Insulin Regulates Glut4 Confinement in Plasma Membrane Clusters in Adipose Cells

<div><p>Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin’s effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60–240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters ∼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.</p> </div

GLUT4 Interaction with Pre-existing Clusters.

<p>GLUT4-EOS association with (a) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s008" target="_blank">Movie S5</a>) and dissociation from (b) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s009" target="_blank">Movie S6</a>) pre-existing GLUT4 clusters. Activated GLUT4-EOS molecule, detected in the red channel, colocalizes with the cluster visible in green channel (non-activated GLUT4-EOS). The last frame is shown for an association event (a), and the first frame is shown for dissociation event (b). Trajectory of the activated GLUT4-EOS molecule is shown in white; white circles depict the site of the cluster. All bars, 2 µm. (c) and (d) show graphs of the Mean Square Displacement (MSD) for association and dissociation events depicted in (a) and (b) correspondingly.</p

Tracking GLUT4-EOS Molecules.

<p>Trajectories of GLUT4-EOS molecules exhibiting different types of motion: (<b>a</b>) directed motion corresponding to vesicular transport (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s005" target="_blank">Movie S2</a>)<b>;</b> (<b>b</b>) free lateral diffusion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s006" target="_blank">Movie S3</a>); (<b>c</b>) constrained diffusion within plasma membrane cluster (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s007" target="_blank">Movie S4</a>). All bars, 2 µm. (<b>d</b>) Graphs of the Mean Square Displacement (MSD) of GLUT4-EOS molecules for three distinct types of motion: directed movement (red), free lateral diffusion (blue), and constrained diffusion within a cluster (orange). Dashed lines correspond to 95% confidence intervals obtained from simulation. (<b>e</b>) Percentage of trajectories categorized into three types of motion observed, based on the analysis of MSD. Trajectories containing at least 30 time points were scored as directed motion or constrained diffusion if five or more points were above or below 95% confidence interval bounds. At least 150 trajectories were analyzed for each condition. Error bars are SEM, N = 15 cells. *p<0.05; **p<0.01.</p

GLUT4 Dwell Time at the Clusters: Rates of Dissociation and Endocytosis.

<p>(<b>a</b>) and (<b>b</b>) - histograms of dwell time for individual GLUT4-EOS molecules localized within the clusters in control cells (gray) and cells treated with KCN (red). Fat cells treated with 2 mM KCN for 15 min showed complete inhibition of GLUT4 endocytosis due to ATP depletion (Kono, Robinson et al. 1977; Quon, Guerre-Millo et al. 1994). Dwell time data was pooled from at least 15 cells for each condition. The dwell time of GLUT4-EOS molecule was measured as the time between appearance or activation of GLUT4-EOS at the cluster site and the time when the molecule was lost for more than three consecutive frames. Exponential fit of the histogram data was used to calculate the rates of disappearance under different illumination protocols for at least 5 cells in each condition (basal, insulin, with and without KCN). The combined rate of disappearance was considered to be a sum of three independent processes (bleaching, lateral dissociation from cluster and endocytosis): K(p) = p*K_b+K_d+K_e, where K_b – rate constant of bleaching, p – relative exposure to excitation light, K_d and K_e– rate constants for dissociation and endocytosis from clusters respectively. To control for bleaching, we measured the rates of disappearance at four different illumination protocols with constant exposure t_exp = 200 ms but with different intervals between acquisitions (t_int = 0.2, 0.5, 1.0, and 2 sec), which correspond to the relative exposure to excitation light p = t_exp/t_int : 1.0; 0.4; 0.2; and 0.1. (<b>c</b>) and (<b>d</b>) show graphs of combined rate of disappearance K(p) as a function of the relative exposure for basal and insulin stimulated conditions. Red circles correspond to cells pre-treated with KCN for 15 min. The combined rate of disappearance for KCN-treated cells was assumed to be K(p) = p*K_b+K_d, with the rate constant of endocytosis (K_e) being essentially zero. The rate constants of dissociation K_d and endocytosis K_e from clusters were determined from intersection of linear fit of the data with ordinate axis.</p

Formation of the Clusters via GLUT4-Specific Retention During Exocytosis.

<p>Sequences of consecutive time-lapse frames showing examples of GLUT4 vesicle fusion resulting in complete dispersal of GLUT4 molecules “fusion-with-dispersal” (a) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s010" target="_blank">Movie S7</a>) and formation of GLUT4 cluster “fusion-with-retention” (b) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s011" target="_blank">Movie S8</a>). Lower panels show corresponding graphs for time-lapse fluorescence changes at the site of fusion. Mean fluorescence intensity was calculated for circular regions of 1 µm radius. Red channel corresponds to activated GLUT4-EOS molecules; green channel corresponds to IRAP-pHluorin. IRAP-pHluorin fluorescence spikes correspond to luminal pH equilibration upon fusion pore opening (black arrows). Note that IRAP-pHluorin leaves the site of fusion when GLUT4 forms a cluster. Frames are shown immediately before and after fusion with 200 ms interval. All bars, 1 µm. (c) and (d) show cartoon depictions of GLUT4 molecules leaving site of fusion during “fusion-with-dispersal” and forming a cluster during “fusion-with-retention”.</p

Photo-Activation of GLUT4-EOS in the Plasma Membrane Clusters.

