KR101572162B1 - Method for regulation of brown adipocyte differentiation using - Google Patents

Abstract

More particularly, the present invention relates to a method for regulating the differentiation of brown adipocytes using protein tyrosine dephosphorylase. More particularly, the present invention relates to a method for regulating differentiation of brown adipocytes using PTP-RE (protein tyrosine phosphatase receptor type E) -RE overexpression inhibits the differentiation of brown adipocytes and decreases insulin receptor phosphorylation. Thus, the PTP-RE can be usefully used to control the differentiation of brown adipocytes.

Description

Methods for regulating brown adipocyte differentiation using PTP-RE {

The present invention relates to a method for controlling the differentiation of brown adipocytes using protein tyrosine phosphatase receptor type E (PTP-RE).

Adipocytes play an important role in energy homeostasis and are closely related to metabolic diseases such as obesity and diabetes. There are two types of adipocytes in mammals: white adipocytes and brown adipocytes. While white adipocytes store excess energy, such as triglycerides, in the form of lipid droplets, brown adipocytes emit energy in the form of heat through thermogenesis and thus correspond to obesity . Brown fat is brown because it possesses a large amount of cytochrome by retaining a large amount of mitochondria (the heme group of cytochrome strongly absorbs visible light). There is a unique protein called the thermogenin (uncoupling protein) in the inner part of the mitochondria of brown fat, which passes hydrogen through the mitochondrial matrix without passing through the F0F1 complex. Therefore, the oxidation energy is not used for the production of ATP but is released as heat to maintain the body temperature. Hibernating animals also maintain their body temperature during the hibernation period using brown fat mitochondria (Lehninger: Principles of Biochemistry Ch. 19). Both white adipocytes and brown adipocytes are derived from mesenchymal stem cells (MSCs), but there are distinct differences in the development of the two adipocytes. In particular, the molecular mechanisms of brown adipocytes have not been studied as accurately as those of white adipocytes.

Phosphorylation of tyrosine residues plays an important role in many biological steps such as cell death, proliferation and differentiation which are directly or indirectly related to eukaryotic or human diseases. Reversible tyrosine phosphorylation is controlled by the balanced action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). In addition, many PTPs functioned more positively than negative controls and displayed unique and complex trait expressions in PTPs knockout mice. In addition, the human genome further contains PTPs genes rather than PTKs genes.

The phosphorylation-dephosphorylation process of protein tyrosine is used as one of the most important means in the intracellular signal transduction system. Protein tyrosine phosphatase (PTP) is an enzyme that hydrolyzes phosphoryl groups in the tyrosine residues in proteins. There are 107 kinds of PTP in humans. Approximately 80 to 90 species of these enzymes act as PTP (Alonso A., et al., Cell, 117, 699-711, 2004).

Since destruction of the intracellular signaling pathway leads directly to disease, it has been shown that many PTPs are actually involved in disease and some PTPs have been the target of new drug development (Current Topics in Med. Chem. 3: 739-835 , 2003).

Recently, PTP-1B has been shown to be useful for the treatment of autoimmune diseases, inflammatory diseases and tissue graft rejection through the use of RPTP, a CD45 inhibitor. Among them, PTP-1B has been used as a drug for the treatment of diabetes and obesity (Zhang et al., Annu. Rev. Pharmacol. Toxicol. 42, 209-234, 2002). In addition, not only PTPs such as PRL-3 exhibit cancer-specific expression, but the Weizmann Institute shows the ability to control the signaling pathway of cancer (Experimental Cell Research, 294, 236243, 2004) .

The present inventors have investigated a method of regulating differentiation of brown adipocytes and found that the expression of PTP-RE (protein tyrosine phosphatase receptor type E) is decreased in the early stage of differentiation of brown adipose cells and that of PTP-RE Overexpression to inhibit the differentiation of brown adipocytes and reduce insulin receptor phosphorylation. Thus, the present inventors have completed the present invention by confirming that the PTP-RE can be effectively used for the control of brown adipocyte differentiation.

It is an object of the present invention to provide an agent for regulating the differentiation of brown adipocytes comprising PTP-RE (protein tyrosine phosphatase receptor type E) or its expression promoter, or an inhibitor of PTP-RE.

Another object of the present invention is to provide a method for regulating the differentiation of brown adipocytes comprising PTP-RE (protein tyrosine phosphatase receptor type E) or its expression promoter, or an inhibitor of PTP-RE.

Another object of the present invention is to provide a method for monitoring the degree of differentiation of brown adipocytes by measuring the expression level of PTP-RE in brown adipocytes.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting the differentiation of brown adipocytes comprising a vector comprising a PTP-RE (protein tyrosine phosphatase receptor type E) protein or a gene encoding the PTP-RE to provide.

The present invention also provides a pharmaceutical composition for promoting differentiation of brown adipocytes containing an antibody against PTP-RE protein, or a vector comprising antisense nucleotide, siRNA or shRNA against said PTP-RE.

