Plin2 Inhibits Cellular Glucose Uptake through Interactions with SNAP23, a SNARE Complex Protein

Although a link between excess lipid storage and aberrant glucose metabolism has been recognized for many years, little is known what role lipid storage droplets and associated proteins such as Plin2 play in managing cellular glucose levels. To address this issue, the influence of Plin2 on glucose uptake was examined using 2-NBD-Glucose and [(3)H]-2-deoxyglucose to show that insulin-mediated glucose uptake was decreased 1.7- and 1.8-fold, respectively in L cell fibroblasts overexpressing Plin2. Conversely, suppression of Plin2 levels by RNAi-mediated knockdown increased 2-NBD-Glucose uptake several fold in transfected L cells and differentiated 3T3-L1 cells. The effect of Plin2 expression on proteins involved in glucose uptake and transport was also examined. Expression of the SNARE protein SNAP23 was increased 1.6-fold while levels of syntaxin-5 were decreased 1.7-fold in Plin2 overexpression cells with no significant changes observed in lipid droplet associated proteins Plin1 or FSP27 or with the insulin receptor, GLUT1, or VAMP4. FRET experiments revealed a close proximity of Plin2 to SNAP23 on lipid droplets to within an intramolecular distance of 51 Å. The extent of targeting of SNAP23 to lipid droplets was determined by co-localization and co-immunoprecipitation experiments to show increased partitioning of SNAP23 to lipid droplets when Plin2 was overexpressed. Taken together, these results suggest that Plin2 inhibits glucose uptake by interacting with, and regulating cellular targeting of SNAP23 to lipid droplets. In summary, the current study for the first time provides direct evidence for the role of Plin2 in mediating cellular glucose uptake

Dose response and effect of cytochalsin B and insulin on 2-NBD-Glucose uptake into L cells.

<p>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.</p

Schematic diagram for SNARE-mediated regulation of glucose transport in Plin2 overexpression cells.

<p>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].</p

Relative expression levels of key proteins involved in lipid droplet formation, glucose uptake and transport.

<p>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).</p

Effect of altered Plin2 expression on 2-NBD-Glucose uptake in L cells.

<p>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.</p

Co-immunoprecipitation of SNAP23 with GLUT1, Plin2, or Plin1.

<p>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.</p

Co-localization and FRET imaging between Cy3-labeled SNAP23 and Cy5-labeled GLUT1.

<p>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).</p

Effect of Plin2 overexpression in transfected Lcells.

<p>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.</p

Structural and Functional Assessment of Perilipin 2 Lipid Binding Domain(s)

Although perilipin 2 (Plin2) has been shown to bind lipids with high affinity, the Plin2 lipid binding site has yet to be defined. This is of interest since Plin2’s affinity for lipids has been suggested to be important for lipid droplet biogenesis and intracellular triacylglycerol accumulation. To define these regions, mouse Plin2 and several deletion mutants expressed as recombinant proteins and in mammalian cells were assessed by molecular modeling, fluorescence binding, circular dichroic, and fluorescence resonance energy transfer techniques to identify the structural and functional requirements for lipid binding. Major findings of this study indicate (1) the N-terminal PAT domain does not bind cholesterol or stearic acid; (2) Plin2 residues 119–251, containing helix α4, the α–β domain, and part of helix α6 form a Plin3-like cleft found to be important for highest affinity lipid binding; (3) both stearic acid and cholesterol interact favorably with the Plin2 cleft formed by conserved residues in helix α6 and adjacent strands, which is common to all the active lipid-binding constructs; and (4) discrete targeting of the Plin2 mutants to lipid droplets supports Plin2 containing two independent, nonoverlapping lipid droplet targeting domains in its central and C-terminal sequences. Thus, the current work reveals specific domains responsible for Plin2–lipid interactions that involves the protein’s lipid binding and targeting functions