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 4

PHB regulates FA uptake in adipocytes through interaction with ANX2.

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PHB regulates FA uptake in adipocytes through interaction with ANX2. (A)...
(A) 3T3-L1 adipocytes after differentiation were transfected with control untargeted siRNA or siRNA targeting PHB. As assessed by PHB and β-actin (loading control) immunoblotting 72 hours after transfection, a substantial reduction of PHB protein expression is observed for siRNA clones 12 and 13. (B) Upon treatment with 0.3 μM boron-dipyrromethene–conjugated (BODIPY-conjugated) C16 fatty acid (FA) (green), anti-PHB immunofluorescence (red) reveals FA uptake by the majority of control PHB-expressing adipocytes (arrows), while for clone 12, FA uptake is not observed for adipocytes (a) that lost PHB expression. Nuclei are blue. The graph quantifies percentage of adipocytes with detectable FA fluorescence in n = 5 view fields. Error bars ± SEM; *P < 0.05 (Student’s t test, WT vs. PHB knockdown). Scale bar: 50 μm. (C) 3T3-L1 adipocytes expressing GFP-tagged WT ANX2 or ANX2 mutant engineered to lack PHB binding were treated with 1 μM BODIPY-FL-C12 for 30 min. Note the abundant FA uptake (red lipid droplets) by adipocytes (a) expressing WT ANX2 on the cell membrane (arrows) and lack of FA uptake in adipocytes expressing ANX2 mutant, which fails to localize to cell membrane. (D) Anxa2–/– cells were differentiated into adipocytes and then transduced with GFP-tagged WT ANX2 or GFP-tagged ANX2 mutant engineered to lack PHB binding or with empty GFP vector. Note GFP signal in transduced adipocytes, which is absent in nontransduced (N.T.) cells. Arrows indicate cell membrane WT ANX2 localization. Nuclei are blue. Scale bar: 50 μm. (E) Fluorescent and bright-field microscopy on cells from D treated with 0.3 μM BODIPY-FL-C12 for 30 min. Note abundant FA uptake (red lipid droplets) by adipocytes (a) expressing WT ANX2 but not ANX2 mutant. Nuclei are blue. Scale bar: 50 μm. (F) Real-time measurement of FA uptake. Cells from D were plated in triplicate, prestarved in 1% FBS/DMEM-low glucose for 2 hours, and then subjected to the QBT assay. Intracellular long-chain FA accumulation is monitored by measuring fluorescence (excitation 485 nm/emission 515 nm). Plotted are mean values; error bars ± SEM; *P < 0.01 (Student’s t test, WT ANX2 vs. ANX2 mutant).