KR101698068B1 - PPAR-gamma acrivation or expression regulating composition comprising galectin-3 - Google Patents

Abstract

The present invention relates to the treatment of metabolic diseases using galectin-3, and more particularly, to the use of galectin-3 for the expression and activity of peroxisome proliferator-activated receptor gamma (PPARγ) And a method for treating or preventing a metabolic disease using the same.

Description

Gamma activity or expression regulating composition comprising galectin-3 as an active ingredient (PPAR-gamma acrivation or expression regulating composition comprising galectin-3)

The present invention relates to a composition for regulating the activity or expression of peroxisome proliferator-activated receptor gamma (PPARγ) comprising galectin-3 as an active ingredient.

The main role of white adipose tissue (WAT) is the maintenance of homeostatic energy through energy storage. When excessive food energy is supplied, the remaining energy is stored in the form of triglycerides in white adipose tissue and increases the weight of white adipose tissue. Too much white fat tissue causes metabolic diseases such as type 2 diabetes, hypertension, arteriosclerosis, and hyperlipidemia.

The peroxisome proliferator-activated receptor is a nuclear receptor that is a ligand of peroxisome proliferator, which is a compound capable of increasing the number of peroxisomes. It is activated in the ligand, and the PPAR response element PPRE) to increase the expression of the target gene, and the homozygous forms of PPAR [alpha], PPAR [beta], PPAR [gamma] and PPAR [delta] are known [Biochem. Biophys. Res. Commun., 224, 431, 1996].

PPARs mainly regulate the expression of genes involved in the differentiation of genes or fatty cells (asipocyte) involved in fat metabolism. PPARγ is mainly expressed in adipocytes, immune cells, adrenals, spleen, and small intestine. It is known as a major transcription factor. As described above, since PPAR plays an important role in lipid metabolism, many studies are being conducted to develop a therapeutic agent for metabolic diseases targeting PPAR. U.S. Patent No. 6,939,875 relates to a composition that can be used to treat a PPAR mediated disease wherein said composition is said to be effective in Type 2 diabetes, obesity, eating disorders, metabolic syndrome, etc. U.S. Patent No. 6,967,212 Discloses that a composition comprising an azole acid derivative acting as a PPAR agonist is effective for insulin resistance, obesity, hyperlipidemia, metabolic syndrome, and the like. However, since the method using the PPAR ligand as described above is accompanied with various side effects including hepatotoxicity and hypoglycemic symptoms, a new method for controlling PPARγ without using a ligand is required.

Galectins are thiol-binding lectins that are generally galactose-binding, and have been found to be tissue-specific expression proteins in animal tissues such as nematodes and sponge mammals. Galectin-1, -3, and -8 are distributed in various organs in vivo, whereas Galectin-2, -4, -5, and -7 are specific organ or cell Are known to be limited.

Most studies on galectin-3 have been focused on cancer and inflammatory diseases, and the role of galectin-3 in obesity has not been well studied. Pang J et al. Reported that obesity systemic inflammation and abnormal glucose metabolism were increased in galectin-3 knockout mice, and increased intestinal mass, blood glucose and insulin levels [PolS one, 2013; 8 (2): e57915].

Accordingly, a problem to be solved by the present invention is to provide a composition for regulating transcription activity or expression of peroxisome proliferator-activated receptor gamma (PPARγ) comprising galectin-3 as an active ingredient.

Another object to be solved by the present invention is to provide a method for reducing the activity or expression of peroxisome proliferator activated receptor-gamma by inhibiting or eliminating galectin-3.

The present invention provides a composition for controlling the activity or expression of peroxisome proliferator-activated receptor gamma (PPARγ) comprising galectin-3 as an active ingredient.

According to an embodiment of the present invention, the galectin-3 may act on the peroxidase proliferator-activated receptor gamma to increase transcription activity or expression level,

Preferably, the galectin-3 binds to peroxidase proliferator-activated receptor gamma to increase the activity or expression of peroxisome proliferator activated receptor gamma.

The present invention also provides a composition for controlling adipocyte differentiation comprising galectin-3 as an active ingredient.

According to one embodiment of the present invention, galectin-3 may induce adipocyte differentiation.

The present invention also provides a composition for inhibiting the expression of adipose triglyceride lipase (ATGL) comprising galectin-3 as an active ingredient.

