JP2004016074A - Amp activation protein kinase activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an AMPK activator in which 5'-AMP activation protein kinase (AMPK) activation has the same effect as motility of fatty acid combustion, etc., and which is useful as a therapeutic agent for obesity and type 2 diabetes. <P>SOLUTION: The AMP activation protein kinase activator comprises a C-terminal side spherical domain of adiponectin, which is one of hormones secreted from fat cell and plays a central roll in regulation of energy homeostasis, glucose and lipid metabolism, adiponectin or their gene as an active ingredient. It is elucidated that phosphorylation and activity of AMPK in vitro and in vivo are raised by administration of adiponectin. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】Ｃ２Ｃ１２筋細胞におけるアディポネクチンによる脂肪燃焼作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較、または示された２群間での比較）。
【図２】Ｃ２Ｃ１２筋細胞におけるアディポネクチンによるグルコース取り込み作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較、または示された２群間での比較）。
【図３】Ｃ２Ｃ１２筋細胞におけるアディポネクチンによるＡＭＰＫ及びＡＣＣのリン酸化に及ぼす作用を示す図である（ａ、ｂ、ｃ；ＡＰＭＫのリン酸化、ｄ、ｅ；ＡＣＣのリン酸化）。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図４】ヒラメ筋におけるアディポネクチンによるＡＭＰＫαサブユニットのリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図５】ヒラメ筋におけるアディポネクチンによるα１ＡＭＰＫ（ａ）及びα２ＡＭＰＫ（ｂ）のリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図６】ヒラメ筋におけるアディポネクチンによるＡＣＣのリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図７】Ｃ２Ｃ１２筋細胞におけるアディポネクチン刺激によるＡＭＰ濃度に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図８】ヒラメ筋におけるアディポネクチン刺激によるＡＭＰ濃度に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体との比較）。
【図９】Ｍｏｃｋ又はＤＮ−α２ＡＭＰＫ挿入レトロウイルス感染Ｃ２Ｃ１２筋細胞におけるアディポネクチンによるＡＭＰＫ及びＡＣＣリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１０】Ｍｏｃｋ又はＤＮ−α２ＡＭＰＫ挿入レトロウイルス感染Ｃ２Ｃ１２筋細胞におけるアディポネクチンによる脂肪酸燃焼作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１１】Ｍｏｃｋ又はＤＮ−α２ＡＭＰＫ挿入レトロウイルス感染Ｃ２Ｃ１２筋細胞におけるアディポネクチンによるグルコース取り込みに及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１２】Ｍｏｃｋ又はＤＮ−α２ＡＭＰＫ挿入レトロウイルス感染Ｃ２Ｃ１２筋細胞におけるアディポネクチンによる乳酸産生に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１３】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクション単離肝細胞におけるＡＭＰＫ及びＡＣＣのリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１４】アディポネクチンのＣ２Ｃ１２筋細胞との結合性を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１５】アディポネクチンの単離肝細胞との結合性を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝５〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；示された２群間での比較、もしくはｇＡｄとＡｄの比較）。
【図１６】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウスの肝におけるアディポネクチンによるＡＭＰＫαサブニユットのリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図１７】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウス肝における、アディポネクチンによるα１ＡＭＰＫ活性に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図１８】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウス肝における、アディポネクチンによるα２ＡＭＰＫ活性に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図１９】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウスの肝におけるアディポネクチンによるＡＣＣのリン酸化に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図２０】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウスを指定時間、アディポネクチンで処理（１８４μｇ／ｇ体重）した場合のアディポネクチン耐性試験時の血漿グルコースレベルを示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図２１】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウスをアディポネクチンで４時間処理（８４μｇ／ｇ体重）した場合の肝（上段）及び骨格筋（下段）のＤＮ−α１ＡＭＰＫ活性に及ぼす作用を示す図である。各バーは平均±ｓ．ｅ．（ｎ＝８〜１０）を示す。（^＊Ｐ＜０．０５；^＊＊Ｐ＜０．０１；媒体のみとの比較）。
【図２２】ＬａｃＺ又はＤＮ−α１ＡＭＰＫ挿入アデノウイルスでトランスフェクションしたマウスを、アディポネクチンで４時間処理（８４μｇ／ｇ体重）した場合の、肝中のＰＥＰＣＫおよびＧ６Ｐａｓｅ　ｍＲＮＡ量を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an AMP-activated protein kinase activator.
