Epigenetic mechanisms translating cellular energy homeostasis to differentiation of the intestinal epithelium

Abstract

Background and aims: The small intestine has many important functions, which constitute to the welfare of the whole organism. These vital functions are mediated by community of intestinal epithelial cells. However, the luminal environment that these cells are exposed to, makes it hard for them to survive. This leads to the situation in which fully maturated epithelial cells located in finger-like projections, called villi, are going through apoptosis and that is why new cells are needed daily to fill the cap created by the cell death. Small population of intestinal stem cells that are lying in base of invagination of small intestine, called crypts, are asymmetrically dividing and producing progenitor cells. These cells can further divide and differentiate to any cells of the epithelium while migrating upwards along the villi to fill the cap created by the apoptosis. Recently, it was shown that Polycomb Repressive Complex 2 is maintaining the epigenetic polarization between the intestinal stem cells and fully differentiated cells. Polycomb Repressive Complex 2 must be silenced for the time that progenitor cells can differentiate. It was found with the fibroblast, that metabolic related AMP-activated protein kinase can phosphorylate, and in this way inactivate the Polycomb Repressive Complex 2. The first aim of this thesis is to investigate whether this AMP-activated protein kinase mediated inactivation of Polycomb Repressive Complex could affect cell differentiation in small intestinal epithelium. The second aim is to define metabolic phenotypes of intestinal epithelial cells to gain more knowledge how these cells produce their energy.

Methods: This study models epithelium of small intestine with organotypic mouse mini-gut organoid cultures. Intestinal organoids are cultured under various differentiation medias and, AMPK inhibitors and activators. Quantitative polymerase chain reaction is used to investing the changes on cell differentiation during treatments. Western blot is used to examine the protein levels of intestinal organoids to see whether fully activated AMP-activated protein kinase can phosphorylate Polycomb Repressive Complex 2. Finally, energy metabolism of different types of intestinal organoids is investigated with Agilent`s Seahorse- technique.

Results: This study shows that AMP-activated protein kinase is not behind phosphorylation of Polycomb Repressive Complex 2 in small intestine. Interestingly, both inhibition and activation of AMP-activated protein kinase are creating less differentiated intestinal organoids. Metabolic phenotypes analyses show, that intestinal stem cells are more dependent on oxidative phosphorylation while fully differentiated intestinal epithelial cells are capable to use oxidative phosphorylation and glycolysis to fill their energy needs.

Conclusions: More studies are needed to figure out how Polycomb Repressive Complex 2 is regulated in intestinal epithelial cells and why both inhibition and activation of AMP-activated protein kinase created organoids with significant increment of intestinal stem cells. Also, the procedure of Agilent`s Seahorse measurement must be optimized further to obtain more reliable OCAR and ECAR values.