20131107Epac2A&AdipoRon

Report
Journal Club
Takahashi T, Shibasaki T, Takahashi H, Sugawara K, Ono A, Inoue N, Furuya
T, Seino S.
Antidiabetic Sulfonylureas and cAMP Cooperatively Activate Epac2A.
Sci Signal. 2013 Oct 22;6(298):ra94.
Okada-Iwabu M, Yamauchi T, Iwabu M, Honma T, Hamagami KI, Matsuda K,
Yamaguchi M, Tanabe H, Kimura-Someya T, Shirouzu M, Ogata H, Tokuyama
K, Ueki K, Nagano T, Tanaka A, Yokoyama S, Kadowaki T.
A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity.
Nature. 2013 Oct 30. doi: 10.1038/nature12656.
2013年11月7日 8:30-8:55
8階 医局
埼玉医科大学 総合医療センター 内分泌・糖尿病内科
Department of Endocrinology and Diabetes,
Saitama Medical Center, Saitama Medical University
松田 昌文
Matsuda, Masafumi
Glutamate
Prentki M, Matschinsky FM, Madiraju SR.: Metabolic signaling in fuel-induced
insulin secretion. Cell Metab. 18:162-85, 2013.
sulfonylureas
Glipizide
Meglitinides (glinides)
nateglinide
Chlorpropamide
Tolbutamide
mitiglinide
Acetohexamide
Glibenclamide (glyburide)
Tolazamide
repaglinide
Glimepiride
Gliclazide
http://www.jst.go.jp/pr/announce/20090731/
1Division
of Molecular and Metabolic Medicine, Kobe University Graduate
School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
2Division of Diabetes and Endocrinology, Kobe University Graduate School
of Medicine, Kobe 650-0017, Japan. 3Division of Cellular and Molecular
Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017,
Japan. 4PharmaDesign Inc., 2-19-8, Hatchobori, Chuo-ku, Tokyo 104-0032,
Japan.
Sci Signal. 2013 Oct 22;6(298):ra94.
BACKGROUND
Sulfonylureas are widely used drugs for
treating insulin deficiency in patients with
type 2 diabetes. Sulfonylureas bind to the
regulatory subunit of the pancreatic b cell
potassium channel that controls insulin
secretion. Sulfonylureas also bind to and
activate Epac2A, a member of the Epac
family of cyclic adenosine monophosphate
(cAMP)–binding proteins that promote
insulin secretion through activation of the
Ras-like guanosine triphosphatase Rap1.
MATERIALS AND METHODS 1
Reagents Glibenclamide, tolbutamide, chlorpropamide, acetohexamide, and glipizide were
purchased from Sigma. Gliclazide was from LKT Laboratories Inc. [3H]Glibenclamide was from
PerkinElmer. 8-pCPTwas from BioLog Life Science Institute.
Molecular docking simulation and calculation of binding affinities MOE (CCG Inc.) and MOEASEDock (Ryoka Systems Inc.) (25) were used in docking studies of sulfonylureas to the active
form of cNBD-A of Epac2A as well as in constructing the active form of cNBD-A of Epac2A. We
constructed two types of cNBD-A models: one with the His124 side chain toward the inside of the
pocket, and the other with the side chain toward the outside. We then used ASEDock to dock
sulfonylureas against both cNBD-A models. For free energy calculations of sulfonylurea binding, we
used the docked structures with His124-inside models. Using NAMD (43) for optimization of docked
structures, we calculated the free energy of binding using solvated interaction energies (44).
Cell culture and transfection MIN6-K8 b cells, Epac2A (Rapgef4)–deficient mouse clonal
pancreatic b cells, and COS-1 cells were grown in Dulbecco’s modified Eagle’s medium containing
10% heat-inactivated fetal bovine serum and maintained in a humidified incubator with 95% air and
5% CO2 at 37℃ (12, 20, 24). Two days before FRETmeasurements, cells were transiently
transfected with plasmids encoding FRET sensor, using Effectene Transfection Reagent (Qiagen).
