Role of Myristate and Sphingolipids on Intestinal ER Stress and Lipid Metabolism

Diets high in saturated fat have been implicated in disturbing intestinal homeostasis via multiple mechanisms; however, the contribution of specific saturated fatty acids (SFAs) is largely unknown. Therefore, the goal of this proposal is to examine the effects of specific dietary fatty acids (FAs) on sphingolipid metabolism and their influence on intestinal pathobiology. Specifically, we aim to define the role of ceramide generating enzymes, ceramide synthase (CerS) 5 and 6, in endoplasmic reticulum (ER) stress, inflammation, and lipid metabolism in the intestines. Excess SFAs and a subsequent increase in ceramide generation induce ER stress in multiple tissues. Biopsy samples from inflammatory bowel disease (IBD) patients exhibited dysregulation of sphingolipid enzymes and increased activation of all three ER stress pathways; however, whether a relationship exists between the two is only beginning to be elucidated. In the intestines we and others have implicated CerS as regulators of lipid metabolism and ER stress. Canonically, ER stress is triggered by accumulation of mis-folded proteins leading to activation of effector proteins IRE1α, PERK, and ATF6. Activation of these pathways halts generation of new proteins, leads to degradation of mRNA and proteins, and if left unresolved leads to inflammation and apoptosis. IRE1α activation has been implicated in regulating sphingolipid enzymes and other lipid metabolic proteins, such as stearoyl-CoA desaturase 1 (SCD1). Although there have been a few investigations into the role of SCD1 in intestinal inflammation and colon cancer; there is contention in the literature on whether SCD1 is protective or detrimental. Individually ER stress, sphingolipids, and SCD1 have been implicated in IBD and CRC; but the interplay of these factors in intestinal biology is not well understood. Much of the literature examining the effects of a high fat diets (HFDs) has focused primarily on palmitate, a C16 SFA found in red meat and processed foods. However, we have previously shown that treatment with myristate, a C14 SFA rich in milk-fat based diets, increased activation of IRE1α in a CerS5/6-dependent manner in rat small intestinal epithelial cells (IEC6). Our current work extends these findings in human colon epithelial cells (HCECs). In HCECs chemical inhibition of all six CerS isoforms suppressed myristate-induced ER stress however, knockdown of CerS5/6 exacerbated ER stress. Our novel findings led us to the hypothesis that loss of CerS5/6 reduces SCD1 protein levels both basally and after myristate treatment in an IRE1a-dependent manner. To this end we propose the following specific aims: Specific Aim 1: Define the role of CerS5/6 in milk-fat diet induced IRE1α activation in intestinal biology in vivo. Specific Aim 2: Elucidate the mechanism of CerS5/6 regulation on SCD1 in intestinal biology.