Fatty acid mobilization from adipose tissue is mediated by CD36 posttranslational modifications and intracellular trafficking
Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin
Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin
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Research Article Cell biology Metabolism

Fatty acid mobilization from adipose tissue is mediated by CD36 posttranslational modifications and intracellular trafficking

Abstract

The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific KO mouse models, we showed that CD36 in adipocytes and endothelial cells mediated both LCFA deposition into and release from adipose tissue. We demonstrated the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by adipose tissue–derived LCFAs. We showed that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediated intercellular LCFA transport. We demonstrated that lipolysis induction in adipocytes triggered CD36 deacylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB) and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S-acylation and its interactions with PHB and ANX2.

Authors

Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin

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Figure 4

Acylation/deacylation of CD36 mediates LCFA transport.

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Acylation/deacylation of CD36 mediates LCFA transport. (A) Western blott...
(A) Western blotting on extracts from 4T1.2 cells nontransduced or transduced with WT CD36 or CD36 mutant lacking S-acylated cysteines. Arrow: glycosylated CD36, also expressed in 3T3-L1 adipocytes. NS, nonspecific band. (B) 4T1.2 cells transduced with WT CD36 or CD36 mutant lacking S-acylated cysteines grown in 2D were preinduced to undergo lipogenesis as in Figure 2G and then treated with BODIPY-FL-C16 for 10 minutes and imaged to visualize uptake by lipid droplets (arrows). Scale bar: 50 μm. (C) Intercellular fatty acid transfer from 3T3-L1 adipocytes (not plotted) preloaded with BODIPY-FL-C16 (green) to adjacent RFP+ 4T1 cells detected by flow cytometry with 530 nm (BODIPY) and 610 nm (RFP) lasers. Histograms at the bottom are provided to compare double-positive (BODIPY-FL-C16+/RFP+) population in 4T1 cells expressing WT versus mutant CD36. (D) 3T3-L1 adipocytes treated with 300 μM palmitic acid for 0 to 120 minutes analyzed by acyl biotin exchange (ABE) assay (Supplemental Figure 4A). Extracted proteins were alkylated with hydroxylamine (HA) where indicated, de–S-acylated, biotinylated, and removed with streptavidin beads. Remaining proteins were immunoblotted with indicated antibodies. Note progressive accumulation of nonacylated glycosylated (arrow) and nonglycosylated (arrowhead) CD36 upon LCFA treatment. PHB immunoblot indicates constant PHB acylation and equal loading. Quantification is on the right. (E) bEND.3 endothelial cells treated with 300 μM palmitic acid for 0 to 120 minutes analyzed by ABE assay. Extracted proteins were alkylated (HA), de–S-acylated, biotinylated, and removed with streptavidin beads. The remaining proteins were immunoblotted with indicated antibodies. Note accumulation of nonacylated glycosylated (arrow) and nonglycosylated (arrowhead) CD36 upon LCFA treatment. ANX2 immunoblot indicates constant ANX2 acylation and equal loading. Quantification is on the right.