JP2007039367A - Composition for promoting cytodifferentiation and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technique for promoting differentiation of fat cells useful for treatment of diabetes and amelioration of sugar metabolism. <P>SOLUTION: The composition comprises leucine and promotes differentiation of fat cells. Leucine has ameliorating effect on sugar metabolism at a solid level. Consequently, leucine is extremely effective for treating, ameliorating or preventing diabetes. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a composition that promotes differentiation of adipocytes that can be used as a pharmaceutical product, an insulin resistance improving composition, a composition for treating diabetes, foods and drinks containing these compositions, and the like.

  Diabetes mellitus is a disease in which genetic factors and environmental factors are complicatedly involved. Diabetes can be broadly classified into type 1 diabetes caused by a lack of absolute amount of insulin and type 2 diabetes caused by a lack of action of insulin. In addition, type 2 is overwhelmingly common in the incidence of diabetes.

  In recent years, the number of patients with diabetes, particularly type 2 diabetes, is increasing rapidly. Diabetes is known to cause complications such as arteriosclerosis, neuropathy, and infections, and the health problems are very large. Diabetes is also becoming a social problem such as an increase in medical costs.

  It is believed that the lack of action of insulin, which is the cause of type 2 diabetes, is largely due to a decrease in the response of peripheral tissues, particularly adipose tissues, to insulin (aggravation of insulin resistance).

To date, thiazolidine-based drugs such as thiazolidine derivatives are known to be effective in improving insulin resistance. Thiazolidine drugs are agonists of peroxisome proliferator-activated receptor γ (PPARγ), and differentiate preadipocytes into adipocytes via PPARγ. Since adipocytes are abundantly expressed with insulin receptors and sugar transport carriers (GLUT4), it is considered that insulin resistance is improved by thiazolidine drugs.
S. Nishitani et al., Am. J. Physiol Gastrointest Liver Physiol. 288: G1292-G1300, 2005 Luc JC Van Loon et al., Diabetes Care, 26: 625-630, 2003

  However, thiazolidine drugs are problematic because they have side effects such as hepatitis, edema, and excessive obesity due to increased appetite.

  The present invention has been made in view of the above-mentioned conventional problems, and an object thereof is to provide a new technique for promoting differentiation of adipocytes.

As a result of intensive studies to solve the above-mentioned problems, the present inventors can promote the differentiation of adipocytes by leucine. Therefore, a composition containing leucine is effective in improving insulin resistance and treating diabetes. The present invention has been uniquely found to be usable, and the present invention has been completed. The present invention has been completed based on the above-described novel findings and includes the following inventions.
(1) A composition comprising leucine and promoting differentiation of cells into adipocytes.
(2) The composition according to the above (1), which promotes differentiation of cells into adipocytes without increasing the amount of neutral fat accumulated in adipocytes.
(3) A composition for improving insulin resistance comprising the composition described in (1) above.
(4) A composition for treating diabetes comprising the composition according to (1) above.
(5) A composition comprising leucine and inhibiting the accumulation of neutral fat in adipocytes.
(6) Food / beverage products containing the composition in any one of said (1)-(5).
(7) A method of promoting differentiation of cells into adipocytes by leucine.

  As described above, the present invention includes a composition that includes leucine and promotes differentiation of cells into adipocytes. This composition can be used as a pharmaceutical agent for improving insulin resistance and treating diabetes. In addition, foods and drinks containing this composition can also be used for the treatment of diabetes and the like.

  The present inventors have also newly found that leucine has an action of inhibiting the accumulation of neutral fat in adipocytes. Therefore, in the present invention, neutral fat to adipocytes containing leucine is included in the present invention. Are also included.

  Since leucine can be ingested as a food, the present invention can be used not only in the medical field but also in the field of food production, and can be used very suitably for the prevention and improvement of diabetes.

The present invention provides a composition that contains leucine and promotes differentiation of cells into adipocytes, and further provides techniques such as a composition for improving insulin resistance and a composition for treating diabetes that are typical uses thereof. It is. This will be described in more detail below.
<1. Differentiation promoting composition>
The composition for promoting differentiation of adipocytes according to the present invention (hereinafter sometimes simply referred to as a composition for promoting differentiation) contains leucine and exhibits an action of promoting differentiation of cells into adipocytes. The content of leucine, the production method, the composition contained elsewhere, the mechanism of action thereof, the target cells, etc. are not particularly limited.

