JP2011019615A - Method for inducing expression of ucp1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inducing expression of UCP1 which does not require a treatment by a physician or administration of medicaments. <P>SOLUTION: The method for inducing expression of UCP1 is characterized by triggering an increase in the expression of UCP1 by imparting a thermal stimulus to a local or to the entire body to increase heat-producing ability. An energy expenditure by heat like this suppresses fat accumulation in a living body since burning of surplus fat and sugar is accelerated and leads to the prevention or improvement of various pathological conditions and diseases caused by obesity or fat accumulation. The application of a thermal load to regions (a body part, an organ, or tissues) where the increase in the expression of UCP1 is desired in a human or an animal or to the entire body from the outside is preferred as the thermal stimulus. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for inducing the expression of UCP1 (Uncoupling Protein 1) involved in fat burning and heat production.

  Uncoupling protein (UCP) has long been known as a protein (UCP1) present in brown adipose tissue of small hibernating mammals, but its homolog molecule UCP2 was discovered in humans in 1997 ( Non-patent document 1). Thereafter, UCP3 and UCP4 were cloned one after another (Non-Patent Documents 2 and 3), and it was shown that homologous proteins exist in amoeba and fungi (Non-Patent Documents 4 and 5). UCP is localized in mitochondria, and its function is uncoupling of oxidative phosphorylation as the name suggests (Non-patent Document 6). That is, the electron transport system and ATP synthesis in mitochondria are closely coupled by a proton concentration gradient through the inner membrane. UCP is a six-transmembrane channel that short-circuits and eliminates this proton concentration gradient. When activated, the chemical energy of the oxidized substrate is not used for ATP synthesis but converted to heat. Because of this function, UCP is also called a heat-producing protein and is considered to be involved in body temperature regulation and energy metabolism.

  Fat is mainly responsible for energy storage in the living body, but it is a part called white adipose tissue (WAT) that does this. On the other hand, fat that consumes energy as heat also exists in the living body, and this is called brown adipose tissue (BAT).

  UCP1, UCP2, and UCP3 are expressed in the adipose tissue. Among these, UCP1 is characteristically expressed in BAT and is known to be a central molecule for heat energy production in BAT. When a cold stimulus (4 ° C.) is given to a rodent animal such as a rat, non-tremor heat production centering on BAT occurs, which is due to UCP1 whose expression is strongly induced by the cold stimulus. In other words, it is believed that under low temperature conditions, the production of thermal energy by UCP1 is enhanced, and BAT is considered to act as a living body warming device. In fact, body temperature cannot be maintained under low temperature conditions in UCP1 knockout mice. (Non-Patent Documents 7 and 8).

  In addition, UCP1 is implicated in energy consumption as heat because its expression increases during overeating and causes dietary heat production. In fact, when mice with reduced expression of UCP1 were produced, the heat production ability during cold exposure was reduced, and a decrease in normal oxygen consumption (energy consumption) was also observed, and the body fat mass was doubled. From these results, UCP1 is considered to play a role of consuming surplus energy by heat production (Non-patent Document 9). Conversely, in the transgenic mice in which UCP1 is overexpressed specifically in adipocytes, the amount of fat is decreased, and this is thought to be due to increased energy consumption by UCP1. (Non-Patent Document 10). Furthermore, in vitro tests also suggest that UCP1 increases energy consumption by heat production. Originally, it has been obtained that when UCP1 is overexpressed in liver cells with very low UCP1 expression, ATP production is decreased. This is because the increase in UCP1 results in oxidative phosphorus in liver cells. It has been reported that a part of the oxidation reaction is transferred to the heat production pathway (Non-patent Document 11). From the above, it is considered that increasing the gene expression level of UCP1 promotes heat production, and as a result, increases the consumption of surplus energy and leads to suppression of fat accumulation and obesity.

  Moreover, the free fatty acid cut out from neutral fat (triglyceride) is mainly utilized for energy consumption by such UCP1, and this is converted into heat energy. Therefore, it is considered that an increase in UCP1 leads to promotion of fat burning in the living body (Non-patent Document 12).

