WO2025143788A1 - Composition for enhancing bioenergy metabolism containing kestose - Google Patents

Abstract

The present invention relates to: a composition for enhancing cellular energy metabolism, comprising kestose as an active ingredient; and use thereof for the prevention, treatment or amelioration of diseases related to cellular energy metabolism.

Description

Composition for promoting bioenergy metabolism containing ketose

The present invention relates to a use for promoting energy metabolism or a use for improving energy metabolism reduction, which comprises kestos as an effective ingredient.

It is well known that the energy of cells is adenosine triphosphate (ATP). During anabolism, the energy derived from the metabolism of nutrients is transferred to the high-energy phosphate bond of ATP. The energy of this bond is consumed in the energy consumption phase.

To increase ATP levels, various nutritional supplements that rely on creatine compounds are known in the art. Creatine has been reported to be effective in stimulating ATP production because it plays a role in anaerobic ATP production during short/intensive exercise via the creatine kinase system. These supplements often fail to achieve the desired increase in ATP production, especially intramuscular ATP production.

Although numerous compositions and methods for increasing energy metabolism and ATP production are known in the art, all or nearly all of them suffer from one or more drawbacks. Accordingly, there still remains a need to provide improved compositions and methods for increasing energy metabolism and ATP production.

One example of the present invention is to provide a composition for promoting energy metabolism in an individual, comprising kestos as an active ingredient.

Another example of the present invention relates to a composition for promoting adenosine triphosphate (ATP) production in tissues having mitochondria, comprising kestose as an active ingredient, and more particularly, to provide a composition for promoting mitochondrial biogenesis.

Another example of the present invention is to provide a composition for promoting protein biosynthesis in mitochondria in tissues having mitochondria, comprising kestose as an active ingredient.

Another example of the present invention is to provide a composition for promoting sugar absorption, comprising kestose as an active ingredient.

Another aspect of the present invention provides a composition for preventing, improving and/or treating energy metabolism-related diseases, comprising kestose as an active ingredient.

Another example of the present invention is to provide a method for enhancing energy metabolism in a subject, comprising the step of administering kestos to a subject in need of enhancing energy metabolism.

Another example of the present invention is to provide a method for preventing, improving and/or treating an energy metabolism related disease, comprising the step of administering kestos to a subject in need of prevention, improvement and/or treatment of an energy metabolism related disease.

Another example of the present invention provides a use of Kestos for promoting energy metabolism or for preventing, improving and/or treating diseases related to energy metabolism.

Another example of the present invention is to provide a use of kestos for preparing a composition for promoting energy metabolism in a subject or a composition for preventing, improving and/or treating a disease related to energy metabolism.

In this specification, "enhancing energy metabolism" means a process of enhancing energy production, utilization, and storage at the cellular and biological levels, and specifically, may be at least one selected from the group consisting of promoting adenosine triphosphate (ATP) production in tissues having mitochondria (specifically, promoting mitochondrial biogenesis), promoting protein biosynthesis within mitochondria in tissues having mitochondria, promoting insulin secretion, and promoting sugar absorption, but is not limited thereto.

Specifically, the composition of the present invention containing kestose as an active ingredient has uses such as promoting bioenergy metabolism, promoting adenosine triphosphate (ATP) production in tissues having mitochondria, promoting protein biosynthesis within mitochondria in tissues having mitochondria, promoting insulin secretion, and promoting sugar absorption.

A composition containing Kestos according to the present invention as an active ingredient may be, for example, a pharmaceutical composition or a food composition.

A composition containing ketose according to the present invention as an active ingredient can be used to promote energy metabolism in a subject or an individual, and the subject or individual can be a mammal, including a human.

In addition, the present invention can be used for promoting adenosine triphosphate (ATP) production in a tissue having mitochondria, promoting protein biosynthesis in mitochondria in a tissue having mitochondria, or promoting sugar absorption in a tissue, wherein the individual or subject can be a mammal including a human.

In one embodiment of the present invention, the present invention relates to a composition and method for promoting energy metabolism, and more particularly, to a composition for increasing ATP production. The present invention provides a food composition for promoting or improving the energy level of a subject. The composition according to the present invention can substantially increase ATP and energy levels, and in particular, it has been confirmed that the increase in ATP production in muscle tissue is higher, and in particular, it has the effect of promoting ATP and energy levels in muscle cells.

Such entities may be mammals, including humans, for example, humans suffering from diseases that reduce intracellular adenosine triphosphate (ATP), humans engaged in excessive physical activity, such as athletes or workers, and humans seeking to increase their energy levels. Other mammals, such as dogs or cats, are also included in the present method.

Administration of a composition according to the present invention increases the level of ATP within muscle cells, prolongs the time and intensity for which a mammal can exercise, and mammals that do not exercise and mammals that expend higher than normal levels of energy while recovering from physical insults such as trauma, burns and sepsis also benefit from administration of the composition of the present invention.

