFD+9-37 group increased less than that of mice in HFD group, but there was no statistical difference in body weight between the two groups of mice during the 12-week observation period. 7-37 / Fc can significantly reduce the increase of body weight induced by high-fat diet in mice. From the 7th week, the body weight of mice in HFD+7-37 group was significantly lower than that of mice in HFD+9-37 group and continued to the end of 12-week feeding (Figure 14A). The change (%) of food intake of mice after 12-week intervention relative to the baseline food intake was calculated, taking the baseline food intake of mice as 100%. Both 7-37 / Fc and 9-37 / Fc can significantly reduce the food intake of HFD mice (Figure 14B). The above results show that 7-37 / Fc can prevent the increase of body weight induced by high-fat diet in mice, while the effect of 9-37 / Fc is weaker.

[0635] 9-37 / Fc improves insulin resistance induced by high-fat diet in mice, but has no obvious effect on glucose tolerance

[0636] After 12 weeks of intervention, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (IPITT) were performed to evaluate the effects of 7-37 / Fc and 9-37 / Fc on glucose metabolism and insulin sensitivity in HFD mice. The results of IPGTT showed that the glucose tolerance level was significantly impaired in HFD mice and 9-37+HFD mice compared with NCD mice, and there was no significant difference between the two groups. The glucose tolerance level of 7-37+HFD mice was significantly lower than that of HFD and 9-37+HFD mice, and almost consistent with that of NCD mice (Figure 15A). The results of analysis of the area under the curve further confirmed that 7-37 / Fc could improve the impaired glucose tolerance in HFD mice, while 9-37 / Fc could not (Figure 15B). The results of IPITT showed that the slope of blood glucose decrease from 0 min to 15 min after insulin injection was significantly increased in 9-37+HFD and 7-37+HFD mice compared with HFD mice, suggesting that insulin sensitivity was enhanced (Figure 15C). Although there was no statistical difference in the area under the curve between 9-37+HFD and HFD groups, a lower trend was observed in the former. 7-37 / Fc could significantly improve insulin resistance in HFD mice (Figure 15D).

[0637] 9-37 / Fc reduces blood lipid and transaminase levels in HFD-induced mice

[0638] After 12 weeks of intervention, the mice in each group were sacrificed and serum was isolated to determine blood lipid and transaminase levels. The results showed that the levels of triglyceride (TG), cholesterol (TC), low-density lipoprotein (LDL) and non-esterified fatty acid (NEFA) were significantly reduced in 9-37+HFD and 7-37+HFD mice compared with HFD mice. The above indicators were comparable in 9-37+HFD and 7-37+HFD groups (Figures 16A-D). The above results showed that 9-37 / Fc and 7-37 / Fc could reduce blood lipid levels and improve systemic lipid metabolism disorder in HFD mice. At the same time, 9-37 / Fc and 7-37 / Fc could significantly reduce the level of ALT in serum (Figure 16E), and 9-37 / Fc could more significantly reduce the level of AST than 7-37 / Fc (Figure 16F), suggesting that 9-37 / Fc and 7-37 / Fc could improve liver damage in HFD-induced mice and play a liver protection role.

[0639] 9-37 / Fc reduces liver lipid accumulation in HFD-induced mice

[0640] Results showed that compared with NCD group mice, the liver, epididymal white adipose tissue (eWAT), inguinal subcutaneous adipose tissue (iWAT) and pancreas tissue weight of HFD group mice were significantly increased, while 9-37 / Fc could reduce the weight of the above tissues with statistical significance. Except for the pancreas, 7-37 / Fc could also significantly reduce the weight of the above tissues, and more significantly reduce the weight of eWAT and iWAT than 9-37 / Fc, which was consistent with the result that 7-37 / Fc played a stronger weight loss effect (Figure 17A).

[0641] The liver index of each group of mice was calculated and compared, and the results showed that the liver index of 9-37+HFD group and 7-37+HFD group mice was significantly lower than that of HFD group, and the liver index of 9-37+HFD group mice had a lower trend than that of 7-37+HFD (Figure 17B). It is suggested that 9-37 / Fc and 7-37 / Fc can improve the weight gain of liver of high-fat mice and prevent liver enlargement.

[0642] After 12 weeks of intervention, the liver tissue of mice was taken and the general morphological changes were observed. The liver of NCD group mice was reddish brown, the edge was sharp, and there was no greasy feeling; the liver volume of HFD group mice increased significantly, which was milk yellow, the edge was blunt, the section was greasy, and the tissue fragility increased; the liver of 9-37+HFD group and 7-37+HFD group mice was similar to that of NCD group mice in volume and shape, which was reddish brown (Figure 17C).

[0643] HE staining results showed that the liver lobule of NCD group mice had complete structure, the liver cells were arranged regularly, the morphology was normal, and the cells were regular polygon, with large and round nucleus in the center, uniform cytoplasm, and no lipid droplets. The liver cells of HFD group mice had obvious fatty degeneration, the cells were swollen and round, a large number of vacuole-like lipid droplets were visible in the cytoplasm, the ballooning was obvious, and the liver cord arrangement was disordered. The morphology of liver cells and liver cord arrangement of 9-37+HFD group and 7-37+HFD group mice tended to be normal, and the lipid droplets and ballooning in the cytoplasm were significantly reduced (Figure 17D). According to the HE staining, the NAFLD activity score was calculated from three aspects of liver tissue fatty degeneration, hepatocyte ballooning and lobular inflammation. The results showed that the NAFLD activity score of 9-37+HFD group and 7-37+HFD group mice was significantly lower than that of HFD group, indicating that 9-37 / Fc and 7-37 / Fc could play a liver protection role (Figure 17H).

[0644] Compared with NCD group mice, the TG and TC content of liver of HFD group mice increased significantly, while the TG content of liver of 9-37 / Fc and 7-37 / Fc treatment group mice decreased significantly compared with HFD group (Figure 17E), and 9-37 / Fc also significantly reduced the TC content of liver of HFD mice (Figure 17F).

[0645] The lipid deposition in the liver of mice was further detected by oil red O staining. The results showed that a small amount of red lipid droplets existed in the hepatocytes of NCD group mice, while a large amount of red lipid droplets accumulated in the hepatocytes of HFD group mice, and the lipid deposition was obvious; 9-37 / Fc and 7-37 / Fc intervention significantly reduced the content of red lipid droplets in hepatocytes (Figure 17G). The percentage of accumulated area of red lipid droplets in the liver tissue of each group of mice was quantitatively calculated and compared by Image-Pro Plus 6.0 software, and the results showed that 9-37 / Fc and 7-37 / Fc could significantly reduce the lipid content in the liver of high-fat mice (Figure 17I).

[0646] 9-37 / Fc increases the activities of antioxidant enzymes GSH-Px and SOD in the liver of high-fat diet mice and improves oxidative stress

[0647] Malondialdehyde (MDA) is a lipid peroxidation product formed by the reaction of oxygen free radicals and lipids in the body. The content of MDA represents the degree of lipid peroxidation and is an important marker of cell damage caused by oxidative stress. In order to detect whether 7-37 / Fc and 9-37 / Fc reduce the oxidative stress response in the liver of high-fat diet mice, the content of MDA in the liver tissue of each treatment group was detected by chemical colorimetry. The results showed that there was only a small amount of MDA in the NCD group, the content of MDA in the liver tissue of HFD group mice increased significantly, and the content of MDA in 9-37+HFD group and 7-37+HFD group mice decreased significantly, which had statistical significance (Figure 18A). The level of oxidative stress in liver tissue was further detected by DCFH-DA (2,7-Dichlorodihydrofluorescein diacetate, 2,7-dichlorofluorescein diacetate) fluorescent probe. Intracellular reactive oxygen species generate fluorescent DCF from DCFH-DA, and detecting the fluorescence intensity of DCF can reflect the level of reactive oxygen species. The results showed that the DCF fluorescence intensity of 7-37 / Fc and 9-37 / Fc treatment groups was significantly lower than that of HFD group (Figures 18D, E). The above results suggest that the oxidative stress in the liver of mice in 7-37 / Fc and 9-37 / Fc treatment groups is reduced.

[0648] GSH-Px (Glutathione peroxidase) and SOD (superoxide dismutase) are two important antioxidant metalloenzymes in vivo, which can prevent cells from oxidative damage. The results showed that compared with NCD mice, the GSH-Px and SOD enzyme activities in the liver of HFD group mice were significantly decreased, while the GSH-Px enzyme activity in the liver of 9-37+HFD group and 7-37+HFD group mice was significantly increased, and the SOD enzyme activity in the liver of 9-37+HFD group mice was also significantly increased (Fig. 18B, C). This indicates that the improvement of 7-37 / Fc and 9-37 / Fc in oxidative stress of HFD mice liver may be due to the increase of antioxidant enzymes.