FRET experiments FRET experiments in MIN6-K8 b cells transfected with mouse wild-type
Epac2A, mutant Epac2A, human wild-type Epac1 (45), or mutant Epac1 FRET sensor were
performed as described previously (20). EYFP/ECFP ratio for Epac2A or EYFP/citrine ratio for
Epac1 was normalized to R0 to describe FRET efficiency changes (FRET change = R/R0, where
R0 is the ratio at time 0). FRET changes were acquired every 5 s.
MATERIALS AND METHODS 2
Site-directed mutagenesis Site-directed mutagenesis was carried out with a KOD-PlusMutagenesis kit (Toyobo) according to the manufacturer’s instructions. Mutations were confirmed
with an ABI 3700 PRISM (PerkinElmer Life Sciences) automated sequencer.
Sulfonylurea binding experiments Sulfonylurea binding experiments were performed as
described previously (20). Briefly, COS-1 cells transfected with Epac2A complementary DNA
(cDNA) were resuspended in assay buffer containing 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2,
1.2 mMKH2PO4, 1.2 mMMgSO4, 5 mM NaHCO3, and 20 mM Hepes (pH 7.4). Each aliquot was
incubated for 1 hour at room temperature with [3H]glibenclamide in the absence or presence of
unlabeled glibenclamide at various concentrations. Bound [3H]glibenclamide was separated from
free [3H]glibenclamide by rapid vacuum filtration through Whatman GF/C filters (Whatman
International). The filters were washed with ice-cold buffer, and the radioactivity was determined
with a liquid scintillation counter.
Measurement of Rap1 activity Pull-down assay for Rap1-GTP (guanosine triphosphate) was
performed as described previously (12). Dimethyl sulfoxide was used as vehicle. Precise
quantification was achieved by densitometric analysis of the immunoreactive bands with the
National Institutes of Health Image software. The intensity of the Rap1-GTP signal was normalized
to that of total Rap1. Anti-Rap1 antibody was purchased from Millipore (07-916).
.
MATERIALS AND METHODS 3
Adenovirus-mediated gene transfer Adenovirus-mediated gene transfer was performed as
described previously (20). Briefly, recombinant adenovirus carrying mCherry (Ad-mCherry) or
Epac2A (Ad-Epac2A) was generated according to the manufacturer’s instructions (Invitrogen).
Epac2A-deficient b cells were infected with Ad-mCherry or Ad-Epac2A. After 3 days of culture, the
infected cells were preincubated with 2.8 mM glucose and then incubated with 2.8 mM glucose plus
various stimuli for 15 min. GTP-bound Rap1 was assessed.
Measurement of cAMP Measurement of cAMP was performed as described previously (20). Briefly,
MIN6-K8 b cells were seeded at a density of 6 × 104 cells per well (96-well plate) and cultured for
2 days. After 30 min of preincubation with Krebs- Ringer bicarbonate buffer containing 2.8 mM
glucose, the cells were treated with vehicle or adrenaline in the same buffer for 15min.The
cellswere incubated, and cellular cAMP concentration was determined by HTRF (homogeneous
time-resolved fluorescence assay) with theCisbio cAMPfemto 2 kit (Cisbio International) according
to the manufacturer’s instruction.
Epac2A
Epac1
The cNBD of Epac1
binds cAMP with
high affinity,
whereas in Epac2A,
cAMP binds to the
cNBD-A, the first
cNBD, with low
affinity and to the
cNBD-B, the
second cNBD, with
high affinity
Fig. 1. Effects of 8-pCPT and sulfonylureas on Epac activation as revealed by FRET in MIN6K8 b cells. (A) FRET emission ratio time course of wild-type (WT) Epac2A exposed to the cAMP
analog 8-pCPT. (B) FRET emission ratio time course ofWT Epac2A exposed to the indicated
sulfonylurea at the indicated concentration. (C) FRET emission ratio time course of WT Epac1
exposed to the cAMP analog. (D) FRET emission ratio time course of WT Epac1 exposed to the
indicated sulfonylurea at the indicated concentration. FRET change = R/R0. Data are presented as
means ± SEM (n = 4 to 9 experiments for each point).
