Application of polypeptide in preparation of medicine, food or health care product for treating obesity and complications thereof

Abstract

The invention discloses application of a polypeptide in preparation of medicines, foods or health-care products for treating obesity and complications thereof, and belongs to the technical field of biological medicines. The polypeptides are human GPR15L polypeptides or mouse GPR15L polypeptides and polypeptide conjugates of the human GPR15L polypeptides or the mouse GPR15L polypeptides. The obesity treatment comprises the steps of reducing fat accumulation, promoting fat heat production and decomposition, increasing energy consumption and reducing body weight. Obesity complications include chronic inflammation, fatty liver, steatohepatitis, metabolic diseases, cardiovascular and cerebrovascular diseases, osteoarticular diseases, Alzheimer's disease, depression, tumors and skin diseases. The blood sugar and the body weight are reduced through a brand new action mechanism, so that the obesity and complications thereof are effectively treated.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to the use of a class of polypeptides in the preparation of medicines, foods or health products for treating obesity and its complications. Background Art

[0002] Obesity is a condition caused by an imbalance between the body's energy intake and expenditure. When energy intake consistently exceeds energy expenditure over a long period of time, the excess energy is stored as fat, leading to a gradual increase in adipose tissue and, consequently, obesity and its associated complications.

[0003] With economic development and rising living standards, overweight and obesity have become a global epidemic, posing a serious threat to human health and a widespread problem in recent decades. Obesity significantly increases the incidence of metabolic diseases (such as fatty liver and hyperlipidemia), cardiovascular and cerebrovascular diseases (such as hypertension, myocardial infarction, and shock), osteoarthritis (such as femoral osteoarthritis), Alzheimer's disease, depression, and even certain cancers (such as breast, pancreatic, and liver tumors). Furthermore, obesity can reduce quality of life, diminish work productivity, and lead to unemployment, significantly increasing the global health burden. Therefore, addressing obesity is imperative.

[0004] Currently, the main treatments for obesity include exercise, medication, healthcare therapies, and surgical intervention. Among these, medications have attracted significant attention due to their high compliance and proven efficacy. The development of weight loss drugs has gradually become a key focus of global pharmaceutical research and development. Human glucagon-like peptide-1 (GLP-1) analogs have achieved significant success in weight loss applications. These drugs lower blood sugar by enhancing insulin secretion and inhibiting glucagon secretion. They also slow gastric emptying and intestinal motility, prolonging satiety, and affecting the hypothalamus, which controls appetite, thereby reducing food intake. According to Novo Nordisk's 2022 annual report, its GLP-1 business generated annual revenue of approximately US$11.812 billion, a year-on-year increase of 56%. Of this, semaglutide sales reached US$8.465 billion, a year-on-year increase of 77%. According to estimates by CITIC Securities, the market size of GLP-1 receptor agonist weight loss drugs in China is expected to reach approximately RMB 38.3 billion by 2030, with approximately RMB 25.5 billion dedicated to obese individuals and approximately RMB 12.9 billion dedicated to overweight individuals. These data fully demonstrate that the field of blood sugar reduction / weight loss has broad development prospects and huge market potential.

[0005] However, despite the overwhelming demand for weight loss, current options for weight loss medications remain very limited. The list of obesity treatments approved by the U.S. Food and Drug Administration (FDA) and the China Food and Drug Administration (CFDA) is limited. FDA-approved drugs include Roche's orlistat, Vivus's Qsymia phentermine / topiramate, Arena's Belviq lorcaserin, Orexigen's Contrave (bupropion / naltrexone), Novo Nordisk's liraglutide (Saxenda) and semaglutide (Wegovy), and Eli Lilly's Tirzepatide. The latter three are all GLP-1 receptor agonists. In China, the only obesity medication approved is Roche's orlistat (which only inhibits fat absorption, not carbohydrates, requiring a low-carb diet and resulting in limited weight control effectiveness). Current weight loss medications primarily work by reducing food intake and absorption, which presents several challenges. First, as a chronic disease medication, long-term adherence is required. Once patients stop taking it, they may regain about two-thirds of their lost weight. Furthermore, drugs that reduce food intake primarily act on GLP-1 receptors in the gastrointestinal tract and central nervous system, increasing satiety and reducing appetite. This often leads to abdominal distension, decreased concentration, loss of appetite, rapid heartbeat, dry mouth, bitter taste in the mouth, and even nausea, vomiting, severe diarrhea, and other uncomfortable symptoms. Recent reports suggest that GLP-1 receptor agonists may be associated with some risks of depression and suicidal thoughts. Furthermore, the effectiveness of these drugs is often affected by the patient's lifestyle and dietary habits, resulting in less than satisfactory weight loss results. Therefore, the development of new weight loss drug targets will help meet the enormous demand for weight loss and has important clinical significance and market value.

