TW201422226A - Use of PPAR-α activator for enhancing ATGL protein expression and reducing fat deposition - Google Patents

Abstract

The present invention relates to using peroxisome proliferator-activated receptor alpha (PPAR-α) activator for enhancing adipose triglyceride lipase (ATGL) protein expression, in which the enhancement of ATGL expression is achieved by enhancing the ATGL transcription activity by stimulating FoxO1 transposition to enter the cell nucleus. The present invention further relates to a PPAR-α activator for reducing body fat deposition through the PPAR-α/AMPK/FoxO1/ATGL signaling pathway.

Description

PPARα activator increases ATGL protein performance and reduces fat accumulation

The present invention relates to the use of PPARα activators to increase the expression of ATGL proteins, and more particularly to the use of PPARα activators to penetrate PPAR-α/AMPK/FoxO1/ATGL message pathways in organisms to reduce serum fat accumulation.

Due to social progress, modern people's diet is refined and the amount of exercise is insufficient. The problem of obesity in Chinese people is becoming more and more serious, and the related diseases are followed. The metabolic syndrome is a syndrome derived from obesity, including the medium-sized body and fasting blood sugar. Excessively high, triglycerides exceed the standard value, etc., the proportion of the disease is increasing and the age layer is gradually decreasing. The metabolic syndrome is derived from many chronic diseases such as hypertension, type 2 diabetes, hyperlipidemia, etc. It is the suffering of the patient itself, and it is also a burden on the cost of social medical care.

The Peroxisome Proliferator Activated Receptor type alpha (PPAR-α) family belongs to the nuclear receptor superfamily, and such members include estrogen, thyroxine, vitamin D, and glucocorticoid receptors. The PPARs family includes three subtypes: PPAR-α, PPAR-β, PPAR-γ, which play an important role in energy metabolism (Tyagi et al., J Adv Pharm Technol Res 2 : 236-240, 2011). PPARα is expressed in multiple organs and is highly expressed in the liver, which can enhance the β-oxidation of fatty acids in the mitochondria and provide energy to the surrounding tissues (Lefebvre et al., J Clin Invest 116 :571-580, 2006). The literature indicates that PPAR-α is involved in lipid and lipoprotein metabolism regulation, thus reducing dyslipidemia and metabolic syndrome (Griese et al., J Mol Biol 361 , 140-152, 2006).

Fenofibrate is a fibric acid derivative. It is known that its mechanism of regulating blood lipids is to activate PPAR-α (Frazier-Wood et al., Pharmacogenomics J, 2012), and fenofibrate can activate lipoprotein lipids. Lipoporotein lipase (LPL) and apoprotein (apo) C-III production, lipid lysis and excretion of triglyceride-rich particles in blood atherosclerosis, fenofibrate can reduce very low density Lipoprotein (Very Low Density Lipoporotein, VLDL), Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL) (Yetukuri et al., PLoS One 6 , e23589, 2011), Among them, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are common indicators in patients with high-risk coronary artery disease. In clinical trials, treatment with 200 mg/day fenofibrate can reduce total body temperature by 20-25%. Cholesterol, 40-55% triglyceride, increased 10-30% high-density lipoprotein (HDL), due to its obvious efficacy in low-density lipoprotein (LDL) and triglyceride (TG) (Bijland et al ., J Biol Chem 285 , 25168-25175, 2010), with fenofibr It is beneficial to treat patients with high cholesterol or patients with type 2 diabetes (Davis et al., Diabetologia 54 , 280-290, 2011).

Thus, the present invention takes fenofibrate as an example to investigate the mechanism of action of PPARα activator to promote β-oxidation of intracellular free fatty acids.

Accordingly, the present invention provides a composition for promoting the expression of an Adiopose Triglyceride Lipase (ATGL) protein comprising a PPARα activator as an active ingredient. In one embodiment of the invention, the PPARα activator is fenofibrate.

The PPARα activator of the present invention increases the transcriptional activity of ATGL by stimulating the translocation of FoxO1 into the nucleus. Moreover, the PPARα activator of the present invention increases the ATGL expression by activating the PPAR-α and AMPK message pathways, and promotes lipolysis and β-oxidation of free fatty acids to reduce body weight and serum triglyceride. The amount, as well as the accumulation of fat in the liver and muscles.

