Cytoplasmic Lipid Droplets in Metabolic Disease

<p>Metabolic diseases associated with conditions of the metabolic syndrome (MetS) are on the rise in the United States. MetS develops as a consequence of dysfunctional nutrient metabolism, leading to hypertriglyceridemia, insulin resistance, and obesity. These conditions contribute to the development of more serious diseases such as Type 2 diabetes, cardiovascular disease, non-alcoholic/metabolic associated fatty liver disease (NAFLD/MAFLD), and cancer. Therefore, it is important to understand the cellular and molecular factors contributing to metabolic dysfunction and disease progression. A common feature of metabolic disease and its contributing conditions is abnormal lipid metabolism, specifically the accumulation of neutral lipid in cellular cytoplasmic lipid droplets (CLDs). The objective of this dissertation is to examine the role of cytoplasmic lipid droplets in metabolic disease.</p> <p> First, we investigated CLDs in metastatic breast cancer. CLD accumulation in breast cancer cells is positively associated with cancer aggressiveness; however, the functional consequence of this phenomenon is unclear. The function of CLDs is often reflected by their associated proteins, which regulate both cellular and CLD metabolism. However, the proteome of CLDs in metastatic breast cancer cells has not been described. In this study, we characterized the proteome of CLDs in the human metastatic breast cancer cell line, MCF10CA1a, for the first time. We identified a novel CLD proteome with both similarities and differences to CLDs of other cell types. Overall, this study is the first to analyze the proteins associated with CLDs in metastatic breast cancer cells and in turn produced a hypothesis-generating list of potential proteins involved breast cancer metastasis that can be applied to future studies in order to define the role of CLDs and their proteins in breast cancer metabolism.</p> <p> Next, we investigated the characteristics and proteome of CLDs in enterocytes of the proximal, middle, and distal regions of the small intestine in the response to dietary fat. Enterocytes of all three regions of the small intestine are capable of packaging and secreting dietary fat on chylomicrons to contribute to blood triacylglycerol (TAG) levels, although to different extents. All regions can also store dietary fat in CLDs, however whether CLDs serve different roles or are differentially metabolized in each region is not clear. Further, obesity has been shown to influence the rate at which dietary fat is absorbed and stored in the middle region of the small intestine, however, whether obesity influences dietary fat storage in the other regions is not known. Therefore, we examined the effect of intestine region and obesity on the characteristics and proteome of CLDs in the proximal, middle, and distal regions of the small intestine in response to dietary fat to determine potential differences in lipid processing, storage, or CLD metabolism. We found dietary fat storage and CLD proteins varied in each region of the small intestine in lean and diet-induced obese mice, which may indicate differences in dietary fat processing or CLD metabolism in each intestine region. Overall, this study helped to characterize the dynamics of dietary fat absorption along the length of the small intestine and provides insight as to how the process of dietary fat absorption or enterocyte lipid metabolism may be altered in obesity.</p> <p> Third, we investigated the molecular mechanisms of intestinal lipid mobilization by the enteroendocrine hormone, glucagon-like peptide-2 (GLP-2). GLP-2 has been shown to briefly stimulate the secretion of TAG in chylomicrons from the small intestine hours after the consumption of dietary fat, contributing to blood TAG concentrations. Multiple intestinal TAG pools are potentially mobilized by GLP-2, including those in the lamina propria or lymphatics. However, the exact pool mobilized is not clear. Therefore, we assessed the presence and size of CLDs in human enterocytes as well as the proteome of intestine biopsies to identify the TAG storage pool mobilized by GLP-2 and/or the molecular mediators of GLP-2’s effects on TAG mobilization. We identified no differences in CLD characteristics in GLP-2 biopsies compared to placebo, supporting a role for GLP-2 in mobilizing TAG pools outside of enterocytes. Further, we identified several proteins potentially involved in mediating the intestinal response to GLP-2. Overall, this study helped characterize the effect of a novel physiological stimulus on intestinal TAG secretion which has implications in the development of treatment strategies to reduce hyperlipidemia and prevent cardiovascular disease. </p> <p>Last, we identified and compared the tissue proteome and phosphoproteome of liver in obesity-associated hepatosteatosis to that of lean liver in the postprandial state. The liver plays a central role in the maintenance of systemic nutrient homeostasis during both the fasted and fed states by tightly regulating its cellular metabolic pathways. During obesity, development of hepatosteatosis alters hepatic nutrient utilization, contributing to metabolic dysfunction. The molecular factors that contribute to this metabolic dysfunction, particularly in the fed state, are unclear. Therefore, we performed proteome and phosphoproteome analysis of liver from DIO compared to lean mice in the postprandial state after a lipid meal in order to determine the effect of obesity-associated hepatosteatosis on the liver proteome during the postprandial state. We identified significant differences in the relative levels of proteins involved in major nutrient metabolic pathways in livers of obese compared to lean mice, indicating changes in hepatic nutrient utilization in obese mice in the postprandial state. Overall, this study helped characterize the liver proteome and phosphoproteome of DIO and lean mice in a controlled postprandial state and uncovered potentially disrupted metabolic pathways contributing to the disorders present in obesity-associated hepatosteatosis.</p> <p>The four research projects included in this dissertation apply proteomic methods to understand the role of CLDs in metabolic disease. Proteomics is used to characterize the molecular landscape of an experimental model, and we capitalize on the untargeted nature of proteomics in these projects to generate protein datasets that contain numerous candidate proteins contributing to metabolic disease. This research expands our knowledge about the CLD proteome in metastatic breast cancer and in enterocytes during the process of dietary fat absorption in lean and obese states, as well as the liver proteome during obesity-associated hepatosteatosis. As proteins are the core constituents of metabolic pathways in the form of enzymes, transcription factors, and regulatory proteins, identifying the proteome of CLDs and tissues offers a large-scale untargeted molecular view of cellular components that may contribute to metabolic abnormalities and disease. The identification of these factors will allow for the development of targeted therapies modulating cellular lipid storage and its associated consequences present in metabolic disease.</p><br>