[0006] GPR15L is an orphan heterotrimeric guanine nucleotide binding protein (G protein) coupled receptor ligand and a 9KDa polypeptide containing 57 amino acid residues. Its gene is called C10orf99 (chromosome 10 open reading frame 99) in humans and 2610528a11Rik in mice. C10orf99 is more highly expressed in the colon than in the small intestine. This receptor ligand was recently named orphan G protein coupled receptor (GPCR) 15 ligand (GPR15L). The polypeptide sequences are SEQ ID NO.1 (human) and SEQ ID NO.3 (mouse) and its gene sequences are SEQ ID NO.2 (human) and SEQ ID NO.4 (mouse). GPR15L contains four cysteines and a net charged amphiphilic short peptide with a molecular weight of approximately 6.52kD. Our previous studies have found that GPR15L is most highly expressed in the intestine ( Figure 4). Reports indicate that its main activity lies in the C-terminal 11 amino acids (aa71-81), but its potency is about 40 times lower than that of the full-length peptide.

[0007] Currently, there are limited reports on the functions of GPR15L, primarily manifesting in the following areas. Under pathophysiological conditions, GPR15L has anti-inflammatory effects. GPR15L knockout in mice leads to exacerbated skin inflammation, autoantibody-mediated disease, or innate immune cell-mediated colonic inflammation. Therefore, some have hypothesized that GPR15L represents a novel signaling axis that controls intestinal homeostasis and inflammation through immune cell migration. However, other reports suggest a more complex role for GPR15L. It controls the specific homing of FoxP3 Tregs to the large intestinal lamina propria, and its expression is regulated by the gut microbiota and TGF-β. It has also been shown to be an epithelial inflammatory pruritogen and to be involved in regulating the proinflammatory response of keratinocytes and skin barrier formation. It can also contribute to the development of psoriasis by promoting keratinocyte proliferation, regulating lymphocyte recruitment to epithelial cells, acting on the blood and vasculature, and possessing antimicrobial properties. However, there are also reports suggesting that it promotes intestinal cancer by recruiting T cells. However, whether and how GPR15L plays a regulatory role in metabolic homeostasis is completely unclear.

[0008] Currently, information on the receptor for GPR15L, G protein-coupled receptor 15 (GPR15), remains limited. GPR15 is a class A G protein-coupled receptor (GPCR) that was first cloned in 1996. GPR15 is an "orphan" receptor with relatively low sequence similarity to its paralogs. It is similar to the potential orphan receptor GPR25, with a maximum similarity of 36%, slightly higher than that of the angiotensin II receptor, apelin receptor, and other chemokine receptor members (haps: / / www.ensembl.org). GPR15 has essentially no homology with the GLP receptor (<10%; Figure 3 ), suggesting that they exert their respective functions through completely different mechanisms of action. GPR15 is also unique in that its NH2-terminal region and third extracellular loop lack cysteines, which are thought to form disulfide bonds and are required for optimal ligand binding and / or receptor activation in many GPCRs. However, the NH2-terminal region of GPR15 carries multiple tyrosine and acidic residues, a feature shared by many chemokine receptors. Studies have shown that the sulfated Tyr residue at the NH2-terminus of GPR15 is required for optimal binding of GPR15L.

