Regulation of Intestinal Lipid Storage and Mobilization

Dietary fat is an energy dense nutrient that also provides essential fatty acids and aids in the absorption of fat soluble vitamins. The process of dietary fat absorption regulates the amount and rate at which dietary fat enters circulation, and can therefore contribute to diseases such as obesity, diabetes and cardiovascular disease when it becomes dysregulated. Triacylglycerol (TAG), the major form of dietary fat, is efficiently absorbed (\u3e95%) even when high amounts of fat are consumed. The digestive products of dietary fat are taken up by enterocytes, the absorptive cells of the small intestine, and rapidly re-synthesized into TAG. This re-synthesized TAG can either be packed onto chylomicrons and secreted into circulation for delivery to peripheral tissues, or temporarily stored within cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to investigate the regulation of enterocyte lipid stores and their contribution to dietary fat absorption. First, we determined differences in enterocyte CLD-associated proteins in genetic models with alterations in intestinal TAG synthesis. Acyl-CoA: diacylglycerol acyltransferase 1 (Dgat1) and Dgat2 catalyze the final, committed step of TAG synthesis. Mouse models with intestine-specific overexpression of Dgat1 (Dgat1Int) or Dgat2 (Dgat2Int), or a whole body Dgat1 deficiency (Dgat1-/-), have previously been shown to exhibit alterations in intestinal TAG storage and secretion. We isolated CLDs from enterocytes of these models after an acute dietary fat challenge and identified proteins present in the enterocyte CLD-enriched fraction using LC-MS/MS. We identified a total of 158 proteins, 53 of which were common to all four models (Dgat1Int, WT, Dgat2Int, and Dgat1-/- mice). In addition, 13% of the proteins identified are associated with lipid metabolism, and several of these lipid metabolism related proteins were differentially present among the models. This analysis has identified novel potential regulators of intestinal lipid storage and mobilization. We also assessed differences in enterocyte CLD morphology and protein composition in models resistant compared to susceptible to high fat diet-induced obesity (DIO). Whole body Dgat1-/- mice exhibit alterations in intestinal lipid metabolism that are thought to contribute to their resistance to DIO and insulin resistance. In the current study we found that in response to chronic high fat feeding Dgat1-/- enterocytes contain a greater number of larger sized CLDs compared to enterocytes from WT, diet-induced obese mice. In addition, we identified a total of 125 proteins in the enterocyte CLD-enriched fractions from these models, 52 of which were only identified in the Dgat1-/- model. Furthermore, a greater number of proteins associated with the mitochondria and fatty acid oxidation were identified in the enterocyte CLD fraction from Dgat1-/- mice, and this was accompanied by more alterations in enterocyte mitochondrial morphology in this model. Overall, this study increases knowledge about alterations in enterocyte CLD metabolism in Dgat1-/- mice that may be contributing to their beneficial DIO-resistant phenotype. Finally, we investigated the effects of oral nutrient exposure and ingestion on intestinal lipid stores in both humans and mice. Glucose ingestion was shown to mobilize lipid stored in the intestine from a previous high fat meal in humans. We observed a decrease in enterocyte CLD number and a greater number of smaller sized CLDs in response to glucose compared to water ingestion. In addition, we identified a total of 2919 proteins present within human duodenal biopsy tissues in response to this dietary challenge, 134 of which were differentially present in response to glucose compared to water ingestion. Consistent with these human studies, we found that glucose compared to water ingestion (but not oral exposure) also decreases intestinal lipid stores in mice. Previous studies in humans have shown that sham feeding of fat and oral glucose ingestion induce a peak in plasma CM-TAG levels, however no detectable differences in plasma TAG levels were observed in mice in response to an oral fatty acid or glucose exposure, or to glucose ingestion in the present studies. This work confirms and expands upon previous work in humans by identifying the specific enterocyte lipid pools mobilized by glucose ingestion, as well as identifying glucose regulated proteins in the small intestine that have the potential to contribute to the observed glucose stimulated lipid mobilization. Taken together, this work identifies novel potential regulators of intestinal lipid storage and mobilization. Further investigation into their contributions to dietary fat absorption will help to provide novel dietary and therapeutic strategies for the management of obesity, hypertriglyceridemia, and related metabolic diseases