Fig 1.
AICAR and metformin increased AAK phosphorylation in a time-dependent manner.
AAK phosphorylation at Thr243 (equivalent to Thr172 of mammals) was detected using an anti-pAMPK antibody in N2 adult nematodes treated with A) 1 mM AICAR or B) 50 mM metformin. In both cases, the top panel shows a representative Western blot of phosphorylated AAK (pAAK), where the intensity of the bands represents the phosphorylation level, corresponding to kinase activation. C) Expression levels of aak-2 and aak-1 by qRT-PCR in N2 and aak-2/ok524 strains exposed to 1 mM AICAR for 12 h or 50mM Metformin for 24 h. The graphs show the mean ± SEM of three independent experiments *p<0.05, **p<0.01 vs. 0 h, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.
Fig 2.
AAK activation by AICAR and metformin enhances oxidative metabolism in C. elegans.
N2 (white box) and aak-2/ok524 (black box) nematode strains were grown to the adult stage and exposed to 1 mM AICAR for 12 h or 50 mM metformin for 24 h. A) Mitochondrial and non-mitochondrial oxygen consumption. B) Lactate concentration and C) Total triglyceride content. Each graph shows the mean ± SEM of three independent experiments. The aak-2 mutant groups showed no change between them. *p<0.05, **p<0.01, ***p<0.001 vs. untreated group, one-way ANOVA with Bonferroni's post hoc test, using GraphPad Prism.
Fig 3.
AICAR and metformin modify the fatty acid profile in C. elegans.
Individual fatty acids were determined by gas chromatography analysis. Control wild-type (WT) N2 nematodes (white box) were grown in 1 mM AICAR (black box) or in 50 mM metformin (gray box). A) Individual fatty acids: 14:0, myristic acid; 16:0, palmitic acid; 18:0, stearic acid; 16:1n9, 7-palmitoleic acid; 16:1n7, 9-palmitoleic acid; 18:1n9, oleic acid; 18:1n7, vaccenic acid; 18:2n6, linoleic acid; 20:3n6, dihomo-γlinolenic acid (DGLA); 20:4n6, arachidonic acid; 20:4n3, eicosatetraenoic acid; 20:5n3, eicosapentaenoic acid (EPA). B) Fatty acids grouped as saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids. C) Elongase activity indirectly measured from substrate/product ratios. D) Desaturase activity indirectly measured from substrate/product ratios. Each graph shows the mean ± SEM of three independent experiments, *p<0.05, **p<0.01, ***p<0.005 vs. untreated group, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.
Fig 4.
AAK activation-dependent changes of selected transcripts involved in energy metabolism regulation in C. elegans.
Gene expression analysis was performed using qRT-PCR on wild-type N2 (WT) and aak-2/ok524 nematodes grown until adults and exposed to 1 mM AICAR for 12 h. A) nhr-49, B) mdt-15, C) sbp-1, D) fat-7, E) fat-2, F) fasn-1, G) acs-2, H) acdh-2 and I) elo-2. The relative expression of each gene was normalized to endogenous 18S rRNA gene expression. Data shown are the mean ± SEM of three independent experiments, *p<0.05, **p<0.01, ***p<0.001 vs. WT control and ##p<0.01, ###p<0.001 vs. WT AICAR, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.
Fig 5.
AICAR modifies AAK phosphorylation in mutant strains lacking NHR-49, MDT-15 or SBP-1 transcription factors without altering the expression of both aak-2 and aak-1.
Adult worms of the mutant strains nhr-49/ok2165, mdt-15/tm2182 and sbp-1/ok2363 lacking the proteins NHR-49, MDT-15 and SBP-1, respectively, were exposed to 1 mM AICAR for 12 h. A) AAK phosphorylation at Thr243; top panel shows a representative Western blot of phosphorylated AAK (pAAK), the graph represents the densitometric analysis. B-D) Relative expression of aak-2 and aak-1 genes normalized to endogenous 18S rRNA gene expression. Each graph shows the mean ± SEM of three independent experiments, **p<0.01 vs. untreated group of its own strain, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.
Fig 6.
NHR-49, MDT-15 and SBP-1 are required to modulate some metabolic markers upon AICAR exposure.
Adult worms of the mutant strains nhr-49/ok2165, mdt-15/tm2182 and sbp-1/ok2363 lacking the proteins NHR-49, MDT-15 and SBP-1, respectively, were exposed to 1 mM AICAR for 12 h. A) Oxygen consumption rate. B) Lactate concentration and, C) Triglyceride content. Each graph shows the mean ± SEM of three independent experiments, **p<0.01 vs. untreated group of its own strain, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.
Fig 7.
Metformin reverses the deleterious effects of dietary glucose in C. elegans in an AAK-dependent manner.
A-C) Adult wild-type (WT) or D-E) aak-2/ok524 nematodes were grown on 100 mM glucose with or without 50 mM metformin for 24 h. A) the top panel is a representative Western blot of AAK phosphorylated (pAAK) at Thr243, where the intensity of bands represents the phosphorylation level, corresponding to kinase activation. B and D) triglyceride content and C and E) oxygen consumption rates. The graphs represent the mean ± SEM of three independent experiments, *p<0.05, ***p<0.001 vs. WT control and ##p<0.01, ###p<0.001 vs. WT glucose, one-way ANOVA with Bonferroni's post hoc test using GraphPad Prism.