  The promotion of differentiation of cells into adipocytes is preferably promotion of differentiation from preadipocytes into adipocytes.

  The method of using the composition for promoting differentiation according to the present invention is not particularly limited. However, preadipocytes have an increased ability to process sugars and lipids by promoting differentiation into adipocytes. Moreover, it also acquires sensitivity to hormones such as insulin. Therefore, as a typical example, it can be suitably used for a composition for improving insulin resistance, a composition for treating diabetes, and the like. The composition for promoting differentiation, the composition for improving insulin resistance, and the composition for treating diabetes according to the present invention can be used as a pharmaceutical for prevention, improvement, etc. as well as treatment of diabetes.

  In addition, the present inventors can promote the differentiation of preadipocytes into adipocytes without increasing the amount of triglyceride (TG) accumulated in adipocytes. I found.

Normally, when a cell differentiates into an adipocyte, the amount of TG accumulated in the cell increases. However, the composition for promoting differentiation of the present invention can also be used as a composition for promoting differentiation without increasing the amount of TG accumulated in fat cells. Therefore, the composition for promoting differentiation of the present invention can improve insulin resistance or cause glucose metabolism without causing weight gain, which is one of the side effects of conventional drugs that promote adipocyte differentiation such as thiazolidine-based drugs. Can also be improved. The phrase “not accompanied by an increase in the amount of TG accumulated” may simply mean that the amount of TG accumulated in the cell is not increased, or may be that the amount of TG accumulated in the fat cell is decreased.
<2. Composition for inhibiting TG accumulation>
The present invention also includes a composition that contains leucine and inhibits the accumulation of TG in adipocytes (hereinafter, a composition for inhibiting TG accumulation). By utilizing the effect of reducing the amount of TG accumulated in fat cells by leucine, the composition for inhibiting TG accumulation of the present invention can be used for antihyperlipidemic drugs and the like.

The composition for inhibiting TG accumulation can also prevent or ameliorate excessive obesity, which is a side effect of conventional therapeutic agents for diabetes. Therefore, the composition for inhibiting TG accumulation according to the present invention can also be used in combination with conventional therapeutic agents for diabetes.
<3. Use of Leucine-Containing Composition>
As already described, the composition described in the above <1> column, that is, a differentiation promoting composition, and a composition containing the differentiation promoting composition, such as a composition for improving insulin resistance and a composition for treating diabetes Furthermore, the TG accumulation-inhibiting composition described in the above <2> column (hereinafter, these leucine-containing compositions are collectively referred to as leucine-containing compositions) can be used as a pharmaceutical product.

  Examples of pharmaceutical dosage forms using the above-described leucine-containing composition include oral preparations such as tablets, capsules (including soft capsules and microcapsules), powders, granules, syrups, injections, and suppositories. And parenterals such as pellets and drops.

  Moreover, the food / beverage products containing at least 1 of the above-mentioned leucine containing composition are also contained in this invention. In addition, food-drinks refer to foodstuffs and beverages, and the form, composition contained elsewhere, etc. are not particularly limited. Furthermore, food and drink materials include food and beverage materials.

  Since the food-drinks concerning this invention should just contain the said composition, the kind is not specifically limited. Specifically, bread, Japanese and Western confectionery (including frozen confectionery, etc.), prepared foods, dairy products, cereals, tofu and fried foods, noodles, bento, seasonings, processed agricultural products such as flour and meat, long-term preserved foods General foods such as soups such as canned foods (frozen foods, frozen foods, retort foods, etc.), soft drinks, milk drinks, soy milk, and potage soups are not particularly limited. The method for adding the oil / fat composition to these general foods is not particularly limited, and any known appropriate method can be employed depending on the type of the general food.

  In addition, examples of the food according to the present invention include functional foods used for specific uses other than general foods such as health foods and nutritional foods. Specific examples include dietary supplements such as various supplements and foods for specified health use. In the case of supplements and the like, the above-described leucine-containing composition can be used as it is by simply processing it into an appropriate shape, or a third component can be added as necessary.

  The organisms to be used for the above-mentioned pharmaceuticals and foods and drinks are not particularly limited and may be any organisms, but are typically humans. Other than that, pets, domestic animals , And experimental animals.

