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Diabetes mellitus typ-2 (DMT-II) is a major cause of morbidity and mortality worldwide. Diabetes belongs to age-related diseases and is associated with chronic inflammation. The underlying molecular mechanisms responsible for the chronic inflammation are still largely unknown and patients suffer from little therapeutic possibilities to effectively treat chronic inflammation. Recent research indicates that chronic inflammation is closely linked to changes in energy metabolism via lysine acetylation of both histone and none-histone proteins.
Protein acetylation is carefully balanced by a network of lysine acetyl transferases (KAT, also known as histone acetyl transferases (HAT)) and lysine deacetylases (KDACs, also known as histone deacetylases (HDACs)). Reversible protein acetylation regulates a number of important cellular processes including gene expression, via acetylation of histones and transcription factors such as NFkB. Acetyl-CoA represents the substrate for KATs to acetylate lysine residues. Changes in glycolysis, tricarboxylic acid (TCA) cycle and beta-oxidation of fatty acids induce changes in cellular acetyl-CoA concentrations and may represent an important link between changes in energy metabolism and gene expression. Since DMT-II is associated with changes in energy metabolism, we hypothesize that regulation of protein acetylation and deacetylation is misbalanced resulting in disease-specific gene expression changes. To investigate how changes in energy metabolism affect protein acetylation profiles, a methodology will be developed to monitor changes in cellular metabolites (e.g. cellular acetyl-CoA) on the one hand and dynamics of protein acetylation patterns on the other hand and correlate them to changes in gene expression. Experiments will be carried out in perfused tissue slices, notably precision cut liver slices, in a dedicated microfluidics device allowing precise control over nutrient delivery as well as sample collection. We will use this method to study the anti-inflammatory effects of KDAC inhibitors by investigating changes in protein acetylation and deacetylation dynamics.
The aim of this research project is to identify molecular mechanisms linking changes in energy metabolism to pro-inflammatory gene expression in DMT-II. The project follows the hypothesis that aberrant changes in the levels of cellular energy metabolites, notably acetyl-CoA, lead to a change in the relative levels of acetylated protein species driving pro-inflammatory gene expression. To this end we will establish methodology based on liquid chromatography – mass spectrometry (LC-MS) in conjunction with microfluidics and stable isotope labelling.
Experiments will be carried out in microfluidic perfusion tissue slice culture in collaboration with the group of Sabeth Verpoorte. For microfluidic perfusion tissue slice culture different tissues relevant to DMT-II, such as precision cut liver slices, will be used. To investigate how changes in energy metabolism affect protein acetylation profiles, a methodology will be developed to monitor changes in cellular metabolites and protein acetylation patterns using metabolic and chemical labeling with stable isotopes. This methodology will include analysis of cellular metabolites (e.g. citrate and acetyl-CoA) by mass spectrometry (MS) and NMR on the one hand and MS analysis of acetylation dynamics of histones and none-histone proteins on the other hand. To investigate histone acetylation dynamics, a combination of metabolic (tracer molecules of energy metabolism such as glucose, glutamine, octanoic acid) and chemical stable isotope labeling (acetic-anhydride) will be used. This combination will enable us to investigate changes in histone acetylation dynamics upon treatment with high glucose, high lipid and high insulin concentrations with or without KDAC inhibitors. The kinetics of histone acetylation and deacetylation will be correlated to changes in gene expression of both pro- and anti-inflammatory cytokines, and to changes in cellular metabolite concentrations. Non-histone protein-acetylation dynamics will be investigated by differential proteomics/acetylomics using isobaric tandem mass tags (TMT). Upon treatment with high glucose, high lipid and high insulin concentrations with or without KDAC inhibitors, proteins will be isolated from tissue slices at different time points. Differential proteomics/acetylomics experiment will be carried out in collaboration with the group of Hartmut Schlüter (University Medical Center Hamburg-Eppendorf).
The proposed research project will make a significant contribution to our understanding of how changes in energy metabolism are linked to chronic inflammation in DMT-II. The research project will elucidate molecular mechanisms at the protein species level that can be ultimately exploited as drug targets for development of therapeutics that can be addressed by KAT and/or KDAC inhibitors. Responders/Non-Responders based on changes in acetylation patterns, which may serve as novel, mechanism-based biomarkers.
The proposed research will elucidate disease mechanisms linking aberrant energy metabolism to chronic inflammation in DMT-II by assessing the balance between protein acetylation and deacetylation protein. The proposed research will identify key protein species that may ultimately serve as starting points for the development of future therapeutics in conjunction with diagnostic assays. The project is therefore linked to the ProminenT domains “drug development” and “drug application” via the domain ''disease mechanisms''.