Fig. 2. Structural model of the active form of the cNBD-A of Epac2A.
(A) Diagram of the domain structures of Epac2A and Epac1. The regulatory region contains one or two cNBDs
and a DEP domain. The catalytic region contains the REM, RA domain, and CDC25-HD. The numbers of the
amino acid residues indicate the approximate domain boundaries within the primary structures.
(B) Superimposition of the active form of cNBD-B in Epac2A and the inactive form of cNBD-A in Epac2A. Magenta,
active form of cNBD-B in Epac2A (PDB ID: 3CF6); green, inactive form of cNBD-A in Epac2A (PDB ID: 1O7F).
(C) Structural model of the active form of cNBD-A in Epac2A. We constructed a structural model of the active form
of cNBD-A with the inactive form of cNBD-A (amino acid residues 44 to 139, PDB ID: 1O7F) and the active form of
cNBD-B (amino acid residues 450 to 477, PDB ID: 3CF6). AMBER99 force field was applied for refinement of the
structure. Yellow, b sheet; red, a helix. Labeled residues were predicted to interact with sulfonylureas.
(D) Structural model of the active form of RA domain in Epac2A (PDB ID: 3CF6). The predicted amino acid
residues that interact with sulfonylureas by docking simulation are shown in (C) and (D).
Fig. 3. Predicted models of interactions of sulfonylurea and Epac2A. (A) Core structure of the sulfonylureas.
(B) Model of the interaction between Epac2A cNBD-A and the inactive sulfonylurea. (C) Model of the interaction
between Epac2A cNBD-A and sulfonylureas requiring a relatively high concentration to activate Epac2A. (D)
Model of sulfonylureas requiring a relatively low concentration to activate Epac2A. (E) Model of the interaction
between Epac2A cNBD-A and sulfonylurea. (F) Model of the interaction between Epac2A RA domain and
sulfonylurea. (G) Predicted affinities for the sulfonylurea binding to Epac2A. Ball-and-stick indicates sulfonylureas;
wire diagram indicates amino acids of Epac2A. Dotted lines indicate predicted interactions. Carbon, nitrogen,
oxygen, and sulfur atoms are colored green, blue, red, and yellow, respectively. For tolbutamide, only those
interactions subsequently confirmed by mutagenesis are shown. PDB files of models for the interactions between
Epac2A and sulfonylureas are provided in the Supplementary Materials.
Fig. 4. Effects of Epac mutations on Epac activation as revealed by FRET in MIN6K8 b cells. (A) FRET emission ratio time courses of WT Epac2A or mutant Epac2A in
cells treated with the indicated activators. Data are presented as means ± SEM (n = 4
to 7 experiments for each point). (B) Alignment of the amino acid sequences of the
cNBD-A of Epac2A and cNBD of Epac1. Residues predicted to be involved in
sulfonylurea binding in Epac2A and the corresponding residues in Epac1 are in red. (C)
FRET emission ratio time courses of triple-mutant Epac1 (T302C, L313S, and A322H) in
cells treated with the indicated activators. The FRET response of WT Epac1 in response
to 8-pCPT is shown for comparison. Data are presented as means ± SEM (n = 4 to 9
experiments for each point).
Fig. 4. Effects of Epac mutations on Epac activation as revealed by FRET in MIN6K8 b cells. (A) FRET emission ratio time courses of WT Epac2A or mutant Epac2A in
cells treated with the indicated activators. Data are presented as means ± SEM (n = 4
to 7 experiments for each point). (B) Alignment of the amino acid sequences of the
cNBD-A of Epac2A and cNBD of Epac1. Residues predicted to be involved in
sulfonylurea binding in Epac2A and the corresponding residues in Epac1 are in red. (C)
FRET emission ratio time courses of triple-mutant Epac1 (T302C, L313S, and A322H) in
cells treated with the indicated activators. The FRET response of WT Epac1 in response
to 8-pCPT is shown for comparison. Data are presented as means ± SEM (n = 4 to 9
experiments for each point).
