Irisin polypeptide product and use thereof

Abstract

The present invention provides an irisin polypeptide, as well as its expression nucleic acid, recombinant vector, recombinant cell, pharmaceutical composition, food composition, and use thereof in preparing drugs for treating and preventing metabolic-related diseases in subjects; and the irisin polypeptide plays an obvious role in regulating body metabolism, controlling blood sugar levels, enhancing muscle strength, reducing body weight, and reducing appetite, and can be widely used in the fields of medicine, food, health care products, etc.

Description

Technical Field

[0001] The present invention relates to an irisin polypeptide product and application thereof, belonging to the field of macromolecular medicine and food health care. Background Art

[0002] Metabolic disorders include a range of health disorders or risks that increase the risk of morbidity and loss of quality of life. Examples include diabetes, obesity, including central obesity (disproportionate fat tissue in and around the abdomen), atherogenic dyslipidemia (a range of dyslipidemias, such as high triglycerides, low HDL cholesterol, and high LDL cholesterol, which promote plaque formation in the vascular system, including artery walls), high blood pressure (130 / 85 mmHg or higher), insulin resistance or glucose intolerance (inability to use insulin or blood sugar properly), chronic prothrombotic states (e.g., characterized by high fibrinogen or plasminogen activator inhibitor 1 levels in the blood), and chronic proinflammatory states (e.g., higher than normal levels of high-sensitivity C-reactive protein in the blood).

[0003] Irisin was discovered in 2012. It is a myogenic cytokine that is secreted by skeletal muscle under exercise stimulation and can induce brown cell-like changes in white fat cells. It increases BAT activity, can convert fat types, and increases the metabolic rate of fat cells and muscle cells (A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis, Nature volume 481, pages463–468 (2012)). Although the content of brown adipose tissue in adults is small, its heat production efficiency is extremely high. A small piece of brown fat can burn a lot of calories. The discovery of irisin provides new ideas for the treatment of metabolic disorder-related diseases, obesity, and blood sugar control. Some research results show that the increase in irisin levels in the human body after exercise can promote the browning of white adipose tissue (i.e., the appearance of multi-vacuolar small fat droplets in white fat cells and the increase in UCP1 protein expression), and further determine that PGC-1α can induce muscle cells to secrete specific factors to induce intracellular heat production. Exercise can promote the expression of skeletal muscle PGC-1α, which then stimulates the expression of skeletal muscle fibronectin type III domain protein 5 (FNDC5), which is cleaved and processed to produce irisin. Irisin can promote the expression of related thermogenic genes in white adipocytes by promoting p38 mitogen-activated protein kinase; in addition, exercise-promoted myogenic irisin release can further activate adipose tissue browning through SIRT1-dependent PPAR deacetylation, and its increased level can stimulate WAT browning and enhance energy consumption. These research results demonstrate the potential role of irisin in the treatment of obesity, and can also improve insulin resistance and have a certain protective effect on pancreatic β-cell function. Summary of the invention

[0004] Based on the above technical problems, the present invention provides an isolated irisin polypeptide.

[0005] 1Irisin peptide and its variants

[0006] Including irisin polypeptide, the irisin polypeptide is an irisin parent peptide or a variant peptide thereof, and the variant peptide is obtained by modifying the parent peptide;

[0007] Specifically, the variant peptide is obtained by truncating 1-15 amino acids from the parent peptide, preferably 2-10 amino acids, preferably 3-8 amino acids, preferably 3-6 amino acids;

[0008] Specifically, the variant peptide is based on the parent peptide, with 1-10 amino acids modified, preferably 2-6 amino acids, more preferably 3-5 amino acids, and more preferably 2-5 amino acids modified.

