Composition for glucose regulation containing an atypical PKC activator

Abstract

The present invention relates to a composition for glucose regulation that regulates the amount of glucose metabolized in the small intestine. According to the present invention, by improving glucose metabolism in the small intestine and promoting the excretion of blood glucose into feces, blood glucose concentration can be reduced, and metabolic diseases can be effectively prevented, ameliorated or treated.

Description

[Technical field]

[0001] The present invention relates to a composition for glucose regulation that regulates the amount of glucose metabolized in the small intestine, and more specifically, to a composition for glucose regulation containing an atypical PKC activator as an active ingredient, and a pharmaceutical composition for the prevention or treatment of metabolic diseases containing an atypical PKC activator as an active ingredient. [Background technology]

[0002] Diabetes is a chronic metabolic disease caused by various triggers and characterized by prolonged high blood glucose levels (hyperglycemia). Insulin is required for blood glucose to be utilized by cells, and is secreted from the islets of Langerhans in the pancreas and has the effect of lowering blood glucose levels after meals. Insulin is secreted from the beta cells of the pancreas and is a hormone that regulates carbohydrate and fat metabolism, stimulating target cells (liver, muscle, and fat cells, etc.) to inject and store glucose from the bloodstream. In normal people, insulin converts blood glucose into energy and lowers it, whereas in diabetic patients, insulin secretion is not performed normally or, even if insulin is secreted normally, problems with the receptors occur, causing the body to lose its ability to control blood glucose and blood glucose levels to rise. Thus, diabetes can be broadly divided into two types. In type 1 diabetes or insulin-dependent diabetes mellitus (IDDM), patients produce little or no insulin, the hormone that regulates glucose utilization. It occurs due to the destruction of pancreatic beta cells by an autoimmune mechanism and the resulting insulin deficiency. Thus, blood glucose cannot be absorbed into cells, leading to diabetes. On the other hand, in type 2 diabetes or non-insulin-dependent diabetes mellitus (NIDDM), insulin is still produced in the body. It is an acquired form of diabetes and accounts for more than 80% of all diabetes cases. Despite the pancreatic β cells secreting sufficient amounts of insulin, the cells are unable to accept it, resulting in a hyperglycemic state. Type 2 diabetes patients have resistance to the effects of insulin in stimulating glucose and lipid metabolism in the major insulin-sensitive tissues, which are muscle, liver and adipose tissue. This insufficient responsiveness to insulin leads to insufficient insulin-mediated activation of glucose absorption, oxidation and storage in muscle and inadequate insulin-mediated suppression of lipolysis in adipose tissue and glucose production and secretion in the liver.

[0003] More than 150 million people worldwide suffer from diabetes, and the number of diabetic patients has been increasing rapidly in recent years. According to data collected by the WHO, it is predicted that there will be more than 360 million diabetic patients by 2030. In addition, lifestyles are changing in Korea as well, with an increase in meat-centered eating habits and lack of exercise, resulting in a rapid rise in the incidence of diabetes patients and a lower age of onset. After being diagnosed with diabetes, dietary restrictions are imposed in order to regulate blood sugar levels, lowering quality of life and raising the risk of various complications, so technology development is ongoing to regulate blood sugar levels to the normal range. For example, Korean Patent Registration No. 10-1983982 discloses a diabetes treatment technique using desPro36 exendin-4(1-39)-Lys6-NH2 and glitazones, and Korean Patent Publication No. 10-2006-0020548 discloses a diabetes treatment technique using a crude polysaccharide extract of Tremella fuciformis. However, the related prior art techniques only transport sugar that is already present in high concentrations in the blood to other parts of the body, and are unable to excrete sugar from the body.

[0004] Therefore, in the present invention, the inventors made extensive efforts to select a drug that induces an increase in the glucose absorption rate in the small intestine, and as a result, they selected PKC activators prostratin and ingenol 3-angelate as compounds with glucose regulation ability, and confirmed that the glucose absorption rate in the small intestine of diabetic model mice administered with prostratin increased, and the glucose excretion rate in the feces increased, thereby completing the present invention. Summary of the Invention

[0005] An object of the present invention is to provide a composition for glucose regulation that induces an increase in the glucose absorption rate in the small intestine.

[0006] Another object of the present invention is to provide a composition for preventing or treating metabolic diseases, which comprises a glucose regulating compound as an active ingredient.

[0007] Another object of the present invention is to provide a functional food composition for preventing or improving metabolic diseases, which contains a glucose regulating compound as an active ingredient.

[0008] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those having ordinary skill in the art from the following description.

[0009] In order to achieve the above object, the present invention provides a composition for glucose regulation comprising an atypical PKC activator as an active ingredient.

[0010] The present invention also provides a composition for preventing or treating a metabolic disease, which comprises an atypical PKC activator as an active ingredient.

[0011] The present invention also provides a method for preventing or treating a metabolic disease, comprising the step of administering an atypical PKC activator.