[0009] Studies have shown that GPR15L receptors i / o path rather than through G s , G q / 11 and / or G12 / 13 GPR15 is a cytokine that is coupled to the cytokine signaling pathway for signal transduction and has been shown to be a common receptor for infection with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIMV). In humans, GPR15 mRNA is highly expressed in the colon and lymphoid tissues, including peripheral blood lymphocytes and the spleen. GPR15 protein has been detected in many other tissues, such as the colon, small intestinal mucosa, lymphocytes, testis, liver, prostate, as well as in the vascular endothelium and skin. In peripheral blood, GPR15 is expressed in T cells (primarily CD4+) and at lower levels in B cells, monocytes, and neutrophils. Evidence suggests that GPR15 plays an important role in immune diseases such as ulcerative colitis, skin diseases, and multiple sclerosis. These findings suggest that GPR15 is a potential therapeutic target for a variety of diseases. However, whether GPR15 is involved in the regulation of metabolic homeostasis is completely unknown. Summary of the Invention

[0010] Based on the current state of the art, the present invention has discovered for the first time that GPR15L is indeed involved in regulating metabolic homeostasis. Therefore, the first objective of the present invention is to provide a class of polypeptides for use in preparing at least one of a glucose-lowering, weight-reducing, and insulin-secreting preparation. The polypeptides include the human polypeptide set forth in SEQ ID No. 1, the murine polypeptide set forth in SEQ ID No. 3, or polypeptides having similar functions after mutation or truncation of these two polypeptides; preferably, GPR15L (1-30):

[0011] KRRPAKAWSGRRTRLCCHRVPSPNSTNLKG;

[0012] Furthermore, it also includes a polypeptide conjugate in which an IgG Fc region is linked to the polypeptide;

[0013] In particular, the polypeptide conjugate is connected to the Fc region of human IgG1, 3, and 4.

[0014] Having similar functions means at least one of lowering blood sugar, reducing weight, and inducing insulin secretion.

[0015] In the above-mentioned use, the IgG Fc region includes CH2, CH3 domains and hinge region.

[0016] A second objective of the present invention is to provide the use of the polypeptide in the preparation of a medicament, food, or health product for treating obesity and its complications. The present invention utilizes a novel target and mechanism of action, namely, by activating the GPR15 receptor-mediated signaling pathway to reduce obesity, thereby effectively treating obesity and its complications.

[0017] The above-mentioned use for treating obesity includes at least one of reducing fat accumulation, promoting fat heat production and decomposition, increasing energy consumption, and reducing body weight.

[0018] The above-mentioned uses, obesity complications include various diseases caused by obesity.

[0019] Furthermore, various diseases caused by obesity include: chronic inflammation, fatty liver, fatty liver hepatitis, metabolic diseases, cardiovascular and cerebrovascular diseases, bone and joint diseases, Alzheimer's disease, depression, tumors, diabetic complications, and skin diseases.

[0020] Furthermore, metabolic diseases include: insulin resistance, impaired glucose tolerance, diabetes, diabetic complications, fatty liver disease, metabolic syndrome, hypercholesterolemia, hypertriglyceridemia; cardiovascular and cerebrovascular diseases include: atherosclerosis, hypertension, stroke, coronary heart disease, myocardial infarction and shock; osteoarticular diseases include hip arthritis; tumors include: breast tumors, pancreatic and liver tumors; diabetic complications include diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis and late-stage renal disease such as albuminuria.

[0021] In the above-mentioned use, the drug is administered orally or by subcutaneous injection.

[0022] In the above-mentioned use, the medicine further comprises pharmaceutically acceptable excipients.

[0023] In the above-mentioned use, the auxiliary material is selected from at least one of a filler, a binder, a disintegrant, a solubilizer, and a solvent.

[0024] The drug may also be subjected to amino acid modifications, including fatty acid acylation, artificial amino acid substitution or chiral amino acid replacement.

[0025] The preparation of the present invention has no significant impact on normal eating, digestion, and dietary habits. Furthermore, as a metabolite of the host's intestinal flora, GRP15L has virtually no side effects and can be used to prepare drugs, health supplements, or foods for treating obesity and its complications. Therefore, the use of GRP15L described in the present invention in the preparation of weight-loss-related drugs, health supplements, or foods has promising development prospects.

[0026] Through experimental research, this application confirms that GPR15L has the functions of lowering blood sugar and losing weight, and increasing the body's energy consumption. It has shown good effects in lowering blood sugar, reducing weight, inducing insulin secretion and treating obesity and its complications in animal models. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the embodiments of this application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only the embodiments described in this application. For those of ordinary skill in the art, all other embodiments obtained without inventive effort are within the scope of protection of this application. Other drawings can also be obtained based on these drawings.