According to one embodiment of the present invention, the galectin-3 may be one that increases the expression of a fatty liver producing gene.

In one embodiment of the present invention, the fatty liver gene is selected from the group consisting of peroxidase proliferator-activated receptor gamma (PPARγ), ccaat-enhancer-binding protein alpha (C / EBPα), fatty acid binding protein (FABP4) FAS). ≪ / RTI >

The present invention also provides a method for inhibiting the expression or elimination of galectin-3 by knocking galectin-3, thereby reducing the activity or expression of peroxisome proliferator-activated receptor gamma (PPARγ) .

In addition, the present invention provides a method for reducing the expression of a fatty liver producing gene by knocking galectin-3.

Also, the present invention provides a method for reducing fat cell size comprising knocking galactin-3 to increase the expression of adipose triglyceride lipase (ATGL).

The present invention relates to a method of regulating the activity or expression of a peroxidase proliferating receptor-gamma gene, which is a transcriptional gene known as an adipocyte differentiation index, by using galectin-3, For example, metabolic diseases, and the like.

FIG. 1 shows the results of measuring the white adipose tissue reducing effect of galectin knockout (lgals3 ^{- / -} ) mice and wild type (lgals3 ^{+ / +} ) mice at 17 months of age consuming high fat diets,
FIG. 1A is a graph showing an image and body weight of a body size of an Igal3 ^{- / -} mouse and an Igal3 ^{+ / +} mouse.
FIG. 1B is a graph showing images and weights comparing sizes of epididymal white adipose tissue (eWAT) of Igal3 ^{- / -} mice and Igal3 ^{+ / +} mice.
FIG. 1C is a graph comparing the weight of epididymal white adipose tissue (eWAT), the weight of brown adipose tissue (BAT), and the liver weight of each of lgals3 ^{- / -} and lgals3 ^{+ / +} mice.
Figure 1d is lgals3 ^{- / -} mice and lgals3 ^{+ / +} Galectin-3, in the epididymal white adipose tissue (eWAT) of mouse peroxisome proliferator-activated receptor gamma (hereinafter, 'PPARγ' referred to), ccaat-enhancer-binding protein alpha (Hereinafter referred to as 'C / EBP alpha'), fatty acid binding protein (hereinafter referred to as 'FABP4') and fatty acid synthase to be.
FIG. 1E shows the results of real-time RT-PCT analysis of expression amounts of IL-10, INFγ and TNFα in epididymal white adipose tissue (eWAT) of Igal3 ^{- / -} mice and Igal3 ^{+ / +} mice.
FIG. 1F shows the results of real-time RT-PCT analysis of the expression levels of Galectin-3, PPARγ, C / EBPα, FABP4 and FAS in lgals3 ^{- / -} mice and lgals3 ^{+ / +} mice.
FIG. 1g shows the results of real-time RT-PCT analysis of the expression levels of IL-10, INFγ and TNFα in the liver of lgals3 ^{- / -} and lgals3 ^{+ / +} mice.
Expression of mRNA was normalized to β-actin and all data were expressed as mean ± SE (independent sample t-test). The number of experimental animals was 5 (n = 5).
* P &lt; 0.05, ** P &lt; 0.01, *** P < 0.001.
FIG. 2 is a graphical representation of the fat content of mouse embryonic fibroblasts (MEFs) of galectin knockout mice (lgals3 ^{- / -} ) and wild type (lgals3 ^{+ / +} ) mice at 17 months of age fed a high fat diet As a result of measuring the delayed cell differentiation,
Figures 2a and 2b show the adipocyte differentiation of lgals3 ^{- / -} and lgals3 ^{+ / +} mouse MEFs treated with DMI and rosiglitazone, respectively.
MEFs were treated with DMI and rosiglitazone for 14 days, respectively, and fat cells were stained with oil-red-O.
FIG. 2C is a result of measurement of lipid accumulation amount.
FIG. 2d shows Western blot analysis of lipogenic factors (Galectin-3, PPARγ, C / EBPα, C / EBPβ and FABP4) in Igal3 ^{- / -} and Igal3 ^{+ / +} mouse MEFs.