The present invention also provides a method for inhibiting the differentiation of brown adipocytes, which comprises treating a brown adipocyte with a PTP-RE protein.

The present invention also provides a method for promoting the differentiation of brown adipocytes, which comprises treating a brown adipocyte with an antibody against a PTP-RE protein.

In addition,

1) preparing an expression vector comprising a gene encoding a PTP-RE protein; And

2) introducing the expression vector of step 1) into brown adipocytes.

In addition,

1) preparing a vector comprising an antisense nucleotide, siRNA or shRNA for a gene encoding PTP-RE; And

2) introducing the vector of step 1) into brown adipocytes.

The present invention also provides a kit for confirming the degree of differentiation of brown adipocytes comprising an antibody against a PTP-RE protein or a primer specific to a gene encoding the PTP-RE protein.

In addition, the present invention provides a method for determining the degree of differentiation of brown adipocytes, comprising the step of measuring the expression level of PTP-RE in brown adipocytes using the kit.

The present invention is based on the finding that the expression of PTP-RE (protein tyrosine phosphatase receptor type E) is decreased in the early stage of differentiation of brown adipocytes and overexpression of PTP-RE inhibits the differentiation of brown adipocytes and reduces insulin receptor phosphorylation , The PTP-RE can be usefully used for controlling the differentiation of brown adipocytes.

Fig. 1 is a graph showing brown fat saturation differentiation of HIB-1B cell line.
A shows the accumulation of fat in cells using morphological changes of cells and Oil-Red-O staining. B shows the expression of brown adipose-specific gene UCP-1 using real time-PCR Fig. 1 shows the expression of the brown adipocyte-specific gene UCP-1 using C. western blot. Fig.
Figure 2 shows the PTPs mRNA profile during the brown fat saturation process of the HIB-1B cell line.
A shows the expression level of PTPs mRNA using RT-PCR, and B shows the expression level of PTPs mRNA using real time-PCR (data are shown as means ± standard deviation (n = 3 ; * P <0.05, ** P <0.01).
FIG. 3 is a graph showing that overexpression of PTP-RE suppresses brown fat saturation of HIB-1B cell line. FIG.
A shows the observation of GFP in a fluorescence microscope, B shows the cytoplasmic expression of PTP-RE using Western blot, C shows the cell-overexpressed cells of PTP-RE or mutant overexpressed cells using differentiation medium After differentiation for 6 days, Oil-Red-O staining was used to confirm accumulated fat. D is the result of quantification of the stained cells using a staining extraction buffer solution.
Figure 4 shows the regulation of phosphorylation of the insulin receptor (IR) of PTP-RE.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for inhibiting differentiation of brown adipocytes containing a vector comprising a PTP-RE (protein tyrosine phosphatase receptor type E) protein or a gene encoding the PTP-RE.

The present invention also provides a pharmaceutical composition for promoting differentiation of brown adipocytes containing an antibody against PTP-RE protein, or a vector comprising antisense nucleotide, siRNA or shRNA against said PTP-RE.

The PTP-RE protein preferably has the amino acid sequence of SEQ ID NO: 1, but is not particularly limited to the above amino acid sequence. The gene coding for the PTP-RE preferably has the nucleotide sequence shown in SEQ ID NO: 2, but is not particularly limited to the nucleotide sequence shown above.

The vector comprises a plasmid vector, a cosmid vector, a bacteriophage vector or a recombinant viral vector, and is preferably a lentiviral vector or a retroviral vector.

In a specific example of the present invention, the brown adipose precursor cell line, HIB-1B, was induced to differentiate into brown adipocytes using growth and differentiation media. To determine whether the adipocytes were differentiated, 0, 3, After 6 days, the cells were stained with Oil-Red-O staining, which is a stain-specific staining method. As a result, it was confirmed that fat globules accumulated according to progress of differentiation of brown adipose cell lines were increased (see FIG. 1A) Real-time PCR and Western blotting were performed to confirm the expression of UCP-1 as an anti-marker marker at the RNA level. As a result, the expression of UCP-1, a brown fat cell-specific marker, (See Figs. 1B to 1C).

Further, RT-PCR and real-time PCR were performed on the cells to identify the PTP mRNA profiles of 0, 2, 4 and 6 days of the brown adipose cell line. As a result, PTP-RE and PTPN6 tended to decrease. Especially, PTP-RE showed a significant decrease at the 4th day of differentiation, and PTP-RT, PTPN11 and CDC14A showed an increasing tendency, -RE, PTP-RT, PTPN6, PTPN11 and CDC14A (see FIG. 2).

In order to confirm the role of the identified PTP-RE in the brown adipocyte differentiation process, a HTP-1B cell line was transfected with a construct having a cytoplasmic domain of PTP-RE using a retrovirus system (cytoPTP-RE-IRES-GFP ), And differentiation into brown adipocytes was induced. On the 6th day of differentiation, cells were confirmed by Western blotting using PTP-RE specific antibody and Oil-Red-O staining, and the stained cells were stained with a staining extraction buffer solution The PTP-RE-transfected cells were transformed with PTP-RE-CS mutant (catalytically inactive mutant (PTP-RE-CS mutant) ) Showed low fat accumulation as compared to the transformed cells (Figure 3B-3D).

