Use of antifreeze protein in losing weight and lowering glucose levels

Abstract

The present invention belongs to the technical field of biopharmaceuticals and specifically relates to use of an antifreeze protein in losing weight and lowering glucose levels. The present invention successfully constructs an NPAFP transgenic mouse model by means of genetic engineering technology and verifies the potential application value of the NPAFP protein in losing weight and lowering glucose levels. Further, on the basis of the model, the NPAFP protein has been found to significantly reduce fat accumulation and improve blood glucose levels by regulating brown adipose metabolism and hepatic lipid droplet content. Compound development or protein engineering based on the NPAFP protein of the present invention can provide novel drugs and strategies for the treatment of obesity and diabetes. Moreover, the molecular mechanism of the NPAFP protein is further explored using multi-omics technology, which helps promote clinical translation and application thereof.

Description

Application of an antifreeze protein in weight loss and blood sugar control Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an antifreeze protein in weight loss and blood sugar reduction. Background Technology

[0002] As people's living standards improve, their dietary structures are also changing. Irregular eating habits have led to a year-on-year increase in the incidence of metabolic syndrome (MS), with a trend towards affecting younger people. Once it develops, metabolic syndrome can cause various health problems, such as obesity, insulin resistance, hyperinsulinemia, diabetes, coronary heart disease, hypertension, stroke, and even certain cancers, including pancreatic cancer, liver and gallbladder cancer, and colon cancer. Weight loss and blood sugar control are important topics in current research on metabolic diseases. Obesity is a modifiable risk factor for many diseases, especially since visceral adipose tissue can modulate pro-inflammatory cytokines (such as interleukin-6), leading to inflammation, oxidative stress, and insulin resistance, thereby increasing the risk of chronic diseases (Thomas DM, Brdau C, Bosy-Wesiphal A, et al. Relationships between body roundness with body fat and viseeral adipose tissue e-menging from a new geometrical model[J]. Obesity (Silver Spring), 2013, 21(11): 2264-2271.). The main characteristics of type 2 diabetes mellitus (T2DM) are hyperglycemia caused by insulin resistance and pancreatic β-cell dysfunction, as well as persistent low-grade inflammation in peripheral tissues. Obesity, unhealthy eating habits, lack of exercise, and various pathophysiological disorders caused by environmental and genetic factors can lead to impaired glucose homeostasis in type 2 diabetes mellitus (DeFronzo RA, Ferrannini E, Groop L, et al. Type 2 diabetes mellitus. Nat Rev Dis Primers. 2015 Jul 23; 1:15019.). Studies have shown that the prevalence of type 2 diabetes mellitus increases proportionally with weight gain, starting from a normal BMI of 25 kg / m². 2 Increased to 30kg / m 2The risk of death associated with type 2 diabetes mellitus (T2DM) is more than doubled (Magkos F, Hjorth MF, Astrid A. Diet and exercise in the prevention and treatment of type 2 diabetes mellitus. Nat Rev Endocrinol. 2020 Oct; 16(10):545-555.). In addition, excessive carbohydrate intake can lead to insulin resistance, which may bring forward the onset of T2DM in people with a genetic predisposition. In patients with T2DM and obesity, a weight loss of about 15 kg can alleviate the condition of 80% of T2DM patients (Jao CL, Hung CC, Tung YS, et al. The development of bioactive peptides from dietary proteins as a dipeptidyl peptidase IV inhibitor for the management of type 2 diabetes. Biomedicine (Taipei). 2015 Sep; 5(3):14.).

