JPWO2008032450A1 - Preventive and / or therapeutic agent for fatty liver - Google Patents

Abstract

The present invention relates to an agent for preventing and / or treating fatty liver containing an agonist of natriuretic peptide receptor GC-A. The medicament of the present invention not only can prevent and treat fatty liver, but can also prevent metabolic syndrome represented by obesity and diabetes.

Description

  The present invention relates to a prophylactic and / or therapeutic agent for fatty liver.

  Among the items that are regarded as abnormal test findings in medical examinations, liver dysfunction is the second most common after hyperlipidemia, and most of the liver dysfunction is fatty liver. Fatty liver is a state in which lipids mainly composed of neutral fat are stored in hepatocytes and the weight ratio is 5% or more. Neutral fat is a fatty acid ester of glycerin (triacylglycerol). When the lipid is 5% or more, lipid droplets appear in more than one third of the hepatocytes (liver parenchymal cells) of the liver lobule. There are various causes of fatty liver, such as alcohol consumption, obesity, diabetes, drugs, and the like, which are roughly classified into alcoholic fatty liver and non-alcoholic fatty liver. Non-alcoholic fatty liver is increasing with an increase in lifestyle-related diseases such as diabetes, hyperlipidemia and obesity.

  Alcoholic fatty liver is likely to progress to hepatitis, cirrhosis, and liver cancer, and its treatment is centered on alcohol cessation. On the other hand, nonalcoholic fatty liver disease (NAFLD) has been considered to be reversible, which is improved by diet and exercise therapy and does not shift to a particularly serious disease. In recent years, it has been pointed out that if left untreated, the liver function is lowered and there is a risk of progressing to cirrhosis. In particular, among non-alcoholic steatohepatitis (NASH) with inflammation, NAFLD has inflammation similar to alcoholic hepatitis in liver histology, and has a high risk of progression from cirrhosis to hepatocellular carcinoma It has become known to be a disease. Therefore, more than ever, appropriate treatment of NAFLD, particularly NASH, is considered to be a very important medical issue.

  Metabolic syndrome, on the other hand, is a syndrome that is associated with the accumulation of visceral fat (intraperitoneal fat) and multiple risks for cardiovascular diseases such as insulin resistance, dyslipidemia, and hypertension. It is understood that this is a pathological condition that leads to various lifestyle-related diseases brought about by the accumulation of. In patients with metabolic syndrome, NAFLD containing NASH is observed more frequently than patients with non-metabolic syndrome, and it is known that many patients have concurrent NAFLD containing NASH.

  That is, patients with non-alcoholic fatty liver disease may have metabolic syndrome associated with basic diseases such as obesity, hypertension, diabetes and hyperlipidemia. In such patients, not only treatment of fatty liver Treatment of those underlying diseases may also be necessary. Therefore, for the treatment of fatty liver, it is desirable to use drugs that do not adversely affect such underlying diseases, and further drugs that can simultaneously treat these underlying diseases.

  As dietary therapy for fatty liver, alcohol consumption restriction, caloric restriction, and intake of low-fat / high-protein diet are mainly used. As exercise therapy, measures are taken to consume fat as energy by aerobic exercise or the like. At that time, as it is known that fatty liver is also caused by malnutrition such as anorexia, sudden weight loss is not preferable because extreme muscle restriction drops muscles and increases fat on the contrary, A weight loss of about 1-2 kg / month is desirable. Thus, the treatment of fatty liver requires a controlled diet and perseverance with a considerable length of aerobic exercise.

  Because modern life is full of stress and satiety, and is prone to causing fatty liver, it can be used instead of, or in addition to, the above-mentioned persistent treatment of fatty liver. New therapies that can be used in combination are desired.

  By the way, in patients with obesity and hypertension, an excessive burden is placed on circulatory organs such as the heart and kidneys. In such a situation, it is well known that the production and secretion of a peptide called a natriuretic peptide is enhanced, and a compensatory action that reduces the burden on the circulatory system organ is working. As peptides called natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are known. ANP is a peptide having a cyclic structure consisting of 28 amino acids produced and secreted by atrial cells, exhibits diuretic action in the kidney, and relaxes / expands vascular smooth muscle in blood vessels. ANP also acts antagonistically on the renin-vasopressin-aldosterone system. These actions generally work to reduce the burden on the heart through a reduction in blood pressure and a reduction in the amount of body fluid. BNP is a peptide having a cyclic structure consisting of 32 amino acids, and is produced and secreted mainly by ventricular cells and has the same action as ANP. Both ANP and BNP bind to the receptor NPR-A (also known as GC-A) having a guanylate cyclase domain, and promote the production of cGMP to express the above action. In fact, ANP promotes secretion with increasing atrial fullness pressure in congestive heart failure or the like, and functions to reduce symptoms such as congestive heart failure by the above action. BNP also promotes secretion during myocardial infarction, and functions to relieve various symptoms associated with myocardial infarction by the above-described action (Non-patent Document 1).

