The pro-drug C13 activates AMPK by two distinct mechanisms

Abstract

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in almost all eukaryotic cells. In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the α1 (but not the α2) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.

Keywords: AMPK; C13; formaldehyde; mitochondria; mitochondrial respiration.

Figure 1.. C13 activates AMPK in human and rat cells.

(A) Human osteosarcoma (U2OS) cells, either wild type or double AMPK-α1/-α2 knockout made using CRISPR/Cas9 (DKO), were incubated with the indicated concentrations of vehicle (DMSO) or C13 for 1 h, cell lysates prepared and samples containing equal amounts of lysate protein analyzed by Western blotting with probing using antibodies against pThr172, total AMPK-α, pACC (pS80) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, loading control), or with streptavidin to detect total ACC. (B) quantification of (A) (pThr172/AMPK-α). (C) quantification of (A) (pACC/ACC). (D) AMPK activity measured in anti-α1/-α2 immunoprecipitates (wild type cells only). (E) Rat myoblast (L6) cells, either wild type or with AMPK-α1 knockdown made using shRNA, were incubated with the indicated concentrations of vehicle (DMSO) or C13 for 1 h, cell lysates prepared and samples containing equal amounts of lysate protein analyzed by Western blotting as in (A). (F) quantification of (E) (pThr172/AMPK-α). (G) quantification of (E) (pACC/ACC). (H) AMPK activity measured in anti-α1/-α2 immunoprecipitates (wild type cells only). For bar charts, results are mean ± SEM with individual points shown, with n = 2 for (B), (C), (F) and (G) and n = 4 for (D) and (H); mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2.. Formaldehyde is generated from C13 in U2OS cells, and associates with mitochondria.

(A) U2OS cells were incubated with 5 µM R6-FA for 20 min and then with either vehicle (DMSO), formaldehyde (600 µM), MK-8722 (200 nM) or C13 (300 µM) for 1 h and analysed by fluorescence microscopy. (B) L6 myoblasts were incubated with 5 µM R6-FA and 200 nM Mitospy for 20 min prior to the addition of either vehicle (DMSO) or the indicated concentrations of C13 or formaldehyde for 1 h and analysed by fluorescence microscopy. The top three rows show fluorescence of the R6-FA (647 nm) or Mitospy (488 nM) probes or a merge of the two, while the bottom row shows false color images of the ratio of R6-FA fluorescence to that of Hoechst 3342 DNA-binding dye. (C) Fluorescence intensity of R6-FA in U2OS cells after the indicated treatments. (D) Pearson's correlation of fluorescence from R6-FA and the Mitospy mitochondrial marker in U2OS cells after the indicated treatments. (E) Fluorescence intensity of R6-FA in L6 myoblasts after the indicated treatments. (F) Pearson's correlation of fluorescence from R6-FA and Mitospy dye in L6 myoblasts after the indicated treatments. In (C) to (F), results are mean ± SEM (n = 5–10) with individual data points shown, and mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3.. C13 and free formaldehyde both inhibit mitochondrial function in U2OS and L6 cells.

(A) Effect of increasing concentrations of C13 added at time zero (open circles, DMSO control; filled squares, 30 µM; filled triangles, 100 µM; filled inverted triangles, 300 µM) on oxygen consumption rate (OCR) in U2OS cells. At the point shown by arrows, the ATP synthase inhibitor oligomycin (1 µM), the uncoupler BAM15 (U2OS cells, 1 µM) or FCCP (L6 cells, 1 µM), or the respiratory chain inhibitors antimycin A and rotenone (2 and 1 µM) were added as indicated and OCR recorded. (B) Effect of increasing concentrations of C13 on basal OCR (before addition of oligomycin) in U2OS cells. (C) Effect of increasing concentrations of C13 (data from (A)) on ATP-linked OCR (OCR_basal - OCR_oligomycin) in U2OS cells. (D) Effect of increasing concentrations of C13 on maximal OCR (OCR_BAM15 - OCR_{antimycin/rotenone}) in U2OS cells. (E) As (A) but after treatment with formaldehyde (60, 200 and 600 µM). (F) to (H): as (B–D) but after treatment with formaldehyde (data from (E)). (I): as (A) but in L6 cells, in which FCCP was used in place of BAM15. (J) to (L): as (B) to (D) but with L6 cells (data from (I)). (M): as (E) but in L6 cells. (N) to (P): as (F) to (H) but in L6 cells (data from (M). (Q): as (A) but adding increasing concentrations of MK-8722 (30, 200, 500 nM) in place of C13 (U2OS cells). (R) to (T): as (B) to (D) but using MK-8722 (data from (Q)). For the bar charts results are mean SEM with individual data points shown (n = 3–6), and mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 4.. Effect of C13 or formaldehyde on mitochondrial membrane potential in (A) U2OS cells or (B) L6 cells.

Cells were treated with the mitochondrial uncoupler FCCP (1 µM, positive control), with the ADaM site activator MK-8722 (200 nM, negative control), or with the indicated concentrations of C13 or free formaldehyde (FA). Results are mean ± SEM with individual data points shown (n = 3–8) and mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5.. Effect of free formaldehyde on phosphorylation of AMPK and ACC and AMPK activity in U2OS and L6 cells.

Wild type or AMPK-α1/-α2 double knockout (DKO) U2OS cells ((A) to (D)), or wild type or AMPK-α1 knockdown L6 cells ((E) to (H)) were incubated for 1 h with the indicated concentrations of formaldehyde and analyzed by Western blotting using the indicated antibodies or other probes. (B), (C), (F) and (G) show quantification of pT172 to total AMPK ratio and pACC (Ser79/80) to total ACC ratios, while (D) and (H) show AMPK activities measured in anti-AMPK-α immunoprecipitates. Results in bar charts are mean ± SEM with individual data points shown (n = 2 for Western blots and n = 3 for kinase assays), and mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 6.. Effect of increasing concentrations of C13 and formaldehyde on nucleotide ratios and AMPK activities in U2OS or HEK-293 cells.

(A) AMP:ATP and ADP:ATP ratios in U2OS cells after treatment for 1 h with the indicated concentrations of C13 or formaldehyde (FA). (B): as (A) but in HEK-293 cells. (C) AMPK activity assessed by immunoprecipitate kinase assay in U2OS cells. (D): as (B) but in HEK-293 cells. Results are mean ± SEM with individual data points shown (n = 3) and mean values that are significantly different from DMSO controls by one-way ANOVA are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 7.. AMPK activation by both C13 and formaldehyde in HEK-293 cells are abolished by an R531G mutation in AMPK-γ2.

Cells were incubated for 1 h with DMSO (control), A23187 (3 µM), MK-8722 (200 nM), C13 (300 µM) or formaldehyde (600 µM). (A) AMPK activity in anti-FLAG immunoprecipitates in HEK-293 cells expressing wild type γ2 or an R531G γ2 mutant. Results are mean ± SEM with individual data points shown (n = 3), and mean values that are significantly different from the DMSO controls for that genotype by two-way ANOVA are indicated (*P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant). (B) Phosphorylation of Thr172 on AMPK-α, and levels of AMPK-α, -β and -γ2 (FLAG-tagged) in HEK-293 cells expressing wild type γ2 or an R531G γ2 mutant.