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).