Protein expression of fat-producing factors was normalized to β-actin and all data were expressed as mean ± SE (independent sample t-test).
FIG. 3 shows the results of confirming the inhibition of adipocyte differentiation by galectin-3-deficient 3T3-L1 cells.
FIG. 3A shows the results of measuring the expression level of galectin-3 mRNA in adipocyte differentiation.
FIG. 3B shows the results of measuring the level of galectin-3 protein in adipocyte differentiation.
FIG. 3c shows the results of 3T3-L1 cells treated with puromycin and galectin-3 shRNA lentivirus so as not to express galectin-3, and FIG. 3d shows the result of differentiating adipocytes using the same. Cells were treated with DMI 8 days after differentiation and stained with Oil-Red-O
3E is a result of measurement of the amount of lipid axes.
Figure 3f shows results of western blot analysis of adipogenic factors (Galectin-3, PPARgamma, C / EBPa, C / EBP [beta] and FABP4).
Protein expression of fat-producing factors was normalized to β-actin and all data were expressed as mean ± SE (independent sample t-test).
* P < 0.05, ** P < 0.01, *** P &lt; 0.001.
FIG. 4 shows the results of confirming the interaction between galectin-3 and PPAR gamma and the increase of transcriptional activity,
Figure 4A shows that cell lysates were immunoprecipitated using anti-PLAG beads as a result of transformation of HEK293 cells with FLAG-labeled galectin and HA-labeled PPARy.
FIG. 4B shows the result of confirming endogenous interactions between galectin-3 and PPARγ.
4C shows the results of PPRE luciferase activity in HEK293 cells. HEK293 cells were transformed with sh-galectin-3, PPRE, and β-gal and treated with 20 μM rosiglitazone. Luciferase activity was normalized to? -Gal activity.
FIG. 4d shows immunocytochemical results of galectin-3 and PPARγ in 3T3-L1 cells treated to inhibit galectin-3 expression.
All data were expressed as mean ± SE (independent sample t-test).
* P &lt; 0.05, ** P < 0.01, *** P < 0.001.
FIG. 5 shows the results of obesity resistance of lgals3 ^{- / -} mice and lgals3 ^{+ / +} mice on high fat diet,
FIG. 5A is a graph showing changes in body weight of lgals3 ^{- / -} mice and lgals3 ^{+ / +} mice fed high fat diets containing 60% fat for 12 weeks.
5B is a graph showing an average food intake per day.
FIG. 5c shows eWAT weight, weight of eWAT, weight of brown fat, and liver weight of lgals3 ^{- / -} mice and lgals3 ^{+ / +} mice consuming high fat diets, respectively.
FIG. 5d shows blood glucose levels and plasma-free fatty acids concentrations measured after fasting for 5 hours in Igal3 ^{- / -} mice and Igal3 ^{+ / +} mice, respectively, which were fed a high fat diet.
All data were expressed as mean ± SE (independent sample t-test).
* P < 0.05, ** P < 0.01, *** P < 0.001.
FIG. 6 shows the effect of galectin-3 knockout mice on the regulation of obesity and ATGL expression through eWAT of lgals3 ^{- / -} and lgals3 ^{+ / +} mice,
Figure 6a is an image comparing the fat cell size of Igal3 ^{- / -} mouse and Igal3 ^{+ / +} mouse eWAT.
FIG. 6B shows the result of measuring the triglyceride content of IgAL3 ^{- / -} mouse and IgAL3 ^{+ / +} mouse eWAT.
FIG. 6C shows the results of real-time RT-PCR measurement of expression levels of galectin-3 and ATGL in IgAL3 ^{- / -} mouse and IgAL3 ^{+ / +} mouse eWAT.
FIG. 6d shows microarray analysis results for PPARγ, C / EBPα, FABP4, FAS, Me1, Acaa, Acyl and Slc25a1 of Igal3 ^{- / -} and Igal3 ^{+ / +} mouse liver tissues.
FIG. 6E shows the results of real-time RT-PCR analysis of galectin-3, PPARγ, C / EBPα, FABP4 and FAS genes of Igal3 ^{- / -} mouse and Igal3 ^{+ / +} mouse liver tissues.
Protein expression of fat-producing factors was normalized to β-actin and all data were expressed as mean ± SE (independent sample t-test).
* P < 0.05, ** P < 0.01, *** P < 0.001.

Hereinafter, the present invention will be described in detail.

