Role of Sphingolipid Metabolism in Intestinal Stem Cell Differentiation

Abstract

Epidemiological studies have demonstrated correlations between pro-obesity diets high in saturated fats and colorectal cancer (CRC) incidence. The effects of fatty acid palmitate are well supported in both in vivo and in vitro work to induce obesity and disease like states. The gastrointestinal tract plays a vital role in nutrient absorption and acts as a barrier from harmful pathogens. The intestinal epithelium can regenerate and repair due to the self-renewal and proliferative properties of intestinal stem cells (ISCs). ISCs increase proliferation in response to high fat diet, augmenting tumor initiation capacity; however, the mechanisms by which this occurs are not fully understood. Organoids are self-renewing, self-organizing structures grown from stem cells or intestinal crypts that allow for investigation of intestinal biology. While the effects of palmitate (C16) have been shown to increase stemness and self-renewal capacity in organoids, little is documented about the effects of additional fatty acids. Our lab has previously implicated myristate (C14) in the induction of ER stress in small intestinal cell lines and in mice fed a diet high in milkfat, a dietary source of C14. ER stress has also been shown to increase differentiation in the intestine. Sphingolipids are bioactive lipids that reside within cell membranes and participate in cell signaling. Our lab has implicated sphingolipids in ER stress and inflammation in the intestines; however, very little is known about the role that sphingolipids play in stem cell metabolism. Ceramide synthases (CerS) 5 and 6 are responsible for integrating fatty acids myristate and palmitate into the acyl chain of the central sphingolipid molecule ceramide. Therefore, we examined the effects of myristate on stemness and differentiation in small intestinal (SI) organoid models. To understand the effects of myristate treatment in CerS5 and CerS6 knockout organoids, we quantified sphingolipid species and measured stemness and differentiation markers. We found that species of C14 sphingolipids decreased in CerS6 knockout organoids. We then quantified morphological changes and sphingolipid species in response to inhibiting enzymes in the sphingolipid metabolic pathway. We found that inhibiting CerS decreased branching in SI organoids. Future studies in CerS5 andCerS6 knockout SI organoid models will help to elucidate the role of sphingolipids in stem cell biology and sphingolipid metabolism.