JP5354866B2 - Pancreatic β-cell proliferation promoter, blood insulin level-increasing agent, blood sugar level-lowering agent, and diabetes treatment / prevention agent - Google Patents

Description

  The present invention relates to a pancreatic β-cell proliferation promoter, a blood insulin concentration-increasing agent, a blood glucose level-lowering agent, and a diabetes therapeutic / preventive agent.

  Type 1 diabetes occurs when beta cells that synthesize and secrete insulin in the islets of Langerhans in the pancreas are destroyed and disappeared, resulting in insulin deficiency. Thus, patients with type 1 diabetes need insulin treatment throughout their lives. However, in fact, it is difficult to control the blood glucose level even if the injection is repeated 3-4 times a day, and the development of drugs and treatment methods for treating type 1 diabetes has been desired.

  In recent years, treatment methods such as pancreas transplantation and islet transplantation for type 1 diabetes patients have been developed (see, for example, Non-Patent Document 1). However, pancreas transplantation and islet transplantation have problems such as rejection and an absolute shortage of donors, and it is expected that such transplantation is difficult to perform. Therefore, it was necessary to develop drugs and treatment methods for treating type 1 diabetes other than pancreas transplantation and islet transplantation.

In addition, recently, it has been shown that the number of pancreatic β cells is decreased in patients with type 2 diabetes. From this, not only type 1 diabetes but also type 2 diabetes which occupies a large number of diabetic patients, there is a long-awaited development of a treatment method that causes an increase in pancreatic β cells and an increase in insulin secretion.
Ryan EA, Paty BW, Senior PA, Bigam D, Alfadhli E, Kneteman NM, Lakey JR, Shapiro AM: Five-year follow-up after clinical islet transplantation. Diabetes 54: 2060-2069, 2005

  An object of the present invention is to provide a novel drug effective for promoting pancreatic β cell proliferation, increasing blood insulin concentration, decreasing blood glucose level, and treating / preventing diabetes.

  The present inventors introduced a gene encoding an active MEK protein into the liver of a normal mouse using adenovirus, and examined the effect thereof. In the glucose tolerance test on the third day after virus administration, the sugar reactivity was confirmed. It was found that the blood insulin concentration increased and the blood glucose level decreased during the glucose tolerance test. In addition, the inventors found that the pancreatic insulin content of the activated MEK protein-introduced mice on the 16th day after virus administration increased to about 3 times the pancreatic insulin content of the mice introduced with the LacZ gene. Furthermore, histological examination showed a significant increase in the ratio of BrdU positive cells in islet cells in activated MEK protein-introduced mice, indicating that β cells proliferate in islets of activated MEK protein-introduced mice. The headline and the present invention were completed.

  That is, the pancreatic β-cell proliferation promoter, blood insulin concentration-increasing agent, blood sugar level-lowering agent, diabetes preventive / therapeutic agent, and pancreatic vagus nerve stimulation according to the present invention are characterized by enhancing the function of ERK protein in the liver. And Here, any of the aforementioned drugs may activate the endogenous MEK protein in the liver. In addition, any of the above-mentioned drugs may contain an active MEK protein or an active MEK protein expressing substance. The active MEK protein expression substance may be an expression vector that expresses a gene encoding the active MEK protein. Examples of diabetes to be prevented and treated include type 1 diabetes, type 2 diabetes, diabetes due to pancreatic β cell loss, diabetes due to pancreatic β cell disorder, and the like.

  Furthermore, the method for promoting pancreatic β-cell proliferation, the method for increasing blood insulin concentration, the method for lowering blood glucose level, the method for preventing or treating diabetes, the method for stimulating pancreatic vagus nerve according to the present invention include ERK in the liver in human or non-human vertebrates. It is characterized by enhancing the function of a protein. Here, in any of the above methods, endogenous MEK protein may be activated in the liver. In any of the above methods, an active MEK protein or an active MEK protein-expressing substance may be introduced into the liver. The active MEK protein-expressing substance may be an expression vector that expresses a gene encoding the active MEK protein in the liver. Examples of diabetes to be prevented and treated include type 1 diabetes, type 2 diabetes, diabetes due to pancreatic β cell loss, diabetes due to pancreatic β cell disorder, and the like.

  According to the present invention, a novel drug effective for promoting pancreatic β-cell proliferation, increasing blood insulin concentration, decreasing blood glucose level, and treating / preventing diabetes can be provided.

  Hereinafter, the present invention will be described specifically and in detail with reference to examples, but the present invention is not limited thereto.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

=== Pharmacological action ===
As shown in the Examples below, the present inventors incorporated a gene encoding an active MEK protein into an adenovirus that can efficiently introduce a gene into the liver, and using this adenovirus, normal mice (C57Bl / 6N mice) promoted the proliferation of pancreatic β cells, increased pancreatic insulin content, and lowered blood glucose levels by introducing active MEK protein into the liver.

