KR20120067190A - A composition comprising materials of inhibiting ahnak for preventing and treating obesity - Google Patents

Abstract

PURPOSE: A composition containing a material which suppresses Ahnak gene expression is provided to effectively prevent or treat obesity. CONSTITUTION: A composition for preventing or treating obesity contains a material which suppresses Ahnak gene expression. The material is an antisense oligonucleotide, siRNA, or aptamer. The composition additionally contains pharmaceutically acceptable carrier. A food composition for treating obesity contains the composition. A method for screening a therapeutic agent for obesity comprises: a step of treating a subject suspicious for obesity with a candidate material for treating obesity; and a step of comparing mRNA or protein expression level of a control group and Ahnak gene.

Description

A composition comprising materials of inhibiting Ahnak for preventing and treating obesity

The present invention relates to a composition for preventing or treating obesity, comprising a substance for inhibiting the expression of the Ahnak gene, and more particularly, a composition comprising an antisense oligonucleotide, siRNA, aptamer, and an antibody that inhibits the expression of the gene. A method for preventing or treating obesity using a composition, a method for screening obesity treatments comprising the step of comparing the expression level of the Ahnak gene after treating the food composition for improving obesity and the candidate substance for treating obesity It is about.

There have been many changes in eating habits due to recent economic growth and changes in Western lifestyle. In particular, busy modern people are increasing overweight and obesity due to high calorie diet and fast exercise. Obesity is a chronic disease that increases the morbidity and mortality of various diseases due to excessive accumulation of fat tissue due to abnormal energy balance or hypernutrition. Obesity and related diseases are common and very serious public health issues in the United States and around the world, and according to the World Health Organization (WHO), more than one billion adults are overweight worldwide, at least three million of them It is clinically seen as obese. It is also markedly increasing in the United States and Europe, with an estimated 250,000 deaths per year in Europe and more than 25,000 people worldwide associated with overweight (World Health Organization, Global Strategy on Diet, Physical Activity and Health, 2004).

Overweight and obesity increase blood pressure and cholesterol levels, causing various diseases such as heart disease, diabetes, arthritis, and increasing the incidence of various adult diseases. In addition, overweight and obesity have been a factor in increasing the incidence of various adult diseases such as arteriosclerosis, hypertension, hyperlipidemia or heart disease in children as well as adolescents.

As described above, obesity is a serious disease that causes various diseases as a worldwide disease, but is a disease that cannot be easily treated. Among medical experts, the cause of obesity is complex and due to various factors, and there is a growing awareness that obesity is not a simple matter of self-control, but a complex disease involving appetite control and energy metabolism. However, the most common cause of obesity is that the energy consumed is continuously excessive compared to the energy consumed. Therefore, an effective and safe medicine that can be used in combination with diet and exercise is required in the long term to prevent or treat obesity. It is true.

Ahnak (Ahnak nucleoprotein) is a 700kDa nuclear protein whose central repeat unit (CRU) binds to and activates PLC-γ1 and PKC (protein kinase C) to affect tumor proliferation. Results (JBC 279: 26645-53, 2004; JBC 283: 6312-20, 2008; Tuberculosis and Respiratory Disease, 47 (3) 1999), and play a role in calcium signaling related to T cell activation associated with immunity ( Immunity 28; 64, 2008), but no association with obesity has been disclosed.

Against this background, the present inventors have made diligent efforts to find a safe substance that does not affect the body as a prophylactic or therapeutic agent for obesity. As a result, the mice knocked out of the Ahnak gene have no weight gain in high fat diet-induced obesity. Although the size of the cells is also reduced, the respiratory volume, energy consumption, activity was observed that no change, and confirmed that Ahnak can be used as a target of obesity treatment, the present invention was completed.

One object of the present invention is to provide a composition for preventing or treating obesity, including a substance for inhibiting the expression of Ahnak gene.

Another object of the present invention is to provide a method for preventing or treating obesity comprising administering the composition to a subject.

Another object of the present invention to provide a food composition for improving obesity comprising the composition.

