KR102073759B1 - Composition comprising NADH for preventing or treating metabolic disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases, a health functional food composition, and a cosmetic composition by including nicotinamide adenine dinucleotide reduced (NADH); and an in vitro composition for inhibiting the differentiation of preadipocytes into adipocytes and an in vitro composition for producing adipocytes exogenous reactive oxygen species by including NADH. The NADH of the present invention induces the formation of exogenous reactive oxygen species through OXPHOS I existing on the surface of preadipocytes; induced exogenous reactive oxygen species inhibits the differentiation of preadipocytes into adipocytes and induces the aging of cells; and the present invention can be useful for the purpose of preventing or treating various metabolic diseases related to proliferation and differentiation of fat cells by suppressing the signal transduction pathway by insulin.

Description

Composition comprising NADH for preventing or treating metabolic disease

The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases including NADH (Nicotinamide Adenine Dinucleotide reduced), a dietary supplement, a cosmetic composition and an in vitro composition for inhibiting differentiation of adipocytes into adipocytes, including NADH, fat It relates to an in vitro composition for producing progenitor exogenous reactive oxygen species.

Recently, mortality rates due to metabolic diseases such as obesity and diabetes are continuously increasing due to westernization of eating habits and lack of exercise. Obesity is a metabolic disease caused by an imbalance between calorie intake and consumption and is morphologically caused by hypertrophy or hyperplasia of fat cells in the body. Obesity is not only the most common malnutrition disorder in Western society, but also the importance of treatment and prevention in the recent trend of rapid increase in obesity due to the improvement of dietary life and westernization of lifestyle in Korea. have. Obesity is an important factor that not only psychologically shrinks individuals but also increases the risk of developing various adult diseases. Obesity is known to be directly related to the increased prevalence of various adult diseases, such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease, and it is defined as metabolic syndrome or insulin resistance syndrome by tying together diseases related to obesity. These are known to be the cause of atherosclerosis and cardiovascular disease.

It has been reported that fat cells are involved in inducing obesity. Adipose tissue is not just an energy storage organ, but an important endocrine organ that regulates homeostasis of energy by releasing adipokine such as adiponectin, leptin and resistin. As a result, it is expected to elucidate the regulatory mechanisms of fat cells and to target various metabolic diseases such as obesity. In obese patients, fat cells are increased. It is known that the increased fat cells are derived from differentiation of preadipocytes in the body. Therefore, attempts have been made to treat obesity through the regulation of fat cells.

The oxidative phosphorylation (OXPHOS) system has recently been found to be present in the cell membranes of various mammalian cell lines. However, the physiological function of the OXPHOS system in the cell membranes is still unknown. OXPHOS includes NADH-coenzyme Q oxidoreductase (complex I), succinate-Q oxidoreductase (complex II), Q-cytochrome c oxidoreductase (complex III), cytochrome c, cytochrome c oxidase (complex IV), and ATP synthase (complex V) It is composed of the same subunits, but it is unknown what role they play in the cell line's membranes, in particular in relation to adipocytes.

While the present inventors are studying a method for treating metabolic diseases including obesity targeting adipose progenitor cells, exogenous reactive oxygen species are formed by OXPHOS I present on the surface of adipose progenitor cells during NADH treatment. The present invention has been completed by inhibiting the differentiation of adipocyte progenitors into adipocytes and inducing senescence of cells.

Therefore, an object of the present invention is to inhibit the differentiation of adipocytes into adipocytes, including NADH (Nicotinamide Adenine Dinucleotide reduced) as an active ingredient, a pharmaceutical composition for preventing or treating metabolic diseases, health food composition, cosmetic composition and NADH It is to provide an in vitro composition for producing an in vitro composition for producing exogenous reactive oxygen species of adipocytes.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, including NADH (Nicotinamide Adenine Dinucleotide reduced).

The present invention also provides a dietary supplement for preventing or improving metabolic diseases, including NADH (Nicotinamide Adenine Dinucleotide reduced).

