Composition for increasing insulin sensitivity or ameliorating insulin resistance, comprising 1,1-diethoxyethane as active ingredient, and use thereof

Abstract

The present invention relates to a composition for increasing insulin sensitivity or ameliorating insulin resistance, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient, and a use thereof. More specifically, the present invention relates to a composition for increasing insulin sensitivity or ameliorating insulin resistance, comprising 1,1-diethoxyethane as an active ingredient as a PTEN oxidative inhibitor, and a use thereof, for example, a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition as a composition for preventing, treating, or ameliorating diseases related to insulin resistance.

Description

Composition for increasing insulin sensitivity or improving resistance containing 1,1-diethoxyethanein as an active ingredient and use thereof

[0001] The present invention relates to a composition for improving insulin resistance comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient and its use. More specifically, the present invention relates to a composition for increasing insulin sensitivity or improving resistance comprising 1,1-diethoxyethane as an active ingredient as a PTEN oxidative inhibitor, and its use, for example, as a composition for the prevention, treatment, or improvement of diseases related to insulin resistance, such as a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition.

[0002] The PI3K / Akt (phosphoinositide-3-kinase / protein kinase B) pathway is an important signaling system that regulates various cellular processes such as proliferation, survival, and differentiation. When ligands such as growth factors bind to RTKs (receptor tyrosine kinases), PI3K is activated, and PIP2 (phosphatidylinositol (4,5)-bisphosphate) is phosphorylated to PIP3 (phosphatidylinositol (3,4,5)-trisphosphate). When PIP3 accumulates in the cell membrane, Akt is activated, which regulates various downstream effectors.

[0003] PTEN (phosphatase and tensin homolog deleted on chromosome 10) possesses lipid phosphatase function by dephosphorylating PIP3 to PIP2, playing an important role as a negative regulator of the PI3K / Akt signaling pathway. PTEN is regulated by various post-translational modifications, and changes such as oxidation, acetylation, or s-nitrosylation can inhibit PTEN's lipid phosphatase function. For example, the Cys124 residue, a catalytic nucleophile of PTEN, is readily oxidized by reactive oxygen species (ROS), which leads to the formation of a disulfide bond between the Cys124 and Cys71 residues, thereby inhibiting PTEN's function.

[0004] Inhibition of PTEN can increase the concentration of PIP3 and activate the PI3K / Akt pathway through Akt activation. This can lead to enhanced insulin signaling and improved insulin sensitivity. This mechanism presents new possibilities for the treatment of metabolic diseases, and related research is actively underway.

[0005] The PI3K / Akt pathway plays a particularly important role in regulating insulin sensitivity. When insulin binds to the transmembrane RTK IR (insulin receptor), the PI3K / Akt pathway is activated through autophosphorylation. Through this, Akt activation promotes glycolysis and glycogen synthesis, and increases the cellular uptake and metabolism of glucose.

[0006]

[0007] Prior art literature

[0008] [Non-patent Document 1] Vivanco, I. and CL Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer, 2002. 2(7): p. 489-501.

[0009] [비특허문헌 2] He, Y., et al., Targeting PI3K / Akt signal transduction for cancer therapy. Signal Transduct Target Ther, 2021. 6(1): p. 425.

[0010] [비특허문헌 3] Lee, Y.R., M. Chen, and P.P. Pandolfi, The functions and regulation of the PTEN tumour suppressor: new modes and prospects. Nat Rev Mol Cell Biol, 2018. 19(9): p. 547-562.

[0011] [비특허문헌 4] Lee, S.R., et al., Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem, 2002. 277(23): p. 20336-42.

[0012] [비특허문헌 5] Han, S.J., et al., Assay of the redox state of the tumor suppressor PTEN by mobility shift. Methods, 2015. 77-78: p. 58-62.

[0013] [비특허문헌 6] Trinh, V.H., et al., Redox Regulation of Phosphatase and Tensin Homolog by Bicarbonate and Hydrogen Peroxide: Implication of Peroxymonocarbonate in Cell Signaling. Antioxidants (Basel), 2024. 13(4).

[0014] [비특허문헌 7] Nguyen Huu, T., et al., The Role of Oxidative Inactivation of Phosphatase PTEN and TCPTP in Fatty Liver Disease. Antioxidants (Basel), 2023. 12(1).

[0015] [비특허문헌 8] Huang, X., et al., The PI3K / AKT pathway in obesity and type 2 diabetes. Int J Biol Sci, 2018. 14(11): p. 1483-1496.

[0016] [비특허문헌 9] Savova, M.S., et al., Targeting PI3K / AKT signaling pathway in obesity. Biomed Pharmacother, 2023. 159: p. 114244.

[0017] [비특허문헌 10] Hopkins, B.D., M.D. Goncalves, and L.C. Cantley, Insulin-PI3K signalling: an evolutionarily insulated metabolic driver of cancer. Nat Rev Endocrinol, 2020. 16(5): p. 276-283.

[0018] [비특허문헌 11] Marcq, P. and P. Schieberle, Characterization of the Key Aroma Compounds in a Commercial Fino and a Commercial Pedro Ximenez Sherry Wine by Application of the Sensomics Approach. J Agric Food Chem, 2021. 69(17): p. 5125-5133.

[0019] [비특허문헌 12] Moreno, J., et al., Use of a flor velum yeast for modulating colour, ethanol and major aroma compound contents in red wine. Food Chem, 2016. 213: p. 90-97.

[0020] [비특허문헌 13] Tan, C., et al., The Effects of Ultrasonic and Gamma Irradiation on the Flavor of Potato Wines Investigated by Sensory Omics. Foods, 2023. 12(15).

[0021] [비특허문헌 14] Dunkel, A., et al., Nature's chemical signatures in human olfaction: a foodborne perspective for future biotechnology. Angew Chem Int Ed Engl, 2014. 53(28): p. 7124-43.

[0022] [비특허문헌 15] Kawaguchi, D., H. Ogihara, and H. Kurokawa, Upgrading of Ethanol to 1,1-Diethoxyethane by Proton-Exchange Membrane Electrolysis. ChemSusChem, 2021. 14(20): p. 4431-4438.

[0023]

[0024] Accordingly, the inventors made diligent efforts to investigate the physiological functions of 1,1-diethoxyethane (1,1-DEE) and confirmed that 1,1-DEE induces oxidative inactivation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) through the formation of disulfide bonds between Cys124 and Cys71 residues (see Experimental Examples 2 and 3), that oxidative inactivation of PTEN induces increased phosphorylation at Ser473 and Thr308 of Akt, thereby activating Akt (see Experimental Examples 4 and 5), and that activation of Akt increases phosphorylation at the Ser483 residue of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2), thereby promoting the rate of glycolysis (see Experimental Example 6).

[0025] In addition, it was confirmed that combined treatment with 1,1-DEE and insulin can increase Akt activation and improve insulin sensitivity, and that 1,1-DEE can also have the effect of alleviating palmitic acid-induced insulin resistance (see Experimental Example 7), thereby completing the present invention.