Recently, the expression of the natriuretic peptide receptor GC-A has also been confirmed in adipose tissue, and reports that ANP and BNP not only act on the cardiovascular system but are also involved in the promotion of lipolysis (Non-Patent Document 2). The lipolysis promoting action of ANP and BNP is such that cGMP produced by guanylate cyclase present in the intracellular part of natriuretic peptide receptor GC-A acts on cGMP-dependent protein kinase I (cGKI), It is thought to be by activating phosphorylation of hormone-sensitive lipase. Activated hormone sensitive lipase hydrolyzes triglycerides to free fatty acids. However, it has been reported that the action of natriuretic peptides on adipose tissue as described above is not involved in obesity in young boys (Non-patent Document 3). In addition, it is not known how the effect of promoting lipolysis in adipose tissue by natriuretic peptides such as ANP and BNP affects hepatocytes. That is, no effect has been clarified on the effect of natriuretic peptides such as ANP and BNP directly or indirectly on fat accumulation and metabolism in hepatocytes that are essentially different from adipose tissue. In fact, a recent review on natriuretic peptides also describes in detail the therapeutic application to cardiovascular disease, but does not suggest any application for the prevention and / or treatment of fatty liver (Non-Patent Document 4).
European J.M. Endocrinology, 135, 265, 1996 FASEB, 14, 1445, 2000 J. et al. Lipid Res. 42, 536, 2001 Endocrine Review, 27, 47, 2006

  An object of the present invention is to find a drug useful for the prevention and / or treatment of fatty liver. Furthermore, it is a problem to find a drug useful for the prevention and / or treatment of fatty liver in patients with metabolic syndrome who are likely to cause cardiovascular diseases such as myocardial infarction.

  The inventors of the present invention have proposed an agent capable of preventing and / or treating fatty liver, as well as fatty liver (or metabolic syndrome associated with metabolic syndrome that is likely to cause cardiovascular diseases such as myocardial infarction). As a result of intensive studies on drugs capable of preventing and / or treating (hepatic steatosis), it was found that natriuretic peptides have weight loss, visceral fat reduction, improved glucose tolerance, and fatty liver suppression. . Furthermore, it has been found that activation of cGMP-dependent protein kinase I (cGKI) has the effects of reducing body weight, reducing visceral fat, improving glucose tolerance, and inhibiting fatty liver, similar to natriuretic peptide. Thus, the present invention has been completed.

  Specifically, a mouse that overexpressed BNP in a liver-specific manner (hereinafter referred to as a BNP-Tg mouse) was produced in a manner similar to the situation where BNP was continuously administered, and the effect of BNP was examined by giving a high fat diet. As a result, it was found that body weight gain, visceral fat increase, liver weight increase, and hepatic neutral fat increase were significantly suppressed in BNP-Tg mice compared to wild-type mice. In addition, in BNP-Tg mice fed with a high fat diet, the increase in blood glucose level is significantly reduced compared to wild-type mice during glucose tolerance and insulin administration, so that the natriuretic peptide is glucose tolerance and insulin sensitivity. Also found to improve.

  Furthermore, mice overexpressing cGMP-dependent protein kinase I (cGKI) activated by cGMP produced by the action of natriuretic peptide on natriuretic peptide receptor GC-A (hereinafter referred to as “systemic overexpression”). (cGK-Tg mice) were fed a high fat diet, resulting in increased body weight, increased visceral fat, increased liver weight, and increased neutral fat in the liver, as in BNP-Tg mice. Was found to be significantly suppressed. Moreover, in cGK-Tg mice fed with a high fat diet, the increase in blood glucose level is significantly reduced compared to wild-type mice during glucose tolerance and insulin administration. It has been found that sensitivity is also improved. In addition, as a result of giving a high fat diet to GC-A hetero knockout mice, it was found that obesity and fatty liver formation were greater and the degree of impaired glucose tolerance was greater than in wild type mice.