In another aspect, the present invention provides a cosmetic composition for preventing or improving metabolic diseases, including NADH (Nicotinamide Adenine Dinucleotide reduced).

The present invention also provides an in vitro composition for inhibiting the differentiation of adipocytes into adipocytes, including NADH (Nicotinamide Adenine Dinucleotide reduced).

The present invention also provides an in vitro composition for producing adipose progenitor cell exogenous reactive oxygen species (ROS) comprising NADH (Nicotinamide Adenine Dinucleotide reduced).

NADH of the present invention induces the formation of exogenous reactive oxygen species through OXPHOS I present on the surface of adipocytes, the induced exogenous oxygen species inhibit the differentiation of adipocytes into adipocytes, Induces and inhibits the signal transduction pathway by insulin can be useful for the purpose of preventing or treating various metabolic diseases associated with the proliferation and differentiation of fat cells.

1 is a diagram showing the results of confirming the change in reactive oxygen species (ROS) according to NADH or NADH + NAC treatment through fluorescence analysis. Figure 1 A is a diagram showing the change in ROS production level for 60 minutes, Figure 1 B is a view showing the result of confirming the ROS production level after 5 minutes of NADH or NADH + NAC treatment.
Figure 2 shows the results confirming that NDUFV1 expression is suppressed by siRNA treatment for NDUFV1, a subunit of OXPHOS I.
Figure 3 is a diagram showing the results confirmed the reduction of ROS production in NDUFV1 knockdown cells.
Figure 4 is a diagram showing the results of confirming the adipocyte differentiation of adipocytes cultured in NADH-containing medium (500μM and 1000μM) through oil red staining. Figure 4 A is a diagram showing the results of confirming the adipocyte differentiation by light microscopy after oil red staining. Figure 4 B is a view showing a quantitative determination of the relative oil red dye amount according to the NADH treatment.
FIG. 5 shows adipoQ (adiponectin) and PPAR which are adipocyte differentiation marker genes according to NADH concentration-specific treatment. Fig. 3 shows the results of confirming the γ expression change.
Figure 6 is a diagram showing the results confirmed by Western blot phosphorylation inhibitory effects of IRS-1, Akt, Erk according to NADH treatment.
Figure 7 is a diagram showing the results confirmed by the senescence-associated beta-galactosidase (SA-B-gal) of the fat precursor cell senescence according to NADH treatment.
8 is a schematic diagram showing the mechanism of inhibiting adipocyte differentiation and promoting cell senescence of NADH.

The present invention relates to a pharmaceutical composition, nutraceutical composition, cosmetic composition for preventing or treating metabolic diseases including NADH (Nicotinamide Adenine Dinucleotide reduced).

NADH of the present invention is a coenzyme found in cells and is a reduced form of NAD. NADH is converted to oxidative phosphorylation (OXPHOS) I on the surface of adipose progenitor cells when processed on adipose progenitor cells to generate free radicals, which inhibit the differentiation of adipocytes into adipocytes and May promote aging.

Hereinafter, the present invention will be described in detail.

NADH of the present invention may be included in the composition at 130 to 20,000 μM, preferably 500 to 15,000 μM. When NADH is included below 125 μM, NADH may rather promote the differentiation of adipocytes into adipocytes, activate the insulin signaling pathway, and cause toxicity if included in excess of 20,000 μM.

NADH of the present invention can induce an increase in extracellular reactive oxygen species (ROS). In the present invention, "exogenous reactive oxygen species" refers to reactive oxygen species induced by exogenous NADH stimulation, and exogenous reactive oxygen species are generated by OXPHOS (oxidative phosphorylation) I on the surface of adipocytes and changed to H ₂ O ₂ . It may be characterized by.

