Peptides with antidiabetic activity and uses thereof

Abstract

The present invention relates to novel peptides with antidiabetic activity, which improve glucose metabolism and insulin resistance, increase muscle mass while inhibiting muscle atrophy, and protect kidney function, and may therefore be used for the treatment and amelioration of diabetes and diabetic complications.

Description

[Technical Field]

[0001] The present invention relates to peptides having antidiabetic activity and pharmaceutical compositions containing the same for treating diabetes. [Background technology]

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by insufficient or impaired insulin secretion. It is characterized by hyperglycemia (high blood glucose levels), which leads to various symptoms and signs, including excretion of glucose in the urine. Diabetes is classified into insulin-dependent diabetes (type 1 diabetes), non-insulin-dependent diabetes (type 2 diabetes), and malnutrition-related diabetes mellitus (MRDM). Type 2 diabetes, which accounts for more than 90% of diabetes patients in Korea, is a metabolic disease characterized by hyperglycemia. It is reported to develop due to reduced insulin secretion from pancreatic beta cells or increased insulin resistance in peripheral tissues due to genetic, metabolic, and environmental factors. Increased body fat due to obesity is associated with decreased insulin sensitivity, and abdominal fat accumulation in particular is known to be associated with glucose intolerance. Furthermore, obesity and insulin resistance are closely correlated in patients with type 2 diabetes, with the severity of obesity being associated with more severe insulin resistance. Diabetes also causes complications such as angina pectoris, myocardial infarction, diabetic retinopathy, and diabetic nephropathy. Therefore, it is important to treat diabetes by not only controlling blood glucose levels but also managing complications. Summary of the Invention [Problem to be solved by the invention]

[0003] Under these circumstances, the present inventors have discovered a novel functional peptide and confirmed that this peptide has antidiabetic effects that improve insulin resistance and promote glucose metabolism.

[0004] Therefore, an object of the present invention is to provide a peptide having antidiabetic activity and a pharmaceutical composition containing the same for preventing or treating diabetes. [Means for solving the problem]

[0005] To achieve the above object, one aspect of the present invention provides a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14.

[0006] According to one example of the present invention, the amino acid sequences of SEQ ID NOs: 1, 4, 6, 7, and 8 commonly contain the sequence of SEQ ID NO: 1, and the amino acid sequences of SEQ ID NOs: 2, 4, 7, and 8 commonly contain the sequence of SEQ ID NO: 2. Furthermore, the sequences of SEQ ID NOs: 3, 7, and 8 commonly contain the sequence of SEQ ID NO: 3. The sequences of SEQ ID NOs: 5 and 6 commonly contain the sequence of SEQ ID NO: 5.

[0007] The peptides disclosed herein may include peptides having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence homology. Furthermore, the peptides disclosed herein may include peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 8, or fragments thereof, and peptides in which one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, or seven or more amino acids have been changed.

[0008] Meanwhile, mouse sequences corresponding to the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 8 are as shown in Table 1 below.

[0009] [Table 1]

[0010] As shown in Table 1, the sequence of SEQ ID NO: 1 is identical between humans and mice, and is also included in SEQ ID NOs: 10, 12 to 14. Furthermore, the sequence of SEQ ID NO: 9 is included in SEQ ID NOs: 10, 13, and 14. The sequence of SEQ ID NO: 10 is included in SEQ ID NOs: 13 and 14, and the sequence of SEQ ID NO: 11 is included in SEQ ID NO: 12. Meanwhile, the sequence of SEQ ID NO: 3 is also identical between humans and mice, and is included in SEQ ID NOs: 13 and 14. In one embodiment of the present invention, amino acid changes are those that alter the physicochemical properties of the peptide. For example, amino acid changes may be made to improve the thermostability of the peptide, change its substrate specificity, or change the optimal pH.

[0011] As used herein, the term "amino acid" includes not only the 22 standard amino acids naturally incorporated into peptides, but also D-isomers and modified amino acids. Thus, in one embodiment of the present invention, a peptide may contain a D-amino acid. Meanwhile, in another embodiment of the present invention, a peptide may contain a non-standard amino acid that has undergone post-translational modification. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, chemical changes (e.g., beta-elimination deimidation, deamidation), and structural changes (e.g., disulfide bridge formation). Also included are amino acid changes, such as changes in amino groups, carboxyl groups, or side chains, that occur due to chemical reactions that occur during the conjugation process with a crosslinker to form a peptide conjugate.