The composition for promoting bioenergy metabolism, the method for promoting energy metabolism in a subject, and/or the method for preventing, improving, and/or treating diseases related to energy metabolism according to the present invention stimulate and promote ATP synthesis in a mammal, and specifically, kestose is orally administered in an amount effective to enhance the energy of the mammal before, during, and after a period of high ATP demand. The subject administered kestose can exercise longer, achieve higher intensities, and subjectively have more energy than a mammal not administered kestose. The present invention provides a method for stimulating ATP synthesis by administering kestose, and provides a kestose-containing composition that is particularly beneficial for mammals experiencing high energy demand or mammals with chronically low energy levels.

The above enhancement of energy metabolism can occur in muscle cells or muscle tissue.

In general, mitochondrial biogenesis pathway activators activate or increase genes of the pathway. Increase or activation of the mitochondrial biogenesis pathway can be observed by increased activity of genes of the pathway. AMPK senses the amount of energy (ATP) to maintain energy balance and plays a role in activating energy and mitochondrial biogenesis metabolic pathways.

In particular, as activators of the mitochondrial biogenesis pathway, SIRT1 (sirtuin1), PGC1α (peroxisome proliferator-activated receptor gamma coactivator), TFAM (Mitochondrial transcription factor A), NRF1 (Nuclear respiratory factor 1), IRS-1 (Insulin receptor substrate 1), Akt, MyoD (Myogenic differentiation 1), MyoG (Myogenin), etc. are linked to the mitochondrial biogenesis metabolic pathway. Therefore, AMPK, SIRT1, PGC1α, TFAM, NRF1, IRS-1 (Insulin receptor substrate 1), Akt, MyoD (Myogenic differentiation 1), MyoG (Myogenin) are energy metabolism regulatory enzymes and genes that follow the mitochondrial biogenesis signal transduction pathway. Promotion or enhancement of mitochondrial biogenesis can be determined by the expression level and/or expression of related genes. Therefore, activation of the AMPK and SIRT1 pathways includes stimulation of PGC1α, TFAM, NRF1, IRS-1, Akt, MyoD, MyoG and/or stimulation of mitochondrial biogenesis. In addition, when AMPK is activated, it is known that it increases the movement of GLUT4 to the cell membrane within the cell, and the GLUT4 in the cell membrane moved by AMPK promotes glucose uptake. The composition according to the present invention, the method for promoting energy metabolism in a subject, and/or the method for preventing, improving and/or treating a disease related to energy metabolism can promote the expression of one or more factors selected from the group consisting of AMPK, SIRT1, PGC1α, TFAM, NRF1, IRS-1, Akt, MyoD and MyoG.

The composition according to the present invention relates to a composition for promoting insulin secretion or sugar absorption of a tissue, wherein the insulin secretion or sugar absorption may be muscle tissue or intestinal tissue. Sugar absorption of an individual, tissue or cell may be promoted by various hormones or physiological factors together with insulin. For example, exercise induces sugar absorption in skeletal muscle through an insulin-independent pathway, and α1-adrenaline or endothelin A receptors also increase the sugar absorption rate through an insulin-independent pathway. Insulin is secreted from pancreatic cells and binds to insulin receptors on cell membranes to send signals so that blood sugar can be absorbed into cells, and causes sugar metabolism and energy metabolism of each tissue to occur so that blood sugar can be maintained at a constant level through body metabolism. If insulin secretion decreases due to a decline in pancreatic beta cell function, or insulin resistance of peripheral tissues such as muscles, liver and blood vessels increases, blood sugar cannot be used as an energy source and is excreted from the body, which leads to various complications. Therefore, it also contributes to promoting energy metabolism by promoting glucose absorption to supply glucose used as an energy source in muscle cells or intestinal cells.

In particular, as factors promoting sugar absorption, there are GLUT2 (glucose transporter 2), SGLT1 (Sodium-Glucose Cotransporter 1), GCK (Glucokinase), etc., and as factors related to inducing insulin secretion, there are GLP-1 (Glucagon-Like Peptide-1), CREB (cAMP Response Element-Binding Protein), etc. Specifically, when insulin is secreted, the expression of GLP-1 increases, the expression of CREB decreases, and the decrease in CREB expression may occur due to the induction of insulin secretion by the phosphorylation of CREB. The composition according to the present invention, the method for promoting energy metabolism in an individual, and/or the method for preventing, improving, and/or treating a disease related to energy metabolism can promote the expression of one or more factors selected from the group consisting of GLUT2, SGLT1, GCK, and GLP-1, and can suppress the expression of the CREB factor.

Another example of the present invention provides a composition for treating energy metabolism-related diseases, comprising kestose as an active ingredient. In the present specification, the energy metabolism-related diseases may be at least one selected from the group consisting of diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, diseases that may be caused by mitochondrial disease, and precocious puberty, but is not limited thereto. The diseases that may be caused by the mitochondrial disease include degenerative brain diseases (e.g., dementia, Parkinson's disease, etc.), diabetes, cardiovascular diseases, cancer, etc.

