Figure 1.
Dose response and effect of cytochalsin B and insulin on 2-NBD-Glucose uptake into L cells.
Representative confocal images of L cells labeled with different doses of 2-NBD-Glucose (50-300 µM) are shown (A). Uptake of 2-NBD-Glucose (B) and the time-course parameters Fmax (C) and initial rate (D) were calculated as described in the Methods section. The effect of the glucose transport protein inhibitor, cytochalsin B, insulin, and unlabeled glucose on 2-NBD-Glucose uptake after 30 min incubation was determined (E). Increases in fluorescence intensity were calculated and expressed as arbitrary units (a.u.). Values represent mean ± SEM. (*) indicates p < 0.05 as compared to control.
Figure 2.
Effect of Plin2 overexpression in transfected Lcells.
L cells were transfected with empty vector to show little to no fluorescence in lipid droplets (A) that were evident by light microscopy (B). Lipid droplet targeting of the CFP-Plin2 construct was confirmed by confocal microscopy showing CFP-labeled lipid droplets in Plin2 overexpressing cells (C) that were also visible by light microscopy (D). Relative expression of Plin2 (E) and neutral lipid content (F) in control (open bar) and Plin2 overexpression (closed bar) cells were measured as described in Methods. (*) indicates p<0.05 as compared to control. Insets: Representative Western blots showing relative protein expression of Plin2 and GAPDH in control and Plin2 overexpression cells.
Figure 3.
Effect of altered Plin2 expression on 2-NBD-Glucose uptake in L cells.
2-NBD-Glucose uptake (A) in Plin2 overexpression (closed circles) and control (open circle) cells was measured as an increase in fluorescence intensity expressed in arbitrary units (a.u.) as described in the Methods section. [3H]-2-deoxyglucose uptake (B) was measured in control (open bar) and Plin2 overexpression (closed bar) cells as a fold-change in radioactivity relative to control cells. Relative expression levels of Plin2 after siRNA-mediated Plin2 knock down (C) and 2-NBD-Glucose uptake (D) in cells that were untransfected (closed circles), treated with control siRNA (open circle), or Plin2 siRNA (closed square) was also measured, normalized to the housekeeper gene GAPDH. Values represent mean ± SEM (n=3). (*) indicates p < 0.05 as compared to control. Insets: Representative Western blots showing relative protein expression of Plin2 and GAPDH.
Figure 4.
Relative expression levels of key proteins involved in lipid droplet formation, glucose uptake and transport.
Cell homogenates from Plin2 overexpressing and control cells were probed with antibodies against the following proteins: insulin receptor (A), Plin1 (B), GLUT1 (C), FSP27 (D), SNAP23 (E), and syntaxin-5 (F). Expression levels were quantified as described in the Method section, normalized to the housekeeper gene GAPDH. Values represent mean ± SEM (n=3-5). (*) indicates p < 0.01 and (**) indicates p < 0.001 as compared to control. Insets: Representative Western blots showing relative protein expression of proteins of interest and housekeeping gene (GAPDH or actin).
Figure 5.
Co-localization and FRET imaging between Cy3-labeled SNAP23 and Cy5-labeled Plin2.
Confocal images of Cy3-labeled SNAP23 and Cy5-labeled Plin2 were examined to determine co-localization (A) and FRET efficiencies, E between the fluorescently labeled proteins. The extent of co-localization was shown graphically in a pixel fluorogram (B) to reveal Cy3-SNAP23 (arbitrarily placed in the green channel) co-stained with Cy5-Plin2 (red channel) in yellow-to-orange areas where both probes co-localized. FRET efficiency maps were generated from the following images: donor emission image of Cy3-SNAP23 co-labeled with Cy5-Plin2 before acceptor (Cy5-Plin2) photobleaching (C); donor emission image of Cy3-SNAP23 co-labeled with Cy5-Plin2 after acceptor photobleaching (D); donor emission image of Cy3-SNAP23 after photobleaching overlaid with a pseudo-colored FRET image (E); and acceptor emission image of Cy5-Plin2 after photobleaching (F). Cells were imaged and FRET efficiency images generated as described in the Method section. The FRET overlay was pseudo-colored to visualize regions of higher and lower FRET as shown by the inset color scale (E).