Still another object of the present invention is a method for treating obesity in a subject that is suspected of being obese; And (b) to provide a method for screening a therapeutic agent for obesity comprising the step of comparing the expression level of the mRNA or protein of the Ahnak gene with the control.

As one aspect for achieving the above object, the present invention relates to a composition for preventing or treating obesity, comprising a substance for inhibiting the expression of Ahnak gene.

As used herein, the term "Ahank gene" (NCBI Gene ID: 79026) is a gene encoding a nuclear protein of 700 kDa. The protein is a protein that activates PLCγ in the presence of AA (arachidonic acid) in neural tissues, such as Tau of neural tissues. Ahnak phosphorylated by PKC (protein kinase C) as an activator of PLCγ-1 (phospholipase C) is known to increase the activity of PLC-γ than Ahnak, which is not phosphorylated. Ahnak protein purified from lung tissue seems to have a very large difference in size from 70 kDa to 130 kDa because of the instability of proteolytic enzymes. In general, 128 amino acids are repeated about 30 times in one unit. Of these, even if only one unit exists, PLCγ can be activated, and up to four units of Ahnak protein may exist. Ahnak's function has been studied a lot until now, and it binds and activates PLC-γ1 and PKC (protein kinase C) to affect tumor proliferation or calcium signal related to T cell activation related to immunity. Although there is an indication that it plays a role in delivery, the association between the gene and obesity has not been disclosed so far, the present inventors first identified that obesity can be treated by inhibiting the gene.

As used herein, the term "substance that inhibits the expression of Ahnak gene" refers to a substance that reduces the expression or activity of the Ahnak gene in a cell, and specifically, acts directly on the Ahnak gene or Substances that act indirectly on higher regulators to reduce the expression of the Ahnak gene at the transcriptional level, to increase the degradation of the expressed Ahnak gene, or to interfere with its activity, thereby reducing the expression level of the Ahnak gene or its activity it means. This includes, but is not limited to, biological molecules such as nucleic acids or polypeptides, compounds, extracts isolated from bacteria or plants or animals that can be processed into cells through standard techniques known in the art that can inhibit such genes, For example, it may be an antisense oligonucleotide that binds to the Ahnak gene, an siRNA, an aptamer, or an antibody that binds to the Ahnak protein. Since the sequence of the Ahnak gene of the present invention can be easily obtained by those skilled in the art from GenBank, a known database, antisense oligonucleotides, siRNAs, aptamers, and antibodies capable of inhibiting the genes can be prepared by a standard standard technique. Can be.

As used herein, the term “antisense oligonucleotide” refers to oligomers or polymers of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar linkages. The term also encompasses modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof that function similarly. Incorporation of substituted oligomers is based on factors including increased cell uptake or increased nuclease resistance and is selected as known in the art. The entire oligonucleotide or portion thereof may contain substituted oligomers. In addition, the antisense oligonucleotides of the invention are oligomeric mimetics, such as peptides, modified by methods known in the art to increase their affinity for their target and to provide resistance to mismatch of the target sequence. Nucleic acids (PNA) and locked nucleic acids (LNA). In addition, the antisense oligonucleotide of the present invention is a natural oligonucleotide, phosphorothioate oligodeoxyribonucleotide, phosphorodithioate oligodeoxyribonucleotide, methylphosphonate oligodeoxyribonucleotide, phosphor Amidate oligodeoxyribonucleotides, H-phosphonate oligodeoxyribonucleotides, triester oligodeoxyribonucleotides, alpha-anomeric oligodeoxyribonucleotides, peptide nucleic acids, other artificial nucleic acids and nucleic acid-modifications Modified oligonucleotide forms, including, but not limited to: Antisense oligonucleotide molecules complementary to the Ahnak sequence of the present invention may be isolated or prepared using standard molecular biology techniques, such as chemical synthesis or recombinant methods, or may be commercially available.

As used herein, the term "siRNA" refers to a double-chain RNA that can induce RNA interference (RNAi) by cleaving Ahnak RNA by specifically acting on the Ahnak gene. The siRNA of the present invention is composed of a sense RNA strand comprising a sequence homologous to part or all of the nucleic acid sequence of the Ahank gene and an antisense RNA strand comprising a complementary sequence thereof, and a nucleotide sequence capable of hybridizing to Ahnak RNA in a cell. It is preferable to include.