PPARs mainly regulate the expression of genes involved in the differentiation of adipocytes or adipocytes involved in lipid metabolism. PPARγ is mainly expressed in adipocytes, immune cells, adrenals, spleen and small intestine, and adipocyte differentiation It is known as a major transcription factor. The inventors of the present invention found that galectin-3, which is a biomolecule, binds to PPARγ to directly activate PPARγ and leads to differentiation into adipocytes. Thus, the present inventors have found that galectin-3 is effective as an active ingredient, And the present invention has been completed.

Specifically, the present invention provides a composition for regulating PPARgamma activity or expression comprising Galectin-3 as an active ingredient.

According to the present invention, galectin-3 acts on PPARγ to increase the activity or expression level of PPARγ,

Specifically, galectin-3 can directly or intrinsically and externally directly interact with or bind to PPARγ to increase the activity or expression level of PPARγ.

According to the present invention, the transcriptional activity of PPARγ was reduced in the absence of galectin-3 regardless of the presence or absence of rosiglitazone, an agonist of PPARγ. This means that galectin-3 binds directly to PPARy to activate or express PPARy. With these characteristics, the composition for controlling the activity or expression of PPARy comprising gallontin-3 as an active ingredient of the present invention can be effectively used for the treatment or prevention of metabolic diseases caused by the expression of PPARy.

The metabolic diseases may include, for example, at least one selected from obesity, diabetes, hyperlipidemia and non-alcoholic fatty liver.

In addition, according to the present invention, galectin-3 is characterized in that it does not participate in an inflammatory reaction. In other words, according to the present invention, the galectin-3-deleted or overexpressed environment and the inflammatory reaction are independent of each other, and the induction of a metabolic disease such as obesity is not caused by an inflammatory reaction or hyperglycemia, And may be induced by adipocyte differentiation by activated PPARy.

According to the present invention, a composition comprising galectin-3 as an active ingredient can be used for controlling adipocyte differentiation. Specifically galectin-3 may induce adipocyte differentiation. In other words, it is possible to inhibit or delay adipocyte differentiation in an environment in which the removal or expression of galectin-3 is inhibited. Therefore, it is possible to prevent or delay diseases caused by adipocyte differentiation, for example, metabolic diseases or the like It can be applied to therapy.

In addition, according to the present invention, a composition comprising galectin-3 as an active ingredient can inhibit the expression of adipose triglyceride lipase (ATGL). Specifically, the triglycerolase catalyzes the hydrolysis rate of triglyceride in the step of hydrolyzing the triglyceride. In the absence of ATGL, the triglyceride and white adipose tissue may be increased in various tissues. According to the present invention, ATGL can be increased by suppressing the elimination or expression of galectin-3, and ATGL can hydrolyze the triglyceride, thereby reducing the triglyceride in the body.

In addition, according to the present invention, the triglycerolytic enzyme can reduce the size of adipocytes, thereby preventing the increase in the cell size of adipocytes by suppressing the elimination or expression of galectin-3.

According to the present invention, a composition comprising galectin-3 as an active ingredient can control the expression of a fatty liver producing gene. 3 can be eliminated or expression of galectin-3 can be inhibited, thereby suppressing the expression of the genes of PPARγ, C / EBPα, FABP4 and FAS, which are genes that induce fatty liver, thereby reducing the production of fatty liver.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example

Mouse and Diet

Male and female C57BL / 6 mice with galectin-3 wild-type allele (homozygous + / +) or galectin-3 knockout allele (homozygous - / - FT Liu (UC Davis). Wild and galectin-3 deficient mice were housed and maintained in a C57BL / 6 genetic background. Six weeks old wild-type males (lgals3 ^{+ / +} ) and galectin-3-deficient (lgals3 ^{- / -} ) mice received 60% fat for 12 weeks while maintaining 12 h / High-fat diet. The experiments were performed independently on female lgals3 ^{+ / +} and lgals3 ^{- / -} mice. The same age group mice were used in the study. Body weight and food intake were measured once a week. The tissues of the mice used in the experiment were frozen using liquid nitrogen, stored at -80 ° C and used for analysis.