[0012] The present invention also provides the use of an atypical PKC activator for the prevention or treatment of a metabolic disease.

[0013] The present invention also provides the use of an atypical PKC activator for the manufacture of a medicament for the prevention or treatment of a metabolic disease.

[0014] The present invention also provides a functional food composition for preventing or ameliorating metabolic diseases, which contains an atypical PKC activator as an active ingredient. [Brief description of the drawings]

[0015] [Figure 1] This shows the classification of PKC subtypes, which are broadly divided into the classical group, the novel group, and the atypical group, and shows the respective genes, protein names and domains, and respective ligands. [Diagram 2]This shows the results of Western blotting to confirm protein changes in cell lysates after treating IEC-6 and IEC-18 cells with prostratin and ingenol 3-angelate. [Diagram 3] 2 shows the results of confirming the 2-DG absorption rate by treating IEC-6 and IEC-18 cells with prostratin and ingenol 3-angelate. [Figure 4] 1 shows the results of confirming increased activity of PKC zeta and GLUT1 by treatment of IEC-18 cells with prostratin and ingenol 3-angelate. [Diagram 5] (A) shows the results of confirming the expression of PKC zeta, p-GLUT1, and GLUT1 in PKC zeta-transfected IEC-6 cells and IEC-18 cells, (B) shows the results of confirming the expression of PKC zeta, p-GLUT1, and GLUT1 in PKC zeta-transfected HEK293 cells, and (C) shows the results of confirming the 2-DG uptake rate in PKC zeta-transfected IEC-18 cells. [Figure 6] The glucose uptake rate was measured after treatment of EC-6 cells and IEC-18 cells treated with prostratin and ingenol with a PKC zeta inhibitor (ZIP). [Figure 7] The IEC-6 cell line was transfected with siRNA against PKCα, PKCδ, PKCζ, and PKCι, respectively, and then each cell line was treated with prostratin. The changes in the expression of p-GLUT1 and GLUT1 were confirmed by Western blotting. [Figure 8] The IEC-6 cell line was transfected with siRNA against PKCα, PKCδ, PKCζ, and PKCι, respectively, and then each cell line was treated with prostratin and ingenol, after which the change in 2-DG uptake rate was confirmed. [Figure 9]Panel A shows the results of IP glucose tolerance test after a single subcutaneous injection of ingenol or prostratin into C57BL6 mice and intraperitoneal administration of 2 g glucose per kg mouse (2 g / kg glucose). Panel B shows the results of IP glucose tolerance test after two weeks of prostratin administration into C57BL6 mice and intraperitoneal administration of 2 g glucose per kg mouse (2 g / kg glucose). [Figure 10] The figures show the results of body weight (left) and intraperitoneal glucose tolerance test (right) after daily intraperitoneal injection of prostratin (0.5 mg / kg) and ingenol (1 μg / kg) to diabetic model mice for 40 days. [Figure 11] (A) shows the results of administering prostratin to diabetic model mice for one month, then removing the intestines and performing radiography for 18FDG. (B) shows the results of measuring the amount of 18FDG in removed cow fecal samples. (C) shows the results of measuring the amount of 18FDG uptake in the duodenum, jejunum, and ileum of the small intestine using a gamma counter. [Figure 12] After administering prostratin to a diabetic model mouse for one month, the small intestine was removed and the duodenum, jejunum, and ileum were immunohistochemically stained with an antibody against PKC zeta. The left side is a 100x optical microscope image, and the right side is a 400x optical microscope image. [Figure 13] This is an electron microscope photograph of the apical membrane of the lumen in the jejunum of the small intestine that was excised from a diabetic model mouse after administering prostratin for one month. Detailed Description of the Invention and Preferred Embodiments

[0016] 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. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0017] In the present invention, we performed AI-based drug screening to select drugs that show similar or better effects than Hb-EGF, an EGFR ligand that induces an increase in glucose absorption in the small intestine, and selected prostratin and ingenol 3-angelate (hereinafter referred to as "ingenol") as candidate drugs. Both prostratin and ingenol 3-angelate act as activators of PKC (protein kinase C).

[0018] In one embodiment of the present invention, intestinal epithelial cell lines (IEC-6 and IEC-18) treated with prostratin and ingenol were found to have increased expression of p-GLUT1 and GLUT1, proteins involved in glucose absorption in the cell membrane, and also to have increased uptake of the glucose analog 2-DG.

[0019] In another embodiment of the present invention, it was confirmed that administration of prostratin to diabetic model mice (db / db mice) increased intraperitoneal glucose tolerance and improved glucose metabolism, and that the body weight of mice administered prostratin was significantly reduced.

[0020] Therefore, from one aspect, the present invention relates to a composition for glucose regulation containing an atypical PKC activator as an active ingredient.

[0021] In the present invention, the atypical PKC can be characterized as being PKC zeta (PKC ζ) or PKC iota (PKCι).