[0026]

[0027] Accordingly, the present invention aims to provide a composition for increasing insulin sensitivity or improving resistance comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0028] The present invention also aims to provide a pharmaceutical composition for the prevention or treatment of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0029] The present invention also aims to provide a cosmetic composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0030] The present invention also aims to provide a food composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0031] The present invention also aims to provide a feed composition for the prevention or improvement of diseases related to insulin resistance.

[0032]

[0033] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0034] The present invention discloses a composition for increasing insulin sensitivity or improving resistance comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient, and a composition for the use thereof, for example, a pharmaceutical composition, a cosmetic composition, a food composition, or a feed composition for the prevention, treatment, or improvement of diseases related to insulin resistance.

[0035] In the present invention, the 1,1-diethoxyethane (1,1-DEE) has the molecular formula C6H 14 It is represented by the following structural formula 1 as O2, and is also called acetaldehyde diethyl acetal or ethyllidene diethyl ether.

[0036] [Structural Formula 1]

[0037]

[0038] According to one embodiment,

[0039] A composition for increasing insulin sensitivity or improving resistance is disclosed, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0040] In the present invention, the 1,1-diethoxyethane can induce oxidative inactivation of PTEN through the formation of disulfide bonds between Cys124 and Cys71 residues of PTEN.

[0041] In the present invention, the 1,1-diethoxytain can activate Akt by increasing phosphorylation at Ser473 and Thr308 of Akt.

[0042] In the present invention, the 1,1-diethoxyethane can promote glycolysis by increasing phosphorylation at the Ser483 residue of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2).

[0043]

[0044] According to another embodiment,

[0045] A pharmaceutical composition for the prevention or treatment of diseases associated with insulin resistance is disclosed, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0046] In the present invention, the diseases associated with insulin resistance include metabolic disorders such as Type 1 diabetes, Type 2 diabetes, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), hyperglycemia, postprandial hyperglycemia, polycystic ovary syndrome (PCOS), hyperlipidemia, hypertension, overweight, obesity, and metabolic syndrome; and blood glucose control disorders such as a decrease in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c and an improvement in blood glucose control. Diabetes progression-related disorders, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;It may include one or more selected from insulin-related disorders, including maintenance and / or improvement of insulin sensitivity, and prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0047] In the present invention, the pharmaceutical composition may be administered by one or more administration methods selected from the group consisting of oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, lung administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernous injection, intrathecal injection, epidural injection and rectal administration, arterial injection, lymphatic administration, intraosseous injection, lacrimal duct drug delivery.

[0048]

[0049] According to another embodiment,

[0050] A cosmetic composition for the prevention or improvement of diseases related to insulin resistance is disclosed, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0051] In the present invention, the cosmetic composition may be formulated into one or more selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a perfume, a pack, a softening water, a lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing product, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0052]

[0053] According to another embodiment,

[0054] A food composition for the prevention or improvement of diseases related to insulin resistance is disclosed, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0055] In the present invention, the food may include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, stamina drinks, alcoholic beverages, or vitamin complexes.

[0056]

[0057] According to another embodiment,

[0058] A feed composition for the prevention or improvement of diseases related to insulin resistance is disclosed, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0059] In the present invention, the feed may include powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, or raw feed.

[0060] 1,1-diethoxyethane (1,1-DEE) according to the present invention can provide the following effects.

[0061] (1) 1,1-diethoxytaine can induce oxidative inactivation of PTEN, which increases Akt activation and increases Ser483 phosphorylation of PFKFB2, thereby promoting glycolysis.

[0062] (2) 1,1-diethoxytain can improve insulin sensitivity and palmitic acid-induced insulin resistance.

[0063] Accordingly, 1,1-diethoxyethane according to the present invention is expected to be usefully utilized as a pharmaceutical composition, cosmetic composition, food composition, or feed composition for the prevention, treatment, or improvement of diseases related to insulin resistance.

[0064]

[0065] Meanwhile, the scope of the present invention is not limited by the effects described above.

[0066] Figure 1 shows 1,1-DEE identified in various ethanol batches: (A) HepG2 cells were treated with 100 mM E1, E2, or E3 for 10 minutes. (B) Lyophilization process flow of 100 mM E1, E2, and E4. (C) Cells were treated with 100 mM E1, E2, or E4 or the corresponding lyophilized samples described in (B) for 10 minutes. After treatment, cell extracts were alkylated with 10 mM NEM, followed by non-reducing or reducing electrophoresis and staining with PTEN and β-actin antibodies. (D) Presence and chemical structure of 1,1-DEE confirmed by GC-MS analysis of E1.

[0067] Figure 2 indicates that 1,1-DEE can induce concentration-dependent PTEN oxidation: (A) C2C12, MEF, AC16, and Ea.hy926 cells were treated with various concentrations of 1,1-DEE (0–50 mM) for 10 minutes. (B) C2C12 cells were treated with 1,2-DEE at the same concentrations as in (A). After alkylating the cell lysates with 10 mM NEM, Western blot analysis was performed using PTEN and GAPDH antibodies to determine the levels of PTEN oxidation. (C) HepG2 cells were transfected with HA-tagged pCGN PTEN WT, C71S, C124S, or C71S / C124S. After transfection, the cells were treated with 10 mM 1,1-DEE for 10 minutes. After treatment, the cells were lysed and alkylated with 10 mM NEM, and then immunoblotting was performed using an antibody against the HA-tag.

[0068] Figure 3 shows that 1,1-DEE can induce time-dependent PTEN oxidation: (A) C2C12, MEF, AC16, and EA.hy926 cells were treated with 10 mM 1,1-DEE for 120 minutes at various time intervals. (B) C2C12 cells were treated with 10 mM 1,2-DEE under the same conditions as in (A). After alkylating the cell lysates with 10 mM NEM, reductive or non-reductive electrophoresis was performed, and immunoblot analysis was conducted using PTEN antibodies.

[0069] Figure 4 indicates that PTEN oxidation induced by 1,1-DEE can activate the Akt signaling pathway: (A) C2C12 cells were treated with various concentrations of 1,1-DEE for 10 minutes. (B) C2C12 cells were treated with 10 mM 1,1-DEE for 120 minutes at various time intervals. Immunoblot analysis was performed using cell lysates and antibodies against phosphorylated Akt and total Akt at Ser473 and Thr308.

[0070] Figure 5 illustrates that PTEN oxidation induced by 1,1-DEE is mediated by ROS generation: (A) HepG2 cells were treated with 10 mM 1,1-DEE or 0.5 mM H2O2 for 10 minutes. Subsequently, the cells were stained with 10 μM DCFH-DA for 30 minutes, and representative images of the stained cells were obtained using a fluorescence microscope (10x magnification). (B) HepG2 cells were treated with 0.5 mM H2O2 for 10 minutes or with 10 mM 1,1-DEE for 30 minutes at various time intervals. After staining the cells with 10 μM DCFH-DA, flow cytometry was performed using the FITC channel at 10,000 events per sample. (C) HepG2 cells were pretreated with 10 mM NAC for 120 minutes, followed by treatment with 10 mM 1,1-DEE for 120 minutes at various time intervals. After alkylating the cell lysate with 10 mM NEM, non-reducing or reducing immunoblotting was performed and analyzed using antibodies against PTEN, phosphorylated Akt, Akt, and GAPDH.

