Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue
Ahmad Salameh, … , Wadih Arap, Mikhail G. Kolonin
Ahmad Salameh, … , Wadih Arap, Mikhail G. Kolonin
Published July 7, 2016
Citation Information: JCI Insight. 2016;1(10):e86351. https://doi.org/10.1172/jci.insight.86351.
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Research Article Metabolism Vascular biology

Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue

Abstract

We have previously identified prohibitin (PHB) and annexin A2 (ANX2) as proteins interacting on the surface of vascular endothelial cells in white adipose tissue (WAT) of humans and mice. Here, we demonstrate that ANX2 and PHB also interact in adipocytes. Mice lacking ANX2 have normal WAT vascularization, adipogenesis, and glucose metabolism but display WAT hypotrophy due to reduced fatty acid uptake by WAT endothelium and adipocytes. By using cell culture systems in which ANX2/PHB binding is disrupted either genetically or through treatment with a blocking peptide, we show that fatty acid transport efficiency relies on this protein complex. We also provide evidence that the interaction between ANX2 and PHB mediates fatty acid transport from the endothelium into adipocytes. Moreover, we demonstrate that ANX2 and PHB form a complex with the fatty acid transporter CD36. Finally, we show that the colocalization of PHB and CD36 on adipocyte surface is induced by extracellular fatty acids. Together, our results suggest that an unrecognized biochemical interaction between ANX2 and PHB regulates CD36-mediated fatty acid transport in WAT, thus revealing a new potential pathway for intervention in metabolic diseases.

Authors

Ahmad Salameh, Alexes C. Daquinag, Daniela I. Staquicini, Zhiqiang An, Katherine A. Hajjar, Renata Pasqualini, Wadih Arap, Mikhail G. Kolonin

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Figure 6

FA treatment induces plasmalemmal interaction of ANX2, PHB, and CD36.

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FA treatment induces plasmalemmal interaction of ANX2, PHB, and CD36. (A...
(A) SVF stimulated to undergo adipogenesis were subjected (without permeabilization) to immunofluorescence with CD36 (red) and PHB (green) or ANX2 (green) antibodies. Both ANX2 and PHB are colocalized (yellow) with CD36 in adipocytes (a) and in vascular structures (arrows). Scale bar: 50 μm. (B) Membrane proteins immunoprecipitated (IP) with CD36 antibodies from WT or ANX2-null mouse–derived adipocytes were subjected to Western blotting (WB) with antibodies against CD36, ANX2, or PHB. Note that CD36 antibodies coimmunoprecipitate PHB and ANX2 from WT but not from ANX2-null extracts. (C) Membrane proteins immunoprecipitated with non–immune IgG or ANX2 antibodies from WT or ANX2-null mouse–derived adipocytes were subjected to immunoblotting with antibodies against CD36, ANX2, or PHB. Note that ANX2 antibodies coimmunoprecipitate PHB and CD36 from WT but not from ANX2-null extracts. Expression of CD36 in ANX2-null cells is confirmed by whole extract (input) immunoblotting. (D) Proteins precipitated with PHB or ANX2 antibodies (or control IgG) from surface-biotinylated 3T3-L1 adipocytes either untreated (0) or pretreated with 1% Intralipid fatty acids (FA) for the indicated time were subjected to anti-PHB or anti-ANX2 WB (Total) or streptavidin-conjugated IRDye-800CW (Surface). Note the increase in biotinylated PHB recovery upon FA treatment. (E) Cell surface proteins precipitated with streptavidin beads from surface-biotinylated 3T3-L1 adipocytes either untreated (-) or treated with FA (1% Intralipid) for 1 hour (+) were subjected to immunoblotting with antibodies against CD36 or PHB. Note that FA treatment increases the amount of CD36 and PHB (arrows) but not of other proteins (nonspecific bands) at the surface. (F) 3T3-L1 adipocytes either untreated or treated with 1 μM lauric acid for 10 minutes were fixed and subjected to immunofluorescence with ANX2 (red), PHB (green), and CD36 (blue) antibodies. Note plasmalemmal colocalization of the 3 proteins (white) in adipocytes (a) upon FA treatment (arrows). Scale bar: 50 μm. (G) A working model: ANX2/PHB/CD36 complex assembly on the cell surface is induced by the postprandial extracellular FA influx and contributes to the uptake of FA from the circulation by the endothelium and FA transfer into adipocytes.