In order to confirm the function of insulin reporter (IR) phosphorylation of PTP-RE in the lipid differentiation process of brown adipose cell line, a cell line overexpressing PTP-RE or PTP-RE- The cells were treated with brown adipogenic cocktails at indicated times and immunoprecipitated using agarose with anti-phosphotyrosine antibody (4G10; anti-pY) Western blot analysis was performed using an anti-insulin receptor (IR-β). As a result, it was found that PTP-RE-transfected HIB-1B cells had a brown Although IR tyrosine phosphorylation was significantly reduced during adipocyte differentiation, catalytic inactive mutant transfected HIB-1B cells were found to have no change in IR phosphorylation level (See Fig. 4).

Therefore, the expression of PTP-RE (protein tyrosine phosphatase receptor type E) of the present invention is decreased in the early stage of differentiation of brown adipocytes and overexpression of PTP-RE inhibits the differentiation of brown adipocytes and reduces insulin receptor phosphorylation , The PTP-RE can be usefully used for the control of brown adipocyte differentiation.

The pharmaceutical composition of the present invention may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutically auxiliary agent such as a salt for controlling osmotic pressure, a buffer, and other therapeutically useful substances, Oral or parenteral administration.

Examples of formulations for oral administration include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, Sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, sorbents, coloring agents, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods. Also typical of parenteral administration formulations are isotonic aqueous solutions or suspensions which are suitable for injectable use.

In addition, the composition of the present invention may be administered in combination with an existing therapeutic agent, and may be administered simultaneously or sequentially. The compositions of the present invention can be administered to a subject in any manner that delivers to the surface. Suitable parenteral routes of administration include, but are not limited to, intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intraluminal bolus injection, intramuscular injection, catheter instillation into the vasculature), subcutaneous injection deposition (e.g., using an osmotic pump), peri-and per-tissue administration or deposition, and the like.

The dosage of the pharmaceutical composition of the present invention can be easily determined by those skilled in the art in consideration of various factors such as sex, age, health condition, body weight, administration method and number of times, target tissues and cells. The total dose required for each treatment may be administered in several doses or doses.

The present invention also provides a method for inhibiting the differentiation of brown adipocytes, which comprises treating a brown adipocyte with a PTP-RE protein.

The present invention also provides a method for promoting the differentiation of brown adipocytes, which comprises treating a brown adipocyte with an antibody against a PTP-RE protein.

In addition,

1) preparing an expression vector comprising a gene encoding a PTP-RE protein; And

2) introducing the expression vector of step 1) into a brown adipocyte, thereby inhibiting the differentiation of brown adipocytes.

In addition,

1) preparing a vector comprising an antisense nucleotide, siRNA or shRNA for a gene encoding PTP-RE; And

2) introducing the vector of step 1) into brown adipocytes.

Methods of inhibiting the expression of PTP-RE protein are largely knock-out and knock-down. The double knockdown method is a method of using RNA interference (hereinafter referred to as RNAi), and is actively used due to the specificity of the base sequence and the function of inducing gene expression suppression more simply and more rapidly than the conventional gene knockout method. RNAi is a mechanism that inhibits gene expression by degrading a specific RNA molecule or by interfering with the transcription of a specific gene, and is induced by miRNA (microRNA) or siRNA (small interfering RNA). The siRNA is a double stranded RNA (dsRNA) molecule having 20 to 25 nucleotides, which forms an RNA-induced silencing complex (RISC) and then binds complementarily to the target mRNA to induce degradation of the target mRNA do. In vivo, an enzyme called dicer converts long dsRNA or shRNA (small hairpin RNA) into siRNA. In addition, the siRNA may be introduced into cells by various transfection methods such as a method of directly synthesizing siRNA in vitro and then introducing the siRNA into cells, a method of introducing an siRNA expression vector prepared to express siRNA in cells, .

The antibody is a term known in the art and refers to an active fragment of a molecule or molecule that binds to a known antigen. Examples of active fragments that bind to known antigens include Fab and F (ab) 2 fragments. The active fragments can be derived from the antibodies of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved by enzymes such as pepsin and subjected to HPLC gel filtration. Suitable fractions containing Fab fragments can be collected and concentrated by membrane filtration or the like. A description of common techniques for the isolation of active fragments of antibodies can be found in Khaw, B.A. et al., J. Nucl. Med. 23: 1011-1019 (1982). In addition, the antibody includes bispecific and chimeric antibodies.

In the present invention, the component that induces or inhibits the expression of the PTP-RE protein may be a nucleic acid, a protein, an extract or a natural product. Particularly, the component that inhibits the expression of the PTP-RE protein is preferably PTP-RE and siRNA (small interfering RNA) having a sequence complementary to mRNA.