  These results not only indicate that administration of a natriuretic peptide such as BNP can prevent and / or treat fatty liver, but also fatty liver (or obesity associated with metabolic syndrome such as obesity and diabetes). Or fatty liver with metabolic syndrome represented by diabetes, etc. can be prevented and / or treated. Furthermore, it shows that metabolic syndrome and obesity can be prevented and / or treated. Furthermore, for example, it is considered that a compound known to function as an agonist of GC-A like BNP, such as ANP, has the same effect as BNP. Similarly, not only shows that cGKI can be activated to prevent and / or treat fatty liver, but also fatty liver associated with metabolic syndrome such as obesity and diabetes (or represented by obesity and diabetes). (Hepatic steatosis with metabolic syndrome) can be prevented and / or treated. Furthermore, it shows that metabolic syndrome and obesity can be prevented.

Therefore, the present invention provides a method for preventing and / or treating fatty liver by administering an agonist of natriuretic peptide receptor GC-A.
The present invention also provides a method for preventing and / or treating fatty liver by administering an activator of cGMP-dependent protein kinase I (cGKI).
The present invention also provides a prophylactic and / or therapeutic agent for fatty liver containing a gene for natriuretic peptide.
The present invention also provides a preventive and / or therapeutic agent for fatty liver containing the gene for cGMP-dependent protein kinase I (cGKI).
The present invention also provides a prophylactic and / or therapeutic agent for fatty liver containing an agonist of natriuretic peptide receptor GC-A.
The present invention also provides a preventive and / or therapeutic agent for fatty liver containing an activator of cGMP-dependent protein kinase I (cGKI).
The present invention also provides a prophylactic and / or therapeutic agent for fatty liver in patients with metabolic syndrome containing an agonist of natriuretic peptide receptor GC-A.
The present invention also provides a kit for treating fatty liver containing an agonist of natriuretic peptide receptor GC-A.
The present invention also provides a preventive and / or therapeutic agent for metabolic syndrome, which contains an agonist of natriuretic peptide receptor GC-A.
The present invention also provides a prophylactic and / or therapeutic agent for obesity containing an agonist of natriuretic peptide receptor GC-A.
The present invention also provides use of a natriuretic peptide gene for the production of a prophylactic and / or therapeutic agent for fatty liver.
The present invention also provides the use of a gene for cGMP-dependent protein kinase I (cGKI) for the production of a prophylactic and / or therapeutic agent for fatty liver.
The present invention also provides the use of an agonist of natriuretic peptide receptor GC-A for the production of a prophylactic and / or therapeutic agent for fatty liver.
The present invention also provides use of an activator of cGMP-dependent protein kinase I (cGKI) for producing a prophylactic and / or therapeutic agent for fatty liver.
The present invention also provides the use of an agonist of natriuretic peptide receptor GC-A for the production of a prophylactic and / or therapeutic agent for fatty liver in patients with metabolic syndrome.
The present invention also provides the use of an agonist of natriuretic peptide receptor GC-A for the production of a preventive and / or therapeutic agent for metabolic syndrome.
The present invention also provides the use of an agonist of natriuretic peptide receptor GC-A for the production of a prophylactic and / or therapeutic agent for obesity.

  The present invention enables prevention and / or treatment of fatty liver using a medicament containing an agonist of natriuretic peptide receptor GC-A. In addition, the present invention enables prevention and / or treatment of fatty liver associated with metabolic syndrome (or fatty liver associated with metabolic syndrome) using a medicament containing an agonist of natriuretic peptide receptor GC-A. . Furthermore, it enables prevention and / or treatment of metabolic syndrome and obesity.

