Fig 1.
Analysis of AMPK and PPARδ interactions in primary human macrophages with overexpression of AMPK catalytic or regulatory subunits.
Primary human macrophages were transduced with control lentivirus (CV) or lentiviral particles coding for a constitutively active AMPKα1 (AMPK OE) for 48 hours and treated with 100 nM GW501516 for additional 24 hours. A Venn diagram showing numbers of genes regulated by AMPK OE, GW501516, or their combination. B Validation of microarray analysis by quantitative PCR. C mRNA expression of PDK4, CPT1a, and PLIN2 in macrophages infected with control adenovirus (AdTrack) or AMPKγ1 R70Q adenovirus for 48 hours prior to 24 hour-treatment with 100 nM GW 501516. D Western blot showing ACC phosphorylation and its quantification in macrophages infected with control adenovirus (AdTrack) or AMPKγ1 R70Q adenovirus for 48 hours. Values represent averages ± 95% Confidence Interval. *, p<0.05; **, p<0.01 (n = 3).
Table 1.
Major regulated pathways by Gene Set Enrichment Analysis.
Table 2.
20 strongest induced genes upon combined AMPK/PPARδ activation.
Fig 2.
Pharmacological AMPK and PPARδ activation affects expression of FAO genes, FAO and VLDL-induced lipid accumulation in primary human macrophages.
A Western analysis of macrophages treated with indicated concentrations of A-769662 for 1 hour (n = 4). C, untreated cells. B, C mRNA (B) and protein (C) expression of PDK4, CPT1a, and PLIN2 in macrophages treated with 500 μM A-769662 and/or 100 nM GW501516 for 24 hours (mRNA) or 48 hours (protein) (n = 3). D Etomoxir-sensitive respiration in human macrophages following 48 hour-treatment with 100 nM GW501516 and 100 μM A-769662. E Triglyceride content of primary macrophages treated with 100 nM GW501516 and/or 250 μM A-769662 for 48 hours prior to VLDL (20 μg/ml) stimulation for 24 hours (n = 4). Values represent averages ± 95% Confidence Interval.*, p<0.05; **, p<0.01.
Fig 3.
AMPK increased the expression of PPARδ target genes through PPARδ.
Primary macrophages were transfected with 50 nM siControl (siC), AMPKα1 or PPARδ siRNA for 72 hours and treated with 250 μM A-769662 or 100 nM GW501516 for additional 24 hours. A mRNA expression of PPARδ and AMPKα1. B mRNA expression of PDK4, CPT1a, and PLIN2 in macrophages treated as indicated above. C Protein expression of phospho-ACC, ACC, PDK4, CPT1a, PPARδ, and AMPKα1 in macrophages treated as indicated above. Values represent averages ± 95% Confidence Interval. §, p<0.05 (n = 5–8).