[0002]
Problems to be solved by the prior art and the invention
5'-AMP-activated protein kinase (AMPK) inhibits acetylcoenzyme A carboxylase (ACC) activity, thereby strongly stimulating fatty acid oxidation in muscle and promoting glucose utilization including glucose uptake and lactate production. (Ann. Rev. Biochem. 67, 821-855 (1998), Am. J. Physiol. 277, E1-E10 (1999)). That is, ACC-β isoform (ACC-β), which is a major isoform of skeletal muscle, is an indispensable regulator for fatty acid oxidation. AMPK activated in muscle contraction or hypoxia has been shown to phosphorylate ACC-β, and inhibition of ACC activity by this causes a decrease in malonyl-CoA content. This further reduces carnitine palmitoyltransferase 1 activity and increases fatty acid burning. Activation of AMPK stimulates glucose uptake independent of the effect of AMPK on fatty acids.
Therefore, activation of AMPK has the same effect as exercise such as fatty acid burning, is useful as a therapeutic agent for obesity and type 2 diabetes, and development of an AMPK activator is desired.
[0003]
[Means for Solving the Problems]
The present inventor has studied the pharmacological action of adiponectin, which is one of the hormones secreted from adipocytes and plays a central role in regulating energy homeostasis and glucose / lipid metabolism. The inventors have found that activation is stimulated by adiponectin, and completed the present invention.
[0004]
That is, the present invention provides an AMPK activator comprising a C-terminal globular region of adiponectin, adiponectin or a gene thereof as an active ingredient.
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
The adiponectin used in the present invention has already been cloned [Maeda, K et al, Biochem. Biophys. Res. Commun. 221, 286-296 (1996); Nakano, Y .; et al, J.A. Biochem. (Tokyo) 120, 802-812 (1996)]. SEQ ID NO: 1 shows the amino acid sequence and base sequence of human adiponectin. Adiponectin is composed of an N-terminal collagen-like sequence (cAd; 45 to 109) and a C-terminal spherical region (gAd; 110 to 247). The C-terminal spherical region (gAd) has a skeleton. In muscle, it has a stronger AMPK activating effect than full-length adiponectin. In the present invention, not only a protein having the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence showing the gAd region, but also an amino acid sequence in which one or more amino acids of these amino acid sequences are substituted, deleted or added. Even proteins can be used as long as they have an action as adiponectin.
[0006]
Examples of the gene used in the present invention include a gene encoding adiponectin shown in SEQ ID NO: 1 and a gene encoding gAd. Further, a gene having a nucleotide sequence capable of hybridizing with these genes under stringent conditions can also be used.
[0007]
Adiponectin or some polypeptides thereof (including gAd) can be isolated from cells in which it is present, but since the gene encoding adiponectin has already been cloned, DNA recombination techniques, that is, It may be prepared using transformed cells by using the expression vector prepared using the above method.
[0008]
As shown in the Examples below, phosphorylation and activation of AMPK is stimulated by globular adiponectin in skeletal muscle and by full-length adiponectin in liver. Adiponectin also stimulates ACC phosphorylation, fatty acid burning, glucose uptake and lactate production in muscle cells, phosphorylation of ACC and reduction of gluconeogenesis-related molecules in liver, and in vivo glucose levels in parallel with AMPK activation. It also stimulates the decline. Blocking AMPK with a dominant negative mutant inhibits all of these effects, indicating that glucose utilization and fatty acid burning by adiponectin occur through AMPK activation. These results indicate a novel paradigm in which adiponectin, one of adipocyte-derived hormones, directly regulates in vitro and in vivo glucose metabolism and insulin sensitivity by activating AMPK.