  UCP2 is widely expressed throughout the body, such as fat, skeletal muscle, lungs, and heart. Recently, it is considered that one of the roles is to suppress the generation of active oxygen and prevent oxidative damage in vivo. (Non-patent Document 12). Although the degree of contribution to energy consumption as heat is often unclear, the results of human analysis indicate that UCP2 gene expression in adipose tissue is inversely proportional to fat mass (Non-patent Document 13), obesity It has been reported that the amount of UCP2 in the skeletal muscles of the elderly has a significant correlation with the body fat percentage (Non-patent Document 14), and these results suggest that UCP2 is involved in obesity.

  UCP3 is highly expressed in skeletal muscle, and is also expressed in cardiac muscle and brown fat, and recently, like UCP2, it is thought to play a role of preventing oxidative damage to the living body (non-patent document). 12). Regarding the involvement of UCP3 in obesity, studies on transgenic mice that highly express UCP3 have reported that they are leaner than in control mice and that glucose metabolism is improved (Non-patent Documents). 15). In addition, in a study targeting Pima Indians, it was reported that a negative correlation was found between the expression level of UCP3 and BMI, and a positive correlation was found in the resting metabolic rate (Non-patent Document 16). These facts suggest that UCP3 is involved in a part of energy consumption and metabolism.

  As described above, UCP is considered to be involved in obesity and energy metabolism. In other words, the increase and activation of UCP will improve energy metabolism, and various pathologies and diseases (insulin resistance, type 2 diabetes, hypertension, hyperlipidemia, ischemic heart, etc. caused by obesity and fat accumulation) It is thought to lead to prevention and improvement of diseases, etc.).

  In fact, fucoxanthin, which is abundant in seaweeds such as seaweed, has been reported to activate UCP1 in fat cells and promote fat burning (Non-patent Document 17), and fucoxanthin is effective as an anti-obesity active agent Has been reported (Patent Document 1). In addition, it has been reported that conjugate unsaturated isomerized polyunsaturated fatty acids exhibit an anti-obesity / visceral accumulated fat reducing function by activating UCP (Patent Document 2).

  In addition to oral ingestion, a slimming skin external preparation that activates UCP by applying it to the skin (Patent Document 3), and a fragrance diet composition that UCP is activated by odor stimulation (patent) Reference 4).

  UCP is involved not only in obesity but also in other lipid metabolism-related diseases because of its fat burning action. In fact, the dalmagiku extract known to have an anti-obesity action activates UCP, and thus has been shown to be effective in preventing and improving hyperlipidemia and cardiovascular diseases (Patent Literature) 5).

  From the above, it is speculated that increasing or activating UCP is useful for the prevention and improvement of hyperlipidemia and diabetes, including obesity, visceral fat syndrome, and lifestyle-related diseases caused by them. The In addition, as a method for improving / preventing such symptoms, it has been desired to establish a method for controlling UCP simply and safely without using drugs.

JP 2008-280281 A JP 2000-144170 A Japanese Patent Laid-Open No. 2003-63977 JP 2009-23964 A Special table 2008-533196

Fleury C, et al. (1997) Nat Genet. 15: 269-272 Boss O, et al. (1997) FEBS Lett. 408: 39-42 Mao W, et al. (1999) FEBS Lett. 443: 326-330 Jarmuszkiewicz W, et al. (1999) J Biol Chem. 274: 23198 -23202 Jarmuszkiewicz W, et al. (2000) FEBS Lett. 467: 145-149 Ricquier D, Bouillaud F. (1997) Prog Nucleic Acid Res Mol Biol. 56: 83-108 Lowell BB, Spiegelman BM. (2000) Nature. 404: 652-660 Enerback S, et al. (1997) Nature. 387: 90-94 Lowell BB, et al. (1993) Nature. 366: 740-742. Kopecky J, et al. (1995) J Clin Invest. 96: 2914-2923. Gonzalez-Muniesa P, et al. (2005) J Physiol Biochem. 61: 389-393 Masayuki Saito, Noriyasu Sasaki (1996) Experimental Medicine. 14: 222-227 Kogure A, et al. (1998) Diabetologia. 41: 1399 Echtay KS. (2007) Free Radic Biol Med. 43: 1351-1371. Pinkney JH, et al. (2000) J Clin Endocrinol Metab. 85: 2312-2317. Simoneau JA, et al. (1999) Int J Obes Relat Metab Disord. 23: S68- S71 Clapham JC, et al. (2000) Nature. 406: 415-418

  The present invention relates to a method for inducing UCP1 in a simple, safe and effective manner and promoting heat production and fat burning.