[0649] 9-37 / Fc reduces the levels of inflammatory factors such as IL-6 and TNF-α in serum and liver of high-fat diet mice

[0650] Western blot results showed that compared with NCD group mice, high-fat diet for 12 weeks significantly increased the protein and gene levels of inflammatory factors IL-6 and TNF-α in the liver of mice, while the protein and gene levels of IL-6 and TNF-α in the liver of mice after 7-37 / Fc and 9-37 / Fc intervention for 12 weeks were significantly reduced, with statistical significance (Fig. 19A-D). The results of serum IL-6 and TNF-α levels and liver in each group were consistent (Fig. 19E, F). The above results suggest that 7-37 / Fc and 9-37 / Fc inhibit the activation of inflammatory response in the liver of high-fat diet induced mice and the systemic inflammatory response.

[0651] 3.2.4. 9-37 / Fc treatment effect on high-fat diet induced fatty liver in mice

[0652] 9-37 / Fc does not affect the body weight of high-fat diet induced mice

[0653] After 12 weeks of normal diet or high-fat diet, the mice in the high-fat diet group were randomly divided into 3 groups and received intraperitoneal injection of normal saline or 7-37 / Fc or 9-37 / Fc fusion protein, respectively. The body weight and food intake of mice in each group were monitored every week. The results showed that the body weight of mice in the HFD group was significantly increased compared with that in the NCD group from the 6th week until the end of the experiment. After 2 weeks of intervention, the body weight of mice receiving 7-37 / Fc injection was significantly lower than that of the HFD group and the HFD+9-37 / Fc group until the end of the experiment. The body weight of mice receiving 9-37 / Fc injection for 4 weeks was consistent with that of the HFD group (Figure 20A). The food intake of mice after 16 weeks of high-fat diet (before administration of 7-37 / Fc or 9-37 / Fc) was taken as 100%, and the change (%) in food intake of mice after 4 weeks of administration relative to that before administration was calculated. During the 4-week administration of fusion proteins, 7-37 / Fc significantly reduced the food intake of HFD mice, while 9-37 / Fc had no significant effect on food intake (Figure 20B).

[0654] 9-37 / Fc improves insulin resistance in high-fat diet-induced mice without significant effect on glucose tolerance

[0655] The mice in each group were subjected to IPGTT and IPITT at the 16th week, respectively. The results of IPGTT showed that the blood glucose values of mice in the 7-37+HFD group were significantly lower than those in the HFD group at each time point, and the blood glucose values of mice in the 9-37+HFD group were significantly lower than those in the HFD group at 15 and 30 minutes after sugar loading (Figure 21A). The results of area under the curve analysis showed that 7-37 / Fc improved glucose intolerance in high-fat diet mice, while 9-37 / Fc showed a trend of improving glucose intolerance in high-fat diet mice, but there was no statistical significance (Figure 21B). The results of IPITT showed that 4 weeks of injection of 7-37 / Fc and 9-37 / Fc significantly improved insulin resistance in high-fat diet mice, indicating that 7-37 / Fc and 9-37 / Fc improved the sensitivity of peripheral insulin response organs such as liver to insulin (Figures 21C, D).

[0656] 9-37 / Fc reduces blood lipid and transaminase levels in high-fat diet-induced mice

[0657] Results showed that compared with HFD group mice, serum TG, TC, LDL and NEFA levels were significantly reduced after 9-37 / Fc and 7-37 / Fc intervention for 4 weeks, suggesting that 9-37 / Fc and 7-37 / Fc can reduce the blood lipid levels of high-fat diet mice and improve systemic lipid metabolism disorder (Fig. 22A-D). At the same time, 9-37 / Fc and 7-37 / Fc can significantly reduce the level of ALT in serum, suggesting that 9-37 / Fc and 7-37 / Fc can improve the liver injury of high-fat diet induced mice (Fig. 22E). 9-37 / Fc more significantly reduced the AST level than 7-37 / Fc (Fig. 22F).

[0658] 9-37 / Fc reduces liver lipid accumulation in high-fat diet induced mice

[0659] Results showed that compared with HFD group mice, 7-37 / Fc can significantly reduce the weight of liver, eWAT, iWAT and muscle tissue, which has statistical significance. 9-37 / Fc can significantly reduce the weight of liver tissue, and has no obvious effect on the weight of other tissues (Fig. 23A). The liver index of each group of mice was calculated and compared, and the results showed that the liver index of 9-37+HFD group and 7-37+HFD group mice was significantly lower than HFD, and the liver index of 9-37+HFD group was lower than that of 7-37+HFD group mice (Fig. 23B).

[0660] After 16 weeks of intervention, the liver of the mouse was taken to observe the general morphological change. The liver of NCD group mice was reddish brown, the edge was sharp, and there was no greasy feeling; the liver volume of HFD group mice increased significantly, which was dark yellow, the edge was blunt, and the section was greasy; the color, texture and volume of the liver of 9-37+HFD group and 7-37+HFD group mice were between NCD and HFD groups (Fig. 23C). Further observation and comparison of the pathological changes of liver tissue of each group of mice were made by HE staining. The results showed that the liver lobule of NCD group mice was clear with complete structure, and no lipid droplets were found in the cytoplasm. A large number of vacuole-like lipid droplets were observed in the cytoplasm of hepatocytes of HFD group mice, and balloon-like degeneration was obvious with inflammatory cell infiltration. The lipid droplets, balloon-like degeneration and inflammatory cell infiltration in the cytoplasm of hepatocytes of 9-37+HFD group and 7-37+HFD group mice were significantly reduced (Fig. 23D). The NAS score of 9-37+HFD group and 7-37+HFD group mice was significantly lower than that of HFD group (Fig. 23H).

[0661] The lipid content in the liver was further determined. Compared with the NCD group of mice, the TG and TC contents in the liver of the HFD group of mice were significantly increased, while the 9-37 / Fc and 7-37 / Fc treatments could significantly reduce the TG and TC contents in the liver of the high-fat diet mice (Fig. 23E, F). The lipid deposition in the liver of the mice was further detected by oil red O staining. The results showed that a small amount of red lipid droplets existed in the hepatocytes of the NCD group of mice, while a large amount of red lipid droplets accumulated in the hepatocytes of the HFD group of mice; the 9-37 / Fc and 7-37 / Fc interventions significantly reduced the content of red lipid droplets in the hepatocytes (Fig. 23G). The percentage of the accumulated area of red lipid droplets in the liver tissue of each group of mice was calculated and compared, and the results showed that the 9-37 / Fc and 7-37 / Fc could significantly improve the lipid content in the liver of the high-fat mice (Fig. 23I).

[0662] 9-37 / Fc increased the antioxidant enzyme GSH-Px and SOD activities in the liver of high-fat diet mice and improved oxidative stress

[0663] The contents of lipid peroxidation product MDA, the activities of antioxidant enzymes GSH-Px and SOD in the liver tissue of each treatment group were detected by colorimetry. The results showed that the MDA content in the liver tissue of the HFD group of mice was significantly higher than that of the NCD group, while the 9-37 / Fc and 7-37 / Fc interventions significantly reduced the MDA content in the liver of the high-fat diet mice (Fig. 24A). Compared with the NCD mice, the GSH-Px and SOD enzyme activities in the liver of the HFD group of mice were significantly decreased, while the SOD enzyme activity in the 9-37+HFD group and the 7-37+HFD group of mice was significantly increased, and the GSH-Px enzyme activity in the 9-37+HFD group of mice was also significantly increased (Fig. 24B, C).

[0664] The level of oxidative stress in the liver tissue was further detected by DCFH-DA immunoprobe. The results showed that the DCF fluorescence intensity of the 7-37 / Fc and 9-37 / Fc treatment groups was significantly lower than that of the HFD group (Fig. 24D, E). The above results suggest that the oxidative stress in the liver of the mice in the 7-37 / Fc and 9-37 / Fc treatment groups is weakened, accompanied by an increase in the activities of the antioxidant enzymes GSH-Px and SOD.

[0665] 9-37 / Fc reduced the levels of inflammatory factors such as IL-6 and TNF-α in the serum and liver of high-fat diet mice

[0666] Western blot results showed that the protein and gene expression levels of inflammatory factors IL-6 and TNF-a in the liver of mice fed with high-fat diet for 16 weeks were significantly increased compared with NCD group mice, while the protein and gene expression levels of IL-6 and TNF-a in the liver of high-fat diet mice after 4 weeks of 9-37 / Fc intervention were significantly reduced. Although 7-37 / Fc reduced the gene expression of IL-6 and TNF-a in the liver of high-fat diet mice, no obvious change was found in the protein level (Fig. 25A-D). 7-37 / Fc and 9-37 / Fc reduced the levels of IL-6 and TNF-a in the serum of high-fat diet mice (Fig. 25E, F). The above results suggest that 7-37 / Fc and 9-37 / Fc inhibit the systemic inflammatory response induced by high-fat diet in mice, and 9-37 / Fc also inhibits the activation of liver inflammatory response induced by high-fat diet.

