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. 2016 Oct 4;126(11):4273–4288. doi: 10.1172/JCI85510

Figure 7. IP6 and IP6K1 differentially regulate AMPK stimulatory phosphorylation.

Figure 7

(A) Immunoblot demonstrates that overexpression of GST-IP6K1 diminishes AMPK and ACC phosphorylation levels in HEK293 cells under basal (+), glucose-deprived (–), and glucose-reintroduced (–/+) conditions. Data represent results from at least 3 independent experiments. (B) IP6, at increasing concentrations, stimulates AMPKα2/β1/γ1 (AMPKα T172) phosphorylation mediated by LKB1-only (120 ng/reaction). Data represent results from at least 3 independent experiments. (C) Quantification of B reveals that IP6 leads to 6- to 7-fold enhancements in LKB1-mediated AMPK phosphorylation in vitro. Data represent results from at least 3 independent experiments (1-way ANOVA). (D) Among inositol phosphates, IP6 most potently stimulates LKB1-only–mediated AMPKα2/β1/γ1 phosphorylation (IP6 > IP5 > IP4 = IP5* > IP3 > 5-IP7). IP5 displays partial stimulatory effect. IP6K1 products IP5* and 5-IP7 are ineffective. Data represent results from duplicate experiments. (E) IP6K1 inhibits IP6-mediated AMPKα2/β11 phosphorylation by LKB1-only, whereas the inactive IP6K1 mutant is much less efficient. Data represent results from 3 independent experiments. (F) 5-IP7 reverses IP6-mediated AMPKα2/β1/γ1 phosphorylation by LKB1-only. Data represent results obtained from 3 independent experiments. (G) IP6 also enhances LKB1-complex–mediated AMPKα2/β11 phosphorylation. Other inositol polyphosphates are ineffective. Data represent results obtained from duplicate experiments. (H) IP6 enhances CaMKKβ-mediated AMPKα21/γ1 phosphorylation. Data represent results obtained from duplicate experiments. (I) IP6 does not enhance LKB1-mediated phosphorylation of heat-denatured AMPKα2/β1/γ1. Data represent results obtained from 3 independent experiments. (J) IP6-mediated enhancement of AMPK phosphorylation is largely similar when AMPKα is used alone or in a complex with AMPKα2/β1/γ1. Data represent results obtained from 3 independent experiments. (K) AMP stimulates LKB1-mediated AMPKα2/β11 phosphorylation at 50- to 100-μM concentrations. Data represent results obtained from duplicate experiments. (L) IP6 or 5-IP7 does not influence active AMPK’s activity on its target SAMStide (n = 3; 1-way ANOVA). Data are expressed as mean ± SEM. §P < 0.0001.