[0028] Figure 1 : Human Fc' structure and sequence.

[0029] Figure 2 :Structures of human and mouse GPR15L-Fc' fusion proteins.

[0030] Figure 3 : Sequence comparison of human GLP1 with human and mouse GPR15L.

[0031] Figure 4 : Identification of GPR15L as a secreted molecule enriched in the mouse colon;

[0032] GPR15L mRNA or protein expression in human or mouse metabolic tissues was determined by qPCR (A and C) or Western blotting (B and D), respectively.

[0033] Figure 5 :GPR15L levels in the serum of diabetic patients and mice were significantly decreased;

[0034] (A) Western blot analysis of serum GPR15L levels in normal controls, obese / normal glucose tolerance, and obese T2D males. Transferrin was used as an internal control (n = 4 per group). Western blot data were semiquantified using Image J (**p < 0.01). Serum GPR15L levels were assessed by Western blot in male HFD-fed mice (B), obese diabetic mice with a spontaneous leptin receptor mutation (dB / dB) (C), and STZ-induced hyperglycemia mice (D) (n = 3 per group). GPR15L mRNA (E) or protein (F) expression levels in various tissues of male mice fed a normal (N) or high-fat diet (H). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

[0035] Figure 6 :GPR15L selectively binds to different mouse tissues and human pancreatic tissue;

[0036] Binding assay of alkaline phosphatase (SEAP) or alkaline phosphatase-GPR15L fusion protein to various mouse frozen tissues and TD human pancreatic sections. Pancreas and liver showed high affinity binding.

[0037] Figure 7 : Synthetic mouse GPR15L has hypoglycemic and weight-reducing effects;

[0038] High-fat-fed mice were continuously injected with synthetic GPR15L or Fc protein control for 8 days; changes in mouse body weight and random blood glucose were recorded;

[0039] GPR15L polypeptide has significant hypoglycemic effect ( Figure 7 A) and weight loss ( Figure 7 GPR15L injection also increased glucose tolerance in mice (B); Figure 7 C) and insulin sensitivity ( Figure 7 D).

[0040] Figure 8 :Fc'-fused mouse GPR15L protein has a good hypoglycemic effect;

[0041] (A) Mice were injected intraperitoneally with 2 mg / kg GPR15L-Fc protein, serum samples were collected at different time points, and its serum half-life was determined by Western blotting; (B) Relative concentrations of GPR15L-Fc protein in serum at different time points; (C) HFD-fed male mice (n=3 / treatment group) were intraperitoneally injected with PBS or different concentrations of GPR15L-Fc, and the mice's random blood glucose levels were measured at different time points; (D) Schematic diagram of the structure of the long-acting GPR15L-Fc' fusion protein; (E) 2 mg / kg of long-acting GPR15L-Fc' fusion protein was intraperitoneally injected, and serum samples were collected at different time points, and the half-life of GPR15L-Fc' protein in serum was determined by Western blotting; (F) Male high-fat-fed obese mice (n=3 / group) were intraperitoneally injected (250 μg / every other day) with long-acting GPR15L-Fc' fusion protein; the mice's body weight (F) and random blood glucose concentration (G) were measured at different time points.

[0042] Figure 9 :Fc'-fused human GPR15L has a good hypoglycemic effect;

[0043] (A) Male mice were intraperitoneally injected with 2 mg / kg mouse or human GPR15L-Fc' protein (250 μg /

[0044] (A) Male mice were given 2 mg / kg mouse or human GPR15L-Fc' protein by gavage; serum samples were taken at different time points to detect the random blood glucose levels of the mice. (B) Male mice were given 2 mg / kg mouse or human GPR15L-Fc' protein by gavage; serum samples were taken at different time points to detect the random blood glucose levels of the mice.