[0667] 3.3. Summary

[0668] GLP-1(9-37) plays an important regulatory role in hepatic glucose and lipid metabolism. GLP-1(9-37) inhibits hepatic gluconeogenesis; improves liver steatosis, oxidative stress and inflammation without affecting body weight and glucose tolerance in high-fat-fed mice, and reduces liver enzymes and blood lipid levels, thereby playing a liver-protecting role in both prevention and treatment. GLP-1(9-37) but not GLP-1(7-37) improves palmitic acid-induced lipid accumulation in HepG2 cells, demonstrating the direct regulatory effect of GLP-1(9-37) on hepatic lipid metabolism, and suggesting that the direct biological effect of GLP-1 in the liver is mainly exerted through GLP-1(9-37).

[0669] Example 4. Mechanism study of ACSL1-mediated 9-37 / Fc improving hepatic glucose and lipid metabolism disorders

[0670] In the study of Example 3, it was confirmed that GLP-1(9-37) can improve liver steatosis in high-fat-fed mice. Moreover, in vitro, GLP-1(9-37) also improved palmitic acid-induced lipid accumulation in HepG2 cells, demonstrating the direct regulatory effect of GLP-1(9-37) on hepatic lipid metabolism. Therefore, the specific mechanism of how GLP-1(9-37) regulates lipid metabolism disorders in liver cells needs further study.

[0671] It is proposed that GLP-1(9-37) binds to a new receptor on the cell membrane surface, inhibits the ERK signaling pathway, on the one hand, inhibits the expression of lipid synthesis-related genes such as SREBP-1, and on the other hand, promotes the expression of lipid decomposition-related genes such as PPARa, thereby improving liver lipid metabolism disorders. To verify this hypothesis, the liver cell membrane proteins are first isolated, and 9-37 / Fc is used for pull-down experiments, and mass spectrometry is used to find possible receptors of 9-37 / Fc. To verify the specificity of the receptor, after knocking down or overexpressing the receptor in HepG2 cells, ligand-receptor binding experiments are performed to detect whether the binding force of 9-37 / Fc to HepG2 cells changes. Then construct a palmitic acid-induced HepG2 cell high-fat model, and explore the regulatory effect and mechanism of 9-37 / Fc on liver cell lipid metabolism through the receptor at the in vitro level. The experimental tests include oil red staining, triglyceride content, p-ERK expression, and changes in lipid synthesis and decomposition-related genes. At the same time, the regulatory effect of 9-37 / Fc on the above signaling pathways is verified in the animal experiment of Example 3.

[0672] 4.1. Experimental method

[0673] 4.1.1 Sucrose density gradient centrifugation to separate liver cell membrane proteins

[0674] Prepare solutions

[0675] (1) Tissue lysis solution (8.3% w / w)

[0676] 0.25 mol / L sucrose, 10 mM Tris, 1 mmol / L MgCl2, 1X protease inhibitor, pH 7.5.

[0677] (2) 2 mol / L sucrose solution (55% w / w)

[0678] 2 mol / L sucrose, 10 mM Tris, 1 mmol / L MgCl2, 1X protease inhibitor, pH 7.5.

[0679] (3) 1.42 mol / L sucrose solution (41% w / w)

[0680] 1.42 mol / L sucrose, 10 mM Tris, 1 mmol / L MgCl2, 1X protease inhibitor, pH 7.5.

[0681] Operation steps

[0682] (1) The rats were fasted overnight and then anesthetized with an intraperitoneal injection of 10% chloral hydrate. The blood was collected by removing the eyeball and the rat was perfused with 0.9% saline through the heart. The liver was removed immediately after perfusion. The liver was rinsed three times with 4°C PBS buffer (pH = 7.4) to remove the blood completely, and then dried with filter paper and weighed.

[0683] (2) The tissue was cut into small pieces with ophthalmic scissors and then put into a dounce glass homogenizer. The tissue was homogenized with 5 times the volume of lysis buffer. The homogenate was ground 10 times at 4°C to make the tissue into a homogenate. The homogenate was adjusted to 20% (w / v) with lysis buffer and filtered through four layers of wet gauze. The homogenate was centrifuged at 280 x g for 5 min at 4°C. The supernatant was collected and the precipitate was resuspended with half the volume of the first lysis buffer and ground 3 times at 4°C. The precipitate was centrifuged at 280 x g for 5 min at 4°C. The supernatant was collected and centrifuged at 1500 x g for 15 min at 4°C. The supernatant was collected and used for total protein analysis of the tissue.

[0684] (3) The precipitate was resuspended with an appropriate amount of lysis buffer (about 8-9 mL for 25 g of liver). The sucrose concentration was adjusted to 1.42 mol / L by adding 2 mol / L sucrose solution. The total solution was added to 200 mL of suspension.

[0685] (4) In the ultracentrifuge tube, 35 mL of suspension was added to the lower layer, and 2 mL of lysis buffer was carefully added to the upper layer, taking care not to disrupt the layering between the two. The membrane layer between the 1.42 mol / L and 0.25 mol / L sucrose concentrations was carefully extracted with a 1 mL syringe.

[0686] (5) The membrane layer was resuspended with an appropriate amount of lysis buffer to change the sucrose solution density to 1.05 g / mL, and centrifuged at 1500 x g for 10 min at 4°C.

[0687] (6) The precipitate was resuspended in 4 mL of 1% Triton X-100 solution (containing 1% Triton X100, 10 mM Tris, 1 x protease inhibitor, pH 7.5), and after shaking to dissolve, centrifuged at 2,0000 x g for 1 h at 4°C. The supernatant was collected, diluted 5 times, and the supernatant was the membrane protein. After aliquoting, it was frozen at -80°C or subjected to Coomassie brilliant blue, silver staining and Western blot analysis.

[0688] 4.1.2 Coomassie Brilliant Blue Staining and Western Blot

[0689] The same as in Example 2, Section 2.1.2 and 2.1.3.

[0690] 4.1.3 Silver Staining SDS-PAGE Gel

[0691] Solution preparation

[0692] (1) Fixing solution: 5 mL acetic acid was added to 25 mL ethanol and 20 mL double distilled water, and mixed well.

[0693] (2) 30% ethanol: 15 mL ethanol was added to 35 mL double distilled water, and mixed well.

[0694] (3) 1x silver staining sensitizing solution: 0.5 mL silver staining sensitizing solution was added to 49.5 mL double distilled water, and mixed well, and used within 2 hours after preparation.

[0695] (4) 1x silver solution: 0.5 mL silver solution (100x) was added to 49.5 mL double distilled water, and mixed well, and used within 2 hours after preparation.

[0696] (5) 1x silver staining developing solution: 10 mL 5x silver staining basic developing solution was added to 40 mL double distilled water, and then 0.025 mL silver staining developing accelerating solution (2000x) was added, and mixed well, and used within 20 minutes.

[0697] (6) 1x silver staining termination solution: 2.5 mL silver staining termination solution (20x) was added to 47.5 mL double distilled water, and mixed well, and used on the same day.

[0698] Operation steps

[0699] (1) After the electrophoresis was completed, the gel was placed in 50 mL of fixing solution, and shaken at room temperature at a speed of 60 rpm for 40 min.

[0700] (2) The fixing solution was poured off, 50 mL of 30% ethanol was added, and shaken at room temperature at a speed of 60 rpm for 10 min.

[0701] (3) The 30% ethanol was poured off, 100 mL of double distilled water was added, and shaken at room temperature at a speed of 60 rpm for 10 min.

[0702] (4) The double distilled water was poured off, 100 mL of 1x silver staining sensitizing solution was added, and shaken at room temperature at a speed of 60 rpm for 2 min.

[0703] (5) The silver staining sensitizing solution was poured off, 100 mL of double distilled water was added, and shaken at room temperature at a speed of 60 rpm for 1 min, and repeated twice.

[0704] (6) The double distilled water was poured off, 50 mL of 1x silver solution was added, and shaken at room temperature at a speed of 60 rpm for 10 min.

[0705] (7) The silver solution was poured off, 50 mL of double distilled water was added, and shaken at room temperature at a speed of 60 rpm for 1-1.5 min.

[0706] (8) Discard the double-distilled water, add 50 mL silver staining developing solution, shake at 60 rpm for 3-10 min at room temperature until the expected protein bands appear.