[0009] Specifically, the modification may be modification of positions 19-68 of the parent peptide, preferably modification of positions 21-67, preferably modification of positions 21-63, preferably modification of positions 22-59, preferably modification of positions 21-48, preferably modification of positions 7-61, preferably modification of positions 7-62, preferably modification of positions 23-67, preferably modification of positions 25-68, preferably modification of positions 7-62, preferably modification of positions 7-62, preferably modification of positions 54-68;

[0010] The modification may be a truncation of the N-terminus and / or the C-terminus, or the modification may be a charged amino acid modification; specifically, aspartic acid (Asp, D) may be mutated to alanine (Ala, A), or glutamic acid (Glu, E) may be mutated to alanine (Ala, A), or lysine (Lys, K) may be mutated to glutamine (Gln, Q), or lysine (Lys, K) may be mutated to arginine (Arg, R), etc.

[0011] Specifically, the modification is selected from at least one of E7, D19, E21, E22, D23, E25, E54, K48, K59, K61, D62, E63, K67R, K67, and E68;

[0012] Specifically, the modification is selected from at least one modification of E7A, D19A, E21A, E22A, D23A, E25A, E54Q, K48Q, K48R, K59Q, K61Q, D62A, E63A, K67R, K67Q, E68A;

[0013] Specifically, the modification is selected from at least one combination of

[0014] D19A, D23A, D62A;

[0015] E7A, E21A, E22A;

[0016] E7A, E21A, E22A, E68A;

[0017] E7A, D23A, E25A;

[0018] E7A, D23A, E25A, K61Q, K67Q;

[0019] E7A, D23A, E25A, E54Q, K67Q;

[0020] E7A, E21A, E22A, K59Q, E68A;

[0021] E7A, D23A, E25A, K67R;

[0022] E7A, E25A, K48Q, K67R;

[0023] E7A, D23A, E25A, K48Q, K67R;

[0024] E7A, E21A;

[0025] E7A, E21A, E22A, D62A, E63A;

[0026] E7A, D23A, E25A, K48R;

[0027] E7A, E25A;

[0028] E7A, D23A, E25A, K48Q;

[0029] D19A, D23A, K48Q, D62A;

[0030] The irisin parent peptide is shown in SEQ ID NO: 47, and the above modifications are numbered based on SEQ ID NO: 47 as the parent.

[0031] Specifically, the irisin variant polypeptide can be selected from at least one of SEQ ID NOs: 1-16, or a sequence having 85%, 90% or more identity thereto.

[0032] In another embodiment, the irisin variant polypeptide may further comprise a heterologous polypeptide selected from a signal peptide, a peptide tag, a prolongation fragment, a dimerization domain, an oligomerization domain, an antibody or an antibody fragment, for example, the N-terminal signal peptide shown in SEQ ID NO: 45, the C-terminal fragment domain shown in SEQ ID NO: 46, or the human serum albumin shown in SEQ ID NO: 23, or the Fc fragment shown in SEQ ID NO: 17-18 as the heterologous polypeptide;

[0033] Specifically, the signal peptide shown in SEQ ID NO: 45 can be set at the N-terminus of the irisin peptide, and the C-terminal fragment domain shown in SEQ ID NO: 46 can be set at the C-terminus; specifically, the human serum albumin shown in SEQ ID NO: 23 can be connected to the N-terminus or C-terminus; specifically, the long-acting fragment, such as the Fc fragment shown in SEQ ID NO: 17-18, SEQ ID NO: 40-41, can be directly or through a linker connected to the N-terminus or C-terminus of the irisin peptide to form a fusion molecule comprising the irisin peptide as shown in SEQ ID NO: 19-22.

[0034] In a specific embodiment, the polypeptide has a human irisin sequence.

[0035] The linker may be (GGGS) 3-4 A fusion peptide, preferably a fusion peptide of (GGGS) 4, but it is not used to limit the scope of protection.

[0036] The fusion molecule may be an enhancer that enhances the effect of irisin, or a long-acting fragment, or other functional fragment;

[0037] The long-acting fragment may be selected from Fc, CTP, human serum albumin HSA, transferrin, and the like.