[0012] The peptides disclosed herein may be wild-type peptides identified and isolated from natural sources. Alternatively, the peptides disclosed herein may be artificial mutants, which contain an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted compared to a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8. Amino acid changes in wild-type polypeptides, as well as artificial mutants, include conservative amino acid substitutions that do not significantly affect protein folding and / or activity. Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine, and methionine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). Amino acid substitutions that generally do not alter specific activity are known in the art. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and their opposites.

[0013] Practical modifications in the biological properties of peptides are achieved by selecting substituents that differ significantly in (a) their effectiveness in maintaining the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical conformation, (b) their effectiveness in maintaining the charge or hydrophobicity of the molecule at the target site, or (c) their effectiveness in maintaining the bulk of the side chain. Natural residues are divided into the following groups based on common side chain properties:

[0014] (1) Hydrophobic: norleucine, met, ala, val, leu, ile,

[0015] (2) Neutral hydrophilicity: cys, ser, thr,

[0016] (3) Acidic: asp, glu,

[0017] (4) Basic: asn, gln, his, lys, arg,

[0018] (5) Residues that influence chain orientation: gly, pro, and

[0019] (6) Aromatics: trp, tyr, phe.

[0020] Non-conservative substitutions are made by exchanging a member of one of these classes for yet another. Any cysteine ​​residue not involved in maintaining the proper conformation of the peptide may generally be replaced with a serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds can be added to the peptide to improve its stability.

[0021] Another type of amino acid variant of a peptide is one that has an altered glycosylation pattern, which may represent the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites not present in the peptide.

[0022] Glycosylation of peptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of the carbohydrate residue to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of the sugar N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0023] Glycosylation sites are conveniently added to the peptide by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such alteration may also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0024] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 5 (an "anti-diabetic peptide").

[0025] The inventors have confirmed that when muscle cells are treated with peptides selected from SEQ ID NOs: 1 to 8, phosphorylation of Ampk and Akt, which promote glucose metabolism, increases, glucose tolerance improves, and lipid metabolism also improves.

[0026] AMPK (AMP-activated protein kinase) is an enzyme that plays an important role in maintaining cellular energy homeostasis and is an AMP sensor that detects the AMP / ATP ratio in cells. When ATP consumption increases ADP and subsequently AMP, AMP binds to the binding site of the AMPK gamma subunit, exposing the catalytic domain of the alpha subunit. AMPKK, an upstream phosphorylation enzyme of AMPK, then phosphorylates threonine-172, activating AMPK. AMPK promotes fatty acid oxidation, glucose influx, and other functions.

[0027] Akt is a protein that is present in the intermediate process of insulin signal transduction, and when activated by phosphorylation, it promotes glucose metabolism.

[0028] Therefore, peptides selected from SEQ ID NOs: 1 to 8 that increase the phosphorylation of Ampk and Akt may be usefully used to improve / treat diabetes.

[0029] The term "prevention" as used in the present invention means any action of suppressing the progression of a disease or delaying its onset by administering the pharmaceutical composition according to the present invention.

[0030] The term "treatment" as used herein means any action in which the symptoms of a disease are ameliorated or beneficially altered by administering a pharmaceutical composition according to the present invention.

[0031] According to one embodiment of the present invention, the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 may be a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 4, 6, 7 or 8.

[0032] Furthermore, the peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 5 may be a peptide consisting of an amino acid sequence selected from the group consisting of 2, 3, and 5, respectively.

[0033] Meanwhile, according to one embodiment of the present invention, the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 have antidiabetic activity. Therefore, the sequences of SEQ ID NO: 10 and 12 to 14, which include the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, may also have antidiabetic activity.

[0034] According to one example of the present invention, the pharmaceutical composition may comprise a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 8, a peptide having 80% or more sequence identity with the amino acid sequence, or a peptide that is a fragment thereof.

