Extended Data Fig. 11. Aldolase-TRPV act as a separate route from PEN2-ATP6AP1.
a, b, Inhibition of v-ATPase by conA activates AMPK in cells lacking the PEN2 and ATP6AP1 axis. PEN2-/- MEFs (a) or ATP6AP1-/- MEFs re-introduced with full length (FL) ATP6AP1 or its Δ420-440 mutant (b; expressed at close-to-endogenous levels) were treated with 5 μM conA for 2 h, followed by determining p-AMPKα and p-ACC levels by immunoblotting. c, Liver-specific ALDOA-D34S transgenic (Tg) mice renders hepatic AMPK inactive under starvation. Tg mice (6-week-old) expressing ALDOA-D34S (expressed under an ApoE promoter and its hepatic control region included in the pLiv-Le6 vector), which can still bind FBP in low glucose, and therefore mimics a high glucose state in which AMPK is inactivated (see cell-based data in ref. 6), were starved for 16 h. P-AMPKα and p-ACC levels in livers were then determined by immunoblotting. d, Aldolase is dispensable for low metformin-induced AMPK activation in HEK293T cells. Cells stably expressing HA-tagged ALDOA-D34S mutant, or wildtype ALDOA were treated with 300 μM metformin (low concentration) or 5 mM (high concentration, as a control) for 12 h, followed by analysis of p-AMPKα and p-ACC levels by immunoblotting. e, Aldolase is dispensable for low metformin-induced AMPK activation in mouse liver. Mice with Tg-ALDOA-D34S at 6 weeks old were treated with metformin in drinking water (1 g/l) for another 7 days. At the day 8, mice were sacrificed, and hepatic p-AMPKα and p-ACC levels were determined by immunoblotting. f, g, TRPV is dispensable for low metformin-induced AMPK activation. Experiments pertaining to TRPV dependency were performed in MEFs (f) as well as in mouse livers (g) as described in d (except 200 μM metformin was used) and e, respectively, except that MEFs with quadruple knockout of TRPV1-4 (f, TRPV-QKO), or the TRPV1-/- mice injected with a combination of AAV-carried siRNAs against TRPV2, TRPV3 and TRPV4 (g, siTRPV2-4, at 8 weeks old, which had been validated to show little expression of TRPVs, see ref. 25), were used (viruses were injected at 4 weeks old, and metformin was supplied at 8 weeks old). Experiments in this figure were performed three times, except d five times.