The majority of bile acids are absorbed in the intestine via Apical Sodium Dependent Bile Acid Transporter, ASBT. Disturbances in hASBT function have been shown to alter the level of plasma cholesterol and implicated in the phathophysiology of various cholesterol-related disorders. To date, however, the molecular mechanisms of ASBT regulation in the human intestine are not well understood. In order to define the molecular link between bile acid absorption and cholesterol-related disorders, it is critical to elucidate the mechanism(s) of regulation of hASBT under normal conditions as well as in conditions of hormonal imbalance as seen in diabetes mellitus. In this regard, extensive preliminary studies from our laboratory have demonstrated the modulation of hASBT function and expression in response to both short-term and long-term exposure to insulin and glucagon, utilizing human intestinal Caco2 cells as an in vitro cellular model. The proposed studies are designed to extensively investigate the signal transduction pathways, membrane trafficking events as well as alterations in the expression of hASBT in response to insulin and glucagon in Caco-2 cells.
In specific aim 1, we will focus on delineating the signaling pathways involved in ASBT regulation by short-term exposure to insulin and glucagon by utilizing specific inhibitors of protein kinases, siRNA and immunoprecipitation techniques. Studies proposed in specific aim 2 will examine the distribution of hASBT indifferent subdomains (lipid rafts) of the plasma membrane and the role of membrane trafficking events in their regulation by insulin and glucagon. Lipid rafts will be isolated by floatation of solubilized plasma membrane on Optiprep density gradient. For membrane trafficking studies, green fluorescent protein fused to ASBT will be transfected in Caco2 cells followed by live cell imaging utilizing confocal microscopy.
Specific aim 3 will focus on elucidating the molecular mechanisms underlying the altered expression of ASBT in response to insulin and glucagon. Promoter analysis and gel shift assays will yield important information on the transcriptional regulation of ASBT by insulin and glucagon. Given the central role for these endocrine hormones in the integration of metabolic processes, the proposed studies would provide critical novel data about the regulation of hASBT and enhance our understanding of the molecular basis for the link between bile acid absorption and the lipid and cholesterol homeostasis. A better understanding of the role of bile acid absorption in cholesterol related disorders may help advance new therapeutic modalities to counter diseases such as hypercholesterolemia and atherosclerosis. ? ?
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