[0022] The PKC subfamily is classified into the classical group (PKCα, PKCβ, and PKCγ), the novel group (PKCδ, PKCε, PKCη, and PKCθ), and the atypical group (PKCζ and PKCλ / ι), and the ligands that activate each subtype of PKC vary depending on the subfamily group. The classical group is activated by Ca2+, DAG, and PIP2, while the novel group is activated by DAG and lipid. Atypical PKC lacks the C2 and C1 domains and is not activated by Ca2+ or DAG. It also contains the Phox and Bem 1 (PB1) domain and is known to interact with proteins such as polarity regulator (PAR6) (Figure 1).

[0023] In one embodiment of the present invention, siRNAs against conventional PKC (PKCα), novel PKC (PKCδ) and atypical PKC (PKCζ and PKCι) were transfected into IEC-6 cells, and after 48 hours, prostratin (1μM) was treated, total protein was eluted, and Western blot was performed. As a result, it was confirmed that the expression of GLUT1 (p-GLUT1) increased by prostratin in the negative control group (siNeg), but did not increase in the cell group treated with siRNAs against atypical PKCs PKCζ and PKCι (Figure 7). Therefore, it was confirmed that PKCζ and PKCι play important roles in the mechanism of increased activity of GLUT1 (p-GLUT1) by prostratin.

[0024] In the present invention, glucose regulation can be characterized as regulating sugar absorption or sugar excretion in the small intestine.

[0025] In one embodiment of the present invention, prostratin (0.5 mg / kg) was administered to diabetic model mice (db / db mice) for one month, and the results of radiography after isolating the intestines confirmed that glucose absorption in the small intestine of the prostratin-treated db / db mice was significantly higher than that of the control group (vehicle group). Furthermore, it was confirmed that more glucose was excreted from the fecal wash of the mice administered prostratin (Figure 11).

[0026] In the present invention, the atypical PKC activator may be selected from the group consisting of prostratin or a salt thereof, a prostratin derivative or a salt thereof, ingenol 3-angelate or a salt thereof, and an ingenol 3-angelate derivative or a salt thereof.

[0027] The "prostratin" is a compound having the structure of Chemical Formula 1, and is known to have promising therapeutic potential as a regulator of protein kinase C against cancer and other diseases such as Alzheimer's disease. It has been reported that orally administered prostratin inhibits human pancreatic tumors (Wang, Man-Tzu et al., Cell. 16: 1237, 2015). [ka]

[0028] The "Ingenol 3-angelate" used in the present invention has the structure of Chemical Formula 2, is known as "ingenol mebutate", and is sold under the brand name "Picato". It is a substance found in the sap of the Euphorbia peplus plant and is known to induce cell death, and a gel formulation of the drug has been approved by the U.S. Food and Drug Administration (FDA) and the European Medical Agency (EMA) for the topical treatment of actinic keratosis. [ka]

[0029] In the present invention, the glucose regulating composition can induce absorption of blood glucose into small intestinal cells in the small intestine, preferably in the duodenum, jejunum, or ileum of the small intestine, and excretion of glucose into feces.

[0030] In the present invention, the "ileum" refers to the terminal part of the small intestine, located at the junction of the jejunum and large intestine. The small intestine is a digestive organ located between the stomach and large intestine, and anatomically consists of three parts: the duodenum, jejunum, and ileum. The small intestine of a human being aged 5 or older is usually about 7m long, 4-5 times longer than the large intestine, but is much thinner at 2.5-3cm compared to the large intestine, which has an average diameter of 7.6cm. The duodenum is about 25-30cm long, and the jejunum is about 2.5m long at the middle part of the small intestine. The ileum is about 3.6m long and is connected to the colon by the ileocecal valve. The ileocecal valve allows the contents to flow into the large intestine and prevents reflux into the small intestine. During the digestive process, nutrients are diffused and transmitted into the bloodstream through protruding ridges inside the small intestine. The mucous membrane in the small intestine has many wrinkles and has villi on its surface. Usually, most of the chyme that enters from the stomach is digested and absorbed in the duodenum and jejunum, which are the first and middle parts of the small intestine. In contrast, it is known that digestion and absorption in the ileum is relatively low (Diabetes Management Interactive Case Study, 2016). The composition of the present invention has the effect of increasing glucose absorption in the duodenum, jejunum, and ileum.

[0031] In addition, the glucose regulating composition of the present invention not only promotes the excretion of blood glucose into feces, but also has the effect of improving glucose metabolism, and can be used to effectively prevent, ameliorate or treat metabolic diseases.

[0032] Therefore, from another aspect, the present invention relates to a composition for preventing or treating a metabolic disease, which comprises an atypical PKC activator as an active ingredient.

[0033] In the present invention, the atypical PKC can be characterized as being PKC zeta (PKCζ) or PKC iota (PKCι).

[0034] In the present invention, glucose regulation can be characterized as regulating sugar absorption or sugar excretion in the small intestine.

