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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

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.
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pone.0130893.g002: 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.

Mentions: Next, we questioned whether pharmacological AMPK activation similarly enhanced PPARδ target gene mRNA expression as AMPK overexpression. In these experiments we used the allosteric AMPK activator A-769662. Analysis of the phosphorylation status of AMPK, its substrate ACC and ribosomal protein S6, which served as readout of mTOR (mechanistic target of rapamycin) activity, revealed that significant ACC phosphorylation was already observed at 50 μM A-769662, and continued to increase up to 500 μM A-769662 (Fig 2A). In contrast, significant down-regulation of phospho-S6 and up-regulation of phospho-AMPK was achieved only at 250 μM and 500 μM A-769662, respectively. Therefore, we used 500 μM of the drug in subsequent experiments. Measuring intracellular ATP did not show changes after exposure to these concentrations of A-769662, ruling out a loss of viability (data not shown). As shown in Fig 2B, treatment of primary human macrophages with A-769662 induced mRNA expression of the PPARδ target genes PDK4, CPT1a, and PLIN2 to a similar extent as the exposure of cells to the PPARδ ligand GW501516. Importantly, A-769662 significantly augmented PPARδ target gene expression in GW501516-treated cells. Similar results were obtained when analyzing mRNA expression of PPARδ target genes following macrophage treatment with GW501516 and salicylate, which has recently been recognized as another direct allosteric AMPK activator [37] (S1 Fig). Analysis of PPARδ target gene expression also revealed cell type-specific differences. Whereas the well-known PPARδ target gene angiopoietin-like 4 (Angptl4), which is a lipoprotein lipase inhibitor, was robustly induced in the THP-1 macrophage cell line, it was not induced in primary macrophages (S2 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)

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.
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pone.0130893.g002: 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.
Mentions: Next, we questioned whether pharmacological AMPK activation similarly enhanced PPARδ target gene mRNA expression as AMPK overexpression. In these experiments we used the allosteric AMPK activator A-769662. Analysis of the phosphorylation status of AMPK, its substrate ACC and ribosomal protein S6, which served as readout of mTOR (mechanistic target of rapamycin) activity, revealed that significant ACC phosphorylation was already observed at 50 μM A-769662, and continued to increase up to 500 μM A-769662 (Fig 2A). In contrast, significant down-regulation of phospho-S6 and up-regulation of phospho-AMPK was achieved only at 250 μM and 500 μM A-769662, respectively. Therefore, we used 500 μM of the drug in subsequent experiments. Measuring intracellular ATP did not show changes after exposure to these concentrations of A-769662, ruling out a loss of viability (data not shown). As shown in Fig 2B, treatment of primary human macrophages with A-769662 induced mRNA expression of the PPARδ target genes PDK4, CPT1a, and PLIN2 to a similar extent as the exposure of cells to the PPARδ ligand GW501516. Importantly, A-769662 significantly augmented PPARδ target gene expression in GW501516-treated cells. Similar results were obtained when analyzing mRNA expression of PPARδ target genes following macrophage treatment with GW501516 and salicylate, which has recently been recognized as another direct allosteric AMPK activator [37] (S1 Fig). Analysis of PPARδ target gene expression also revealed cell type-specific differences. Whereas the well-known PPARδ target gene angiopoietin-like 4 (Angptl4), which is a lipoprotein lipase inhibitor, was robustly induced in the THP-1 macrophage cell line, it was not induced in primary macrophages (S2 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