Since leucine is an amino acid and is a substance widely contained in living organisms, the composition and food and drink of the present invention are considered to have low side effects and high safety. In addition, exercise therapy and diet therapy are commonly used to improve diabetes, but it is very difficult to continue exercise therapy in the modern lifestyle. For this reason, it is desirable to prevent and improve diabetes through foods taken daily. The differentiation promoting composition, the insulin resistance improving composition, and the diabetes treatment composition described above are easily taken on a daily basis by adding them to food and drink. In addition, it is preferable that the food / beverage products according to the present invention exhibit at least one effect among adipocyte differentiation promotion, insulin resistance improvement, and diabetes improvement.
<4. Method for promoting differentiation into adipocytes>
The present invention also includes a method for promoting differentiation of cells into adipocytes (hereinafter sometimes simply referred to as differentiation promoting method). As long as this method is a method of promoting differentiation of adipocytes with leucine, the action route, the reagent used, etc. are not particularly limited. As the differentiation promoting method, it is preferable to use the composition for promoting differentiation described in the section <1>. The method for administering leucine to cells is not limited, and may be appropriately changed according to the purpose.

  It has been reported so far that leucine may improve glucose metabolism. However, leucine increases GLUT4 translocation in the muscle via the insulin pathway (Non-patent Document 1), or pancreatic β This is because the amount of insulin released from the cells is increased (Non-patent Document 2). That is, the present invention is based on a completely new finding that leucine promotes differentiation of adipocytes.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. That is, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

Hereinafter, although an example is given and the present invention is explained more concretely, the present invention is not limited to this. The operations not particularly mentioned were performed using techniques generally performed by those skilled in the art. Each instrument and reagent were used according to the attached instruction manual unless otherwise specified.
(Example 1) Differentiation promotion of adipocytes (A) Differentiation treatment (differentiation induction treatment and differentiation promotion treatment)
The following operations were performed using mouse-derived preadipocytes 3T3-L1.

  The cell culture temperature was 37 ° C., and DMEM (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum (FBS) was used as the normal medium. DMEM contains 0.8 mM leucine. Hereinafter, all concentrations of additives to the medium are expressed as final concentrations.

  Differentiation induction processing was performed on the cells that had been cultured in a normal medium and reached confluence. That is, the cells were cultured for 2 days in a differentiation-inducing medium in which 0.25 mM dexamethasone, 0.5 mM isobutylmethylxanthine, and 10 μg / ml insulin were added to a normal medium. The differentiation induction start date is the 0th day.

  On the second day from the start of differentiation induction, the medium was replaced with a differentiation promotion medium, and differentiation promotion treatment was performed. Further, the medium was changed twice every two days and cultured for 4 days (from the start of differentiation induction to the sixth day). The differentiation promoting medium is obtained by adding 5 μg / ml insulin to a normal medium.

In order to examine the influence of leucine on cell differentiation, cells cultured for 0 to 6 days after adding leucine to the differentiation induction medium and differentiation promotion medium so that the leucine concentration was increased by 1 mM from normal DMEM were used. And used for the following operation (B). Further, as a control, the following operation (B) was performed using cells cultured from 0 to 6 days in a medium not added with leucine.
(B) Evaluation of differentiation promoting effect The differentiation promoting effect by leucine was evaluated.

  Cells from day 6 after differentiation induction are collected, mRNA is extracted from the cells with Sepasol-RNAI (registered trademark, manufactured by Nacalai Tesque), and then reverse transcribed using an oligo dT primer to obtain a cDNA sample. It was. The target gene (aP2 or leptin) in this cDNA sample was quantified with LightCycler (trademark, manufactured by Roche). aP2 and leptin are genes commonly used as differentiation markers for adipocytes.

(Differentiation promoting effect by leucine)
The differentiation promoting effect was evaluated using the aP2 and leptin mRNA amounts measured in this manner as a ratio to the amount of 36B4 mRNA transcribed constantly regardless of differentiation. The results are shown in FIGS. 1 (a) and (b). In the figure, + Leu represents the addition of leucine, and -Leu represents the absence of leucine.

FIGS. 1A and 1B are graphs showing the amounts of aP2 and leptin mRNA relative to the amount of 36B4 mRNA, respectively. As shown in FIGS. 1 (a) and 1 (b), the addition of leucine increased the amount of differentiation marker. That is, leucine promoted differentiation of preadipocytes into adipocytes.
(C) Effect of PPARγ ligand Furthermore, the differentiation promoting effect of leucine in the presence of pPARlitazone, a PPARγ ligand, was examined. Differentiation treatment was performed in the same manner as in the above (A) except that pioglitazone was added to the differentiation induction medium to a final concentration of 10 μM and cultured from day 0 to day 2. For these cells, the amount of differentiation marker was measured in the same manner as in (B) above, and the results are shown in FIG.