[0035] In the present invention, the atypical PKC activator may be selected from the group consisting of prostratin or a salt thereof, a prostratin derivative or a salt thereof, ingenol 3-angelate or a salt thereof, and an ingenol 3-angelate derivative or a salt thereof.

[0036] In the present invention, the "metabolic disease" refers to a disease caused by excessive synthesis or accumulation of fat due to abnormal energy metabolism in the body caused by various causes such as excessive energy intake or hormonal imbalance. Specifically, the metabolic disease may be obesity, diabetic disease, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, fatty liver, or arteriosclerosis.

[0037] In the present invention, the "diabetic disease" refers to diabetes and its complications due to acute hyperglycemia, including, but not limited to, diabetic ketoacidosis, diabetic acidosis, diabetic xanthoma, diabetic amyotrophy, diabetic ketosis, diabetic coma, diabetic gastric disorder, diabetic gangrene, diabetic ulcer, diabetic complications, diabetic diarrhea, diabetic microangiopathy, diabetic uterine body sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, bullosis diabeticorum, diabetic cataract, diabetic dermopathy, diabetic scleredema, diabetic retinopathy, necrobiosis lipoidica diabeticorum, or diabetic blood circulation disorder, etc.

[0038] In the present invention, the term "diabetes" or "diabetes" refers to a disease caused by an increase in blood glucose concentration, and the longer the duration of diabetes, the more the incidence of chronic complications such as blindness, end-stage renal failure, neurological disease, lower limb amputation, and infectious diseases increases rapidly. In particular, cerebrovascular disease and cardiovascular disease are the most common diabetic complications, and about 3 / 4 of disease deaths are due to diabetic complications, and it has been reported that the risk of death from cardiovascular complications increases by 24% for every 10-year increase in the duration of diabetes. It has been reported that diabetic patients have twice the prevalence of coronary artery disease and about three times the prevalence of peripheral vascular disease compared to normal subjects, and there are various known causes of atherosclerosis in diabetes, such as hyperglycemia, abnormal lipid metabolism, hyperinsulinemia, hypertension, and changes in blood coagulation mechanisms. The etiology of type 2 (non-insulin-dependent) diabetes, which accounts for more than 95% of diabetes, is known to be a complex disorder of insulin secretion and insulin resistance due to two causes. In other words, diabetes is a disease that causes chronic hyperglycemic symptoms due to these complex disorders.

[0039] In the present invention, the "diabetic ketoacidosis" is the most important acute metabolic complication occurring in diabetic patients, and is a disease caused by the excessive accumulation of acid metabolites in the bloodstream and the loss of water and sugar caused by the use of fat rather than sugar as the energy required by the body. Diabetic ketoacidosis occurs due to resistance to insulin or the absence of insulin. When there is little insulin, glucose cannot enter the cells and accumulates in the blood. As a result, the cells are not supplied with glucose and use fat as an energy source. Fat metabolism produces fatty acids and glycerol, and while glycerol provides some energy to cells, fatty acids are metabolized to keto acids, resulting in acid toxicity. Acid toxicity increases the movement of potassium into the bloodstream within the cells, which causes hyperkaliuria due to the diuretic effect, resulting in a state of potassium depletion throughout the body.

[0040] In the present invention, the term "obesity" refers to a condition or disease in which excess body fat is present due to energy imbalance, and refers to a disease in which fat is excessively synthesized or accumulated due to improper energy metabolism in the body due to various causes.

[0041] From another aspect, the present invention relates to a method for preventing or treating a metabolic disease, comprising the step of administering an atypical PKC activator.

[0042] From another aspect, the present invention relates to the use of an atypical PKC activator for the prevention or treatment of a metabolic disease.

[0043] From another aspect, the present invention relates to the use of an atypical PKC activator for the manufacture of a medicament for the prevention or treatment of a metabolic disease.

[0044] From another viewpoint, the present invention relates to a functional food composition for preventing or ameliorating metabolic diseases, which contains an atypical PKC activator as an active ingredient.

[0045] In the present invention, the atypical PKC can be characterized as being PKC zeta (PKCζ) or PKC iota (PKCι).

[0046] In the present invention, glucose regulation can be characterized as regulating sugar absorption or sugar excretion in the small intestine.

[0047] In the present invention, the atypical PKC activator may be selected from the group consisting of prostratin or a salt thereof, a prostratin derivative or a salt thereof, ingenol 3-angelate or a salt thereof, and an ingenol 3-angelate derivative or a salt thereof.

[0048] In one embodiment of the present invention, "prevention" may include, without limitation, any action that can block, suppress or delay symptoms caused by a metabolic disease using the composition of the present invention.

[0049] In one embodiment of the present invention, "improvement" includes, without limitation, any action of improving or benefiting symptoms caused by a metabolic disease using the composition of the present invention.

[0050] In one embodiment of the present invention, "treatment" refers to a set of activities undertaken to alleviate and / or ameliorate a desired disease. For the purposes of the present invention, treatment includes activities that significantly increase the amount of glucose absorbed in the intestine and improve glucose metabolism.