It is a graph which shows the weight transition of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth when a high fat diet is administered. It is a graph which shows the liver weight of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth when a high fat diet is administered. It is a graph which shows the neutral fat content of the liver of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a standard diet and a high fat diet. Subcutaneous adipose tissue weight (A), mesenteric adipose tissue weight (B), total adipose tissue weight (C), and BNP-Tg mice and wild type (Wt) mice when standard diet and high fat diet were administered, and It is a graph which shows the average area (D) of the fat cell which comprises the subcutaneous fat tissue of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth when a high fat diet is administered. It is a graph which shows the glucose tolerance of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a high fat diet. It is a graph which shows the insulin sensitivity of a BNP-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a high fat diet. It is a graph which shows the weight transition of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a standard diet and a high fat diet. It is a graph which shows the liver weight of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth when a high fat diet is administered. It is a graph which shows the neutral fat content of the liver of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a standard diet and a high fat diet. Subcutaneous adipose tissue weight (A), mesenteric adipose tissue weight (B), total adipose tissue weight (C), and cGK-Tg mice and wild type (Wt) mice when standard diet and high fat diet were administered, and It is a graph which shows the average area (D) of the fat cell which comprises the subcutaneous fat tissue of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth when a high fat diet is administered. It is a graph which shows the glucose tolerance of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a high fat diet. It is a graph which shows the insulin sensitivity of a cGK-Tg mouse | mouth and a wild type (Wt) mouse | mouth at the time of administering a high fat diet. It is a graph which shows the gene (PGC-1 (alpha) and UCP-1) expression in the interscapular brown adipose tissue of a cGK-Tg mouse | mouth and a wild type mouse | mouth at the time of administering a high fat diet (beta-actin of each gene expression level) The ratio to the gene expression level is shown with the wild type as 100%. ** p <0.01 vs. wild type.) It is a graph which shows gene (PGC-1, UCP-2, UCP-3, PPAR (alpha) and PPAR (delta)) expression in the quadriceps muscle of a cGK-Tg mouse | mouth and a wild type mouse | mouth at the time of administering a high fat diet (each gene) The ratio of the expression level to the β-actin gene expression level is shown with the wild type as 100%. * P <0.05, ** p <0.01 vs. wild type. It is a graph which shows the gene (PPAR (alpha) and PPAR (delta)) expression in the liver of a cGK-Tg mouse | mouth and a wild type mouse | mouth at the time of administering a high fat diet (The ratio with respect to (beta) -actin gene expression level of each gene expression level is shown in a wild type) Is expressed as 100%. * P <0.05, ** p <0.01 vs. wild type.). It is a graph which shows the weight transition of GC-A ^<+/-> mouse ^| mouth and a wild type mouse ^| mouth at the time of administering a high fat diet. It is a graph which shows the body weight at the time of 20-week-old of a GC-A ^<+/-> mouse ^| mouth and a wild type mouse ^| mouth at the time of administering a high fat diet, a liver weight body weight ratio, and a liver triglyceride content. It is a graph which shows the total fat weight of GC-A ^<+/-> mouse ^| mouth and a wild type mouse ^| mouth at the time of administering a high fat diet. It is a graph which shows the food intake at the time of 18-week-old of GC-A ^<+/-> mouse ^| mouth and a wild type mouse ^| mouth at the time of administering a high fat diet. It is a graph which shows the glucose tolerance test result of the intraperitoneal administration at the age of 18 weeks of GC-A ^+/- mouse and wild type mouse when a high fat diet is administered.

  Although the present invention is intended for the prevention and / or treatment of fatty liver, the target patient may be a patient with fatty liver without other underlying diseases, and for example, fat associated with metabolic syndrome It may be a patient with liver (or fatty liver with metabolic syndrome). Further, the present invention may treat patients with metabolic syndrome and obese patients as treatment targets.

  The agonist of natriuretic peptide receptor GC-A used in the present invention is not particularly limited as long as it is a compound that binds to GC-A and has an action of activating guanylate cyclase. However, a typical example is a natriuretic peptide. Typical examples of natriuretic peptides are ANP and BNP, but are not limited to these natriuretic peptides, and those in which one or more amino acids in the amino acid sequence are substituted with other amino acids. In addition, one or more amino acids of those amino acid sequences may be deleted, one or more amino acids of the amino acid sequence may be deleted, and one or more amino acids of the amino acid sequence may be other amino acids. In addition, one or a plurality of amino acids may be chemically synthetically substituted with an artificial amino acid-like one. Further, it may be a low molecular weight compound derived from chemical synthesis or a natural product.

  As ANP, for example, Biochem. Biophys. Res. Commun. 118, 131, 1984, human-derived α-hANP can be used, and this ANP is sold under the general name carperitide (trade name: HAMP, HANP). α-hANP is also commonly known as Human pro-ANP [99-126]. As BNP, for example, Biochem. Biophys. Res. Commun. 159, 1420, 1989, and this BNP is sold under the generic name nesiritide (trade name: Natrecor).

Examples of the substitution of one or more amino acids of the natriuretic peptide with other amino acids include, for example, rat α-rANP (Biochem. Biophys. Res. Commun., Met at position 12 of human ANP replaced with Ile). 121, 585, 1984). Examples of deletion of one or more amino acids include ANP in which N-terminal Ser-Leu-Arg-Arg-Ser-Ser is deleted. Such ANP or BNP derivatives include, for example, a series of derivatives described in Medicinal Research Review, Vol. 10, p. 115, 1990, which can be obtained by the methods described in the cited references or It can be manufactured.
In addition, as an example in which one or more amino acids in the amino acid sequence are deleted and one or more amino acids in the amino acid sequence are substituted with other amino acids, for example, mini-ANP (Science, 270) consisting of 15 amino acid residues. Volume, 1657, 1995).