[0003] Current treatment options include lifestyle interventions, medication, and metabolic surgery. The 2017 AACE / ACE consensus statement newly proposed that weight loss should be a long-term goal. For overweight or obese patients with prediabetes and type 2 diabetes, behavioral interventions and weight-loss medications should be used when necessary, emphasizing the importance of weight loss in disease management. Weight loss can reduce insulin resistance, increase blood glucose lowering effects, improve lipid metabolism, and improve blood pressure. The 2017 AACE / ACE consensus statement prioritizes lifestyle interventions, with weight-loss medications added when necessary. Weight-loss medications can be used for all patients with a BMI ≥ 27 kg / m² who are receiving intensive therapy combined with lifestyle interventions. 2 Patients with complications or BMI ≥ 30 kg / m² 2Patients (Garber AJ, Handelsman Y, Grunberger G, et al. Consensus State by the American Association of Clinical Endocrinologists and American College of Endocrinology on the Comprehends-Type Two Diabets Management Algorithm-2020 Excipient Summary. Endocr Pract. 2020 Jan; 26(1):107-139.). Studies have shown that the mechanisms of action of weight-loss and blood sugar-lowering compounds involve multiple biological pathways, mainly focusing on the regulation of lipid metabolism, regulation of glucose metabolism, and the balance of gut microbiota. Naturally derived compounds, such as polyphenols and flavonoids, have been extensively studied due to their low toxicity and multi-target effects. For example, resveratrol and green tea polyphenols can promote lipolysis and enhance insulin sensitivity by activating the AMPK signaling pathway; quercetin and astragaloside A significantly improve insulin resistance and lipid metabolism by regulating the expression of metabolism-related genes. Furthermore, synthetic compounds, such as the GLP-1 receptor agonist smegglutide, and metabolites derived from gut microbiota (such as short-chain fatty acids), have also shown unique roles in lipolysis, glucose metabolism, and gut microbiota regulation. These studies reveal the complexity of metabolic regulation and suggest that multi-target strategies may be key to future drug development. However, existing treatments suffer from limited efficacy, significant side effects, or limited applicability. Therefore, developing more efficient and safer weight-loss and glucose-lowering compounds and exploring their mechanisms of action has become a hot research topic.

[0004] Antifreeze proteins (AFPs) are a collective term for various structural ice-binding proteins that enhance the antifreeze ability of organisms. They were initially discovered in Antarctic fish and defined as antifreeze agents. To date, AFPs have been found in various organisms, primarily including bony fish, insects, plants, fungi, nematodes, amphibians, bacteria, and diatoms. The structures of antifreeze proteins from different species vary significantly, and no obvious homology has been found from a genetic perspective; they only maintain consistency in their antifreeze function. The adsorption process of antifreeze proteins on ice crystal surfaces is specific; different types of antifreeze proteins may bind to crystal faces with different crystal orientations on the ice crystal surface. After binding to the ice crystal surface, antifreeze proteins can lower the temperature of the ice crystal growth point. In this way, antifreeze proteins can limit the growth of ice crystals and inhibit the recrystallization of ice crystals (Graham LA, Hobbs RS, Flet alher GL, et al. Helical Antifreeze Proteins Have Independently Evolved in Fishes on Four Occasions[J]. Plos One, 2013, 8(12): 81285.). The protective function of AFPs is due to their properties, including ice surface affinity, thermal hysteresis, inhibition of ice recrystallization, and temporary binding of organisms to ice. Different properties produce different antifreeze effects. Although multiple antifreeze properties of AFPs can usually be detected, their antifreeze effect is mainly based on one property. Generally, all these properties or effects stem from the protein's ability to bind to ice (Xiang H, Yang X, Ke L, et al. The properties, biotechnologies, and applications of antifreeze proteins. Int J Biol Macromol. 2020 Jun 15; 153:661-675.). In recent years, with the gradual clarification of the properties and mechanisms of action of AFP, its applications have become increasingly widespread, including cryomedicine, agriculture, food industry, environmental monitoring, and anti-icing materials.

[0005] Antifreeze proteins can be broadly classified into four categories based on their species origin: fish antifreeze proteins, insect antifreeze proteins, plant antifreeze proteins, and microbial antifreeze proteins. Among these, antifreeze proteins isolated from fish are further classified into six types based on differences in their primary structure and conformation: type I, II, III, and IV antifreeze proteins (AFPⅠ, AFPⅡ, AFPⅢ, AFPⅣ), antifreeze glycoproteins (AFGPs), and hyperactive-AFP (Guo Tinghe. Irreversible Random Adsorption Inhibition Model of Thermal Hysteresis Activity of Antifreeze Proteins [D]. Inner Mongolia: Inner Mongolia University, 2022.). Antifreeze protein III (AFPⅢ) is currently one of the most widely used cryoprotectants and has not yet been found to be toxic. There are reports on adding AFPⅢ for freezing ovaries, sperm, and oocytes, but these studies primarily focus on developmental aspects. The structures of antifreeze proteins from different species differ significantly, and no obvious homology has been found from a genetic perspective; they only maintain consistency in their antifreeze function.