[0038] The fusion molecule comprising irisin peptide may further comprise an enhancer, which may be a cytokine capable of enhancing irisin, including but not limited to FGF-21, GLP-1, IL-6, GM-CSF, IL2, IL12, IL15, IL21, IL23, etc.

[0039] 2 Nucleic Acids

[0040] The present invention further provides a nucleic acid molecule capable of expressing the irisin polypeptide, wherein the nucleic acid molecule may further be a DNA molecule or an RNA molecule, preferably a DNA molecule; the DNA molecule does not need to be particularly limited and can be obtained by conventional techniques in the art according to general codon preference rules;

[0041] If specifically limited, it may be a sequence shown in at least one of SEQ ID NOs: 24-44, or a sequence maintaining 85%, 90% or more identity thereto, but is not limited to the above sequences.

[0042] Nucleic acid molecules also include nucleic acid sequences encoding heterologous polypeptides, as well as nucleic acid sequences encoding fusion molecules.

[0043] 3 Compositions and mixtures

[0044] The present invention further provides a composition or mixture, which comprises at least one of the above-mentioned irisin and its variant polypeptide or fusion molecule; specifically, it can be a composition comprising only irisin variant peptide, or a composition comprising at least one irisin variant peptide and further comprising irisin polypeptide fusion molecule, or a composition comprising only irisin polypeptide fusion molecule; the ratio of the composition does not need to be specifically limited. If it needs to be limited, it can be preferably mixed in equal proportions, for example, an equal ratio of 1:1, or 1:1:1.

[0045] The irisin composition administered to the subject in a therapeutically effective amount may further comprise a composition that increases the plasma or serum level of irisin (e.g., metformin, statins, or a vector containing a nucleic acid encoding FNDC5 and upregulating muscle cell FNDC5 expression), and a therapeutic agent selected from at least one of the following: a chemotherapeutic agent, a HER antibody, an antibody against a tumor-associated antigen, a cardioprotective agent, an anti-hormone compound, a cytokine, an EGFR targeted drug, an anti-angiogenic agent, a tyrosine kinase inhibitor, a COX inhibitor, a nonsteroidal anti-inflammatory drug, a farnesyl transferase inhibitor, an antibody that binds to the oncofetal protein CA125, a HER2 vaccine, a HER targeted therapy, a Raf or RAS inhibitor, doxorubicin (e.g., liposomal doxorubicin), topotecan, a taxane, a dual tyrosine kinase inhibitor, TLK286, and EMD-7200, etc.

[0046] 4 Recombinant

[0047] The present invention further relates to a recombinant comprising an irisin polypeptide, and a recombinant comprising a DNA expressing an irisin polypeptide; the recombinant vector may be a recombinant cloning vector or a recombinant expression vector comprising a DNA molecule expressing the irisin polypeptide; the recombinant bacteria / recombinant cells expressing the irisin polypeptide may also be host cells, and the host cells may be eukaryotic cells or prokaryotic cells (bacterial cells). In other embodiments, the host cells are genetically engineered to express a selection marker.

[0048] The term "recombinant" refers to the preparation, expression, production or isolation of exogenous proteins by recombinant methods based on genetic engineering, such as proteins expressed by recombinant expression vectors transfected into host cells.

[0049] 5. Pharmaceutical Composition

[0050] The present invention further provides a pharmaceutical composition comprising the irisin polypeptide.

[0051] The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, carriers, adjuvants, vehicles. Examples of excipients, carriers, adjuvants, vehicles include, but are not limited to, anti-adherents, adhesives, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, aromatics, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients, carriers, adjuvants, vehicles include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0052] The pharmaceutical composition can be prepared in the form of an injection preparation or an oral preparation. The injection preparations are classified according to physical state, including liquid injections, powders for injection, and tablets for injection; according to the injection part, they are classified into intradermal injections, subcutaneous injections, intramuscular injections, intravenous injections, and spinal cavity injections; preferably, the solvent of the injection preparation includes water for injection or physiological saline.