[0009]
In order to administer the medicament of the present invention to mammals including humans, pharmaceutically acceptable carriers can be added to the above-mentioned active ingredients to prepare various administration forms of pharmaceutical compositions. Such administration forms include oral administration preparations and injection preparations, with injection preparations being preferred. Pharmaceutically acceptable carriers include distilled water, solubilizers, stabilizers, emulsifiers, buffers and the like. The dose of these drugs varies depending on the disease, sex, body weight, etc., but may be about 0.1 μg to 100 mg / day as adiponectin or gAd amount.
[0010]
【Example】
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
[0011]
A. Method
(1) Animals
Male C57BL6 mice (8-10 weeks old) were purchased from Japan CREA. For in vivo AMPK activity measurement, mice were intravenously injected with the indicated amount of recombinant murine diponectin or 1 g / kg (body weight) of AICAR from the inferior vena cava catheter. At each time point, the skeletal muscle and liver were immediately excised and frozen in liquid nitrogen.
[0012]
(2) Preparation of recombinant adiponectin
Murine full-length adiponectin (Ad) and globular adiponectin (gAd) expressed in bacteria were purified by previously reported methods (Nat. Med. 7, 941-946 (2001)). Ad and gAd produced by the mammalian expression system were isolated from NIH-3T3 cells stably expressing Ad and gAd by the method already described (Nat. Med. 7, 947-953 (2001)). -Purified. AntiClean Etox affinity columns (Sterogene Bioseparations) were used to remove endotoxins that could be contaminated. The stimulatory effects on AMPK and ACC phosphorylation of C2C12 myocytes and isolated hepatocytes and on fatty acid oxidation, glucose uptake, or lactate production of C2C12 myocytes were determined by bacterially expressed gAd.Ad and gAd. No significant difference was found between Ad.
[0013]
(3) AMPK activity measurement
To measure skeletal muscle or liver isoform-specific AMPK activity, a specific antibody against the α1 or α2 catalytic subunit bound to Protein G / Sepharose beads (FEBS Lett. 397, 347-351 (1996)) was used. AMPK was immunoprecipitated from a muscle or hepatocyte lysate (protein amount 100 μg). Kinase activity was measured using a synthetic “SAMS” peptide and [γ-32P] ATP (Diabetes. 49, 527-531 (2000)).
[0014]
(4) Western blot analysis
Phosphorylation of AMPKα1 and α2 subunits and ACC in soleus muscle, liver or cell lysate (protein amount 40 μg) was carried out by using SDS acrylamide gel with a concentration gradient of 4 to 15% and around Thr172 of human AMPKα subunit (Cell Signaling). And an antibody against a phosphorylated peptide based on the amino acid sequence around Ser79 of rat ACC (Upstate Biotech). The expression level of DN-AMPK was measured using an anti-myc antibody (9E10; “Cell Center, University of Pennsylvania Medical School”).
[0015]
(5) RNA preparation and Northern blot analysis
Total RNA was prepared from tissues using TRIzol (GIBCO-BRL) according to the manufacturer's instructions. Total RNA from 5-10 mice in each group was pooled and a portion was used for Northern blot analysis (Nat. Med. 7, 941-946 (2001)) using mouse PEPCK and G6Pase probes. After quantifying the radioactivity of each band and correcting the difference in the amount of loading by measuring the amount of 28S rRNA, the fold change of each mRNA was calculated. One data out of three independent experiments is shown as a representative.
[0016]
(6) Lipid and glucose metabolism
[1- ¹⁴ C] from palmitic acid ¹⁴ C] CO ₂ The measurement of the production amount was performed using the cell lysate by the method already described (Nat. Med. 7, 941-946 (2001)). The lactic acid concentration was measured by a colorimetric method (Lactate C; Wako Pure Chemical Industries, Ltd.). Glucose uptake was measured by the method already described (Mol. Cell. Biol. 16, 3074-1308 (1996)).