As shown in FIG. 1 (c), differentiation was promoted by adding leucine even in the presence of pioglitazone.
(D) Influence of insulin Furthermore, the differentiation promoting effect of leucine in the absence of insulin was examined. Differentiation treatment was performed in the same manner as in the above (A) except that insulin was not added to the differentiation induction medium. For these cells, the amount of differentiation marker was measured in the same manner as in (B) above, and the results are shown in FIG.

  As shown in FIG. 1 (d), differentiation was promoted by adding leucine even in the absence of insulin.

That is, as shown in FIGS. 1 (a) to 1 (d), differentiation from preadipocytes to adipocytes was promoted by leucine regardless of the presence or absence of insulin or pioglitazone.
(E) Differentiation stages affected by leucine Next, in order to clarify the differentiation stages affected by leucine, 0-2, 2-4, 4-6, The effect on differentiation markers when leucine was added on days 0 to 6 was examined.

In addition, as shown in FIG.1 (c), since the differentiation promoting effect of leucine had appeared strongly in the presence of pioglitazone, pioglitazone was also added to the differentiation induction medium this time. The measurement result of the differentiation marker is shown in FIG. In FIG. 2, + pio represents the addition of pioglitazone, and -pio represents the absence of pioglitazone. As shown in FIG. 2, leucine was thought to act particularly at the early and late stages of differentiation.
(F) Involvement of P13K and PKC in the differentiation promoting action of leucine When luciferase assay was performed using full-length PPARγ, leucine did not affect the transcriptional activity of PPARγ (data not shown). This suggests that leucine affects cell differentiation through a pathway that does not depend on PPARγ.

  As an action pathway of leucine, the present inventors paid attention to the insulin pathway shown in FIG. In this example, in order to prevent signal transduction of the insulin pathway by insulin, differentiation treatment was performed in a non-insulin-containing medium as in (D) above. Unlike the above (D), differentiation treatment was performed from day 0 to day 6 under the conditions where LY294002 and / or GF109203X was added to the differentiation induction medium and differentiation promotion medium, or these inhibitors were not added.

  As shown in FIG. 3, LY294002 is a specific inhibitor of P13K and GF109203X is a specific inhibitor of PKC.

As shown in FIG. 4, the amount of aP2 mRNA increased in the absence of an inhibitor (same as (D) above). Even when LY294002 was added, the differentiation promoting effect by leucine was observed. However, the differentiation promoting effect of leucine disappeared in the presence of GF109203X. From the above, it is considered that leucine activates PKC through a pathway different from P13K and affects the differentiation of 3T3-L1.
(Example 2)
(G) Measurement of the amount of TG in cells It is generally known that the amount of TG accumulated in fat cells increases as the differentiation of fat cells proceeds. Therefore, the amount of TG accumulated in cells whose differentiation was promoted by leucine was measured.

  As the cells, the cells subjected to the differentiation treatment of the above (A) or (D) were used. Cells were lysed with 1% Triton X100 to obtain a TG sample. Measurement was performed by adding 250 μl of TG E test (manufactured by Wako Pure Chemical Industries, Ltd.) to 16 μl of TG sample. Usually, 300 μl of TG E test is added to 2 μl of sample, and measurement is performed by a modified method in this example because of the low TG concentration of the TG sample and the problem of capacity.

  The measurement result of the TG accumulation amount is shown in FIG. FIG. 5 (a) shows the TG content of the cells subjected to the differentiation process of (D), that is, the cells subjected to the differentiation process in the absence of insulin, and (b) shows the differentiation of (A) above. The TG content of the cells that have been treated, that is, the cells that have been differentiated in the presence of insulin is shown.

  As shown in FIGS. 5A and 5B, the amount of TG accumulated was not increased by leucine regardless of the presence or absence of insulin. In particular, in the absence of insulin, there was a significant tendency for leucine to reduce the amount of TG accumulation.

From the above, it was found that leucine increases the amount of mRNA of a gene expressed with the differentiation of adipocytes, while not increasing TG accumulation. That is, it is considered that sugar metabolism can be improved without causing weight gain, which is one of the side effects of thiazolysin drugs.
(Example 3) Improvement of glucose metabolism in diabetic model mice by leucine The effect of leucine on glucose metabolism in diabetic model mice was examined.