NADH of the present invention can induce the production of reactive oxygen species and inhibit the differentiation of adipocytes into adipocytes, and can down-regulate the expression of various transcription factors related to the differentiation of adipocytes into adipocytes. For example, NADH is a peroxisome proliferator-activated receptor (PPARγ), CCAAT-enhancer-binding protein α (C / EBP α), adipocyte fatty acid-binding protein-2 (aP-2), adiponectin (adiponectin, adipoQ) in adipose progenitor cells. , Downregulation of ADD1 / SREBP1c (adipocyte determination and differentiation factor 1 / sterol regulatory element binding protein), Fas (fatty acid synthase) or resistin, preferably PPARγ and adiponectin (adiponectin, adipoQ). Expression can be down-regulated in a concentration dependent manner.

In addition, the NADH of the present invention may be characterized by promoting the aging of fat precursor cells. The exogenous reactive oxygen species produced when the NADH of the present invention is treated to adipose progenitor cells are H ₂ O ₂ It can enter the cells in the form, induce DNA damage and promote cellular aging. Adipose progenitor cells, which promote cellular senescence, can be suppressed from differentiation into adipocytes.

In addition, the NADH of the present invention can more effectively inhibit the differentiation of adipocytes into adipocytes by inhibiting the insulin signaling pathway that is essential for adipocytes to differentiate into adipocytes.

Metabolic disease in the present invention may include without limitation a variety of diseases involving fat cells, can be used interchangeably with metabolic syndrome or insulin resistance syndrome. Metabolic disease in the present invention is preferably at least one selected from the group consisting of obesity, type 2 diabetes, fatty liver, hyperlipidemia, cardiovascular disease, lipid-related metabolic syndrome and atherosclerosis, more preferably obesity, second At least one selected from the group consisting of type diabetes mellitus, lipid-related metabolic syndrome and atherosclerosis.

As mentioned above, when the present invention is used as a medicament, the composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, Lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic Aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In the present invention, the composition may be administered in various oral or parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used It can be prepared using a suitable formulation known in the art, it is preferable to use those disclosed in the literature. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or sucrose. It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. In addition, the liquid preparations for oral administration include suspensions, solvents, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. This may be included.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. The parenteral administration can be made using external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, with an effective dose level of the individual type and severity, age, sex, type of virus infected, drug Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

As used herein, the term "administration" means providing a subject with a predetermined composition of the invention in any suitable manner.

In the present invention, the pharmaceutical composition may be administered to the individual by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

The pharmaceutical compositions of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the treatment of metabolic diseases.

In another aspect, the present invention provides a dietary supplement or food composition for preventing or improving metabolic diseases, including NADH (Nicotinamide Adenine Dinucleotide reduced).

In the present invention, the health functional food is preferably a bio-arm rhythm control having a food group or a food composition that has added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It is a food processed and designed to express the body's regulatory functions related to disease prevention and recovery to the living body and should be harmless to the human body when taken for a long time.

In the present invention, the nutraceutical may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of nutraceuticals.

The nutraceutical composition or food composition of the present invention may be used alone or as a food additive or adjuvant, and when used as an additive or adjuvant, the composition may be added as it is or used with other food or food ingredients, It can be suitably used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the manufacture of food or beverages the compositions of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the type of dietary supplement or food. Examples of health functional foods or foods to which the substance may be added include dairy products including ice creams, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes. It includes both food groups that give added value to function and express a specific purpose, or foods designed to sufficiently express the body's regulatory functions on the body, such as biodefense control, disease prevention and recovery, etc.

In addition to the above, the nutraceutical composition or food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, Preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the food composition of the present invention may include a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In another aspect, the present invention provides a cosmetic composition for preventing or improving metabolic diseases, including NADH (Nicotinamide Adenine Dinucleotide reduced).