Fig. 5. Dependence of Rap1
activation by glibenclamide
on Epac2A. (A) Alignment of
the amino acid sequences in
the cNBD of Epac2A, Epac1,
and PKA regulatory subunits.
Asterisk indicates Ala-to-His
substitution at position 124 in
Epac2A. (B and C) Effects of
glibenclamide and 8-pCPT on
Rap1 activation in Epac2Adeficient b cells expressing
mCherry (control) or
exogenous WT Epac2A or
Epac2A H124A mutant
(H124A). Quantification of
chemiluminescent signals is
shown with corresponding
bars positioned under the
bands. Data are presented as
means ± SEM (n = 4 to 7
experiments for each point).
*P < 0.05; **P < 0.01; NS, not
significant (Dunnett’s
method).
Fig. 6. cAMP-dependent activation of Epac2A and Rap1 by glibenclamide. (A to C) Graphs
showing the direct binding of glibenclamide to Epac2A. [3H]Glibenclamide binding was assessed in
COS-1 cells transfected with either WT Epac2A or Epac2A H124A. DPM, disintegration per minute.
Data are presented as means ± SEM (n = 4 to 6 experiments for each point). Data are presented
as means ± SEM (n = 4 to 7 experiments for each point). **P < 0.01; NS, not significant (Dunnett’s
method).
Fig. 6. cAMP-dependent activation of Epac2A and Rap1 by glibenclamide. (D) FRET emission
ratio time courses of WT Epac2A in MIN6-K8 b cells exposed to the indicated compounds. (E)
FRET emission ratio time courses of Epac2A H124A in MIN6-K8 b cells exposed to the indicated
compounds. Data are presented as means ± SEM (n = 5 to 9 experiments for each point). Data
are presented as means ± SEM (n = 4 to 7 experiments for each point). **P < 0.01; NS, not
significant (Dunnett’s method).
Fig. 6. cAMP-dependent activation of Epac2A and Rap1 by glibenclamide. (F and G) Rap1
activation assays after treatment with the indicated combinations of 8-pCPT and glibenclamide in
MIN6-K8 b cells (F) or Epac2A-deficient b cells expressing mCherry (control) or exogenous WT
Epac2A or Epac2A H124A (G). Data are presented as means ± SEM (n = 4 to 7 experiments for
each point). **P < 0.01; NS, not significant (Dunnett’s method).
Fig. 7. Effect of depletion of endogenous cAMP by adrenaline on the activation of
Epac2A by sulfonylureas. (A and B) FRET emission ratio time courses of Epac2A in
MIN6-K8 b cells treated with adrenaline and glibenclamide or tolbutamide. Time of
application of the compounds is indicated by the bars. Data are presented as means ±
SEM (n = 3 to 9 experiments for each point). Data are presented as means ± SEM (n =
6 experiments for each condition). ***P < 0.001. Statistical analysis was performed with
Tukey’s method.
Fig. 7. Effect of depletion of endogenous cAMP by adrenaline on the activation of
Epac2A by sulfonylureas. (A and B) FRET emission ratio time courses of Epac2A in
MIN6-K8 b cells treated with adrenaline and glibenclamide or tolbutamide. Time of
application of the compounds is indicated by the bars. Data are presented as means ±
SEM (n = 3 to 9 experiments for each point). Data are presented as means ± SEM (n =
6 experiments for each condition). ***P < 0.001. Statistical analysis was performed with
Tukey’s method.
Fig. 7. Effect of depletion of
endogenous cAMP by
adrenaline on the activation
of Epac2A by sulfonylureas.
(C) Rap1 activation assays in
MIN6-K8 b cells treated with the
indicated combinations of
adrenaline and glibenclamide.
Data are presented as means
± SEM (n = 6 experiments for
each condition). ***P < 0.001.
Statistical analysis was
performed with Tukey’s method.