[0045] Figure 10:GPR15L promotes glucose-induced insulin secretion (GSIS) in mouse β cells;

[0046] (A) MIN6 cells were treated with mouse GPR15L protein (50 ng / ml) for 24 hours, and mRNA changes were detected by RT-PCR. (B) MIN6 islets were treated with different doses of mouse GPR15L for 30 minutes under low glucose (2.8 mM) or blood glucose (16.7 mM) conditions, and glucose-stimulated insulin secretion (GSIS) was measured. Values ​​represent the mean ± standard error of the mean (SEM) of 6 independent experiments, and three measurements were performed in each independent experiment. (C) MIN6 cells were treated with different doses of mouse GPR15L in the presence of 25 mM KCl (30 minutes), and then changes in GSIS were measured. Islets obtained from wild-type mice (D) or leptin-mutant obese diabetic mice (ob / ob) (E) were treated with different doses of mouse GPR15L in the presence of different concentrations of glucose for 30 minutes, and GSIS was measured. (F) Human islets were treated with synthetic human GPR15L peptide for 30 minutes, and then changes in GSIS were measured. Significance level: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0047] Figure 11 : Truncated human GPR15L can significantly lower blood glucose in mice treated with STZ.

[0048] Figure 12 : GPR15L has a different mechanism of action from the currently used clinical weight loss drug GLP-1 agonist (liraglutide).

[0049] Figure 12 SCD1: Stearoyl-CoA Desaturase 1; ACC: Acetyl-CoA Carboxylase; PPARα: Peroxisome Proliferator-Activated Receptor Alpha; SREBP1: Sterol Regulatory Element-Binding Protein 1; AOX: Acyl-CoA Oxidase

[0050] The English and abbreviations in the attached drawings are translated as follows:

[0051] Obese: obese

[0052] ND: Normal feed

[0053] Transferrin: transferrin

[0054] HFD: High Fat Diet

[0055] IHC: Immunohistochemistry

[0056] HE: Hematoxylin and eosin staining

[0057] Basal: Baseline

[0058] Body weight: body weight

[0059] Blood glucose level:

[0060] Brain:

[0061] BAT: brown adipose tissue

[0062] Colon: Colon

[0063] eWAT: visceral fat

[0064] Heart:

[0065] Islets: pancreatic islets

[0066] Kidney:

[0067] Liver

[0068] Lung:

[0069] Muscle:

[0070] NS: No significant difference

[0071] OCR: oxygen consumption rate

[0072] Rel.mRNA level: relative expression level of mRNA

[0073] Skin:

[0074] Small intestine (SI)

[0075] Spleen

[0076] Stomach:

[0077] sWAT: subcutaneous white adipose tissue

[0078] Pancreas

[0079] *: indicates P<0.05, **: indicates P<0.01 and the difference is significant. DETAILED DESCRIPTION

[0080] In order to help those skilled in the art better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0081] All animal models used in this study were established using wild-type C57BL / 6 mice, purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. All mice were housed in a specific pathogen-free (SPF) barrier animal laboratory room maintained at an ambient temperature of 24°C and a daily light and dark cycle of 12 h each (i.e., light cycle: 6:00–18:00; dark cycle: 18:00–6:00).

[0082] Example 1: GPR15L is highly expressed in the intestine

[0083] To determine the potential role of GPR15L, we examined the expression of GPR15L in different tissues of humans and mice. We found that GPR15L mRNA was expressed in different human tissues, but was most highly expressed in tissues such as the small intestine, colon, and skin. Figure 4 A). Consistent with this result, GPR5L protein is also highly expressed in the small intestine and colon ( Figure 4 B). GPR15L mRNA and protein are also expressed in different tissues of mice, but the highest expression is in the small intestine and colon ( Figure 4 These results suggest that, similar to GLP-1, the main source of GPR15L may also be the intestine.

[0084] Example 2: GRP15L levels are downregulated in the serum of obese and diabetic patients and mice

[0085] To determine the role of GPR15L in metabolic diseases, we measured the levels of GPR15L protein in the blood of obese people, people with type 2 diabetes, and healthy people. We found that GPR15L levels were high in the blood of healthy people, but significantly decreased in the blood of obese and diabetic people ( Figure 5 A). Further studies have shown that compared with normal control mice, the serum GPR15L content in high-fat-induced obese mice ( Figure 5 B), dB / dB obese mice ( Figure 5C), and STZ-induced diabetic mice ( Figure 5 This result was confirmed by immunoblotting experiments in the blood of obese humans and different obese mice.