  The above ANP, BNP, and derivatives thereof may be produced using genetic engineering / cell engineering techniques, may be chemically synthesized, and may be further treated with an enzyme treatment or the like. The amino acid residue may be modified by chemical treatment or a part of the amino acid sequence may be removed.

  The natriuretic peptide such as ANP or BNP as an agonist of the natriuretic peptide receptor GC-A may be a pharmaceutically acceptable salt, for example, a salt with an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid. Or a salt with an organic acid such as formic acid, acetic acid, butyric acid, succinic acid or citric acid. Moreover, it is good also as metal salts, such as sodium, potassium, lithium, and calcium, and is good also as salts with organic bases, such as a triethylamine. In addition, natriuretic peptides such as ANP and BNP or their salts are mixed with pharmaceutically acceptable excipients, isotonic agents, pH adjusters, diluents, etc., administered intravenously, intramuscularly, It is preferable to administer by parenteral administration methods such as subcutaneous administration, nasal administration, pulmonary administration, and sublingual administration. In addition, these natriuretic peptides or salts thereof may be administered parenterally by encapsulating them in liposomes or microcapsules, or may be implanted in a patient's body as a sustained release preparation.

  When administering a natriuretic peptide such as ANP or BNP or a salt thereof, for example, a lyophilized preparation is dissolved in water for injection and a micro infusion pump (in the case of absence, a micro infusion set for children) is used. What is necessary is just to administer continuously. Since natriuretic peptide has the action of relaxing and dilating blood vessels and lowering blood pressure as described above, it is preferable to administer at a rate that does not lower blood pressure more than necessary for the prevention and / or treatment of fatty liver. It is recommended to monitor blood pressure during and immediately after administration. The daily dose of natriuretic peptides such as ANP and BNP or their salts is in the range of 0.1 μg / kg to 100 mg / kg, preferably 0.5 μg / kg to 5 mg / kg. When the administration method is described in detail, when ANP is administered, for example, 1000 μg of ANP is dissolved in 10 mL of water for injection, and the rate of “body weight × 0.06 mL / hour” (0.1 μg / kg / min) is obtained. It is conceivable to administer at an administration rate). The administration rate is not limited to the above-mentioned rate, and may be 1/2 or 1/4 of the rate. When administering BNP, it is conceivable to administer 0.01 μg / kg continuously after bolus administration of 2 μg / kg. Also in this case, it is preferable to administer the blood pressure at a rate that does not lower the blood pressure more than necessary, and it is recommended to monitor the blood pressure at the time of administration and immediately after administration. If ANP or BNP does not significantly affect blood pressure, heart rate, etc. at the above administration rate, the administration rate may be further increased. For other ANP derivatives or BNP derivatives, the administration rate may be determined in consideration of the activity and persistence.

  In addition, when any of the above drugs is used, the administration period is preferably until a therapeutic effect for fatty liver is obtained, and may be administered continuously or intermittently. Good. The therapeutic effect of fatty liver may be determined by measuring blood neutral fat, cholesterol level, GOT, GPT by hematological examination, but is preferably judged by ultrasonic examination or CT examination.

  In addition, as a kit for treating fatty liver containing an agonist of natriuretic peptide receptor GC-A, a vial in which a natriuretic peptide such as ANP or BNP or a salt thereof is enclosed as a lyophilized preparation and its dissolution are used. Injectable water is used as a kit, and a syringe for use in dissolution and administration may be combined therewith, and a microinfusion pump or a pediatric microinfusion set may be combined.

  As an activator of cGKI, cGMP itself can be used, but a chemically synthesized cGMP derivative such as stable 8-bromocGMP may be used, and further, stability against phosphodiesterase that degrades 8-bromocGMP. (Pharmacol. Ther., 87, 199, 2000) may be used. Moreover, you may use the activator of the low molecular weight cGKI which has a chemical structure different from such a cGMP derivative.

  As the gene for ANP, those described in Science, 226, 1206, 1984 may be used. In addition, as a gene of BNP, Biochem. Biophys. Res. Commun. 165, 650, 1989 may be used. As a gene for cGMP-dependent protein kinase I (cGKI), cDNA described in Hypertension, 27, 552, 1996 may be used. When treatment is performed using the above gene, the gene may be introduced by intramuscular injection or local injection using a retrovirus, adenovirus, adeno-associated virus or an artificial vector as a vector. Moreover, you may introduce | transduce a gene in the form of a plasmid, without using the above vectors. As a specific gene therapy method, a method described in Experimental Medicine, Vol. 12, page 303, 1994, or a method cited in the literature may be used.

