FIG. 1.

Trien opposes the action of metformin on AMPK and signaling to S6. A: Graph of the available stability constants for EDTA, triethylaminetetramine (trien), and the cumulative constant for a 2:1 dimethylbiguanide (metformin)/metal complex (data obtained from Refs. 17,25,26). Biguanides do not form stable complexes with zinc or alkaline earth metals (17). B: Cells placed in serum-free medium treated with (bottom)/without (top) 10 mM trien for 18 h were treated with the copper-binding probe followed by washout and image capture on the confocal microscope. Scale bar, 10 μm. C: Preincubation of cells with trien inhibits metformin’s effects on AMPK Thr¹⁷² phosphorylation (pAMPK), ACC Ser⁷⁹ phosphorylation (pACC), and S6 Ser²⁴⁰/²⁴⁴ phosphorylation (pS6). H4IIE cells were grown in serum-free medium for 18 h in the presence or absence of 10 mM trien, followed by stimulation with or without 2 mM metformin. Lysates were prepared as described in research design and methods then subjected to SDS-PAGE, followed by immunoblotting with the antibodies indicated. D: Cells were treated as in C, except 1 mM metformin (Met) was used, before lysis, immunoprecipitation, and AMPK assay as described in research design and methods. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc test, ***P < 0.001 with respect to control. The significance of other column-to-column differences are presented next to a horizontal line identifying the two columns. Errors are SEM. E: Cells were treated as in C but in the presence of a dose response of trien. (A high-quality digital representation of this figure is available in the online issue.)