[0086] Example 3: GPR15L binds to human and mouse pancreatic β cells and liver with high selectivity

[0087] To identify the target tissues of GPR15L, we constructed a fusion protein (AP-GRP15L) that binds to secreted alkaline phosphatase (AP) and mouse GPR15L. We tested the binding of AP-GPR15L to different mouse tissues and found that this fusion protein binds to various tissues such as liver, kidney, brain, heart, lung, and spleen, but has the highest binding affinity to mouse pancreas and pancreatic islets. Figure 6 In addition to the pancreas, GPR15L binding to liver tissue also showed stronger binding signals than other tissues, suggesting the potential role of GPR15L in regulating the functions of these tissues.

[0088] Example 4: Synthetic GPR15L polypeptide has weight loss and blood sugar lowering effects

[0089] To determine the function of GPR15L, we first synthesized a mouse GPR15L peptide through Shanghai Kaijing Pharmaceutical Co., Ltd. This peptide or Fc control was used to continuously treat high-fat-fed obese mice for 8 days (2 mg / kg / day), and it was found that this peptide had a significant hypoglycemic effect ( Figure 7 A) and weight loss ( Figure 7 GPR15L injection also increased glucose tolerance in mice (B); Figure 7 C) and insulin sensitivity ( Figure 7 D).

[0090] Example 5: GPR15L-Fc fusion protein or modified GPR15L-Fc′ fusion protein has weight loss and blood sugar lowering effects

[0091] Given that Fc fusion proteins can avoid lysosomal degradation in plasma and exhibit a longer half-life, we prepared a fusion protein of murine GPR15L and human IgG1 Fc antibody (GPR15L-Fc). In mice, the half-life of recombinant GPR15L-Fc fusion protein with a His tag (2 mg / kg body weight) was approximately 8 hours after intraperitoneal injection ( Figure 8 A and 8B). In HFD-fed male obese mice, injection of different doses of GPR15L-Fc significantly reduced the blood glucose levels of the mice ( Figure 8 C), indicating improvement in glucose tolerance.

[0092] The anti-obesity and anti-hyperglycemic effects of GPR15L-Fc may be achieved by reducing food intake and improving tissue and cell function. To increase the stability of the anti-obesity peptide, we modified the GPR15L-Fc fusion protein by removing three amino acids (Met252; Ser254;

[0093] The long-acting GPR15L-Fc fusion protein (GPR15L-Fc') was prepared by point mutation of Thr256 to Tyr252, Thr254 and Glu256 respectively. Figure 8 D). Western blot experiments showed that the half-life of long-acting GPR15L-Fc' was significantly increased compared with GPR15L-Fc ( Figure 8 E). HFD-fed mice were injected with Fc' or murine GPR15L-Fc' fusion protein for 21 days (injection every other day; each injection dose is 250 μg / mouse). The body weight and random blood glucose concentration of mice were recorded at different time points. Compared with mice injected with Fc'-control, mice injected with GPR15L-Fc' fusion protein showed significantly reduced body weight ( Figure 8 F) and blood sugar levels ( Figure 8 G), confirmed the weight loss and hypoglycemic effects of GPR15L.

[0094] Example 6: Human GLP15L has a significant hypoglycemic effect

[0095] To confirm that human GPR15L also has the function of improving metabolism, we prepared human GPR15L-Fc' fusion protein. GLP1 receptor humanized mice (B6 / JGpt-Glp1r em1Cin(hGLP1R) / Gpt; T053832; Jicui Yaokang). We found that intraperitoneal injection of human GPR15L-Fc' fusion protein significantly reduced blood glucose in mice ( Figure 9 A). Gavage of human GPR15L-Fc' fusion protein to mice also had a good hypoglycemic effect ( Figure 9 B).

[0096] Example 7: GPR15L promotes glucose-induced insulin secretion in mouse β cells

[0097] To determine the mechanism of GPR15L's hypoglycemic effect, we treated pancreatic β-cell line MIN6 cells with a synthetic mouse GPR15L peptide. We found that GPR15L treatment significantly increased the expression of genes related to islet function, such as Glut2, Ins1, and Pdx1. Figure 10A). Under low glucose (2.8 mM) conditions, mouse GPR15L had little effect on insulin secretion, but under high glucose (16.7 mM) conditions, GPR15L treatment significantly stimulated insulin secretion ( Figure 10 B). In MIN6 cells, treatment with mouse GPR15L also increased KCl-induced insulin secretion ( Figure 10 C), indicating that GPR15L acts downstream of K ion channels. We also further examined the role of mouse GPR15L in isolated pancreatic islets. We found that GPR15L can stimulate the expression of K ion channels in islets isolated from normal mice ( Figure 10 D) or from ob / ob obese mice ( Figure 10 E) of the pancreatic islets isolated; we also tested the effect of the synthetic human GPR15L peptide on glucose-stimulated insulin secretion in human pancreatic islets and found that human GPR15L significantly promoted insulin secretion ( Figure 10 F).