[0017]
(7) Examination using C2C12 myocytes and isolated hepatocytes
A cDNA encoding α1AMPK containing a mutation from aspartic acid to alanine at residue 157 was used as DN-α1AMPK (Mol. Cell. Biol. 20, 6704-6711 (2000)). This mutation inactivates the kinase activity but does not affect the ability to bind to the β and γ subunits inside the complex. A cDNA encoding α2AMPK containing a lysine to arginine change at residue 45 was used as DN-α2AMPK (Mol. Cell. 7, 1085-1094 (2000)). This mutation also inactivates the kinase activity.
[0018]
A DN-α2AMPK (Mol. Cell. 7, 1085-1904 (2000)) expression vector for retrovirus-mediated gene transfer was constructed by ligation to the EcoRI / NotI site of pMX-puro (Mol. Cell. Biol). 18, 3871-3879 (1996)). The retrovirus (Mock vector) or DN-α2AMPK (Mol. Cell. 7, 1085-1094 (2000)) into which a control was inserted, with a slight modification to the method already described (Cell. 79, 1147-1156 (1994)). )) The inserted retrovirus was used to infect C2C12 cells at the same titer. Differentiation induction was performed according to the method already described (Proc. Natl. Acad. Sci. USA. 98, 2005-2010 (2001)). Five days later, cells were treated with the indicated concentrations of adiponectin and examined for AMPK and ACC phosphorylation, fatty acid burning, glucose uptake, and lactate production. Adenovirus into which LacZ or DN-α1AMPK (Mol. Cell. Biol. 20, 6704-6711 (2000)) was inserted was transfected into the isolated hepatocytes (Mol. Cell. Biol. 20, 6704-6711 (2000)). Was taken. Cells were treated with the indicated concentrations of adiponectin and examined for AMPK and ACC phosphorylation.
[0019]
(8) Adenovirus-mediated gene transfer in vivo and adiponectin resistance test
As already described (J. Clin. Invest. 105, 1437-1445 (2000)), the adenovirus into which LacZ or DN-α1AMPK (Mol. Cell. Biol. 20, 6704-6711 (2000)) was inserted was used. 3 × 10 per gram weight ⁸ Mice were injected at a concentration of plaque forming units (pfu). A solution in which the virus was suspended in 200 μL of PBS was intraperitoneally injected. Three to five days after injection, mice were fasted for 3 hours before testing. Glucose was measured at 0, 4, 8, and 12 hours or 4 hours after intraperitoneal administration of adiponectin (84 μg / g body weight), and mouse tissues were collected and used for Northern blot analysis. No significant changes were found in the liver of mice injected with adenovirus, including morphology.
[0020]
(9) Binding of adiponectin to C2C12 myocytes or isolated hepatocytes
Recombinant globular (gAd) or full-length (Ad) adiponectin was biotinylated with NHS-LC-Biotin (Pierce). C2C12 myocytes or isolated hepatocytes (5 × 10 6 in 96-well plates) ⁴ Cells / well) were incubated for 1 hour at 37 ° C. with the indicated concentrations of biotinylated gAd or Ad. Cell surface binding of biotinylated gAd or Ad was quantified by ELISA using streptavidin-conjugated horseradish peroxidase (HRP) and o-phenylenediamine dihydrochloride. Absorbance was measured at 492 nm. The data are shown by subtracting the binding concentration in the presence of 100-fold excess of unlabeled gAd or Ad from the total binding.
[0021]
(10) Measurement of AMP, ADP and ATP contents
Soleus muscle or C2C12 myocytes treated with adiponectin were transferred to ice-cold HClO ₄ After homogenization in a KRB buffer solution containing NaOH, NaOH was added to neutralize the cell lysate. The AMP, ADP and ATP contents in the supernatant were measured using a bioluminescence assay kit as previously described (J. Biol. Chem. 276, 2979-2985 (2001)).