SUMMARY
Using molecular docking simulation, we identified
amino acid residues in one of two cyclic nucleotide–
binding domains, cNBD-A, in Epac2A predicted to
mediate the interaction with sulfonylureas. We
confirmed the importance of the identified residues by
site-directed mutagenesis and analysis of the
response of the mutants to sulfonylureas using two
assays: changes in fluorescence resonance energy
transfer (FRET) of an Epac2A-FRET biosensor and
direct sulfonylurea-binding experiments. These
residues were also required for the sulfonylurea
dependent Rap1 activation by Epac2A. Binding of
sulfonylureas to Epac2A depended on the
concentration of cAMP and the structures of the drugs.
CONCLUSION
Sulfonylureas and cAMP cooperatively
activated Epac2A through binding to cNBDA and cNBD-B, respectively. Our data
suggest that sulfonylureas stabilize Epac2A
in its open, active state and provide insight
for the development of drugs that target
Epac2A.
Message
Epac2Aについては再現性
が問題あるとされていた
が、次第に確立されてき
ているようである。
SU薬の結合はスルホニル
骨格とベンザミド骨格が
SURの細胞の内側部位にそ
れぞれ結合するとされる。
今回Epac2Aにスルホニル
骨格が結合しインスリン
分泌を促すことが示され
た!
グリニド薬はベンザミド
骨格しかないが、更に、
Epac2Aにも結合しない。
SU薬の標的は、これまではSU受容体が唯一
知られており、SU薬によるインスリン分泌は
SU受容体を介するメカニズムのみで説明され
ていた(青部分)。本研究によりSU薬の作用に
は、Epac2/Rap1を介するメカニズムも重要で
あることが解明された(赤部分)。Epac2はイン
クレチンによるインスリン分泌の増強にも重要
であることが示されている。
要約:2型糖尿病患者におけるインスリン欠乏の治療には、スルホニル尿素
が広く使用されている。スルホニル尿素は、インスリン分泌を制御している膵
β細胞のカリウムチャネルの調節サブユニットに結合する。またスルホニル尿
素は、Ras様グアノシントリホスファターゼRap1の活性化を介してインスリン
分泌を促進している、環状アデノシン一リン酸(cAMP)結合タンパク質の
EpacファミリーのメンバーであるEpac2Aにも結合し、活性化している。
われわれは分子ドッキングシミュレーションを用い、Epac2Aの2つの環状ヌク
レオチド結合ドメインのうち1つ(cNBD-A)に、スルホニル尿素との相互作用
を媒介すると予測されるアミノ酸残基を特定した。さらに、この特定された残
基の重要性を、部位特異的変異導入法と、以下の2つのアッセイを用いたス
ルホニル尿素に対する突然変異体の反応の解析により確認した。すなわち、
Epac2A-FRETバイオセンサーの蛍光共鳴エネルギー移動(FRET)の変化、
および直接のスルホニル尿素結合実験である。
これらの残基は、Epac2Aによるスルホニル尿素依存性のRap1活性化にも
必要であった。Epac2Aに対するスルホニル尿素の結合は、cAMP濃度と薬
物の構造に依存していた。スルホニル尿素とcAMPは、それぞれcNBD-Aと
cNBD-Bに結合することでEpac2Aを協調的に活性化していた。われわれの
データは、スルホニル尿素がEpac2Aを開放型の活性化状態で安定化するこ
とを示唆し、さらにEpac2Aを標的とする薬物の開発の手がかりを示している。
Fig. 1 Schematic representation of known
and assumed pathways for adiponectin
signal transduction. Adiponectin receptor 1
(AdipoR1) has a high affinity for globular
adiponectin and a low affinity for full-length
adiponectin, whereas adiponectin receptor 2
(AdipoR2) has an intermediate affinity for fulllength and globular adiponectin. T-cadherin is a
truncated receptor that can bind the hexameric
and high molecular weight (HMW) oligomeric
forms of adiponectin. AdipoR1 and AdipoR2
interact with the adaptor protein containing a
pleckstrin homology domain, a phosphotyrosine
domain and a leucine zipper motif (APPL1),
which binds the N-terminal intracellular domains
of the receptors. The binding of adiponectin to
its receptors provokes the activation of
adenosine monophosphate (AMP)-activated
protein kinase (AMPK), and the activation of
various signaling molecules such as p38
mitogen-activated protein kinase (p38 MAPK),
peroxisome proliferator-activated receptor-α
(PPARα), the RAS-associated protein Rab5,
phosphatidylinositol 3-kinase (PI3K) and the vakt murine thymoma viral oncogene homolog
(Akt). Activation of AMPK can also block the
nuclear factor κ B (NFκB) signaling, known to
be a mediator of inflammation in endothelial
cells. ACC acetyl coenzyme-A carboxylase;
Brochu-Gaudreau K, Rehfeldt C, Blouin R, Bordignon V, Murphy
BD, Palin MF.: Adiponectin action from head to toe. Endocrine.