Another example of the present invention is to provide a method for enhancing energy metabolism in a subject, comprising the step of administering kestos to a subject in need of enhancing energy metabolism.

Another example of the present invention is to provide a method for preventing, improving and/or treating an energy metabolism related disease, comprising the step of administering kestos to a subject in need of prevention, improvement and/or treatment of an energy metabolism related disease.

The above object may be at least one selected from the group consisting of, but is not limited to, primates such as humans and monkeys, rodents such as mice, rats and rabbits, mammals including dogs, cats, cows, pigs, sheep, horses and goats, birds including chickens, ducks and geese, reptiles including snakes, lizards, turtles and crocodiles, amphibians and vertebrates such as fish.

As used herein, "treatment" may be used to mean alleviation or improvement of a symptom (e.g., diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, precocious puberty, etc.), delay or alleviation of the progression of a disease (e.g., diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, precocious puberty, etc.), improvement, alleviation or stabilization of a disease state or symptom, partial or complete recovery or elimination, and other beneficial treatment results. "Improvement" may be used to mean alleviation or improvement of a symptom (e.g., diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, precocious puberty, etc.), delay or alleviation of the progression of a disease (e.g., diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, precocious puberty, etc.), improvement, alleviation or stabilization of a disease state or symptom, and the like. “Prevention” is used to mean any mechanism and/or effect that acts on a subject who does not have a particular disease to prevent the development of said particular disease or to delay the onset of said disease.

The pharmaceutical composition provided herein may be administered by any commonly used route, for example, oral administration, or parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, local administration to a lesion site, or intraperitoneal injection.

The pharmaceutical composition above can be administered in a pharmaceutically effective amount. The dosage of the pharmaceutical composition above can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. The administration dosage can vary depending on the patient's age, weight, sex, administration form, health condition, and disease severity, and can be administered once a day or several times at regular intervals according to the judgment of a doctor or pharmacist.

The dosage form of medicines, quasi-drugs, or supplements containing Kestos is not particularly limited, and a dosage form suitable for the administration method can be appropriately selected. For example, in the case of oral administration, it can be in the form of solid or liquid formulations such as powder, tablet, sugar, capsule, granule, dry syrup, liquid, syrup, drop, or drink.

The composition according to the present invention comprises kestose or 1-kestose as an effective ingredient, and kestose can be used alone or as a sugar composition containing it. Kestose can be used in the form of a liquid or powder, and the powder can be amorphous or crystalline. The kestose can be purchased and used as a commercial product, or can be manufactured using a predetermined raw material.

An example of the above sugar composition may be a kestose-containing fructooligosaccharide (FOS). FOS consists of a linear chain having 1 to 9 fructose residues linked to a sucrose molecule by β2→1 linkages. The above kestose-containing fructooligosaccharide preferably has 1-kestose as its main component, and specifically, the sugar composition may include kestose and a mixture of at least one saccharide selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4), that is, the kestose may be provided as a mixture of at least one saccharide selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4). For example, it may contain a high content of 1-kesotse (GF2), and may contain at least one selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4).

When the above kestose is provided as a mixture with at least one sugar selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4), the kestose content may be 50 wt% or more, 60 wt% or more, 65 wt% or more, 70 wt% or more, 75 wt% or more, 80 wt% or more, or 85 wt% or more, and may be 99.9 wt% or less, 99.5 wt% or less, or 99 wt% or less, but is not limited thereto.

The kestose or sugar composition containing kestose that can be used in the present invention is not particularly limited, and can be produced using sugar as a substrate, usually by using an enzyme having kestose conversion activity or a microorganism producing the enzyme.

The enzyme having the above kestose conversion activity is an enzyme having an activity of converting a fructooligosaccharide containing kestose from a substrate containing sugar, and may be an enzyme derived from at least one selected from the group consisting of, for example, an Aspergillus niger strain, a Pichia farinose strain, a Yarrowia lipolytica strain, a Millerozyma farinose, and an Aspergillus oryzae strain.

In the present invention, as the intake (administration) of kestose, specifically, in the method for promoting energy metabolism of the subject and/or the method for preventing, improving and/or treating energy metabolism-related diseases, the intake (administration) of kestose is, for example, 0.01 to 0.34 g/kg body weight, preferably 0.01 to 0.30 g/kg body weight, more preferably 0.01 to 0.24 g/kg body weight, 0.03 to 0.34 g/kg body weight, 0.05 to 0.34 g/kg body weight, 0.06 to 0.34 g/kg body weight, 0.03 to 0.24 g/kg body weight, 0.05 to 0.24 g/kg body weight, 0.06 to 0.24 g/kg body weight, 0.03 to 0.30 g/kg body weight, 0.05 to 0.30 g/kg body weight, 0.06 to 0.30 g/kg body weight, 0.03 to 0.26 g/kg body weight, 0.05 to 0.26 g/kg body weight, or 0.06 to 0.26 g/kg body weight. These intake amounts are not limited to once a day, but may be divided into multiple times and consumed.

