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。 Endocrinology, May 2012, 153(5):2130– /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 AMPK Enhances Insulin-Stimulated GLUT4 Regulation via Lowering Membrane Cholesterol 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 anti- phospho- 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. 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。 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. 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 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 nine to 13 independent experiments. *, P < 0.05 vs. control (or control-basal) groups; #, P < 0.05 vs. control-insulin group. ns, nonsignificant 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 の活性化はインスリンシグナルの活性化には関与しな い。 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。 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。 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).

FIG. 6. AMPK activation protects against hyperinsulinemia- induced 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). 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。 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。 ・ 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。 まとめ Supplement Fig.2