[0053] In some embodiments, administration comprises administering a composition described herein intramuscularly, intravenously (e.g., in the form of a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, transperitoneally, subcutaneously, subconjunctivally, intracapsularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as a tablet, capsule, caplet, caplet-shaped tablet, or syrup), topically (e.g., in the form of a cream, gel, lotion, or ointment), topically, by inhalation, by injection, or by infusion (e.g., continuous infusion in the form of a cream or lipid composition, local perfusion, catheterization, lavage directly bathing target cells).

[0054] Furthermore, the present invention provides a stable formulation comprising irisin polypeptide, which, in addition to irisin polypeptide, also comprises a surfactant, a stabilizer, a buffer, a filler, etc.; further, the stable formulation may comprise PB, mannitol, sucrose, polysorbate, etc.; the stable formulation may further comprise, for example, 20-50 mM PB, 0.1-1% mannitol, 1-3% sucrose, 0.005-0.05% polysorbate 80.

[0055] 6 Food composition

[0056] The present invention further provides a food composition comprising irisin polypeptide, wherein the food composition has irisin polypeptide as an effective ingredient;

[0057] Specifically, it can be used as a probiotic, a health food, a nutritional supplement, etc.;

[0058] Composition: The food composition can be in the form of a composition that can be prepared in the prior art, including but not limited to solid food, liquid decoction food, sugar, and can also contain amino acids, soy protein powder, whey protein powder, pea protein powder, flavoring, oligofructose, collagen, vitamins, microbial agents, etc. that assist in their functions;

[0059] Stabilizer: It may further include one or more stabilizers selected from edible pectin, propylene glycol alginate, sodium carboxymethyl cellulose, sodium alginate, microcrystalline cellulose, xanthan gum, citric acid monoglyceride, sucrose ester, monostearate, succinic acid monoglyceride, mono- and di-glycerides, sucrose fatty acid ester and unsaturated fatty acid monoglyceride.

[0060] A food composition for preventing or improving metabolic diseases, comprising the above-mentioned irisin peptide;

[0061] A food composition that can be used for reducing body weight, comprising the irisin peptide.

[0062] 7. Uses of Irisin Peptide

[0063] Irisin polypeptides and variants and fusion proteins thereof, and compositions comprising the same, use in treating and preventing metabolic-related diseases, and use in preparing drugs for treating and preventing metabolic-related diseases in subjects;

[0064] Use of irisin polypeptide and its variants, fusion proteins, and compositions containing the same in preparing food compositions; the food compositions have certain health care effects of treating and preventing metabolic-related diseases, and the food compositions have the effect of controlling body weight;

[0065] Irisin polypeptide and its variants, fusion proteins, and compositions containing the same are used in regulating body metabolism, controlling blood sugar levels, enhancing muscle strength, controlling body weight, and controlling appetite.

[0066] The weight control mainly refers to weight loss; the blood sugar level control further refers to the effect of lowering blood sugar, and further refers to the effect of enhancing glucose tolerance; the appetite control refers to reducing appetite.

[0067] In another embodiment, the subject is a human.

[0068] The metabolic disease is caused by changes in irisin expression levels, and is specifically selected from insulin resistance, hyperinsulinemia, obesity, diabetes, type II diabetes, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, fatty liver and arteriosclerosis.

[0069] Beneficial Effects

[0070] The present invention provides a novel irisin variant polypeptide, as well as its expression nucleic acid, recombinant vector, recombinant cell, pharmaceutical composition, and food composition, and provides its use in preparing drugs for treating and preventing metabolic-related diseases in subjects, such as related treatments for diabetes; and it plays a significant role in regulating body metabolism, controlling blood sugar levels, enhancing muscle strength, reducing body weight, and reducing appetite, and can be widely used in the fields of medicines, foods, health products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Western-blot results of irisin peptide preparation and expression.

[0072] Figure 2 Detection of Ucp1 mRNA expression level in mouse white adipocytes.

[0073] Figure 3 Detection of Pgc1α mRNA expression level in mouse white adipocytes.