[0051] In one embodiment of the present invention, the term "pharmaceutical composition" refers to a composition administered for a specific purpose. For the purposes of the present invention, the pharmaceutical composition of the present invention is intended to prevent or treat diabetes or complications caused by diabetes, and may include a compound involved therein and a pharma- ceutical acceptable carrier, excipient, or diluent.

[0052] The pharmaceutical composition according to the present invention contains the active ingredient of the present invention in an amount of 0.1 to 50% by weight based on the total weight of the composition. Carriers, excipients and diluents that may be included in the composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0053] In one embodiment of the present invention, "administration" refers to introducing the composition of the present invention into a patient in some suitable manner, and the route of administration of the composition of the present invention can be any common route as long as it can reach the target tissue.

[0054] The composition may be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, intranasally, intrapulmonary, intestinal, intracavitary, or intrathecally, but is not limited thereto. In the present invention, the effective amount may be adjusted depending on various factors, including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, the age, weight, general health condition, sex, and diet of the patient, the administration time, the administration route, the secretion rate of the composition, the treatment period, and drugs used simultaneously. In addition, the pharmaceutical composition of the present invention may be administered alone or together with other therapies known in the art, such as chemotherapy and surgery, for the treatment of the metabolic disease of interest. In addition, the pharmaceutical composition of the present invention may be administered in combination with other therapies designed to promote blood glucose reduction, such as those known in the art. Other standard delivery methods, such as biolistic delivery or ex vivo treatment, may also be used.

[0055] In one embodiment of the present invention, the "food composition" is a food composition that is used in various ways to prevent or improve metabolic diseases, which is the object of the present invention. The food composition containing the composition of the present invention as an active ingredient may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectioneries, rice cakes, breads, etc. The food composition of the present invention is an improved version of an existing food ingestible product that has almost no toxicity or side effects, so it can be used safely even when taken for a long period of time for preventive purposes. When the composition of the present invention is contained in the food composition, the amount of the composition may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there are no special restrictions other than the inclusion of the food composition in the indicated proportion, and various flavoring agents or natural carbohydrates, etc., may be contained as additional ingredients, as in ordinary beverages. That is, the natural carbohydrates may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, ordinary sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, and the like. These components can be used independently or in combination. The ratio of these additives is generally selected from the range of 0.1 to 100 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto. EXAMPLES

[0056] The present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and that the scope of the present invention is not limited by these examples.

[0057] Example 1: Screening of drugs that increase glucose absorption rate in the ileum of the small intestine To screen for drugs that show similar or better effects than Hb-EGF, an EGFR ligand that induces an increase in glucose absorption rate in the ileum, we performed Arontier's AI-based drug screening (Arontier, Korea). Candidate compounds similar to Hb-EGF were selected using the REMEDY platform developed by Alontia (Lamb et al., Science, 313:1929~1935, 2006; Subramanian et al., Cell, 171:1437~1452, 2017). As a result, prostratin and ingenol 3-angelate, which act as atypical PKC activators, were selected.

[0058] Example 2: Confirmation of the expression level of glucose transporter (GLUT transporter) by treatment with prostratin and ingenol (Ingenol 3-angelate) in small intestinal cell line Intestinal epithelial cells, IEC-6 cells (ATCC CRL-1592) and IEC-18 cells (ATCC CRL-1589), were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin at 10% CO. 2 The cells were cultured at 37°C in a humidified atmosphere containing 10% CO and 90% air, and the stock cultures were subcultured every 3–4 days. For the experiments, IEC-6 and IEC-18 cells were cultured at 2 x 10 5Cells were seeded into 6-well plates at a density of 1.5 x 10 cells / dish. 5 cells / cm 2 The culture medium was used 6 days after plating. Prostratin was purchased from Sigma-Aldrich (Cat. No. P0077). Ingenol was purchased from Sigma-Aldrich (Cat. No. SML1318). IEC-6 cells and IEC-18 cells were treated with prostratin and ingenol at 30 nM, 100 nM, and 300 nM, respectively, for 30 minutes, and then cell lysates were obtained and the protein expression levels were confirmed by Western blotting. Western blots were performed as follows. Total cell protein lysates were prepared and Western blot analysis was performed according to standard procedures. Briefly, cells were chilled on ice, washed twice with ice-cold phosphate-buffered saline, and lysed in buffer containing 1 mM phenylmethylsulfonyl fluoride and 1x protease inhibitors (Sigma-Aldrich). Protein concentration was measured with a Bradford assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to membranes incubated overnight at 4°C with primary antibodies, anti-PKCα (Cell signaling Technology, USA), anti-PKCδ (Cell signaling Technology, USA), anti-PKCζ (Cell signaling Technology, USA), anti-p-GLUT1 (Abcam, UK), anti-GLUT1 (Abcam, UK), and anti-GLUT2 (Novus Biological, USA), respectively. Blots were washed three times with TBST (Tris-buffered saline containing 0.05% Tween 20) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 25°C. Donkey anti-rabbit IgG-HRP antibody (1:5000, Santa Cruz) and donkey anti-mouse IgG-HRP antibody (1:5000, Santa Cruz) were used as secondary antibodies. Immunoreactivity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA). As a result, we confirmed that ingenol 3-angelate and prostratin promoted GLUT1 phosphorylation at all concentrations (30, 100, 300 nM) in IEC-6 and IEC-18 cells, as shown in Figure 2. We confirmed that prostratin and ingenol 3-angelate increased the expression of the glucose transporters p-GLUT1 and GLUT1, in relation to the protein quantification marker Na / K ATPase.