In the present invention, the cosmetic composition may further contain one or more known active ingredients having the effect of preventing or improving metabolic diseases together with the NADH. Cosmetic compositions in the present invention may include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

Cosmetic compositions in the present invention can be prepared in any formulation commonly prepared in the art, for example solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

In the present invention, when the formulation of the cosmetic composition is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. And the like can be used. In the present invention, when the cosmetic formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, and in the case of a spray, additionally, chlorofluorohydrocarbons. Propellant, such as propane / butane or dimethyl ether. In the present invention, when the cosmetic formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as the carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol Fatty acid esters of 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used. When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

The present invention also provides an in vitro composition for inhibiting the differentiation of adipocytes into adipocytes, including NADH (Nicotinamide Adenine Dinucleotide reduced).

NADH of the present invention can effectively inhibit the differentiation of fat precursor cells into adipocytes when treated or co-cultured to adipose progenitor cells in vitro. In the present invention, NADH may be included in the composition at 130 to 20,000 μM, and preferably at 500 to 15,000 μM. When NADH is included below 125 μM, NADH may rather promote the differentiation of adipocytes into adipocytes, activate the insulin signaling pathway, and cause toxicity if contained in excess of 20,000 μM.

In another aspect, the present invention provides an in vitro composition for producing adipocyte progenitor exogenous reactive oxygen species comprising NADH (Nicotinamide Adenine Dinucleotide reduced).

In the present invention, when NADH, which is an exogenous stimulus, is co-cultured with adipose progenitor cells in vitro, or co-culture, the generation of exogenous active species can be induced in a short time. It can inhibit the differentiation of progenitor cells.

The exogenous reactive oxygen species may be generated by OXPHOS (oxidative phosphorylation) I on the surface of the fat progenitor cell surface. In one embodiment of the present invention, when NDUFV1, which is a subunit, is knocked down, NADH is treated. It was confirmed that free radical species were not induced.

The description of the composition of the present invention described above omit the description in order to avoid excessive complexity by the description of the overlapping content, terms not otherwise defined herein have the meaning commonly used in the art to which the present invention belongs. To have.

Hereinafter, preferred examples and formulation examples are provided to aid in understanding the present invention. However, the following examples and formulations are provided only to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example  One. NADH  According to treatment Reactive oxygen species  Confirm creation

When NADH was treated to pre-pre-adipocytes, an experiment was performed to determine whether reactive oxygen species (ROS) are generated in a NADH dependent manner. 3T3L1 (American Type Culture Collection, Manassas, VA, USA) was used as adipose progenitor cells, and NADH 1mM was treated and ROS generation was confirmed through luminescence analysis and the amount of its production was measured. It was. More specifically, 3T3L1 was incubated in a 100 mm culture dish for 48 hours using DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% fetal bovine serum with or without NADH 1 mM. 3T3L1 cells were washed three times with Krebs-HEPES buffer (25 mM HEPES, pH 7.4, 25 mM glucose, 140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ ) and floated in a culture dish to transfer to a 96 well plate. After that, the cells were incubated with Krebs-HEPES buffer containing 10 μM lucigenin and 1000 μM NADH for 60 minutes. Luminescent luminescence analysis was then performed using a SpectraMAX I3x multiplate reader (Molecular Devices, Sunnyvale, Calif., USA), and the measurement was determined.

In addition, NAC (N-acetyl-cysteine, 10 mM) was simultaneously treated to confirm whether the generation of ROS induced by NADH treatment was eliminated by ROS scavenger treatment, and the change in ROS production was confirmed accordingly. Is shown in FIG.

As shown in FIG. 1A, as a result of treating NADH to 3T3L1 adipocytes, it was confirmed that ROS were rapidly generated outside the cells within 10 minutes, and the generated ROS was reduced by NAC treatment. FIG. 1B shows that the extracellular ROS, which was hardly present when NADH was not treated, increased about 6000 times after 5 minutes after NADH treatment.

Example  2. NADH  According to treatment Reactive oxygen species  Verify creation path

In order to determine whether the adipose precursor extracellular ROS increase following NADH treatment is induced, siRNA was treated for NDUFV1, a subunit of oxidative phosphorylation (OXPHOS) I. siRNA oligomer targeting NDUFV1 (si-NDUFV1) and scrambled oligomers (si-con) were purchased from Ambion® (Thermo Fisher Scientific) and used. The siRNA was introduced into the cells according to the manufacturer's method using Lipfectamine 3000 reagent (Thermo Fisher Scientific), and the results are shown in FIG. 2.

