KR101689803B1 - Composition for preventing or treating of diabetes or obesity containing Apelin-16 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes or obesity comprising apelin-12, apelin-16 or apelin-36 as an active ingredient. More particularly, the resent invention relates to a composition for preventing or treating diabetes or obesity comprising apelin-12, apelin-16 or apelin-36 which stimulates secretion of insulin caused by a high concentration of sugar. According to the present invention, apelin-12, apelin-16 or apelin-36 shows not only the effects of reducing blood cholesterol, triglyceride levels and blood sugar through the stimulation of secretion of insulin but also excellent anti-diabetic and anti-obesity effects, including inhibition of fat accumulation, fatty liver and kidney damages, and thus is useful as an agent for preventing or treating diabetes, diabetes complications or obesity.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating diabetes or obesity comprising apelin-16 as an active ingredient.

The present invention relates to a composition for preventing or treating diabetes or obesity comprising apelin-12, apelin-16 or apelin-36 as an active ingredient And more particularly to a composition containing apelin-12, apelin-16 or apelin-36, which further promotes insulin secretion by high glucose concentration and exhibits a synergistic effect, The present invention relates to a composition for preventing or treating diabetes or obesity.

Type 2 diabetes is a disease that affects patients' mortality by inducing various complications due to the inadequate utilization of glucose absorbed into the body. In practice, it is difficult to control blood sugar, which is the most important part of diabetes treatment. Only about 40% of diabetic patients have reached the target of HbA1c of 7.0% or less. In recent years, incretin hormone-based therapeutics have been widely used to treat such diabetes.

Incretin hormone is a hormone secreted from the small intestine by ingesting food, helping to secrete insulin from the pancreas and is recently seen as a new alternative to diabetes treatment. The most important advantages of treatment with incretin hormone are that it has few side effects such as hypoglycemia and weight gain, which are the blind points of insulin hormone therapy, and also beta cell of pancreas damaged by diabetes is regenerated, to be. Glucagon-like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP) are known to be produced and secreted in the intestine in response to nutrients. GLP-1 and GIP are glucose-dependent methods that lower blood glucose levels through increased insulin secretion and decreased secretion of glucagon, delay gastric emptying rate, increase satiety and thus not affect weight gain Lt; / RTI > However, since both hormones are rapidly degraded by dipeptidyl peptidase-4 (DPP-4), they are difficult to use for therapeutic purposes.

To overcome this problem, GLP-1 analogs that have not been degraded by DPP-4 inhibitors or DPP-4 have been developed. The DPP-4 inhibitor is known to inhibit the degradation of active GLP-1 by nutrients, improving HbA1c by up to 0.7%, and the GLP-1 analogue by up to 1% in HbA1c. However, the presently used GLP-1 analogue or DPP-4 inhibitor has been reported to have low efficacy or risk of pancreatitis or pancreatic cancer, and thus a new therapeutic agent for diabetes needs to be developed.

It is known that Akt, AS (Akt substrate) 160, PKCzeta, etc. are mainly involved in the regulation of glucose control and act mainly on the transport of glucose transporter protein. In addition, the pathogenesis at the molecular level for glucose, sugar, and urine is considered to be the cause of the disease rather than the defect of the insulin receptor itself, since the signal transduction between proteins through the receptor-mediated process, especially the PI-3 kinase (phosphoinositide-3 kinase) have. Therefore, since the therapeutic effect of insulin on diabetes is limited, it is important to discover new target proteins and their molecular mechanisms related to glucose control.

Accordingly, the present inventors have made intensive efforts to develop new type 1 and type 2 diabetes, diabetic complications, or obesity treatment drugs targeting blood glucose control. As a result, apelin-12, apelin- 16 or apelin-36 promotes insulin secretion, and in particular, shows a synergistic effect of insulin secretion under hyperglycemic conditions, thereby completing the present invention.

It is an object of the present invention to provide a method for the treatment of diabetes mellitus comprising diabetes mellitus comprising apelin-12, apelin-16 or apelin-36 which can regulate blood glucose by promoting insulin secretion, And to provide a pharmaceutical composition and food for preventing or treating complications or obesity.