Cell culture and adipocyte differentiation analysis

HEK293 cells were purchased from Korean Cell Line Bank (KCLB) and cultured. Heterozygous (lgals3 ^+/- ) female and male N6 mice crossed to produce lgals3 ^{+ / +} and gals3 ^{- / -} mice. 3T3-L1 cells were grown in fusion state and differentiation was induced by high glucose DMEM containing 10% FBS, insulin (5 g / ml), isobutyl methyl xanthate (520 M) and dexamethasone Respectively. The medium was replaced every 2 days with high glucose DMEM containing 10% FBS and insulin, and replaced until day 8. MEF differentiation was induced using high glucose DMEM containing 10% FBS, insulin (10 g / ml), isobutyl methyl xanthate (520 M), dexamethasone (1 M) and rosiglitazone (1 M). MEF medium was changed every 2 days with high glucose DMEM containing 10% FBS, insulin (10 g / ml) and rosiglitazone (1 M) and replaced until day 14. Lipid accumulation was detected using an oil-red-O staining reagent (O0625, Sigma-Aldrich).

Galectin -3 is stable Knocked down  3 T3 - L1  Cell formation

Galectin-3 shRNA lentivirus carriers were purchased from Sigma-Aldrich and Galectin-3 shRNA lentiviruses were prepared by the method of Kim (J. Control Release 2011; 21 (4) 594-603). Specifically, HEK293FT cells were infected with lentivirus and packaging carriers using Lipofectamine 2000 (Invitrogen). After 48 hours, the medium containing the lentivirus was filtered using a 0.45-μm syringe filter. Lentivirus was transfected into 50% confluent adipose precursor cells 3T3-L1. 3T3-L1 cells infected with lentivirus were selected by puromycin (2 g / ml).

Western blot  analysis

Cell lysate extracts were dissolved in RIPA buffer (1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl pH 7.5 and 2 mM EDTA pH 8.0) and protease inhibitor cocktail (Genedepot) Respectively. The lysates were incubated at 4 ° C for 20 minutes and centrifuged at 13,200 rpm for 25 minutes at 4 ° C. The concentration of the supernatant was measured with a Quibit 2.0 fluorometer (Invitrogen). Proteins were loaded onto SDS PAGE gels and then transferred to PVDF membranes (GE Healthcare). Followed by blocking with 5% skim milk. Membranes were incubated overnight at 4 ° C on a rotator using primary antibodies (Galectin-3, C / EBPα, C / EBPβ, PPARγ, AFABP, HA (Santa Cruz), and Flag (Sigma-Aldrich) Respectively. Then, the membrane was washed with PBTS and then cultured with HRP-conjugated secondary antibody (Santa Cruz). Next, images were detected using a LAS-300 (Fujifilm) detector according to the manufacturer's instructions. Anti-beta-actin (Santa-Cruz) was used for the normalization control.

Immune sedimentation

Cell disruption extraction was performed using IP buffer and protease inhibitor cocktail (Genedepot). After centrifugation (13,200 rpm at 4 DEG C for 25 minutes), the protein in the supernatant was added to A / G agarose beads (Santa Cruz) and cultured in rotors at 4 DEG C for 30 minutes. After centrifugation, anti-Flag beads (Sigma-Aldrich), antigalectin-3, anti-PPAR and normal IgG (negative control) were separately added to the supernatant and cultured overnight at 4 ° C. Immunoprecipitation was performed twice with IP buffer, followed by addition of 2X SDS sample buffer and boiling at 95 ° C for 5 minutes. After centrifugation (13,200 rpm for 25 minutes at 4 DEG C), the supernatant was subjected to Western blot analysis.

Luciferase  Reporter Analysis

PPRE-TK-Luc, shgalectin-3 and? -Gal were transfected with HEK293 cells using lipofectamine 2000 (Invitrogen). After 48 hours, the cells were harvested and the luciferase activity was measured using the luciferase assay system (Promega). Luciferase activity was normalized using a beta-gal enzyme assay system (Promega).

Immune cell chemistry

The cells of the chamber slices were fixed with 4% formaldehyde for 30 minutes at 4 ° C, washed with 1X PBS buffer, and permeabilized with 0.5% Triton X-100 for 10 minutes. Cells were incubated with primary antibodies (galectin-3 and PPARγ) at 4 ° C and cultured with FITC anti-mouse and cy5 anti-rabbit secondary antibody (Invitrogen) and DAPI staining solution (Vector Laboratories). Images were analyzed by confocal microscopy (LSM 700).