  KKAy mice (6 weeks old, females), which are diabetes model mice, were grouped so that the average body weights were equal after 6 weeks of preliminary breeding (6 groups, each n = 3). Low fat diet (fat content 10%), high fat diet (fat content 60%), high fat diet + thiazolidine derivative diet (including 20% energy ratio protein in each diet), leucine or casein 2% It was added and ingested.

  Before the start of intake (day 0) and on the 20th day (day 20), the blood glucose level was measured after fasting for 4 hours to confirm that the patient had diabetes. An oral glucose addition test (OGTT) was performed on the 21st day (10 weeks of age) of ingestion, and sacrifice and dissection were performed on the 35th day (12 weeks of age).

  In all diets, the leucine intake group showed a lower blood glucose level than the casein group in the 4-hour fast. This tendency was most noticeable in the low-fat diet group. Only the results of the low fat diet group are shown in Table 1 and FIG.

  In addition, after fasting for 16 hours, a 20% glucose solution was orally administered to mice so that the body weight was 100 μl / 10 g body weight (20 mg glucose / 10 g body weight). The blood glucose level was lowered. This tendency was also most prominent in the low-fat diet group. Only the results of the low fat diet group are shown in FIG. In addition, the horizontal axis | shaft of FIG.6 (b) represents time (minute) after glucose oral administration.

  Thus, leucine has improved glucose metabolism when fasted for 16 hours after long-term administration. At this time, it is considered that the leucine concentration in the blood is lowered to the basal value due to fasting (Can J Physiol Pharmacol. 1999 Nov; 77 (11): 827-34). That is, leucine may not only temporarily improve the symptoms of diabetes but also treat diabetes itself. Thus, the present inventors have discovered for the first time that glucose metabolism is improved when leucine is administered for a long period of time and the blood leucine concentration is low.

  The gist of the present invention is to promote differentiation into adipocytes, and the action mechanism of leucine is not limited. That is, the mechanism of action of leucine described in the above-mentioned examples is a hypothesis shown to explain the effect of leucine found by the present inventors, and the present invention is not limited to this route of action. Therefore, even if it is an action route other than the above-described route, what is included in the gist of the present invention that leucine promotes differentiation of adipocytes is not limited to the composition and method, but is included in the present invention. Not too long.

  The composition comprising leucine according to the present invention and promoting the differentiation of cells into adipocytes can improve glucose metabolism and is used for pharmaceuticals for improving insulin resistance and for treating diabetes, or for foods and drinks. be able to.

It is a graph which shows the adipocyte differentiation promotion effect of leucine in the Example of this invention, and shows the amount of mRNA which is a differentiation marker. (A) and (b) show the amount of aP2 and leptin mRNA in the presence of insulin, (c) shows the amount of aP2 mRNA in the presence of pioglitazone, and (d) shows the amount of aP2 mRNA in the absence of insulin. It is a graph which shows the influence which it has on the differentiation stage of the leucine in the Example of this invention. It is drawing which shows the signal transduction pathway of insulin. It is drawing which shows the influence of the insulin pathway inhibitor with respect to the adipocyte differentiation promotion effect of leucine in the Example of this invention. It is a graph which shows the amount of TG accumulation | storage in the fat cell in the Example of this invention, (a) shows the TG accumulation amount of the cell which each differentiated in presence of insulin, (b) in the presence of insulin. In the Example of this invention, it is a graph which shows the glucose metabolism improvement effect of the mouse | mouth by leucine, (a) is the blood glucose level after a 4-hour fast before (20 days) before leucine or casein ingestion, ( b) represents the result of the OGTT test on day 21 of leucine or casein intake.

Claims (7)

  A composition comprising leucine and promoting differentiation of cells into adipocytes.   The composition according to claim 1, which promotes differentiation of cells into adipocytes without increasing the amount of neutral fat accumulated in the adipocytes.   A composition for improving insulin resistance comprising the composition according to claim 1.   A composition for treating diabetes comprising the composition according to claim 1.   A composition comprising leucine, which inhibits the accumulation of neutral fat in adipocytes.   Food / beverage products containing the composition of any one of Claims 1-5.   A method of promoting differentiation of cells into adipocytes by leucine.

Images