Figure 6.
Co-localization and FRET imaging between Cy3-labeled SNAP23 and Cy5-labeled GLUT1.
Confocal images of Cy3-labeled SNAP23 and Cy5-labeled GLUT1 were examined by laser scanning confocal microscopy. Co-localization of Cy3-SNAP23 (green) with Cy5-GLUT1 (red) revealed yellow-to-orange areas where both probes overlapped (A). The extent of co-localization was shown in a pixel fluorogram (B). FRET efficiency maps were generated from the following images: donor emission image of Cy3-SNAP23 co-labeled with the acceptor Cy5-GLUT1 before photobleaching (C); donor emission image of Cy3-SNAP23 co-labeled with Cy5-GLUT1 after acceptor photobleaching (D); donor emission image of Cy3-SNAP23 after photobleaching overlaid with a pseudo-colored FRET image (E); and acceptor emission image of Cy5-GLUT1 after photobleaching (F). Cells were imaged and FRET efficiency images generated as described in the Method section. The FRET overlay was pseudo-colored to visualize regions of higher and lower FRET as shown by the inset color scale (E).
Figure 7.
Co-immunoprecipitation of SNAP23 with GLUT1, Plin2, or Plin1.
The native proteins in cell homogenates from Plin2 overexpressing and control cells were co-immunoprecipitated as described in the Methods section. To examine SNAP23/GLUT1 interactions, GLUT1 was immunoprecipitated using anti-GLUT1. Levels of SNAP23 (A) in the immunoprecipitate from control (lane 1) and PLIN2 overexpressing cells (lane 2) were analyzed by immunoblotting with anti-SNAP23. Equal immunoprecipitation of GLUT1 was verified by immunoblotting with anti-GLUT1 (B). The ratio of SNAP23 immunoprecipitated with GLUT1 was calculated from the integrated density values from Western blots (C). To analyze SNAP23/Plin2 interactions, Plin2 was immunoprecipitated with anti-Plin2 and levels of SNAP23 (D) were detected by immunoblotting with anti-SNAP23. Equal immunoprecipitation of Plin2 was verified by immunoblotting with anti-Plin2 (E). The ratio of SNAP23 immunoprecipitated with GLUT1 was calculated from the integrated density values from Western blots (F). Reverse immunoprecipitation experiments were also performed. SNAP23 was immunoprecipitated with anti-SNAP23 and levels of Plin2 (G), GLUT1 (H), and Plin1 (I) in the immunoprecipitate were analyzed by Western blotting. Equal immunoprecipitation of SNAP23 was verified by immunoblotting (J). The ratio of Plin2, GLUT1 and Plin1 immunoprecipitated with SNAP23 was calculated from the integrated density values from Western blots (K). Immunoprecipitate obtained using secondary IgG antibodies (lane 3) were used as negative controls for each set. (*) indicates p < 0.05 as compared to control.
Figure 8.
Schematic diagram for SNARE-mediated regulation of glucose transport in Plin2 overexpression cells.
Key regulators involved in glucose uptake and transport are illustrated. Glucose transporters (GLUT) are involved in the entry of glucose into the cells. In the absence of insulin stimulation, these transporters predominantly reside in vesicular structures that move slowly from the cytoplasm to PM. Upon insulin stimulation GLUT-containing vesicles translocate, dock, and fuse with the plasma membrane through the action of SNARE fusion machinery proteins including SNAP23, Syntaxin-5, and VAMP4. Similar proteins (SNAP23, Syntaxin-4, and VAMP2) are also present on the lipid droplet surface along with Plin2 which directly interacts with SNAP23. Under conditions of excess lipid storage that increase lipid droplet formation and Plin2 expression, SNAP23 is retained on the lipid droplet by enhanced interaction between Plin2 and SNAP23. A decreased interaction between SNAP23 with other SNARE proteins at the plasma membrane results in decreased glucose uptake. * Studies that show SNAP23 participate in Plin2-coated lipid droplet fusion are described in [32,72].