The MEK protein is a component of the ERK signal transduction system and is also called ERK kinase, and has a function of phosphorylating and activating the ERK protein in the cell. Thus, since MEK protein functions through ERK protein, substances that enhance the function of ERK protein in the liver can promote the proliferation of pancreatic β cells, increase blood insulin concentration, decrease blood glucose level, and diabetic It can be used for prevention or treatment. Since the ERK signal transduction system governs the basic functions of cells, its functions are preserved in a wide range of animal species, so the components of the ERK signal transduction system such as MEK protein and ERK protein are derived. The animal species is not particularly limited.
Specifically, it is as follows.

(1) Pancreatic β-cell proliferation promoter The present inventors introduced a gene encoding active MEK into the liver of a normal mouse, and then measured the number of pancreatic β-cells and the islet area. It was clarified that the proliferation of β cells was promoted. Therefore, a substance that enhances the function of ERK protein in the liver is useful as a pancreatic β cell proliferation promoter.

(2) Blood insulin concentration-increasing agent At the same time, the present inventors introduced a gene encoding active MEK into the liver of a normal mouse and then measured pancreatic insulin content. As a result, the pancreatic insulin content increased. It revealed that. Here, since insulin synthesized in the pancreas is subsequently secreted into the blood, when the pancreatic insulin content is increased, the blood insulin concentration is increased. In fact, the glucose tolerance test revealed that glucose-responsive insulin secretion increased. Therefore, a substance that enhances the function of ERK protein in the liver is useful as a blood insulin level increasing agent.

(3) Blood sugar level-lowering agent In addition, the present inventors introduced a gene encoding active MEK into the liver of a normal mouse and then measured the fasting blood glucose level. It was revealed that it declined. In addition, using the same method as described above, a gene encoding active MEK was introduced into the liver of a normal mouse, and then a glucose tolerance test was performed to measure the blood glucose level. Was found to decrease to normal values. Here, the “normal value” means a blood glucose level of a healthy mouse having no disease or the like. Therefore, a substance that enhances the function of ERK protein in the liver is useful as a blood glucose level lowering agent.

(4) Diabetes preventive / therapeutic agents The present inventors are a type 1 diabetes model mouse (STZ mouse: a diabetes model mouse that produces hyperglycemia by destruction of pancreatic β cells), a diabetes model that causes progressive pancreatic β cell loss. Mice (AKITA mice: diabetes model mice in which pancreatic β cells undergo apoptosis due to endoplasmic reticulum stress and hyperglycemia) and model mice that develop type 2 diabetes due to obesity (C57BL / KSJ-db / db mice: due to leptin receptor mutations) Pancreatic insulin content increases when pancreatic insulin content and fasting blood glucose level are measured after introduction of a gene encoding active MEK into the liver of a diabetic model mouse with genetic obesity and insulin resistance) And that the fasting blood glucose level decreased to a normal level. Therefore, the substance that enhances the function of ERK protein in the liver of the present invention is useful as an agent for preventing or treating diabetes.

  Here, in general, type 1 diabetes is a type in which deficiency of insulin proceeds due to destructive lesions of pancreatic β cells, and in many cases, it is said to fall into absolute deficiency of insulin. On the other hand, type 2 diabetes is the most common type that accounts for 95% or more of diabetic patients, and it is said that decreased insulin secretion and decreased insulin sensitivity are involved in the pathogenesis. However, recently, it has been found that in the onset of type 2 diabetes, insulin deficiency is caused by pancreatic β-cell damage, closely related to the insulin resistance load. Therefore, the substance that enhances the function of ERK protein in the liver of the present invention not only serves as a preventive / therapeutic agent for type 1 diabetes caused by pancreatic β cell destruction / disappearance, but also causes pancreatic β cell damage 2 It is also useful as a prophylactic / therapeutic agent for type 2 diabetes (for example, diabetes due to obesity / insulin resistance). Here, pancreatic β-cell disorders include pancreatic β-cell dysfunction (insulin secretion failure, pancreatic β-cell differentiation abnormality, pancreatic β-cell proliferation abnormality), pancreatic β-cell number decrease (the vulnerability of existing pancreatic β-cells, existing pancreatic β-cells) Pancreatic β-cell proliferation disorder), pancreatic β-cell fatigue, and the like.

  In addition, the diabetes preventive / therapeutic agent of the present invention is useful for secondary diabetes caused by acute pancreatitis, chronic pancreatitis, necrotizing pancreatitis, pancreatic cancer, congenital anomalies, autoimmune diseases, side effects due to drug administration, environmental factors, etc. is there.

  In addition to the administration of the agent of the present invention, in addition to the administration of the agent of the present invention, insulin therapy or the like is used for type 1 diabetes, α-glucosidase inhibitor (αGI), fast-acting for type 2 diabetes. Administration of postprandial hyperglycemia inhibitors such as type I insulin secretagogues, diet therapy, exercise therapy and the like may be combined.

