Abstract
Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy.
Highlights
In many cell types, including liver cells, autophagy is inhibited by amino acids, in synergy with insulin, and this inhibition is mediated, at least in part, by mTOR3-dependent signaling [1]
Inhibition of AMPactivated protein kinase (AMPK) activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICA riboside (AICAR) is not related to its ability to activate AMPK
In the present study, using different mammalian cell types, we have examined the possible role of AMPK in the control of autophagy in more detail
Summary
Plasmid purification kit Nucleobond AX and nitrocellulose membranes kinase; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; p70S6K, p70S6 kinase; MDC, monodansylcadaverine; CC, compound C; ACC, acetylCoA carboxylase; eIF4E, eukaryotic initiation factor 4E; GFP, green fluorescent protein; gdw, gram dry weight of cells; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid; Met, metformin. Mouse anti-FLAG (2EL-1B11) was from Euromedex (Souffelweyersheim, France). After removal of the precipitated protein by centrifugation in a microcentrifuge (1 min; 10,000 ϫ g), the clear supernatant was neutralized to pH 7 with a small volume of a mixture of 2 M KOH plus 0.3 M MOPS
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