One method is to administer Kestos orally to humans or animals as is or in the form of food or medicine.

In the above Kestos composition, Kestos as an active ingredient may be included in a total daily dosage of 1 g to 30 g, 1 g to 20 g, 1 g to 15 g, 2 g to 15 g, 1 g to 10 g, 2 g to 10 g, 1 to 7 g, or 2 to 7 g based on a 60 kg adult, but is not limited thereto.

In addition, the above-mentioned kestose composition may be a composition containing the combined content of kestose, nystose, and fructofuranosylnystose of 400 mg/g or more, 500 mg/g or more, 800 mg/g or more, or 900 mg/g or more.

The kestose usable in the present invention may be in either liquid (e.g., sugar syrup) or powder form, and may be included in the composition according to the present invention in various amounts. The kestose may be used as a single component, or may be used in a mixed composition containing other sugars, for example, fructooligosaccharide (FOS).

The above-mentioned liquid or powdered kestose may be a composition having a content of 50 (w/w) % or more, 60 (w/w) % or more, 70 (w/w) % or more, 80 (w/w) % or more, preferably 85 (w/w) % or more based on the total solid content. In addition, the crystalline kestose may be a composition having a content of 98 % or more based on the total solid content.

Kestose can be used by adding it to the normal manufacturing processes of various foods, food additives, and animal feed. The sweetness of 1-kestose is 30, and its quality of taste, physical properties, and processability are close to sucrose, so it can be used in various foods, beverages, food additives, medicines, or feeds by handling it in the same way as sugar, such as by replacing part or all of the sugar with 1-kestose in the manufacturing process of various foods.

In the food composition provided herein, the term "food" means an edible natural product or processed product containing one or more nutrients, and may be used in its usual sense to mean at least one selected from the group consisting of various general foods, health functional foods, beverages, food additives, and beverage additives. The term "food composition" may mean a combination of materials for producing the food.

Specific embodiments of the composition according to the present invention include, for example, beverages, dairy products, granules for food use, pastes, seasonings, retort foods, baby foods, fermented foods, preserved foods, processed foods such as processed aquatic products, processed meat products, and processed grain products, food additives, health foods, animal feed, etc.

The present invention relates to a use for promoting energy metabolism, or a use for preventing, treating or improving a disease related to decreased energy metabolism, comprising kestose as an active ingredient.

Figure 1 is a graph comparing the expression levels of sugar absorption promoting factors for inducing energy metabolism in muscle cells (C2C12). After treating muscle cells (C2C12) with kestose, glucose, and maltose, RNA was extracted from the cells, cDNA was synthesized, and this was confirmed through real-time PCR.

Figure 2 is a graph showing the content of sugar absorbed into muscle cells (C2C12). It is the result of confirming the content of sugar within the cells by ELISA after treating muscle cells (C2C12) with kestose, glucose, and maltose.

Figure 3 is a graph comparing the expression level of mitochondrial biogenesis promoting factors that produce energy sources (ATP) using sugar absorbed in muscle cells (C2C12). This is the result of treating muscle cells (C2C12) with kestose, glucose, and maltose, extracting RNA from the cells, synthesizing cDNA, and confirming it through real-time PCR.

Figure 4 is a graph showing the quantitative analysis of mitochondrial proteins that produce energy sources (ATP) used for energy metabolism in muscle cells (C2C12). The results are obtained by treating muscle cells (C2C12) with kestose, glucose, and maltose, extracting mitochondria from the cells, and confirming the protein concentration of the mitochondria using BSA.

Figure 5 is a graph that confirms the amount of energy source (ATP) produced and utilized for energy metabolism in muscle cells (C2C12).

Figure 6 is a graph showing the change in body weight according to the type of administered substance and administration time for normal mice and obese mice.

Figure 7 is a graph comparing the expression levels of mitochondrial biogenesis promoting factors in normal mouse and obese mouse muscles.

Figure 8 is a graph comparing the expression levels of glucose absorption promoting factors in the intestines of normal and obese mice.

Figure 9 is a graph comparing the expression levels of insulin secretion inducing factors and glucose absorption factors in the pancreas of normal and obese mice.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

Example 1: Cell culture and differentiation induction

Muscle cells C2C12 were cultured in an incubator using DMEM medium (Lonza, USA) containing 1% penicillin/streptomycin and 10% FBS. Cells were passaged when they were 70–80% confluent. After washing twice with PBS, cells were detached using 0.25% Trypsin-EDTA and centrifuged at 1,000 rpm for 5 min to obtain cells. The obtained cells were passaged using fresh cell medium. In this experiment, additional experiments were performed using cells that had been passaged more than twice.