  The prevention and treatment of fatty liver according to the present invention can be applied to all fatty livers regardless of their origin, but can be suitably used for the prevention and treatment of fatty liver induced by metabolic syndrome. In addition, the description regarding the prevention and treatment of fatty liver containing the natriuretic peptide receptor GC-A agonist is the same in the prevention and treatment of metabolic syndrome and obesity.

  Hereinafter, the present invention will be described with reference to test examples.

[Test Example 1]
A normal diet and a high-fat diet were given to wild-type mice and mice overexpressing BNP specifically in the liver (hereinafter referred to as BNP-Tg mice), and the effect of inhibiting the progression of fatty liver was examined. BNP-Tg mice are Clinical Invest. 93, page 1911, 1994.
A standard diet and a high fat diet (60% in terms of calorie ratio: Research diet, catalog number: D12492) were given to wild-type mice and BNP-Tg mice (10 mice each) after 10 weeks of age for 10 weeks. Body weight was measured every week from 6 weeks of age. After the administration of the standard diet and the high fat diet, the liver was removed and its weight was measured. Moreover, after homogenizing the liver, lipids were extracted by the chloroform-methanol method, and the neutral fat content in the liver was measured using a neutral fat measurement kit (Wako Pure Chemical Industries, catalog number: 432-40201).
In addition, the subcutaneous and mesenteric adipose tissue was separated and weighed. Further, a tissue section was prepared from the collected subcutaneous adipose tissue, this was subjected to HE staining, 4 fields (100 times field) were observed per mouse, and the average area of adipocytes was measured.

<Result>
In the standard diet administration group, there was no difference in body weight between the wild type mouse (Wt) and the BNP-Tg mouse, but in the high fat diet administration group, the body weight of the BNP-Tg mouse was higher than that of the wild type mouse. The value was significantly lower than 3 weeks after the start of administration (FIG. 1).
In the high-fat diet administration group, the liver weight of the BNP-Tg mice and the neutral fat content in the liver were significantly lower than those of the wild-type mice (FIGS. 2 and 3).
In the high fat diet administration group, the amount of subcutaneous adipose tissue and the amount of mesenteric adipose tissue of BNP-Tg mice were lower than those of normal mice. Furthermore, the total weight of the adipose tissue obtained by adding the subcutaneous adipose tissue and the mesenteric adipose tissue was less in the BNP-Tg mice when either the standard diet or the high fat diet was fed (FIGS. 4A-C). . Furthermore, the average area of adipocytes in the mesenteric adipose tissue of the high fat diet administration group was significantly smaller in the BNP-Tg mice. (Fig. 4D)

[Test Example 2]
Wild type mice (12 mice) and BNP-Tg mice (8 mice) after 10 weeks of age were given a high fat diet (Research diet, catalog number: D12492) at a calorie ratio for 10 weeks. After fasting for 24 hours, glucose 1.0 g / kg was administered intraperitoneally, and blood glucose levels were measured before administration of glucose and at 15, 30, 60 and 120 minutes after administration.
In addition, as described above, mice fed with a high fat diet were fasted for 6 hours, and then insulin 0.75 U / kg was administered intraperitoneally, before insulin administration, at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after administration. The blood glucose level was measured.
Blood was collected from the tail vein and blood glucose level was measured using Dexter ZII (Bayer).

<Result>
The blood glucose level of BNP-Tg mice was significantly lower after glucose administration and after insulin administration compared to wild type mice (FIGS. 5 and 6). This result suggests improved glucose tolerance and insulin sensitivity in BNP-Tg mice.

[Test Example 3]
A standard diet and a high-fat diet were given to wild-type mice and mice in which cGMP-dependent protein kinase I was overexpressed systemically (hereinafter, cGK-Tg mice), and the effect of inhibiting the development of fatty liver was examined. cGK-Tg mice were obtained from Proc. Natl. Acad. Sci. USA, 100, 3404, 2003.
A standard diet and a high fat diet (60% in terms of calorie ratio: Research diet, catalog number: D12492) were given to 10-week-old wild-type mice and cGK-Tg mice (12 mice each) for 10 weeks. Body weight was measured every week from 6 weeks of age. After the administration of the high fat diet, the liver was removed and its weight was measured. Moreover, after homogenizing the liver, lipids were extracted by the chloroform-methanol method, and the neutral fat content in the liver was measured using a neutral fat measurement kit (Wako Pure Chemical Industries, catalog number: 432-40201). Further, the weight of each adipose tissue and the average area of adipocytes were measured by the same method as in Test Example 1.