[0035] The pharmaceutical composition according to one example of the present invention may be applied to all animals, including humans, dogs, chickens, pigs, cows, sheep, guinea pigs or monkeys.

[0036] The pharmaceutical composition according to an embodiment of the present invention may contain additives such as diluents, excipients, lubricants, binders, boric acid dispersants, buffers, dispersants, surfactants, colorants, flavors, or sweeteners, as needed. The pharmaceutical composition according to an embodiment of the present invention may be manufactured by a conventional method in the art.

[0037] In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0038] Pharmaceutical compositions according to one embodiment of the present invention may be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intraosseously, intrathecally, or subcutaneously, and the like.

[0039] Dosage forms for oral administration may include, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions, while dosage forms for parenteral administration may include, but are not limited to, injections, drops, gels, suspensions, emulsions, suppositories, patches, or sprays.

[0040] The pharmaceutical compositions may be in the form of a sterile injectable preparation as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, any non-irritating, fixed oil, including synthetic mono- or diglycerides, may be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injectable preparations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated versions.

[0041] Parenteral administration of pharmaceutical compositions according to the present invention is particularly useful when the desired treatment involves areas or organs easily accessible by topical application. Carriers for topical administration of the compositions of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.

[0042] The active ingredient of the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the recipient, the pathological condition and its severity, the route of administration, and the discretion of the prescriber. Determining the dosage based on these factors is within the skill of those skilled in the art. The daily dosage may be, for example, 10 ng / kg / day to 10 mg / kg / day, specifically 0.1 μg / kg / day to 1 mg / kg / day, more specifically 1 μg / kg / day to 100 μg / kg / day, and even more specifically 2 μg / kg / day to 50 μg / kg / day. However, if there are differences in efficacy depending on the dose, this may be appropriately adjusted. A pharmaceutical composition according to one embodiment of the present invention may be administered once to three times daily, but is not limited thereto.

[0043] The terminology used herein is intended only for the purpose of describing particular embodiments and is not intended to limit the invention. The use of omitted numbers before a noun does not attempt to limit the quantity, but rather indicates the presence of one or more of the referenced noun item. The terms "comprise," "have," and "contain" are to be construed as inclusive terms (i.e., meaning "including but not limited to").

[0044] Reciting a range of values ​​is merely a shorthand alternative to individually reciting each separate value falling within the range, and unless expressly stated otherwise, each separate value is incorporated herein by the same token as if it were individually set forth in the specification. All range endpoints are included within the range and are independently combinable.

[0045] All methods referred to herein may be performed in any suitable order unless otherwise specified or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as"), unless included in the claims, is merely to better describe the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0046] Another aspect of the present invention provides a method for treating diabetes, comprising administering the pharmaceutical composition for preventing or treating diabetes to an individual in need thereof. The pharmaceutical composition and the method for administering the same are as described above.

[0047] On the other hand, diabetic patients may develop complications such as angina pectoris, myocardial infarction, diabetic retinopathy, and diabetic nephropathy due to prolonged hyperglycemia. In particular, sarcopenia has recently emerged as a diabetic complication (Lancet Diabetes Endocrinol 2013;1:106-14).

[0048] According to one embodiment of the present invention, the antidiabetic peptide has the activity of suppressing the increase in glomerular size caused by diabetes, protecting kidney function, and suppressing fat accumulation in muscle tissue. Therefore, the antidiabetic peptide of the present invention may be used as a pharmaceutical composition for preventing or treating diabetic complications, or a health functional food composition for preventing or ameliorating diabetic complications.

[0049] In the present invention, the diabetic complication may be selected from the group consisting of sarcopenia, diabetic nephropathy, diabetic neuropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, hyperlipidemia, fatty liver, and diabetic foot ulcer.

[0050] According to one embodiment of the present invention, the diabetic complication may be sarcopenia or diabetic nephropathy.