[0071] Figure 6 shows that 1,1-DEE can promote glycolysis through Akt activation: (A) AC16 cells were pretreated with 10 μM MK-2206 for 24 hours, followed by treatment with 10 mM 1,1-DEE for 30 minutes. (B) AC16 cells were pretreated with 10 μM Ebselen for 60 minutes, followed by treatment with 10 mM 1,1-DEE for 30 minutes at various time intervals. Cell lysates were alkylated with 10 mM NEM, and Western blot analysis was performed using antibodies against PTEN, phosphorylated Akt, Akt, phosphorylated PFKFB2, PFKFB2, and GAPDH.

[0072] Figure 7 indicates that 1,1-DEE can improve insulin sensitivity and alleviate palmitic acid-induced insulin resistance: (A) C2C12 cells were treated with various concentrations of 1,1-DEE (0–10 mM) for 10 minutes in the presence or absence of 20 nM insulin. (B) C2C12 cells were pretreated with 500 μM palmitic acid for 24 hours, followed by treatment with 10 mM 1,1-DEE, 20 nM insulin, or both for 10 minutes. Cell lysates were alkylated with 10 mM NEM, and Western blot analysis was performed using antibodies against PTEN, phosphorylated Akt, Akt, and GAPDH.

[0073] Hereinafter, a composition for increasing insulin sensitivity or improving resistance containing 1,1-diethoxyethane as an active ingredient according to a specific embodiment of the invention and the use thereof will be described in detail. However, this is presented as one example of the invention and does not limit the scope of the invention, and it is obvious to those skilled in the art that various modifications to the embodiment are possible within the scope of the invention. Throughout this specification, unless otherwise specifically stated, "includes" or "contains" refers to the inclusion of any component (or constituent) without particular limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0074] As used herein, the term "treatment" means any form of treatment or prevention that provides effects, including improvement of the individual's condition, delay of disease progression, delay of symptom onset, or slowing of symptom progression, to an individual who suffers from a disease or is at risk of developing a disease. Accordingly, the term "treatment" includes preventive treatment of the individual that prevents the onset of symptoms. Furthermore, the terms "treatment" and "prevention" are not intended to mean the cure or complete elimination of symptoms.

[0075] As used in this specification, the term "improvement" may mean any action that at least reduces parameters related to the alleviation or treatment of a condition, such as the degree of symptoms.

[0076] As used herein, the term “object” means an animal including animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs. For example, the object may be a mammal, particularly a human.

[0077]

[0078] 1. Composition for increasing insulin sensitivity or improving resistance

[0079] The present invention

[0080] We intend to provide a composition for increasing insulin sensitivity or improving resistance comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0081] In the composition for increasing insulin sensitivity or improving resistance according to the present invention, the 1,1-diethoxyethane can induce oxidative inactivation of PTEN through the formation of disulfide bonds between Cys124 and Cys71 residues of PTEN.

[0082] In the composition for increasing insulin sensitivity or improving resistance according to the present invention, the 1,1-diethoxyethane can activate Akt by increasing phosphorylation at Ser473 and Thr308 of Akt.

[0083] In the composition for increasing insulin sensitivity or improving resistance according to the present invention, the 1,1-diethoxyethane can promote glycolysis by increasing phosphorylation at the Ser483 residue of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2).

[0084] In the composition for increasing insulin sensitivity or improving resistance according to the present invention, the 1,1-diethoxyethane may be used for the prevention, treatment, or improvement of diseases related to insulin resistance. The composition for increasing insulin sensitivity or improving resistance may include one or more selected from metabolic disorders, blood glucose control disorders, prevention and reversal of diabetes progression, diabetes complications, weight management disorders, protection and improvement of pancreatic beta cells, liver disease, and insulin-related disorders.

[0085] In one embodiment, the metabolic disorder may include, but is not limited to, Type 1 diabetes, Type 2 diabetes, Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Hyperglycemia, Postprandial Hyperglycemia, Polycystic Ovary Syndrome (PCOS), Hyperlipidemia, Hypertension, Overweight, Obesity, or Metabolic Syndrome.

[0086] In one embodiment, the blood glucose control disorder may include, but is not limited to, a decrease in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c or an improvement in blood glucose control.

[0087] In one embodiment, the diabetes progression-related impairment may include, but is not limited to, prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome.

[0088] In one embodiment, the diabetes complications may include, but are not limited to, cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, or vascular restenosis.

[0089] In one embodiment, the weight management disorder may include weight loss, prevention of weight gain, or promotion of weight loss, but is not limited thereto.

[0090] In one embodiment, the pancreatic beta cell-related disorder may include, but is not limited to, prevention, slowing, delaying, or treatment of pancreatic beta cell degeneration and / or pancreatic beta cell dysfunction, improvement and / or recovery of pancreatic beta cell function, and / or recovery of pancreatic insulin secretion function.

[0091] In one embodiment, the liver-related disease may include, but is not limited to, the prevention, slowing, delaying, or treatment of a disease or condition caused by abnormal accumulation of liver fat.

[0092] In one embodiment, the insulin-related disorder may include, but is not limited to, the maintenance and / or improvement of insulin sensitivity, and the prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0093]

[0094] 2. Use of a composition for increasing insulin sensitivity or improving resistance

[0095] The present invention aims to provide a pharmaceutical composition, cosmetic composition, food composition, or feed composition for the prevention, treatment, or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0096] As used herein, the term "insulin resistance" refers to a condition in which the function of insulin in lowering blood sugar is impaired, preventing cells from effectively burning glucose. High insulin resistance may refer to a state in which the human body produces excessive insulin, which can lead to the development of various diseases.

[0097] As used herein, the term “treatment of insulin resistance-related diseases” refers to enabling insulin to function properly within the body in a state of insulin resistance. In this sense, the treatment of insulin resistance-related diseases may be used to describe the improvement of insulin resistance, the regulation of insulin resistance, the enhancement of insulin sensitivity, or the regulation of insulin sensitivity; any action corresponding to a state of insulin resistance that enables recovery may be included within the scope of the present invention.

