Chloropupukeananin (RN56‐6) and Pestalofone C (RN56‐49), isolated from the culture of the plant endophytic fungus Pestalotiopsis fici, have been shown cytotoxic, anti‐HIV, and antimicrobial activities. However, the underlying mechanism of their regulatory roles in autophagy remains unknown. In the present study, we revealed that both compounds increased the formation of autophagosome and enhanced autophagic flux. While RN56‐6 upregulated the expression of HK2, one of the key rate‐limiting enzymes of glycolysis, the inhibition of glycolysis chemically attenuated RN56‐6‐induced autophagy. On the contrary, RN56‐49 downregulated the expression of HK2, while the suppression of glycolysis promoted RN56‐49‐dependent autophagic flux. Moreover, the knockdown of AMPKβ1, a scaffolding subunit of AMPK, decreased autophagy induced by these two compounds. Collectively, these findings revealed that RN56‐6 and RN56‐49 regulated autophagic process through AMPK and glycolytic pathway.
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