  The present inventors examined a UCP1 expression control method that can be easily carried out with a small load on the living body, and found that the expression of UCP1 can be induced by applying a thermal stimulus at a safe temperature to the living body.

That is, the present invention relates to the following inventions 1) to 4).
1) A method for inducing UCP1 expression, which comprises applying thermal stimulation locally or systemically.
2) A method for promoting the production of heat in adipose tissue, characterized by applying a thermal stimulus locally or throughout the body.
3) A method for promoting fat burning in adipose tissue, characterized by applying thermal stimulation locally or systemically.
4) The method according to 1) to 3) above, wherein the thermal stimulation is a thermal load at 40 to 43 ° C. for 30 to 60 minutes.

The method of the present invention has a small burden on a living body, does not require treatment by a doctor and does not require drug administration, and can be easily implemented by ordinary citizens, estheticians, and the like. Therefore, according to the present invention, it is possible to induce UCP1 expression in a local or whole body adipose tissue easily and safely.
In addition, through the production of heat due to increased UCP1 expression, it is possible to promote consumption of surplus energy, to reduce local fat and systemic fat such as subcutaneous fat, and to develop pathological conditions caused by fat accumulation Can prevent and improve.

The figure which shows the time-dependent expression level of UCP1 in BAT when a thermal stimulus is added The figure which shows the time-dependent expression level of UCP1 in WAT when a thermal stimulus is added

  The method for inducing UCP1 expression of the present invention is intended to prevent or improve symptoms caused by a decrease in local subcutaneous fat or fat accumulation through an increase in energy consumption by heat production. The purpose is mainly for the purpose of beauty and dieting, and does not include so-called medical practice.

  As shown in Examples below, by applying a thermal stimulus to mice, the gene expression of UCP1 in BAT and WAT increases. Therefore, it is possible to promote heat production in the adipose tissue in the living body, that is, promote fat burning by thermal stimulation.

  As the thermal stimulation used in the present invention, it is preferable to apply a thermal load from the outside to a local area of human or animal, that is, a site (organ / organ / tissue) in which UCP1 expression increase is desired, or the whole body. In the case of humans, it is preferable to apply a thermal load of at least 38 ° C. or more for 30 minutes or more, preferably 40 to 43 ° C. for 30 to 60 minutes.

  The thermal stimulation is performed at least once a week, preferably once every three days, more preferably every day, and can be performed by repeating this thermal stimulation one or more times.

  The thermal stimulation may be performed using a thermal load means capable of inducing the expression of UCP1, but preferably the above-described thermal load is applied using an existing thermal load device or instrument.

  As the heat load device / apparatus, any heat load device or tool may be used as long as it can apply a thermal stimulus to a local or whole body of a human or an animal and its heat amount and temperature can be controlled.

The heat source of the thermal load device / apparatus is usually gas, electricity or the like, and examples of the heat transfer means produced by the heat source include pipes and electric wires. Examples of the medium for transferring such heat to humans, animals and the like include gases, liquids, solids having heat transfer properties, and the like. For example, in the case of an air incubator that requires a power source as a thermal load device, heat generated by electricity may be conveyed by electric wires or pipes, and heat may be transmitted to the living body using gas as a medium. Moreover, ultrasonic vibration, a thermal load by microwaves, and the like can be used as the thermal load method of the present invention.
Also, commercially available thermal warmers that generate heat, thermal patch, steam thermal patch, and the like can be used.

  As test animals, C57BL / 6J mice (6 weeks old pupa) (CLEA Japan) were used. One week before the start of the test, the animals were preliminarily reared under a light / dark cycle (light period; AM7: 00 to PM7: 00) of room temperature 23 ± 2 ° C., humidity 55 ± 10%, and 12 hours. During the breeding period, all mice were allowed to freely eat the CE-2 solid food (Clea Japan) and tap water freely.