[0098] SEQ ID No. 1: Human GPR15L polypeptide amino acid sequence

[0099] MRLLVLSSLLCILLLCFSIFSTEGKRRPAKAWSGRRTRLCCHRVPSPNSTNLKGHHVRLCKPCKLEPEPRLWVVPGALPQV

[0100] SEQ ID No. 2: Human GPR15L gene nucleotide sequence

[0101] ATGAGGCTTCTAGTCCTTTCCAGCCTGCTCTGTATCCTGCTTCTCTGCTTCTCCATCTTCTCCACAGAAGGGAAGAGGCGTCCTGCCAAGGCCTGGTCAGGCAGGAGAACCAGGCTCTGCTGCCACCGAGTCCCTAGCCCCAACTCAACAAACCTGAAAGGACATCATGTGAGGCTCTGTAAACCATGCAAGCTTGAGCCAGAGCCCCGCCTTTGGGTGGTGCCTGGGGCACTCCCACAGGTGTAG

[0102] SEQ ID No.3: Mouse GPR15L polypeptide amino acid sequence

[0103] MRLLALSGLLCMLLLCFCIFSSEGRRHPAKSLKLRRCCHLSPRSKLTTWKGNHTRPCRLCRNKLPVKSWVVPGALPQI

[0104] SEQ ID No.4: Mouse GPR15L gene nucleotide sequence

[0105] ATGAGACTTCTAGCCCTTTCCGGTCTGCTCTGCATGCTGCTCCTCTGTTTCTGCATTTTCTCCTCAGAAGGGAGAAGACATCCTGCCAAGTCCTTGAAACTCAGGCGCTGCTGTCACCTATCTCCTAGATCCAAGCTGACAACCTGGAAAGGAAACCACACAAGGCCCTGCAGACTCTGCAGAAACAAGCTACCAGTCAAGTCATGGGTGGTGCCTGGGGCTCTCCCACAGATATAG.

[0106] SEQ ID No.5: Hinge region of human IgG1 Fc used in the present invention

[0107] EPKSCDKTHTCP (C forms disulfide bonds)

[0108] SEQ ID No.6:

[0109] The present invention uses a modified human IgG1 Fc CH2 region (Fc'; M, S, and T at amino acid positions 252, 254, and 256 are replaced by Y, T, and E, respectively (the mutated amino acids are underlined))

[0110] PCPAPELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0111] SEQ ID.No.7:

[0112] The CH3 region of human IgG1 Fc' used in the present invention is

[0113] GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0114] Example 8:

[0115] In order to determine the function of the N-terminal truncated GPR15L (1-30), we constructed a human GPR15L-N-terminal (1-30) amino acid-IgG4 (FC) fusion protein eukaryotic expression vector (human serum albumin secretory peptide-GPR15L-N (1-30)-IgG4 (FC)-pCDNA3.4), transiently transferred into 293F suspension cells by PEI, and suspended in a carbon dioxide shaker for one week, and the supernatant was harvested. After the fusion protein was purified by rProteinA, mice were treated with 250ug intraperitoneal injection and the blood glucose changes were detected at different time points. It was found that the truncated human GPR15L (1-30) IgG4 (FC) fusion protein can significantly reduce the blood glucose of mice treated with STZ. The results are shown in Figure 11 and Table 1.

[0116] The sequence of the human N-terminal GPR15L (1-30) fusion protein is as follows:

[0117] GPR15L(1-30): KRRPAKAWSGRRTRLCCHRVPSPNSTNLKG; SEQ ID No.8

[0118] IgG4(FC) sequence:

[0119] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA

[0120] KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE

[0121] EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK, SEQ ID No. 9.