[0707] (9) Discard the silver staining developing solution, add 50 mL lx silver staining stop solution, shake at 60 rpm for 10 min at room temperature.

[0708] (10) Discard the silver staining stop solution, add 50 mL double-distilled water, shake at 60 rpm for 2-5 min at room temperature.

[0709] (11) The silver staining gel can be stored in double-distilled water for 3-4 days and analyzed by mass spectrometry as soon as possible.

[0710] 4.1.4 Pull-down experiment and LC-MS / MS mass spectrometry analysis

[0711] Solution preparation

[0712] (1) The binding buffer is prepared as follows: add fresh 10% Nonidet P-40 solution each time, and store at 4°C:

[0713] (2) The 2x PBS buffer is prepared as follows:

[0714] Dissolve the above powder in 1 L double-distilled water, and store at 4°C for one month.

[0715] Fusion protein and agarose bead connection

[0716] (1) Take 100 μL agarose beads, add lx PBS. Centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant. Repeat twice. Finally, after the supernatant is aspirated, add 100 μL 0.1% BSA solution (dissolved in lx PBS) to prepare a 50% agarose bead suspension.

[0717] (2) Mix well at 4°C for 1 hour.

[0718] (3) Take out the ep tube containing the 50% agarose bead suspension, centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant.

[0719] (4) Add the binding buffer at a ratio of 1:1, mix well at 4°C for 10 min. Centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant.

[0720] (5) Dilute the 7-37 / Fc, 9-37 / Fc or IgG-Fc protein to 0.3 mg / mL with 1-2 mL of binding buffer.

[0721] (6) Add 970 μL of diluted protein to 30 μL of agarose beads and mix well at 4°C overnight.

[0722] (7) Remove the protein-agarose beads mixture and centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant.

[0723] (8) Wash the beads with binding buffer and centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant. Repeat 3 times. The binding buffer wash is to reduce non-specific protein binding.

[0724] (9) Resuspend the fusion protein-coated beads with an equal volume of binding buffer to make a 50% agarose beads-fusion protein suspension.

[0725] Membrane protein pretreatment

[0726] (1) Add 1 mL (about 300 μg) of membrane protein to the 30 μL agarose beads suspension from Step 5 above.

[0727] (2) Mix well at 4°C for 1 hour.

[0728] (3) Remove the membrane protein-agarose beads suspension and centrifuge at 14,000 x g for 10 min at 4°C.

[0729] (4) Discard the agarose beads and keep the supernatant for further use.

[0730] Co-immunoprecipitation

[0731] (1) Add 300 μg (1 mL) of pretreated membrane protein to 30 μL of fusion protein-coated agarose beads.

[0732] (2) Mix well at 4°C for 4 hours.

[0733] (3) Remove the mixture and centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant.

[0734] (4) Wash the beads with binding buffer and centrifuge at 500 x g for 5 min at room temperature. Discard the supernatant. Repeat 3 times.

[0735] (5) Add 100 μL of IgG elution buffer (pH = 2.8), centrifuge to collect the supernatant, and then immediately add 10 μL of 1 M Tris buffer (pH = 9.0) to adjust the pH to neutral.

[0736] (6) Perform Western blot or silver staining to detect the pulled-down proteins.

[0737] 4.1.5 Flow cytometry to detect intracellular ROS levels

[0738] The intracellular ROS level was detected by using fluorescent probe DCFH-DA. After entering the cell, the DCFH-DA probe was hydrolyzed by esterase to generate DCFH. In the presence of ROS, DCFH will be oxidized to DCF with green fluorescence. Therefore, the level of ROS can be represented by the fluorescence intensity of DCF.

[0739] (1) HepG2 cells were inoculated in a 6-well plate, and after overnight adhesion, different drug intervention treatments were given for 24 hours.

[0740] (2) The DCFH-DA mother liquor was diluted 1:1000 with serum-free DMEM medium, thoroughly mixed, and prepared into a DCFH-DA working solution with a final concentration of 10 μmol / L.

[0741] (3) The Blank group was not added with the DCFH-DA working solution and was not given any treatment, and the positive control group was intervened with Rosup (50 mg / mL) diluted 1:1000 with serum-free DMEM medium for 30 min.

[0742] (4) The 6-well plate was taken out, and the original culture solution was discarded. PBS was used to rinse twice, and 1 mL of DCFH-DA working solution was added to each well except for the Blank group.

[0743] (5) The culture plate was placed in a 37°C incubator for incubation for 60 min in the dark.

[0744] (6) The original DCFH-DA working solution was discarded, and the cells were washed with serum-free DMEM medium for 3 times to remove the probes that did not enter the cells.

[0745] (7) 1 mL of 0.25% trypsin was added to each well for 2 min of digestion, and 1 mL of HepG2 complete medium was added to terminate the digestion. The cells were blown apart, collected into a 1.5 mL EP tube, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded.

[0746] (8) The cells were resuspended with 1 mL of ice PBS, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded. Repeat twice. Finally, the cells were resuspended with 200 μL of ice PBS, and the sample was placed on ice for machine detection; the whole process was operated in the dark.

[0747] (9) The proportion of DCF positive cells to all cells was detected by flow cytometry.

[0748] 4.1.6 Endocytosis experiment

[0749] (1) Sterile 24-well plates were seeded in advance, and HepG2 cells were evenly seeded into the 24-well plates (density of 60%-70%).

[0750] (2) After the cells adhere to the plate, take out the plate and incubate in 4°C refrigerator for 30 min. Then take out and place on ice, and add 100 nM 7-37 / Fc or 9-37 / Fc, respectively, and incubate at 4°C for 4 h.

[0751] (3) Then take out and place on ice, and rinse twice with 4°C PBS. Two wells are directly fixed with 4% paraformaldehyde for 5 min. The other wells are added with 500 μL of preheated medium and incubated at 37°C for 5, 10, 15 or 30 min, respectively.

[0752] (4) After the intervention of each treatment group is completed, discard the medium, and wash each well with 1x PBS for 3 times.

[0753] (5) Fixation: add 500 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min (except for the wells directly fixed).

[0754] (6) Membrane breaking: after the fixation of the cells is completed, wash with PBS containing 1% glycine for 3 times, and then add 500 μL of PBS containing 0.1% Triton X-100 to each well, and place at room temperature for 15 min (4°C directly fixed wells are not done)

[0755] (7) Remove the 0.1% Triton X-100 PBS solution, and wash with PBS for 3 times.

[0756] (8) Blocking: take out the slides, and add 500 μL of 10% goat serum diluted with PBS to each well, and block at room temperature for 30 min.

[0757] (9) Incubation of primary antibody: first, attach a layer of sealing film on the slide, then add 50 μL of 1% BSA diluted primary antibody to the sealing film, take out the slide, and place it upside down on the slide, and then place it in a wet box. Incubate at 4°C overnight.

[0758] (10) Take the slide back to the well plate, recover the primary antibody, and freeze in -20°C refrigerator.

[0759] (11) Incubation of secondary antibody: wash each well with 1x PBS for 3-4 times, 10 min each time. Take out the slide and place it on the slide, and add 50 μL of secondary antibody (of the same species as the primary antibody) diluted with 1% goat serum to each slide (also upside down, same as the primary antibody). Incubate at room temperature for 1 hour in the dark.

[0760] (12) Nuclear staining: wash with PBS for 4 times, 15 min each time; add 100 μL of PBS buffer containing 100 ng / mL DAPI, and incubate at room temperature for 8 min in the dark.

[0761] (13) Mounting: PBS wash 3 times, 5 min each. Remove the slide and drop 10 μΐ of antifluorescence quencher on the coverslip. Then gently invert the slide onto the coverslip to avoid air bubbles. Place in the dark at room temperature overnight, take a picture immediately or store at 4°C in the dark for long-term storage.

[0762] (14) Confocal microscope imaging.

[0763] 4.1.7 Cell immunofluorescence

[0764] Refer to Example 3.

[0765] 4.1.8 Cell RNA extraction

[0766] (1) Cells were seeded in 12-well plates and incubated overnight. Different drugs were added to the cells and incubated overnight.

[0767] (2) Discard the old medium and wash the cells with PBS twice. Discard the PBS.

[0768] (3) Add 500 μΐ of TRIzol reagent pre-cooled at 4°C to each well and incubate on ice for 10 min to lyse the cells.

[0769] (3) After mixing the cells with a pipette several times, transfer the liquid to a 1.5 mL EP tube without RNase.

[0770] (4) Add 100 μΐ of chloroform to each tube and mix well on a vortex instrument.