  After one week of preliminary breeding, C57BL / 6J mice were divided into two groups, a non-warming group and a warming group, so that the average body weights were equal. The mice in the warming group were given a thermal stimulus by placing them in a 41 ° C. heat chamber for 1 hour, dissected 12, 24, 36, and 48 hours after the stimulus (N = 6), and interscapular white fat ( WAT) and interscapular brown fat (BAT). The non-warming group was used as a control, and no thermal stimulation was given, and dissection was performed after 0, 12, 24, 36, and 48 hours (N = 6), and WAT and BAT were collected. In order to avoid dehydration caused by thermal stimulation, the mice in the warming group were allowed to freely take water at 41 ° C. in the heat chamber. The collected adipose tissue was homogenized in 1 ml of ISOGEN (Nippon Gene), immediately placed in liquid nitrogen, and stored frozen.

Preparation of RNA from frozen tissues was according to ISOGEN manual.
The prepared RNAs were prepared at the same concentration, heat-treated at 65 ° C. for 10 minutes, and used after rapid cooling. For reverse transcription, RNA corresponding to 125 ng was used, and 20 μl of reaction solution (1 × PCR buffer II (Roche), 5 mM MgCl 2 (Roche), 1 mM dNTP mix (Takara), 2.5 mM Oligo d (T) 18 mRNA) primer (New England Biolabs), 1 U / μl RNase inhibitor (Takara)) was prepared. The reaction was performed at 42 ° C., 60 minutes → 52 ° C., 30 minutes → 99 ° C., 5 minutes → 4 ° C., and the obtained cDNA was stored at −20 ° C. until use. In addition, as a standard for quantitative PCR, 500 ng of RNA was used, and reverse transcription was performed in the same reaction system.

Quantitative PCR was performed with the ABI PRISM7500 Real-time PCR System (Applied Biosystems) using the cDNA obtained by the reverse transcription reaction as a template. Quantification was performed based on a standard curve prepared using a standard cDNA diluted in 7 steps as a standard. The obtained analysis results were corrected by using the expression level of 36B4 as an internal standard and expressed as a relative mRNA expression level. The reaction solution was prepared as follows: Power SYBR Green PCR Master Mix (Applied Biosystems) 25 μl, 100 μM Forward primer 1 μl, 100 μM Reverse primer 1 μl, dH2O 22 μl, cDNA 1 μl. The temperature conditions for quantitative PCR were 50 ° C. for 2 minutes and 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.
The primer sequences used in quantitative PCR are shown below.

  The test results were obtained by calculating a relative average with the gene expression level of the non-warming group at each point 0, 12, 24, 36, and 48 hours after thermal stimulation as 1, and calculating an average value ± standard deviation (Average (Ave) ± Standard deviation (SD)). Moreover, the significant difference test performed t-test between the non-warming group and the warming group of each time point, and the thing with p value less than 0.05 was considered to have a statistical significant difference.

  When heat stimulation was given to mice, the gene expression of UCP1 in BAT began to increase 12 hours after the stimulation, and a significant increase in expression was observed after 24 hours (FIG. 1). Furthermore, the high expression state of UCP1 was maintained even 48 hours after the thermal stimulation. From this, it became clear that thermal stimulation increases the expression of UCP1 in BAT. Therefore, it is considered that thermal stimulation is effective for enhancing production of thermal energy.

  Heat stimulation also increased UCP1 gene expression in WAT (FIG. 2). A significant increase in the UCP1 gene was observed 12 hours after the thermal stimulation, and this significant increase was also observed after 48 hours. From this, it was shown that thermal stimulation promotes the increase in UCP1 expression and enhances the production of thermal energy even in WAT.

  From the above, it was shown that thermal stimulation increases the expression of UCP1 and improves the heat production ability. Such energy consumption by heat promotes the burning of excess fat and sugar, leading to suppression of fat accumulation in the living body. Therefore, it is considered that the thermal stimulation that promotes the induction of UCP1 expression is effective in controlling obesity and lifestyle-related diseases.

Claims (4)

  A method for inducing UCP1 expression, comprising applying thermal stimulation locally or systemically.   A method for promoting heat production in adipose tissue, characterized by applying a thermal stimulus locally or systemically.   A method for promoting fat burning in adipose tissue, characterized by applying a thermal stimulus locally or systemically.   The method according to claim 1, wherein the thermal stimulation is a thermal load at 40 to 43 ° C. for 30 to 60 minutes.

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