The composition comprising the PPARα activator of the present invention may be formulated into a pharmaceutical composition or may be used as a food additive.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention. In view of the various embodiments of the present invention, the various instruments, devices, methods, and related results described below are not intended to be limited to the scope of the present invention.

Example Fenofibrate regulates the performance of lipid metabolism-related proteins and reduces lipid droplet accumulation

First, in order to confirm whether Fenofibrate can affect the lipid reduction phenomenon through the regulation of ATGL, the differentiated rat myotubes (C2C12 myotubes) were given fenofibrate for 24 hours, and then the Western blotting method was used to observe the performance of ATGL protein. It was found that ATGL protein showed a dose-dependent increase and was statistically significantly different (Fig. 1A).

Next, the cells were cultured in a medium containing high glucose (50 mM glucose) to promote intracellular fat accumulation, and then treated with fenofibrate for 24 hours to observe fenofibrate for fatty acid synthase (FAS) and cholesterol regulating protein associated with lipid breakdown. The effect of the amount of expression of (Sterol regulatory element-binding protein-1c, SREBP-1c). It was found that fenofibrate inhibited the expression of fatty acid synthase and cholesterol-regulating protein, and there was a statistically significant difference (Fig. 1B).

In order to further confirm the effect of fenofibrate on lipid accumulation, the cells were cultured in high glucose medium (50 mM glucose) to promote intracellular fat accumulation, and then treated with fenofibrate for 24 hours, followed by oil red O staining. It was found that in the case of high sugar, the accumulation of oil droplets in the cells was obvious; and after 24 hours of treatment with 10, 30, 100 μM of fenofibrate, it was found that the lipid oil droplets accumulated with concentration. The increase and decrease (Fig. 2) indicate that fenofibrate can reduce the accumulation of intracellular triglycerides even in the case of high sugar (50 mM).

Fenofibrate increases AMPK, ACC phosphorylation, and palmitic beta oxidation

The AMPK message pathway is a natural response to reducing dyslipidemia and insulin resistance in vivo, and thus the present invention further confirms the effect of fenofibrate on AMP-activated kinase (AMPK) and its downstream protein Acetyl-CoA carboxylase (ACC). Treatment with different concentrations of fenofibrate revealed that AMPK phosphorylation and ACC phosphorylation gradually increased with increasing concentrations, and there was a statistically significant difference (Fig. 3A).

Since fenofibrate is a known PPAR-α activator, the C2C12 myotube cells were pre-treated with the AMPK inhibitor Compound C (20 μM) and the PPAR-α inhibitor GW9662 (10 μM) for 1 hour. The fenofibrate treatment was carried out for 1 hour, and the effects on AMPK phosphorylation and ACC phosphorylation were observed. It was found that fenofibrate increased AMPK and ACC phosphorylation, while both AMPK inhibitor and PPAR-α inhibitor inhibited fenofibrate-induced AMPK and ACC phosphorylation (Fig. 3B).

From past studies, we can know that when inhibiting ACC activity, it can increase the performance of carnitine palmitate transferase-1 (CPT1), which is related to fat β oxidation, and promote the combustion of free fatty acids to generate energy. Therefore, in this experiment, we use After 24 hours of treatment of C2C12 myotubes with different concentrations of fenofibrate, it was found that fenofibrate increased CPT1 expression (Fig. 4A) and fatty acid oxidation in a concentration-dependent manner (Fig. 4B).

From the above results, it is inferred that the PPARα activator fenofibrate loses its activity by stimulating AMPK activation and phosphorylating its downstream protein ACC. Originally, Acetyl-CoA will become Malonyl-CoA via ACC, while Malonyl-CoA will inhibit CPT1 protein on the mitochondrial membrane, causing free fatty acids to pass through CPT1 and enter the mitochondrial body to produce energy, so once ACC Because of the inactivation of phosphorylation, Acetyl-CoA cannot form Malonyl-CoA, and CPT1 is not inhibited, and free fatty acids can be transferred into the mitochondria for β-oxidation to generate energy. In summary, AMPK phosphorylation will affect The protein ACC inactivates its phosphorylation, which in turn causes the free fatty acids decomposed by ATGL to be carried into the mitochondria, and the β-oxidation burns to generate energy without accumulating in the body to cause insulin resistance.