Muscle cells (C2C12) were differentiated using horse serum (Gibco, USA). Muscle cells seeded in a well plate using DMEM media with 1% penicillin/streptomycin and 10% FBS were grown in an incubator until 90% confluent and differentiation was induced. The differentiation medium used was DMEM media containing 1% penicillin/streptomycin and 2% horse serum, and the differentiation medium was replaced with fresh differentiation medium once every two days. The differentiation medium was replaced three times over a total of 6 days. On the 6th day after the initiation of differentiation, 1-kestose, glucose, or maltose dissolved in the differentiation medium were treated to the muscle cells at a concentration of 14 mM each and reacted for 24 hours.

Example 2: Measurement of expression levels of genes related to sugar absorption for energy metabolism and the content of absorbed sugar

Example 2-1: Measurement of expression levels of factors that induce sugar uptake and utilize absorbed sugar to induce energy metabolism in muscle cells (C2C12)

To measure the mRNA expression levels of factors that promote sugar uptake and are involved in the utilization of absorbed sugar in differentiated muscle cells, RNA was extracted from muscle cells treated with 1-kestose, glucose, or maltose in Example 1 using Trizol (Invitrogen, USA). The isolated RNA was used to synthesize cDNA using a cDNA synthesis kit (Takara, Japan). The expression levels of factors that promote sugar uptake (GLUT4, GCK) in differentiated muscle cells were measured by performing real-time PCR quantitative analysis using the synthesized cDNA.

The primer sequence information used in performing the above PCR is shown in Table 1 below, and the results of measuring the expression levels of GLUT4 and GCK are shown in Figures 1 and 2, respectively.

Target gene explanation order SEQ ID NO GLUT4 Forward (5′-3′) CTTGGCTCCCTTCAGTTTG 1 GLUT4 Reverse (5′-3′) TGCCTTGTGGGATGGAAT 2 GCK Forward (5′-3′) CTGTGAAAGCGTGTCCACTC 3 GCK Reverse (5′-3′) GTGATTTCGCAGTTGGGTGT 4 AMPK Forward (5′-3′) TCACCGGACATAAAGTGGCT 5 AMPK Reverse (5′-3′) TGATGATGTGAGGGTGCCTG 6 SIRT1 Forward (5′-3′) AGTTCCAGCCGTCTCTGTGT 7 SIRT1 Reverse (5′-3′) CTCCACGAACAGCTTCACAA 8 PGC1α Forward (5′-3′) CGGAAATCATATCCAACCAG 9 PGC1α Reverse (5′-3′) TGAGGACCGCTAGCAAGTTTG 10 TFAM Forward (5′-3′) GGGTATGGAGAAGGAGGCCC 11 TFAM Reverse (5′-3′) TCCCTGAGCCGAATCATCCT 12 NRF1 Forward (5′-3′) AGGGCGGTGAAATGACCATC 13 NRF1 Reverse (5′-3′) CGGCAGCTTCACTGTTGAGG 14 IRS-1 Forward (5′-3′) TGAACCTCAGTCCCAACCATAA 15 IRS-1 Reverse (5′-3′) TCCGGCACCCTTGAGTGT 16 Act Forward (5′-3′) ACTCATTCCAGACCCACGAC 17 Act Reverse (5′-3′) CCGGTACACCACGTTCTTCT 18 MyoD Forward (5′-3′) TGGGATATGGAGCTTCTATCGC 19 MyoD Reverse (5′-3′) GGTGAGTCGAAACACGGATCAT 20 MyoG Forward (5′-3′) AGCATCACGGTGGAGGATATG 21 MyoG Reverse (5′-3′) CAGTTGGGCATGGTTTCGT 22 GLUT2 Forward (5′-3′) TTACTCTCCATTTCAGTCCTTTGT 23 GLUT2 Reverse (5′-3′) TAGAGCAGCTCTTTATTCCAGATTT 24 SGLT1 Forward (5′-3′) ACTGCCACCGATGCACCCAT 25 SGLT1 Reverse (5′-3′) AAACATGGCCCACAGCCCGA 26 GCK Forward (5′-3′) CTGTGAAAGCGTGTCCACTC 27 GCK Reverse (5′-3′) GTGATTTCGCAGTTGGGTGT 28 GLP-1 Forward (5′-3′) CGGAGTGTGAAGAGTCTAAGCG 29 GLP-1 Reverse (5′-3′) ATGGCTGAAGCGATGACCAAGG 30 CREB Forward (5′-3′) CAGGTATCCATGCCAGCAGCTC 31 CREB Reverse (5′-3′) AGGCCTCCTTGAAAGGATTTCCC 32

As a result of measuring the expression level of sugar uptake promoting factors in the differentiated muscle cells, the expression level of sugar uptake promoting factors (GLUT4, GCK) was high in the muscle cells treated with 1-kestose.

