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Elucidation of lipid metabolism pathways mediated by AMPK and nuclear receptors PPARδ and LXRα in human macrophages

 
: Kemmerer, M.

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Fulltext (PDF; )

Frankfurt/M., 2016, 149 pp.
Frankfurt/M., Univ., Diss., 2015
URN: urn:nbn:de:hebis:30:3-390935
English
Dissertation, Electronic Publication
Fraunhofer IME ()

Abstract
Disturbances in lipid metabolism are responsible for many chronic disorders, such as type 2 diabetes and atherosclerosis. Regulation of lipid metabolism occurs by activated transcription factors peroxisome proliferator-activated receptor δ (PPARδ) and liver X receptor α (LXRα) mediating transcription of different target genes involved in regulation of fatty acid uptake and oxidation or cellular cholesterol homeostasis. This is especially relevant for the macrophages, since pathways regulated by PPARδ and LXRα affect foam cell formation, a process driving the progression of atherosclerotic lesion. AMP-activated protein kinase (AMPK) plays a central role in energy homeostasis in every type of eukaryotic cell, but its role in human macrophages, particularly with regard to lipid metabolism, is not precisely defined yet. Thus, I investigated the impact of AMPK activity on PPARδ and LXRα and the expression of their target genes involved in fatty acid oxidation (FAO) and cholesterol metabolism.

As PPARδ has been described as a potential target for prevention and treatment of several disorders and AMPK as interesting drug target for diabetes and metabolic syndrome, the aim of the first part of my studies was to investigate their interaction in primary human macrophages. Completing the first challenge successfully, I was able to establish a lentiviral transduction system for constitutively active AMPK (consisting of a truncated catalytic AMPKα1 subunit bearing an activating T198D mutation) in primary human macrophages.

Using genome-wide microarray analysis of gene expression, I demonstrate FAO as the strongest affected pathway during combined AMPKα1 overexpression and PPARδ activation.

The most influenced genes were validated by quantitative PCR as well as by Western analysis. I found that AMPK increases the expression of FAO-associated genes targeted by PPARδ. Corroborating the results obtained using AMPKα1 overexpression, PPARδ target gene expression was increased not only by PPARδ agonist GW501516, but also by pharmacological allosteric AMPK activator A-769662. Additional enhancement of target gene mRNA expression was achieved upon co-activation of PPARδ and AMPK. Silencing PPARδ expression increased basal expression of target genes, confirming the repressive nature of ligand-free PPARδ, abolishing the increased target gene expression upon AMPK or PPARδ activation. Measurements of triglyceride contents of human macrophages incubated with VLDL following PPARδ activation demonstrated a reduction of intracellular triglyceride accumulation in cells, which may reflect the enhancement of fat catabolism.

In the second part of my studies, I concentrated on the regulation of cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) expression by AMPK. ABCA1 facilitates

cholesterol efflux from macrophages thus, preventing atherosclerosis progression. For the first time, AMPK implication in the regulation of the ABCA1 pathway could be presented. Both AMPK overexpression and activation lead to significantly increased ABCA1 expression, whereas AMPKα1 knock-down strongly reduced this effect. Besides, I was able to prove an enhanced activity of ABCA1 during AMPK activation in human THP-1 macrophages by measuring cholesterol efflux into apolipoprotein AI-containing medium.

Previous findings showed regulation of ABCA1 by LXRα. I confirmed these results by silencing experiments indicating an essential role of LXRα in ABCA1 regulation pathway.

Here, ABCA1 mRNA as well as protein expression were positively mediated by LXRα. LXRα activation elevated ABCA1 levels, whereas its silencing down-regulated this effect.

Interestingly, ABCA1 was found to be regulated only by LXRα and not through LXRα. At the same time, knock-down of PPARδ, -γ or -δ, which may be also involved in the regulation of LXR/ABCA1 axis, did not influence the activation of ABCA1 expression by an AMPK activator. To confirm that LXRE on Abca1 promoter is essential for ABCA1 regulation, I performed luciferase reporter assay using constructs based on Abca1 promoter with or without LXRE mutation. Mutation of LXRE abolished reporter activity, whereas AMPK activation increased luciferase activity of wild-type LXRE construct. Furthermore, I demonstrate AMPK-dependent LXRα binding to the LXRE site of Abca1 promoter using the method of chromatin immunoprecipitation. AMPK activation significantly increased, whereas silencing of AMPK significantly attenuated LXRα binding, indicating AMPK as one of the most important regulators of ABCA1 expression.

In summary, I provided an evidence for AMPK involvement into lipid and cholesterol metabolism in human macrophages showing the regulation of PPARδ and LXRα target genes. The understanding of AMPK and PPARδ interaction allows the development of new approaches for treatment of metabolic syndrome and related diseases. Increased FAO during the activation of both proteins may exhibit better therapeutic benefit. On the other hand, I have shown the impact of AMPK activation on ABCA1 via LXRα up-regulation leading to increased cholesterol efflux in human macrophages for the first time. These findings thus may impact future improving of anti-atherosclerosis therapies.

 

Akuter Herztod ist mit 4-5 Millionen Fällen pro Jahr weltweit eines der größten Todesrisiken, das durch eine bestimmte ventrikuläre Herzrhythmusstörung verursacht wird. 80% dieser Fälle sind mit koronaren Arterienerkrankungen assoziiert, u.a. durch das Metabolische Syndrom. Das Metabolische Syndrom kann mehrere Stoffwechsel-erkrankungen, inklusive Typ 2 Diabetes sowie Atherosklerose, einschließen. Beide Störungen werden durch einen Entzündungsprozess hervorgerufen. Durch die Ausschüttung pro-inflammatorischer Chemokine migrieren die Monozyten zu der Entzündungsstelle, wandern anschließend ins entzündliche Gewebe ein und differenzieren zu Makrophagen. Makrophagen gehören zu Immunzellen des angeborenen Immunsystems und entfernen während jedes entzündlichen Krankheitsverlaufs apoptotische und immunsystemfremde Körper aus dem Gewebe mittels Phagozytose und aktivieren das adaptive Immunsystem durch Zytokin-Ausschüttung. Der Lipidmetabolismus spielt bei diesen Prozessen eine große Rolle. Aus diesem Grund sind die Untersuchungen des Lipidstoffwechselwegs in Makrophagen notwendig, um neue Therapien entwickeln zu können.

: http://publica.fraunhofer.de/documents/N-439336.html