Both PPAR-α and AMPK inhibitors inhibit the effect of Fenofibrate on reducing lipids

In order to further confirm the relationship between AMPK, PPARα and ATGL, differentiated myotube cells were pretreated with Compound C (20 μM) or GW9662 (10 μM) for 1 hour, and then 100 μM of fenofibrate was added for 24 hours. The experimental results showed that both inhibitors inhibited the ATGL performance caused by fenofibrate, and there were statistically significant differences (Fig. 5A). On the other hand, we used high glucose medium to culture myotubes to differentiate them to induce lipid-related protein FAS and SREBP expression. fenofibrate (100 μM) treatment for 24 hours reduced the protein expression of FAS and SREBP, and the inhibitor (Compound C) Or GW9662) pretreatment, the FAS and SREBP expressions suppressed by fenofibrate will be recovered (Fig. 5B), and the oil red O staining results also show that after the addition of the inhibitor, the fenofibrate inhibits the accumulation of oil droplets. Will be replied (Fig. 5C), so it is inferred from the above results that fenofibrate reduces the effect of lipid accumulation through the PPARα/ATGL pathway.

Fenofibrate causes FOXO1 to enter the nucleus and bind to the ATGL promoter

The transcription factor FoxO1 plays a key role in regulating the body's energy balance. The entry and exit of the nucleus is negatively regulated by the insulin/PI3K message pathway, and the transcription of the target gene is stopped ( Zheng et al., Mol Pharmacol 62: 225-233 , 2002 ). In the past, FoxO1 entered the nucleus to regulate the performance of ATGL in adipocytes, and insulin inhibited this phenomenon ( Smimova et al., EMBO Rep 7 , 106-113 , 2006 ). Therefore, this experiment used immunofluorescence staining to observe the entry and exit of FoxO1 into the nucleus. Differentiated C2C12 myotube cells were treated with 100 nM insulin, 100 μM fenofibrate (Feno) and 20 μM Ly294002 (Ly) for 3 hours. The fixed cells were stained with FoxO1 antibody, and Alexa Fluor 555-conjugated donkey anti-rabbit antibody, and observed under a fluorescent microscope (red).

It was found that treatment with insulin caused FoxO1 to move out of the nucleus, whereas treatment with fenofibrate or PI3K inhibitor (LY294002) revealed that FoxO1 would enter the nucleus (Fig. 6A). If pretreated with AMPK inhibitor (Compound C) and PPAR-α inhibitor (GW9662) and then treated with fenofibrate, it was found that FoxO1 was distributed outside the nucleus (Fig. 6B), so the translocation of FoxO1 was inferred from the results. Will be regulated by the PPAR-α/AMPK pathway.

In addition, it was confirmed by chromatin immunoprecipitation whether FoxO1 binds to the ATGL promoter. The differentiated C2C12 cells were treated with serum-free (Starvation), insulin (insulin), and Fenofibrate (Feno) for 4 hours, respectively, and then the protein was seized and precipitated by anti-FoxO1 antibody, and the supernatant was subjected to ATGL promoter primer: (5) '-ATCTTTAAAAGGTACCTAAGCTGGGGGCCTC-3' and 5'-AAGTCCAGGTCCTCGAGATGTGCCCAAGTACC-3') A PCR reaction was carried out to amplify a 221-bp DNA fragment located between the promoter nucleotides -1004 to _1225. From the electrophoresis results of the PCR products, it was found that fenofibrate treatment increased FoxO1 binding to the ATGL promoter (Fig. 7A).