[0098]

[0099] (1) Pharmaceutical composition

[0100] According to one embodiment,

[0101] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0102] In a pharmaceutical composition according to the present invention, the disease associated with insulin resistance comprises metabolic disorders including Type 1 diabetes, Type 2 diabetes, Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Hyperglycemia, Postprandial Hyperglycemia, Polycystic Ovary Syndrome (PCOS), Hyperlipidemia, Hypertension, Overweight, Obesity, and Metabolic Syndrome; Impaired blood glucose control, including a reduction in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c and improved blood glucose control; impairments related to the progression of diabetes, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;It may include, but is not limited to, one or more selected from insulin-related disorders, including, maintenance and / or improvement of insulin sensitivity, and prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0103] In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered by oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernous injection, intrathecal injection, epidural injection, and rectal administration. When administered orally, for example, the pharmaceutical composition may be formulated as a tablet, or the active agent may be coated or protected from degradation in the stomach. Additionally, the composition may be administered by any device capable of delivering the active substance to target cells. The route of administration may vary depending on the general condition and age of the subject being treated, the nature of the treatment conditions, and the selected active ingredient.

[0104] In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be carried on a carrier, and the carrier may include one or more selected from virus particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes, but is not limited thereto.

[0105] In the pharmaceutical composition according to the present invention, the suitable dosage of the pharmaceutical composition varies depending on factors such as the formulation method, the mode of administration, the patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the pharmaceutical composition may be administered as a single or multiple doses, or divided into 1 to 4 doses per day. For example, the pharmaceutical composition may contain 0.01 mg / kg to 100 mg / kg, preferably 0.02 mg / kg to 90 mg / kg, and more preferably 0.03 mg / kg to 80 mg / kg per adult.

[0106] In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention pertains. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer. Furthermore, the pharmaceutical composition may be administered in the form of a suppository, spray, ointment, cream, gel, inhalant, or skin patch. Additionally, the pharmaceutical composition may be prepared for administration to mammals, more preferably for administration to humans.

[0107] In the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier may be a solid or a liquid and may be one or more selected from excipients, antioxidants, buffers, bacteriostatic agents, dispersants, adsorbents, surfactants, binders, preservatives, disintegrants, sweeteners, flavoring agents, lubricants, release regulators, wetting agents, stabilizers, suspending agents, and lubricants. Additionally, the pharmaceutically acceptable carrier may be selected from saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof.

[0108] In one embodiment, suitable fillers may include, but are not limited to, sugars (e.g., dextrose, sucrose, maltose and lactose), starch (e.g., corn starch), sugar-alcohols (e.g., mannitol, sorbitol, maltitol, erythritol and xylitol), starch hydrolysates (e.g., dextrin and maltodextrin), cellulose or cellulose derivatives (e.g., microcrystalline cellulose).

[0109] In one embodiment, suitable binders may include, but are not limited to, povidone, copovidone, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, gelatin, gums, sucrose, starch, or mixtures thereof.

[0110] In one embodiment, suitable preservatives may include, but are not limited to, benzoic acid, sodium benzoate, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, chlorbutol, gallate, hydroxybenzoate, EDTA, or mixtures thereof.

[0111] In one embodiment, suitable disintegrant may be sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked carboxymethylcellulose, starch, microcrystalline cellulose, or a mixture thereof, but is not limited thereto.

[0112] In one embodiment, suitable sweeteners may include, but are not limited to, sucralose, saccharin, sodium or potassium or calcium saccharin, acesulfame potassium or sodium cyclamate, mannitol, fructose, sucrose, maltose, or mixtures thereof.

[0113] In one embodiment, suitable glidant may be silica, colloidal silicon dioxide, talc, etc., but is not limited thereto.

[0114] In one embodiment, suitable lubricants may include, but are not limited to, long-chain fatty acids and their salts, such as magnesium stearate and stearic acid, talc, glyceride wax, or mixtures thereof.

[0115]

[0116] (2) Cosmetic composition

[0117] According to one embodiment,

[0118] The present invention aims to provide a cosmetic composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxyethane (1,1-DEE) as an active ingredient.

[0119] In a cosmetic composition according to the present invention, the disease associated with insulin resistance comprises metabolic disorders including Type 1 diabetes, Type 2 diabetes, Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Hyperglycemia, Postprandial Hyperglycemia, Polycystic Ovary Syndrome (PCOS), Hyperlipidemia, Hypertension, Overweight, Obesity, and Metabolic Syndrome; Impaired blood glucose control, including a reduction in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c and improved blood glucose control; impairments related to the progression of diabetes, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;It may include, but is not limited to, one or more selected from insulin-related disorders, including, maintenance and / or improvement of insulin sensitivity, and prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0120] In the cosmetic composition according to the present invention, the cosmetic composition may additionally include a dermatologically acceptable carrier. The dermatologically acceptable carrier may include, but is not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, hygroscopic agent, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, preservative, lower alcohol, etc., and its type and concentration may vary and may include parts that a person skilled in the art can modify within the scope of the present invention.

[0121] In the cosmetic composition according to the present invention, in addition to the active ingredient of the present invention, the cosmetic composition may include functional substances as needed, such as whitening agents, moisturizers, anti-inflammatory agents, antibacterial agents, antifungal agents, vitamins, sunscreens, antibiotics, anti-acne agents, perfumes, and dyes, and these may be included in the cosmetic composition according to the present invention in amounts commonly used in the field of cosmetics. To enhance the functional effect, the cosmetic composition of the present invention may additionally contain one or more moisturizing active ingredients exhibiting the same or similar functions.

[0122] In the cosmetic composition according to the present invention, the cosmetic composition may be prepared in the form of a general emulsion formulation and a solubilizing formulation. Cosmetics in the form of an emulsion include nourishing lotions, creams, essences, etc., and cosmetics in the form of a solubilizing formulation include softening lotions. In addition to the active ingredient of the present invention, the cosmetic composition may also be prepared in the form of an adjuvant for topical or systemic application commonly used in the art by containing a dermatologically acceptable medium or base. Suitable cosmetic formulations may be provided, for example, in the form of a solution, gel, solid or paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranulocyte, or an ionic (liposome) or non-ionic vesicular dispersant, or in the form of a cream, skin toner, lotion, powder, ointment, spray, or conceal stick. Additionally, it may be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

[0123] In the cosmetic composition according to the present invention, the cosmetic composition may be formulated into one or more selected from the group consisting of a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a perfume, a pack, a softening water, a lotion, a makeup base, an essence, a soap, a liquid cleanser, a bath additive, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing product, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray.

[0124]

[0125] (3) Food composition

[0126] As used in this specification, the term “food” refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone some degree of processing to become ready for direct consumption, and in a conventional sense may include food, food additives, functional foods, and beverages.

[0127] As used in this specification, the terms “functional food” or “health functional food” refer to a group of foods to which added value has been added to the food by using physical, biochemical, or biotechnological methods to act or manifest the function of the food for a specific purpose, or to foods designed and processed to sufficiently express in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery, which are inherent in the food composition; specifically, they may be health functional foods. The functional food may include food science-acceptable food additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods. The types of health functional foods may include, but are not limited to, powder, granules, tablets, capsules, or beverage forms.

