The role of lipid droplet protein Perilipin 2 in lipid metabolism and inflammation
Perturbations in lipid droplet function have been implicated in promoting several diseases including type-2 diabetes, atherosclerosis, and cardiovascular disease, yet few studies have focused on the role of lipid droplets and associated proteins in the regulation of inflammation. The most abundant lipid droplet protein in liver, Perilipin 2 (Plin2), has been shown to promote lipid accumulation, lipid droplet proliferation, and to augment inflammation in macrophages, yet the current approaches used to study Plin2 are generally descriptive and lack details on the mechanism of action. The objective of this study is to establish a mechanistic link between Plin2’s action on the lipid droplet and the onset of lipid-based inflammation by examining recombinant Plin2, overexpression-Plin2 cells, and Plin2 liver-specific knockout mice in the context of hepatic steatosis and inflammation. Using molecular modeling, fluorescence binding, circular dichroic, and FRET techniques, we demonstrated that Plin2 contains a lipid binding site that consists of a 4-helix bundle and unique / domain. We found that Plin2 residues 119−251 are critical for highest affinity lipid binding. Both stearic acid and cholesterol interact favorably with the Plin2 cleft formed by conserved residues in helix α6 and adjacent strands within the 4-helix bundle. Findings that Plin2 contains specific domains responsible for Plin2−lipid interactions and binding are significant because it suggests a mechanism by which Plin2 may retain pro-inflammatory lipids on the lipid droplet surface to promote inflammation. To support this hypothesis we treated cells with LPS to induce inflammation. Using a novel technique that traps eicosanoids at the site of synthesis, we found that Plin2-coated lipid droplets are the site of PGE2 production. These findings are consistent with our results showing that Plin2 actively recruits COX2 to the lipid droplet surface promoting eicosanoid biosynthesis. Taken together, our findings indicate that Plin2 may exert a significant role in COX-mediated inflammation through direct interactions on the lipid droplet surface. To establish Plin2’s inflammatory effect in vivo we challenged Plin2 liver-specific knockout mice and their respective wild type controls with a methionine-choline-deficient (MCD) diet to induce a NASH phenotype of increased hepatic steatosis, inflammation, and fibrosis. Results on liver weights, body weights, fat tissue mass, and histology in wild type and Plin2 null mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation however; these effects were blunted in the Plin2 null mice. Levels of PC and VLDL were unchanged and hepatic steatosis was reduced by hepatic ablation of Plin2 due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine methyl transferase (PEMT). We also found that Plin2 ablation influences the hepatic miRNA-biome. Two of the miRNAs affected by Plin2 ablation (miRNAs-1894 and -711) were predicted to target genes associated with eicosanoid biosynthesis and inflammation. Luciferase reporter assays and Western blotting demonstrated that miRNAs-1894 and -711 directly target COX1, COX2, and human PTGIS and inhibit protein expression of the target genes. Overall, results from several studies indicate that Plin2 may play a key role in the development of inflammation and inflammation-related diseases.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Najt, Charles
- Thesis Advisors
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Atshaves, Barbara P.
- Committee Members
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Kaguni, Laurie S.
Hoffmann-Benning, Susanne
Hoogstraten, Charles G.
Yang, Chengfeng
- Date
- 2016
- Program of Study
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Biochemistry and Molecular Biology - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- xviii, 234 pages
- ISBN
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9781369431445
1369431449