In the present invention, the PTP-RE protein inhibits the differentiation into adipocytes when the expression level of the PTP-RE protein increases in brown adipose cells, and promotes the differentiation into adipocytes when the expression level thereof is decreased. Thus, Or the degree of differentiation into a brown adipocyte from the stage of differentiation from stem cells to adipocytes is measured, and when the level of differentiation into the adipocytes is above or below a preset reference value, an adipocyte differentiation promoter or an adipocyte differentiation inhibitor is added, And the adipocyte differentiation promoter and the adipocyte differentiation inhibitor can be regulated to a desired level to control the differentiation into brown adipocytes.

As a method of injecting an adipocyte differentiation promoter or an adipocyte differentiation inhibitor into cells, a method of preparing an adipocyte differentiation promoter or an adipocyte differentiation inhibitor in the form of a pharmaceutical preparation and administering it to a human body in a conventional manner can be used. A method using a vector system which is a genetic engineering technique, and the like.

The present invention also provides a kit for confirming the degree of differentiation of brown adipocytes comprising an antibody against a PTP-RE protein or a primer specific to a gene encoding the PTP-RE protein.

In addition, the present invention provides a method for determining the degree of differentiation of brown adipocytes, comprising the step of measuring the expression level of PTP-RE in brown adipocytes using the kit. The kit provides an antibody that is immunologically reactive with the PTP-RE protein or epitope thereof. The antibodies provided in the kits of the invention can be produced in animals by inoculating cells expressing the PTP-RE protein or epitope thereof by methods known in the art. The protein can be isolated from the cells at various levels of homogeneity by conventional biochemical methods. Synthetic peptides prepared by in vitro synthetic methods and arbitrarily conjugated to heterologous amino acid sequences are also included in the methods for producing antibodies of the present invention. The animals used for the inoculation include cattle, sheep, pigs, mice, rats, rabbits, hamsters, chlorine and primates. Preferred animals for inoculation include rodents, such as mice, rats, and hamsters. The most preferred animal is a mouse.

In addition, the kit of the present invention provides a monoclonal antibody that is immunologically reactive with the PTP-RE protein of the present invention present on the surface of the above-mentioned cells. Such antibodies are prepared using methods and techniques known to those skilled in the art. The monoclonal antibodies provided by the present invention are also produced by recombinant genetic engineering methods known to those skilled in the art, and the invention is directed to methods of producing the PTP-RE proteins by the methods described above which are immunologically reactive with the epitopes of the PTP- Lt; / RTI &gt;

In addition, the present invention includes fragments of such antibodies, including, but not limited to, Fab and F (ab) 2. Such fragments are produced by any method including, but not limited to, protein hydrolytic cleavage, chemical synthesis or the production of such fragments by genetic engineering methods. The invention may also include antibodies that are immunologically reactive with the PTP-RE protein, prepared by methods known to those skilled in the art. The invention also encompasses chimeric antibodies comprising light and heavy chain peptides that are immunologically reactive with a PTP-RE derived epitope. The chimeric antibodies provided herein include natural antibodies as well as chimeric antibodies made by methods of genetic engineering techniques known to those skilled in the art.

In addition, reagents used in immunological assays in the kit include suitable carriers, detection labels capable of producing detectable signals, solubilizers, and detergents. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers (e.g., PBS) or insoluble carriers known in the art such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile , Fluorocarbon resin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles coated with metal on lactate, other paper, glass, metal, agarose, and combinations thereof.

The antibody of the present invention may be coupled with a detectable substance, i.e., a detection labeling agent, to facilitate detection. Examples of the detection labeling agent include various enzymes, a constituent element, a fluorescent substance, a luminescent substance, a bioluminescent substance, a radioactive substance and the like. Detection markers can be coupled or conjugated directly to a resistive monoclonal antibody of the invention using techniques known in the art, or indirectly coupled via an intermediate (e. G., A linker known in the art) Or may be coupled or conjugated to a secondary antibody that is another polyclonal antibody that recognizes a resistin protein or that is an antibody that recognizes a resistin antibody. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, peroxidase, alkaline phosphatase,? -D-galactosidase, glucose oxidase, malate dehydrogenase, glucose- Examples of suitable subunit complexes including dihydrogenase, invertase and the like include streptavidin / biotin and avidin / biotin, and examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothio Examples of the luminescent material include luminol, iso-lucinol, lucigenin, examples of the bioluminescent material include luciferase, luciferase, luciferase, luciferase, Examples of suitable radiation materials include 125 I, 131 I, 111 In, 99 Tc, 14 C or 3 H, and the like. .

In addition, the method of confirming the degree of differentiation of brown adipocytes using the kit is as follows: when the level of PTP-RE expression in brown adipocytes is decreased compared with the level of PTP-RE expression in the control group, it is in the early stage of differentiation, . In addition, the level of PTP-RE expression is measured by PCR, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and enzyme-linked immunosorbent assay (ELISA) do.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are intended to illustrate the present invention in detail, and the content of the present invention is not limited by the examples and the experimental examples.

