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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

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).
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pone.0130893.g001: 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).

Mentions: Previous studies revealed interactions of AMPK and PPARδ in muscle cells, causing a transcriptional reprogramming to increase fatty acid oxidative metabolism [28]. We questioned functional interactions of AMPK and PPARδ in primary human macrophages by analyzing alterations of the macrophage transcriptome following single and combined treatments to activate AMPK and PPARδ. To avoid off-target effects associated with pharmacological AMPK activation we overexpressed a lentiviral construct coding for a truncated, constitutively active human AMPKα1 subunit (AMPK OE) in primary human macrophages, followed by 24 hour-treatments with 100 nM of the selective PPARδ agonist GW501516. Genome-wide mRNA expression profiling was then performed using Illumina HT12v4 bead arrays (EMBL-EBI Array Express accession number E-MTAB-2524). As illustrated by the Venn diagram (Fig 1A), 238 genes were regulated by AMPK overexpression (107 up, 131 down, log2(fold change)≥0.58), while 79 genes changed their expression in response to GW501516 (46 up, 33 down) with an overlap of 8 genes (3 up, 5 down). Combined AMPK/PPARδ activation altered the expression of 322 genes (128 up, 194 down). Testing the cooperativity of gene regulation by AMPK and PPARδ we did not find any probe showing >50% difference in intensity after co-activation of AMPK and PPARδ compared with single stimulations, indicating no synergistic effects.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476747&req=5

pone.0130893.g001: 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).
Mentions: Previous studies revealed interactions of AMPK and PPARδ in muscle cells, causing a transcriptional reprogramming to increase fatty acid oxidative metabolism [28]. We questioned functional interactions of AMPK and PPARδ in primary human macrophages by analyzing alterations of the macrophage transcriptome following single and combined treatments to activate AMPK and PPARδ. To avoid off-target effects associated with pharmacological AMPK activation we overexpressed a lentiviral construct coding for a truncated, constitutively active human AMPKα1 subunit (AMPK OE) in primary human macrophages, followed by 24 hour-treatments with 100 nM of the selective PPARδ agonist GW501516. Genome-wide mRNA expression profiling was then performed using Illumina HT12v4 bead arrays (EMBL-EBI Array Express accession number E-MTAB-2524). As illustrated by the Venn diagram (Fig 1A), 238 genes were regulated by AMPK overexpression (107 up, 131 down, log2(fold change)≥0.58), while 79 genes changed their expression in response to GW501516 (46 up, 33 down) with an overlap of 8 genes (3 up, 5 down). Combined AMPK/PPARδ activation altered the expression of 322 genes (128 up, 194 down). Testing the cooperativity of gene regulation by AMPK and PPARδ we did not find any probe showing >50% difference in intensity after co-activation of AMPK and PPARδ compared with single stimulations, indicating no synergistic effects.

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