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AMPK Enhances Insulin-Stimulated GLUT4 Regulation via Lowering
Membrane Cholesterol
Endocrinology, May 2012, 153(5):2130–2141
AMPKは細胞膜のコレステロール量を低下させることで、インスリン刺激によるGLUT4の膜
移行を促進する。
2015/06/01 M1 眞野僚
【背景と目的】
AMPKは様々なシグナル伝達に関わり、コレステロール生合成の律速酵素となるHMGRを阻害する作用を
持つことが知られている。また骨格筋における糖取り込みの上昇には、インスリン刺激に応じたGLUT4の
膜移行が重要であるが、細胞膜のコレステロール量とGLUT4の膜移行が正の相関を持つことが報告され
ている。そこで本研究では、筋管細胞においてAMPKの活性化が細胞膜のコレステロール量とGLUT4の膜
移行にどのような影響を及ぼすか、AMPKの活性化が高インスリン状態時に増加する細胞膜のコレステ
ロールを抑制するかどうかについて検討を行った。
【略語】
ACC, Acetylcoenzyme A carboxylase
AICAR, 5-aminoimidazole-4-carboxamide-1-D-ribonucleoside
AMPK, AMP-activated protein kinase
βCD, methyl-β-cyclodextrin
βCD:Chol, βCD preloaded with cholesterol
2-DG, 2-deoxy-D-glucose
DNP, 2,4-dinitrophenol
F-actin, filamentous actin
HBP, hexosamine biosynthesis pathway
HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase
PIP2, phosphatidylinositol 4,5 bisphosphate SREBP, sterol-regulatory element-binding proteins
FIG. 1. AMPK activation in L6 myotubes. Myotubes were left untreated or treated with AICAR (1
mM, 45 min) or DNP (200 µM, 30 min). Total cell extracts were prepared, and protein
concentrations were determined by the Bradford method. Equivalent protein amounts
were separated by 7.5% SDS-PAGE. The resolved fractions were transferred to nitrocellulose, and
immunoblot analyses were performed using anti-phospho-AMPK and anti-pan-AMPK (A) and antiphospho- ACC and anti-pan-ACC antibodies (B). Quantification of these analyses represent the
ratio of immunoblot band intensities detected with antiphosphorylation antibodies [i.e. -pAMPK,
-pACC] to that measured with specific protein antibodies [i.e. -AMPK, -ACC].
Values are means ± SE of six to 12 independent experiments, *, P <0.05 vs. control group.
AICAR、DNPの添加によってAMPKの活性化が確認できた。
また、AMPKの下流に位置するACCの活性化も確認できた。
FIG. 2. AMPK activation increases plasma membrane GLUT4. Myotubes were left untreated or
treated with AICAR (1 mM, 45 min) or DNP (200 M, 30 min) in the absence (basal, panels and bars 1,
3, and 5) or presence (insulin, panels and bars 2, 4, and 6) of acute insulin stimulation (100 nM, 20
min). After treatment, cells were fixed and left unpermeabilized. The samples were then labeled with
anti-myc antibody, washed, and incubated with an infrared-conjugated secondary antibody (LI-COR)
or with fluorescein isothiocyanate - conjugated antimouse (confocal). Immunofluorescent intensity
was normalized to intensity from Syto60, a fluorescent nucleic acid stain. Images were collected and
quantified with the Odyssey system. Representative -myc immunofluorescent and Syto60 staining
images of treated myotubes are shown (A). Values are means SE of -myc/Syto 60 signal
quantification from 20–40 independent experiments (B). *, P < 0.05 vs. control-basal group;
#, P < 0.05 vs. control-insulin group.
AICAR、DNPの添加により、糖取り込みが増加することを確認できた。
また、インスリン刺激によってさらに増加することも確認できた。
FIG. 3. AMPK acts independently of insulin. Myotubes were left untreated or treated with
AICAR (1 mM, 45 min) or DNP (200 M, 30 min) in the absence (basal, panels and bars 1, 3, and
5) or presence (insulin, panels and bars 2, 4, and 6) of acute insulin stimulation (100 nM,
5 min). Total cell extracts were prepared and protein concentrations were determined via the
Bradford method. Equivalent protein amounts were separated by 7.5% SDS-PAGE. The
resolved fractions were transferred to nitrocellulose, and immunoblot analyses were
performed using phospho-IRS-1 and phospho-Akt2 (A) and phospho-AS160 (B) antibodies.
Equal protein loading was confirmed by Ponceau staining and by immunoblot analysis with
anti-actin antibody. Representative immunoblots are shown from three to six independent
experiments (A). Quantification of these analyses represent the ratio of immunoblot band
intensities detected with antiphosphorylation antibody [i.e. -pAS160] to that measured with
specific protein antibody [i.e. -AS160]. Values presented in panel B are means ± SE from six
independent experiments. *, P < 0.05 vs. control-basal group. Ins., Insulin.