<Example 1> Fatty differentiation induction of brown fat cell line

The brown lipid progenitor cell line HIB-1B was cultured in 100% confluence in growth medium containing 10% fetal bovine serum (FBS), 1 nM 3,3 ', 5-triiodo-L-thyronine (T3) and 20 nM insulin. (IBMX), 0.5 μM dexamethasone, 20 nM insulin, 1 nM T3, and 1 mM T3 to induce lipid differentiation after 5% CO 2 uptake at 37 ° C. Differentiation was induced (differentiation 2 days) in 10% FBS / DMEM differentiation medium containing 125 M indomethacin for 2 days and induced differentiation and maturation in growth medium containing 20 nM insulin and 1 nM T3 (6 days of differentiation) for 6 days while changing the growth medium every 2 to 3 days.

< Example  2> Identification of differentiation into brown adipocytes

<2-1> Oil - Red Identification of adipocyte differentiation using -O dyeing

In order to confirm the differentiation of the brown adipose precursor cell line HIB-1B of Example 1 into adipocytes, cells were stained with Oil-Red-O staining, which is an intracellular triglyceride-specific staining method, on days 0, 3 and 6 after differentiation Respectively.

Specifically, the cells were fixed with 10% formalin for 30 minutes, followed by reaction with 0.3% Oil-Red-O solution for 30 minutes. After the reaction, the cells were washed twice with distilled water and observed under an optical microscope.

As a result, as shown in Fig. 1A, it was confirmed that fat globules accumulated according to the progress of differentiation of the brown adipose precursor cell line were increased (Fig. IA).

<2-2> Real time - PCR Using mRNA  Confirmation of adipocyte differentiation at the level

Real-time PCR was performed to confirm the expression of UCP-1, a brown adipocyte-specific marker, at the RNA level in the differentiation of the brown lipid precursor cell line HIB-1B of Example 1 into adipocytes.

Specifically, 5 μl of 40 ng of cDNA, 18 μl of distilled water and 25 μl of Power SYBR Green PCR Master Mix (2X) (ABI, USA) were used to make a final volume of 50 μl, The mixture was subjected to final 40 &lt; RTI ID = 0.0 &gt; Cycle reaction.

As a result, as shown in Fig. 1B, the expression of UCP-1, a brown adipocyte-specific marker, was significantly increased with progress of differentiation of brown adipose cell line (Fig. 1B).

<2-3> Western The western blot  Identification of adipocyte differentiation at the level of protein used

Western blotting was performed to identify the expression of UCP-1, a brown adipocyte-specific marker, at the protein level in the differentiation of the brown adipose precursor cell line HIB-1B of Example 1 into adipocytes .

Specifically, to prepare a protein sample, an intracellular lysis solution (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris) was added thereto, and the mixture was crushed at 10 ° C. for 10 seconds at 4 ° C. using a sonicator . The cell lysate was centrifuged at 4,000 rpm for 20 minutes at 13,000 rpm, and the supernatant was recovered. Protein content of recovered supernatant was quantified by Bradford method. 20 μg of the protein sample described above was loaded on an 11-12% acrylamide gel, and the protein was separated by size by electrophoresis (SDS-PAGE). The separated proteins were transferred to PVD (polyvinylidene fluoride) membrane in 1X transient buffer (20% methanol, 0.025 M Tris base, 0.19 M glycine) and reacted at 4 ° C for 12 hours using a specific antibody of UCP-1 . The PVDF membrane reacted with the primary antibody was thoroughly washed with 1X TBST for at least 3 times for 15 minutes, and HRP (goat anti rabbit IgG-HRP) of anti-rabbit immunoglobulin G isolated from the secondary antibody, chlorine serum, Lt; / RTI &gt; After washing three times for 15 minutes with 1X TBST, the film was developed with ECL treatment. In order to further clarify the difference in expression, β-actin was used as a reference.

As a result, as shown in Fig. 1B, the expression of UCP-1, a brown adipocyte-specific marker, was significantly increased with progress of differentiation of brown adipose cell line (Fig. 1B).

< Experimental Example  1> In the lipid differentiation process of brown fat cells PTP mRNA  Verify your profile

RT-PCR and real-time PCR were performed on the cells to identify PTP mRNA profiles of 0, 2, 4 and 6 days of the brown adipose cell line differentiated by the method of Example 1 above.

Specifically, RT-PCR extracted mRNA at each indicated differentiation date of the cells in the differentiation process of the adipocyte precursor cell line HIB-1B into adipocytes. 2 μl of RNA and 2 μl of Oligo dT primer (500 μg / ml) were reacted at 70 ° C for 5 minutes, and then RT-premix kit (Bioneer kit, , Korea) was used to synthesize complementary cDNA. Polymerase chain reaction was analyzed using PCR Premix Kit (Binoeer, Korea). 5 μl of cDNA, 10 pM primers, 2 μl and 13 μl of distilled water was reacted at 95 ° C for 5 minutes and then subjected to 30 cycles of reaction at 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds. Then, 1.5% agarose Gel electrophoresis and confirmed by EtBr (ethidium bromide) staining. The list of primers used is shown in Table 1 below. In addition, the above results were confirmed again using real-time PCR, and real-time PCR was performed using the method of Example 2.