[0074] Figure 4 Detection of Cidea mRNA expression level in mouse white adipocytes.

[0075] Figure 5 Detection of adiponectin mRNA expression level in mouse white adipocytes.

[0076] Figure 6 Results of fasting blood glucose test in mice.

[0077] Figure 7 Results of the mouse glucose tolerance GTT test.

[0078] Figure 8Results of the forelimb and limb grasping strength test of mice.

[0079] Fig. 9 Mouse body weight monitoring results. DETAILED DESCRIPTION

[0080] Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available conventional reagents and are available through public channels.

[0081] 1 Expression and preparation of irisin variant peptides and fusion proteins

[0082] The WT sequence was retrieved and the protein sequence is shown in SEQ ID NO: 47

[0083] MLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE

[0084] According to the mutation design rules, the DNA sequence and amino acid sequence corresponding to the irisin peptide variant design, as well as the fusion protein DNA sequence and amino acid sequence are obtained.

[0085]

[0086] (Note: The starting point for calculating the mutation position is the N-terminus containing the signal peptide)

[0087] The above exogenous fragments were synthesized to obtain DNA and cloned into the cloning vector pGEM-T-easy ( pGEM ® -TEasyVectorSystems, Promega ) to obtain the pGEM-T-easy-foreign gene recombinant plasmid; the reaction conditions are: 4.0℃ for 16h. The ligation product was transformed into E. coli competent cells TOP10 (TOP10 source: TIANGEN Tiangen Biochemical Technology (Beijing) Co., Ltd. CB104-02TOP10 competent cells), and the plasmid was extracted (the extraction method was according to the kit: TIANGEN Tiangen Biochemical Technology (Beijing) Co., Ltd. DP103-02 Plasmid Mini Extraction Kit), and identified by double restriction digestion with BamHI (NEBR0136L) + HindIII (NEBR0104L) and single restriction digestion with EcoRV (NEBR0195L), and the presence of the target fragment was observed. The positive clones screened were sequenced in both directions (sequencing was completed by Shanghai Sangon Biotechnology Co., Ltd.).

[0088] After the sequenced pGEM-T-easy-foreign gene recombinant plasmid was digested with BamHI and HindIII to recover the target gene, the pET20B (+) plasmid (plasmid source: Shanghai Qincheng Biotechnology Co., Ltd., catalog number QCP0489) was connected with the fragments recovered by BamHI and HindIII double digestion gel. The digestion system was: plasmid 1μg (1μl), BamHI (0.5μl), HindIII (0.5μl), buffer 10X (2μl), H2O16μl. Reaction at 37 degrees for 2 hours. The recombinant expression vector pET20B (+)-foreign gene was constructed.

[0089] Single enzyme digestion (NotI) and double enzyme digestion (EcoRI / BstI) different from the enzyme digestion system were used to verify the accuracy of the construction.

[0090] The constructed recombinant expression vector pET20B (+) -exogenous gene plasmid was heat-shock transformed into E. coli BL21 Rosetta (DE3) PlysS host Escherichia coli, and then cultured on 100 mg / L ampicillin-50 mg / L chloramphenicol-LB plates at 37°C overnight. The transformants were selected: recombinant bacteria pET20B (+) -exogenous gene / E. coli BL21 Rosetta (DE3) PlysS, and cultured in TB medium: glycerol 5g / L, peptone 12g / L, yeast extract 24g / L, K2HPO4 12.54g / L and KH2PO4 2.31g / L at 37°C liquid overnight, then inoculated into TB fermentation liquid medium and cultured at 37°C for 3h, induced with 4mg / L isopropylthio-βD-galactoside, cooled to 24°C and cultured for a certain period of time, and samples were taken to lyse the bacteria to detect protein expression.