<p>(<b>a</b>) Simultaneous TIRF/FPALM imaging of GLUT4-EOS. Non-activated GLUT4-EOS is detected in the green channel and shows abundant plasma membrane clusters. Individual activated GLUT4-EOS molecules are detected in the red channel. Bar, 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057559#pone.0057559.s004" target="_blank">Movie S1</a> (<b>b</b>) Zoomed region from (A) depicting the sequential activation of a GLUT4-EOS. Bar, 2 µm. (<b>c</b>) A plot of mean fluorescence of activated GLUT4-EOS from region shown in (<b>b</b>) (<b>d</b>) Series of frames depicting single GLUT4-EOS molecule photo-bleaching event (upper panel); lower panel shows corresponding step-wise drop in fluorescence intensity measured within circular region. Bar, 1 µm.</p

Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic <i>Drosophila melanogaster</i>

<div><p>The fruit fly <i>Drosophila melanogaster</i> is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of <i>Drosophila</i> Insulin-Like Peptides resulted in ‘diabetic’ flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether <i>Drosophila</i> fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic <i>Drosophila</i> expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that <i>Drosophila</i> fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in <i>Drosophila</i>, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.</p></div

Trafficking, tethering and fusion of GLUT4 to the plasma membrane in <i>Drosophila</i> fat.

<p>(A) Time-lapse frames from TIRFM recording showing trafficking of GLUT4 in fat tissue isolated from sugar-restricted larvae. The white line indicates linear GLUT4 movement from one point to another, possibly along a microtubule network. Movement of two individual vesicle-like particles following the same trajectory is indicated by the white lines. The movement of the first particle is shown on the frames 0.0–5.0 s; the movement of the second particle is presented on the frames 6.0–9.0 s. Arrows indicate positions of the particles. (B) Time-lapse frames from TIRFM recording showing tethering and fusion of HA-GLUT4-GFP. White circles indicate the final position where tethering (1–7) and fusion takes place (8–10). Fluorescence intensity is shown in pseudocolor. Scale bars, 1 µm.</p

Expression and localization of HA-GLUT4-GFP in <i>Drosophila</i> fat.

<p>Confocal imaging of fat bodies from animals expressing: (A–D) <i>HA-GLUT4-GFP</i>; (E–H) negative control, <i>UAS-lacZ</i>. (A, E) GFP fluorescence (green); (B,F) Immunostaining with anti-GLUT4 (red); (C,G) Merge of images shows overlap of GFP and GLUT4 in transgenic fat (A–C) and little background in controls (E–G). (D, H) DIC images show abundant lipid droplets within each cell. (Panel D inset), enlarged image of fat body cells with multiple whitish lipid droplets (white arrows). Scale bars, 100 µm. (I–K) High resolution imaging of HA-GLUT4-GFP by TIRFM. Live fat body tissue was isolated and distribution of HA-GLUT4-GFP monitored using 488 nm laser illumination in TIRF mode. Scale bar, 5 µm. Note network of GLUT4 surrounding lipid droplets (I, white arrows) and presence of GLUT4 in punctate structures. Fig. 1J, representative projection image obtained by averaging 60 time frames from TIRF recordings, showing GLUT4 localized at the membrane. Scale bar, 10 µm. (K) Magnification of boxed region from (J) shows at a higher resolution, vesicular distribution of GLUT4, as well as GLUT4 associated with the plasma membrane. Scale bar, 5 µm.</p

Insulin stimulates GLUT4 trafficking in <i>Drosophila</i> fat cells.

<p>(A–F) Projection images representing in TIRFM the number of trafficking HA-GLUT4-GFP particles in fat cells from transgenic larvae under basal conditions (A, C, E) or after insulin addition (B, D, F). (A, B) Animals were grown on standard food; (C, D) starved for 24 h; or (E, F) grown on sugar-restricted food. To enhance clarity images were inverted so that HA-GLUT4-GFP particles appear dark on a light background. Scale bar, 2 µm. Note increased number of trafficking particles for sugar-restricted larvae. (G) Quantification of trafficking (number of trafficking particles/100 µm²/min) under basal conditions (light gray bars) or 5 min after insulin stimulation (dark gray bars). Fat body tissue was analyzed from at least three independent animals for each condition. Statistical significance: *** p<0.001. On standard food, the difference between basal and insulin trafficking was not statistically significant (p>0.05). The total number of trajectories counted for 4 regions (10×10 µm) for each cell used to generate data for starved (basal-48; insulin 16); sugar-restricted (basal-48, insulin 16); standard (basal-48; insulin 16).</p