=== Method for enhancing function of ERK protein ===
In order to enhance the function of the ERK protein in the cell, it is not particularly limited as long as the ERK signal transduction system can be activated, and the endogenous ERK protein in the cell may be activated. An ERK protein expressing substance may be introduced. Therefore, the substance that enhances the function of the ERK protein is not particularly limited as long as it can activate the ERK signal transduction system.

(1) Method for introducing active ERK protein or active ERK protein expressing substance The method for introducing active ERK protein into cells is not particularly limited, and protein transduction domains (PTD) fusion proteins with TAT or VP22 A fusion protein may be used, and an active MEK protein synthesized in vitro may be introduced into a cell by, for example, a protein introduction reagent such as BioPorter ^™ or Chariot ^™, or injection.

  The active ERK protein expression substance introduction method is not particularly limited, such as calcium phosphate method, lipofection method, electroporation method, microinjection method, virus infection method, and various other drug delivery systems (DDS).

(2) Method for activating endogenous ERK protein To activate the endogenous ERK protein in the cell, for example, a drug that activates the ERK protein such as TPA may be used, and the upstream of the ERK signal transduction system is activated. May be used.

  In order to activate the upstream of the ERK signal transduction system in the cell, for example, the function of the MEK protein may be enhanced. As the method, an endogenous MEK protein may be activated, or an active MEK protein or an active MEK protein expression substance may be introduced.

  In order to activate the endogenous MEK protein in the cell, for example, the upstream of the MEK protein may be activated in the ERK signal transduction system, and the method is not particularly limited, but for example, Ras protein or Raf What is necessary is just to activate MEK kinases, such as protein and MEKK-1 protein. The activation method is not particularly limited. For example, when an activator of an ERK signal transduction system such as EGF is administered to the cell, or an active Raf protein or an active Raf protein expression substance is introduced into the cell. Good. The method is not particularly limited, and the active Raf protein may be injected into the cell, or an expression vector encoding the active Raf protein may be introduced into the cell by microinjection, lipofection, virus infection, or the like.

  In order to introduce an active MEK protein or an active MEK protein-expressing substance, the method described for the ERK protein in (1) may be applied to the active MEK protein.

(3) Active protein and active protein-expressing substance Here, the active Raf protein, active MEK protein, or active ERK protein are pre-acidified amino acids that are phosphorylated when each protein is activated. It can be produced by substitution with an amino acid. Specifically, for example, 338th serine for Raf protein, 218th serine and 222th serine for MEK protein, 183th threonine and 185th tyrosine for ERK protein are added to aspartic acid and glutamic acid. By substituting, each protein can be activated.

  The active protein-expressing substance is not particularly limited as long as it is a substance that expresses the active protein in the cell, and examples thereof include mRNA encoding the active protein and an expression vector expressing the gene encoding the active protein. It is done.

  The expression vector that expresses the gene encoding the active protein may be any vector, plasmid vector, or viral vector as long as it has a promoter that functions in the host cell to be introduced. In addition, when a gene is introduced, even if it is a transient expression after the gene is introduced, the expression vector is a permanent expression in which the expression vector is integrated into the chromosome. Also good.

=== Method for Producing the Drug ===
As a pharmaceutically acceptable carrier that can be used for a drug that enhances the function of the ERK protein, various organic or inorganic carrier substances that are conventionally used as pharmaceutical materials may be used. For example, excipients and lubricants in solid formulations , A binder and a disintegrant, or a solvent, a solubilizer, a suspending agent, a tonicity agent, a buffering agent, a soothing agent and the like in a liquid preparation. Further, if necessary, additives such as usual preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like may be appropriately contained. As the dosage form, oral preparations include, for example, tablets, capsules, granules, powders, fine granules, syrups, sustained-release tablets / capsules / granules, cashews, chewable tablets or drops. Examples of injections include solution injections, emulsion injections, and solid injections.

  In addition, a carrier necessary for the administration method may be contained, for example, a lipid molecule in the lipofection method.

=== Method of Administration of the above Drug in an Animal Individual ===
Administration of the agent of the present invention to an animal individual can be performed by applying the above administration method to cells.

  The dose of the drug of the present invention varies depending on age, weight, indication, or administration / intake route, but is not particularly limited as long as the above-described effects can be exhibited and the side effects to be produced are within an acceptable range.

  The administration route of the drug of the present invention is not particularly limited as long as it is a route that can enhance the function of the ERK protein in the liver.

  As a situation where the drug of the present invention is administered, for example, in a human or non-human vertebrate, a fasting blood glucose level or a blood glucose level 2 hours after a glucose tolerance test is measured, and these blood glucose levels show abnormally high levels An agent that enhances the function of the ERK protein of the present invention is administered.

In the case of borderline diabetes, when there are typical symptoms of diabetes (dry mouth, heavy drinking, polyuria, weight loss), or Hb _A1c is determined to be higher than normal (eg 6.5% or more) In the case where complications such as diabetic retinopathy are present, the agent of the present invention may be used as prevention of the onset of diabetes or treatment of diabetes.