[0091] Proteins were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the protein in the bacterial lysate was verified by Western Blot (see results). Figure 1), 1-16 in the figure represent the irisin polypeptides IR230018, IR230027, IR230044, IR230054, IR230055, IR230060, IR230063, IR230070, IR230072, IR230070, IR240113, IR240118, IR240132, IR240133, IR240139, IR240146 shown in SEQ ID NO: 19-21 in Table 1, with a molecular weight of about 23.1 KDa; 17-19 represent the irisin fusion proteins IR240113- IgG1 Fc, IR230072- IgG1 Fc, IR230070- IgG1 Fc, molecular weight is about 49.2KDa (determined by gel electrophoresis under deglycosylation, reduction and denaturation conditions, see Figure 1 The mycelium fermentation broth was centrifuged to separate the fermentation broth supernatant, and the supernatant was purified by Ni-NTA affinity chromatography to obtain a crude human irisin protein, and FPLC protein purification was performed to collect the purified protein with a complete sequence detection peak. The purity of the protein was more than 99.6%, which could be used for medicine and met the quality standards for medicine.

[0092] 2. Animal model construction and group setting

[0093] Experimental animals: SPF-grade healthy male C57 BL / 6NCrj mice (SPF) were purchased from Kaixue Biotechnology (Shanghai) Co., Ltd., weighing 20-25g. After one week of pre-feeding, animals aged 7 / 8 weeks were used in formal experiments. The mice were kept in a laboratory with a temperature control of 23±2℃ and a humidity of 55±10%, and the lighting cycle was lights on (8:00 to 20:00) and lights off (20:00 to 8:00). During the pre-feeding period, the experimental animals were allowed to eat animal feed and sterile water freely. When used for induction experiments, the mice were fasted; the control group was always given a normal diet.

[0094] Streptozotocin (STZ) (purchased from Sigma) was dissolved in 10mM sodium citrate solution (pH5.5) to obtain a 20mg / ml solution. STZ solution was prepared for use and used within 5 minutes after dissolution. For mice pre-fed with a high-sugar and high-fat diet, the prepared STZ solution was administered intraperitoneally at a dose of STZ 100mg / kg to reduce insulin secretion. On the second day, the mice were given STZ solution again. Three days after the second administration of STZ, the blood glucose levels and body weights of all mice were measured, and the animals were grouped according to the results, and random blood glucose levels above 16.7mmol / L were screened as the successful standard for modeling type 2 diabetes. Mice that did not show hyperglycemia and mice that were obviously in poor condition were excluded in this experiment. Thus, mice with induced diabetes model (type II diabetes induced model) were obtained.

[0095] Irisin group: The irisin stock solution was diluted with normal saline in a gradient manner and injected intravenously into the experimental mice at a dose of 0.5 mg / kg / day for 15 consecutive days.

[0096] The mice were randomly divided into groups, with 5 mice in each group.

[0097]

[0098] 3. Detection of white adipocyte biomarkers

[0099] The white adipose tissue of mice was extracted by fat extraction. The mice were placed with their backs facing up. The incision was made along the line connecting the roots of the upper limbs and the skin was cut open. The skin above and below the incision was pinched with both hands and pulled apart on both sides. The symmetrical white fat in the inguinal area was visible. The white fat was digested and separated. The total RNA was extracted by TRIzol (Invitrogen™, 15596026CN) method for isolated cells. The cDNA of each sample was controlled at 20ng using a reverse transcription kit. Real-time PCR (SYBR® Green quantitative RT-qPCR kit, Sigma-Aldrich, QR0100) was used. The reaction volume was 20μl, 10μl of SYBR Premix ExTaqTMII (2×), and 5μl of primer (single). PCR amplification conditions: 95℃ denaturation for 30s, 45 cycles (95℃ 5s, 57℃ 15s, 72℃ 15s), using 2 -DCt The gene expression level was calculated by the method, and the housekeeping gene β-actin was used to detect the expression level of each sample.

