Loss of ceramide synthases 5 and 6 exacerbate myristate-induced colonic IRE1α activation.

High-fat diets (HFDs) are a major modifiable risk factor for intestinal health. Current research focuses primarily on palmitate (C16:0); however, myristate (C14:0, rich in dairy products) has been minimally investigated. HFDs increase ceramide generation which drives endoplasmic reticulum (ER) stress; with both sphingolipids and ER stress being key contributors to intestinal biology. Whether different fatty acids uniquely impact sphingolipid metabolism and ER stress in intestinal biology has not been well defined. Human colon epithelial cells were utilized to determine the role of ceramide synthases (CerS) 5 and 6 on myristate-induced ER stress using pharmacologic inhibitors and siRNA. Intestinal epithelial cell specific CerS5 and/or CerS6 knockout mice of both sexes were fed a control, high milk-fat, or high lard-fat diet for 16 weeks. Cells and colon tissues were analyzed for lipids, mRNA, and protein. Myristate treatment increased d18:1/C14:0-ceramide and induced IRE1α-dependent ER stress. Inhibition of CerS suppressed these effects, yet knockdown of CerS5/6, the primary enzymes generating d18:1/C14:0-ceramide, unexpectedly exacerbated IRE1α activation both in vitro and in vivo, potentially due to depletion of dihydro(dh)sphingosine. Altogether, our data suggest that loss of CerS5/6 exacerbate myristate-induced intestinal ER stress in colon epithelial cells and in vivo and accumulation of dhsphingosine may provide protection.