EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake

Abstract

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Keywords: EHD2 ATPase; caveolae; fatty acid uptake; lipid metabolism; obesity.

Fig. 1.

Loss of EHD2 results in increased lipid accumulation in white and brown adipose tissue. (A) Generation of the EHD2 KO mouse model. A targeting vector containing a pGK-Neomycin (neo) cassette and loxP sites flanking exon 3 was placed in the EHD2 wild-type allele. EHD2 del/del mice were obtained by breeding with Cre-deleter mouse strain (diphtheria toxin A [DTA]). (B) EHD2 mRNA level in EHD2 del/+ and EHD2 del/del mice (mRNA from BAT, n = 5). (C) Western blot analysis of WAT of EHD2 +/+, ^+/−, and ^−/− mice against EHD2, Cav1, and Cavin1. (D) EHD2 immunostaining in BAT cryostat sections from EHD2 +/+, del/+, and del/del mice. (E) The body weight was monitored over 12 mo (n = 7). (F) EHD2 del/+ and EHD2 del/del mice during preparation. (G) Masson trichrome staining of WAT paraffin sections of EHD2 del/+ and EHD2 del/del. Analysis of the adipocyte cell size [n(del/+) = 172/3, n(del/del) = 199/3]. (H) EHD2 del/del mice showed increased amount of beige BAT in the neck region. In addition, white adipocytes were infiltrated into the BAT depots (white arrowheads). (Left) Masson trichrome staining of EHD2 del/+ and del/del BAT paraffin sections. (Right) BAT cryostat sections were stained against the LD coat protein Perilipin1 to visualize LDs. (I) LD size, number per cell, and cell area were measured in BAT cryostat sections [n(del/+) = 118/3, n(del/del) = 104/3]. (J–M) Triglyceride (TG; J and K), total cholesterol, and cholesterol ester (L and M) concentrations were determined for WAT (n = 5 to 8) and BAT (n = 5 to 6) of EHD2 del/+ and del/del mice. Line graph represents mean ± SE, column bar graphs show mean + SE or each replicate with mean ± SE, normal distributed groups were analyzed by t test, not normally distributed values with Mann–Whitney U test, *P < 0.05, ***P < 0.0001. For comparison to C57BL6/N, see also SI Appendix, Figs. S1 and S2.

Fig. 2.

EHD2 del/del adipocytes show increased LD size and faster fatty acid uptake. (A–D) Analysis of LD size in EHD2 del/del and EHD2 del/+ adipocytes by staining with BODIPY (A and B) [untreated: n(del(+) = 74/3, n(del/del) = 60/3); differentiated: n(del/+) = 132/3, n(del/del) = 95/3]. A 3D reconstruction of EHD2 del/del differentiated adipocyte (C). Green, LDs; blue, nucleus. (D) LD number per cell of differentiated EHD2 del/+ and del/del adipocytes [H, n(del/+) = 26/3; n(del/del) = 31/3]. (E and F) FA uptake assay in differentiated EHD2 del/+ and EHD2 del/del adipocytes was measured by FACS analysis. The R1 population indicates positively stained cells (illustrated in red in graph E). R2 populations correspond to higher BODIPY staining intensity in cells and represent adipocytes with increased BODIPY-FA12 uptake (shown in blue in E). The percentages of R1 and R2 cell numbers are shown in F. See also SI Appendix, Fig. S6. Column bar graphs illustrate mean ± SE, or each replicate with mean ± SE. A t test or Mann–Whitney U test was used to calculate significance, *P < 0.05; ***P < 0.0001. a.u., arbitrary unit; FCS-A, forward cytometry standard-area.

Fig. 3.

Loss of EHD2 results in detached caveolae in vivo. (A and B) Representative EM images of BAT from EHD2 del/+ and del/del mice and systematic analysis [caveolae number: n(del/+) = 140/3, n(del/del) = 100/3; caveolae size and diameter: n(del/+) = 201/3, n(del/del) = 171/3]. White arrowheads indicate caveolae. (C and D) EM images of EHD2 del/+ and del/del WAT [caveolae number: n(del/+) = 108/3, n(del/del) = 124/3; caveolae size and diameter: n(del/+) = 151/3, n(del/del) = 185/3]. White arrowheads indicate membrane-bound caveolae, black arrowheads detached caveolae. (E) Representative image for EM gold immunolabeling against Cav1. Control labeling did not reveal specific staining. (F and G) Electron tomogram of a 150-nm EHD2 del/del BAT section (F). White box indicates segmentation and visualization of displayed cell area in G. The 3D model contains the plasma membrane (G, green) and the detached caveolae (violet). Detachment of caveolae was observed by changing the viewing angle (white arrow indicates the direction). Closer inspection of cell membrane and caveolae showed displacement of caveolae from the membrane. The 3D model also revealed attachment of some caveolae to the membrane (arrowhead). Graphs illustrate each replicate with mean ± SE; column bar graphs illustrate mean + SE. A t test or Mann–Whitney U test was used to calculate significance, **P < 0.001; ***P < 0.0001. See also Movie S1.

Fig. 4.

Enhanced caveolar mobility in cells lacking EHD2. (A and B) TIRF live-imaging of EHD2 +/+ and del/del MEFs expressing pCav1-EGFP (in A, white boxes indicate magnified cell areas in which regions of interest, illustrated in yellow boxes, were analyzed). Line scan analysis of the recorded Cav1 intensities revealed for fixed, nonmoving caveolae lines and for fast moving caveolae single sparks (as illustrated in a and b). n(^+/+) = 90/3; n(del/del) = 92/3; each replicate is represented with mean ± SE, ***P < 0.0001. t, time; x, scale; numbers 1–10 in single line scan panels indicate successive regions within the yellow rectangles. See also SI Appendix, Fig. S7 and Movies S2–S4.