[0022]
B, result
(1) Action of adiponectin in vitro
C2C12 myocytes were incubated with adiponectin in vitro. Treatment of C2C12 myocytes with adiponectin for 60 minutes stimulated fatty acid oxidation in vitro (FIG. 1). Actinomycin D (5 μg / ml) is a PPARα agonist Wy-14,643 (10 ^-5 M) Suppressed fatty acid oxidation by stimulation but did not affect the increased fatty acid oxidation seen in adiponectin-treated cells (FIG. 1), suggesting that these effects of adiponectin may be independent of transcriptional regulation. . Treatment of C2C12 myocytes with adiponectin also increased glucose uptake in vitro (FIG. 2). Furthermore, insulin-stimulated glucose uptake was inhibited by malt mannin (100 mM), but adiponectin-induced increase in glucose uptake was hardly affected (FIG. 2). These results indicate that these effects of adiponectin are expressed via a pathway independent of phosphatidylinositol 3 (PI3) kinase.
[0023]
Treatment of C2C12 myocytes with the cell-permeable AMPK activator AICAR (5-aminoimidazole-4-carboxamide 1-D-ribofuranoside), which is converted to the AMP analog ZMP, results in the persistence of Thr172 in the α subunit of AMPK. Phosphorylation occurred. In contrast, treatment with globular adiponectin or full-length adiponectin resulted in a rapidly potent and transient increase in AMPK phosphorylation (FIG. 3a). 0.1 and 0.5 μg / ml globular adiponectin and 10 and 25 μg / ml full-length adiponectin near the physiological concentration range enhance AMPK phosphorylation continuously (FIG. 3b) and dose-dependent (FIG. 3c). did. Next, it was examined whether adiponectin also stimulates ACC phosphorylation following AMPK phosphorylation. Treatment of C2C12 myocytes with globular or full-length adiponectin resulted in a continuous (FIG. 3d) and dose-dependent (FIG. 3e) increase in ACC phosphorylation. At full-length adiponectin at a concentration of 25 μg / ml, the effect reached a maximum of 2.2-fold after 15 minutes, comparable to the increase by AICAR (FIG. 3e) and returned to baseline by 60 minutes. (FIG. 3d).
[0024]
(2) Action of adiponectin in vivo
To examine whether adiponectin could stimulate AMPK phosphorylation of skeletal muscle in vivo, mice were administered adiponectin. When globular adiponectin or full-length adiponectin was administered to mice, AMPK phosphorylation in soleus muscle increased in a dose-dependent manner (FIGS. 4a, b). The effect reached a maximum of twice after 5 minutes and returned to the baseline value by 60 minutes (FIG. 4b). The effect of adiponectin on AMPK phosphorylation was more pronounced in the red muscle (slow muscle, due to oxidative phosphorylation) with a higher fatty acid oxidation rate than in the white muscle (immediate muscle, due to glycolysis), but not in the white gastrocnemius muscle. Oxidation tended to increase 5 minutes after administration of adiponectin.
[0025]
Phosphorylation of Thr172 within the AMPKα subunit involves AMPK activation of both α1 and α2. Administration of globular adiponectin to mice significantly increased α1 and α2 AMPK activity, and full-length adiponectin also significantly increased α2 AMPK activity in soleus muscle (FIGS. 5a, b). Spherical adiponectin, which has a higher binding affinity to the muscle cell surface (see FIG. 14), had a more pronounced effect on AMPK activation of skeletal muscle than full-length adiponectin (FIGS. 5a, b). Next, it was examined whether adiponectin also stimulates ACC phosphorylation following skeletal muscle AMPK phosphorylation in vivo. Administration of globular to full-length adiponectin increased soleus muscle ACC phosphorylation at 15 minutes (FIGS. 6a, b). At a concentration of about 50 μg / ml of spherical adiponectin, the maximum reached 2.2-fold at 15 minutes, which was greater than the increase by AICAR (FIG. 6b), and returned to the baseline value by 60 minutes (FIG. 6b). .