[0059] Example 3: Confirmation of intracellular glucose absorption rate by treatment with prostratin and ingenol (Ingenol 3-angelate) in small intestinal cell line The 2-DG (2-deoxy-D-glucose) uptake rate was measured in cell lysates obtained from IEC-6 and IEC-18 cells treated with prostratin and ingenol (100 nM) for 10 minutes. Glucose concentration was measured to confirm the change in glucose absorption rate (2-DG uptake) due to treatment with prostratin and ingenol. Glucose levels were measured in low glucose medium and serum-free DMEM after 24 h of prostratin and ingenol treatment. IEC-6 and IEC-18 cell glucose levels were detected using a Glucose Assay Kit (Cayman Chemical) according to the manufacturer's instructions. Absorbance signals were measured with a Gen 5 luminescence spectrometer (BioTek, Winooski, VT, USA). As a result, as shown in FIG. 3, it was confirmed that intracellular glucose (2-DG) absorption rate was increased by treatment with prostratin and ingenol.

[0060] Example 4: Confirmation of PKC and GLUT1 activation by prostratin and ingenol (Ingenol 3-angelate) in small intestinal cell lines After treating IEC-18 cells with prostratin (2 μM) and ingenol (0.1 μM) for 0, 15, and 60 minutes, respectively, the protein expression of PKC and GLUT1 was confirmed by Western blotting. As a result, we confirmed that prostratin and ingenol increased the activity of PKC zeta and GLUT1, as shown in Figure 4. We also confirmed that prostratin translocated PKC and GLUT1 to the cell membrane more quickly than ingenol.

[0061] Example 5: Confirmation that PKC zeta induces GLUT1-mediated glucose absorption in small intestinal cell lines PKC zeta was transiently overexpressed in IEC-6 cells, IEC-18 cells and HEK293 (ATCC CRL-1573) cells via transfection to generate transfected cell lines. A plasmid expressing rat PKC zeta (Korean Gene Bank) was subcloned into pCDNA3.1 (Addgene, USA). IEC-6 and IEC-18 cells were cultured at 0.5 x 10 per 6-well plate one day before transfection. 6 The cells were plated at a density of 1000 x 1000 and 1000 x 1000. Cells were transfected with expression plasmids using Polyjet transfection reagent (SignaGen) according to the manufacturer's instructions. Transfected cells were cultured at 37°C for 48 h before lysis and treatment with ingenol (0.1 μM). Cell lysates were obtained and Western blots were performed for empty vector-infected cells as a control, PKC zeta-transfected cells, wild-type cells treated with ingenol, and transfected cells treated with ingenol. In Western blot, Na / K ATPase was used as a loading control for the membrane. As a result, as shown in Figure 5A and Figure 5B, PKC zeta was activated and moved to the cell membrane in the ingenol-treated group compared to the empty vector-treated negative control group, and translocation of GLUT1 was confirmed. In the cell line overexpressing PKC zeta, even in the group not treated with ingenol, p-GLUT1 and GLUT1 were expressed more than in the negative control group. We also measured the 2-DG uptake rate in IEC-18 cells transfected with PKC zeta overexpression, and found that the 2-DG uptake rate was significantly increased in IEC-18 cells overexpressing PKC zeta compared to the control group, as shown in Figure 5C. Therefore, it was confirmed that the 2-DG uptake rate was significantly increased in cell lines with high atypical PKC expression.

[0062] Example 6: Confirmation of the inhibition of glucose absorption by prostratin and ingenol by treatment with a PKC zeta inhibitor IEC-6 and IEC-18 cells in which atypical PKC was activated by treatment with prostratin (100 nM) and ingenol (100 nM) were treated with ZIP (pseudosubstrate-derived PKC ζ-inhibitory peptide, R&D system, USA) at a concentration of 1 μM, which specifically inhibits PKC zeta, and glucose uptake was measured. As a result, as shown in Figure 6, intracellular glucose-6-phosphate increased when IEC-6 and IEC-18 cells were treated with prostratin (100 nM) and ingenol (100 nM), but when co-treated with ZIP, a PKC zeta inhibitor, the intracellular glucose-6-phosphate uptake rate decreased to the same level as the negative control group (NT).