<Result>
In the standard diet administration group and the high fat diet administration group, the body weight of the cGK-Tg mice was significantly lower than that of the wild type mice (FIG. 7).
Further, in the cGK-Tg mice administered with the high fat diet, the liver weight and the neutral fat content in the liver were significantly lower than those in the wild type mice (FIGS. 8 and 9). The cGK-Tg mice showed lower values for the subcutaneous fat tissue weight, the mesenteric adipose tissue weight, and the total adipose tissue weight, which is the sum of them, regardless of the content of the meal (FIGS. 10A-D). ).

[Test Example 4]
A 60% high fat diet (Research diet, catalog number: D12492) was given to a wild-type mouse and a cGK-Tg mouse (each 8 mice) after 10 weeks of age for 10 weeks, respectively. After fasting for 24 hours, glucose 1.0 g / kg was administered intraperitoneally, and blood glucose levels were measured before administration of glucose and at 15, 30, 60 and 120 minutes after administration.
In addition, as described above, mice fed with a high fat diet were fasted for 6 hours, and then insulin 0.75 U / kg was administered intraperitoneally, before insulin administration, at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after administration. The blood glucose level was measured.
Blood was collected from the tail vein and blood glucose level was measured using Dexter ZII (Bayer).

<Result>
The blood glucose level of cGK-Tg mice was significantly lower after glucose administration and after insulin administration compared to wild type mice (FIGS. 11 and 12). This result suggests improved glucose tolerance and insulin sensitivity in cGK-Tg mice.

[Test Example 5]
Wild-type mice and cGK-Tg were raised on a normal diet (F-2, Funabashi Farm) until 10 weeks of age. After being raised on a high fat diet (60 kcal% fat, D12492, Research Diet) from 10 to 20 weeks of age, dissected under pentobarbital anesthesia under free feeding in the light period, interscapular brown adipose tissue, The quadriceps tissue and liver were removed. Immediately frozen in liquid nitrogen and stored at -80 ° C. Total RNA was extracted from the cryopreserved tissue with RNeasy mini kit for lipid tissue (Qiagen), and was converted to cDNA with ExScript RT reagent kit (Takara BIO). Gene expression was examined in ABI7300 Real-Time PCR System (Applied Biosystems) using a primer set specific for each gene (Takara BIO) and Syber Premix ExTaq (Takara BIO).
In the interscapular brown adipose tissue of cGK-Tg, peroxisome proliferators-activated receptor γ co-activator (PGC) -1α and uncoupling protein (UCP) -1 gene expression is enhanced by increasing heat production and increasing energy consumption. It was about 3 times as high as that of type mice (FIG. 13). In the skeletal muscle of cGK-Tg, PPARα that activates fatty acid oxidation in addition to increased expression of genes (PGC-1α, UCP-3) that increase heat consumption and increase energy consumption compared to wild type And PPARδ gene expression was also enhanced (FIG. 14). Also in the liver of cGK-Tg, an increase in PPARδ gene expression was observed compared to the wild type (FIG. 15). From the above, at least part of the mechanism of anti-fatty liver action by overexpression of cGK, which is an effector molecule of the GC-A system, is due to energy consumption in brown adipose tissue, skeletal muscle, and liver centered on enhancement of fatty acid oxidation. It was suggested that it was due to enhancement.

[Test Example 6]
Wild-type mice and GC-A hetero knockout mice (GC-A ^+/- ) were bred on a normal diet (F-2, Funabashi Farm) until 8 weeks of age. The animals were reared on a high fat diet (45 kcal% fat, D12451, Research Diet) from 8 to 20 weeks of age, and dissected under free feeding in the light period at 20 weeks of age.
After 14 weeks of age, weight gain was significantly increased in GC-A ^+/− mice compared to wild type (FIG. 16). At 20 weeks of age, GC-A ^+/− mice were significantly larger in body weight than the wild-type mice, and the liver weight-weight ratio and liver triglyceride content tended to be large (FIG. 17). Total fat weight was significantly increased in GC-A ^+/− mice compared to wild type (FIG. 18). There was no difference in food intake between wild-type mice and GC-A ^+/− mice (FIG. 19). Moreover, the deterioration of glucose tolerance due to a high-fat diet was enhanced in GC-A ^+/− mice compared to the wild type (FIG. 20). From the above, it was shown that in GC-A ^+/− mice, obesity and fatty liver formation due to a high-fat diet were larger than in wild-type mice, and the degree of impaired glucose tolerance was large.