[0026]
To elucidate the mechanism by which adiponectin activates AMPK, the intracellular concentration of AMP known as an AMPK activator was measured. The AMP content of C2C12 myocytes (FIG. 7) and soleus muscle (FIG. 8) increased about 2-fold 5 minutes after adiponectin administration. ATP or ADP concentrations did not change upon administration of adiponectin. Therefore, it is considered that the activation of AMPK that occurred 5 minutes after administration of adiponectin was due to an increase in intracellular AMP concentration.
[0027]
α2AMPK is the major isoform in skeletal muscle. To investigate whether the effect of adiponectin on ACC phosphorylation, fatty acid metabolism, and glucose metabolism in muscle cells requires the AMPK pathway, it has a dominant negative effect on α1 and α2 AMPK activity in skeletal muscle (Mol. Cell). 7, 1085-1094 (2000)), AMPK activation was blocked by the AMPKα2 subunit lacking catalytic activity. Both globular and full-length adiponectin resulted in a 1.8-fold increase in AMPK phosphorylation in control C2C12 myocytes (FIG. 9, middle panel). These effects were clearly blocked by dominant negative (DN) -α2AMPK (FIG. 9, middle panel). Both spherical and full-length adiponectin increased ACC phosphorylation of C2C12 myocytes by 2.3 times (FIG. 9, bottom), fatty acid burning by 1.8 times (FIG. 10), and glucose uptake by 2 times (FIG. 11). Lactic acid production was further increased by a factor of 1.3 (FIG. 12). These effects were blocked by DN-α2AMPK. Therefore, activation of AMPK is required for stimulation of muscle cell ACC phosphorylation, fatty acid burning, glucose uptake and lactate production by adiponectin.
[0028]
Next, in order to examine whether adiponectin also activates AMPK in hepatocytes, a search was also conducted for the phosphorylation of AMPK and ACC in the isolated hepatocytes. Only full-length adiponectin had a phosphorylation-stimulating effect on AMPK (FIG. 13, middle panel) and ACC (FIG. 13, lower panel) of isolated hepatocytes, and globular adiponectin did not have this effect.
[0029]
α1 AMPK is the major isoform in the liver. To investigate whether the effect of adiponectin on ACC phosphorylation in isolated hepatocytes requires the AMPK pathway, a lack of catalytic activity with a dominant negative effect on both AMP1 activity of α1 and α2 in hepatocytes was examined. AMPK activation was blocked by the missing AMPKα1 subunit. Full-length adiponectin resulted in a 2.3-fold increase in AMPK phosphorylation in control isolated hepatocytes (FIG. 13, middle panel). These effects were clearly blocked when expressing DN-α1 AMPK by adenovirus-mediated gene transfer (FIG. 13, upper and middle panels). Full-length adiponectin resulted in a 1.7-fold increase in ACC phosphorylation in control isolated hepatocytes (FIG. 13, bottom). These effects were markedly reduced by DN-α1 AMPK (FIG. 13, lower panel). Therefore, activation of AMPK is required for stimulation of ACC phosphorylation of isolated hepatocytes by adiponectin.
[0030]
To determine the mechanism by which different forms of adiponectin work effectively in liver and muscle, we measured the binding affinity of spherical and full-length adiponectin to C2C12 myocytes or hepatocyte surfaces. The binding affinity to the C2C12 myocyte surface was higher for globular adiponectin than for full-length adiponectin (FIG. 14), and for hepatocyte surfaces, full-length adiponectin had a higher binding affinity than globular adiponectin. (FIG. 15). These are consistent with data on AMPK phosphorylation and activity in liver and muscle cells.