[0100] The genes of brown adipose tissue markers, peroxisome proliferator-activated receptor gamma cofactor 1-α (Pgc1α), Ucp1 (Uncoupling protein-1) and Cidea, are the standards for the conversion of white fat into brown fat. This experiment uses the expression levels of UCP1, Pgc1α and Cidea to verify whether the irisin variant polypeptide prepared in the experiment has the browning activity of adipocytes. Adiponectin is a representative adipocyte secreted adipocyte. It has anti-diabetic and anti-obesity effects by improving insulin resistance and other metabolic calculations. As a marker, it can be used to prove that the irisin variant polypeptide prepared in the experiment has the function of controlling systemic metabolism through adipocyte factors. This experiment uses the expression level of adiponectin to verify whether the irisin variant polypeptide prepared in the experiment has the function of regulating metabolism.

[0101] The experimental results are presented as relative values, with the Control group positioned at 1.00, and the other groups compared with it to obtain relative values. The experimental results showed that the expression levels of Ucp1, Pgc1α, and Cidea were significantly decreased in the white fat of diabetic mice, and were significantly increased in the irisin peptide treatment group. The irisin variant peptide was also significantly increased relative to the Control group, and some MT groups were better than the WT group.

[0102] Among the Ucp1 expression results, the increases in IRMT-4, IRMT-5, IRMT-9, and IRMT-13 groups were the most significant, while the increases in IRMT-6, IRMT-7, IRMT-8, IRMT-10, IRMT-11, IRMT-12, and IRMT-14 groups were slightly less, but still higher than the IRWT group. Among them, the increases in IRMT-1, IRMT-2, IRMT-3, IRMT-15, and IRMT-16 groups were lower than the IRWT group.

[0103] Among the results of Pgc1α expression, the increases in IRMT-4, IRMT-5, IRMT-6, IRMT-7, IRMT-9, and IRMT-13 groups were the most significant, the increase in IRMT-8 group was slightly less and was the same as that in IRWT group, and the increases in IRMT-1, IRMT-2, IRMT-3, IRMT-10, IRMT-11, IRMT-12, IRMT-14, IRMT-15, and IRMT-16 were lower than those in IRWT group.

[0104] Among the Cidea expression results, the IRMT-3, IRMT-4, IRMT-9, IRMT-13, and IRMT-14 groups showed the most significant increases, while the increases in the IRMT-5 and IRMT-8 groups were slightly less, but still higher than the IRWT group. Among them, the IRMT-1, IRMT-2, IRMT-6, IRMT-7, IRMT-11, IRMT-12, and IRMT-16 groups were lower than the IRWT group.

[0105] In the results of adiponectin expression levels, all groups were significantly improved compared with the control group, among which IRMT-4, IRMT-5, and IRMT-6 were able to achieve better expression levels (see the results). Figure 2-5 )

[0106] (Note: After testing, there was no significant difference in the data before irisin peptide treatment in each experimental group, before treatment in the diabetic saline treatment group, and after treatment in the diabetic saline treatment group. Figure 2-5 The data of the control group-2 is the data detected synchronously with the experimental group after injection of normal saline)

[0107] The results showed that irisin variant peptides could improve the tissue function of brown fat insulin-deficient diabetic mice and further improve the body's metabolism. In summary, IRMT-4, IRMT-5, IRMT-6, IRMT-9, and IRMT-13 belonged to the group with the best overall performance.

[0108] 4 Fasting blood glucose test (FBG)

[0109] The fasting blood glucose of diabetic mice was detected after irisin treatment. The fasting blood glucose of diabetic mice was also detected. The detection method was as follows: fasting for 6-8 hours without food or water, and the blood glucose level of the tail vein blood of the experimental mice was measured with Antsence Ⅱ blood glucose analyzer (Sankyo, Bayer). The blood glucose level was analyzed by Turkey test. The test results are shown in Figure 6 .