=== Method of stimulating pancreatic vagus nerve ===
When the vagus nerve that controls the pancreas of normal mice is cut, symptoms observed in normal mice with the gene encoding active MEK protein introduced into the liver (proliferation of pancreatic β cells, increased blood insulin concentration, and blood glucose level) ) Is almost completely not recognized. Thus, activating the ERK protein in the liver stimulates the vagus nerve that governs the pancreas. Therefore, in order to stimulate the vagus nerve that controls the pancreas in humans or non-human vertebrates, the function of the ERK protein in the liver may be enhanced. Here, in the case of humans, the “vagus nerve that controls the pancreas” corresponds to the X-th cranial nerve originating from the medullary cerebral nerve cell. The method of “enhancing the function of ERK protein in the liver” is as described above.

  Hereinafter, although the embodiment described above will be specifically described using examples, this is an exemplification, and the present invention is not limited to these examples.

<Example 1: Effect of introduction of active MEK gene into liver of normal mouse>
In this example, a gene encoding active MEK was introduced into the liver of a normal mouse (wild-type mouse), and the blood glucose level, serum insulin level, pancreatic cell count / islet count in the mouse were measured.

(1) Experimental animal In this Example, 8-week-old male C57Bl / 6N mice (Kudo Co., Ltd.) were used as normal mice.

(2) Production of genetically modified adenovirus Xenopus MEK1 protein (SEQ ID NO: 1 shows the base sequence of MEK1 cDNA and SEQ ID NO: 2 shows the amino acid sequence of MEK1) (Fukuda M, Gotoh I, Adachi M, Gotoh Y , Nishida E: A novel regulatory mechanism in the mitogen-activated protein (MAP) kinase cascade. Role of nuclear export signal of MAP kinase kinase. J Biol Chem 272: 32642-32648, 1997). In addition, a gene encoding an active MEK1 protein in which 222nd serine is converted to glutamic acid and 11th and 37th leucine to alanine (hereinafter referred to as CAM gene) (SEQ ID NO: 3 shows the nucleotide sequence of CAM cDNA) SEQ ID NO: 4 shows the amino acid sequence of CAM.). Specifically, using a cDNA encoding Xenopus MEK1 protein as a template, a PCR reaction is performed using a primer having a substitution base for introducing the amino acid mutation, and a double-stranded DNA containing a mutation-containing sequence is obtained. A full-length CAM gene was prepared by replacing and ligating with a restriction enzyme and replacing part of the wild-type cDNA with a DNA fragment having a mutation. Furthermore, the CAM gene is incorporated into a cosmid substituted so that the adenovirus genome E1 region can be expressed under the CAG promoter, cut with the restriction enzyme EcoT22I, and then transfected into HEK293 cells with the DNA-terminal protein complex (COS- TPC method (Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus Proc Natl Acad Sci USA, 93: 1320-1324, 1996), an adenoviral vector expressing CAM gene (hereinafter referred to as CAM-Ad) was prepared. For control, a genetically modified adenovirus vector (hereinafter referred to as LacZ-Ad) produced by incorporating a β-galactosidase gene was used.

(3) Introduction of CAM gene into normal mouse liver (i) Activation of ERK pathway
1.5 × 10 ⁸ PFU / body of adenovirus (CAM-Ad and lacZ-Ad) was injected into the tail vein of normal mice (C57Bl / 6N) to infect the liver with the virus. On the third day after virus administration, the degree of phosphorylation of ERK in the liver was confirmed by Western blotting. The method is as follows.

First, the mouse liver was removed and the liver was buffered (100 mM Tris pH 8.5, 250 mM NaCl, 1% BP-40, 1 mM EDTA, 40 mM Glycerol 2-phosphate, 50 mM NaF, 1 mM Na ₃ VO ₄ , 350 μg / ml phenylmethane) (sulfonyl fluoride, 2 μg / ml Aprotinin, 2 μg / ml Leupeptin) and homogenized on ice. The homogenized tissue was centrifuged at 14,000 × g for 10 minutes at 4 ° C., and the supernatant containing the protein extract (180 μg total protein) was boiled in Laemmli buffer containing 10 mM dithiothreitol. Next, protein amount was quantified using Protein assay kit (Bio-Rad, Heracules, CA), and 15 μg of protein per sample was subjected to SDS-PAGE. Thereafter, the protein was blotted on a membrane by a conventional method, and ERK phosphorylation was performed using ECL plus a Western Blotting Detection System Kit (Amersham Buckinghamshire, UK) and anti-phosphorylated ERK antibody (# 4376, Cell Signaling Technology, Danvers, MA). Oxidation was detected. For detection of ERK protein expression level, an anti-ERK antibody (# 9102, Cell Signaling Technology) was used.

  As a result, the liver of the mouse into which the CAM gene was introduced (hereinafter referred to as CAM mouse) was significantly different from that of the mouse into which the LacZ gene had been introduced (hereinafter referred to as LacZ mouse). Increased phosphorylation was observed (Fig. 1a). This indicated that CAM-Ad was infected in the liver of mice.