Example 2-2: Measurement of Glucose Uptake in Muscle Cells (C2C12)

Muscle cells (C2C12) passaged more than twice in Example 1 were seeded at a concentration of ^1x104 cells/well/200mL in a 96-well plate, and then muscle cells were differentiated for 6 days using DMEM containing 2% horse serum.

The absorbed sugar content was measured using a glucose uptake cell-based assay kit (Cayman Chemical Co., USA). Differentiated C2C12 cells were simultaneously treated with glucose-free medium containing 150 mg/mL 2-NBDG, a fluorescent glucose analogue, and 1-kesotse, glucose, or maltose as sugars at a concentration of 14 mM in a 96-well plate for 4 h. After the 4-h reaction, the fluorescence absorbance was measured at a wavelength of excitation 485 nm/emission 650 nm, and the results were calculated as a percentage of the control group that was not treated with sugars, and the results are shown in Fig. 3.

As a result of measuring the sugar content absorbed by the differentiated muscle cells, the sugar content absorbed by the muscle cells treated with 1-ketose significantly increased.

Example 3: Measurement of expression levels of genes related to mitochondrial biogenesis and concentration of mitochondrial proteins

Example 3-1: Measurement of the expression level of a mitochondrial biosynthesis promoting factor that produces energy source (ATP) by utilizing sugar absorbed by muscle cells (C2C12)

To measure the mRNA expression level of mitochondrial biogenesis promoting factors in differentiated muscle cells (C2C12), RNA was extracted from muscle cells treated with 1-kestose, glucose or maltose in Example 1 using Trizol (Invitrogen, USA). The isolated RNA was used to synthesize cDNA using a cDNA synthesis kit (Takara, Japan).

The expression levels of mitochondrial biogenesis promoting factors (AMPK, SIRT1, PGC1α, TFAM, NRF1) were measured using real-time PCR analysis for the synthesized cDNA.

The primer sequence information used in performing PCR is shown in Table 1 above, and the results of measuring the expression level of mitochondrial biogenesis promoting factors are shown in Figure 3.

As a result of measuring the expression levels of mitochondrial biogenesis promoting factors, the expression levels of AMPK, SIRT1, PGC1α, TFAM, and NRF1 were measured to be higher in the group treated with 1-kestose compared to the other groups.

Example 3-2: Measurement of protein concentration in mitochondria that generate energy source (ATP) used for energy metabolism in muscle cells (C2C12)

Muscle cells (C2C12) passaged more than twice in Example 1 were seeded at a concentration of ^5x104 cells/well/mL in a 12-well plate, and the muscle cells were differentiated for 6 days using DMEM containing 2% horse serum in substantially the same manner as in Example 1, and 1-kestose, glucose, or maltose was treated to the muscle cells to react.

To isolate mitochondria in the muscle cells, a Mitochondrial isolation kit (Thermo Fisher Scientific; Rockford, IL, USA) was used. Differentiated muscle cells (C2C12) were obtained with a scraper and washed twice with cold PBS. Reagent A included in the Mitochondrial isolation kit was added to the cells and mixed. Then, isolation reagent B was added and mixed. After that, the mixture was mixed at maximum speed per minute for 5 minutes at 4°C. Isolation reagent C was added, the tube was inverted, and centrifuged (700 g, 10 minutes) at 4°C. After removing the supernatant, the sediment was suspended in isolation reagent C and centrifuged at 4°C (12,000 g, 5 minutes) to obtain mitochondria. The protein concentration of mitochondria was measured using BCA protein assay kit (Thermo Fisher Scientific, USA) using BSA (Bovine Serum Albumin) as a standard protein, and the results are shown in Figure 4.

As a result of measuring the protein concentration of the above mitochondria, the group treated with 1-kestose showed an increase in the protein concentration of the mitochondria.

Example 4: Measurement of the amount of energy source (ATP) produced for energy metabolism in muscle cells (C2C12)

In the above Example 1, the muscle cells passaged more than twice were seeded in a 12-well plate at a concentration of ^5x104 cells/well/mL, and differentiated for 6 days using DMEM containing 2% horse serum in substantially the same manner as in Example 1. On the 6th day, 1-kestose, glucose, or maltose were each treated at a concentration of 14 mM as sugars and reacted for 24 hours. After the reaction, the cells were washed twice with PBS, and the amount of ATP production was measured using an ATP colorimetric assay kit (Abcam, UK), and the concentration of energy sources inside the cells was analyzed by the ELISA method. The results are shown in Fig. 5.

When measuring the amount of ATP production, the group treated with 1-kestose showed increased production of the intracellular energy source (ATP), resulting in a higher concentration.