[0006] In a previous study (“Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was first discovered and demonstrated that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein, AFPIII, through neofunctionalization during the process of escaping adaptive conflict, and named it LdAFPII. This protein evolved from SAS-B and has multiple repetitions of functional domains (such as LdAFPIII-1 with one repetition of functional domain, LdAFPIII-4 with four repetitions of functional domain, LdAFPIII-12 with twelve repetitions of functional domain, etc.), with each AFPIII domain containing 62 amino acids. The sequence of the LdAFPII protein was published in a previous study (“Cloning and evolutionary analysis of the multimeric type III antifreeze protein gene of Antarctic eel Pout (Lycodichthys dearborni)”, Yu et al., Acta Genetica Sinica, 2005, 32(8): 789-794.). Summary of the Invention

[0007] This invention successfully constructed an NPAFP transgenic mouse model using genetic engineering techniques, and discovered that NPAFP protein has a significant effect on reducing fat accumulation and improving blood sugar levels by regulating brown adipose tissue metabolism and liver lipid droplet content. Based on this, this invention was completed.

[0008] In a first aspect, the present invention provides a pharmaceutical composition for weight loss and blood sugar reduction, wherein the active ingredient of the pharmaceutical composition is NPAFP protein.

[0009] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0010] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0011] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0012] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0013] Furthermore, the diabetes can be divided into insulin-dependent type 1 diabetes (T1DM) and non-insulin-dependent type 2 diabetes (T2DM).

[0014] Secondly, this invention provides the application of NPAFP protein in the preparation of weight loss and blood sugar reduction products.

[0015] Furthermore, the products include, but are not limited to, health supplements, weight loss foods, or functional beverages.

[0016] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0017] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0018] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0019] Thirdly, the present invention provides the use of NPAFP protein in the preparation of medicaments for treating obesity and / or diabetes.

[0020] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0021] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0022] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0023] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0024] Furthermore, the drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, and / or suppositories.

[0025] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, and / or a controlled-release formulation.

[0026] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0027] Furthermore, the drug can be administered by injection, cavity, or respiratory tract.

[0028] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; the cavity administration includes rectal or vaginal administration; and the respiratory administration includes nasal administration.

[0029] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0030] Furthermore, the diabetes can be divided into insulin-dependent type 1 diabetes (T1DM) and non-insulin-dependent type 2 diabetes (T2DM).

[0031] Fourthly, this invention provides the application of NPAFP protein in constructing animal models of obesity and / or diabetes.

[0032] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0033] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0034] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5.

[0035] Furthermore, the animals in the animal model include rodents, large mammals, and non-human primates.

[0036] Furthermore, the rodents include, but are not limited to, mice, rats, and rabbits.

[0037] Furthermore, the large mammals include, but are not limited to, pigs and dogs.

[0038] Furthermore, the non-human primates mentioned include, but are not limited to, rhesus monkeys and cynomolgus monkeys.

[0039] Furthermore, the obesity is divided into primary obesity and secondary obesity.

[0040] Furthermore, the diabetes can be divided into insulin-dependent type 1 diabetes (T1DM) and non-insulin-dependent type 2 diabetes (T2DM).

[0041] Fifthly, the present invention provides a method for constructing an animal model of obesity and / or diabetes, wherein the animal model is an NPAFP transgenic mouse expressing different numbers of AFP III functional domains by inserting the NPAFP gene into the H11 gene locus of a mouse using gene editing technology.

[0042] Furthermore, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0043] Preferably, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12.

[0044] Furthermore, the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP2 protein is shown in SEQ ID NO.2; the nucleotide sequence of the NPAFP3 protein is shown in SEQ ID NO.3; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.5. Beneficial effects

[0045] 1. This invention successfully constructed an NPAFP transgenic mouse model using genetic engineering technology, verifying the potential application value of NPAFP protein in weight loss and blood sugar reduction.

[0046] 2. This invention has found that NPAFP protein has a significant effect on reducing fat accumulation and improving blood sugar levels by regulating brown adipose tissue metabolism and liver lipid droplet content.

[0047] 3. The development of compounds or protein engineering based on the NPAFP protein of this invention can provide novel drugs and strategies for the treatment of obesity and diabetes. Furthermore, combining multi-omics technologies to further explore the molecular mechanisms of the NPAFP protein will help promote its clinical translation and application. Attached Figure Description

[0048] Figure 1 shows the method for constructing NPAFP mice.

[0049] Figure 2 shows how NPAFP protein regulates brown fat metabolism.

[0050] Figure 3 shows how NPAFP reduces the density of brown fat droplets.

[0051] Figure 4 shows how NPAFP protein reduces the accumulation of lipid droplets in the liver.