(Ii) Glucose tolerance test A glucose tolerance test was performed on CAM mice and LacZ mice prepared in the same manner as in (i). First, on the third day of virus administration, 2 g / kg body weight glucose was intraperitoneally administered, blood was collected from the tail vein before glucose administration (0 min), 15 min, 30 min, 60 min, 120 min after administration, Blood glucose levels and serum insulin levels were measured. Insulin levels were measured using a highly sensitive mouse insulin assay kit or an ultrasensitive mouse insulin assay kit (Otsuka Pharmaceutical Co., Ltd.).

As shown in FIG. 1b, both fasting blood glucose and post-load blood glucose were significantly decreased in CAM mice compared to LacZ mice. On the other hand, as shown in FIG. 1c, the serum insulin value measured simultaneously showed a significant increase at the 15 minute value after glucose loading in CAM mice, and glucose-responsive insulin secretion increased in CAM mice. It was shown that the blood glucose level decreased during the stress test.
In addition, the pancreatic insulin content on days 4, 11, 16, and 20 of virus administration was measured as follows. First, about 2/3 of the entire pancreas was collected from the mouse, centering on the pancreatic tail, homogenized in Acid ethanol, and stored in a -20 ° C freezer. After sonication for 5 minutes on the next day and the day after next, the supernatant was collected by centrifugation at 10,000 rpm for 10 minutes, and the insulin level was measured with the above kit. The obtained value was corrected with the collected pancreas weight to obtain the pancreatic insulin concentration.
As shown in FIG. 1d, the pancreatic insulin content gradually increased after virus infection, and increased to about twice the pancreatic insulin content of LacZ mice on the 16th day after virus administration.
Thus, by expressing active MEK in the liver, fasting blood glucose and post-load blood glucose are reduced, and glucose-responsive insulin secretion is enhanced.

(Iii) Histological examination Furthermore, the pancreatic tissue after virus administration was observed. Specifically, it is as follows.
First, the pancreas was excised from the mice on days 3, 9, 15, and 21 of virus administration. The pancreas was cut into 3 μm sections every 500 μm, stained with insulin, and the total number of islets on the sections was counted. The number of islets measured was standardized by dividing by the number of tissue sections, and the number of islets per unit volume was compared.
As shown in FIG. 2a, in this histological examination, the number of islets increased with time in the CAM mice, and after the 15th day after virus administration, a significant increase was observed as compared with the LacZ mice.

  In addition, BrdU staining was performed using a BrdU assay kit (BD bioscience, San Jose, Calif.) To examine cell proliferation. Specifically, 3 days, 9 days, 15 days, and 21 days after virus administration, 1 ml of BrdU (1 mg / ml) was injected intraperitoneally, and 24 hours later, the pancreas was excised, The whole was performed as described above and subjected to immunostaining for BrdU as follows. First, the section was heated to 89 ° C. by autoclaving in the mixed solution of solution 1 and solution 2 attached to the kit to activate the antigen. Thereafter, a 10-fold diluted biotinylated anti-BrdU antibody in the kit was dropped on the section, and an antigen-antibody reaction was performed at room temperature for 1 hour. Furthermore, Streptavidin-HRP in the kit was added and reacted at room temperature for 30 minutes, and then reacted with 3,3′-diaminobenzidine (DAB) chromogen solution for 5 minutes to develop color.

  As shown in FIG. 2b, a significant increase in the ratio of BrdU positive cells in the islet cells of CAM mice was observed from the third day after virus administration. Although a significant increase in the ratio of BrdU positive cells continued on day 9 after virus administration, no difference from LacZ mice was observed on day 15 after virus administration (FIG. 2c). From this, it was shown that in the CAM mouse islets, there are cells whose proliferation is enhanced immediately after virus administration.

  Furthermore, in order to examine what kind of cell proliferation in the islet is enhanced, double staining of BrdU and insulin was performed using a pancreatic section of the CAM mouse on the third day after virus administration. Double staining is performed by BrdU staining as described above, and after the color development reaction of the first color using DAB solution (originally brown, but shown in gray in FIG. 2d), anti-insulin antibody (Sigma, St .Louis, MO) for 1 hour at room temperature and peroxidase-labeled secondary antibody (Nichirei Co., Ltd.) for secondary antibody reaction for 1 hour at room temperature. AEC) A second color development reaction (originally red but shown in black in FIG. 2d) was performed using a substrate kit (Nichirei).

  As a result, as shown in FIG. 2d, 97.6% of the cells with a gray signal have a black signal, that is, 97.6% of the BrdU positive cells in the islets are also positive for insulin. It became clear to show.

  Since the ERK pathway has an important effect on cell proliferation (Roux PP, Blenis J: ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68: 320 -344, 2004), it was suggested that CAM Ad may directly act on β cells to promote proliferation.

  Therefore, Western blotting was performed on the isolated islets of LacZ mice and CAM mice on the third day after virus administration using a phosphorylated ERK antibody as follows to examine the degree of phosphorylation of ERK.