As used herein, the term “aptamer” refers to a nucleic acid ligand molecule that binds to and has an antagonistic effect on the Ahnak gene by its ability to adopt a particular three-dimensional conformation. Typically the aptamers may be small nucleic acids of 15-50 bases in length that are folded into defined secondary and tertiary structures, such as stem-loop structures. Aptamers are preferably bound to the target molecule with a kd of less than 10 ⁻⁶ , 10 ⁻⁸ , 10 ⁻¹⁰ , or 10 ⁻¹² . Aptamers can bind to target molecules with very high specificity. In addition, aptamers may be composed of multiple ribonucleotide units, deoxyribonucleotide units, or a mixture of two types of nucleotide residues. Aptamers may further comprise one or more modified base, sugar or phosphate backbone units.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site capable of binding to Ahnak protein and inhibiting Ahnak activity. For the purposes of the present invention, antibody refers to an antibody that specifically binds to Ahnak protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Since the Ahnak protein has been identified as a target for the treatment of obesity as described above, the production of antibodies using the same can be easily prepared using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for injecting the Ahnak protein antigen described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

Monoclonal antibodies are well known in the art of the hybridoma method (European Jounral of Immunology 6: 511-519, 1976), or phage antibody libraries (Nature, 352: 624-628, 1991; J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. The antibodies of the present invention also include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, and includes Fab, F (ab '), F (ab') 2 and Fv.

As used herein, the term "obesity" generally means a lot of weight, but even if it is not obese, a lot of muscle means a lot of weight because the fat tissue in the body means an excessive state. Obesity, as well as weight gain, overeating, overeating and bulimia, hypertension, diabetes, increased plasma insulin levels, insulin resistance, hyperlipidemia, metabolic syndrome, insulin resistance syndrome, obesity related gastroesophageal reflux, arteriosclerosis, hypercholesterolemia, Treatment of such diseases using the composition of the present invention may cause obesity-related diseases such as uric acid hyperemia, lower back pain, cardiac hypertrophy and left ventricular hypertrophy, lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease or polycystic ovary syndrome. It can be done at the same time. In addition, the subjects of treatment for such obesity-related diseases include those who have a desire to lose weight.

As used herein, the term "treatment" refers to any action by which administration of a composition according to the present invention improves or advantageously alters the symptoms of obesity or obesity-related disease. As used herein, the term "prevention" refers to any action that inhibits or delays the development of obesity by administration of a composition according to the present invention.

According to one embodiment of the present invention, the present inventors found that the mice knocked out of the Ahnak gene do not have an increase in weight in obesity induced by a high fat diet (FIGS. 3 and 4), and the size of adipose tissue is reduced (FIG. 5). ), It was confirmed that the size of the adipocytes was also reduced (FIG. 6), and it was found that a composition including a substance that suppresses the expression of the gene can prevent or treat obesity. In addition, even when the gene is knocked out, it is confirmed that there is no significant difference in comparison with the wild type that does not suppress appetite, respiratory rate, energy consumption, and activity amount (FIG. 7). None was found.

On the other hand, the composition of the present invention may further comprise a pharmaceutically acceptable carrier, it may be formulated with a carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition comprising a substance that inhibits the expression of the Ahnak gene of the present invention and a pharmaceutically acceptable carrier may be applied in any formulation including the same as an active ingredient, and may be prepared in an oral or parenteral formulation. It may be formulated in unit dosage form for ease of administration and uniformity of dosage. Pharmaceutical formulations of the invention may be oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). And forms suitable for administration by inhalation or insufflation.

Oral dosage forms containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated as a rectal composition such as suppositories or body enema including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

As another aspect, the present invention relates to a method for preventing or treating obesity comprising the step of administering a composition for preventing or treating obesity comprising a substance for inhibiting the expression of Ahnak gene.

As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner, and when the composition of the present invention is introduced into an individual, there is no change in weight gain and a decrease in the size of adipose tissue In addition, fat cell size reduction can prevent or treat obesity.