[0063] Example 7: Confirmation of the role of atypical PKC (zeta + iota) in prostratin-mediated GLUT1 activation To confirm whether GLUT1 activation by prostratin and ingenol is induced through which PKC subtype, siRNAs against conventional PKC (PKCα), novel PKC (PKCδ), and atypical PKCs (PKCζ and PKCι) were transfected into IEC-6 cells at 50 nM concentration using RNAiMax (Invitrogen). After 48 h, prostratin (1 μM) was treated, and total protein was eluted and subjected to Western blotting. The siRNA sequences used are shown below. PKCα siRNA: GGGAUGUCAGAGAGCAUGCCUUCUU (SEQ ID NO: 1) PKCδ siRNA: CUCACAGUACUUCCUCUGU (SEQ ID NO: 2) PKCζ siRNA: GCUGAGAUCUGUAUCGCUCUCAACU (SEQ ID NO: 3) PKCι siRNA: GGACAAUGUACUGCUAGACUCUGAA (SEQ ID NO: 4) As a result, as shown in Figure 7, it was confirmed that the expression of GLUT1 (p-GLUT1), which was increased by prostratin in the negative control group (siNeg), did not increase even when treated with prostratin in the cell groups treated with siRNA for the atypical PKCs PKCζ and PKCι. Therefore, it was confirmed that PKCζ and PKCι play important roles in the mechanism by which prostratin increases GLUT1 (p-GLUT1) activity.

[0064] Example 8: Confirmation of the role of atypical PKC (zeta + iota) in increasing glucose absorption rate by prostratin and ingenol To confirm which subtype of PKC is responsible for the increased glucose uptake (2DG uptake) in IEC cells induced by treatment with prostratin and ingenol, IEC-6 cells were transfected with siRNAs against conventional PKC (PKCα), novel PKC (PKCδ), and atypical PKCs (PKCζ and PKCι) as in Example 7, and after 48 hours, prostratin (1 μM) or ingenol (100 nM) was treated to confirm 2DG uptake. As a result, as shown in Figure 8, 2DG absorption was increased by treatment with prostratin and ingenol in the negative control group (siNeg), but in the group in which atypical PKC (PKCζ and PKCι) were knocked down, 2DG absorption did not increase even with treatment with prostratin and ingenol. Thus, we confirmed that PKCζ and PKCι all play important roles in glucose absorption induced by prostratin and ingenol treatment.

[0065] Example 9: Confirmation of the glucose metabolism improving effect by administration of prostratin and ingenol in a mouse animal model 9-1: Confirmation of improvement in glucose metabolism by single administration Twelve C57BL6 mice were fasted for 12 hours, and then intraperitoneal glucose tests were performed to measure fasting blood glucose. Then, the ingenol group (4 mice) and the prostratin group (4 mice) were given a single subcutaneous injection of ingenol (4ug / kg) and prostratin (1mg / kg), respectively, and 2g of glucose per kg mouse (2g / kg glucose) was administered intraperitoneally in saline. Then, IP glucotolerance tests were performed at 15, 30, 60, 90, and 120 minutes, respectively. As a result, as shown in Figure 9A, it was confirmed that glucose metabolism was significantly improved in the prostratin-administered group compared to the negative control group (vehicle) at 30 and 60 minutes, and that glucose metabolism was also improved in the ingenol-administered group compared to the negative control group. 9-2: After 2 weeks of administration, improvement in glucose metabolism was confirmed C57BL6 mice were administered prostratin at concentrations of 0, 0.5, or 1 mg / kg using an osmotic pump for 2 weeks, fasted for 12 hours, and then intraperitoneally administered 2 g glucose per kg mouse (2 g / kg glucose) diluted in saline. IP glucotolerance tests were then performed 15, 30, 60, 90, and 120 minutes after administration. As a result, as shown in FIG. 9B, it was confirmed that both the blood glucose change and the area under the glucose change curve over a 2-hour period were improved.

[0066] Example 10: Confirmation of the effect of administration of prostratin and ingenol on improving body weight and glucose metabolism in diabetic model mice db / db mice (Jackson laboratory), known as a representative diabetic model lacking leptin receptors, were intraperitoneally injected with prostratin (0.5 mg / kg) and ingenol (1 μg / kg) every day for 40 days, and then their body weights were measured. As a result, as shown in Figure 10, when the concentration of ingenol was increased to 2 μg / kg on the 28th day of administration, weight loss showed statistical significance on the 30th day. The group administered 0.5 mg / kg of prostratin showed statistically significant weight loss from the 11th day of administration compared to the control group (vehicle group). An intraperitoneal glucose tolerance test (IPGTT) was performed 17 days after drug administration. After fasting for 12 hours before the IPGTT, 2g / kg IPGTT was performed 15 minutes after prostratin and ingenol injections, and the results are shown in Figure 10. In the 1 μg / kg ingenol administration group, glucose improvement was observed 240 minutes after glucose administration. In the prostratin (0.5 mg / kg) administration group, glucose improvement was confirmed to be superior to the control group (vehicle group) over the entire time period from 30 to 240 minutes.

