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

Insulin-stimulated GLUT4 translocation in <i>Drosophila</i> fat is enhanced by sugar restriction.

<p>(A) Surface exposure of HA-GLUT4 upon insulin stimulation. Fat bodies were collected from larvae reared on different dietary regimes, as indicated. After immunostaining of non-permeabilized fat body cells, values were determined by measuring fluorescence and averaging calculations of corrected integrated density. Values are expressed as percent of HA-GLUT4 fluorescence of insulin stimulation over basal conditions. Fat bodies were collected from three animals for each dietary regime. (B–G) Confocal microscopy of HA-GLUT4-GFP-expression in fat body cells from animals reared on a sugar-restricted diet in the absence or presence of insulin. (B, C, D) Basal conditions; (E, F, G) after addition of 0.1 U/ml insulin. GLUT4 was visualized in non-permeabilized cells by GFP fluorescence (green, B, E); or with anti-HA antibody (red C, F) to monitor membrane translocation; merged images (D,G). Scale bar is 5 µm. Note increase in GLUT4 at the cell surface (red, F).</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

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

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

Two-colour live-cell nanoscale imaging of intracellular targets

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution