EHD2 regulates adipocyte function and is enriched at cell surface-associated lipid droplets in primary human adipocytes

Abstract

Adipocytes play a central role in energy balance, and dysfunctional adipose tissue severely affects systemic energy homeostasis. The ATPase EH domain-containing 2 (EHD2) has previously been shown to regulate caveolae, plasma membrane-specific domains that are involved in lipid uptake and signal transduction. Here, we investigated the role of EHD2 in adipocyte function. We demonstrate that EHD2 protein expression is highly up-regulated at the onset of triglyceride accumulation during adipocyte differentiation. Small interfering RNA-mediated EHD2 silencing affected the differentiation process and impaired insulin sensitivity, lipid storage capacity, and lipolysis. Fluorescence imaging revealed localization of EHD2 to caveolae, close to cell surface-associated lipid droplets in primary human adipocytes. These lipid droplets stained positive for glycerol transporter aquaporin 7 and phosphorylated perilipin-1 following adrenergic stimulation. Further, EHD2 overexpression in human adipocytes increased the lipolytic signaling and suppressed the activity of transcription factor PPARγ. Overall, these data suggest that EHD2 plays a key role for adipocyte function.

FIGURE 1:

(A) Correlation between EHD2 and caveolin-1 mRNA expression in epididymal fat tissue during 14 d of HFD feeding of C57BL6/J mice, n = 20 biological data points. (B) Protein expression of EHD2 and caveolin-1 in epididymal adipose tissue during 14 d of HFD, n = 3–4/group, data normalized to β-actin. (C) Western blot analysis showing EHD2 protein expression in isolated primary adipocytes and epididymal adipose tissue from mice fed chow or 14 d of HFD, HSP90 used as a loading control, ap2 used as an adipocyte marker. (D) Western blot analysis showing caveolin-1 and EHD2 protein expression in epididymal adipose and lung tissues from WT- and caveolin-1 KO mice. n = 3 animals/group; HSP90 used as a loading control. (E) Representative blots showing protein expression of FAS, EDH2, caveolin-1, and C/EBPα during 3T3-L1 differentiation, n = 4 independent experiments; each sample was collected and is presented as a technical duplicate. HSP90 used as a loading control. (F) Representative immunofluorescence microscopy images of differentiating 3T3-L1 cells –2, 4, and 11 d after initiating differentiation. Cells were stained with Hoechst (blue signal, nuclei), BODIPY (yellow signal, neutral lipids) (top panel), and EHD2 antibody (bottom panel). (G) Same as in F but stained for caveolin-1 instead of EHD2 (caveolin-1 shown in bottom panel, Cav1). (H) Representative images showing a projection, bottom and middle sections of 3T3-L1 cells, 8 d after differentiation, costained with direct-conjugated antibodies toward EHD2 and caveolin-1, Hoechst, and BODIPY. Data in B are presented as mean ± SD. #,*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 represent significance compared with 0 d of HFD. Scale bar = 10 µm.

FIGURE 2:

(A) Representative Western blots of EHD2 and transcriptional targets in 3T3-L1 cell lysates collected 72 h after gene silencing with siRNA control (SCR) or EHD2 siRNA (siEHD2). HSP90 was used as a loading control. Arrow indicates PPARγ2. (B) Quantification of protein expression in A normalized to HSP90. n = 4 independent experiments; each sample was collected and presented as a technical duplicate. (C) Representative Western blots of targets involved in lipid metabolism in 3T3-L1 cell lysates were collected 72 h after gene silencing with siRNA control (SCR) or siEHD2. HSP90 was used as a loading control. (D) Quantification of protein expression in C normalized to HSP90. n = 4 independent experiments; each experiment was run in duplicate. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3:

(A) Representative immunofluorescence images of 3T3-L1 cells: control (SCR, left panels) or EHD2-silenced (siEHD2, right panels), stained for nuclei (Hoechst, blue signal), neutral lipids (BODIPY, yellow signal), and EHD2 (gray scale). Scale bar = 50 µm. (B) Average intensity in arbitrary units (AU) of neutral lipids (BODIPY) in control (SCR) and EHD2-silenced (siEHD2) cells. (C) Quantification of lipid droplet size (relative size expressed as pixel area) in control (SCR) or EHD2-silenced (siEHD2) cells. Data are in presented as mean ± SEM.*p < 0.05, ***p < 0.001. (D) Representative immunofluorescence images of 3T3-L1 cells: control (SCR, left panels) or EHD2-silenced (siEHD2, right panels) that were stained for nuclei (Hoechst, blue signal), neutral lipids (BODIPY, yellow signal), and caveolin-1. Scale bar = 50 µm.