[0067] Example 11: Confirmation of the effect of prostratin administration on improving glucose absorption and excretion in diabetic model mice Intestines of db / db mice (Jackson laboratory) were administered 0.5 mg / kg prostratin for one month, and radiographs were performed using 18FDG (Fluorodeoxyglucose, DuChemBio). 18FDG was purchased from DuChemBio and injected at 200 μCi per mouse via the tail vein. In addition, the amount of 18FDG in fecal samples obtained by washing the excised small intestine with 5 mL of PBS was measured, and the amount of 18FDG uptake in the duodenum, jejunum, and ileum of the small intestine was measured using a gamma counter. As a result, as shown in FIG. 11A, it was confirmed from the radiographic results that glucose absorption in the small intestine of db / db mice treated with prostratin was much higher than that in the control group (vehicle group). In addition, when glucose excretion was examined in fecal samples, more glucose was excreted from fecal washes of mice administered prostratin, as shown in Figure 11B, and glucose absorption was increased by more than two-fold in the duodenum, jejunum, and ileum of the small intestine, as shown in Figure 11C.

[0068] Example 12: Confirmation of the membrane translocation effect of atypical PKC by administration of prostratin in the jejunum and ileum of diabetic model mice After administration of prostratin (0.5 mg / kg) or vehicle to db / db mice for one month, the small intestine was removed, and the duodenum, jejunum, and ileum were fixed in formalin and then immunohistochemically stained with an antibody against PKC zeta (Abcam, USA) and photographed under an optical microscope at 100x and 400x magnification. As a result, as shown in Figure 12, there was no significant difference between the prostratin-administered group and the control group (vehicle) in the duodenum, but in the jejunum and ileum, the location of PKC zeta tended to concentrate from the cytoplasm to the apical membrane of the lumen in the prostratin-administered group.

[0069] Example 13: Electron microscopic observation of the lumen of the jejunum of diabetic model mice administered prostratin After administration of prostratin (0.5 mg / kg) or vehicle to db / db mice for one month, the small intestine was excised and the apical membrane of the lumen in the jejunum was observed under an electron microscope. As a result, as shown in Figure 13, it was confirmed that more glycocalyx was secreted near the microvilli in the jejunum in the prostratin-administered group compared to the control group (vehicle), and the number of mitochondria in the small intestinal cells of the prostratin-administered group was increased, indicating active intracellular metabolism. [Industrial Applicability]

[0070] According to the present invention, by improving glucose metabolism in the small intestine and promoting the excretion of blood glucose into feces, blood glucose concentration can be reduced, and metabolic diseases can be effectively prevented, ameliorated or treated.

[0071] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for glucose regulation comprising as an active ingredient an atypical PKC activator selected from the group consisting of prostratin or a salt thereof, and ingenol 3-angelate or a salt thereof.

2. 2. The composition for regulating glucose according to claim 1, wherein the atypical PKC is PKC zeta (PKCζ) or PKC iota (PKCι).

3. 2. The composition for glucose regulation according to claim 1, wherein the glucose regulation is characterized by regulating sugar absorption or sugar excretion in the small intestine.

4. A pharmaceutical composition for preventing or treating a metabolic disease, comprising as an active ingredient an atypical PKC activator selected from the group consisting of prostratin or a salt thereof, and ingenol 3-angelate or a salt thereof.

5. 5. The pharmaceutical composition for preventing or treating a metabolic disease according to claim 4, wherein the atypical PKC is PKC zeta (PKC ζ) or PKC iota (PKCι).

6. 5. The pharmaceutical composition for preventing or treating a metabolic disease according to claim 4, which regulates sugar absorption in the small intestine.

7. 5. The pharmaceutical composition for preventing or treating a metabolic disease according to claim 4, which regulates sugar absorption or sugar excretion in the small intestine.

8. 5. The pharmaceutical composition for preventing or treating a metabolic disease according to claim 4, wherein the metabolic disease is diabetic disease, obesity, hypertension, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, arteriosclerosis or fatty liver disease.

9. 9. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 8, wherein the diabetic disease is selected from the group consisting of diabetes, diabetic ketoacidosis, diabetic acidosis, diabetic xanthomas, diabetic amyotrophy, diabetic ketosis, diabetic coma, diabetic gastrointestinal disorders, diabetic gangrene, diabetic ulcers, diabetic complications, diabetic diarrhea, diabetic microangiopathy, diabetic uterine sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic renal failure, diabetic blisters, diabetic cataracts, diabetic skin diseases, diabetic edema sclerosis, diabetic retinopathy, diabetic necrobiosis lipoidica and diabetic blood circulation disorders.

10. A functional food composition for preventing or improving a metabolic disease, comprising as an active ingredient an atypical PKC activator selected from the group consisting of prostratin or a salt thereof, and ingenol 3-angelate or a salt thereof.

11. 11. The functional food composition for preventing or improving metabolic diseases according to claim 10, characterized by regulating sugar absorption or sugar excretion in the small intestine.

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