2010 Feb;37(1):11-32.
Figure 2 Regulation of adiponectin transcription by upstream signals
In obesity, increased pro-inflammatory cytokines such as TNFα, IL-6 and IL-18 negatively regulate adiponectin gene transcription by activating
several pathways such as the JNK and ERK1/2 pathways. High-fat-diet (HFD)-induced obesity also suppresses adiponectin expression by
increasing cellular levels of catecholamine and PKA-mediated activation of CREB [85]. Insulin has been suggested to positively regulate
adiponectin gene expression by activating PPARγ via suppressing FoxO1 activity in vitro [50]; however, a negatively correlated relationship has
been documented between insulin and adiponectin levels in vivo (see the text). FoxO1 could have a positive effect on adiponectin transcription
via interaction with C/EBP [49,169]. Regulation of FoxO1 by insulin and Sirt1 may provide a mechanism to dynamically regulate adiponectin
gene expression. IR, insulin receptor; IRS, insulin receptor substrate; P-FoxO1, phosphorylated FoxO1; PI3K, phosphoinositide 3-kinase; RXR,
retinoid X receptor; TNFR-1, TNFα receptor 1. An animated version of this Figure can be seen at
http://www.BiochemJ.org/bj/425/0041/bj4250041add.htm
1Department
of Diabetes and Metabolic Diseases, Graduate School of Medicine, The
University of Tokyo, Tokyo 113-0033, Japan. 2Department of Integrated Molecular
Science on Metabolic Diseases, 22nd Century Medical and Research Center, The
University of Tokyo, Tokyo 113-0033, Japan. 3Department of Molecular Medicinal
Sciences on Metabolic Regulation, 22nd Century Medical and Research Center, The
University of Tokyo, Tokyo 113-0033, Japan. 4RIKEN Systems and Structural Biology
Center, Tsurumi, Yokohama 230-0045, Japan. 5Graduate School of Comprehensive
Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan. 6Open Innovation
Center for Drug Discovery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan. 7Graduate School of Science, The University of Tokyo, Bunkyo-ku,
Tokyo 113-0033, Japan.
Background
Adiponectin secreted from adipocytes binds to
adiponectin receptors AdipoR1 and AdipoR2, and
exerts antidiabetic effects via activation of AMPK
and PPAR-a pathways, respectively. Levels of
adiponectin in plasma are reduced in obesity,
which causes insulin resistance and type 2
diabetes. Thus, orally active small molecules that
bind to and activate AdipoR1 and AdipoR2 could
ameliorate obesity-related diseases such as type
2 diabetes.
Content
Here we report the identification of orally active synthetic
small-molecule AdipoR agonists. One of these
compounds, AdipoR agonist (AdipoRon), bound to both
AdipoR1 and AdipoR2 in vitro.
Extended Data Figure 1 |
Phosphorylation of AMPK in
C2C12 myotubes.
Phosphorylation of AMPK
normalized to the amount of AMPK
in C2C12 myotubes treated for 5
min with 15 μgml-1 adiponectin or
the indicated small molecule
compounds (10 mM).
#, AdipoRon;
##, no. 112254;
###, no. 165073.
Figure 1 |
Smallmolecule
AdipoR
agonist
AdipoRon
binds to both
AdipoR1 and
AdipoR2, and
increases
AMPK
activation,
PGC-1a
expression
and
mitochondrial
biogenesis in
C2C12
myotubes.