FIGURE 4:

(A) Seventy-two hours after EHD2 gene silencing (scrambled [SCR] or siEHD2), 3T3-L1 cells were nonstimulated or stimulated with insulin (0.01, 0.1, or 10 nM) for 30 min followed by Western blot analysis to detect total and phosphorylated protein levels of IRS-1 (pY612), PKB (pS473), and EHD2. HSP90 used as a loading control. Levels of PKB (pT308) are shown in the bottom panel. Representative blots from n = 34 independent experiments. Quantifications of Western blot analysis in A are shown in B. Seventy-two hours after gene silencing (SCR or siEHD2-treated), 3T3-L1 cells were either nonstimulated or stimulated with ISO (10 or 100 nM) for 30 min; glycerol concentration in medium (mM) is shown in C, and Western blot analysis for detection of total and phosphorylated protein levels of HSL (pS563) and perilipin-1 (pS522) are shown in D. Representative blots from n = 4 independent experiments. (E) Quantification of HSL in D, expressed as HSL pS563/total HSL, n = 4 independent experiments; each sample was run in duplicate. Data in B, C, and E are shown as mean ± SD. *p < 0.05, ****p < 0.0001. (F) Representative immunofluorescence images of 3T3-L1 cells scrambled (SCR) or EHD2-silenced (siEHD2) that were either nontreated (CTRL, left panel) or ISO-treated (100 nM, right panel) for 30 min. After treatment, cells were fixed and stained for nuclei (Hoechst, blue signal), neutral lipids (BODIPY, yellow signal), EHD2 (green signal), and perilipin-1 pS522 (red signal). Scale bar = 10 µm.

FIGURE 5:

(A) Confocal microscopy images of primary human adipocytes that were fixed and stained for nuclei (Hoechst, blue signal) and neutral lipids (BODIPY, yellow); merged image is shown in the top left panel. The same cells were also stained with EHD2 (bottom left panel) and caveolin-1 (bottom right panel). Note that, the top right panel shows the merged image of EHD2 (green) and caveolin-1 (Cav1, red), and colocalization appears as a yellow signal. The insert shows a line drawn along the plasma membrane, and the fluorescence intensity profile (AU) is shown at the bottom to illustrate colocalization. Green represents EHD2, and red represents caveolin-1 (Cav1). (B) Same as in A but costained against cavin1 (Cavin-1, red). Scale bar = 20 µm.

FIGURE 6:

(A) The top panel illustrates native EDH2 antibody labeling of primary human adipocytes that were either nontreated (CTRL) or treated with ISO (100 nM, 30 min) or insulin (Ins, 100 nM, 30 min) prior to fixation. Staining with antibodies against EHD2 (green signal) and perilipin-1 pS522 (red signal). The perilipin-1 pS522 (P-perilipin) signal is also displayed in inverted gray scale below each image. The same conditions in EHD2WT-GFP overexpressing cells are shown in the bottom panel, where EHD2 was detected with GFP (green signal). Scale bar = 20 µm. (B) Protein expression assessed by Western blot in cell lysates from primary human adipocytes transduced with control (Ad-GFP) or Ad-EHD2 (Ad-EHD2WT) virus for 68–94 h, whereafter they were nontreated or treated with ISO (100 nM, 30 min). HSP90 was used for normalization. Data are expressed as fold expression compared with control (Ad-GFP) and presented as mean ± SD, n = 5–6 independent experiments. *p < 0.05, **p < 0.01. (C) Representative confocal microscopy images of primary human adipocytes expressing caveolin-1-RFP (Cav1-RFP) and either EHD2 WT (nonstimulated) or EHD2 mutants with single amino acid substitutions (all EHD2 constructs were GFP-tagged). Cells were also stained with LipidTOX to detected lipid droplets prior to imaging. The two top panels illustrate the distribution at the single cell level, scale bar = 50 µm. Magnified regions (white squares) showing distribution of EHD2 and caveolin-1 in lipid droplets in the surface proximal region are shown in the bottom panels, scale bar = 5 µm.

FIGURE 7:

(A) Representative TIRF microscopy images of primary human adipocytes expressing EHD2 WT (nonstimulated) or EHD2 mutants (all stimulated with ISO 100 nM for 30 min prior fixation). In the top panel, the cells were stained with antibody against perilipin-1 pS522 (red), and EHD2 was detected by GFP signal (green). In the bottom panel, cells were costained against perilipin-1 pS522 (red) and AQP7 (green). Scale bar = 20 µm. Insets show zoom of lipid droplet structures for increased visibility. EHD2 and AQP7 are shown in green in each panel, and perilipin-1 pS522 is shown in red. Scale bar = 2 µm. (B) Fluorescence signal of perilipin-1 pS522 was quantified for each EHD2 mutant using an region of interest around each lipid cluster. Four clusters per cell and ∼16 cells per EHD2 mutant were measured. ****p < 0.0001, ####p < 0.0001 significance levels compared with EHD2WT, nonstimulated (WT-NoStim).

FIGURE 8:

(A) Confocal cross-section of primary human adipocyte showing distribution of EHD2 stained with antibody (Abcam, red) and Hoechst (blue). Scale bar = 10 µm. (B, C) Levels of PPRE luciferase activity in response to overexpression of caveolin-1, EHD2, or EHD2 mutants with single amino acid substitutions. All PPRE activity levels were normalized to the levels of pRL null using a dual luciferase assay. In B, data are presented as fold of the PPRE luciferase activity in the pcDNA3-transduced control sample within each experiment, n = 3 independent experiments. Data are presented as mean ± SD. In C, the data are expressed as fold of the PPRE luciferase activity in the EHD2-transduced sample within each experiment. Data are presented as mean ± SD, n = 3–7 independent experiments/group. *p < 0.05, ***p < 0.001. (D) Graphic summary illustrating the localization of EHD2, together with caveolin-1, close to cell surface–associated lipid droplets that display lipolytic activity in primary human adipocytes. The dynamics of lipid transport across the cell membrane are dependent on EHD2, likely in a caveolae-dependent manner, which indirectly affects the transcriptional activity. Altogether, these data suggest that EHD2 plays a key role in maintaining adipocyte function.