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. 2016 Jul 7;1(10):e86351.
doi: 10.1172/jci.insight.86351.

Prohibitin/annexin 2 Interaction Regulates Fatty Acid Transport in Adipose Tissue

Free PMC article

Prohibitin/annexin 2 Interaction Regulates Fatty Acid Transport in Adipose Tissue

Ahmad Salameh et al. JCI Insight. .
Free PMC article


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.


Figure 1
Figure 1. PHB and ANX2 coexpression on WAT endothelium and adipocytes.
(A) Immunofluorescence (IF) on serial mouse white adipose tissue (WAT) paraffin sections with PHB and ANX2 antibodies or a non–immune IgG (control) and green fluorophore–conjugated secondary antibodies. Endothelium (arrows) is counterstained with isolectin B4 (IB4, red). Green/red channel merging indicates expression of PHB and ANX2 in WAT endothelium (yellow). PHB/ANX2 coexpression is also observed in IB4-negative adipocytes (a). (B) IF analysis of mouse paraffin sections with anti-PHB antibodies/IB4 shows that liver, lung, and kidney PHB expression is not detectable in the endothelium (arrows). (C) 3T3-L1 cells after adipogenesis induction were subjected (without permeabilization) to IF with PHB and ANX2 antibodies. Arrows indicate localization of both PHB and ANX2 in differentiated adipocytes (a), for which lipid droplets are shown in the bright field image. Arrows represent nucleated nondifferentiated cells, in which PHB and ANX2 are not detectable. (D) ANX2 requirement for PHB localization to adipocyte surface. WAT-derived cells from of Anxa2+/+ (WT) and Anxa2–/– (ANX2-null) mice adherent to plastic in tissue culture were subjected to PHB (green)/ANX2 (red) IF (without permeabilization). Lipid droplets in differentiated adipocytes (a) are shown in the bright field images. Note the lack of both ANX2 and PHB signal in ANX2-null adipocytes. Arrows indicate cells not differentiated into adipocytes. Nuclei are blue (BD). (E) Homing of the CKGGRAKDC peptide to WAT is inefficient in the absence of ANX2, as revealed by phage recovery (TU/g of WAT) from visceral WAT extracted from WT and ANX2-null littermates i.v.-injected with 1 × 1010 of CKGGRAKDC-phage transforming units (6 hour circulation). Shown are mean ± SD from n = 4 mice (2 males and 2 females). *P < 0.05 (Student’s t test, WT vs. ANX2-null). Antiphage HRP immunohistochemistry (brown) in sections of WAT demonstrates phage in WAT vasculature in WT mice (arrows). Hematoxylin counterstaining is blue. Scale bars: 50 μm.
Figure 2
Figure 2. ANX2 is required for adipocyte hypertrophy but not for adipogenesis.
(A) Total fat and lean body mass of Anxa2+/+ (WT) males (n = 13) and Anxa2–/– (ANX2-null) males (n = 8) after feeding HFD for 7 weeks. (B) Representative mice with skin removed revealing lower amount of WAT in ANX2-null mice. (C) Immunofluorescence analysis of whole mounts of WAT from WT and ANX2-null mice with caveolin-1 (green) antibodies revealing reduced adipocyte (a) size in ANX2-null mice. Isolectin B4 (red) counterstaining the endothelium (arrows) reveals vasculature. (D) Angiogenesis in an ex vivo sprouting assay. When embedded into collagen-I/methylcellulose matrix, the stromal/vascular fraction from i.p. or s.c. WAT of ANX2-null mice display increased endothelial sprout formation, compared with WT mice. (E) Data in D quantified based on the analysis of n = 10 view fields. (F) ANX2 is not required for adipocyte differentiation; upon adipogenesis induction (5 days), the timing of lipid droplet formation is comparable for WT and ANX2-null SVF. In A and E, error bars ± SEM; * P<0.05 (Student’s t test, WT vs. ANX2-null). Scale bars: 50 μm.
Figure 3
Figure 3. Deficient WAT lipid uptake in ANX2-null mice.
(A) Triglyceride blood concentration analysis upon i.v. Intralipid infusion into prestarved mice shows a delay in clearance for ANX2-null compared with WT mice (n = 4). (B) Abdominal fluorescence of lauric acid boron-dipyrromethene conjugate (BODIPY-FL-C12) measured 15 min. after injection. As quantified for 4 abdominal locations (C), a significantly lower signal is observed for Anxa2–/– mice compared with WT mice. (D) Analysis of individual organs from WT and Anxa2–/– mice 15 min. after injection of palmitic acid (C16) labeled with IRDye-680CW (left) or an equimolar amount of uncoupled IRDye-680 (right). (E) Quantification of D showing a significant reduction of FA localization to s.c. and i.p. WAT of Anxa2–/– mice compared with WT mice (n = 4). FA (but not free fluorophore) accumulation in the liver is higher in Anxa2–/– than in WT mice, while kidney and skeletal muscle accumulation is comparable. (F) SVF from i.p. WAT of WT and Anxa2–/– mice with vascular structures (arrows) were incubated with 0.3 μM BODIPY-FL-C12 (red) and then washed. Note FA uptake (arrows) in WT but not Anxa2–/– endothelium. (G) One hour after red BODIPY-FL-C12 i.v injection into WT and Anxa2–/– mice, adipocytes were isolated after WAT disaggregation using collagenase. Note FA accumulation (arrows) inside WT but not Anxa2–/– nucleated adipocytes. Insets: bright field (BF) images. In A, C, and E, error bars ± SEM; *P<0.05 (Student’s t test, WT vs. Anxa2–/–). Nuclei are blue. Scale bars: 50 μm.
Figure 4
Figure 4. PHB regulates FA uptake in adipocytes through interaction with ANX2.
(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).
Figure 5
Figure 5. PHB/ANX2 complex mediates lipid transport in WAT.
(A) 3T3-L1 adipocytes were incubated for 30 min. in medium containing 0.1 mM blocking or a scrambled peptide and then treated with 0.3 μM BODIPY-conjugated C16 fatty acid (FA) (green). Note reduced FA uptake upon PHB/ANX2 binding blockade. Graph: data quantified based on the analysis of n = 5 view fields. Error bars ± SEM. *P<0.05 (Student’s t test, blocking vs. scrambled). (B) A schematic of the intercellular transfer assay measuring endothelium-adipocyte FA transfer: (1) bEnd.3 endothelial cells (EC) are plated as a monolayer; (2) EC are incubated with BODIPY-FL-C16 (green fluorescence) fatty acid (FA) and washed, after which a part of the plate is scraped off; and (3) 3T3-L1 adipocytes coated with magnetic nanoparticles are added and forced to the bottom using magnetic field in the presence or absence of the blocking peptide. After washing, CD31 immunofluorescence (red) is performed, and adipocytes that received FA from interacting endothelial cells are observed (green). (C) The intercellular transfer assay performed in the presence of 0.1 mM RDAGRSDALVIYEIGKE scrambled peptide control (no blocking) or AKGRRAEDGSVIDYELI (blocking) peptide. Note that peptide blocking the PHB/ANX2 binding, but not the scramble peptide, prevents FA (green) transfer from the endothelium (red) to adipocytes (arrows). Arrowheads indicate nondifferentiated 3T3-L1 cells. Shown below are bright-field images demonstrating adipocytes attached to plastic and to EC monolayer below the scraped area. Scale bar: 50 μm.
Figure 6
Figure 6. FA treatment induces plasmalemmal interaction of ANX2, PHB, and CD36.
(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.

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