Fig. 1Vimentin deficiency prevents diet-induced obesity. (A) Vim−/− mice (n=10) and control wild-type mice (n=10) were fed a high-fat diet (HFD) for 10 weeks. Body weight changes of control and Vim−/− mice on an HFD. Bars indicate mean±standard error of the mean (SEM). (B) Weights of adipose tissues from the experimental mice in (A). Bars indicate mean±SEM. (C) Representative images of adipose tissues and livers harvested from the experimental mice in (A) with hematoxylin-eosin staining. Scale bar: 100 µm. (D) The graph shows comparisons of adipocyte sizes between Vim−/− mice and wide-type mice. Adipocytes from three tissue sections per mouse type were measured. Each bar denotes the mean±SEM. (E) Concentrations of very low density lipoprotein/low density lipoprotein (VLDL/LDL), high density lipoprotein (HDL), triglyceride (TG), non-esterified fatty acid (NEFA), and free glycerol in the sera of the Vim−/− and control mice in Fig. 1A. The graph shows mean±SEM. (F) Representative images of adipose tissues from the experimental mice in (A), stained with antibody for the macrophage marker F4/80. Scale bar: 100 µm. (G) Measurements of the area stained with F4/80 antibody in the crown-like structures of adipose tissues from the experimental mice in (A). WAT, epididymal white adipose tissue; SC, subcutaneous fat; NS, non-significant. aP<0.05, bP<0.01, cP<0.001.
Fig. 2
Vim−/− mice exhibit enhanced glucose tolerance and lower fasting glucose. (A) Glucose tolerance after a 10-week high-fat diet (n=8 for control, n=8 for Vim−/− mice). (B) Area under the curve (AUC) of (A). (C) Blood glucose levels after 16 hours of fasting (n=10 for control, n=10 for Vim−/− mice). (D) Serum insulin concentrations after 16 hours of fasting (n=6 for control, n=6 for Vim−/− mice). The graph shows mean±standard error of the mean). NS, non-significant. aP<0.01, bP<0.001.
Fig. 3Vimentin deficiency reduces CD36 and glucose transporter type 4 (GLUT4) trafficking to the plasma membrane in adipocytes. (A) Representative flow cytometry data of control and Vim−/− mouse adipocytes (left panel). Adipocytes were exposed to TF2-C12 fatty acid 40 µg/mL for 30 minutes. Fluorescence intensity of these cells was measured using flow cytometry (red: control+TF2-C12, green: Vim−/−+TF2-C12, light blue: untreated control adipocytes, yellow: untreated vimentin-null adipocytes). Geometric mean fluorescence intensity (right panel). (B) Western blot analysis for CD36 using the fractionated lysate from control and Vim−/− murine adipocytes. Cytosolic and membrane fractions were separated using a buffer-based protocol. Caveolin-1 was used as a marker for the plasma membrane fraction, and β-actin was used as a marker for the cytosolic fraction. (C) Quantitative real-time polymerase chain reaction to measure CD36 and GLUT4 expression in control and Vim−/− murine adipocytes. The values are normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Plasma membrane-localized CD36 was measured by flow cytometry using allophycocyanin (APC)-conjugated CD36 antibody. Mean fluorescence intensities representing CD36 expression were compared between control and Vim−/− adipocytes. (E) Western blot analysis for GLUT4 using the fractionated lysates from control and Vim−/− murine adipocytes. Cytosolic and membrane fractions were separated using a buffer-based protocol. Caveolin-1 was used as a marker for the membrane fraction, and β-actin was used as a marker for the cytosolic fraction. Quantitation of band intensities (right graph). Cyto, cytosol fraction; Mem., membrane fraction; NS, non-significant. aP<0.01, bP<0.001.
Fig. 4Vimentin deficiency decreased lipolysis via hormone-sensitive lipase (HSL) activity. (A) Western blot analyses for HSL and phosphorylated HSL (p-HSL; Ser563, Ser660, Ser565) using homogenized epididymal fat lysates from the experimental mice in Fig. 1A (control, n=3; Vim−/−, n=3). Quantitation of the band intensities in Western blots (graphs below). (B) Western blot analyses for fatty acid synthase and perilipin using fat lysates from the experimental mice in Fig. 1A (control, n=3; Vim−/−, n=3). Quantitation of the band intensities in Western blots (graphs below). The graph shows mean±standard error of the mean. FAS, fatty acid synthase; NS, non-significant. aP<0.05.