Claims (52)

  A method for preventing and / or treating fatty liver by administering an agonist of natriuretic peptide receptor GC-A.   The prophylactic method and / or therapeutic method according to claim 1, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   The prevention method and / or treatment method according to claim 2, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The method for prevention and / or treatment according to claim 2, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   A method for preventing and / or treating fatty liver by administering an activator of cGMP-dependent protein kinase I (cGKI).   The method for prevention and / or treatment according to any one of claims 1 to 5, wherein the fatty liver is fatty liver induced by metabolic syndrome.   A method for preventing and / or treating metabolic syndrome by administering an agonist of natriuretic peptide receptor GC-A.   The prophylactic method and / or therapeutic method according to claim 7, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   The method for prevention and / or treatment according to claim 8, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The method for prevention and / or treatment according to claim 9, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   A method for preventing and / or treating obesity by administering an agonist of natriuretic peptide receptor GC-A.   The prophylactic method and / or therapeutic method according to claim 11, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   The method for prevention and / or treatment according to claim 12, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The method according to claim 12, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   A prophylactic and / or therapeutic agent for fatty liver comprising an agonist of natriuretic peptide receptor GC-A.   The prophylactic and / or therapeutic agent according to claim 15, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   The prophylactic and / or therapeutic agent according to claim 16, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The prophylactic and / or therapeutic agent according to claim 16, wherein the natriuretic peptide receptor GC-A agonist is atrial natriuretic peptide (ANP).   A prophylactic and / or therapeutic agent for fatty liver containing a gene for natriuretic peptide.   A prophylactic and / or therapeutic agent for fatty liver, comprising an activator of cGMP-dependent protein kinase I (cGKI).   A prophylactic and / or therapeutic agent for fatty liver containing the gene for cGMP-dependent protein kinase I (cGKI).   A preventive and / or therapeutic agent for fatty liver containing cGMP.   The preventive and / or therapeutic agent according to any one of claims 15 to 22, wherein the fatty liver is fatty liver induced by metabolic syndrome.   A kit for the prevention and / or treatment of fatty liver comprising an agonist of natriuretic peptide receptor GC-A.   25. The kit according to claim 24, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   26. The kit of claim 25, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   26. The kit of claim 25, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   A prophylactic and / or therapeutic agent for metabolic syndrome, comprising an agonist of natriuretic peptide receptor GC-A.   29. The prophylactic and / or therapeutic agent according to claim 28, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   30. The prophylactic and / or therapeutic agent according to claim 29, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   30. The prophylactic and / or therapeutic agent according to claim 29, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   A prophylactic and / or therapeutic agent for obesity comprising an agonist of natriuretic peptide receptor GC-A.   33. The prophylactic and / or therapeutic agent according to claim 32, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   The prophylactic and / or therapeutic agent according to claim 33, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The prophylactic and / or therapeutic agent according to claim 33, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   Use of an agonist of natriuretic peptide receptor GC-A for the production of a prophylactic and / or therapeutic agent for fatty liver.   37. Use according to claim 36, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   38. Use according to claim 37, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   38. Use according to claim 37, wherein the natriuretic peptide receptor GC-A agonist is atrial natriuretic peptide (ANP).   Use of a natriuretic peptide gene for the manufacture of a prophylactic and / or therapeutic agent for fatty liver.   Use of an activator of cGMP-dependent protein kinase I (cGKI) for producing a prophylactic and / or therapeutic agent for fatty liver.   Use of the gene for cGMP-dependent protein kinase I (cGKI) for the manufacture of a prophylactic and / or therapeutic agent for fatty liver.   Use of cGMP for the manufacture of a prophylactic and / or therapeutic agent for fatty liver.   44. Use according to any one of claims 36 to 43, wherein the fatty liver is fatty liver induced by metabolic syndrome.   Use of an agonist of natriuretic peptide receptor GC-A for the production of a preventive and / or therapeutic agent for metabolic syndrome.   46. Use according to claim 45, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   47. Use according to claim 46, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   The use according to claim 46, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).   Use of an agonist of natriuretic peptide receptor GC-A for the manufacture of a prophylactic and / or therapeutic agent for obesity.   50. Use according to claim 49, wherein the natriuretic peptide receptor GC-A agonist is a natriuretic peptide.   51. Use according to claim 50, wherein the natriuretic peptide is brain natriuretic peptide (BNP).   51. Use according to claim 50, wherein the natriuretic peptide is atrial natriuretic peptide (ANP).

Images