RNA  Detach and Real Time RT - PCR  analysis

All tissue RNA was prepared using RNA-degradation reagent (5PRIME). The cDNA (1 g) was synthesized from RNA using qPCR RT master mix (TOYOBO). The following primers were used: Galectin-3-F 5'-cagtgctcctggaggctatc-3 ', R 5'-attgaagcgggggttaaagt-3'; PPAR? -F 5'-agggcgatcttgacaggaaa-3 ', R 5'-cgaaactggcacccttgaaa-3'; C / EBP? -F 5'-gtgactttgactacccggga-3 ', R 5'-ggggctcttgtttgatcacc-3'; FABP4-F 5'-catcagcgtaaatggggatt-3 ', R 5'-tcgactttccatcccactTC-3'; FAS-F 5'-tgggttctagccagcagagt-3 ', R 5'accaccagagaccgttatgc-3'; ATGL-F 5'-caacgccactacacctctacg-3 ', R 5'-atgagaggacccaggaa-3'; IL-10-F 5'-atcgatttctcccctgtgaa-3 ', R 5'-ttcggagagaggtacaaacga-3'; IFN? -F 5'-gagccagattatctctctctacc-3 ', R 5'-gttgttgacctcaaacttgg-3'; TNF? -F 5'-CGTcagCCGATTGGTATCT-3 ', R 5'-CGGactCCGCAAAGTTTAAG-3'; and β-actin-F 5'-ggctgtattcccctccatcg-3 ', R 5'-ccagttggtaacaatgccatgt-3'.

Real-time RT-PCR analysis was performed using SYBR Green Master Mix (Toyobo) with ABI instrument. Normalization control used β-actin. The results were compared with the mean expression in wild type (lgals3 + / +) mice.

Microarray analysis

Total RNA was prepared using RNA-degradation reagent (5PRIME). Expression of the genes was analyzed in cell lines using high density oligonucleotide microarrays (HG-U133 plus 2.0, Affymetrix, Santa Clara, Calif., USA) with 20 889 transcripts. The target macroscopic manufacturing and processing procedures were performed according to the Affymetrix GeneChip Expression Analysis Manual (Affymetrix). Gene chip analysis was performed using Microarray Analysis Suite 5.0, Data Mining Tool 2.0 and Microarray Database software (Santa Clara, Calif., USA) and Affymetrix GeneChip manual.

Epididymal white adipose tissue ( epididymal white adipose tissue ; eWAT ) Morphological analysis

Samples of epididymal white adipose tissue (hereinafter "eWAT") were fixed in 4% paraformaldehyde and internalized in paraffin. eWAT paraffin sections were stained with hematocin and eosin. Images were analyzed using ImageJ software.

eWAT Neutral Fat Analysis

eWAT samples were homogenized in chloroform / methanol (2: 1, v / v). 50 mM NaCl was added to the tissue lysate, vortexed for 10 minutes and centrifuged at 4 캜 for 10 minutes. The supernatant and interface were then completely removed from the organic layer. Triton X-100 / chloroform (7.5: 17.5, v / v) and the organic solvent was completely evaporated. TG levels were measured using a TG analysis reagent (Thermo Scientific).

Statistical analysis

Each group was analyzed using t-test and compared. Statistical analysis was performed using GraphPad Prism 5.01. P value <0.05 was considered statistically significant.

result

Galectin-3 Knockout ( LGgS3 ^{- / -} ) Reduction of white adipose tissue in mice

As shown in Figure 1, the 17-month-old male lgals3 ^{- / -} mice (n = 5) were smaller than the wild type (lgals3 ^{+ / +} ) mice (Fig. As a result, eWAT (white epididymal adipose tissue) of the mice was isolated and confirmed that ewAT was much smaller than that of lgals3 ^{- / -} mice (Fig. 1B). In addition, the proportion of lgals3 ^{- / -} mice was much smaller as a result of the ratio of eWAT to body weight. However, brown adipose tissue or liver size was not significantly different between lgals3 ^{- / -} and lgals3 ^{+ / +} mice (Fig. 1c).

As a result, expression of genes related to epididymal white adipose tissue (eWAT) and adipose tissue or fat production in the liver was measured, and it was confirmed that mRNA expression of PPARγ and FABP4 was decreased in eWAT of lgals3 ^{- / -} mice (FIG. 1e) .