[0128] According to one embodiment,

[0129] The present invention aims to provide a food composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0130] In a food composition according to the present invention, the disease associated with insulin resistance is a metabolic disorder including Type 1 diabetes, Type 2 diabetes, Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Hyperglycemia, Postprandial Hyperglycemia, Polycystic Ovary Syndrome (PCOS), Hyperlipidemia, Hypertension, Overweight, Obesity, and Metabolic Syndrome; Impaired blood glucose control, including a reduction in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c and improved blood glucose control; impairments related to the progression of diabetes, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;It may include, but is not limited to, one or more selected from insulin-related disorders, including, maintenance and / or improvement of insulin sensitivity, and prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0131] In the food composition according to the present invention, the food is characterized as being meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, stamina drinks, alcoholic beverages, or vitamin complexes.

[0132] In the food composition according to the present invention, the food composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0133] In the food composition according to the present invention, the term “functional food or health functional food” refers to a group of foods to which added value is imparted by using physical, biochemical, or biotechnological methods to make the function of the food act or manifest for a specific purpose, or a food processed by designing it to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition; specifically, it may be a health functional food. The functional food may include food-scientifically acceptable food auxiliary additives and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0134]

[0135] (4) Feed composition

[0136] According to one embodiment,

[0137] The present invention aims to provide a feed composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0138] In a feed composition according to the present invention, the disease associated with insulin resistance is a metabolic disorder including Type 1 diabetes, Type 2 diabetes, Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Hyperglycemia, Postprandial Hyperglycemia, Polycystic Ovary Syndrome (PCOS), Hyperlipidemia, Hypertension, Overweight, Obesity, and Metabolic Syndrome; Impaired blood glucose control, including a reduction in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin HbA1c and improved blood glucose control; impairments related to the progression of diabetes, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;It may include, but is not limited to, one or more selected from insulin-related disorders, including, maintenance and / or improvement of insulin sensitivity, and prevention or treatment of hyperinsulinemia and / or insulin resistance.

[0139] In the feed composition according to the present invention, the insulin resistance-related disease may include one or more selected from insulin resistance syndrome, hypertension, arteriosclerosis, dyslipidemia, fatty liver, hyperinsulinemia, myocardial infarction, stroke, heart failure, arrhythmia, cataract, diabetic complications, or diabetes, but is not limited thereto.

[0140] In the feed composition according to the present invention, the feed comprises nutrients such as energy, protein, lipids, vitamins, and minerals required by animals, and may be a plant-based feed such as grains, root vegetables, food processing by-products, algae, fibers, oils, starches, meal, grain by-products, or animal-based feed such as proteins, inorganic substances, oils, minerals, oils, and single-cell proteins, but is not limited thereto.

[0141] In the feed composition according to the present invention, the feed may be powder feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc., but is not limited thereto.

[0142] In the feed composition according to the present invention, the feed composition may include binders, emulsifiers, preservatives, etc. added to prevent quality degradation, and the feed composition may include feed additives. To increase utility, amino acid preparations, vitamin preparations, enzyme preparations, flavoring agents, non-protein nitrogen compounds, silicate preparations, buffering agents, extractants, oligosaccharides, etc. may be added to the feed. In addition, feed mixing agents, etc. may be additionally included, but are not limited thereto.

[0143]

[0144] 3. Treatment methods for diseases related to insulin resistance

[0145] The present invention aims to provide a method for preventing or treating diseases related to insulin resistance, comprising the step of administering to an individual a composition for increasing insulin sensitivity or improving resistance according to item 1 above, or a pharmaceutical composition according to item 2 (1) containing 1,1-diethoxytain (1,1-DEE) as an active ingredient.

[0146] In a method for preventing or treating diseases related to insulin resistance according to the present invention, the individual may include, but is not limited to, a human, a cow, a monkey, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit, or a guinea pig.

[0147] In the method for preventing or treating diseases related to insulin resistance according to the present invention, the route of administration, dosage, and frequency of administration of the pharmaceutical composition may be administered to the subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal method of administration, dosage, and frequency of administration may be selected by a person skilled in the art within an appropriate range. In the present invention, the preferred dosage of the exosome or pharmaceutical composition may be in the range of 0.001 mg / kg to 100 mg / kg per day for adults, depending on the patient's condition, body weight, gender, age, severity of the patient, and route of administration. Administration may be performed once a day or divided into several times. Such dosage shall not be interpreted as limiting the scope of the present invention in any aspect.

[0148] Various embodiments are presented below to aid in understanding the invention. The following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of protection of the invention to the following embodiments.

[0149]

[0150] Materials and Methods

[0151] 1. Ingredients

[0152] The four types of ethanol batches used in this study are shown in Table 1 below. 1,1-DEE (A902), N-ethyl maleimide (NEM, E3876), N-acetyl-L-cysteine ​​(NAC, A9165), 2′′ dichlorofluorescein diacetate (DCFH-DA; 35845), and H2O2 solution (88579) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-DEE (D0456) was purchased from Tokyo Chemical Industry (Tokyo, Japan).

[0153] BatchNameChemical Abstracts Service (CAS) #Catalog #Source1E164-17-51.00983.2511Merck2E264-17-51.00983.1011Merck3E364-17-51003304809Sigma-Aldrich4E464-17-51.00983.2511Merck

[0154] 2. Cell Culture, 1,1-DEE Treatment, and Transient Transfection with HA-Tagged pCGN Vector C2C12, MEF, and Ea.hy926 cells were cultured in DMEM (Dulbecco-Modified Eagle's Medium, LM001-05) and supplemented with 10% (v / v) fetal bovine serum (FBS, S001-01) and 1% (v / v) penicillin / streptomycin (P / S, LS202-02) purchased from Welgene (Gyeongsan, Republic of Korea). AC16 cells were cultured in DMEM / Nutrient Mixture F-12 (DMEM / F12, LM002-05) and supplemented with 12.5% ​​(v / v) FBS and 1% (v / v) P / S purchased from Welgene. When the cells reached a density of 70%, they were inoculated into 6-well plates and allowed to attach overnight. 1,1-DEE was diluted to a predetermined concentration in serum-free DMEM prior to cell treatment.

[0155] For transient transfection, cells were seeded into 6-well plates and allowed to attach overnight. Subsequently, cells were transfected with HA-tagged pCGN PTEN vectors containing WT, C71S, C124S, or C71S / C124S using Lipofectamine 2000 (#11608-027, Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. In summary, DNA and Lipofectamine 2000 were mixed separately with Opti-MEM (Ref: 31985-070, Gibco Life Technologies, Grand Island, NY, USA) and incubated at room temperature (RT) for 5 minutes. The mixtures were combined and incubated for an additional 20 minutes at room temperature. Subsequently, cells were treated with the mixture and incubated for 6 hours under 5% CO2 conditions at 37°C. The medium was then replaced with serum-containing DMEM, and the cells were cultured further. After one day following transfection, cells were treated with a predetermined concentration of 1,1-DEE and used for various analyses.