Further confirmation was performed using a secreted embryonic alkaline phosphatase (SEAP) reporter protein assay. C2C12 cells were treated with 100 μM of Fenofibrate for 96 hours, 300 μl of the culture solution was pipetted into 1.5 ml eppendorf, centrifuged (13,000 rpm) for 5 minutes, and then 200 μL of the supernatant was aspirated to a new 1.5 ml eppendorf, and a dilution buffer of 30 μl was added. Liquid (SEAP detection kit, BD Biosciences) to 96 well plate, then add 10 μl of the supernatant to the 96 well plate and fully evenly. Place it in an oven at 60 ° C for 30 minutes, then take it out and let it cool at room temperature. Then, 40 μl of the assay buffer was added for 5 minutes at room temperature, and then 40 μl of dilution CSPD was added in the dark, and after 10 minutes at room temperature, the luminescence value of the reaction was measured using an ELISA data reader. Experimental results show that treating fenofibrate will promote Foxol binding to The ATGL promoter was added to increase the activity of the SEAP reporter protein (Fig. 7B).

In addition, it has been reported in the literature that FoxO1 increases the transcriptional activity of the controlled gene by deacetylation. Therefore, we used immunoprecipitation to differentiate the well-differentiated C2C12 cells with AICAR (AMPK activator) and fenofibrate (Feno). After treatment for 3 hours, the protein was precipitated using an anti-FoxO1 antibody, and the antigen-antibody reaction of the Ac-Lys antibody was detected using a Western blot method. The results of the experiment showed that treatment with fenofibrate did reduce the acetylation of FoxO1 (Fig. 8).

Based on the above results, we infer that the PPARα activator fenofibrate will translocate FoxO1 into the nucleus through AMPK, and FoxO1 entering the nucleus will deacetylate and bind to the ATGL promoter, thereby increasing the transcriptional activity of the ATGL gene, and Increase ATGL protein performance.

Fenofibrate improves body weight, biochemical value and fat accumulation in db/db diabetic rats

Leptin is a hormone secreted by adipose tissue. It functions as an afferent message to the brain's satiety center, and its mutant mice are divided into two types: one is leptin itself ( ob/ob ), and the other is leptin. Receiver is missing ( db/db ) (Tartaglia et al., 1995). These mutant mice exhibit endocrine disorders including diabetes, fatty liver, enlarged liver, infertility, and chills. Among them, fatty liver is one of the indicators for the increase of fat synthesis rate in the liver, so this mutant mouse can be used as an animal model of diabetes.

In order to investigate whether fenofibrate is consistent with the above-mentioned in vitro test results in the animal model, 20-week-old db/db mice were used for oral administration of fenofibrate (100 mg/kg) or the control vehicle for four weeks. During the experiment, the body weight of the mice was measured weekly. At the end of the four-week experimental period, the mice were sacrificed and their accumulation and weight of abdominal fat were observed. In addition, the serum biochemical values, including triacylglycerol, cholesterol, insulin, HDL, LDL, AST, and ALT, and biochemical values of whole blood including HbA1c and glucose concentrations were analyzed by Boochem-Immuno fully autoanalyzer (Brea, CA). Analyze.

From the results listed in Table 1 below, it was found that the fenofibrate group was significantly smaller than the control group, and the body weight was reduced by 12.9% compared with the control group, and visceral fat. Significant differences in the amount of abdominal fat and gonadal fat were 70.7% and 18.8%, respectively, compared with the control group (Table 1). In terms of metabolic parameter values (see Table 2 below), serum triglyceride lipids were significantly reduced compared with the control group, but there was no significant difference in cholesterol content. The liver weight of the fed fenofibrate group was not different from that of the control group. However, serum biochemical values AST and ALT were significantly different from the control group (Table 2).

To confirm whether the in vivo test is consistent with the performance of the protein related to the in vitro test, the liver and muscle tissues of the animal were taken and ground, and then centrifuged at 13,000 rpm for 30 minutes, and the supernatant was taken for analysis of protein expression by Western blotting. The results of the experiment showed that fenbutibrate tissue animals were increased in AMPK and ACC phosphorylation, and ATGL protein expression was increased, while FAS performance was decreased (Fig. 9A). By observing the results of ATGL in muscle tissue sections by immunohistochemical staining, it was found that the ATGL protein was significantly increased in the animal group fed with fenofibrate compared with the control group (Fig. 9B, brown point).