[0051] Another aspect of the present invention provides a method for treating diabetic complications, comprising administering the pharmaceutical composition for preventing or treating diabetic complications to an individual in need thereof. The pharmaceutical composition and the method for administering the pharmaceutical composition are as described above. [Effects of the Invention]

[0052] The peptide of the present invention improves glucose metabolism, ameliorates insulin resistance, increases muscle mass, and suppresses muscle atrophy in diabetic and obese mouse models. It also regulates lipid metabolism and energy metabolism factors in muscle cells and adipocytes, suppresses diabetes-induced increases in glomerular size, protects kidney function, and suppresses fat accumulation in muscle tissue, demonstrating therapeutic and ameliorative effects on diabetes and diabetic complications. [Brief explanation of the drawings]

[0053] [Figure 1] Muscle cells (L6 skeletal muscle cell line) were treated with antidiabetic peptides (No. 1-5, 7-8), and changes in Ampk phosphorylation were observed. [Figure 2] Muscle cells (L6 skeletal muscle cell line) were treated with antidiabetic peptides (No. 1 to 8), and changes in Akt phosphorylation were observed. [Figure 3] Muscle cells (C2C12 skeletal muscle cell line) were treated with antidiabetic peptides (No. 1 to 8) and insulin, and then changes in glucose uptake were observed (*p<0.05 compared with control). [Figure 4](Left) Time course of blood glucose changes observed after administration of antidiabetic peptide (No. 8) to an obese mouse model, and (right) quantitative graphs of blood glucose changes (*p<0.05 compared with control). [Figure 5] This is a quantitative graph showing the degree of improvement in glucose tolerance after administration of antidiabetic peptide (No. 1) to an obese mouse model. [Figure 6] After administering antidiabetic peptides (No. 7 and 8) to a diabetic mouse model, blood glucose metabolism-related indicators were examined, comparing (A) HbA1c (glycated hemoglobin) levels, (B) HbA1c change, (C) fasting blood glucose, (D) insulin levels, and (E) relative HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) values ​​(* p<0.05 compared with control). [Figure 7] The changes in muscle mass were confirmed after administration of antidiabetic peptides (Nos. 7 and 8) to a diabetic mouse model (*p<0.05 compared with control). [Figure 8] Muscle cells (L6 skeletal muscle cell line) were treated with antidiabetic peptides (No. 3, 6, and 7), and changes in S6 phosphorylation were observed. [Figure 9] Muscle cells (L6 skeletal muscle cell line) were treated with antidiabetic peptides (No. 1 to 8), and changes in FoxO1 phosphorylation were observed. [Figure 10] After treating muscle cells (L6 skeletal muscle cell line) with antidiabetic peptides (No. 1, 3, and 8), changes in the expression of PGC-1α and LPL genes, which improve lipid metabolism, were observed. [Figure 11] The anti-diabetic peptides (No. 7 and 8) were administered to a diabetic mouse model, and changes in blood renin and creatinine were confirmed (*p<0.05 compared with control). [Figure 12] After administering antidiabetic peptides (No. 7 and 8) to a diabetic mouse model, fat was stained in muscle tissue. (Left) A representative image of fat staining was shown, and (Right) a graph showing the quantification of fat area. (**p<0.01 compared with control). [Figure 13] Changes in glomerular size after administration of antidiabetic peptides (No. 7 and 8) to a diabetic mouse model were confirmed by histological staining. (Left) Representative images of glomerular staining, and (Right) Quantitative graphs of glomerular area (**p<0.01 compared with control). DETAILED DESCRIPTION OF THE INVENTION

[0054] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0055] peptide The anti-diabetic peptides discovered in this invention are listed in Table 2 below.

[0056] [Table 2]

[0057] Example 1: Confirmation of activation of glucose metabolism promoting signal pathway in muscle cells L6 skeletal muscle cell line cells were cultured in 6-well plates and starvated with 0.5% FBS. They were then treated with anti-diabetic peptides at a concentration of 100 nM. After 1 hour, cells were harvested, proteins were isolated, and activation of the Amp-activated protein kinase (AmpK) and Akt signaling pathways was confirmed by Western blot analysis.

[0058] As a result, it was found that treatment with antidiabetic peptides increased the phosphorylation levels of both Ampk (Figure 1) and Akt (Figure 2) (Figures 1 and 2), confirming the glucose metabolism-promoting function of antidiabetic peptides.