[0110] The results showed that the diabetes-induced model could make the blood glucose of mice reach about 20mmol / L. After treatment with irisin, the fasting blood glucose level could be significantly reduced, basically to 10mmol / L, proving that irisin peptide can effectively control the fasting blood glucose of diabetic mice; specifically, the IRWT group was able to control the blood glucose of diabetic mice at a level of 10mmol / L, the IRMT-4, IRMT-5, IRMT-7, and IRMT-9 groups were able to control the blood glucose of diabetic mice at a level below 9mmol / L, and had better control ability. IRMT-3, IRMT-6, and IRMT-10~15 were able to control the blood glucose of diabetic mice at a level of 9-10mmol / L, and had relatively better control ability. The IRMT-1, IRMT-2, IRMT-8, and IRMT-16 groups controlled the blood glucose of diabetic mice to greater than 10mmol / L, which was equivalent to or slightly inferior to the IRWT group. (Similarly, since there was no significant difference in the values ​​of each irisin-treated experimental group before treatment, the values ​​of the diabetic mouse saline control group before treatment, and the values ​​of the diabetic mouse saline control group after treatment, the figure shows the data of the control group-2 after saline treatment and the experimental group, and the same is true for the rest, so it will not be repeated)

[0111] 5 Glucose tolerance test

[0112] Glucose tolerance test (GTT): After fasting overnight, mice were intraperitoneally injected with glucose (2.0 g / kg), and blood glucose was measured before injection (0 min) and 15 min, 30 min, 60 min, and 120 min after injection. The group with the best fasting blood glucose test was subjected to a glucose tolerance test. The experimental results (see Figure 6 ) It can be seen that compared with the diabetic saline group, the irisin peptide treatment group can effectively enhance the glucose tolerance of mice, and the irisin MT group can basically be on par with or slightly exceed the WT group, ensuring the glucose tolerance of mice.

[0113] 6 Mouse muscle strength test

[0114] The mouse grasping experiment tests the muscle strength of rat limbs, measures the grasping strength of the four limbs, and calculates the average grasping strength of the four limbs (using the Keyue Huacheng rat grasping strength measuring instrument). The experimental results show that the grasping strength of the forelimbs and limbs of diabetic mice is about 30% lower than that of healthy mice, and the grasping strength of diabetic mice is significantly improved by irisin peptide treatment (see Figure 7 ), it can be seen that irisin peptide has a significant effect on improving the muscle strength of diabetic mice.

[0115] 7 Mouse body weight test

[0116] Healthy mice were injected with irisin peptide at a dose of 0.5 mg / kg / single, once every 72 hours (3 days), for a total of 8 injections. The mice were weighed three days after the last injection. The results are shown in Fig. 9 .

[0117] According to the test results, after the injection of irisin peptide, the body weight of healthy mice decreased to varying degrees, with the overall decrease rate between 2% and 8%, which was able to achieve a certain degree of weight loss effect. Among them, the IRMT-5 and IRMT-7 groups decreased the most, but there was no significant difference compared with the IRWT group, and the weight loss ability was limited.

Claims

1. An irisin peptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

4.

2. A nucleic acid molecule encoding the irisin peptide of claim 1, the sequence of which is shown in SEQ ID NO:

27.

3. A recombinant vector comprising the irisin peptide according to claim 1 or the nucleic acid molecule according to claim 2.

4. A recombinant cell comprising the irisin peptide of claim 1 or the nucleic acid molecule of claim 2.

5. A composition comprising the irisin peptide according to claim 1 or the nucleic acid molecule according to claim 2, wherein the composition is a pharmaceutical composition.

6. A composition comprising the irisin peptide according to claim 1 or the nucleic acid molecule according to claim 2, wherein the composition is a health food composition having the function of assisting in lowering blood sugar.

7. Use of the irisin peptide according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 and the composition according to claim 5 in the preparation of a medicament for treating a metabolic-related disease in a subject; the metabolic disease is selected from insulin resistance, hyperinsulinemia, obesity, and diabetes.

8. The use according to claim 7, characterized in that The diabetes is type II diabetes.

9. Use of the irisin peptide according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, the recombinant cell according to claim 4 or the composition according to claim 5 in preparing a health food composition having the function of assisting in lowering blood sugar.

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