Chloroquine increases phosphorylation of AMPK and Akt in myotubes.
Chloroquine increases phosphorylation of AMPK and Akt in myotubes.
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DOI：
10.1016/j.heliyon.2016.e00083
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发表时间：
2016-03
期刊：
影响因子：
4
通讯作者：
Fisher JS
中科院分区：
文献类型：
--
作者：
Spears LD;Tran AV;Qin CY;Hobbs SB;Burns CA;Royer NK;Zhang Z;Ralston L;Fisher JS
有报道称，ataxia毛细血管扩张突变（ATM）可以激活amp活化的蛋白激酶（AMPK）和Akt，这两种激酶在心脏保护和代谢功能中起着不可或缺的作用。我们假设氯喹和白藜芦醇这两种已知的ATM激活剂也会激活AMPK和Akt。在C2C12肌管暴露于氯喹或白藜芦醇后，评估AMPK和Akt的磷酸化。在表达shRNA对抗ATM或存在ATM抑制剂KU55933的细胞中进行了其他实验。用荧光探针钙绿-1 AM评价氯喹对细胞内钙的影响。0.5 mM氯喹使AMPK磷酸化增加近4倍（P < 0.05）， 0.25 mM氯喹使Akt磷酸化增加约1倍（P < 0.05）。氯喹也使ATM的自磷酸化水平提高了50% （P < 0.05）。白藜芦醇（0.15 mM）可使AMPK磷酸化水平升高约3倍（P < 0.05），但与氯喹相比，可显著降低Akt磷酸化水平。氯喹增加了肌管中表达抗ATM shRNA的AMPK和Akt磷酸化，使ATM蛋白水平降低约90%。同样，氯喹刺激的AMPK和Akt磷酸化以及白藜芦醇刺激的AMPK磷酸化未因抑制ATM而改变。氯喹使细胞内钙降低50%，同时葡萄糖转运减少。这些与atm无关的氯喹对AMPK和Akt的影响以及降低细胞内钙的额外作用可能是文献中报道的氯喹的积极代谢作用的部分基础。
There are reports that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK) and also Akt, two kinases that play integral parts in cardioprotection and metabolic function. We hypothesized that chloroquine and resveratrol, both known ATM activators, would also activate AMPK and Akt. Phosphorylation of AMPK and Akt was assessed after C2C12 myotubes were exposed to chloroquine or resveratrol. Additional experiments were done in cells expressing shRNA against ATM or in the presence of the ATM inhibitor KU55933. The effects of chloroquine on intracellular calcium were assessed with the fluorescent probe Calcium Green-1 AM. 0.5 mM chloroquine increased AMPK phosphorylation by nearly four-fold (P < 0.05), and 0.25 mM chloroquine roughly doubled Akt phosphorylation (P < 0.05). Chloroquine also increased autophosphorylation of ATM by ∼50% (P < 0.05). Resveratrol (0.15 mM) increased AMPK phosphorylation about three-fold (P < 0.05) but in contrast to chloroquine sharply decreased Akt phosphorylation. Chloroquine increased AMPK and Akt phosphorylation in myotubes expressing shRNA against ATM that reduced ATM protein levels by about 90%. Likewise, chloroquine-stimulated phosphorylation of AMPK and Akt and resveratrol-stimulated phosphorylation of AMPK were not altered by inhibition of ATM. Chloroquine decreased intracellular calcium by >50% concomitant with a decrease in glucose transport. These ATM-independent effects of chloroquine on AMPK and Akt and the additional effect to decrease intracellular calcium are likely to partially underlie the positive metabolic effects of chloroquine that have been reported in the literature.