[0156]

[0157] 3. Gas Chromatography-Mass Spectrometry (GC-MS)

[0158] Gas sampling

[0159]

[0160] The researchers used a continuous irrigation system for transurethral resection of the prostate (TURP) and vaporization. Gases generated during the cutting and cauterization process were collected in the upper part of the bladder. The mixture of the solution and gas used for perfusion was drawn through tubing into a large vessel connected to a vacuum pump. A portion of the gas collected above the fluid in the vessel was directed from the exhaust gas outlet to tubing connected to a Tenax adsorption tube (Tenax GR; Japan Analytical Industry, Tokyo, Japan). The flow rate of the gas directed into the Tenax adsorption tube was maintained at 0.05 L / min using a gas flow pump (MP-S30; SIBATA, Tokyo, Japan). Measurements using a Tedlar Bag (Sigma-Aldrich) showed that a mixture of gas and room air was generated at a rate of approximately 45 L per hour. For quantification, 1 L of surgical gas was injected into a Tenax adsorption tube at a flow rate of 0.05 L / min.

[0161] Gas analysis

[0162] Gas was purged and trapped using the Automated Purge & Trap Sampler JTD-505III (Japan Analytical Industry). Quantification and qualitative analysis were performed using the GC / MS QP 2010 Plus (Shimadzu, Kyoto, Japan).

[0163] Fuzzy and trap conditions

[0164] The fuzzy and trap conditions are as follows:

[0165] Desorption temperature: 280℃, Desorption time: 30 min, Desorption gas flow rate: 50 mL / min, Cold trap temperature: -40℃, Pyrolysis temperature: 280℃, Transfer line temperature: 280℃, Needle heater temperature: 280℃, Cold trap heater temperature: 200℃, Head press: 86 MPa, Column flow rate: 1.0 mL / min, and Split ratio: 1 / 100.

[0166] GC / MS conditions

[0167] The analysis conditions are as follows:

[0168] Column: DB-624 column (30m × 0.251mm × 1.40μm; Agilent Technologies, Wilmington, DE, USA), Scan range: 30-600 mass, Oven temperature program: Hold at 40°C min), Rise to 10°C to 260°C, then hold for 5 min, Ion source temperature: 200°C, Transmission line temperature: 250°C, Electron energy (EM voltage): 70eV

[0169]

[0170] 4. Freeze-drying

[0171] Ethanol samples were stored in 15 mL Falcon tubes and frozen at -80°C for 5 hours. Afterward, small holes were punctured in the caps of all Falcon tubes. The frozen samples were freeze-dried overnight to completely remove volatile and water-containing components. The following day, the contents of each Falcon tube were reconstituted with serum-free DMEM prior to cell treatment.

[0172]

[0173] 5. DCFH-DA staining for ROS detection

[0174] Total intracellular reactive oxygen species (ROS) levels were evaluated according to a previous study [Kim, H. and X. Xue, Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining. J Vis Exp, 2020(160)]. Briefly, cells were seeded into 6-well plates and cultured overnight under 5% CO2 conditions at 37°C. After treatment, the cells were washed once with serum-free DMEM and cultured with 10 μM DCFH-DA at 37°C for 30 minutes. Subsequently, the cells were washed once with serum-free DMEM and twice more with PBS. Finally, the cells were rapidly imaged using a fluorescence microscope.

[0175]

[0176] 6. Flow cytometry

[0177] Intracellular reactive oxygen species (ROS) and mitochondrial peroxide levels were determined using flow cytometry (FACS CANTO II, BD Biosciences, NJ, USA). After 1,1-DEE treatment, cells were incubated with a predetermined concentration of DCFH-DA at 37°C for 30 minutes. Subsequently, cells were collected with Trypsin-EDTA (#25300-062, Gibco) and washed with cold PBS. Intracellular ROS levels were quantified immediately by excitation at 485 nm and emission at 530 nm, and mitochondrial peroxide levels were quantified by excitation at 510 nm and emission at 580 nm.

[0178]

[0179] 7. Western blot analysis and antibodies

[0180] The redox status of PTEN was analyzed according to the previously described method. In summary, cells were lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% NP-40, a phosphatase inhibitor, a protease inhibitor, and 10 mM NEM. The cell lysates were sonicated and then centrifuged at 13,000 rpm for 10 minutes. The supernatant was then collected, and protein concentrations were measured using the Pierce™ BCA Protein Quantification Kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were mixed with a reducing sample buffer containing 60 mM Tris (pH 6.8), 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.5% bromophenol, or a non-reducing sample buffer excluding 2-mercaptoethanol. Subsequently, SDS-PAGE was performed on the samples, and an immunoblot was conducted using PTEN-specific antibodies.

[0181] To investigate other protein expressions, cell lysates were mixed with the described reduced sample buffer and subjected to SDS-PAGE, followed by immunoblotting with various specific antibodies. The antibodies used in this study were as follows: Akt (#9272S, 1:1000), phospho-Akt473 (#9271S, 1:1000), phospho-Akt308 (#9275S, 1:1000), phospho-PFKFB2 (Ser483) (#13064, 1:1000), PFKFB2 (#13029, 1:1000), and β1:1000. These antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody (LF-PA0212, 1:1000) and anti-rabbit IgG horseradish peroxidase-conjugated antibody (LF-SA8002, 1:5000) were purchased from Ab Frontier (Daejeon, Republic of Korea).

[0182]

[0183] 8. Statistical Analysis

[0184] Western blot protein bands were quantified by densitometric analysis using ImageJ 1.50i (National Institutes of Health, Bethesda, MD, USA). All values ​​were expressed as the mean ± the standard error of the mean (SEM) of three independent experiments. Statistical significance was analyzed using 2-factor ANOVA for multi-group comparisons with GraphPad Prism, version 6 (GraphPad, San Diego, CA, USA). A p-value <0.05 was considered statistically significant.

[0185]

[0186] <Result>

[0187] 1. Evaluation of the Effect of 1,1-DEE on PTEN Oxidation According to Ethanol Batch

[0188] In this study, the effects of various ethanol batches provided by commercial sources on the redox status of PTEN were evaluated. HepG2 cells were used in the experiment, and the effects of ethanol batches at various concentrations on the oxidation rate of PTEN were investigated. As a result, significant differences in the oxidation rate of PTEN were observed depending on the ethanol batch, even at the same concentration. In particular, the E1 batch induced PTEN oxidation more strongly than the E2 and E3 batches. Furthermore, when E1 was mixed with E2 or E3, the level of PTEN oxidation tended to decrease compared to the oxidation effect of E1 (Fig. 1A).

[0189] Additionally, it was confirmed that the odor characteristics of batch E1 differed significantly from other ethanol batches. E1 exhibited strong alcoholic and fruity scents, whereas these scents were relatively less pronounced in batches E2 and E3. This suggests the possibility that specific compounds included in batch E1 may affect the redox state of PTEN.