Example 5: Study on the energy metabolism regulation effect of Kestos through animal testing

Example 5-1: Induction and administration of animal models

Diabetes is a disease in which sugar is not absorbed into cells, and thus the cells cannot use sugar as a sufficient energy source. This sugar absorption disorder is caused by decreased secretion of insulin or insulin resistance. Insulin is secreted from pancreatic cells, and sends a signal by binding to the insulin receptor on the cell membrane so that blood sugar can be absorbed into the cells, and this causes sugar metabolism and energy metabolism in each tissue to occur, so that blood sugar can be maintained at a constant level through metabolism in the body. If insulin secretion decreases due to decreased function of pancreatic beta cells, or insulin resistance in peripheral tissues such as muscles, liver, and blood vessels increases, blood sugar cannot be used as an energy source and is excreted from the body, which leads to various complications. Therefore, insulin secretion and sugar absorption can be monitored using an animal model in which diabetes is induced, and the animal model can analyze the mechanism by which absorbed sugar is converted into an energy source and then metabolized, so it was selected as the subject of this experiment.

To conduct a study on the regulation of kestose energy metabolism, 6-week-old male C57BL/6 mice were separated into groups of 6 each and acclimated for 1 week. Specifically, as shown in Table 2 below, a normal diet group (ND) group fed a normal diet (Zeigler Bros., USA) and a high-fat diet group (HFD) group fed a high-fat diet (Research Diets, USA) were prepared.

To monitor insulin secretion and sugar absorption, and to analyze the mechanism leading to energy metabolism by generating energy sources from the absorbed sugar, T2D (Type 2 diabetes) was induced in a group of mice for 10 weeks, and the test substances for each group were orally administered daily as an amount converted to 30 g of mouse weight by applying 8 g/70 kg based on the daily intake standard for adults, as shown in Table 2 below, during the period of T2D induction. The sugars administered in addition to the diet were kestose, sucrose, or fructooligosaccharide (FOP).

The general diet administered to the above experimental animals was Rodent NIH-41 Open Formula Diet sold by Zeigler Bros., Inc., and its specific composition is shown in Table 3. The high-fat diet was the product D12492 (Rodent Diet With 60 kcal% Fat) sold by Research Diets, Inc., and its specific composition is shown in Table 4.

The experimental animals used in this experiment were divided into negative control group, positive control group, comparison group, and test group (see Table 2).

item Dietary intake Test substance dosage Voice control group Normal diet group * Positive control group High-fat diet group * Comparison group 1 High-fat diet + FOP (Fructooligosaccharides) FOP 3.56mg/d Comparison group 2 High-fat diet + Sucrose intake group Sucrose 3.43mg/d Test group 1 Normal diet + Kestose intake group 1-Kestose 3.45mg/d Test group 2 High-fat diet + Kestose intake group 1-Kestose 3.45mg/d

ingredient Content (weight%) Ground Whole Wheat 34.9 Ground No. 2 Yellow Corn 21 Ground Whole Oats 10 Wheat Middlings 10 Fish Meal (60% Protein) 9 Soybean Meal (47.5% Protein) 5 Soy Oil 2 Alfalfa Meal (17% Protein) 2 Corn Gluten Meal (60% Protein) 2 Dicalcium Phosphate 1.5 Brewers Dried Yeast 1 Premixes 0.6 Limestone 0.5 Salt 0.5 total 100

ingredient Content (weight%) Casein, Lactic, 30 Mesh 25.845% Cystine, L 0.388% Lodex 10 16.153% Sucrose, Fine Granulated 9.408% Solka Floc, FCC200 6.461% Lard 31.660% Soybean Oil, USP 3.231% S10026B 6.461% Choline Bitartrate 0.258% V10001C 0.129% Dye, Blue FD&C #1, Alum. Lake 35-42% 0.006% total 100%

Example 5-2: Measurement of mouse weight gain and food intake and collection of feces

During the T2D induction and test group administration period of Example 5-1 above, the weight change of the mice according to each experimental group administration was recorded to measure the weight change according to T2D induction. The weight measurement for measuring the weight change was performed at the same time before oral administration every week.

In addition, food intake was measured at the same time every week to determine whether obesity due to T2D was present, and the results of analyzing the change in body weight of the mice during the test period are shown in Figure 6.

Analysis of the weight change in mice during the test period showed that 1-kestose alleviated the weight gain caused by the high-fat diet in high-fat diet mice, and compared to sucrose and FOP, the group that consumed 1-kestose had the greatest weight gain alleviation effect.

Example 5-3: Analysis of the regulatory effects of sugar uptake and utilization factors and mitochondrial biogenesis promoting factors in mouse muscle tissue

To measure the mRNA expression level in mouse muscle, the mice whose weight changes were analyzed in Example 5-2 above were sacrificed, muscle tissues were obtained from the sacrificed mice, and RNA was extracted using an RNeasy mini kit (Qiagen, Germany). cDNA was synthesized using the RNA extracted from the muscle tissues using a cDNA synthesis kit (Takara, Japan).