Figure 1 | Small-molecule AdipoR agonist
AdipoRon binds to both AdipoR1 and AdipoR2,
and increases AMPK activation, PGC-1a
expression and mitochondrial biogenesis in
C2C12 myotubes.
a, Chemical structure of AdipoRon.
b–i, l, m, Phosphorylation and amount of AMPK (b–f,
l,m), Ppargc1amRNAlevels (g, h), and mitochondrial
content as assessed by mitochondrial DNA copy
number (i), in C2C12 myotubes after myogenic
differentiation (b–i), in skeletal muscle (l) or in liver
(m) from wild-type (WT) or Adipor1-/- Adipor2 -/double-knockout mice, treated with indicated
concentrations of AdipoRon (b, d–i) or adiponectin (d,
15μgml -1; e, 50μgml-1; i, 10μgml -1), for 5 min (b, d–f),
1.5 h (g, h) and 48 h (i), with or without EGTA (f, h),
25 μM AdipoRon, compound 112254 and 165073, 30
μgml-1 adiponectin for 5 min or 1mMAICAR for 1 h
and transfected with or without the indicated siRNA
duplex (c), or AdipoRon (l, m). j, k, Surface plasmon
resonance measuring AdipoRon binding to AdipoR1
and AdipoR2. AdipoR1 and AdipoR2 were
immobilized onto a sensor chip SA. Binding analyses
were performed using a range of AdipoRon
concentrations (0.49– 31.25 μM). All values are
presented as mean6s.e.m. b, c, e–I, n=4 each; d, l,m,
n=3 each; *P<0.05 and **P<0.01 compared to control
or unrelated siRNA or as indicated. NS, not
significant.
Figure 2: AdipoRon improved insulin resistance, glucose intolerance and
dyslipidaemia via AdipoR.
Figure 2: AdipoRon improved insulin resistance, glucose intolerance and
dyslipidaemia via AdipoR.
Figure 2: AdipoRon improved insulin resistance, glucose intolerance and
dyslipidaemia via AdipoR.
a–g, Plasma AdipoRon concentrations (a), body weight (b), food intake (c), plasma
glucose (d, e, g), plasma insulin (d, e) and insulin resistance index (f) during oral glucose
tolerance test (OGTT) (1.0 g glucose per kg body weight) (d, e) or during insulin
tolerance test (ITT) (0.5 U insulin per kg body weight) (g) in wild-type (WT) and
Adipor1−/− Adipor2−/− double-knockout mice, treated with or without AdipoRon (50 mg per
kg body weight). h, i, Glucose infusion rate (GIR), endogenous glucose production
(EGP) and rates of glucose disposal (Rd) during hyperinsulinaemic euglycaemic clamp
study in wild-type and Adipor1−/− Adipor2−/− double-knockout mice, treated with or
without AdipoRon (50 mg per kg body weight). j, k, Plasma triglyceride (j) and free fatty
acid (FFA) (k) in wild-type and Adipor1−/− Adipor2−/− double-knockout mice, treated with
or without AdipoRon (50 mg per kg body weight). All values are presented as
mean ± s.e.m. a, n = 12–32; b–g, j, k, n = 10 each; h, i, n = 5 each; *P < 0.05 and
**P < 0.01 compared to control or as indicated. NS, not significant.
Figure 4: AdipoRon ameliorated insulin resistance, diabetes and dyslipidaemia in
db/db mice.
a, Plasma glucose levels after intraperitoneal injection of adiponectin (30 µg per 10 g body weight) (left) or after
oral administration of AdipoRon (50 mg per kg body weight) (middle). The area under the curve (AUC) of left and
middle panels is shown on the right. b–i, Body weight (b), food intake (c), liver weight (d), WAT weight (e), treated
with or without AdipoRon (50 mg per kg body weight). All values are presented as mean ± s.e.m. a, n = 6-7; b–i, n
= 10 each from 2–3 independent experiments, *P < 0.05 and **P < 0.01 compared to control or as indicated. NS,
not significant.