The decrease in mRNA expression of PPARγ and FAS in liver of lgals3 ^{- / -} mice shows that galectin-3 deficiency controls the expression of adipocyte and adipogenic genes in both eWAT and liver 1F). It suggests that the increase of inflammation are generated in mice (Fig. 1e, g) ^- On the other hand, lgals3 ^{- / -} in both the liver and eWAT of mouse IL-10, IFNγ, and the level of expression of TNFα is not changed, which lgals3 ^{- /} .

Galectin -3 Knockout ( LGgS3 ^{- / -} ) Of the mouse In embryonic fibroblasts (MEFs)  Identification of inhibition of adipocyte differentiation

It was hypothesized that galectin - 3 might play an important role in lipogenesis because of the decreased expression of adipocytes and fat - producing genes in lgals3 ^{- / -} mice. The embryonic fibroblasts (MEFs) of lgals3 ^{+ / +} and lgals3 ^{- / -} mice were treated with DMI and rosiglitazone to induce adipocyte differentiation, and the results are shown in FIG. The intensity of adipocyte differentiation was measured by staining with oil-red-O (ORO) of lipid droplets (small droplets). Adipogenic differentiation of embryonic fibroblasts (MEFs) of lgals3 ^{- / -} mice for 14 days was poorer than embryonic fibroblasts (MEFs) of control group lgals3 ^{+ / +} mice (Fig. 2a, b). Adipocyte differentiation of embryonic fibroblasts (MEFs) of lgals3 ^{- / -} mice was less than that of embryonic fibroblasts (MEFs) of lgals3 ^{+ / +} mice (Fig. 2c). The expression of PPARγ, C / EBPα, C / EBPβ and FABP4 was found to be reduced in embryonic fibroblasts (MEFs) of adipocyte differentiated lgals3 - / - mice (FIG.

Galectin -3 deficiency 3 T3 - L1  Identification of inhibition of adipocyte differentiation by cells

The effect of galectin-3 on the differentiation of 3T3-L1 cells, a lipid precursor cell, was examined and the results are shown in FIG. The levels of galectin-3 mRNA and protein increased during adipocyte differentiation (Fig. 3a, b). In order to confirm the role of galectin-3 in adipocyte differentiation, galectin-3 was not expressed in 3T3-L1 cells. After 3T3-L1 cells were infected with galectin-3 shRNA lentivirus, galectin-3 was removed from shRNAs 2 and 5. 3T3-L1 cells were induced using DMI and differentiated for 8 days. As a result, galectin-3-deficient 3T3-L1 cells were observed to be differentiated from adipocyte differentiation and fat accumulation in comparison with 3T3-L1 cells of the control group (Fig. 3d, e). As a result, PPARγ, C / EBPα, C / EBPβ and FABP4 showed low expression (FIG. 3f). These results suggest that galectin-3 can regulate adipocyte differentiation.

Galectin -3 and PPAR γ interaction and increase in transcriptional activity

The interaction between galectin-3 and PPARy was confirmed and is shown in FIG. HEK293 cells were transformed with FLAG-labeled galectin-3 and HA-labeled PPARy. After 48 hours, the cells were harvested and the cell lysates were immunoprecipitated using Anti FLAG beads. We have confirmed exogenously expressed interactions between HA-labeled PPARy and FLAG-labeled galectin-3 of HEK-293 cells using Western blot using anti-FLAG and anti-HA antibodies ). In addition, endogenous interactions between galectin-3 and PPARy were confirmed in 3T3-L1 cells (Fig. 4B). In order to confirm the effect of galectin-3 on the transcriptional activity of PPARγ, galectin-3 was transfected by shRNA in HKE293 cells with a PPARγ receptor element (PPRE) receptor transiently transformed in the presence or absence of rosiglitazone, an agonist of PPARγ Knocked down. Galactin-3 depletion significantly reduced PPARg transcriptional activity independent of the presence of rosiglitazone compared to shLacZ. Using immune cells, we found that expression of PPARy and nuclear localization were reduced by absence of galectin-3 in 3T3-L1 cells (Fig. 4d). These results suggest that galectin-3 may regulate the expression and transcriptional activity of PPARγ by direct interaction.

High fat On the diet  About Galectin -3 Knockout ( LGgS3 ^{- / -} ) Obesity resistance effect of mouse

Lectin -3 Knockout go on a high-fat diet (lgals3 ^{- / -)} to evaluate the effect of obesity resistant mice, male a high-fat diet containing 60% fat lgals3 ^{+ / +} mice and lgals3 ^{- / -} mice (each, n = 5) for 12 weeks, and their characteristics and traits were observed, which is shown in FIG.