[0771] (5) Centrifuge at 12,000 rpm for 15 min at 4°C.

[0772] (6) The liquid is divided into three layers. Carefully use a pipette to transfer 200 μΐ of the upper clear solution to a new 1.5 mL EP tube without RNase. Do not take other layers of liquid.

[0773] (7) Add 200 μΐ of isopropanol and mix well. Then place on ice for 30 min.

[0774] (8) Centrifuge at 2,000 rpm for 10 min at 4°C.

[0775] (9) Discard the supernatant and add 1 mL of 75% ethanol prepared with DEPC water to each tube. Mix well and centrifuge at 7,500 rpm for 5 min at 4°C. Discard the supernatant. Repeat twice.

[0776] (10) Open the tube cap and dry at room temperature for 10 min until the precipitate at the bottom of the tube becomes transparent.

[0777] (11) Add 20-30 μΐ of DEPC water to each tube to dissolve the precipitate.

[0778] (12) Take 1 μL RNA to measure the RNA sample concentration and purity with NanoDrop 2000 spectrophotometer. Before detection, use DEPC water to adjust the zero of the spectrophotometer. Wipe the detection point with paper before and after each sample detection. Read the OD260 / 280 and OD260 / 230 ratio. The OD260 / 280 ratio is 1.8-2.0, and the OD260 / 230 ratio is greater than 2.0.

[0779] 4.1.9 Reverse transcription reaction (RT-PCR) and fluorescence quantitative PCR experiment (qPCR)

[0780] The same as the corresponding part of Example 3.

[0781] 4.1.10 Cell protein extraction

[0782] (1) After the end of cell intervention, discard the original culture medium, and add PBS to each well of the 12-well plate for 2 times of washing, and then aspirate the PBS.

[0783] (2) Add 100 μL of RIPA lysis solution containing protease inhibitors and phosphatase inhibitors to each well, and place it on a 4°C shaking table for 30 min of complete lysis.

[0784] (3) After blowing the cells several times with a pipette, transfer the liquid to a 1.5 mL EP tube, centrifuge at 4°C, 12000 rpm for 30 min.

[0785] (4) Aspirate the supernatant into a new EP tube, and perform BCA protein concentration determination. Adjust the protein concentration of each group to be the same. Then add 5X protein loading buffer according to the volume of 4:1, vortex well, and then centrifuge. Heat at 100°C metal bath for 10 min, and then immediately insert into ice. Store in a -20°C refrigerator for standby.

[0786] 4.1.11 Tissue RNA and protein extraction

[0787] The same as the corresponding part of Example 2.

[0788] 4.1.12 ATP content determination

[0789] (1) Prepare cell samples: aspirate the culture solution, and add 200 μL lysis solution to each well of the 6-well plate on ice to lyse the cells. In order to release ATP completely, take 100 μL lysis solution and heat it in a 100°C metal bath for 2 min.

[0790] (2) After lysis or boiling, centrifuge at 4°C, 12000 rpm for 5 min. Take the supernatant for subsequent determination.

[0791] (3) Preparation of standard curve: ATP standard solution (0.5 mM) was diluted with ATP detection lysate to a series of concentration gradients: 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM.

[0792] (4) Preparation of ATP detection working solution: ATP detection reagent was diluted with ATP detection diluent at a ratio of 1:9 to prepare ATP detection working solution for subsequent experiments. ATP detection working solution can be temporarily stored on ice bath.

[0793] (5) Determination of ATP concentration: 100 μL of ATP detection working solution was first added to each well of a white 96-well plate. After 3-5 min at room temperature, the background ATP was completely consumed, thereby reducing the background. Then 20 μL of sample or standard was added to each well on ice, and the mixture was quickly mixed with a pipette, taking care not to have air bubbles.

[0794] (6) The SpectraMax i3x microplate reader was set to chemiluminescence detection (LUM) to read the RLU value of each well.

[0795] 4.1.13 siRNA transfection

[0796] (1) siRNA was purchased from Shanghai Tuoren Biotechnology Co., Ltd., and the sequences of the three primers were as follows:

[0797] (2) After receiving the siRNA powder, centrifuge at 12000 rpm for 5 min at room temperature, gently open the tube cap, add 125 μL of Rnase-free H2O to prepare a 20 μM siRNA stock solution, and store at -80°C after aliquoting.

[0798] (3) Cell transfection (using a 24-well plate as an example):

[0799] (4) HepG2 cells in exponential growth phase were inoculated in a 24-well plate, and transfection was performed when the cell confluence was about 30%-40%.

[0800] (5) Dilute ACSL1 siRNA or Scrambled siRNA with 50 μL of Opti-MEM, mix gently with a pipette, and stand at room temperature for 5 min.

[0801] (6) Dilute 1 μL of Lipofectamine 3000 with 50 μL of Opti-MEM, mix gently with a pipette, and stand at room temperature for 5 min.

[0802] (7) Mix the Opti-MEM containing the transfection reagent with the Opti-MEM containing the siRNA to form a transfection complex, and stand at room temperature for 20 min.

[0803] (8) Within 20 min of waiting, aspirate the original culture medium, rinse the cells twice with PBS, and then add 400 μL Opti-MEM to each well and place it in a 37°C incubator.

[0804] (9) Take out the culture plate, add 100 μL of transfection complex to each well, gently shake the culture plate back and forth, and place it in a 37°C incubator.

[0805] (10) After 6 hours of transfection, aspirate the original culture medium, add 500 μL of HepG2 complete culture medium to each well, and place it in a 37°C incubator for 24 hours before proceeding with the subsequent cell sample processing.

[0806] 4.1.14 Transfection of overexpression plasmid

[0807] (1) The ACSL1 overexpression plasmid was purchased from Shanghai Jikai Gene Medical Technology Co., Ltd. The vector information is shown in Figure 35, and the target gene is as follows:

[0808] (2) Cell transfection (take a 24-well plate as an example):

[0809] (3) Exponentially growing HepG2 cells were seeded in a 24-well plate, and transfection was performed when the cell confluence was about 70%-80%.

[0810] (4) Dilute the ACSL1 overexpression plasmid or empty control plasmid containing GFP with 50 μL Opti-MEM, mix gently with a pipette, and incubate at room temperature for 5 min. Then add HighGene transfection reagent according to the ratio of plasmid (μg) : transfection reagent (μL) 1:3, mix by blowing, and incubate at room temperature for 15 min.

[0811] (5) Within 15 min of waiting, aspirate the original culture medium, rinse the cells twice with PBS, and then add 450 μL of fresh HepG2 complete culture medium to each well and place it in a 37°C incubator.

[0812] (6) Take out the culture plate, add 50 μL of plasmid / HighGene transfection reagent complex to each well of the 24-well cell culture plate, and gently shake the cell culture plate to distribute it evenly.

[0813] (7) After 6 hours of transfection, discard half of the original complete culture medium, add half of the fresh complete culture medium, and place it in a 37°C incubator for 24 hours before proceeding with the subsequent cell sample processing.

[0814] 4.1.15 Cell oil red staining and triglyceride determination

[0815] The same as the corresponding part of Example 3.

[0816] 4.1.16 Ligand-receptor binding assay based on cell ELISA

[0817] The corresponding part of Example 2.

[0818] 4.1.17 Statistical analysis

[0819] The corresponding part of Example 2.

[0820] 4.2. Experimental results

[0821] 4.2.1. 9-37 / Fc inhibits the expression of genes related to lipid synthesis in liver and promotes the expression of genes related to lipid decomposition

[0822] Insufficient fatty acid β-oxidation and excessive lipid synthesis in the liver cause a large accumulation of lipids in hepatocytes, and GLP-1(9-37) can alleviate fatty liver in high-fat diet-fed mice and lipid accumulation in palmitate-induced hepatocytes. Therefore, in the study of this embodiment, first, the effect of GLP-1(9-37) on fatty acid β-oxidation genes and lipid synthesis genes in palmitate-induced HepG2 cells and high-fat diet-fed mice was observed.

[0823] SREBP-1 is an important transcription factor for lipid synthesis in the liver, and CPT1 and PPARα are key rate-limiting enzymes for fatty acid β-oxidation. Western blot results showed that the expression of SREBP-1 protein in the PA treatment group was increased, and 9-37 / Fc could dose-dependently reduce the expression of SREBP-1 (Figure 26A). qRT-PCR results showed that 9-37 / Fc inhibited the expression of lipid synthesis-related genes SREBP-1, ACC, and FAS, and promoted the expression of β-oxidation-related genes ACOX1, PPARα, and CPT1 (Figures 26B-C). 7-37 / Fc had no significant effect on the expression of the above genes in PA-induced HepG2 cells (Figures 26B-C).