The route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhaled, intraocular or intradermal routes.

The prophylactic or therapeutic method of the present invention includes administering a composition of the present invention in a therapeutically effective amount. The therapeutically effective amount means an amount that promotes the effect of weight loss or size reduction of fat cells. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment. The specific therapeutically effective amount for a particular patient may be based on the specific composition, including the type and severity of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health, sex and diet of the patient, time of administration, It is desirable to apply differently depending on the route of administration and the rate of release of the composition, duration of treatment, and similar factors well known in the various factors and medicinal arts. Therefore, the effective amount of the composition for preventing or treating obesity suitable for the purposes of the present invention is preferably determined in consideration of the above-mentioned matters. In addition, in some cases, a known obesity-related disease treatment agent may be co-administered with the composition of the present invention to increase the therapeutic effect of an obesity-related disease including an obesity inhibitory effect.

In addition, the treatment methods of the present invention are applicable to any animal capable of preventing or inhibiting obesity, and animals include humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, and cats.

As another aspect, the present invention relates to a food composition for improving obesity comprising a composition comprising a substance for inhibiting the expression of Ahnak gene. Substances that inhibit the expression of the Ahnak gene are as described above.

The food composition for improving obesity of the present invention includes the form of pills, powders, granules, acupuncture, tablets, capsules or liquids, and the like to which the composition of the present invention can be added, for example, various foods, beverages , Gums, teas, vitamin complexes, and dietary supplements. The composition of the present invention may be used as a food additive due to less side effects such as decreased appetite or activity. There are no particular restrictions on the other ingredients except the composition including a substance that inhibits the expression of the Ahnak gene as an essential ingredient that may be included in the food composition for improving obesity, and various herbal extracts and food supplements, such as conventional foods. Or natural carbohydrate or the like as an additional component. In addition, as mentioned above, food supplement additives may be added as well. Food supplement additives are conventional food additives, such as nutrients, vitamins, minerals (electrolytes), flavoring agents, flavoring agents, and coloring agents. , Fillers, stabilizers and the like. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Other natural fruit juices and fruit juices and fruit juices for the production of vegetable beverages may be contained These ingredients may be used independently or in combination The food composition for improving obesity may be provided in the form of dietary supplements. Can be.

The term "health functional food" in the present invention refers to a food prepared and processed by a method of extracting, concentrating, refining, and mixing a specific ingredient as a raw material or contained in a food ingredient for the purpose of health supplement, It refers to foods that are designed and processed to sufficiently exert bioregulatory functions on the living body such as biological defense, control of biorhythms, prevention and recovery of diseases by the components, and the composition for health foods is related to prevention of obesity and obesity. It may perform a function associated with the recovery of the disease.

In another aspect, the present invention provides a method for treating obesity, comprising: (a) treating an obesity suspect sample with a candidate substance for treating obesity; And (b) comparing the control group with the expression level of the mRNA or protein of the Ahnak gene.

As used herein, the term "suspected obesity sample" refers to an individual or a sample used for screening candidates for the treatment of obesity, which refers to mammals including dogs, cattle, pigs, rabbits, chickens, mice, and humans. Examples include, but are not limited to, whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, samples, and the like isolated from the subject.

As used herein, the term "control" refers to a sample that is not treated with a candidate substance for the treatment of obesity. In addition, the term "candidate" as used in the present invention means a drug to test the expression-changing activity of Ahnak, and as an object for measuring the ability to prevent or treat obesity by directly or indirectly changing the expression level of Ahnak. Such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide variety of compounds. Such candidates also include both natural and synthetic materials.

As used herein, the term "therapeutic agent for obesity" refers to a substance for preventing or treating obesity through weight loss, adipose tissue size reduction, a function of reducing fat cell size, and the like.

The screening method of the present invention compares the expression level of the mRNA or protein of the Ahnak gene with the control group without treatment of the candidate material after treating the suspected obese subject, and expresses the mRNA or protein of the Ahnak gene compared to the control group. It can be carried out by determining that the substance to reduce the obesity treatment.

Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, and DNA chip.

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto.