The present invention relates to a pharmaceutical composition for preventing or treating diabetes, diabetic complications or obesity comprising apelin-12, apelin-16 or apelin-36 as an active ingredient. Lt; / RTI >

The present invention also relates to a method for the prevention or amelioration of diabetes, diabetic complications or obesity comprising as an active ingredient apelin-12, apelin-16 or apelin-36 Food.

The apelin-12, apelin-16 or apelin-36 according to the present invention, through the stimulation of insulin secretion, results in a reduction in blood cholesterol, triglyceride levels and blood glucose In addition, it exhibits excellent antidiabetic and anti-obesity effects such as fat accumulation, inhibition of fatty liver and kidney damage, and thus is very useful as a preventive or therapeutic agent for diabetes, diabetic complication or obesity.

1 is a graph showing the amount of insulin after administration of apelin-16 to pancreatic beta cell line (INS-1).
Figure 2 shows the amount of insulin measured after treatment with apellin-16 in islets isolated from animals.
FIG. 3 shows the effect of increasing apelin-16 induced insulin secretion under high glucose (25 mM glucose) conditions.
FIG. 4 shows the observation of fat accumulation in the abdomen, epididymis, liver, and kidney after administering Apelin-16 to a mouse model of high fat diet.
FIG. 5 is a graph showing the levels of proteinuria after administering apelin-16 to a mouse model in which obesity was induced by high fat diet.
FIG. 6 shows the measurement of insulin levels after administering various types of apelin (36, 16, 12) to the pancreatic beta cell line (INS-1).
FIG. 7 shows the treatment of various types of apellins (36, 16, 12) in islets isolated from animals and then measuring the amount of insulin.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Apelin (APLN) is a human APLN Is an endogenous ligand for the G-protein-coupled APJ receptor (Lee et al., J. Neurochem . , Vol . 251 (2): 471-6, 1988) as a peptide encoded by a gene (Tatemoto K et al . Kleinz et al., Pharmacol . Ther . 107 (2): 198-211, 2005, pp . 74 (1): 34-41, 2000; Szokodi et al., Circ . ). The apelin gene encodes a 77 amino acid preproprotein with a signal peptide at the N-terminal site. After the signal peptide is cleaved and translocated to the endoplasmic reticulum, the 55 amino acid proprotein apelin produces several active fragments, the 36 amino acid peptides corresponding to SEQ ID NOS: 42-77 are Apelin-36, The 17 amino acid peptides corresponding to 77 were named Apelin-17 and the 13 amino acid peptides corresponding to SEQ ID NOS: 65-77 were named Apelin-13. Recently, 46 different apelin peptides from Apelin-55 (proaphelin) to Apelin-12 have been identified (Mesmin C et al., J Proteome Res 10 (11): 5222-31, 2011)

Apelin / APJ pathway is widely expressed in cardiovascular and apelinin has beneficial cardiovascular effects in preclinical models. Administration of acute apelin in humans causes peripheral and coronary vasodilation and increases cardiac output. APJ efficacy is emerging as a therapeutic target for patients with heart failure.

Apelin has also been identified as one of the new myokine proteins (A Basse-Patin et al., Int J Obes . 38 (5): 707-13, 2014). Myocaine is an active substance expressed or synthesized and secreted from skeletal muscle by physical activity , i.e., exercise (Pedersen et al., Journal of Applied Physiology, 103 (3): 1093-98, 2007). IL-6 is known as a representative mycaine, and this myocaine improves immunity, prevents weight control and arteriosclerosis. Therefore, it can be understood that the effect of preventing diabetes or obesity by exercise is due to the secretion of myocaine such as apelin.

In the present invention, islets isolated from the pancreatic beta cell line and mouse were treated with apelin-12, -16, -36, or apelin-12, -16, and -36 under high glucose conditions to confirm insulin secretion promotion. In addition, by confirming the effect of inhibiting lipid accumulation and renal damage, it was confirmed that apelin-12, -16, and -36 were effective for treating or preventing diabetes, obesity or kidney damage.

Accordingly, the present invention relates to a pharmaceutical composition comprising diabetes mellitus, diabetic complications or obesity, which comprises, as an active ingredient, apelin-12, apelin-16 or apelin- And to a pharmaceutical composition for preventing or treating such diseases.

In the present invention, the diabetes mellitus may be a type 1 diabetes mellitus or an early type 2 diabetes mellitus. The diabetic complication is selected from the group consisting of diabetic nephropathy, diabetic retinopathy, hyperlipidemia, fatty liver, arteriosclerosis and hypertension The present invention is not limited thereto.