[0059] To further verify the glucose metabolism-enhancing effect of antidiabetic peptide treatment, we measured the plasma membrane expression of the glucose receptor GLUT4 (glucose transporter type 4), which plays a key role in glucose uptake. GLUT4 is known to translocate from the cytoplasm to the plasma membrane and increase its expression level to promote glucose metabolism. To confirm this, C2C12 skeletal muscle cells were starvated with 0.5% FBS and then treated with peptides or a positive control (insulin) at 100 nM for 1 hour. The cells were fixed with 4% paraformaldehyde and incubated with a GLUT4-specific antibody at 37°C for 30 minutes. Then, the cells were reacted with o-phenylenediamine (OPD) solution and the absorbance was measured and compared with the control group. Significant GLUT4 translocation was observed in all peptide-treated groups, including insulin, compared with the control group, demonstrating the enhanced glucose uptake effect of antidiabetic peptides (Figure 3).

[0060] Example 2: Confirmation of improvement in impaired glucose tolerance 2-1. Construction of an obese mouse model Five-week-old male C57BL / 6N mice were provided by Orient Bio and were used after a one-week adaptation period, during which they were provided with a normal diet. The rearing environment was maintained at 18-24°C and humidity at 50-60%, and the mice had free access to food and water during both the adaptation and experimental periods. After the one-week adaptation period, obesity was induced in the mice using a high-fat diet to induce glucose intolerance. The normal and high-fat diets were prepared and provided as shown in Table 3 below.

[0061] [Table 3]

[0062] 2-2. Check for improvement in glucose tolerance Obesity-induced mice were divided into a control group and an experimental group. All mice were fasted for 12 hours. The control group received PBS, while the experimental groups received peptide No. 1, 7, or 8 (2.5 mg / kg). One hour later, 20% glucose solution was intraperitoneally injected. Blood samples taken at each time point were analyzed using a blood glucose analyzer. Compared to the control group, the experimental group administered peptide No. 8 showed a significant improvement in blood glucose metabolism after baseline correction (Figure 4, left). Similarly, the time-dependent change in blood glucose (GLUAUC) after baseline correction was quantified and compared. The peptide corresponding to SEQ ID NO: 7 reduced blood glucose change by 31.1% compared to the control group, demonstrating that antidiabetic peptides improve glucose tolerance.

[0063] Example 3: Confirmation of antidiabetic efficacy (in vivo) 3-1. Construction of a diabetic mouse model We investigated whether antidiabetic peptides could ameliorate diabetes induced by leptin receptor deficiency. Five-week-old male C57BLKS / J-db / db mice were provided by the Central Laboratory Animal Center and used as an animal model after a one-week adaptation period, following a normal diet. The animals were maintained at 18-24°C and 50-60% humidity, with free access to food and water throughout the adaptation and experimental periods. After completing the adaptation period, mice were divided into a control group (intraperitoneal injection) and a peptide-treated group. The control group received PBS, while the peptide-treated group received antidiabetic peptides (No. 7 and 8; 2.5 mg / kg) intraperitoneally for a total of 8 weeks.

[0064] 3-2. Confirmation of antidiabetic efficacy (tissue examination) After 8 weeks, various tests were performed in the diabetic mouse model.

[0065] Blood tests were performed to examine blood glucose metabolism-related indicators. Measurement of HbA1c (glycated hemoglobin) levels and changes revealed a significant decrease in the peptide-treated group (Figures 6A and 6B), and fasting blood glucose and insulin levels were also observed to be reduced (Figures 6C and 6D). Furthermore, to confirm whether diabetes-induced insulin resistance was improved, the level of HOMA-IR (Homeostatic Model Assessment for Insulin Resistance), an indicator of this, was examined and found to be reduced by peptide treatment (Figure 6E). The results in Figure 6 demonstrate that antidiabetic peptides generally improve diabetes-related blood indicators.

[0066] On the other hand, the progression of diabetes leads to various complications. Muscle tissue is the organ that plays the most important role in glucose metabolism in the human body, and the progression of diabetes can lead to muscle loss and weakening of muscle function. Therefore, by observing changes in muscle tissue, we found that peptide treatment increased muscle mass (quadriceps) (Figure 7).