The present invention newly identified the Ahnak gene as a target for the treatment of obesity. When the composition of the present invention is used, there is no abnormality in energy consumption and activity, and at the same time, the size of fat cells is reduced while preventing weight gain, thereby preventing or treating obesity. It is expected to be able. In addition, when the Ahnak gene is newly presented as a target for the treatment of obesity, screening for a substance inhibiting the gene, it is expected that a novel obesity prevention or treatment can be easily obtained. In addition, the material is expected to be used in various ways to prevent or improve obesity.

Figure 1 is a schematic of the protein of the Ahnak gene used in the present invention.
Figure 2 shows the fabrication and weight of Ahnak knockout mice. A represents a vector for making Ahnak knockout mice. B shows RT-PCR results for the Ahank gene of wild type and knockout mice. C shows Western blotting results for the Ahank gene of wild type and knockout mice. D represents weight-matched group of body weight according to age of wild type and knockout mice.
Figure 3 graphically shows the weight change of wild-type and Ahnak knockout mice over time with high and low fat diets. Ahnak knockout mice can be found to be resistant to obesity induced by a high fat diet.
FIG. 4 graphically plots weight gain (A and B) and relative weight gain (C and D) over time in wild-type and Ahnak knockout mice according to high fat diet (A and C) and low fat diet (B and D). will be. Ahnak knockout mice can be found to be resistant to obesity induced by a high fat diet.
Figure 5 shows the size of the epididymis of wild-type and Ahnak knockout mice according to the high fat diet (A) and low fat diet (B). C is a graph of the weight of epididymal white adipose tissue (EWAT) of wild-type and Ahnak knockout mice according to high-fat diet and low-fat diet, and D is wild-type and Ahnak knock according to high-fat diet and low-fat diet. The weight percentage (%) of epididymal white adipose tissue (EWAT) per body weight of out mice is plotted. Ahnak knockout mice show a decrease in adipose tissue in obesity induced by high fat diet.
Figure 6 shows the size of fat cells of wild-type and Ahnak knockout mice according to low fat diet (A) and high fat diet (B). C and D show the average area and frequency of adipocytes. Ahnak knockout mice show reduced fat cell size in obesity induced by high fat diet compared to wild type.
7 graphically depicts food intake (A), respiratory volume (B), energy expenditure (C) and activity (D) of wild-type and Ahnak knockout mice. Ahnak knockout mice show no change in appetite, respiration, energy intake and activity.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Ahnak Knockout  Mouse manufacturer

A 502 bp genomic fragment corresponding to the N-terminal region of Ahnak was cloned into a pPNT vector, and this targeting vector was inserted into a 129 / SvJ mouse embryonic stem cell (ES) to homologous recombinant. Gene deletions were induced and these positive clones were identified by Southern blotting. Eight positive ES clones out of 252 clones were inserted into C57BL / 6 blastocysts to obtain chimeric mice, and heterozygote mice were obtained by crossing C57BL / 6 with male chimeric mice. Germ line transmission of mutant alleles was confirmed through Southern blotting of genomic DNA of F1 progeny. Then, homozygotes (homozygote) were obtained through cross-crossing of the heterozygotes, and the deficiency of Ahnak protein was confirmed by genotyping and western blotting in the blood of the homozygotes (FIGS. 2B and 2C).

2A shows a vector for constructing an Ahnak knockout mouse and a targeted allele through it.

Example  2: Preparation and Low Fat Diet of Experimental Animals ( Low Fat Diet , LFD ) And high fat diet ( High Fat Diet , HFD )

Each animal was maintained at random with a tap water with a pelleted standard chow diet (Lab Diets, IN, USA) at a room temperature of 24 ° C ± 2 ° C and a 12-hour light-dark cycle. When the mice reach 6 weeks of age, the male Ahnak-/-and wild-type mice each form a weight-matched group, and for 12 weeks, the animals were given a low fat diet (LFD) (70% carbs, 10% fat, 20% protein: D12450B: Research Diets) or a high fat diet (20% carbohydrate, 60% fat, 20% protein: D12492: Research Diets Inc., New Brunswick, NJ).