Figure 4: AdipoRon ameliorated insulin resistance, diabetes and dyslipidaemia in
db/db mice.
plasma glucose (f, left, g), plasma insulin (f, middle) and insulin resistance index (f, right) during oral glucose
tolerance test (OGTT) (1.0 g glucose per kg body weight) (f) or during insulin tolerance test (ITT) (0.75 U insulin
per kg body weight) (g), plasma triglyceride (h) and free fatty acid (FFA) (i) in db/db mice under normal chow
conditions, treated with or without AdipoRon (50 mg per kg body weight). All values are presented as
mean ± s.e.m. a, n = 6-7; b–i, n = 10 each from 2–3 independent experiments, *P < 0.05 and **P < 0.01 compared
to control or as indicated. NS, not significant.
Figure 5: AdipoRon increased mitochondria biogenesis in muscle, reduced tissue triglyceride content and
oxidative stress in muscle and liver, and decreased inflammation in liver and WAT of db/db mice.
a–h, Ppargc1a, Esrra, Tfam,
mt-Co2, Tnni1, Acadm and
Sod2 mRNA levels (a), and
mitochondrial content as
assessed by mitochondrial
DNA copy number (b), tissue
triglyceride content (c) and
TBARS (d) in skeletal muscle,
Ppargc1a, Pck1, G6pc, Ppara,
Acox1, Ucp2, Cat, Tnf and
Ccl2 mRNA levels (e), tissue
triglyceride content (f) and
TBARS (g) in liver, and Tnf, Il6,
Ccl2, Emr1, Itgax and Mrc1
mRNA levels (h) in WAT from
db/db mice on a normal chow
diet, treated with or without
AdipoRon (50 mg per kg body
weight). All values are
presented as mean ± s.e.m. n
= 10, *P < 0.05 and **P < 0.01
compared to control or as
indicated. NS, not significant.
Figure 6: AdipoRon increased insulin sensitivity and glucose tolerance, and at the same time
contributed to longevity of obese diabetic mice.
a–c, Kaplan–Meier survival curves for wild-type, Adipor1−/−, Adipor2−/− and Adipor1−/− Adipor2−/−
knockout mice on a normal chow diet (a) (n = 50, 32, 29 and 35, respectively) or high-fat diet (b) (n
= 47, 33, 35 and 31, respectively), or for db/db mice treated with or without AdipoRon (30 mg per kg
body weight) on a normal chow or high-fat diet (n = 20 each) (c). P values were derived from logrank calculations. d, Scheme illustrating the mechanisms by which AdipoR1 and AdipoR2 agonist
increases insulin sensitivity and glucose tolerance, and at the same time lifespan.
Summary
AdipoRon showed very similar effects to
adiponectin in muscle and liver, such as
activation of AMPK and PPAR-a pathways, and
ameliorated insulin resistance and glucose
intolerance in mice fed a high-fat diet, which was
completely obliterated in AdipoR1 and AdipoR2
double-knockout mice. Moreover, AdipoRon
ameliorated diabetes of genetically obese rodent
model db/db mice, and prolonged the shortened
lifespan of db/db mice on a high-fat diet.
Conclusion
Thus, orally active AdipoR agonists such as
AdipoRon are a promising therapeutic
approach for the treatment of obesityrelated diseases such as type 2 diabetes.
Message
10月29日,東京大学病院糖尿病・代謝内科教授の門脇
孝氏,同科講師の山内敏正氏らは記者会見を開き,同氏
らの研究グループが発見した,アディポネクチン受容体
を活性化させる低分子化合物に関する研究成果について
発表した。2003年にアディポネクチン受容体を同定した
同氏らは,今回,肥満によって発現が減少する同受容体
を活性化させる働きを持つ化合物AdipoRonを同定。マウ
スを用いた実験により,糖尿病をはじめとする生活習慣
病の予防や治療,健康寿命の延長につながる経口薬の種
となる可能性が高いとして,今後5年以内の臨床第Ⅰ相
試験を視野に創薬を目指すと述べた。なお,研究の詳細
は,Nature(2013年10月30日オンライン版)に掲載され
た。
http://mtpro.medical-tribune.co.jp/mtpronews/1310/1310084.html

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