[0031]
Next, mice were administered adiponectin in order to examine whether adiponectin could stimulate AMPK phosphorylation and activation in the liver in vivo. Only full-length adiponectin, which has a higher binding affinity for the hepatocyte surface (FIG. 15), but not globular adiponectin, activates and phosphorylates hepatic α1 (FIG. 17) and α2AMPK (FIG. 18) (FIGS. 16a, b). It had a stimulating effect. The effect reached a maximum of 2.5 fold after 5 minutes and returned to baseline by 60 minutes (FIG. 16b). Next, it was examined whether adiponectin stimulates ACC phosphorylation following AMPK activation in the liver in vivo. Only full-length adiponectin, but not globular adiponectin, had the function of increasing hepatic ACC phosphorylation (FIGS. 19a, b). The effect reached a maximum of 2.2 times after 15 minutes and returned to the baseline value by 60 minutes (FIG. 19b).
[0032]
To investigate whether the AMPK pathway is required for in vivo adiponectin action on glucose metabolism, we injected LacZ or DN-α1 AMPK-inserted adenovirus into mice intraperitoneally, and demonstrated that adiponectin's glucose-lowering effect on both. Compared. Administration of full-length adiponectin reduced glucose levels in mice injected with LacZ-inserted adenovirus by 30% compared to administration of phosphate buffered saline (PBS) in vivo (FIG. 20). Injection of the DN-α1 AMPK-inserted adenovirus partially but significantly attenuated the glucose lowering effect of adiponectin, suggesting that at least a portion of the glucose lowering effect of adiponectin was AMPK-dependent. In order to elucidate the mechanism by which the injection of DN-α1 AMPK-inserted adenovirus into mice attenuates the glucose lowering effect of adiponectin, the expression site of DN-α1 AMPK was first searched. Although expression of DN-α1 AMPK was detected in the liver (FIG. 21, upper panel), expression was not detected in other examined tissues such as skeletal muscle (FIG. 21, lower panel).
[0033]
Next, the mechanism by which DN-α1 AMPK expression in the liver attenuates the glucose lowering effect of adiponectin was examined. Full-length adiponectin reduced expression levels of hepatic gluconeogenesis-related molecules such as PEPCK (phosphoenolpyruvate carboxylase) and G6Pase (glucose-6-phosphatase), as previously reported (FIG. 22). . DN-α1 AMPK expression in the liver blocked these effects of adiponectin (FIG. 22), which was previously reported that activation of AMPK reduced expression levels of PEPCK and G6Pase (Nature 404, 632-). 634 (2000)). Thus, activation of AMPK in the liver is required for adiponectin to reduce the level of expression of hepatic gluconeogenesis-related molecules and cause a decrease in glucose levels in vivo.
[0034]
These results indicate that adiponectin increases glucose uptake and lactate production in C2C12 myocytes and in vivo skeletal muscle, reduces hepatic gluconeogenesis-related molecule expression, reduces in vivo glucose levels, and reduces AMPK levels. Clearly show that it increases the phosphorylation and activity of In addition, adiponectin also enhances ACC phosphorylation and fatty acid burning in C2C12 myocytes and isolated hepatocytes. Dominant negative AMPK blocks adiponectin-stimulated glucose uptake, lactate production, ACC phosphorylation and fatty acid burning in muscle cells, as well as ACC phosphorylation in hepatocytes, suppression of hepatic gluconeogenesis-related molecules, and reduction of glucose levels in vivo. These effects are thought to be mediated by AMPK, since they also block the decline. In other words, the above results show for the first time that adiponectin, an adipocyte-derived hormone, activates AMPK and thereby directly regulates glucose metabolism and insulin sensitivity. Leptin can also activate AMPK, but the involvement of AMPK in glucose metabolism has not been shown for leptin.
[0035]
Furthermore, the above results indicate that, in skeletal muscle, globular adiponectin activates AMPK more than full-length adiponectin and stimulates glucose uptake and fatty acid oxidation more strongly, whereas hepatic activates AMPK. It also shows that it is exclusively full-length adiponectin. These results indicate that spherical adiponectin has a higher binding affinity to the muscle cell surface than full-length adiponectin (FIG. 14), whereas binding affinity to the hepatocyte surface is higher for full-length adiponectin than for spherical adiponectin. This is apparent from the data shown in FIG.
