Intestinal cytoplasmic lipid droplets, associated proteins, and the regulation of dietary fat absorption
Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, and cardiovascular disease. The small intestine absorbs dietary fat through an efficient multi step process of digestion, uptake, metabolism, and secretion or storage. When dietary fat is taken up by the absorptive cells of the small intestine, enterocytes, it can be secreted into circulation where it contributes to blood lipid levels or temporarily stored in cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to investigate the presence and metabolism of CLDs in the intestine. Dietary fat absorption by the small intestine is a multi-step process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. We also investigated the effect of diet induced obesity on CLD morphology and associated proteins. It is currently unclear what effect diet induced obesity (DIO) has on the balance between dietary fat storage and secretion. Specifically, there is limited knowledge of how DIO affects the level and diversity of proteins that associate with CLDs and regulate CLD dynamics. In the current study, we characterize CLDs from lean and DIO mice through histological and proteomic analyses. We demonstrate that DIO mice have larger intestinal CLDs compared to lean mice in response to dietary fat. Additionally we identified 375 proteins associated with CLDs in lean and DIO mice. We identify a subgroup of lipid related proteins which are either increased or unique to the DIO CLD proteome. These proteins are involved in steroid synthesis pathways, TAG synthesis, and lipolysis. This analysis expands upon our knowledge of the effect of DIO obesity on the process of dietary fat absorption in the small intestine. Lastly, we investigated the role of one of the newly identified CLD associated proteins on dietary fat absorption. Little is known of the role of Acsl5 in the metabolism of dietary fat in enterocytes. To understand the role of Acsl5 in dietary fat absorption, we challenged WT and Acsl5-deficient mice with acute and chronic dietary fat challenges. Despite no evidence of quantitative fat malabsorption in the absence of Acls5, we found significantly reduced TAG storage and secretion by enterocytes compared to WT mice. Interestingly, Acsl5-deficient consumed more food, but did not gain more weight compared to WT mice during two weeks of HFD feeding. Furthermore, we found that Acsl5 localizes to the nucleus after an acute dietary fat challenge. Together this data suggests that although Acsl5 is not essential for quantitative dietary fat absorption, it plays a role in intestinal metabolism of dietary fat. The results presented in this dissertation provide new knowledge of regulators of CLD metabolism. The long term objectives of these studies are to identify mechanistic details of the process of intestinal dietary fat absorption. Understanding the process of dietary fat absorption will provide new treatments for the prevention of diseases associated with dyslipidemia.
Buhman, Purdue University.
Molecular biology|Cellular biology|Biochemistry|Nutrition|Physiology
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