In the present invention, the apelin-12, apelin-16 or apelin-36 may be characterized as promoting insulin secretion. Preferably, the apelin- May be characterized by having a synergistic effect for further promoting insulin secretion by the high-concentration saccharide.

In the present invention, the above-mentioned apelin-12, apelin-16 or apelin-36 may be characterized by decreasing total cholesterol or triglyceride in blood And further, the fat accumulation can be suppressed.

In the present invention, the above-mentioned apelin-12, apelin-16 or apelin-36 may be characterized by inhibiting blood glucose due to an increase in insulin secretion And is also characterized by preventing renal toxicity caused by hyperglycemia.

The composition containing apelin-12, apelin-16 or apelin-36 of the present invention can be prepared by adding appropriate carriers, excipients or diluents according to conventional methods . Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, specialty, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing apelin-12, apelin-16 or apelin-36 of the present invention may be formulated into a powder, granule, capsule, , A suspension, a solution, an emulsion, a syrup, a sterilized aqueous solution, a non-aqueous solution, a suspension, a freeze-drying agent, and a suppository.

In the case of formulation, a diluent or excipient such as a filler, a weighting agent, a binder, a wetting agent, a disintegrant, a surfactant, and the like is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 60, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at a daily dose of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. The administration may be carried out once a day, or in several divided doses orally. The dose is not intended to limit the scope of the invention in any way.

The composition of the present invention can be used alone or in combination with methods for the prevention or treatment of diabetes, diabetic complication or obesity, or using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In another aspect, the present invention provides a method of preventing diabetes, diabetic complications or obesity comprising as an active ingredient apelin-12, apelin-16 or apelin-36 Or improving food.

The food of the present invention can be manufactured in all forms such as functional food, nutritional supplement, health food and food additives. For example, as a health food, Rad51 of the present invention can be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated, and powdered. Functional foods also include beverages (including alcoholic beverages), fruits and processed foods (such as canned fruits, bottled, jam, marmalade, etc.), fish, meat and processed foods such as ham, sausages, , Breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, sugar, dairy products such as butter, chitzs, edible vegetable fats, margarine, vegetable protein, , Frozen foods, various kinds of seasonings (for example, soybean paste, soy sauce, sauce, etc.) by adding the apelin of the present invention.

The health functional food also includes various forms of functional food, nutritional supplement, health food, food additive and the like as a food composition, and it can be prepared in various forms according to a conventional method known in the art, for example, 12, apelin-16, or apelin-36 in the form of tea, juice, or drink, granulating, encapsulating, And adding the extract to various foods such as beverage, fruit and processed food, fish oil, meat and processed foods thereof, breads, noodles, seasonings, and the like.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Apelin -16 Insulin secretion measurement

In this Example, in order to confirm increase of insulin secretion by apelin-16, islets isolated from pancreatic beta cell line (INS-1) (ATCC, American type culture collection) -16, and the amount of insulin was measured.

1-1: Insulin secretion in pancreatic beta cell line (INS-1)

2 x 10 ⁶ pancreatic beta cell lines (INS-1) were inoculated into 6-well plates and cultured in RPMI 1640 medium containing 6 mM glucose for 3 days. The cultured pancreatic beta cell line (INS-1) was washed twice with glucose-free KRBS buffer, and 2.5 mM glucose (negative control), 25 mM glucose (positive control) and 10 ng / ml apelin- Diluted and each treated for 1 hour. Then, in order to measure the secreted insulin amount, 1 ml of buffer treated in each cell was taken and the amount of insulin was measured by enzyme immunoassay.

As a result, it was confirmed that insulin secretion was slightly increased by apelin-16 as compared with the control group (2.5 mM glucose-treated group) (Fig. 1).

1-2: Insulin secretion in islets isolated from mouse

The collagenase P solution was injected into the common bile duct of the mouse (Central laboratory animal, Korea), and then the pancreas was removed. The extracted pancreas was reacted in a 37 ° C water bath for 13 minutes and 20 seconds, then filled with 5% FBS-HBSS and centrifuged to remove supernatant. The pellet was resuspended in cold FBS-HBSS, and then 6 ml of different gradients were mixed and centrifuged. Centrifuged, suspended in 10 ml of RPMI1640 medium, and inoculated into 100 mm culture dishes. Insulin secretion was measured in the same manner as in Example 1-1 with respect to the islets of the mice cultured separately.