Although there was no significant difference in the amount of food ingested by the two groups (Fig. 5b), male lgals3 ^{- / -} mice weighed much less than lgals3 ^{+ / +} mice (Fig. 5a). In addition, lgals3 ^{- / -} mice showed less eWAT size and eWAT weight than lgals3 ^{+ / +} mice, whereas brown fat tissue and liver weight did not show a significant difference between the two groups (Fig. 5c).

On the other hand, female lgals3 ^{- / -} mice also showed an obesity resistance effect on high fat diets in lgals3 ^{+ / +} mice. Similar results were obtained in females with respect to body weight, eWAT weight and eWAT ratio per body weight. On the other hand, control of blood glucose and plasma free fatty acids (FFA) in the two groups was confirmed, but there was no significant difference between the weight gain and obesity-induced lgals3 ^{+ / +} mice and lgals3 ^{- / -} mice (Fig. 5D).

Galectin -3 Knockout ( LGgS3 ^{- / -} ) Mouse obesity and ATGL  Confirm expression control effect

EWAT observations of lgals3 ^{- / -} mice fed high fat diets showed that obesity reduction was associated with a decrease in the number of adipocytes. However, as shown in FIG. 6A, it was confirmed that the decrease in obesity in eWAT was not significantly related to the decrease in the number of adipocytes. The triglyceride (TG) levels induced by feeding high fat diets were lower in eWAT of lgals3 ^{- / -} mice (Fig. 6b).

Meanwhile, adipose triglyceride lipase (ATGL) is an enzyme that catalyzes the hydrolysis rate of triglyceride in the stage of triglyceride hydrolysis. Generally, in the absence of ATGL, neutral fat and white fat tissue .

However, it was confirmed that ATGL was significantly increased in the eWAT of lgals3 ^{- / -} mice fed with the high fat diet according to the present invention (Fig. 6C). In addition, we confirmed that the size of reduced fat cells in eWAT of lgals3 ^{- / -} mice fed high fat diets was similar to that of increased ATGL. The above results suggest that galectin-3 has an effect of regulating the expression of ATGL.

Galectin -3 Knockout ( LGgS3 ^{- / -} ) Modulation effect of fatty liver gene expression in mouse

In obesity, fatty liver is a disease that is often found. However, Pharaoh, lgals3 discussed herein ^{- / -} mice and lgals3 ^{- / -} mice between the histological analysis showed no significant difference. NDA microarray analysis was used to measure the expression of lipogenic genes in liver tissues.

MRNA expression in PPARγ, C / EBPα, FABP4, FAS, Me1, Acaca, Acyl and Slc25a1 was found to be decreased in liver tissues of lgals3 ^{- / -} mice (FIG. In addition, analysis of liver tissues of lgals3 ^{- / -} mice performed using real-time RT-PCR analysis revealed that mRNA expression of PPARγ, C / EBPα, FABP4 and FAS was relatively low (FIG. These results suggest that galectin-3 may affect the genes that produce fat in the liver by stimulating systemic obesity.

Claims (12)

delete A biological composition for enhancing the activity or expression of peroxisome proliferator activated receptor gamma including galectin-3 as an active ingredient in vitro. delete delete A biological composition for inhibiting the expression of adipose triglyceride lipase (ATGL) by galectin-3 as an active ingredient in vitro. 6. The method of claim 5,
Wherein the triglycerolytic enzyme reduces the size of adipocytes. delete A biological composition for enhancing the expression of a fatty liver producing gene, comprising galectin-3 as an active ingredient in vitro. 9. The method of claim 8,
The fat-growing gene is at least one selected from among peroxidase proliferator-activated receptor gamma (PPARγ), ccaat-enhancer-binding protein alpha (C / EBPα), fatty acid binding protein (FABP4) and fatty acid synthase &Lt; / RTI &gt; A method for reducing the activity or expression of peroxisome proliferator-activated receptor gamma (PPARγ) by knocking galectin-3 in a mammal other than a human. A method for reducing expression of a fatty liver producing gene by knocking out galectin-3 in a mammal other than a human. Increasing the expression of adipose triglyceride lipase (ATGL) by knocking out galectin-3 in mammals other than humans.

Images