[0824] Mitochondrial dysfunction exists in NAFLD, mainly manifested as decreased beta oxidation, increased ROS production, and reduced ATP content. If the mitochondrial function is impaired and cannot produce its own oxidation, it will further exacerbate the accumulation of lipotoxic products, promote liver insulin resistance and lipid synthesis, and is related to the progression of NASH. Since 9-37 / Fc can increase the expression of beta oxidation-related genes and inhibit the expression of lipid synthesis-related genes, the content of intracellular ROS and ATP was further detected. The content of ROS in HepG2 cells was detected by flow cytometry through a fluorescent probe DCFH-DA. The results showed that 9-37 / Fc can significantly reduce the increase in the proportion of DCFH-DA positive cells in the intracellular PA induced cells (Figure 26H), suggesting that 9-37 / Fc can inhibit the production of ROS in HepG2 cells. At the same time, 9-37 / Fc also improved the PA-induced decrease in ATP content (Figure 261).

[0825] Consistent with the cell results, the expression of SREBP-1 in the liver of mice fed with high-fat diet was increased, while the expression of CPT1 and PPARa was significantly reduced. Both 12 weeks of prophylactic intervention and 4 weeks of therapeutic intervention with 9-37 / Fc can significantly reduce the expression of SREBP-1 and up-regulate the expression of CPT1 and PPARa (Figures 26D, E). 9-37 / Fc also reduces the expression of other lipid synthesis-related genes such as SCD-1 and promotes the expression of beta oxidation-related genes such as P4a14 (Figures 26F, G). 7-37 / Fc can also reduce the expression of SREBP-1 in the liver of mice fed with high-fat diet (Figures 26D-G).

[0826] 4.2.2. 9-37 / Fc inhibits PA and high-fat diet-induced expression of p-ERK

[0827] It was found that phosphorylated ERK can inhibit the expression of PPARa and CPT1, while promoting the expression of SREBP-1. Although the downstream signaling pathway caused by GLP-1(9-37) is not clear at present, some studies have found that GLP-1(9-37) can cause changes in the phosphorylation level of ERK. Therefore, first at the cellular level, HepG2 cells were co-incubated with 300 μM PA and different concentrations of 9-37 / Fc or 7-37 / Fc for 24 hours, and the changes in p-ERK were detected by Western blot. The results showed that PA significantly promoted the expression of p-ERK, and 9-37 / Fc can dose-dependently inhibit the expression of p-ERK. 7-37 / Fc had no obvious change on the expression of p-ERK. At the same time, the changes of the classical GLP-1R downstream signaling pathway PKA were also detected, and the results showed that 9-37 / Fc had no obvious change on the expression of p-PKA (Figure 27A).

[0828] Consistent with the cellular results, in the livers of mice fed with high fat diet, 9-37 / Fc significantly inhibited the HFD-induced ERK activation, while 7-37 / Fc had no significant effect on the ERK activation (Fig. 27B, C).

[0829] 4.2.3. Extraction and identification of hepatocyte membrane proteins

[0830] Previous results showed that GLP-1 (9-37) could inhibit the expression of p-ERK. Moreover, GLP-1 (9-37) was not dependent on the binding of GLP-1R to primary hepatocytes, suggesting that GLP-1 (9-37) might bind to unknown receptors other than GLP-1R on the surface of hepatocyte membranes, causing changes in the downstream p-ERK signaling pathway. Therefore, we first used sucrose density gradient centrifugation to separate rat liver cytoplasmic membrane proteins to identify the potential receptors of GLP-1 (9-37).

[0831] SDS-PAGE silver staining and Coomassie blue staining results showed that the molecular weight of total liver protein (Lysate) was mainly below 70 kD, while the molecular weight of extracted liver cytoplasmic membrane protein (PM) was mainly above 55 kD, and there was a big difference in the distribution position between the two (Fig. 28A). Western blot detection with cell membrane protein marker antibody (Na + / K + ATPase) and cytoplasmic protein marker antibody (GAPDH) showed that there was a large amount of GAPDH protein in Lysate, while the content of Na + / K + ATPase protein was low. There was a large amount of cell membrane protein Na + / K + ATPase in PM, and it did not contain GAPDH (Fig. 28B). The above results suggest that the separation of liver cytoplasmic membrane protein was successful.

[0832] 4.2.4. Mass spectrometry identification of potential receptors of GLP-1 (9-37)

[0833] Pull-down experiment was performed with 9-37 / Fc to find proteins interacting with 9-37 / Fc. 9-37 / Fc, 7-37 / Fc and IgG-Fc (as control) were mixed with protein A agarose beads, based on the high affinity of protein A to IgG, the fusion proteins were pre-bound on protein A agarose beads. Then cell cytosol membrane proteins were added and pull-down experiment was performed. After washing away unbound proteins, the bound proteins were eluted with acidic buffer. The result of silver staining showed that there were specific bands between 70kD-90kD and 40-55kD for proteins binding with 9-37 / Fc (Figure 29A). Then the protein bands shown in white box in Figure 29A were cut and mass spectrometry was performed (Figure 29B).

[0834] Based on the mass spectrometry data, amino acid polypeptide sequences were aligned and a series of proteins that might bind with 9-37 / Fc were found. The top 10 proteins were listed in descending order of Sum PEP score (the possibility of the peptide fragment produced by mass spectrometry is the protein) (Figure 29B). Further use of AlphaFold2 three-dimensional structure simulation tool found that some of the proteins had high structural fit with 9-37 / Fc. Generally, the distance between amino acid residues of interacting proteins with each other would not exceed 5A Therefore, according to whether the amino acid residues within the distance of each other The proteins were judged whether they had structural fit with 9-37 / Fc according to whether they contained amino acid residues within the distance of each other and visualized by chimera software. The results showed that the 316th, 320th, 324th, 409th and 488th amino acids of ACSL1 (Long-chain-fatty-acid—CoA ligase 1, also known as long-chain acyl-CoA synthetase, molecular weight: 78.1kD) (dark blue in Figure 29C) contacted with 9-37 / Fc (rainbow fragment in Figure 29C) to form hydrogen bonds (rainbow residues in Figure 29C).

[0835] By literature research, ACSL1 is a membrane protein with N-terminal transmembrane helix, located on the membrane of adipocytes, with the activity of catalyzing free fatty acid to combine with CoA to generate fatty acyl-CoA (see WEIMAR J D, DIRUSSOC C, DELIO R, et al., The Journal of biological chemistry, 2002, 277(33): 29369-76.). ACSL1 is a key enzyme of fatty acid metabolism, involved in regulating the synthesis and decomposition of liver lipids. In view of the previous finding of the inventors that 9-37 / Fc can regulate the expression of genes related to the synthesis and decomposition of liver lipids, the inventors speculate that ACSL1 may be a potential receptor of GLP-1(9-37), mediating the endocytosis of GLP-1(9-37), and further verification is needed.

[0836] 4.2.5.ACSL1 is located on the membrane of HepG2 cells

[0837] Previous literature reported that ACSL1 is located on the membrane of 3T3-L1 adipocytes (Id.), however, the localization of ACSL1 in hepatocytes is still unclear. The inventors used anti-ACSL1 antibody to label ACSL1 (red) by immunofluorescence staining. The results showed that in HepG2 cells without membrane disruption treatment with Triton X-100, ACSL1 showed red punctate structure along the edge of the cell periphery, with the characteristics of surface staining of antigens (Fig. 30A-C). This staining is specific, because no fluorescent staining distribution was observed in the treatment group using IgG control (Fig. 30D) or adding red fluorescent secondary antibody alone (Fig. 30E) as a control. At the same time, in cells treated with Triton X-100 to disrupt the membrane, ACSL1 staining showed uniform punctate distribution throughout the cell, with a different morphological distribution from that without membrane disruption, further confirming the characteristics of membrane protein staining (Fig. 30F). The above results suggest that ACSL1 is located on the membrane of HepG2 cells.

