This project will determine the localization and function of intestinal caveolae and assess their influence on systemic lipid metabolism. Caveolae are pit-shaped structures formed by oligomers of the protein caveolin-1 (CAV1) in lipid-rich plasma membrane domains. Caveolae regulate cell signaling, internalization of receptors and ligands, inflammation and transcytosis. Cell culture studies suggest that caveolae plasma membrane expression may be asymmetric in polarized cell types such as enterocytes, but data have been conflicting. Further, CAV1 subcellular localization and its functional significance have not been investigated in vivo. As a first step in exploring CAV1, drCav1-GFP zebrafish were created that show in vivo drCav1 localization to the basolateral and lateral plasma membranes of enterocytes, but not to the brush border. Interestingly, in mice, CAV1 interacts with the ABC binding cassette transporter member 1 (ABCA1), which mediates cellular cholesterol efflux to high-density lipoproteins (HDL);mice lacking CAV1 have altered plasma lipids despite normal dietary cholesterol absorption. Thus, intestinal caveolae may play a role in lipid metabolism and cardiovascular disease.
Specific Aim 1 of this proposal is to characterize caveolar localization in enterocytes and hepatocytes. To test the hypothesis that drCav1 is targeted to specific membranes of polarized cells in addition to enterocytes, hepatocyte drCav1-GFP expression will be characterized. To determine if drcav1 mRNA localizes to specific sections of the endoplasmic reticulum prior to asymmetric protein targeting, enterocyte mRNA expression will be determined by in situ hybridization. To ensure that drCav1-GFP localization is representative of wild type drCav1, the subcellular location of intestinal drCav1 will be identifie by immunofluorescence and of caveolae by electron microscopy.
In Specific Aim 2 caveolar endocytosis will be visualized and disrupted. To determine where caveolae-mediated internalization occurs in enterocytes, BODIPY-d-lactosylceramide (LacCer), which has been described in cell culture to be carried by caveolae, will be injected into the intestinal lumen or basolateral side of enterocytes and its uptake visualized in live zebrafish. Intestinal caveolae wil be disrupted in vivo i) via injection of BODIPY-l-LacCer and ii) by creating zebrafish that overexpresses mutated drCav1 that does not oligomerize.
Specific Aim 3 will determine the contribution of intestinal caveolae to lipid metabolism. To test the involvement of intestinal caveolae in lipid transport, dietary lipids transported to the liver in live zebrafish with functioal and disrupted caveolae will be quantified. To investigate the contribution of intestinal caveolae to cellular cholesterol efflux via ABCA1, dietary cholesterol incorporation into HDL in zebrafish with functional and disrupted caveolae will be quantified. This investigation will characterize intestinal caveolae, develop novel methods to study caveolar function in vivo, and determine the contribution of intestinal caveolae to systemic lipid metabolism, providing valuable insights into mechanisms underlying gastrointestinal and metabolic disease.
Caveolae are pits in cellular plasma membrane lipid raft domains formed by the structural protein Caveolin-1;caveolae perform vital cellular internalization and signaling functions. The first goal of this research is to determine the localization of Caveolin-1 and caveolae in intestinal cells and develop novel tools to disrupt thei function in live, optically clear, larval zebrafish. The second goal of this project is to elucidat the role of intestinal caveolae in lipid metabolism and their contribution to cellular cholesterol efflx to high-density lipoproteins, which will improve ability to prevent and treat obesity and cardiovascular disease in humans.
|Otis, Jessica P; Zeituni, Erin M; Thierer, James H et al. (2015) Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake. Dis Model Mech 8:295-309|