As shown in Figure 2, NDUFV1 siRNA treatment disappeared the expression of NDUFV1 on the surface of adipocytes, indicating that effectively inhibited NDUFV1.

 Adipose progenitor cells treated with NDUFV1 siRNA were treated with 1 mM of NADH and the resulting exogenous ROS was confirmed by luminescence analysis and the results are shown in FIG. 3.

As shown in FIG. 3, it was confirmed that the amount of ROS produced was significantly decreased in the cells in which NDUFV1 was suppressed by NDUFV1 siRNA, compared to the siRNA untreated control. This result shows that the induction of ROS generation by NADH treatment is induced by NDUFV1, a cell surface electron transfer system of adipose progenitor cells.

Example  3. NADH  According to treatment Fat precursor cell  Confirmation of fat differentiation inhibitory effect

3.1 Fat precursor cell  Confirm differentiation suppression

In order to confirm that NADH treatment can inhibit the adipocyte differentiation of adipocytes, 3T3L1 adipocytes were cultured in a medium containing NADH 500 to 1000 μM to induce adipose differentiation. Specifically, induction of adipose differentiation was performed after incubation of 3T3L1 adipocytes in DMEM medium containing 10% FBS for 48 hours, followed by 0.25 mM 3-isobutyl-1-methylxanthine (Sigma Aldrich), 0.25 µM dexamethasone (Sigma Aldrich), and 200 nM insulin ( Sigma Aldrich) was performed by further incubation for 48 hours in a medium containing. Fat differentiation was confirmed by observation with an optical microscope after oil red O (Oil-Red O) staining, the results are shown in FIG.

As shown in Figure 4, it was confirmed that the fat progenitor cells cultured in the NADH-containing medium did not cause adipose differentiation compared to the NADH untreated control (A). As a result of measuring and comparing the amount of stained oil red O according to NADH treatment, it was confirmed that the amount of staining in fat progenitor cells cultured in a medium containing NADH was reduced by more than 50% compared to the untreated control (B). These results show that NADH inhibits the proliferation of adipocytes into adipocytes.

3.2 fat Progenitor cells  differentiation Marker  Confirm

In order to confirm that NADH inhibits the proliferation of adipocytes into adipocytes, mRNA amounts of adipoQ (adiponectin) and PPARγ, which are adipocyte differentiation marker genes, were confirmed by real-time RCR. NADH was treated with adipocytes at 500-1000 μM and induced differentiation for 48 hours, and the results of measuring mRNA expression in adipocytes were shown in FIG. 5.

As shown in FIG. 5, when the mRNA expression levels of adipoQ (adiponectin) and PPARγ of cells differentiated without NADH treatment were corrected to 1, it was confirmed that adipocyte differentiation markers decreased in a concentration-dependent manner in the NADH treatment group. It was further confirmed that NADH treatment could inhibit fat differentiation.

Example  4. NADH  Confirmation of Inhibition of Insulin Signaling Following Treatment

To determine whether insulin signaling, the most important signal transduction for adipose differentiation, was altered by NADH treatment, 500 μM and 1000 μM NADH were treated in 3T3L1 adipocytes and phosphorylation of IRS-1, Akt, and Erk by insulin Was confirmed by Western blot. Specifically, 3T3L1 was cultured in NADH 500 μM and 1000 μM containing medium, and cells were further stimulated with 20 nM of insulin for 5 minutes. Thereafter, protein expression levels of IRS-1, phospho-IRS-1 (pIRS-1), Akt, phospho-Akt (pAkt), Erk, phospho-Erk (pErk), and GAPDH were confirmed by immunostaining. The results are shown in FIG.