Real-time PCR quantitative analysis was performed using the synthesized cDNA. The expression levels of mitochondrial biogenesis promoting factors (AMPK, SIRT1, PGC1α, NRF1, IRS-1, Akt, MyoD, MyoG) were measured to analyze the muscle metabolic effect by activated energy metabolism. The PCR primers used to measure the expression levels of the above factors are shown in Table 1, and the analysis results are shown in Fig. 7. Fig. 7 is a graph comparing the expression levels of mitochondrial biogenesis promoting factors that produce energy sources (ATP) using absorbed sugar.

According to the above analysis results, AMPK, SIRT1, PGC1α, NRF1, IRS-1, Akt, MyoD, and MyoG, which are factors promoting mitochondrial biogenesis, showed higher expression levels in the groups treated with a normal diet, a high-fat diet, and 1-kestose compared to the other groups.

Example 5-4: Measurement of the effect of regulating sugar metabolism on energy metabolism in mouse intestinal tissue

In order to measure the mRNA expression level of a glucose absorption regulator in the intestine of an experimental mouse, the mouse whose weight change was analyzed in Example 5-2 was sacrificed, the intestinal tissue was secured from the sacrificed mouse, and RNA extraction, cDNA synthesis and PCR thereof were performed in substantially the same manner as in Example 5-3 to measure the expression level of a glucose absorption promoting factor (GLUT2, SGLT1), thereby analyzing the effect of regulating sugar metabolism for energy metabolism. The primer information used is shown in Table 1, and the analysis results are shown in Fig. 8. Fig. 8 is a graph comparing the expression level of a glucose absorption promoting factor in the intestine of a mouse.

According to the above results, the expression level of glucose absorption promoting factors did not show any difference in expression due to 1-kestose in the general diet administration of test group 1, but in the high-fat diet administration of test group 2, 1-kestose inhibited the expression of glucose absorption promoting factors (GLUT2, SGLT1) that increase postprandial blood sugar levels.

Example 5-5: Evaluation of glucose metabolism regulation ability for energy metabolism in mouse pancreatic tissue

In order to measure the mRNA expression levels of insulin secretion-inducing factors and sugar absorption-promoting factors in the pancreas of experimental mice, the mice whose weight changes were analyzed in Example 5-2 were sacrificed, and pancreatic tissues were obtained from the sacrificed mice, and RNA extraction, cDNA synthesis, and PCR thereof were performed in substantially the same manner as in Example 5-3 to measure the expression levels of pancreatic insulin secretion-inducing factors (GLP-1, CREB) and sugar absorption-promoting factors (GLUT2). The primer information used is shown in Table 1 above.

The results of the above analysis are shown in Figure 9, which is a graph comparing the expression levels of insulin secretion inducing factors and sugar absorption factors in the mouse pancreas.

As shown in Fig. 9, the expression levels of factors related to insulin secretion induction and sugar absorption were measured, and in the normal diet-administered group 1, no difference was observed in the expression of factors related to insulin signaling by 1-kestose. However, in the high-fat diet-administered group 2, the expression of GLP-1, an insulin secretion-inducing factor, increased and the expression of CREB decreased due to 1-kestose, confirming that insulin secretion was induced by the phosphorylation of CREB. In addition, the expression of GLUT2, a sugar absorption-promoting factor, increased compared to other intake groups.

Claims (10)

A composition for promoting energy metabolism in an individual, comprising ketose as an active ingredient. A composition in claim 1, wherein the promotion of energy metabolism is at least one selected from the group consisting of promotion of adenosine triphosphate (ATP) production in tissues having mitochondria, promotion of protein biosynthesis in mitochondria in tissues having mitochondria, and promotion of sugar absorption. A composition in claim 2, wherein the enhancement of energy metabolism occurs in muscle cells or muscle tissue. A composition according to any one of claims 1 to 3, wherein the composition has the following properties (1) or (2): (1) Promotion of expression of one or more factors selected from the group consisting of AMPK, SIRT1, PGC1α, TFAM, NRF1, IRS-1, Akt, MyoD, MyoG, GLUT2, SGLT1, GCK, and GLP-1; (2) Inhibits CREB factor expression. A composition according to any one of claims 1 to 3, wherein the kestose is provided as a mixture with at least one sugar selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4). In claim 5, a composition in which the kestose is provided as a mixture with at least one sugar selected from the group consisting of nystose (GF3) and 1-F-fructosyl nystose (GF4), the kestose content being 50 wt% or more based on 100 wt% of the total mixture. A composition according to any one of claims 1 to 3, wherein the ketose is included in the composition in a total daily dosage of 1 g to 30 g based on a 60 kg adult. A composition according to any one of claims 1 to 3, wherein the kestose is provided in the form of a syrup or powder containing kestose. A composition for preventing or treating energy metabolism-related diseases, comprising kestos as an active ingredient. A composition in claim 9, wherein the energy metabolism-related disease is at least one selected from the group consisting of diabetes, obesity, metabolic syndrome, thyroid disease, mitochondrial disease, diseases that may be caused by mitochondrial disease, and precocious puberty.

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