Research Project:
DETERMINATION OF ENERGY AND INSULIN REGULATION IN AGING
Location: Human Nutrition Research Center on Aging
Title: Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes
Authors
| Granneman, James - WAYNE STATE UNIV. | | Moore, Hsiao-Ping - WAYNE STATE UNIV. | | Granneman, Rachel - WAYNE STATE UNIV. | |
Greenberg, Andrew
| | Obin, Martin - TUFTS/HNRCA | | Zhu, Zhengxian - WAYNE STATE UNIV. |
Submitted to: Journal of Biological Chemistry
Publication Type:
Peer Reviewed Journal
Publication Acceptance Date: December 12, 2006
Publication Date: February 23, 2007
Publisher's URL: http://jbc.org/cgi/content/abstract/282/8/5726
Citation: Granneman, J.G., Moore, H.H., Granneman, R.L., Greenberg, A.S., Obin, M.S., Zhu, Z. 2007. Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes. Journal of Biological Chemistry.282(8): 5726-5735.
Interpretive Summary: This work examined the interactions of the key proteins involved in fat breakdown during brief protein kinase A activation. Analysis of fat cells from a type of genetically engineered mouse, called 3T3-L1, indicated that protein kinase A activation increases the change of hormone-sensitive lipase to the protein perilipin in fat cells and increases the colocalization of fat tissue triglyceride lipase with its co-activator, called Abhd5. Imaging of live 3T3-L1 mouse fat cells showed that hormone sensitive lipase rapidly and specifically translocates to fat droplets in the cell containing perilipin, and that this translocation is partially dependent on perilipin phosphorylation. Hormone sensitive lipase closely, if not directly, interacts with perilipin in contrast to fat tissue triglyceride lipase. The co-activator of fat tissue triglyceride, Abhd5, strongly interacted with perilipin. Protein kinase A activation rapidly decreased between Abhd5 and perilipin, and this decrease depended upon perilipin phosphorylation. Together, these results indicate that perilipin mediates hormone-stimulated cell death via direct and indirect mechanisms. Perilipin indirectly controls fat tissue triglyceride lipase activity by regulating accessibility to Abhd5. In contrast, perilipin directly regulates the access of hormone sensitive lipase to substrate via close, if not direct, interactions.
Technical Abstract: This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormone-sensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET betweeen Abhd5 and Pllin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.
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