[0122] Table 1 Changes of blood glucose in mice over time (mmol / L)

[0123]

[0124] Example 9:

[0125] In primary liver cell experiments, after treatment with PA (palmitic acid, 0.2 mM), the cells were stimulated with different concentrations of mouse GPR15L (full-length) as follows:

[0126] 1. Isolation and culture of primary hepatocytes: WT hepatocytes are isolated, usually using perfusion technology combined with IV collagenase digestion. The isolated hepatocyte suspension is cultured in an appropriate culture medium, usually containing fetal bovine serum (FBS), antibiotics, etc.

[0127] 2. Cell adhesion: Culture the cells in a culture plate or dish until the cells adhere to the wall and grow.

[0128] 3. PA treatment: According to the experimental design, 0.2 mM PA was added to the cell culture medium and treated for 24 h.

[0129] 4. Washing cells after PA treatment: After treatment, wash the cells with an appropriate buffer (such as PBS) to remove unbound PA.

[0130] 5. Stimulation with different concentrations of GPR15L: Culture medium containing different concentrations of GPR15L (100 nM, 500 nM) was added to the washed cells, and the concentration and duration of liraglutide (100 nM) were determined according to the experimental design.

[0131] 6. See the results Figure 12 .

[0132] 7. Data Analysis

[0133] Mouse liver primary cells pretreated with 0.2 mM palmitic acid for 24 hours were treated with 100 nM control drug liraglutide and 100 nM and 500 nM GPR15L in the presence of 0.2 mM palmitic acid for another 24 hours. q-PCR detection showed that different concentrations of GPR15L can significantly promote the significant upregulation of lipolysis-related PPARa and AOX genes in liver primary cells, while the control drug liraglutide had no obvious stimulatory effect due to the lack of corresponding GLP1R in liver cells, suggesting that GPR15L may have a target independent of GLP1R.

Claims

1. A type of polypeptide for use in preparing at least one of a hypoglycemic, weight-reducing, and insulin secretion-inducing preparation, wherein the polypeptide comprises a human polypeptide as shown in SEQ ID No. 1, a mouse polypeptide as shown in SEQ ID No. 3, or a polypeptide having similar functions after mutation or truncation of the two polypeptides; further comprising a polypeptide conjugate in which an IgG Fc region is connected to the polypeptide.

2. The use according to claim 1, characterized in that The polypeptides having similar functions after mutation or truncation of the two polypeptides include GPR15L (1-30): KRRPAKAWSGRRTRLCCHRVPSPNSTNLKG.

3. The use according to claim 1 or 2, characterized in that The IgG Fc region includes CH2, CH3 and hinge region.

4. Use of the polypeptide according to claim 1, 2 or 3 in the preparation of medicines, foods or health products for treating obesity and its complications.

5. The use according to claim 4, characterized in that Treatment of obesity includes at least one of reducing fat accumulation, promoting fat heat production and decomposition, increasing energy expenditure, and reducing body weight.

6. The use according to claim 4, characterized in that Obesity complications include various diseases caused by obesity.

7. The use according to claim 6, characterized in that Various diseases caused by obesity include: chronic inflammation, fatty liver, fatty hepatitis, metabolic diseases, cardiovascular and cerebrovascular diseases, bone and joint diseases, Alzheimer's disease, depression, tumors, diabetic complications, and skin diseases.

8. The use according to claim 6, characterized in that Metabolic diseases include: insulin resistance, impaired glucose tolerance, diabetes, diabetic complications, fatty liver disease, metabolic syndrome, hypercholesterolemia, hypertriglyceridemia; cardiovascular and cerebrovascular diseases include: atherosclerosis, hypertension, stroke, coronary heart disease, myocardial infarction and shock; bone and joint diseases include hip arthritis; tumors include: breast tumors, pancreatic and liver tumors; diabetic complications include diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, glomerulonephritis, glomerular sclerosis, nephrotic syndrome, hypertensive nephrosclerosis and late-stage renal disease such as albuminuria.

9. The use according to claim 4, characterized in that: The drug is administered orally or by subcutaneous injection; The drug also includes pharmaceutically acceptable excipients; the excipients are selected from at least one of fillers, binders, disintegrants, solubilizers, and solvents.

10. The use according to claim 4, characterized in that The drug is subjected to amino acid modification, including fatty acid acylation, artificial amino acid substitution or chiral amino acid substitution.

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