The islets isolated from the mouse were washed twice with glucose-free KRBS buffer and diluted in KRBS buffer (pH 7.4) to give 2.5 mM glucose (negative control), 25 mM glucose (positive control) and 10 ng / ml Apelin-16 Was treated for 1 hour. Then, in order to measure the secreted insulin amount, 1 ml of buffer treated in each cell was taken and the amount of insulin was measured by enzyme immunoassay.

As a result, it was confirmed that apelin-16 increased insulin secretion as compared with the negative control group (2.5 mM glucose-treated group) and the positive control group (25 mM glucose-treated group) (Fig. 2).

Example  2: Insulin secretion induced by high concentration of glucose Apelin Check the effect of -16

In this example, pancreatic beta cell line (INS-1) was cultured in high glucose (25 mM glucose) to confirm insulin secretion by apelin-16 under high glucose concentration.

Insulin secretion was measured in the same manner as in Example 1-1 by treating the pancreatic beta cell line (INS-1) with 10 ng / ml Apelin-16 for 1 hour in each of 2.5 mM glucose condition and 25 mM glucose condition.

As a result, it was found that when apelin-16 was treated at 25 mM glucose condition, insulin secretion was significantly increased as compared with the case where apelin-16 was treated at 2.5 mM glucose (Fig. 3). In other words, the synergistic effect of apelin-16 was confirmed in the insulin secretion induced by high glucose concentration.

Example  3: Apelin -16 to inhibit fat accumulation

In this example, in order to confirm the effect of apelin-16 on obesity, apelin-16 was administered to abdominal cavity of obese-induced obesity-induced mouse, and blood test and fat accumulation in organ were observed . High-fat diet C57BL / 6 mice were reported to develop early stage type 2 diabetes ( Diabetologia 47: 1541-1549, 2004) and were therefore selected as the hyperglycemic animal model of this example.

Six weeks old C57BL / 6 mice were fed with a general diet for 2 weeks and fed 60% high fat diet for 12 weeks from 8 to 20 weeks of age. Ten rats were fed high-fat diets (HFD), while the remaining 10 rats were fed naproxen (ntra-peritoneal) for 12 weeks with aphelin-16 (0.5 μg / g / day) at the same time as the high fat diet (HFD). Twelve weeks old mice that had received 12 weeks of high fat diet and apelin administration were subjected to blood tests and laparotomy.

The results of blood tests showed that total cholesterol (CHO), glucose (GLU) and triglyceride (TG) levels increased in the high fat diet group compared to the control group, while total cholesterol (CHO), glucose GLU) and triglyceride (TG) levels were lower than those of the high fat diet group. In addition, insulin levels were increased in the group treated with apelin-16 compared to the high-fat group (Table 1). That is, it shows that the concentration of sugar is decreased by promoting insulin secretion by apelin-16.

Blood chemistry CHO (mg / dL) GLU (mg / dL) TG (mg / dL) Insulin (uIU / ml) Normal 95.53 301.15 61.04 3.19 HFD 150.70 352.06 102.76 4.70 HFD + Apelin-16 125.23 304.43 82.76 4.99

Next, mice were observed to observe abdominal obesity and fatty liver due to accumulation of fat around the abdomen, epididymal, liver and kidney in the high fat diet group, and the group administered with apelin-16 It was confirmed that the accumulation of fat was suppressed in various organs (Fig. 4). In other words, apelin had an inhibitory effect on fat accumulation.

Example  4: Apelin -16 to confirm the effect on kidney damage

The effect of apelin-16 on diabetic complications, such as diabetic nephropathy, was confirmed by prolonged diabetes with damage to renal tissue and proteinuria due to hyperglycemia. High-fat diet of Example 3 Since C57BL / 6 mice are obese-induced hyperglycemic animal models, the effect on diabetic nephropathy was confirmed in the same experimental group.

Urinary albumin concentration (UAC) was measured one week before the sacrifice of 20 week old high-fat diet and apelin-treated 20-week-old mice in Example 3 by taking urine.