[0190] To characterize the compound properties of batches E1, E2, and E4, volatile compounds in each batch were analyzed. Batch E4 exhibited odor characteristics similar to E1 (Fig. 1B); to confirm this, volatile compounds were evaporated by freeze-drying 100 mM ethanol, and the dried samples were then reconstituted with PBS or 100 mM E2. Subsequently, HepG2 cells were treated for 10 minutes with the ethanol of each batch (100 mM, E1, E2, E4) or the corresponding dried samples. The experimental results showed that batches E1 and E4 strongly induced PTEN oxidation compared to E2 and the control group. However, the dried samples of E1, E2, and E4 did not show similar effects regardless of the reconstitution condition (PBS or E2) (Fig. 1C). This indicates that the volatile compounds evaporated during the freeze-drying process.

[0191] Subsequently, additional compounds were identified in batches E1, E2, E3, and E4 through GC-MS analysis. As a result of the analysis, the aroma compound 1,1-DEE (1,1-diethoxyethane) was found at high concentrations in E1 and E4, but was not detected in E2 and E3. The results of GC-MS-based fragmentation analysis of 1,1-DEE in batches E1 and E4, along with the identified chemical structure, are shown in Figure 1D. Thus, it was demonstrated that 1,1-DEE may play a novel role in cellular functions mediating the redox regulation of PTEN.

[0192]

[0193] 2. Evaluation of whether 1,1-DEE induces PTEN oxidation in a concentration-dependent manner

[0194] To determine whether 1,1-DEE can exhibit novel molecular functions by regulating the redox state of PTEN, cells were treated with various concentrations of 1,1-DEE, and PTEN oxidation levels were evaluated. 1,1-DEE-mediated PTEN oxidation was observed within 10 minutes of treatment in various cell lines, including C2C12, MEF, AC16, and Ea.hy926. In C2C12 and MEF cells, PTEN oxidation was induced after treatment with 1,1-DEE at a concentration of 1 mM, while in AC16 and Ea.hy926 cells, similar levels of oxidation were achieved at 1,1-DEE at a concentration of 5 mM (Fig. 2A). 1,1-DEE-mediated oxidation occurred in a concentration-dependent manner, with oxidation levels increasing with higher concentrations. In contrast, 1,2-DEE, an isomer of 1,1-DEE, failed to induce PTEN oxidation in C2C12 cells at various concentrations. This suggests a specific conformation-dependent function of 1,1-DEE in the redox regulation of PTEN (Fig. 2B).

[0195] To investigate the underlying mechanism of 1,1-DEE-mediated PTEN oxidation, C2C12 cells were transfected with an HA-tagged pCGN vector containing broad-type (WT) PTEN or PTEN mutants (C71S, PTEN C124S, or PTEN C71 / 124S) prior to treatment with 1,1-DEE. As a result, PTEN oxidation was detected in cells transfected with WT PTEN, but not in cells transfected with PTEN C71S, PTEN C124S, or PTEN C71 / 124S (Fig. 2C). This indicates that 1,1-DEE mediates the oxidative inactivation of PTEN by inducing the formation of disulfide bonds between Cys124 and Cys71 residues.

[0196]

[0197] 3. Evaluation of whether 1,1-DEE induces time-dependent PTEN oxidation

[0198] In this study, we investigated the time-dependent regulatory patterns of 1,1-DEE on the redox status of PTEN. Various cell types, including C2C12, MEF, AC16, and Ea.hy926, were treated with 1,1-DEE for 120 minutes. Interestingly, PTEN oxidation levels began to increase just 5 minutes after the start of 1,1-DEE treatment, peaked after 10 minutes, and then gradually returned to basal levels after 120 minutes (Fig. 3A). However, 1,2-DEE failed to induce PTEN oxidation in C2C12 cells even after 120 minutes of treatment (Fig. 3B). Thus, it was demonstrated that specific forms of 1,1-DEE can induce reversible oxidation of PTEN in a time-dependent manner.

[0199]

[0200] 4. Evaluation of whether 1,1-DEE regulates PTEN oxidation and consequently Akt activation

[0201] In this study, Akt activation induced by 1,1-DEE-induced PTEN oxidation was investigated. C2C12 cells were treated with various concentrations of 1,1-DEE for 10 minutes or with 10 mM 1,1-DEE for various durations over 120 minutes. Akt phosphorylation at Ser473 and Thr308 was found to be enhanced after treatment with 1 mM 1,1-DEE for 10 minutes, suggesting increased Akt activity. Akt phosphorylation increased in a concentration-dependent manner due to 1,1-DEE, indicating negative regulation of PTEN in the Akt signaling pathway (Fig. 4A). Furthermore, similar to the reversible oxidation of PTEN mediated by 1,1-DEE, it was confirmed that Akt phosphorylation at Ser473 and Thr308 was also reversible. The levels of phosphorylated Akt473 and Akt308 increased after 5 minutes of 1,1-DEE treatment, peaked at 10 minutes, and then gradually returned to baseline levels after 120 minutes (Fig. 4B). This suggests that oxidative inhibition of PTEN induced by 1,1-DEE may lead to reversible activation of Akt.

[0202]

[0203] 5. Evaluation of whether 1,1-DEE-induced PTEN oxidation is mediated by ROS generation

[0204] In this study, to investigate the underlying mechanism of 1,1-DEE-induced PTEN oxidation, ROS production levels in cells were evaluated after 1,1-DEE treatment. DCFH-DA staining results confirmed that cytoplasmic ROS levels increased 10 minutes after 1,1-DEE treatment compared to the control group (Fig. 5A). Flow cytometry analysis showed that ROS levels increased within 5 minutes of treatment, peaked at 10 minutes, and returned to basal levels after 30 minutes. This indicates that 1,1-DEE can reversibly increase ROS production (Fig. 5B).

[0205] Next, to analyze the relationship between ROS generation and 1,1-DEE-induced PTEN oxidation, cells were pretreated with the ROS scavenger NAC for 120 minutes, followed by treatment with 1,1-DEE. NAC pretreatment was found to reduce 1,1-DEE-induced PTEN oxidation. This implies that ROS generated by 1,1-DEE mediates the oxidative inactivation of PTEN. Additionally, NAC pretreatment reduced the phosphorylation levels of Akt at Ser473 and Thr308 (Fig. 5C). These results suggest a regulatory mechanism of 1,1-DEE-induced ROS generation in the PTEN / Akt signaling pathway.

[0206]

[0207] 6. Evaluate whether 1,1-DEE promotes glycolysis through Akt activation

[0208] Akt activation is known to regulate the rate of glycolysis through the phosphorylation of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2) at Ser483 residues. PFKFB2 is a dual-function enzyme involved in both the synthesis and degradation of Fru-2,6-P2 (fructose-2,6-biphosphate), which controls eukaryotic glycolysis. Phosphorylation of PFKFB2 enhances its activation, generating Fru-2,6-P2 and further upregulating glycolysis. Accordingly, to investigate the effects of Akt activation on glycolysis, phosphorylation of PFKFB2 at Ser483 in AC16 cells was evaluated. As a result, 1,1-DEE treatment was shown to increase PTEN oxidation and Akt activation through phosphorylation at Ser473, and exposure to 1,1-DEE for 5 minutes resulted in phosphorylation of PFKFB2 at the Ser483 residue (Fig. 6A).

