EH Domain-Containing 2 Deficiency Restricts Adipose Tissue Expansion and Impairs Lipolysis in Primary Inguinal Adipocytes

Figures

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  Figure 1. Adipose tissue distribution and adipocyte sizes comparing Ehd2−/− and WT mice. (A) Western blot analysis illustrating expression of EH domain-containing 2 (EHD2) in different tissues; white adipose tissue (WAT), skeletal muscle, lung, brown adipose tissue (BAT), and heart from Ehd2−/− and WT mice. Comparison of WT and Ehd2−/− mice fed chow-diet, showing (B) body weight (BW; n=46–54 animals/group), (C) blood glucose (mmol/L; n=13–15 animals/group), and (D) serum insulin (pM; n=13–15 animals/group). (E) Liver triglycerides (mg/g liver; n=13–15 animals/group), and (F) illustrated by Oil-Red-O-staining, scale bar=100μm. Adipose tissue depots weights; (G) epididymal (EPI; n=46–54 animals/group), (H) inguinal (ING; n=35–38 animals/group), and (I) retroperitoneal (RETRO; 13–15 animals/group). (J,K) Adipose cell size distribution of EPI and ING tissue depots analyzed using coulter counter, 6,000 counts/sample, each sample run in duplicate, data displayed as average from n=3 samples/group. Data in (B–E,G–I) are presented as mean±SD, and *p<0.05 and ***p<0.001 were calculated using unpaired two-tailed Student’s t-test.

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  Figure 2. Body weight gain, liver triglycerides, insulin and free fatty acid (FFA) levels in Ehd2−/− and WT mice following 2weeks of high-fat diet (HFD)-feeding. WT and Ehd2−/− mice (n=18 animals/group) were subjected to HFD-feeding for 2weeks, (A) illustrates body weight gain, and (B) food intake (gram/mouse/day). (C) Final blood glucose (mmol/L; n=11), (D) serum insulin (pM; n=7–8 animals/group), (E) serum levels of FFA (mM; n=8–9 animals/group), and (F) liver triglycerides (mg/g liver; n=18 animals/group). Data in (A–F) are presented as mean±SD.

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  Figure 3. Adipose tissue expansion and adipose cell size distribution in Ehd2−/− and WT mice following 2weeks of HFD-feeding. (A,B) Depot weights of EPI and ING adipose tissue (n=22–23 animals/group) collected after 2weeks of HFD. (C) Correlation of EPI and ING weight, each symbol (gray=WT, black=Ehd2−/−) represent one animal, line shows simple linear regression analysis. Adipose cell size distribution obtained by coulter counter in EPI (D–F) and ING (H–J) depots. Data displayed as average from n=3 samples in chow/group, and n=6 samples in HFD/group. (G,K) Frequency percentage of small cells (cell diameter 20.00–20.55μm) from HFD-fed mice; EPI (G) and ING (K), data obtained from cell size distribution curves, shown in (F,J). Data in (A,B,G,K) are presented as mean±SD, and **p<0.01 and ***p<0.001 were calculated using unpaired two-tailed Student’s t-test.

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  Figure 4. PPARγ activity, FABP4 expression, and caveolin-1 in adipocytes from Ehd2−/− and WT mice. (A) PPARγ activity in primary isolated EPI adipocytes from chow-fed Ehd2−/− and WT mice, non-stimulated or stimulated with rosiglitazone (ROSI) for 24h using transfection and dual luciferase assay. Data were normalized to Renilla reporter (pRLnull), and expressed as percentage of ROSI WT (n=4). (B) Left panel: Confocal image of primary EPI adipocytes from WT and Edh2−/− mice stained for lipids (bodipy, green), mitochondria (Mitotracker, red), and EHD2 (antibody, blue), scale bar=20μm. Right panel: Western blot analysis of EHD2 expression in cell lysates of primary adipocytes isolated from EPI and ING adipose tissue. (C) Representative western blots and quantification of total protein levels of FABP4 and caveolin-1 from primary adipocyte lysates from EPI adipose tissue from chow or HFD-fed mice. HSP90 was used as loading control, n=3-6/group. (D) Same as in (C) with lysates from ING adipose tissue. (E) Electron micrographs showing the structure and distribution of caveolae at the adipocyte cell membrane in EPI and ING tissue from WT and Ehd2−/− animals. Arrows show clearly discernible…

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  Figure 5. Lipolysis in adipocytes isolated from Ehd2−/− and WT mice. (A,B) Primary adipocytes were isolated from EPI or ING depot, and cells in suspension were subjected to non-stimulation (basal) or β adrenergic stimulation using isoprenaline (ISO; 10nM) for 30min. Lipolysis was measured as release of glycerol into the medium in a 10% (v/v) cell suspension. Basal and ISO stimulated lipolysis of EPI (A) and ING (B) adipocytes from chow and HFD fed mice (n=6 chow, n=5 HFD). (C) Western blot showing total and phosphorylated levels of perilipin-1 pS522 and HSL pS563 from ING adipocytes from HFD-fed mice, either non-stimulated or stimulated with isoprenaline (ISO; 30min; n=6-7/group), quantification shows phosphorylation over total protein. HSP90 used as loading control. (D) Western blot analysis displaying representative image and quantification of cell lysates from ING adipocytes after HFD-feeding. Total protein level of perilipin-1, HSL, and adipose triglyceride lipase (ATGL) from non-stimulated cells (n=3–4). (E) Confocal image of lipid droplet clusters in primary ING adipocytes from chow-fed mice. Cells were stained for lipids (bodipy, red) and mitochondria (mitotracker, white). Lower…

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  Figure 6. Lipolysis using cAMP analog and PDE-inhibitors in inguinal adipocytes from Ehd2−/− and WT mice. (A) Lipolysis measured as release of glycerol into the medium in a 10% (v/v) cell suspension using primary adipocytes isolated from the ING adipose tissue depot of HFD-fed mice. Cells in suspension were either non-stimulated (basal), or β adrenergic stimulated using isoprenalin (ISO; 10 or 1,000nM), or stimulated with the non-degradable cAMP analogue dibutyryl cAMP (DcAMP; 100 or 1,000μM) for 30min. (n=5–8/group). (B) mRNA expression of Plin1, Adrb3, and Ehd2 from ING adipose tissue of HFD fed mice, assessed by RT-qPCR, 18S rRNA used as reference gene, data plotted as relative fold of WT (n=11–13/group). (C) Western blot using a phospho protein kinase A (PKA) motif specific antibody [P-RXX(S/T)] in iso-stimulated ING adipocyte lysates from WT (n=3) and Edh2−/− (n=4) mice on HFD. (D) Western blot showing phosphorylated levels of CREB pS133 in ING adipocytes, either non-stimulated or stimulated with isoprenaline (ISO; 30min), isolated from HFD-fed mice (n=3–4/group), HSP90 was used as loading control. (E) Quantification of pCREB pS133 [images shown in (D)], Perilipin-1 pS522 and HSL…