[0052] Figure 5 shows how NPAFP protein reduces blood glucose levels in mice. Detailed Implementation

[0053] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0054] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0055] The NPAFP (NeuroProtect AFP) described in this invention is an antifreeze protein III. The NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0056] Each AFPIII domain contains 62 amino acids. This invention utilizes alphafold3 for protein structure prediction and optimization, changing amino acids at positions 36 and 37 from KL to RI, and amino acid at position 50 from D to E, to increase the flexibility of the protein structure. To explore its application in neurological diseases, it is named NeuroProtectionAFPIII (such as NPAFP1, NPAFP4, NPAFP12, etc.).

[0057] The nucleotide sequence of the NPAFP1 protein described in this invention is shown in SEQ ID NO.1:

[0058] The nucleotide sequence of the NPAFP2 protein described in this invention is shown in SEQ ID NO.2:

[0059] The nucleotide sequence of the NPAFP3 protein described in this invention is shown in SEQ ID NO.3:

[0060] The nucleotide sequence of the NPAFP4 protein described in this invention is shown in SEQ ID NO.4:

[0061] The nucleotide sequence of the NPAFP12 protein described in this invention is shown in SEQ ID NO.5:

[0062] Example 1: Animal Model Construction

[0063] To investigate the regulatory effects of NPAFP protein on lipids and blood glucose in vivo, transgenic mice containing different AFPIII domains were constructed (Figure 1). A plasmid expressing NPAFP protein (Donor vector) was constructed using the CAG promoter. First, the CRISPR / Cas9 system and the Donor vector sample were microinjected into fertilized eggs of mice with a C57BL / 6JGpt background. Using homologous recombination technology, the NPAFP expression plasmid was inserted into the H11 site of the C57BL / 6 mice. Surviving fertilized eggs were then transplanted into pseudopregnant female mice, allowing them to conceive and give birth. Genomic DNA was extracted from the F0 generation pups born to the recipient mice at 5-7 days for PCR and sequencing identification to confirm the genotype. After sexual maturity, the positive F0 generation mice were mated with wild-type background mice, and the resulting F1 generation mice were genomically extracted at 5-7 days for PCR and sequencing identification to confirm the genotype. F1 generation mice were bred with WT mice, and the offspring were genotyped. Three-month-old heterozygous sibling mice and WT mice were perfused and fixed for further section staining and blood glucose measurement.

[0064] During the construction process, specific primers targeting the NPAFP gene were designed at the 5' and 3' ends of the insertion site, as well as upstream and downstream regions of the insertion site (see Figure 1B), to facilitate genotyping of mice using PCR electrophoresis (see Figures 1C-D). Currently, transgenic mice stably expressing NPAFP1, NPAFP4, and NPAFP12 proteins have been successfully obtained. These transgenic mice will be used to verify the role of NPAFP in weight loss and blood sugar-lowering related diseases. Three-month-old heterozygous transgenic mice and WT mice were perfused and fixed for further section staining and blood glucose measurement.

[0065] Table 1. Specific primers for constructing a homozygous mouse model for weight loss and blood sugar reduction

[0066] Table 2. Specific primers for constructing a weight-loss and blood sugar-lowering heterozygous mouse model

[0067] Example 2: Tissue Morphology Detection

[0068] 2.1 HE staining: to examine the histological morphology of liver and brown adipose tissue, and to observe lipid droplet distribution and organelle density.

[0069] Mice were anesthetized with isoflurane and then perfused with PBS and 4% neutral buffered formaldehyde solution, respectively. Fresh liver and brown adipose tissue were collected and fixed with 4% neutral buffered formaldehyde solution. The fixed tissues were dehydrated sequentially with a gradient concentration (70%, 80%, 95%, 100%). The tissues were cleared with xylene. The cleared tissues were then embedded in liquid paraffin to prepare paraffin blocks. The tissue sections were sectioned, laid flat on glass slides, and dried and fixed. The sections were dewaxed with xylene, then rehydrated stepwise with a gradient of ethanol (100%, 95%, 80%, 70%), and then washed with distilled water. The sections were stained with hematoxylin and eosin, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol, and then rinsed thoroughly with tap water to restore blue color. The sections were stained with eosin and rinsed with distilled water. The slides were dehydrated sequentially with gradient ethanol (70%, 80%, 95%, 100%), then cleared with xylene, and finally mounted with neutral resin. After the slides dried, the histological structure and staining effect were observed under a microscope.