As a result, as shown in FIG. 2, PTP-RE and PTPN6 tended to decrease as the differentiation of brown adipose cell line into adipocytes progressed. Particularly, PTP-RE showed a significant decrease at the fourth day of differentiation, PTP-RT, PTPN11 and CDC14A showed an increasing tendency, thereby confirming the profile of PTPs including PTP-RE, PTP-RT, PTPN6, PTPN11 and CDC14A in the brown adipocyte differentiation process (FIG.

< Experimental Example  2> In the lipid differentiation process of brown fat cell line PTP - RE Of lipid peroxidation

<2-1> PTP - RE Lt; RTI ID = 0.0 &gt; transfected &lt; / RTI &gt;

To confirm the role of PTP-RE in the brown adipocyte differentiation process identified in Experimental Example 1, a cytoPTP-RE construct having a cytoplasmic domain using a retrovirus system in HIB-1B cell line, RE-IRES-GFP) and a catalytically inactivated mutant form of PTP-RE-CS (catalytically inactive mutant) was used as a negative control. The transformed cells were separated using a FACSAria cell sorter (BD Biosciences, USA). The separated cells were confirmed by fluorescence microscopy.

As a result, as shown in Fig. 3A, most isolated cells were found to be GFP-positive (Fig. 3A).

<2-2> PTP - RE Of the transfected cells overexpressing

Using the transfected cells, differentiation into brown adipocytes was induced as in the method of Example 1, and cells at 6 days of differentiation were identified by western blotting using PTP-RE specific antibody and Oil-Red-O staining , The stained cells were separated using a staining extraction buffer solution, and the absorbance was measured and compared with the control group.

As a result, as shown in FIG. 3B to FIG. 3D, PTP-RE was transformed without abnormality and protein expression was confirmed, and PTP-RE transformed cells were compared with cells transformed with PTP-RE-CS mutant (Fig. 3B to Fig. 3D).

< Experimental Example  3> In the lipid differentiation process of brown fat cell line PTP - RE Of insulin receptor phosphorylation

To confirm the function of insulin receptor (IR) phosphorylation of PTP-RE in the lipid differentiation process of brown adipose cell line, PTP-RE or PTP-RE-CS was transformed and the cell line overexpressing each protein was transformed into 12 After incubation for a time in a serum-starved state, brown adipogenic cocktails were applied to the cells at indicated times. Whole cell lysates containing the same amount of protein were immunoprecipitated using agarose conjugated with anti-phosphotyrosine antibody (4G10; anti-pY) and then incubated with anti-insulin receptor Western blot analysis was performed using anti-insulin receptor (IR-β).

