Sphingolipids, Dietary Fatty Acids, and Intestinal Pathophysiology

高脂肪饮食与肥胖有关，高脂肪饮食（HFD）与肠道功能的改变有关，包括炎症；然而，特定的HFDs及其主要脂肪酸（FAs）在关键病理生理过程中的作用在很大程度上是未知的。对反式脂肪、猪油、橄榄油、鱼油、植物性脂肪（玉米油、大豆等）的研究表明，特定来源的FAs在疾病中发挥着极其多样化的作用，最常见的是心脏病和代谢性疾病。因此，本研究的长期目标是剖析膳食中特定脂肪酸组分的复杂性及其对肠道病理生物学的影响，并确定其中的机制。具体来说，我们旨在确定两种神经酰胺生成酶，神经酰胺合成酶（CerS） 5和6在肉豆酸盐诱导的肌醇需要酶1α (IRE1α)激活调控中的具体作用，并确定它们在肠道内稳态和病理生理中的关键作用。IRE1α是未折叠蛋白反应（UPR）或内质网应激中的一个关键分支，是一种进化保守的信号机制，可以克服内质网中的应激，导致新蛋白合成的暂停和伴侣蛋白的诱导，恢复正常的内质网功能。在功能上，内质网应激和IRE1αv与肠道内稳态和炎症的改变有关。具体来说，内质网应激基因在小鼠实验性结肠炎模型（il10缺乏）1的肠上皮细胞（IECs）以及炎症性肠病（IBD）患者的IECs或活检中上调1,2。IRE1α以及内质网应激的其他效应物可由几种触发因素启动，包括高脂肪饮食（HFD）或饱和脂肪酸治疗，以及外源性和内源性鞘脂[3-5]。这些重要的文献线索，加上我们对肠道炎症中生物活性鞘脂的研究，促使我们研究特定膳食FAs在鞘脂代谢中的作用及其在肠道内质网应激中的作用和机制。我们非常新颖的观察结果表明，C14饱和FA肉豆蔻酸酯（富含乳基饮食），而不是棕榈酸酯（C16饱和FA），会增加内质网应激，以及体外培养和小鼠肠道组织中的IECs炎症。从机制上讲，神经酰胺合成酶5和6 （CerS 5和6）是激活UPR的IRE1α分支、诱导XBP1剪接和IL66表达所必需的。重要的是，这些数据现在引导我们假设cer5 /6和C14神经酰胺的产生调节肠道中的IRE1α，这可能介导肠道病理生物学中的特异性反应（特别是肠道炎症）。为了验证这一假设，我们提出以下具体目标：确定cer5 /6在肠上皮细胞和肠类器官中调控内质网应激，特别是IRE1α的作用。具体目标2。明确cer5 /6在内质网应激介导的肠道病理生物学中的作用。

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 1α (IRE1α) and define their critical roles in intestinal homeostasis and pathophysiology. IRE1α 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 IRE1αv 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) patients1,2. IRE1α, 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 IRE1α 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 IRE1α 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 IRE1α, in intestinal epithelial cells and intestinal organoids. Specific Aim 2. Define the role of CerS5/6 in ER stress-mediated intestinal pathobiology in vivo.