[0209] To determine whether Ser483 phosphorylation of PFKFB2 is regulated by Akt activation, AC16 cells were pretreated with the Akt inhibitor MK-2206 and then treated with 1,1-DEE. Pretreatment with MK-2206 reduced both Akt activation and PFKFB2 phosphorylation, indicating that glycolysis can be regulated by Akt activation induced by 1,1-DEE (Fig. 6A).

[0210] Next, cells were pretreated with Ebselen to investigate the relationship between 1,1-DEE-induced ROS generation and glycolysis. Interestingly, Ebselen pretreatment reduced the increase in 1,1-DEE-induced PFKFB2 phosphorylation (Fig. 6B). These results suggest that 1,1-DEE-induced ROS generation can further increase glycolysis through the phosphorylation of PFKFB2 by mediating the oxidative inactivation of PTEN and Akt activation.

[0211]

[0212] 7. Evaluate whether 1,1-DEE improves insulin sensitivity and palmitate-induced insulin resistance.

[0213] In this study, the effects of 1,1-DEE on insulin signaling and insulin sensitivity in C2C12 cells were investigated. Treatment with 1,1-DEE induced PTEN oxidation and Akt activation, which were evaluated by phosphorylated-Akt473 and phosphorylated-Akt308 levels. When cells were co-treated with 1,1-DEE and insulin, Akt phosphorylation at Ser473 and Thr308 increased compared to treatment with 1,1-DEE or insulin alone (Fig. 7A). This indicates that 1,1-DEE can enhance insulin signaling and insulin sensitivity.

[0214] To evaluate the effects of 1,1-DEE on the regulation of insulin resistance, an in vitro cellular model of insulin resistance was established using palmitic acid. These cells were treated with either 1,1-DEE or insulin for 10 minutes. It was confirmed that Akt phosphorylation at Ser473 and Thr308 residues in insulin-resistant cells was significantly reduced compared to control cells (Fig. 7B). This indicated that the insulin resistance model was successfully induced through palmitic acid treatment. Importantly, co-administration of 1,1-DEE and insulin upregulated Akt phosphorylation in insulin-resistant cells (Fig. 7B). This suggests that 1,1-DEE can alleviate palmitic acid-induced insulin resistance by mediating Akt activation.

[0215]

[0216] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0217]

[0218] Project ID: 2018R1D1A1B06051438

[0219] Ministry Name: Ministry of Science and ICT

[0220] Research Management Agency: National Research Foundation of Korea

[0221] Research Project Title: Regulation of the Cancer Suppressor Protein PTEN by Alcohol

[0222] Organizing Institution: Chonnam National University

[0223] Participating Company: Lux Anima Co., Ltd.

[0224] Research Period: 2023.03.01.–2024.02.29

[0225]

[0226] Department Name: Jeonnam Technopark Foundation

[0227] Research Project Name: Hwasun Vaccine Industry Special Zone Intellectual Property Capacity Building Support Project

[0228] Research Project Title: IP-R&D

[0229] Organizer: Lux Anima Co., Ltd.

Claims

A composition for increasing insulin sensitivity or improving resistance, comprising 1,1,1-diethoxyethane (1,1-DEE) as an active ingredient.

2. In Paragraph 1, A composition for increasing insulin sensitivity or improving resistance, characterized in that the above 1,1-diethoxyethane induces oxidative inactivation of PTEN through the formation of disulfide bonds between Cys124 and Cys71 residues of PTEN.

3. In Paragraph 1, A composition for increasing insulin sensitivity or improving resistance, characterized in that the above 1,1-diethoxyethane activates Akt by increasing phosphorylation at Ser473 and Thr308 of Akt.

4. In Paragraph 1, A composition for increasing insulin sensitivity or improving resistance, characterized in that the above 1,1-diethoxyethane promotes glycolysis by increasing phosphorylation at the Ser483 residue of PFKFB2 (6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 2). A pharmaceutical composition for the prevention or treatment of diseases related to insulin resistance comprising 5.1,1-diethoxytain (1,1-DEE) as an active ingredient, The above-mentioned diseases associated with insulin resistance include metabolic disorders including Type 1 diabetes, Type 2 diabetes, impaired glucose tolerance (IGT), impaired fasting glucose (IFG), hyperglycemia, postprandial hyperglycemia, polycystic ovary syndrome (PCOS), hyperlipidemia, hypertension, overweight, obesity, and metabolic syndrome; and glycemic control disorders including a decrease in fasting plasma glucose (FPG), postprandial plasma glucose (PPG), and / or glycated hemoglobin (HbA1c) and an improvement in glycemic control. Diabetes progression-related disorders, including prevention, slowing, delaying, or reversal of progression to type 2 diabetes from impaired glucose tolerance (IGT), impaired fasting glucose (IFG), insulin resistance, or metabolic syndrome;Diabetic complications including cataracts, microvascular and macrovascular diseases, nephropathy, retinopathy, neuropathy, tissue ischemia, diabetic foot, atherosclerosis, myocardial infarction, acute coronary syndrome, unstable angina, stable angina, stroke, peripheral artery occlusive disease, cardiomyopathy, heart failure, cardiac arrhythmia, and vascular restenosis; Weight management disorders including weight loss, prevention of weight gain, or promotion of weight loss; pancreatic beta-cell related disorders including prevention, slowing, delaying, or treatment of pancreatic beta-cell degeneration and / or pancreatic beta-cell dysfunction, improvement and / or recovery of pancreatic beta-cell function, and / or recovery of pancreatic insulin secretion function; liver diseases including prevention, slowing, delaying, or treatment of diseases or conditions caused by abnormal accumulation of liver fat;A pharmaceutical composition characterized by comprising any one or more selected from insulin-related disorders, including the maintenance and / or improvement of insulin sensitivity, and the prevention or treatment of hyperinsulinemia and / or insulin resistance.

6. In Paragraph 5, The above pharmaceutical composition is administered by any one or more administration methods selected from the group consisting of oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, epithelial administration, local administration, vaginal administration, pulmonary administration, rectal administration, sublingual administration, buccal administration, transdermal administration, ocular administration, inhalation, intracavernous injection, intrathecal injection, epidural injection and rectal administration, arterial injection, lymphatic administration, intraosseous injection, lacrimal duct delivery.

7. A cosmetic composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

8. In Paragraph 7, The above cosmetic composition is formulated into one or more selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, packs, softening waters, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays.

9. A food composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

10. In Paragraph 9, The above food is a food composition comprising meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, coffee beverages, stamina drinks, alcoholic beverages, or vitamin complexes.

11. A feed composition for the prevention or improvement of diseases related to insulin resistance, comprising 1,1-diethoxytain (1,1-DEE) as an active ingredient.

12. In Paragraph 11, The above feed composition is characterized by comprising powdered feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, or raw feed.

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