Example  3: Weight Change and Adipose Tissue Size Analysis in Low Fat and High Fat Diet Mice

Mice that were fed different low-fat and high-fat diets for 12 weeks measured their weight once a week and reported both relative weight gains based on actual weights measured at the beginning and the first weight gained. It was. After that, mice that had undergone low-fat and high-fat diets for 12 weeks were autopsied to observe the white adipose tissue of the epididymis, and only the fat tissue was isolated and the weight of the fat tissue and its percentage of total body weight were calculated as relative values. .

The results of observing weight change for wild-type and knockout mice following HFD and LFD over a 12-week period are shown in FIGS. 3 and 4. As a result, it was confirmed that there was no weight gain due to obesity induced by fat diet in mice knocked out of both alleles than mice knocked out of wild-type or alleles (FIG. 3). In addition, these results confirm that the obesity caused by a high fat diet is more significant than the obesity caused by a low fat diet (Fig. 3), and confirmed the increase in weight through standardization, obesity induced by a high fat diet It was confirmed that there was no marked weight gain in mice knocked out of both alleles in (Fig. 4C).

In addition, the size change of the white adipose tissue of the epididymis is shown in FIG. 5. As a result, it was confirmed that in obesity induced by high fat diet, the size of the white adipose tissue of the epididymis significantly decreased in mice knocked out of both Ahnak alleles (FIG. 5).

These results suggest that obesity of the Ahnak gene is highly effective in treating obesity.

Example  4: histological analysis of adipose tissue

Epididymal adipose tissue obtained from the experimental animals of Example 2 was fixed in 10% neutral buffered formalin. The tissues were embedded in paraffin after several successive dehydration steps with a graded alcohol series and washing. Tissue sections were cut to 4 μm in thickness and stained with hematoxylin and eosin. To determine the size of white adipocytes, each section of the adipocytes was measured by Opti Pro software (Olympus Co., USA).

The result is shown in FIG. As shown in Figure 6, compared with the wild type, it was confirmed that the size of fat cells significantly reduced in the mouse knocked out both Ahnak allele.

These results suggest that the complete inhibition of Ahnak gene expression reduces the size of adipose tissue and thus is effective in treating obesity.

Example  5: Ahnak Knockout  Analysis of Appetite, Respiration Volume, Energy Consumption and Activity in Mice

Ahnak wild-type and knockout mice were measured for activity, appetite, and energy consumption for three weeks before and after a high fat diet using a Comprehensive animal metabolic monitoring system (CLAMS; Columbus Instruments, Columbus, OH). Energy consumption and respiratory rate (RER) were calculated from gas exchange data [Energy expenditure = (3.815 + 1.232x RER) x VO ₂ ]. RER represents the ratio of VCO ₂ to VO ₂ , depending on the energy source used by the animal. RQ (respiratory quotient) is 1.0 when carbohydrate is the only energy source oxidized, and RQ is 0.7 when fat is the only energy source. The amount of activity was measured by analyzing the data along the x and y axes by measuring the number of passes through the infrared beam during the measurement period. Appetite was found by measuring the weight of the food at and after it was provided. This experiment was conducted using 5 to 6 male mice per group.

The result is shown in FIG. As shown in FIG. 7, Ahnak knockout mice had no significant decrease in appetite compared to wild type (FIG. 7A), respiratory volume (7B), energy consumption (7C), and activity (7D). .

These results suggest that inhibiting the expression of the Ahnak gene does not affect the health and activity of the subject, demonstrating the safety of the composition of the present invention.

Claims (6)

A composition for preventing or treating obesity, comprising a substance for inhibiting the expression of the Ahnak gene.
The composition of claim 1, wherein the substance is an antisense oligonucleotide, siRNA or aptamer that binds to the Ahnak gene.
The composition of claim 1, wherein the substance is an antibody that binds to Ahnak protein.
The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
Obesity improvement food composition comprising the composition of claim 1.
(a) treating the obesity suspect sample with a candidate substance for the treatment of obesity; And
(b) comparing the control and the expression level of the mRNA or protein of the Ahnak gene screening method for the treatment of obesity.

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