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Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

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Fusion of single GLUT4 vesicles in adipose cells. (A) Image sequence (top) showing fusion of the vesicle from Fig. 3 A (green arrow) detected by the spreading of GLUT4 (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). Gaussian fit (bottom) of the vesicle fluorescence radial intensity profile shows a decrease of peak intensity and simultaneous radial widening of the profile (σ represents full-width half-maximum). (B) Time course of peak width (black) and peak intensity (red) acquired from the Gaussian fit, and total fluorescence integrated over a circular region (4-μm-diam) surrounding the vesicle (green); the total fluorescence (green) remains constant as the vesicle diameter (40 nm) is significantly smaller than the characteristic penetration length of the evanescent wave (Loerke et al., 2002). All intensities are normalized to their initial values. (C) Histogram of the time of vesicle fluorescence decay (τ) for insulin-stimulated (red) and basal (blue) cells. The values of τ were obtained by exponential fit of fluorescence time course. In basal cells, the average time of fluorescence decay (77 ± 13 s) was statistically indistinguishable from the rate of bleaching (80 ± 10 s). (D) Vesicle fluorescence before (blue, 23 ± 14 AU, SD, n = 423) and 15 min after (red, 48 ± 18 AU, SD, n = 398) insulin application. The increased brightness indicates closer proximity to the PM as the vesicle fluorescence decreases exponentially with distance from the PM (Fig. 1 B, inset).
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fig5: Fusion of single GLUT4 vesicles in adipose cells. (A) Image sequence (top) showing fusion of the vesicle from Fig. 3 A (green arrow) detected by the spreading of GLUT4 (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). Gaussian fit (bottom) of the vesicle fluorescence radial intensity profile shows a decrease of peak intensity and simultaneous radial widening of the profile (σ represents full-width half-maximum). (B) Time course of peak width (black) and peak intensity (red) acquired from the Gaussian fit, and total fluorescence integrated over a circular region (4-μm-diam) surrounding the vesicle (green); the total fluorescence (green) remains constant as the vesicle diameter (40 nm) is significantly smaller than the characteristic penetration length of the evanescent wave (Loerke et al., 2002). All intensities are normalized to their initial values. (C) Histogram of the time of vesicle fluorescence decay (τ) for insulin-stimulated (red) and basal (blue) cells. The values of τ were obtained by exponential fit of fluorescence time course. In basal cells, the average time of fluorescence decay (77 ± 13 s) was statistically indistinguishable from the rate of bleaching (80 ± 10 s). (D) Vesicle fluorescence before (blue, 23 ± 14 AU, SD, n = 423) and 15 min after (red, 48 ± 18 AU, SD, n = 398) insulin application. The increased brightness indicates closer proximity to the PM as the vesicle fluorescence decreases exponentially with distance from the PM (Fig. 1 B, inset).

Mentions: In insulin-stimulated cells, GLUT4 vesicles lost their fluorescence shortly after attachment; the vesicles approached the PM and stopped, and then the vesicle fluorescence dimmed. Fig. 5 A demonstrates the fluorescence spreading from the same GLUT4 vesicle indicated by the green arrow in Fig. 3 A to the PM; the corresponding radially symmetric Gaussian fits of the fluorescence intensity are placed below each frame (Video 5, available at http://www.jcb.org/cgi/content/full.200412069/DC1). The fits demonstrate that while the peak fluorescence decayed (Fig. 5 B, red curve), the profile of vesicle fluorescence widened (Fig. 5 B, black curve) but the total amount of fluorescence in the circular region (4 μm in diameter) surrounding the vesicle remained constant (Fig. 5 B, green curve), verifying that the vesicle underwent fusion rather than moved away from the PM (Loerke et al., 2002).


Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Fusion of single GLUT4 vesicles in adipose cells. (A) Image sequence (top) showing fusion of the vesicle from Fig. 3 A (green arrow) detected by the spreading of GLUT4 (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). Gaussian fit (bottom) of the vesicle fluorescence radial intensity profile shows a decrease of peak intensity and simultaneous radial widening of the profile (σ represents full-width half-maximum). (B) Time course of peak width (black) and peak intensity (red) acquired from the Gaussian fit, and total fluorescence integrated over a circular region (4-μm-diam) surrounding the vesicle (green); the total fluorescence (green) remains constant as the vesicle diameter (40 nm) is significantly smaller than the characteristic penetration length of the evanescent wave (Loerke et al., 2002). All intensities are normalized to their initial values. (C) Histogram of the time of vesicle fluorescence decay (τ) for insulin-stimulated (red) and basal (blue) cells. The values of τ were obtained by exponential fit of fluorescence time course. In basal cells, the average time of fluorescence decay (77 ± 13 s) was statistically indistinguishable from the rate of bleaching (80 ± 10 s). (D) Vesicle fluorescence before (blue, 23 ± 14 AU, SD, n = 423) and 15 min after (red, 48 ± 18 AU, SD, n = 398) insulin application. The increased brightness indicates closer proximity to the PM as the vesicle fluorescence decreases exponentially with distance from the PM (Fig. 1 B, inset).
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fig5: Fusion of single GLUT4 vesicles in adipose cells. (A) Image sequence (top) showing fusion of the vesicle from Fig. 3 A (green arrow) detected by the spreading of GLUT4 (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). Gaussian fit (bottom) of the vesicle fluorescence radial intensity profile shows a decrease of peak intensity and simultaneous radial widening of the profile (σ represents full-width half-maximum). (B) Time course of peak width (black) and peak intensity (red) acquired from the Gaussian fit, and total fluorescence integrated over a circular region (4-μm-diam) surrounding the vesicle (green); the total fluorescence (green) remains constant as the vesicle diameter (40 nm) is significantly smaller than the characteristic penetration length of the evanescent wave (Loerke et al., 2002). All intensities are normalized to their initial values. (C) Histogram of the time of vesicle fluorescence decay (τ) for insulin-stimulated (red) and basal (blue) cells. The values of τ were obtained by exponential fit of fluorescence time course. In basal cells, the average time of fluorescence decay (77 ± 13 s) was statistically indistinguishable from the rate of bleaching (80 ± 10 s). (D) Vesicle fluorescence before (blue, 23 ± 14 AU, SD, n = 423) and 15 min after (red, 48 ± 18 AU, SD, n = 398) insulin application. The increased brightness indicates closer proximity to the PM as the vesicle fluorescence decreases exponentially with distance from the PM (Fig. 1 B, inset).
Mentions: In insulin-stimulated cells, GLUT4 vesicles lost their fluorescence shortly after attachment; the vesicles approached the PM and stopped, and then the vesicle fluorescence dimmed. Fig. 5 A demonstrates the fluorescence spreading from the same GLUT4 vesicle indicated by the green arrow in Fig. 3 A to the PM; the corresponding radially symmetric Gaussian fits of the fluorescence intensity are placed below each frame (Video 5, available at http://www.jcb.org/cgi/content/full.200412069/DC1). The fits demonstrate that while the peak fluorescence decayed (Fig. 5 B, red curve), the profile of vesicle fluorescence widened (Fig. 5 B, black curve) but the total amount of fluorescence in the circular region (4 μm in diameter) surrounding the vesicle remained constant (Fig. 5 B, green curve), verifying that the vesicle underwent fusion rather than moved away from the PM (Loerke et al., 2002).

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Show MeSH
Related in: MedlinePlus