[0838] 4.2.6. 9-37 / Fc but not 7-37 / Fc binds to ACSL1 on the membrane of hepatocytes and undergoes endocytosis

[0839] Ligand internalization is a receptor-mediated endocytosis process, in which the ligand specifically binds to the cell membrane surface receptor to form a complex, then part of the plasma membrane is invaginated to form a caveolus, the caveolus is separated from the plasma membrane to form a caveosome, and the complex is endocytosed into the cell. The inventors' previous results showed that ACSL1 is located on the HepG2 cell membrane and is distributed in both the cell membrane and the cytoplasm. The inventors speculate that if ACSL1 is the receptor of GLP-1(9-37), then 9-37 / Fc can bind to ACSL1 and cause endocytosis. To verify this hypothesis, first, 100 nM 9-37 / Fc or 7-37 / Fc was incubated with HepG2 cells at 4°C for 4 h, and then switched to 37°C for 5, 15, 30 min, respectively, and stained with fluorescently labeled human IgG antibody (hIgG, green). The results showed that 9-37 / Fc was located on the HepG2 cell membrane at 4°C, while 7-37 / Fc did not show significant binding to HepG2 cells. 9-37 / Fc was rapidly internalized after incubation at 37°C for 30 min, and obvious hIgG staining was observed in the cytoplasm, which was distributed along the perinuclear region (Figure 31A). The results of immunofluorescence co-staining with ACSL1 antibody (red) showed that 9-37 / Fc co-localized with ACSL1 in the cell membrane and cytoplasm, suggesting that ACSL1 mediated the endocytosis of 9-37 / Fc (Figure 31B). The above results suggest that 9-37 / Fc but not 7-37 / Fc binds to ACSL1 on the liver cell membrane and undergoes endocytosis.

[0840] To further investigate whether 9-37 / Fc binds to ACSL1, the affinity of 9-37 / Fc to HepG2 cells was detected after transfecting HepG2 cells with siRNA and plasmid to knock down or overexpress ACSL1, respectively, for 48 hours. Western blot results showed that 100 nM siRNA had the best knockdown efficiency (Figure 31C), therefore, 100 nM siRNA was selected for subsequent experiments. At the same time, it was confirmed that the dosage of ACSL1 overexpression plasmid (ACSL1-OE) used was 0.3 μg, as subsequent studies (Figure 31D). Ligand-receptor binding experiments based on cell ELISA showed that after overexpression of ACSL1, the affinity of 9-37 / Fc to HepG2 cells increased significantly, with an affinity constant (Kd) about 10 times that of the unoverexpressed (3.6 nM vs. 37.1 nM, P<0.05), and the maximum specific binding coefficient (Bmax) also increased significantly (P<0.001). After knocking down ACSL1, the affinity of 9-37 / Fc to HepG2 cells decreased significantly (P<0.05), and the Bmax also decreased significantly (P<0.001). The above results suggest that 9-37 / Fc binds to ACSL1.

[0841] 4.2.7. Knockdown of ACSL1 attenuates the inhibitory effect of 9-37 / Fc on PA-induced lipid accumulation in HepG2 cells

[0842] To investigate whether the improvement of 9-37 / Fc on PA-induced lipid accumulation in HepG2 cells is dependent on ACSL1, siRNA was used to knock down ACSL1 in HepG2 cells, and oil red O staining and triglyceride content determination were performed after incubation with PA alone or in the presence of 9-37 / Fc for 24 hours. The results of oil red staining showed that 9-37 / Fc could reduce PA-induced lipid deposition, and this effect disappeared after knockdown of ACSL1 (Figure 32A). The results of triglyceride also showed that knockdown of ACSL1 significantly inhibited the effect of 9-37 / Fc on improving lipid accumulation (Figure 32B). The above results suggest that ACSL1 plays an important role in the improvement of 9-37 / Fc on liver lipid accumulation.

[0843] 4.2.8. Overexpression of ACSL1 enhances the effect of 9-37 / Fc on reducing PA-induced lipid deposition

[0844] After further overexpression of ACSL1, the effect of 9-37 / Fc on PA-induced liver lipid deposition was detected. The results of oil red staining showed that although the oil red particles in the PA+ACSL1-OE group increased compared with the PA+vector group, the proportion of oil red particle decrease in the PA+ACSL1-OE+9-37 / Fc group and the PA+ACSL1-OE group was greater than that in the PA+vector+9-37 / Fc group and the PA+vector group (Figure 33A). The results of triglyceride were consistent with the results of oil red staining (Figure 33B). The above results show that the effect of 9-37 / Fc on improving PA-induced liver lipid deposition is ACSL1-dependent.

[0845] 4.2.9. 9-37 / Fc inhibits ERK phosphorylation and lipid synthesis in HepG2 cells through ACSL1 and promotes β-oxidation

[0846] It was found that fatty acyl-CoA can bind to nucleoside diphosphate kinase (NME) and inhibit its activity. NME can phosphorylate the 392nd site of KSR (kinase suppressor of RAS, RAS kinase inhibitor), thereby promoting the assembly of Raf / MEK / ERK complex and the activation of ERK signaling pathway. And knockdown of NME on HepG2 cells can significantly activate the ERK signaling pathway.

[0847] To further investigate the role of ACSL1 in the inhibition of ERK phosphorylation and regulation of lipid synthesis and decomposition by 9-37 / Fc in HepG2 cells, after knocking down or overexpressing ACSL1 in HepG2 cells, PA alone or co-incubated with 9-37 / Fc was given for 24 hours, and the expression of ERK phosphorylation, SREBP-1 and PPARa was detected. Since ACSL1 itself has a role in regulating lipid metabolism, a group of simple knockdown or overexpression of ACSL1 without 9-37 / Fc was designed as a control. The results showed that 9-37 / Fc inhibited the expression of p-ERK induced by PA, inhibited the expression of SREBP-1 and promoted the expression of PPARa, and the above effects disappeared after knocking down ACSL1 (Figure 34A). Similarly, compared with the decrease in p-ERK and SREBP1 in the PA+vector+9-37 / Fc group and the PA+vector group, the decrease in p-ERK and SREBP1 in the PA+ACSL1-OE+9-37 / Fc group and the PA+ACSL1-OE group was greater, and the increase in PPARa was higher (Figure 34B). The above results suggest that the inhibition of ERK phosphorylation and lipid synthesis and the promotion of beta oxidation by 9-37 / Fc in HepG2 cells are ACSL1-dependent.

[0848] 4.3. Summary

[0849] GLP-1(9-37) promotes the expression of lipid decomposition genes PPARa and CPT1 and inhibits the expression of lipid synthesis gene SREBP-1 by inhibiting the downstream p-ERK signaling pathway through endocytosis and intracellular co-localization with hepatocyte membrane ACSL1, thereby improving liver lipid accumulation.

Claims

1. A fusion protein comprising a GLP-1 polypeptide and an immunoglobulin Fc domain, wherein, The GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain, wherein the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, and the GLP-1 polypeptide comprises a G22E and / or a R36G substitution relative to a native human GLP-1 polypeptide.

2. The fusion protein of claim 1, wherein, The GLP-1 polypeptide has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and comprises one or more amino acid substitutions relative to a native human GLP-1 polypeptide selected from the group consisting of G22E and R36G.

3. The fusion protein of claim 2, wherein, The GLP-1 polypeptide comprises a G22E and a R36G substitution relative to a native human GLP-1 polypeptide.

4. The fusion protein of claim 3, wherein, The GLP-1 polypeptide is human GLP-1 (9-37), and comprises a G22E and a R36G substitution relative to a native human GLP-1 polypeptide.

5. The fusion protein according to any one of the preceding claims, wherein, The GLP-1 polypeptide has the amino acid sequence set forth in SEQ ID NO:

3.

6. The fusion protein according to any one of the preceding claims, wherein, The immunoglobulin Fc domain comprises or is an IgG2-Fc domain.

7. The fusion protein of claim 6, wherein, The IgG2-Fc domain is an Fc domain from human IgG2.

8. The fusion protein according to claim 6 or 7, wherein, The IgG2-Fc domain comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

9. The fusion protein according to any one of claims 6-8, wherein, The IgG2-Fc domain has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and comprises one or more amino acid substitutions selected from the group consisting of C222S, A330S, and P331S.

10. The fusion protein according to any one of claims 6-9, wherein, The IgG2-Fc domain has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 6, and comprises A330S and P331S substitutions.

11. The fusion protein of claim 10, wherein, The IgG2-Fc domain further comprises a C222S substitution.

12. The fusion protein of claim 11, wherein, The IgG2-Fc domain has the amino acid sequence set forth in SEQ ID NO:

6.

13. The fusion protein according to any one of the preceding claims, wherein, The GLP-1 polypeptide has the amino acid sequence set forth in SEQ ID NO: 3, and the immunoglobulin Fc domain has the amino acid sequence set forth in SEQ ID NO:

6.

14. The fusion protein according to any one of the preceding claims, wherein, The GLP-1 polypeptide is located at the N-terminal or C-terminal end of the immunoglobulin Fc domain.

15. The fusion protein according to any one of the preceding claims, comprising from N- to C-terminal a GLP-1 polypeptide having the amino acid sequence set forth in SEQ ID NO: 3, and an immunoglobulin Fc domain having the amino acid sequence set forth in SEQ ID NO:

6.

16. The fusion protein according to any one of the preceding claims, wherein, The GLP-1 polypeptide is directly covalently linked to the immunoglobulin Fc domain.