As a result, the albumin level in the urine of the mouse group induced by obesity was higher than that of the mouse (control group), whereas the albumin level in the urine of the mouse group treated with apelin-16 decreased to almost the same level as the control (Fig. 5). It can be seen that apelin-16 promotes insulin secretion and reduces the sugar level to some extent to prevent kidney damage caused by high concentrations of sugar.

Example 5: Comparison of various kinds of Apelin effect

To compare the increase in insulin secretion by various kinds of apelin, the present inventors measured the amount of insulin after depleting various kinds of apelin in islets isolated from pancreatic beta cell line (INS-1) and mouse.

5-1: Insulin secretion in pancreatic beta cell line (INS-1)

2 x 10 ⁶ pancreatic beta cell lines (INS-1) were inoculated into 6-well plates and cultured in RPMI 1640 medium containing 6 mM glucose for 3 days. The cultured pancreatic beta cell line (INS-1) was washed twice with glucose-free KRBS buffer and then incubated with 2.5 mM glucose (negative control), 25 mM glucose (positive control) and 10 ng / ml Apelin- -12 were diluted in KRBS buffer and treated for 1 hour each. Then, in order to measure the secreted insulin amount, 1 ml of buffer treated in each cell was taken and the amount of insulin was measured by enzyme immunoassay.

As a result, it was confirmed that the insulin secretion was increased by apelin compared to the control group (2.5 mM glucose-treated group), and apelin-16 was found to have the best insulin secretion ability (Fig. 6). This means that the amino acid sequence between apelin-12 and apelin-16 is important for insulin secretory ability.

5-2: Insulin secretion in islets isolated from mouse

The collagenase P solution was injected into the common bile duct of the mouse and the pancreas was removed. The extracted pancreas was reacted in a 37 ° C water bath for 13 minutes and 20 seconds, then filled with 5% FBS-HBSS and centrifuged to remove supernatant. The pellet was resuspended in cold FBS-HBSS, and then 6 ml of different gradients were mixed and centrifuged. Centrifuged, suspended in 10 ml of RPMI1640 medium, and inoculated into 100 mm culture dishes. Insulin secretion was measured in the same manner as in Example 5-1 by using the islets of the thus separated and cultured mice.

The islets isolated from the mouse were washed twice with glucose-free KRBS buffer and then diluted with 2.5 mM glucose (negative control), 25 mM glucose (positive control) and 10 ng / ml Apelin-36, -16, Diluted in KRBS buffer and treated for 1 hour each. Then, in order to measure the secreted insulin amount, 1 ml of the buffer treated in each cell was taken and the amount of insulin was measured by enzyme immunoassay.

As a result, it was confirmed that the insulin secretion was increased by apelin compared to the negative control group (2.5 mM glucose treated group) and the positive control group (25 mM glucose treated group), and apelin-16 was relatively insulin secretory (Fig. 7). This means that the amino acid sequence between apelin-12 and apelin-16 is important for insulin secretory ability.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A pharmaceutical composition for preventing or treating diabetes, diabetic complication or obesity, comprising apelin-16 as an active ingredient.
The composition of claim 1, wherein the diabetes is type 1 diabetes or an early type 2 diabetes.
The composition according to claim 1, wherein the diabetic complication is one or more diseases selected from the group consisting of diabetic nephropathy, diabetic retinopathy, hyperlipidemia, fatty liver, arteriosclerosis and hypertension.
The composition of claim 1, wherein the apelin-16 promotes insulin secretion.
The composition of claim 1, wherein the apelin-16 reduces blood total cholesterol or triglyceride.
The composition of claim 1, wherein the apelin-16 inhibits fat accumulation.
Food for preventing or improving diabetes, diabetic complications or obesity containing apelin-16 as an active ingredient.
8. The food according to claim 7, wherein the diabetes is type 1 diabetes or an early type 2 diabetes.
The food according to claim 7, wherein the diabetic complication is one or more diseases selected from the group consisting of diabetic nephropathy, diabetic retinopathy, hyperlipidemia, fatty liver, arteriosclerosis and hypertension.
8. The food according to claim 7, wherein the apelin-16 promotes insulin secretion.
8. The food according to claim 7, wherein the apelin-16 reduces blood total cholesterol or triglyceride.
8. The food according to claim 7, wherein the apelin-16 inhibits fat accumulation.

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