[0070] As shown in Figure 2, larger lipid droplets and lower organelle density were observed in the brown adipose tissue cells of the control group WT mice. However, the number of lipid droplets and the relative density of organelles were reduced in the brown adipose tissue cells of NPAFP1 mice. In contrast, the number of lipid droplets in the brown adipose tissue cells of NPAFP4 and NPAFP12 mice was very low, and the density of organelles was significantly increased. These results indicate that NPAFP proteins can significantly improve the metabolic properties of brown adipose tissue, and its effect is positively correlated with the number of AFPIII domains.

[0071] 2.2 Oil Red O staining: to assess the number and size of lipid droplets in the liver and brown adipose tissue.

[0072] Mice were anesthetized with isoflurane and then perfused with PBS and 4% neutral buffered formaldehyde solution, respectively. Freshly ordered liver and adipose tissue were fixed with 4% paraformaldehyde. The fixed tissues were dehydrated in a gradient of 15% and 30% sucrose solutions, embedded in OCT cryoemulation medium, sectioned, and placed on glass slides. The sections were dried at room temperature and then washed with distilled water. The sections were stained with 0.5% Oil Red O working solution. Differentiation was performed with 75% ethanol until the background was clear, and then rinsed with distilled water. The cell nuclei were counterstained with hematoxylin, rinsed with tap water, and then blued back to the original color. The slides were mounted with glycerol gelatin. After the slides dried, the red staining of lipids and the blue background of the cell nuclei were observed under a microscope.

[0073] As shown in Figure 3, the brown adipose tissue of WT mice contains a large number of lipid droplets. However, the number of lipid droplets in the brown adipose tissue of mice expressing the NPAFP gene is significantly reduced, and the lipid droplet density of NPAFP4 and NPAFP12 is lower than that of NPAFP1. This indicates that NPAFP can reduce the lipid droplet content in brown adipocytes, and its effect is positively correlated with the number of AFPIII domains.

[0074] As shown in Figure 4, the liver tissue of WT mice contained a large number and size of lipid droplets; in contrast, the liver lipid droplet content of NPAFP1, NPAFP4, and NPAFP12 mice was significantly reduced, with the NPAF12 group showing the most significant effect. This indicates that NPAFP protein has an inhibitory effect on liver fat accumulation.

[0075] Example 3: Blood Glucose Level Measurement

[0076] Blood was collected from the tail vein of mice, and blood glucose levels were detected using the glucose oxidase method. Prepare a blood glucose meter, test strips, lancets, and alcohol swabs. Before testing, ensure the mice are fasting (fasting blood glucose test), but allow free access to water to avoid affecting blood glucose levels. Collect blood from the marginal ear vein. Insert the blood glucose test strip into the blood glucose meter. After the meter indicates the presence of blood, drop a sufficient amount of blood onto the sampling end of the test strip. The blood glucose meter will display the blood glucose value within seconds. After blood collection, apply pressure to the puncture site to stop bleeding and observe the mouse for any abnormal reactions. Record the blood glucose results accurately, noting the mouse number and the testing time.

[0077] As shown in Figure 5, the blood glucose levels of NPAFP1 mice were not significantly different from those of the WT group, while the blood glucose levels of NPAFP4 and NPAFP12 mice were significantly lower than those of the WT and NPAFP1 groups, and the blood glucose level of NPAFP12 mice was lower than that of NPAFP4 mice. This indicates that the NPAFP protein regulates blood glucose in a dose-dependent manner, and its effect is closely related to the number of AFPIII functional domains.

Claims

1. The application of NPAFP protein in the preparation of weight loss and blood sugar lowering products, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

2. The application as described in claim 1, wherein the product includes, but is not limited to, health supplements, weight loss foods, or functional beverages.

3. The use of NPAFP protein in the preparation of drugs for treating obesity and / or diabetes, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

4. The application as described in claim 3, wherein the drug can be formulated into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, and / or suppositories.

5. Application of NPAFP protein in constructing animal models of obesity and / or diabetes, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP4 and / or NPAFP12; the nucleotide sequence of the NPAFP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the NPAFP4 protein is shown in SEQ ID NO.4; and the nucleotide sequence of the NPAFP12 protein is shown in SEQ ID NO.

5.

6. The application as described in claim 5, wherein the animals in the animal model include rodents, large mammals, and non-human primates.

7. A method for constructing an animal model of obesity and / or diabetes, wherein the animal model is an NPAFP transgenic mouse expressing different numbers of AFP III functional domains by inserting the NPAFP gene into the H11 gene locus of a mouse using gene editing technology.

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