[0067] Example 4: Confirmation of antidiabetic efficacy (in vitro) 4-1. Verification of muscle biosynthesis and muscle atrophy suppression effects L6 skeletal muscle cells were cultured in a 6-well plate and starvated with 0.5% FBS. They were then treated with an anti-diabetic peptide at a concentration of 100 nM for 1 hour, and proteins were collected and subjected to Western blotting.

[0068] We observed that peptides increased the phosphorylation of S6 (mTOR marker) (Nos. 3, 6, and 7, Figure 8), a key indicator of muscle biosynthesis that is known to activate cell signaling pathways, as well as FoxO1 (Figure 9), which exerts muscle atrophy suppression functions. This confirmed the efficacy of peptides in improving muscle loss by promoting muscle biosynthesis and suppressing muscle atrophy. Specifically, peptide No. 1 increased S6 protein phosphorylation by 188%, No. 2 by 365%, No. 4 by 224%, No. 5 by 494%, and No. 8 by 414%. Furthermore, peptide No. 1 increased FoxO1 phosphorylation and also reduced total FoxO1 expression.

[0069] 4-2. Confirmation of lipid metabolism-related gene expression After treating muscle cells with anti-diabetic peptides (No. 1, 3, and 8), changes in the expression of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and LPL (lipoprotein lipase) genes, which are indicators of improved energy metabolism, lipid metabolism, and glucose metabolism, were confirmed by RT-PCR.

[0070] As a result of confirmation, it was found that the expression of PGC-1α and LPL genes was increased by treatment with antidiabetic peptides (FIG. 10).

[0071] Example 5: Verification of the effect of improving diabetic complications To confirm the efficacy of peptide No. 7 or 8 in improving diabetic complications in the db / db diabetic mouse model, various tissue analyses were performed. First, to confirm the muscle loss-related effects, RNA was isolated from muscle tissue and the expression levels of muscle loss-related genes were examined. Treatment with peptide No. 7 or 8 reduced the expression of AP2 (adipocyte protein 2) and C / EBPα (CCAAT enhancer binding protein α), genes associated with lipid accumulation, and increased the expression of PPARα (peroxisome proliferator-activated receptor α), a gene associated with fatty acid oxidation. Furthermore, a reduction in the Murf-1 (Muscle RING Finger-1) gene, which contributes to muscle loss and atrophy, further confirmed the muscle loss-reducing effect (Table 4).

[0072] [Table 4]

[0073] Blood tests observed renin and creatinine, which are excreted as kidney function declines, and confirmed that anti-diabetic peptide treatment significantly reduced renin and creatinine levels (Figure 11).

[0074] H&E tissue staining was performed to confirm the final tissue pathology. The results showed that treatment with antidiabetic peptides improved lipid accumulation in muscle tissue associated with diabetes (Figure 12, left), and that this was quantitatively significant (Figure 12, right). Furthermore, treatment with antidiabetic peptides inhibited the increase in size of glomeruli due to overload in the kidney (Figure 13, left), and that this reduced size quantitatively and significantly (Figure 13, right). The results of this example verified the effectiveness of antidiabetic peptides in improving diabetic complications.

Claims

1. A peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 7 and 8.

2. A polynucleotide encoding the peptide of claim 1.

3. A pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 6, 7 and 8.

4. The pharmaceutical composition for preventing or treating diabetes according to claim 3 , wherein the peptide improves insulin resistance.

5. The pharmaceutical composition for preventing or treating diabetes according to claim 3 , wherein the peptide promotes glucose metabolism.

6. The pharmaceutical composition for preventing or treating diabetes according to claim 3 , wherein the peptide promotes cellular glucose uptake.

7. The pharmaceutical composition for preventing or treating diabetes according to claim 3, wherein the peptide promotes muscle biosynthesis or inhibits muscle atrophy.

8. A pharmaceutical composition for preventing or treating diabetic complications, comprising a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 6, 7 and 8 as an active ingredient.

9. 9. The pharmaceutical composition for preventing or treating diabetic complications according to claim 8, wherein the diabetic complication is selected from the group consisting of sarcopenia, diabetic nephropathy, diabetic neuropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, hyperlipidemia, fatty liver, and diabetic foot ulcer.

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