First, the pancreas was removed from the corresponding mouse and the pancreas was buffered (100 mM Tris pH 8.5, 250 mM NaCl, 1% BP-40, 1 mM EDTA, 40 mM Glycerol 2-phosphate, 50 mM NaF, 1 mM Na ₃ VO ₄ , 350 μg / and homogenized in (ml phenylmethane sulfonyl fuloride, 2 μg / ml Aprotinin, 2 μg / ml Leupeptin). The homogenized tissue was centrifuged at 14,000 × g for 10 minutes at 4 ° C., and the supernatant containing the protein extract (180 μg total protein) was boiled in Laemmli buffer containing 10 mM dithiothreitol. After SDS-PAGE, it was transferred to a membrane, and phosphorylated ERK was detected with an anti-phosphorylated ERK antibody (# 4376, Cell Signaling Technology, Danvers, Mass.). The results were detected using the ECL plus a Western Blotting Detection System Kit (Amersham Buckinghamshire, UK). Western blotting was performed using an anti-ERK antibody (# 9102, Cell Signaling Technology), and it was confirmed that there was no change in the expression level of ERK protein.

  As a result, as shown in Fig. 2e, there was no obvious difference in the degree of phosphorylation of ERK between the two groups, so it was concluded that cell proliferation in the pancreas was not due to CAM Ad acting on the pancreas. It was.

  Thus, in CAM mouse islets, β-cell proliferation is enhanced by mechanisms other than the direct growth-promoting effect of CAM Ad infecting the pancreas, resulting in an increase in the number of islets and pancreatic insulin content. Has increased.

<Example 2: Involvement of vagus nerve governing pancreas in pancreatic β cell proliferation by introduction of CAM gene into liver>
It is known that the pancreas is innervated from the anteroposterior trunk of the vagus nerve that runs on the ventral and dorsal sides of the esophagus. Therefore, first, the abdomen of the mouse was opened through a midline incision to expose the esophagogastric junction, and the anterior vagus nerve trunk running on the esophagus was cut near the esophagogastric junction. Thereafter, the abdominal organs such as the intestinal tract, stomach, and spleen were inverted to the right to expose the vicinity of the celiac artery branching from the abdominal aorta. The vagal nerve branch branching from the posterior trunk of the vagus nerve running on the back side of the esophagus and running along the celiac artery was cut as close as possible to the pancreas (vagotomy (VG)). The virus was administered after a recovery period of 1 week after surgery. The analysis results after virus administration were compared with the group (sham operation (SO)) in which surgery was performed only to expose the esophagogastric junction and the celiac artery after laparotomy.

  In VG-treated mice (VG-CAM mice), the sugars observed on the 3rd day of virus administration in mice administered SO and CAM viruses were administered (SO-CAM mice) During the stress test, the decrease in blood glucose level, the increase in glucose-responsive insulin secretion, and the increase in pancreatic insulin content observed on the 16th day were almost completely not observed (FIGS. 3a, b, c). In VG-CAM mice, no increase in BrdU positive cells in the islets on the third day after virus administration in SO-CAM mice was observed (Fig. 3d).

  This indicates that the increase in glucose-responsive insulin secretion by introducing the CAM gene into the liver and the increase in pancreatic insulin content due to the proliferation of pancreatic β cells occur via the vagus nerve that controls the pancreas, Again, it was shown that CAM pancreatic virus infection was not the cause.

<Example 3: Involvement of hepatic ERK pathway in islet hypertrophy in insulin resistance model mice>
In this example, the involvement of the ERK pathway of the liver in the islet hypertrophy of insulin resistance model mice was examined.

  It has already been reported that phosphorylation of ERK is enhanced in the liver of ob / ob mice (Yang S, Lin HZ, Hwang J, Chacko VP, Diehl AM: Hepatic hyperplasia in noncirrhotic fatty livers: is obesity -Relevant hepatic steatosis a premalignant condition? Cancer Res 61: 5016-5023, 2001), Western blotting was used to determine the degree of ERK phosphorylation in the livers of high fat diet mice (HFD mice) and ob / ob mice I investigated. Note that high-fat diet (32% safflower oil, 33.1% casein, 17.6% sucrose, 5.6% cellulose) loaded mice (HFD mice) were loaded on 5-week-old C57Bl / 6N mice for 4 weeks, ob For / ob mice (Nippon Charles River Co., Ltd.), 8-week old mice were used. As a result, increased phosphorylation of ERK was also observed in the liver of HFD mice, as in ob / ob mice (FIG. 4a).

Similarly, the dominant-negative mutant MEK1 gene (dominant-negative mutant (DNM) (Ueyama T, Kawashima) was developed in db / db (C57Bl / 6 KSJ back gound) mice that exhibited insulin resistance and swollen islets. S, Sakoda T, Rikitake Y, Ishida T, Kawai M, Yamashita T, Ishido S, Hotta H, Yokoyama M: Requirement of activation of the extracellular signal-regulated kinase cascade in myocardial cell hypertrophy.J Mol Cell Cardiol 32: 947- 960, 2000) was similarly infected with 5 × 10 ⁸ PFU / body, ERK phosphorylation in the liver on day 7 after virus administration decreased (Fig. 4b) and pancreatic insulin content Was significantly reduced compared to LacZ mice (Fig. 4c).
Thus, activation of the ERK pathway in the liver was also involved in physiological pancreatic β cell proliferation and islet hypertrophy.