AMPKの活性化はインスリンシグナルの活性化には関与しない。
FIG. 4. AMPK activation decreases
membrane cholesterol, and
AMPKenhanced insulin action is
cholesterol dependent. Myotubes
were left untreated or treated with
AICAR (1 mM, 45 min) or DNP (200
M, 30 min) in the absence or
presence of exogenous cholesterol
(CD:Chol, 45 min).
A plasma membrane-enriched fraction was prepared as described in Materials and Methods. Membrane cholesterol contents (A and B) were
determined by the Amplex Red cholesterol assay and normalized to protein content determined by the Bradford method. Basal and insulinstimulated (100 nM, 20 min) plasma membrane GLUT4 contents (C and D) were determined in myotubes treated as described in Fig. 2. Values
are means ±SE of nine to 13 independent experiments. *, P < 0.05 vs. control (or control-basal) groups; #, P < 0.05 vs. control-insulin group.
ns, nonsignificant
細胞膜のコレステロールの増加は、AMPKが活性化することで促進するインスリン誘導性のGLUT4の膜移
行のみ阻害する。
FIG. 5. AMPK knockdown abrogated DNP-induced membrane cholesterol lowering and increased basal- and insulin-stimulated plasma
membrane GLUT4. Myotubes received transient transfections targeting the AMPK 1 and 2 catalytic subunits as described in
Materials and Methods. Myotubes were then left untreated or treated with AICAR (1 mM, 45 min) or DNP (200 M, 30 min). Total cell
extracts were prepared from nontransfected control, siScrambletransfected (siScr), and siAMPK-transfected myotubes. SDS-PAGE and
immunoblot analyses were performed as described in Fig. 3 using anti-AMPK and antiactin antibodies (A). Membrane cholesterol content was
determined as described in Fig. 4B. Basal and insulin-stimulated (100 nM, 20 min) plasma membrane GLUT4 contents (C and D) were
determined in myotubes treated as described in Fig. 2. Values are means ± SE of six to 11 independent experiments. *, P < 0.05 vs. siScr
(or control-basal) groups; #, P < 0.05 vs. control-insulin groups. ns, Nonsignificant.
AMPKのノックダウンにより、AMPKの活性化によるインスリン誘導性のGLUT4の膜移行の
促進が阻害された。
AMPKの活性化は、高インスリン状態時であってもコレス
テロール量、GLUT4の膜移行を促進する。
FIG. 6. AMPK activation protects against hyperinsulinemiainduced increase in membrane cholesterol, F-actin loss, and
GLUT4 dysregulation. Myotubes were left untreated or
treated with AICAR (1 mM, 45 min) in the absence or
presence of hyperinsulinemia (5 nM, 12 h). Membrane
cholesterol content was determined as described in Fig. 4A.
Cortical F-actin immunofluorescence was determined by
fixing and permeabilizing cells as described in Materials and
Methods. The samples were then labeled with anti-F-actin
antibody, washed, and incubated with an infrared-conjugated
secondary antibody. Immunofluorescent intensity was
normalized to intensity from Syto60 and quantified with the
Odyssey system (B). Basal and insulin-stimulated (100 nM, 20
min) plasma membrane GLUT4 contents (C) were determined
in myotubes treated as described in Fig. 2. Values are
means ± SE of four to eight independent experiments.
*, P < 0.05 vs. control (or control-basal) groups; #, P < 0.05 vs.
control-insulin groups. Fold insulin stimulation for each
treatment condition are presented in panel C inset. Ins.,
Insulin.
FIG. 7. Cholesterol lowering normalizes insulin sensitivity in obese Zucker skeletal muscle. Rat epitrochlearis muscle (A) and soleus muscle (B, C,
and D) from lean/obese Zucker rats were labeled with antibodies against F-actin, imaged by confocal microscopy, and digitally quantitated
using MetaMorph software (A, B, and D). Remaining soleus muscle was fractionated for membrane cholesterol analyses (B) as described in
Materials and Methods. Paired contralateral soleus muscles were subjected to basal and insulin-stimulated 2-DG uptake measurements (C) as
described in Materials and Methods. Subgroups of these muscles were exposed to 2.5 mM CD for 30 min before the 30-min insulin stimulation.
Values are means ± SE from five independent experiments. *, P < 0.05 vs. lean; #, P < 0.05 vs. lean (insulin).
肥満型ラットにおいて、コレステロール量を減少させることで、糖取り込み能が改善した。また、一度破壊
されたF-actinは回復した。
まとめ
・AMPKによるGLUT4の制御には、細胞膜のコレ
ステロールの低下が関わる。
・AMPKの活性化はインスリン抵抗性の改善に
も寄与することができる。
Supplement Fig.2

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