Fig. 5.

EHD2-mediated fatty acid uptake depends on Cav1, Cavin 1, Dyn2, and CD36. (A and B) EHD2 +/+ and del/del MEFs were transfected with either plasmid expressing EGFP (pEGFP) or pEHD2-EGFP, incubated for 48 h, and afterward treated for 6 h with oleic acid, and Nile Red staining was performed to determine LDs [pEGFP: n(^+/+) = 309/3, n(del/del) = 310/3; pEHD2-EGFP: n(^+/+) = 218/4, n(del/del) = 184/4]. Boxes a and b illustrate magnified cell areas. (C) EHD2 +/+ and del/del MEFs were treated with Cav1 or Cavin1 siRNA, and LDs were stained with BODIPY [negative control (negt CTRL): n(^+/+) = 504/3, n(del/del) = 530/3; Cav1 siRNA: n(^+/+) = 521/3, n(del/del) = 558/3; Cavin1 siRNA: n(^+/+) = 366/3, n(del/del) = 362/3]. (D and E) EHD2 +/+ and del/del MEFs were transfected with pGFP-Dyn2-K44A, incubated for 18 h, and afterward treated for 6 h with oleic acid, and Nile Red staining was performed to visualize LDs [pEGFP: n(^+/+) = 309/3, n(del/del) = 233/4; pGFP-Dyn2-K44A: n(^+/+) = 136/3, n(del/del) = 237/4]. Boxes a and b illustrate magnified cell areas. (F and G) LD size after CD36 siRNA knockdown in EHD2 +/+ and del/del MEFs [G, negative control: n(^+/+) = 584/6, n(del/del) = 475/6; CD36 siRNA#1: n(^+/+) = 341/3, n(del/del) = 249/3; CD36 siRNA#2: n(^+/+) = 412/3, n(del/del) = 468/3; CD36 siRNA#3: n(^+/+) = 251/3, n(del/del) = 368/3]. The graph illustrates each replicate with mean ± SE; a two-way ANOVA test was used to calculate significance between siRNA negative CTRL and siRNA, and a t test was used between ^+/+ and del/del data. Box plots indicate mean ± SE, and single replicates of 5% of maximal and minimum values are illustrated; a t test or Mann–Whitney U test was used to calculate significance, ***P < 0.0001. See also SI Appendix, Figs. S7–S9.

Fig. 6.

Caveolae-related fatty acid uptake and trafficking. (A) Time-resolved C11-BODIPY591 FA accumulation in the plasma membrane of EHD2 +/+ and del/del MEFs was recorded by TIRF. (Scale bars: 1 µm.) Cell borders are indicated. In both genotypes, FAs intercalated into the plasma membrane within seconds, and no qualitative difference between the two genotypes was observed. (B and C) Time-resolved TIRF imaging in MEF expressing Cav1-EGFP treated with C11-BODIPY591 FA. (Scale bars: B, 1 µm.) Detailed caveolae movement in EHD2 del/del MEFs treated with C11-BODIPY591 FA (C, TIRF imaging, mobile caveolae depicted in black, magenta, and blue circles; immobile caveolae in white circles). (Scale bars: 200 nm.) Single images represent sequential acquisition frames of the recording (see also Movies S9 and S10). (D and E) C12-BODIPY FA trafficking in EHD2 +/+ (D) and del/del (E) MEFs treated with Cav1 siRNA (endoplasmic reticulum [ER] stained by ER-TrackerRed). (Scale bars: FA and ER, 20 µm; Merge, 5 µm.) The merged panel illustrates the magnified cell area indicated in the FA panel, and arrowheads indicate LDs. (F and G) Fluorescence intensity of cellular C12-BODIPY FA [^+/+: n(Negt CTRL) = 10/3, n(Cav1 si#1) = 12/3, n(Cav1 si#2) = 12/3; del/del: n(Negt CTRL) = 11/3, n(Cav1 si#1) = 12/3, n(Cav1 si#2) = 11/3]. Line graphs indicate mean ± SE. Normal distributed groups were analyzed by t test, and not normally distributed values with Mann–Whitney U test, **P < 0.001, ***P < 0.0001. PM, plasma membrane; AU, arbitrary unit. See also SI Appendix, Fig. S10.

Fig. 7.

Decreased EHD2 expression in obesity. (A) EHD2 expression level was analyzed in fat tissue of ob/ob or NZO mouse models compared to C57BL6/J mice (n = 5). (B and C) Investigation of caveolae by EM [n(ob/ob mice) = 85/2; n(C57BL6/N) = 117/2]. Black arrowheads indicate caveolae. (D and E) EHD2 expression in visceral (E) or subcutaneous WAT (F) in obese patients [n(normal) = 31; n(overweight) = 23; n(obesity grade 1) = 7; n(obesity grade 2) = 17; n(obesity grade 3) = 202]. (F) The model illustrates how FA uptake is affected by caveolae function. In the absence of caveolae, FA uptake is reduced, resulting in smaller LDs compared to normal conditions. In the absence of EHD2, FA is increased, suggesting a regulatory function of EHD2 in caveolae-dependent lipid uptake. FFA, free fatty acid; LD, lipid droplet. Box plots indicate median with whiskers from maximal to minimum value, or each replicate with mean ± SE is represented. Normal distributed groups were analyzed by t test, and not normally distributed values with Mann–Whitney U test, *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001.