[0036]
【The invention's effect】
According to the present invention, it has been revealed that administration of adiponectin increases the phosphorylation and activity of AMPK in vitro and in vivo.
[0037]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the fat burning effect of adiponectin in C2C12 muscle cells. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison with vehicle or comparison between the two groups shown).
FIG. 2 is a graph showing the effect of adiponectin on glucose uptake in C2C12 myocytes. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison with vehicle or comparison between the two groups shown).
FIG. 3 shows the effects of adiponectin on the phosphorylation of AMPK and ACC in C2C12 myocytes (a, b, c; phosphorylation of APMK, d, e; phosphorylation of ACC). Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 4 shows the effect of adiponectin on the phosphorylation of AMPKα subunit in soleus muscle. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 5 is a graph showing the effect of adiponectin on the phosphorylation of α1 AMPK (a) and α2 AMPK (b) in soleus muscle. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 6 is a graph showing the effect of adiponectin on the phosphorylation of ACC in soleus muscle. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 7 is a graph showing the effect of adiponectin stimulation on AMP concentration in C2C12 myocytes. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 8 is a graph showing the effect of adiponectin stimulation on AMP concentration in soleus muscle. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium).
FIG. 9 shows the effect of adiponectin on AMPK and ACC phosphorylation in C2C12 myocytes infected with Mock or DN-α2AMPK inserted retrovirus. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 10 shows the fatty acid burning action of adiponectin in C2C12 muscle cells infected with Mock or DN-α2AMPK inserted retrovirus. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 11 is a graph showing the effect of adiponectin on glucose uptake in C2C12 myocytes infected with Mock or DN-α2AMPK inserted retrovirus. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 12 shows the effect of adiponectin on lactate production in C2C12 myocytes infected with Mock or DN-α2AMPK inserted retrovirus. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 13 shows the effect of LacZ or DN-α1 AMPK-inserted adenovirus on the phosphorylation of AMPK and ACC in isolated hepatocytes transfected with LacZ or DN-α1 AMPK. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 14 shows the binding of adiponectin to C2C12 myocytes. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 15 shows the binding of adiponectin to isolated hepatocytes. Each bar is mean ± s. e. (N = 5 to 10). ( ^* P <0.05; ^** P <0.01; comparison between the two indicated groups or comparison of gAd and Ad).
FIG. 16 shows the effect of adiponectin on the phosphorylation of AMPKα subunits in the liver of mice transfected with LacZ or DN-α1 AMPK inserted adenovirus. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 17 shows the effect of adiponectin on α1 AMPK activity in mouse liver transfected with LacZ or DN-α1 AMPK inserted adenovirus. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 18 shows the effect of adiponectin on α2AMPK activity in mouse liver transfected with LacZ or DN-α1 AMPK inserted adenovirus. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 19 shows the effect of adiponectin on ACC phosphorylation in the liver of mice transfected with LacZ or DN-α1 AMPK-inserted adenovirus. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 20 shows plasma glucose levels in an adiponectin resistance test when mice transfected with LacZ or DN-α1 AMPK-inserted adenovirus were treated with adiponectin (184 μg / g body weight) for a designated time. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 21 shows the effect on liver (upper) and skeletal muscle (lower) DN-α1 AMPK activity of mice transfected with LacZ or DN-α1 AMPK-inserted adenovirus and treated with adiponectin for 4 hours (84 μg / g body weight). FIG. Each bar is mean ± s. e. (N = 8 to 10). ( ^* P <0.05; ^** P <0.01; comparison with medium only).
FIG. 22 is a view showing the amounts of PEPCK and G6Pase mRNA in the liver when mice transfected with LacZ or DN-α1 AMPK-inserted adenovirus were treated with adiponectin for 4 hours (84 μg / g body weight).

Claims (1)

An AMP-activated protein kinase activator comprising a C-terminal globular region of adiponectin, adiponectin or a gene thereof as an active ingredient.

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