17. The fusion protein according to any one of claims 1-15, wherein, The GLP-1 polypeptide is covalently linked to the immunoglobulin Fc domain via a linker.

18. The fusion protein of claim 17, wherein, The linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a flexible linker, a rigid linker, a helical linker, and a non-helical linker.

19. The fusion protein of claim 18, wherein, The linker comprises a connecting peptide connecting the GLP-1 polypeptide and the immunoglobulin Fc domain.

20. The fusion protein of claim 19, wherein, The connecting peptide comprises a glycine and serine containing linker.

21. The fusion protein of claim 20, wherein, The glycine and serine containing linker comprises one, two, three, four or more repeats as set forth in SEQ ID NO: 8 (GGGS), SEQ ID NO: 9 (GGGGS), SEQ ID NO: 10 (GGGGGS), or SEQ ID NO: 11 (GGGGGGGS).

22. The fusion protein according to any one of claims 17-21, wherein, The linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO:

21.

23. The fusion protein of claim 22, wherein, The linker comprises an amino acid sequence as set forth in SEQ ID NO:

7.

24. The fusion protein according to any one of the preceding claims, wherein, The amino acid sequence of the GLP-1 polypeptide is set forth in SEQ ID NO: 3, the amino acid sequence of the immunoglobulin Fc domain is set forth in SEQ ID NO: 6, and the amino acid sequence of the linker is set forth in SEQ ID NO:

7.

25. The fusion protein of any one of the preceding claims, comprising from N-terminus to C-terminus a GLP-1 polypeptide, a linker, and an immunoglobulin Fc domain, the amino acid sequence of the GLP-1 polypeptide is set forth in SEQ ID NO: 3, the amino acid sequence of the linker is set forth in SEQ ID NO: 7, and the amino acid sequence of the immunoglobulin Fc domain is set forth in SEQ ID NO:

6.

26. The fusion protein according to any one of the preceding claims, wherein, The fusion protein has an amino acid sequence as set forth in SEQ ID NO: 24 or comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:

24.

27. The fusion protein of any one of the preceding claims, further comprising a signal peptide.

28. The fusion protein of any one of the preceding claims, wherein the half-life of the fusion protein in a human subject is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days.

29. A dimer comprising two identical peptide chains linked by a disulfide bond, wherein, Each polypeptide chain comprises the fusion protein of any one of claims 1-28.

30. A nucleic acid molecule comprising a polynucleotide encoding the fusion protein of any one of claims 1-28.

31. The nucleic acid molecule of claim 30, comprising a polynucleotide sequence as set forth in SEQ ID NO: 26 or a polynucleotide sequence having at least 70% sequence identity to the polynucleotide as set forth in SEQ ID NO:

26.

32. A vector comprising the nucleic acid molecule of claim 30 or 31.

33. A cell comprising the nucleic acid molecule of claim 30 or 31 or the vector of claim 32.

34. The cell of claim 33, wherein the cell is a prokaryotic cell or a eukaryotic cell.

35. The cell of claim 34, wherein the eukaryotic cell is a mammalian cell.

36. The cell of claim 35, wherein the mammalian cell is a cell derived from a human or a Chinese hamster ovary (CHO) cell.

37. The cell of claim 36, wherein the mammalian cell is a human embryonic kidney cell 293 (HEK293 cell), or a CHO-K1 cell, or a CHO-S cell or a CHO-DG44 cell.

38. A composition comprising the fusion protein of any one of claims 1-28, or the dimer of claim 29, or the nucleic acid molecule of claim 30 or 31, or the vector of claim 32, or the cell of any one of claims 33-37.

39. A method of constructing the cell of any one of claims 33-37, comprising: a) introducing the nucleic acid molecule of claim 30 or 31 into a vector to construct an expression vector; preferably, the vector is a pKN012 vector; b) introducing the expression vector into the cell to obtain a recombinant cell; preferably, the cell is a CHO cell, in particular a CHO-K1 cell.

40. The method of claim 39, comprising: a) inserting the polynucleotide sequence as set forth in SEQ ID NO: 26 into the Ncol and Hindlll sites of the pKN012 vector to generate a pKN012-GLP1-IgG2 / Fc expression vector; b) introducing the pKN012-GLP1-IgG2 / Fc expression vector into a CHO-K1 cell to obtain a recombinant cell.

41. A method of treating or preventing a disease, the method comprising administering to a subject the fusion protein of any one of claims 1-28, or the dimer of claim 29, or the nucleic acid molecule of claim 30 or 31, or the vector of claim 32, the cell of any one of claims 33-37, or the composition of claim 38.

42. The method of claim 41, wherein the disease is selected from the group consisting of metabolic diseases associated with disorders of glucose and / or lipid metabolism, complications of metabolic diseases, central metabolic diseases (e.g., neurological diseases), and other related metabolic diseases.

43. The method of claim 42, wherein the metabolic disease associated with glucose and / or lipid metabolism disorder is a liver metabolic disease associated with glucose and / or lipid metabolism disorder.

44. The method of claim 42 or 43, wherein, The metabolic disease associated with glucose and / or lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, and metabolic syndrome.

45. The method of claim 42, wherein, The complications of the metabolic disease include cardiovascular complications (e.g., heart failure), renal complications (e.g., diabetic nephropathy, chronic kidney disease), or liver complications (e.g., fatty liver, including NAFLD and NASH) caused by the metabolic disease.

46. The method of claim 42, wherein the nervous system disease is a neurodegenerative disease.

47. The method of claim 46, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, motor neuron disease, Huntington’s disease, and Parkinson’s disease.

48. A method of binding to and activating a signaling cascade of long-chain-fatty-acid-CoA-ligase 1 (ACSL1) on a cell membrane of a target cell to improve lipid metabolism, the method comprising exposing the target cell to a GLP-1 polypeptide or a fusion protein comprising a GLP-1 polypeptide selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide, a fusion protein of any one of claims 1-28, or the dimer of claim 29, or the nucleic acid molecule of claim 30 or 31, or the vector of claim 32, the cell of any one of claims 33-37, or the composition of claim 38.

49. The method of claim 48, wherein the target cell is an adipocyte or a hepatocyte.

50. A method of treating or preventing a disease, the method comprising administering to a subject a GLP-1 polypeptide or a fusion protein comprising a GLP-1 polypeptide, wherein, the GLP-1 polypeptide is selected from the group consisting of human GLP-1 (9-37) and human GLP-1 (9-36) amide; the disease is selected from the group consisting of a metabolic disease associated with lipid metabolism disorder, a complication of a metabolic disease, and a nervous system disease and other related diseases.

51. The method of claim 50, wherein, the GLP-1 polypeptide comprises G22E and / or R36G substitutions relative to a native human GLP-1 polypeptide.

52. The method of claim 51, wherein, the GLP-1 polypeptide has at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, and comprises one or more amino acid substitutions relative to a native human GLP-1 polypeptide selected from the group consisting of G22E and R36G.

53. The method of claim 51 or 52, wherein the GLP-1 polypeptide comprises G22E and R36G substitutions relative to a native human GLP-1 polypeptide.

54. The method of any one of claims 49-53, wherein the GLP-1 polypeptide has an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

55. The method of any one of claims 49-54, wherein, The metabolic disease associated with lipid metabolism disorder is a liver disease associated with lipid metabolism disorder.

56. The method of claim 55, wherein the subject has or is at risk of having one or more symptoms selected from the group consisting of lipid deposition, elevated triglyceride level, elevated cholesterol, elevated low density lipoprotein level, elevated free fatty acid level, increased accumulation of fatty acids, insulin resistance, elevated glutamic-pyruvic transaminase level, elevated glutamic-oxalacetic transaminase level, elevated malondialdehyde content, decreased glutathione peroxidase (GSH-Px) activity, decreased superoxide dismutase (SOD) activity, and elevated inflammatory factor level.

57. The method of any one of claims 49-56, wherein, The metabolic disease associated with lipid metabolism disorder is selected from the group consisting of diabetes (e.g., type 2 diabetes, type 2 diabetes with poor glycemic control after diet and exercise intervention), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), obesity, and metabolic syndrome.

58. The method of claim 57, wherein, The NAFLD is non-obese NAFLD.

59. The method of any one of claims 50-54, wherein, The complication of the metabolic disease is selected from the group consisting of cardiovascular disease (CVD) caused by metabolic disease, diabetic kidney disease (DKD), chronic kidney disease (CKD), or liver disease (e.g., fatty liver, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH)).

60. The method of any one of claims 50-54, wherein the nervous system disease is a neurodegenerative disease (e.g., Alzheimer’s disease, motor neuron disease, Huntington’s disease, Parkinson’s disease).

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