<Example 4: Effect of CAM gene introduction on insulin-deficient model mice>
When beta cells are lost, insulin exhibits an absolute or relative deficiency. Therefore, CAM virus infection was performed at 1.5 × 10 ⁸ PFU / body on the liver of mice that developed diabetes, and the effect was examined. In the following experiments, STZ mice that are type 1 diabetes model mice, AKITA mice that progressively decrease insulin secretion, and C57BL / KSJ-db that are type 2 diabetes model mice that develop insulin resistance due to obesity A / db mouse was used.

(1) STZ mice that have been treated with STZ to destroy β cells
(i) Experimental animals
STZ mice were prepared by administering 150 mg / kg of STZ (Sigma) to 8-week-old C57Bl / 6N mice, and the fasting blood glucose level increased to 250 mg / dl or more 16 days after administration. Using.
(Ii) Effects of gene transfer
Mice with a fasting blood glucose level of 250 mg / dl or more on the 16th day after STZ administration were selected, and CAM virus administration to the liver was performed in the same manner (STZ-CAM mice).
The blood glucose level of STZ-CAM mice was significantly decreased from the 4th day after virus administration compared with STZ mice (STZ-LacZ mice) into which LacZ gene was introduced, and the blood glucose levels of STZ-CAM mice were normalized. Furthermore, the decrease continued on day 16 after virus administration (FIG. 5a). Furthermore, the number of BrdU-positive cells in the pancreatic islets on day 3 of virus administration in STZ-CAM mice increased significantly (Fig. 5b), and the pancreatic insulin content on day 16 of virus administration increased significantly compared to STZ-LacZ mice. Recognized (Figure 5c).

(2) AKITA mice in which β-cells fall off due to ER stress (i) Experimental animals
Five-week-old male AKITA mice (Kudo Co., Ltd.) were used for the following experiments.
(Ii) Effects of gene transfer
Similarly, CAM and LacZ genes were introduced into AKITA mice (AKITA-CAM mice and AKITA-LacZ mice). The blood glucose level of AKITA-CAM mice was significantly lower than that of AKITA-LacZ mice from the 4th day after virus administration, and the decrease continued on the 16th day after virus administration (FIG. 6a). In addition, BrdU-positive cells in the islet on day 3 after virus administration in AKITA-CAM mice increased significantly (Fig. 6b), and pancreatic insulin content on day 16 after virus administration increased significantly compared to STZ-LacZ mice. (Fig. 6c).

(3) db / db mice exhibiting marked leptin resistance and insulin resistance due to lack of leptin receptor (i) experimental animals
8-week-old male db / db (C57Bl / 6 KSJ back ground) mice (Nippon Charles River Co., Ltd.) were used.
(Ii) Effects of gene transfer
Similarly, gene transfer of CAM and LacZ was performed for db / db KSJ mice (db / db-CAM mice and db / db-LacZ mice). Compared with db / db-LacZ mice, the blood glucose level of db / db-CAM mice showed a significant decrease from the fourth day after virus administration, and this decrease persisted even on the eighth day after virus administration (FIG. 7a). Furthermore, the number of BrdU positive cells in the pancreatic islets on day 3 after virus administration in db / db-CAM mice increased significantly (Fig. 7b), and the pancreatic insulin content on day 8 after virus administration was significantly higher than in STZ-LacZ mice Increase was observed (FIG. 7c).

(4) Conclusion As described above, the enhancement of the function of ERK protein in the liver decreases the blood glucose level of mice that have developed diabetes due to the loss of β cells, proliferates β cells, and increases the secretion amount of insulin. Therefore, enhancement of the function of ERK protein in the liver is effective for the prevention and treatment of diabetes.