As a result, as shown in FIG. 4, IR phosphorylation was significantly reduced during the differentiation of brown adipocytes in HIB-1B cells transformed with PTP-RE as compared with the control group transfected with vector alone, whereas PTP -RE-CS mutant transfected HIB-1B cells showed no change in IR phosphorylation levels (FIG. 4).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for regulation of brown adipocyte differentiation using          PTP-RE <130> 13P-02-019 <160> 12 <170> Kopatentin 1.71 <210> 1 <211> 642 <212> PRT <213> Homo sapiens <400> 1 Met Ser Asn Arg Ser Ser Phe Ser Arg Leu Thr Trp Phe Arg Lys Gln   1 5 10 15 Arg Lys Ala Val Val Ser Thr Ser Asp Lys Lys Met Pro Asn Gly Ile              20 25 30 Leu Glu Glu Gln Glu Gln Gln Arg Val Met Leu Leu Ser Arg Ser Pro          35 40 45 Ser Gly Pro Lys Lys Tyr Phe Pro Ile Pro Val Glu His Leu Glu Glu      50 55 60 Glu Ile Arg Ile Arg Ser Ala Asp Asp Cys Lys Gln Phe Arg Glu Glu  65 70 75 80 Phe Asn Ser Leu Pro Ser Gly His Ile Gln Gly Thr Phe Glu Leu Ala                  85 90 95 Asn Lys Glu Glu Asn Arg Glu Lys Asn Arg Tyr Pro Asn Ile Leu Pro             100 105 110 Asn Asp His Ser Arg Val Leu Ser Gln Leu Asp Gly Ile Pro Cys         115 120 125 Ser Asp Tyr Ile Asn Ala Ser Tyr Ile Asp Gly Tyr Lys Glu Lys Asn     130 135 140 Lys Phe Ile Ala Ala Gln Gly Pro Lys Gln Glu Thr Val Asn Asp Phe 145 150 155 160 Trp Arg Met Met Trp Glu Gln Lys Ser Ala Thr Ile Val Met Leu Thr                 165 170 175 Asn Leu Lys Glu Arg Lys Glu Glu Lys Cys His Gln Tyr Trp Pro Asp             180 185 190 Gln Gly Cys Trp Thr Tyr Gly Asn Ile Arg Val Cys Val Glu Asp Cys         195 200 205 Val Val Leu Val Asp Tyr Thr Ile Arg Lys Phe Cys Ile Gln Pro Gln     210 215 220 Leu Pro Asp Gly Cys Lys Ala Pro Arg Leu Val Ser Gln Leu His Phe 225 230 235 240 Thr Ser Trp Pro Asp Phe Gly Val Pro Phe Thr Pro Ile Gly Met Leu                 245 250 255 Lys Phe Leu Lys Lys Val Lys Thr Leu Asn Pro Val His Ala Gly Pro             260 265 270 Ile Val Val His Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Phe Ile         275 280 285 Val Ile Asp Met Met Ala Met Met His Ala Glu Gln Lys Val Asp     290 295 300 Val Phe Glu Phe Val Ser Arg Ile Arg Asn Gln Arg Pro Gln Met Val 305 310 315 320 Gln Thr Asp Met Gln Tyr Thr Phe Ile Tyr Gln Ala Leu Leu Glu Tyr                 325 330 335 Tyr Leu Tyr Gly Asp Thr Glu Leu Asp Val Ser Ser Leu Glu Lys His             340 345 350 Leu Gln Thr Met His Gly Thr Thr Thr His Phe Asp Lys Ile Gly Leu         355 360 365 Glu Glu Phe Arg Lys Leu Thr Asn Val Arg Ile Met Lys Glu Asn     370 375 380 Met Arg Thr Gly Asn Leu Pro Ala Asn Met Lys Lys Ala Arg Val Ile 385 390 395 400 Gln Ile Ile Pro Tyr Asp Phe Asn Arg Val Ile Leu Ser Met Lys Arg                 405 410 415 Gly Gln Glu Tyr Thr Asp Tyr Ile Asn Ala Ser Phe Ile Asp Gly Tyr             420 425 430 Arg Gln Lys Asp Tyr Phe Ile Ala Thr Gln Gly Pro Leu Ala His Thr         435 440 445 Val Glu Asp Phe Trp Arg Met Ile Trp Glu Trp Lys Ser His Thr Ile     450 455 460 Val Met Leu Thr Glu Val Gln Glu Arg Glu Gln Asp Lys Cys Tyr Gln 465 470 475 480 Tyr Trp Pro Thr Glu Gly Ser Val Thr His Gly Glu Ile Thr Ile Glu                 485 490 495 Ile Lys Asn Asp Thr Leu Ser Glu Ala Ile Ser Ile Arg Asp Phe Leu             500 505 510 Val Thr Leu Asn Gln Pro Gln Ala Arg Gln Glu Glu Gln Val Arg Val         515 520 525 Val Arg Gln Phe His Phe His Gly Trp Pro Glu Ile Gly Ile Pro Ala     530 535 540 Glu Gly Lys Gly Met Ile Asp Leu Ile Ala Ala Val Gln Lys Gln Gln 545 550 555 560 Gln Gln Thr Gly Asn His Pro Ile Thr Val His Cys Ser Ala Gly Ala                 565 570 575 Gly Arg Thr Gly Thr Phe Ile Ala Leu Ser Asn Ile Leu Glu Arg Val             580 585 590 Lys Ala Glu Gly Leu Leu Asp Val Phe Gln Ala Val Lys Ser Leu Arg         595 600 605 Leu Gln Arg Pro His Met Val Gln Thr Leu Glu Gln Tyr Glu Phe Cys     610 615 620 Tyr Lys Val Val Gln Asp Phe Ile Asp Ile Phe Ser Asp Tyr Ala Asn 625 630 635 640 Phe Lys         <210> 2 <211> 1929 <212> DNA <213> Homo sapiens <400> 2 atgagcaaca ggagtagctt ttcccggctc acctggttca ggaagcagag gaaagctgtg 60 gtcagcacca gcgacaagaa gatgcccaac ggaatcttgg aggagcaaga gcagcaaagg 120 gtgatgctgc tcagcaggtc accctcaggg cccaagaagt attttcccat ccccgtggag 180 cacctggagg aggagatccg tatcagatcc gccgacgact gcaagcagtt tcgggaggag 240 ttcaactcat tgccatctgg acacatacaa ggaacttttg aactggcaaa taaagaagaa 300 aacagagaaa aaaacagata tcccaacatc cttcccaatg accattctag ggtgattctg 360 agccaactgg atggaattcc ctgttcagac tacatcaatg cttcctacat agatggttac 420 aaagagaaga ataaattcat agcagctcaa ggtcccaaac aggaaacggt taacgacttc 480 tggagaatgg tctgggagca aaagtctgcg accatcgtca tgttaacaaa cttgaaagaa 540 aggaaagagg aaaagtgcca tcagtactgg cccgaccaag gctgctggac ctatggaaac 600 atccgggtgt gcgtggagga ctgcgtggtt ttggtcgact acaccatccg gaagttctgc 660 atacagccac agctccccga cggctgcaaa gcccccaggc tggtctcaca gctgcacttc 720 accagctggc ccgacttcgg agtgcctttt acccccattg ggatgctgaa gttcctcaag 780 aaagtaaaga cgctcaaccc cgtgcacgct gggcccatcg tggtccactg tagcgcgggc 840 gtgggccgga cgggcacctt cattgtgatc gatgccatga tggccatgat gcacgcggag 900 cgaaggtgg atgtgtttga atttgtgtct cgaatccgta atcagcgccc tcagatggtt 960 caaacggata tgcagtacac gttcatctac caagccttac tcgagtacta cctctacggg 1020 gacacagagc tggacgtgtc ctccctggag aagcacctgc agaccatgca cggcaccacc 1080 acccacttcg acaagatcgg gctggaggag gagttcagga aattgacaaa tgtccggatc 1140 atgaaggaga acatgaggac gggcaacttg ccggcaaaca tgaagaaggc cagggtcatc 1200 cagatcatcc cgtatgactt caaccgagtg atcctttcca tgaaaagggg tcaagaatac 1260 acagactaca tcaacgcatc cttcatagac ggctaccgac agaaggacta tttcatcgcc 1320 acccaggggc cactggcaca cacggttgag gacttctgga ggatgatctg ggaatggaaa 1380 tcccacacta tcgtgatgct gacggaggtg caggagagag agcaggataa atgctaccag 1440 tattggccaa ccgagggctc agttactcat ggagaaataa cgattgagat aaagaatgat 1500 accctttcag aagccatcag tatacgagac tttctggtca ctctcaatca gccccaggcc 1560 cgccaggagg agcaggtccg agtagtgcgc cagtttcact tccacggctg gcctgagatc 1620 gggattcccg ccgagggcaa aggcatgatt gacctcatcg cagccgtgca gaagcagcag 1680 cagcagacag gcaaccaccc catcaccgtg cactgcagtg ccggagctgg gcgaacaggt 1740 acattcatag ccctcagcaa cattttggag cgagtaaaag ccgagggact tttagatgta 1800 tttcaagctg tgaagagttt acgacttcag agaccacata tggtgcaaac cctggaacag 1860 tatgaattct gctacaaagt ggtacaagat tttattgata tattttctga ttatgctaat 1920 ttcaaatga 1929 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTP-RE Forward primer <400> 3 gccctaagca ggagacagtg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTP-RE reverse primer <400> 4 gccctaagca ggagacagtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTP-RT Forward primer <400> 5 gcagaaggag acccagagtg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTP-RT reverse primer <400> 6 caaggtttgt gaccatgacg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTPN6 Forward primer <400> 7 aaccagctgc taggtccaga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTPN6 reverse primer <400> 8 ctgctgtgtc atgctcccta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTPN11 Forward primer <400> 9 agtccaaagt gacccacgtc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PTPN11 reverse primer <400> 10 ttgagcgcat actcatcagg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mCDC14A Forward primer <400> 11 tcgacgagga gctggtctat 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mCDC14A reverse primer <400> 12 gtcaaggacg gtgaggttgt 20

