Diets high in fat have been linked to obesity and a high fat diet (HFD) has been implicated in altering intestinal function including inflammation; however the contribution of specific HFDs and their predominant fatty acids (FAs) on key pathophysiologic processes is largely unknown. Studies with trans-fats lard olive oil fish oil plant- based fats (corn oil soybean etc.) have demonstrated that specific sources of FAs exert extremely diverse effects in disease most commonly in cardiac and metabolic disease. Therefore the long-term goal of this proposal is to dissect the complexity of components and effects of specific dietary FAs on gut pathobiology and determine the mechanisms involved. Specifically we aim to define the specific roles of two ceramide-generating enzymes ceramide synthases (CerS) 5 and 6 in the regulation of myristate-induced activation of inositol requiring enzyme 1 (IRE1) and define their critical roles in intestinal homeostasis and pathophysiology. IRE1 is a critical branch in the unfolded protein response (UPR) or ER stress and is an evolutionarily conserved signaling mechanism to overcome stress in the ER resulting in a pause of new protein synthesis and induction of chaperone proteins to restore normal ER function. Functionally ER stress and IRE1v have been linked to alterations in intestinal homeostasis and inflammation. Specifically ER stress genes are upregulated in intestinal epithelial cells (IECs) from a murine experimental colitis model (IL10-deficient)1 and in IECs or biopsies from inflammatory bowel disease (IBD) patients12. IRE1 as well as other effectors of ER stress can be initiated by several triggers including high fat diet (HFD) or treatment with saturated FAs as well as exogenous and endogenous sphingolipids3-5. These important clues in the literature together with our studies on bioactive sphingolipids in intestinal inflammation led us to investigate the role of specific dietary FAs in sphingolipid metabolism and their role and mechanism(s) of action in intestinal ER stress. Our very novel observations demonstrate that the C14 saturated FA myristate (rich in milk based diets) but not palmitate (C16 saturated FA) increases ER stress as well as inflammation in IECs in culture and in mouse intestinal tissues in vivo6. Mechanistically ceramide synthases 5 and 6 (CerS 5 and 6) are required for the activation of IRE1 branch of the UPR inducing splicing of XBP1 and expression of IL66. Importantly these data now lead us to the hypothesis that CerS5/6 and the generation of C14 ceramide regulate IRE1 in the intestine and that this may mediate specific responses in intestinal pathobiology (specifically intestinal inflammation). To test this hypothesis we propose the following specific aims: Specific Aim 1. Determine the role of CerS5/6 in the regulation of ER stress specifically IRE1 in intestinal epithelial cells and intestinal organoids. Specific Aim 2. Define the role of CerS5/6 in ER stress-mediated intestinal pathobiology in vivo.