In one Example of this invention, it is a figure which shows the various measurement results when an active MEK gene is introduce | transduced into the liver of a normal mouse. (A) is the result of measuring phosphorylation of ERK protein, (b) is the result of measuring blood glucose level after glucose tolerance test, (c) is the result of measuring serum insulin level at glucose tolerance test (D) shows the results of measuring pancreatic insulin content. In one Example of this invention, it is a figure which shows the histological-analysis result when an active type MEK gene is introduce | transduced into the liver of a normal mouse. (A) is the result of measuring the number of islets over time, (b) is the result of observing BrdU positive cells in the cells in the islets, (c) is the result of measuring the number of BrdU positive cells in the cells in the islets (D) shows the result of double staining of BrdU and insulin in the cells in the islet, and (e) shows the result of measuring phosphorylation of ERK protein in the isolated islet. In one Example of this invention, it is a figure which shows the various measurement results when an active type MEK gene is introduce | transduced into the liver of the mouse | mouth which performed vagotomy. (A) and (b) are the results of measuring blood glucose levels and serum insulin levels during the glucose tolerance test, (c) are the results of measuring pancreatic insulin content, and (d) are BrdU positive in islet cells. The result of measuring the number of cells is shown. In one Example of this invention, it is a figure which shows the measurement result of activation of the ERK pathway in the liver of a type 2 diabetes model mouse. (A) shows the result of measuring phosphorylation of ERK protein in the liver of a type 2 diabetes model mouse. (B) shows phosphorylation of ERK protein in the liver when the dominant-inhibitory mutant MEK1 gene was introduced. The results (c) show the results of measuring the pancreatic insulin content of type 2 diabetes model mice when the dominant inhibitory mutant MEK1 gene was introduced. In one Example of this invention, it is a figure which shows the various measurement results when an active type MEK gene is introduce | transduced into the liver of a type 1 diabetes model mouse (STZ mouse). (A) shows the result of measuring fasting blood glucose level, (b) shows the result of measuring BrdU positive cells in the cells in the islet, and (c) shows the result of measuring pancreatic insulin content. In one Example of this invention, it is a figure which shows the various measurement results when an active type MEK gene is introduce | transduced into the liver of a type 1 diabetes model mouse (AKITA mouse). (A) shows the result of measuring fasting blood glucose level, (b) shows the result of measuring BrdU positive cells in the cells in the islet, and (c) shows the result of measuring pancreatic insulin content. In one Example of this invention, it is a figure which shows the various measurement results when an active type MEK gene is introduce | transduced into the liver of a type 2 diabetes model mouse (db / db ksj mouse). (A) shows the result of measuring fasting blood glucose level, (b) shows the result of measuring BrdU positive cells in the cells in the islet, and (c) shows the result of measuring pancreatic insulin content.

Claims (22)

A method of promoting the proliferation of pancreatic β cells in vertebrates other than humans,
A method for promoting pancreatic β-cell proliferation, comprising enhancing the function of ERK protein in the liver.   2. The method for promoting pancreatic β-cell proliferation according to claim 1, wherein endogenous MEK protein is activated in the liver.   2. The method for promoting pancreatic β cell proliferation according to claim 1, wherein an active MEK protein or an active MEK protein-expressing substance is introduced into the liver.   4. The method of promoting pancreatic β-cell proliferation according to claim 3, wherein an expression vector that expresses the gene encoding the active MEK protein is introduced into the liver. A method for increasing blood insulin levels in vertebrates other than humans,
A method for increasing blood insulin concentration, comprising enhancing the function of ERK protein in the liver.   6. The method for increasing blood insulin concentration according to claim 5, wherein endogenous MEK protein is activated in the liver.   6. The method for increasing blood insulin concentration according to claim 5, wherein an active MEK protein or an active MEK protein-expressing substance is introduced into the liver.   8. The method for increasing blood insulin concentration according to claim 7, wherein an expression vector expressing the gene encoding the active MEK protein is introduced into the liver. In a non-human vertebrate, a method for lowering blood glucose level,
A method for lowering blood glucose level, which comprises enhancing the function of ERK protein in the liver.   The method for lowering blood glucose level according to claim 9, wherein endogenous MEK protein is activated in the liver.   The method for lowering blood glucose level according to claim 9, wherein an active MEK protein or an active MEK protein-expressing substance is introduced into the liver.   The method for lowering blood glucose level according to claim 11, wherein an expression vector for expressing the gene encoding the active MEK protein is introduced into the liver. A method for preventing and treating diabetes in vertebrates other than humans,
A method for preventing and treating diabetes, which comprises enhancing the function of ERK protein in the liver.   The method for preventing or treating diabetes according to claim 13, wherein endogenous MEK protein is activated in the liver.   The method for preventing or treating diabetes according to claim 13, wherein an active MEK protein or an active MEK protein-expressing substance is introduced into the liver.   The method for preventing or treating diabetes according to claim 15, wherein an expression vector expressing the gene encoding the active MEK protein is introduced into the liver. The method for preventing or treating diabetes according to any one of claims 13 to 16, wherein the diabetes is type 1 diabetes or type 2 diabetes.   The method for preventing or treating diabetes according to any one of claims 13 to 16, wherein the diabetes is diabetes caused by pancreatic β cell loss or diabetes caused by pancreatic β cell damage. A method of stimulating the vagus nerve that controls the pancreas in vertebrates other than humans,
A method for stimulating pancreatic vagus nerve, comprising enhancing the function of ERK protein in the liver.   20. The method of stimulating pancreatic vagus nerve according to claim 19, wherein endogenous MEK protein is activated in the liver.   20. The method for stimulating pancreatic vagus nerve according to claim 19, wherein an active MEK protein or an active MEK protein-expressing substance is introduced into the liver.   The method for stimulating pancreatic vagus nerve according to claim 21, wherein an expression vector expressing the gene encoding the active MEK protein is introduced into the liver.