Claims (13)

delete delete delete delete delete A method for inhibiting the differentiation of brown adipocytes, comprising treating the PTP-RE protein with an isolated brown lipid precursor cell or a subject other than a human.
A method for promoting the differentiation of brown adipocytes, comprising the step of treating an antibody that specifically binds to a PTP-RE protein to an individual, except for a brown preadipocyte cell line or a human.
1) preparing an expression vector comprising a gene encoding a PTP-RE protein; And
2) introducing the expression vector of step 1) into a separated brown preadipocyte or a subject other than a human.
1) preparing a vector comprising an antisense nucleotide, siRNA or shRNA that specifically binds to a gene encoding PTP-RE; And
2) introducing the vector of step 1) into a separated brown preadipocyte or a subject other than a human.
A kit for confirming the degree of differentiation of brown adipocytes comprising an antibody that specifically binds to a PTP-RE protein, or a primer specific to a gene encoding the PTP-RE protein.
A method for identifying the degree of differentiation to brown adipocytes in vitro (ex vivo), comprising measuring the level of PTP-RE expression in brown adipocytes using the kit of claim 10.
12. The method according to claim 11, wherein the PTP-RE expression level is an initial stage of differentiation when compared with the PTP-RE protein expression level of the control group, (ex vivo) to determine the degree of differentiation of brown adipocytes.
12. The method of claim 11, wherein the PTP-RE expression level is selected from the group consisting of a polymerase chain reaction (PCR), a reverse transcriptase polymerase chain reaction (RT-PCR), a western blot and an enzyme linked immunosorbent assay The method comprising the step of determining the degree of differentiation of brown adipocytes in vitro (ex vivo).

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