As shown in FIG. 6, the phosphorylation of IRS-1, Akt, and Erk was decreased by NADH treatment, and it was confirmed that insulin signaling was inhibited by NADH. This is a result showing that, unlike liver and muscle tissue, in which the production of ROS is known to increase insulin signaling, treatment of NADH can specifically inhibit insulin signaling in adipocytes.

Example  5. NADH  Induction of cellular aging following treatment

In order to determine whether senescence of adipose progenitor cells is induced by ROS induced when adipose progenitor cells are treated with NADH, 500 μM and 1000 μM of NADH were applied to adipose progenitor cells 3T3L1 and SA-B-gal (senescence-associated beta). The degree of aging of the cells was confirmed by staining. The results are shown in FIG.

As shown in FIG. 7, it was confirmed that the aged cells increased depending on the concentration of NADH treatment, and it was confirmed that NADH could promote adipocyte proliferation.

Through the above results, NADH promotes the production of ROS outside of adipocytes, thereby inhibiting the proliferation of adipocytes into adipocytes, and exogenous ROS is H ₂ O by OXPHOS I, a surface protein of adipocytes. Changed to ₂ to block insulin signaling pathways in adipocytes, thereby inhibiting the differentiation of adipocytes and at the same time confirmed that it can induce cell aging. A schematic diagram of the present invention is shown in FIG. 8, and these results confirm that NADH can be used for the prevention or treatment of metabolic diseases including obesity associated with adipocytes.

Formulation example  1. Manufacturing of medicines

1.1 Powder  Produce

NADH 500 μM

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1.2 Preparation of Tablets

NADH 500 μM

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components and tableting according to the manufacturing method of the conventional tablet to prepare a tablet.

1.3 Preparation of Injection

NADH 500 μM

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1.4 Liquid  Produce

NADH 500 μM

20 g of sugar

20g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1,00 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation example  2. Manufacture of food

NADH 500 μM

Vitamin Mixture

70 μg of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Potassium monophosphate 15 mg

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health functional food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components are mixed according to a conventional health functional food manufacturing method. Then, it can be used for the manufacture of the nutraceutical composition (eg, nutrition candy, etc.) according to a conventional method.

Formulation example  3. Cosmetic  Produce

NADH 500 μM

Glycerin 3.0 mg

Hydrogenated Lecithin 1.0 mg

Cetostearyl Alcohol 2.0

Polysorbate 60 1.5

Antioxidant 0.3

Preservative

Purified water

Claims (11)

A pharmaceutical composition for preventing or treating obesity, comprising NADH (Nicotinamide Adenine Dinucleotide reduced).
The pharmaceutical composition for preventing or treating obesity of claim 1, wherein the NADH induces an increase in extracellular reactive oxygen species (ROS).
The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the extracellular reactive oxygen species is produced by OXPHOS (oxidative phosphorylation) I on the surface of adipose progenitor cells.
The pharmaceutical composition for preventing or treating obesity of claim 1, wherein the NADH inhibits the differentiation of adipocytes into adipocytes.
The pharmaceutical composition for preventing or treating obesity of claim 1, wherein the NADH promotes senescence of fat precursor cells.
delete Health functional food composition for preventing or improving obesity comprising NADH (Nicotinamide Adenine Dinucleotide reduced).
Cosmetic composition for the prevention or improvement of obesity comprising NADH (Nicotinamide Adenine Dinucleotide reduced).
An in vitro composition for inhibiting differentiation of adipocytes into adipocytes comprising NADH (Nicotinamide Adenine Dinucleotide reduced).
In vitro composition for the generation of adipocyte progenitor exogenous reactive oxygen species comprising NADH (Nicotinamide Adenine Dinucleotide reduced).
The in vitro composition for producing adipose progenitor cell exogenous reactive oxygen species according to claim 10, wherein the adipose precursor cell exogenous reactive oxygen species is generated by OXPHOS (oxidative phosphorylation) I on the surface of the fat precursor cell.

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