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AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

Kemmerer M, Finkernagel F, Cavalcante MF, Abdalla DS, Müller R, Brüne B, Namgaladze D - PLoS ONE (2015)

Bottom Line: To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516.The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner.Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.

ABSTRACT
AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0130893.g003: 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).

Mentions: To further dissect the roles of AMPK and PPARδ in regulating FAO-related gene expression, we silenced AMPK α1 catalytic subunit (the predominant isoform in human macrophages) and PPARδ and followed mRNA and protein expression of PPARδ target genes in macrophages treated with A-769662, GW501516 and their combination. Silencing of PPARδ achieved over 90% knockdown (KD) at the mRNA level (Fig 3A) and diminished the expression of PPARδ protein (Fig 3C). It also increased the basal expression of PPARδ target genes PDK4, CPT1a, but not PLIN2, consistent with the known repressor function of ligand-free PPARδ (Fig 3B) [38]. Cells with a PPARδ KD had also a blunted response to A-769662 and GW501516; PLIN2 mRNA expression was unaltered whereas PDK4 was significantly increased only in response to A-769662 and CPT1a was significantly increased only after co-treatment with A-769662 and GW501516. AMPKα1 KD achieved more than 90% reduction of AMPKα1 mRNA levels and over 65% reduction of AMPKα1 protein (Fig 3A and 3C). Accordingly, basal and A-769662-stimulated phosphorylation of the AMPK substrate ACC was significantly attenuated in AMPKα1-silenced cells (Fig 3C). Interestingly, AMPKα1 KD also reduced mRNA and protein levels of PPARδ and increased mRNA expression of PPARδ target genes thus, mimicking the PPARδ target gene mRNA expression changes in PPARδ KD cells (Fig 3A–3C). Similarly increased mRNA expression of PPARδ target genes after AMPKα1 knockdown was observed using unrelated siControl siRNA as well as in THP-1 macrophages stably transduced with unrelated AMPKα1 shRNA lentivirus (data not shown). AMPKα1 KD macrophages also did not show significantly increased mRNA expression of PPARδ target genes after A-769662 treatment. Still, AMPKα1 KD cells responded to PPARδ activation by GW501516 or combined GW501516/A-769662 treatment with increased expression of PLIN2, PDK4 or CPT1a. However, we did not observe any effect of A-769662 on PPARδ mRNA (data not shown). We also did not notice an effect of A-769662 treatment on nuclear PPARδ levels (S3 Fig).


AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

Kemmerer M, Finkernagel F, Cavalcante MF, Abdalla DS, Müller R, Brüne B, Namgaladze D - PLoS ONE (2015)

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).
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pone.0130893.g003: 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).
Mentions: To further dissect the roles of AMPK and PPARδ in regulating FAO-related gene expression, we silenced AMPK α1 catalytic subunit (the predominant isoform in human macrophages) and PPARδ and followed mRNA and protein expression of PPARδ target genes in macrophages treated with A-769662, GW501516 and their combination. Silencing of PPARδ achieved over 90% knockdown (KD) at the mRNA level (Fig 3A) and diminished the expression of PPARδ protein (Fig 3C). It also increased the basal expression of PPARδ target genes PDK4, CPT1a, but not PLIN2, consistent with the known repressor function of ligand-free PPARδ (Fig 3B) [38]. Cells with a PPARδ KD had also a blunted response to A-769662 and GW501516; PLIN2 mRNA expression was unaltered whereas PDK4 was significantly increased only in response to A-769662 and CPT1a was significantly increased only after co-treatment with A-769662 and GW501516. AMPKα1 KD achieved more than 90% reduction of AMPKα1 mRNA levels and over 65% reduction of AMPKα1 protein (Fig 3A and 3C). Accordingly, basal and A-769662-stimulated phosphorylation of the AMPK substrate ACC was significantly attenuated in AMPKα1-silenced cells (Fig 3C). Interestingly, AMPKα1 KD also reduced mRNA and protein levels of PPARδ and increased mRNA expression of PPARδ target genes thus, mimicking the PPARδ target gene mRNA expression changes in PPARδ KD cells (Fig 3A–3C). Similarly increased mRNA expression of PPARδ target genes after AMPKα1 knockdown was observed using unrelated siControl siRNA as well as in THP-1 macrophages stably transduced with unrelated AMPKα1 shRNA lentivirus (data not shown). AMPKα1 KD macrophages also did not show significantly increased mRNA expression of PPARδ target genes after A-769662 treatment. Still, AMPKα1 KD cells responded to PPARδ activation by GW501516 or combined GW501516/A-769662 treatment with increased expression of PLIN2, PDK4 or CPT1a. However, we did not observe any effect of A-769662 on PPARδ mRNA (data not shown). We also did not notice an effect of A-769662 treatment on nuclear PPARδ levels (S3 Fig).

Bottom Line: To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516.The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner.Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.

ABSTRACT
AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

No MeSH data available.


Related in: MedlinePlus