The liver and muscle tissue sections were taken for oil red O staining and Sudan III staining, and the oil droplet content was further observed. From the experimental results, it was found that the liver and muscle tissues of the Fenofibrate-fed animal group showed a significant decrease in oil droplet accumulation in oil red O staining and Sudan III staining compared with the control group (Fig. 9C).

Based on the above experimental results, it can be inferred that the PPARα activator fenofibrate reduces lipid accumulation through the PPAR-α/AMPK/ATGL message pathway. In immunofluorescence staining experiments, fenofibrate can cause FoxO1 translocation into the nucleus and inhibit AMPK. Treatment with PPAR-α inhibitors will motivate FoxO1 is removed from the nucleus.

In addition, it has been found from the foregoing experiments that administration of fenofibrate after treatment with insulin (100 nM) does not affect the nuclear appearance of FoxO1, so it can be speculated that fenofibrate affects the entry of FoxO1 into the nucleus, but this phenomenon is not affected by insulin. influences. Immunoprecipitation and SEAP assay confirmed that Foxo1 binds to the promoter of ATGL gene after treatment with fenofibrate, and deacetylation of Foxo1 increases the transcriptional activity of ATGL protein and reduces intracellular fat. Stacking situation.

Other specific aspects

All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.

From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.

Figure 1 shows the effect of fenofibrate on the lipid metabolism related protein ATGL (A), and the performance of (B) and triglyceride accumulation.

Figure 2 is a microscopic image observed by oil red O staining after treatment with fenofibrate at different concentrations (10, 30, 100 μM) for 24 hours. The original magnification is 1000 x.

Figure 3 shows that Fenofibrate activates AMPK signaling and enhances fatty acid beta oxidation.

Figure 4 shows that fenofibrate increases CPT1 expression (A) in a concentration-dependent manner, as well as fatty acid oxidation (B).

Figure 5 shows that in cell mode, inhibition of PPARα and AMPK by drugs attenuates the fat reduction produced by fenofibrate.

Figure 6 shows that fenofibrate regulates FoxO1 translocation into the nucleus of C2C12 cells (A), and this regulation is inhibited by compound C and GW9662 (B). The fixed cells were stained with a FoxO1 antibody and Alexa Fluor 555-conjugated scorpion anti-rabbit antibody (red), and the DAPI dye (red) was incorporated into a mounting solution.

Figure 7 shows the use of Chip assay (A) and SEAP report assay (B) to show that fenofibrate promotes Foxo1 binding to the ATGL promoter.

Figure 8 shows that immunoprecipitation analysis showed that fenofibrate treatment reduced the acetylation of FoxO1, and the degree of oximation of FoxO1 was measured by Western blot analysis.

Figure 9 is a section of muscle and liver tissue taken from fenofibrate-treated and untreated db/db mice, stained with Oil Red O and Sudan III, respectively, to detect oil droplets in the tissue.

<110> Zhongtai University of Science and Technology

<120> Adiopose Triglyceride Lipase (ATGL) Promoter Sequence for SEAP Analysis of Promoter Activity

<160> 2

<170> PatentIn version 3.2

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<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> ATGL forward primer

<400> 3

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence

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<223> ATGL reverse primer

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Claims (8)

A composition for promoting the expression of an Adiopose Triglyceride Lipase (ATGL) protein comprising a PPARα activator as an active ingredient. The composition of claim 1, wherein the PPARα activator is fenofibrate. The composition of claim 1, wherein the increased ATGL protein expression increases ATGL transcriptional activity by stimulating FoxO1 translocation into the nucleus. The composition according to the first aspect of the patent application is for lowering the triglyceride content in serum. The composition according to the first aspect of the patent application is for reducing fat accumulation in the liver and muscle. The composition according to claim 4 or 5, which promotes lipolysis through the PPAR-α/AMPK/FoxO1/ATGL message pathway. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, diluent or excipient. The composition according to claim 1 of the patent application is a food additive.