Extended Data Figure 3: A glycolytic intermediate(s) is responsible for repression of AMPK in the presence of glucose.

All intact cell experiments in this figure were performed in the presence of 10% serum. a, Summary of glycolysis pathway and pathways branching off it. PPP, pentose phosphate pathway; SSP, serine synthesis pathway; HP, hexosamine pathway. b, c, Knockdown of G6PD (glucose-6-phosphate dehydrogenase) (b) or PHGDH (phosphoglycerate dehydrogenase) (c) has no effect on AMPK activation. HEK293T cells were infected with lentivirus expressing siRNA against G6PD (b) or PHGDH (c), or siRNA against GFP as a control; the cells were then incubated in DMEM medium with (25 mM) or without glucose for 2 h, followed by analysis of p-AMPKα and p-ACC levels. d, The hexosamine pathway is not involved in AMPK regulation under glucose deprivation. MEFs were incubated for 2 h in DMEM medium containing GlcNAc (N-acetylglucosamine), a supplement of the hexosamine pathway which cannot be converted back to glucose³⁵, at indicated concentrations and 25 mM or 0 mM glucose. Cells were then subjected to immunoblotting. Experiments in b and c were performed twice, and that in d three times.