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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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Tethering of mobile GLUT4 vesicles to the PM of primary adipose cells. (A) Sequential frames show two GLUT4 vesicles (arrows) approaching and quickly tethering to the PM. (B) Examples of trajectories (projected onto the x-y “coverslip” plane) of GLUT4 vesicles tethering to the PM in the basal (blue) and insulin-stimulated (red) states; each point corresponds to the center of the vesicle ROI. (C) Histograms of SD of vesicle position from the point of tethering in the x-y plane (amplitude of “wiggling” = \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\sqrt{ \left \left(SD_{x}+SD_{y}\right) \right }\end{equation*}\end{document}); ∼100 consecutive frames were used for the SD calculation for each vesicles; blue, vesicles in the basal state; red, vesicles in the insulin-stimulated state (10 min after insulin treatment). (D) Histograms of the distances a GLUT4 vesicle travels until the first stop in the basal (blue) and insulin-stimulated (red) states.
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fig3: Tethering of mobile GLUT4 vesicles to the PM of primary adipose cells. (A) Sequential frames show two GLUT4 vesicles (arrows) approaching and quickly tethering to the PM. (B) Examples of trajectories (projected onto the x-y “coverslip” plane) of GLUT4 vesicles tethering to the PM in the basal (blue) and insulin-stimulated (red) states; each point corresponds to the center of the vesicle ROI. (C) Histograms of SD of vesicle position from the point of tethering in the x-y plane (amplitude of “wiggling” = \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\sqrt{ \left \left(SD_{x}+SD_{y}\right) \right }\end{equation*}\end{document}); ∼100 consecutive frames were used for the SD calculation for each vesicles; blue, vesicles in the basal state; red, vesicles in the insulin-stimulated state (10 min after insulin treatment). (D) Histograms of the distances a GLUT4 vesicle travels until the first stop in the basal (blue) and insulin-stimulated (red) states.

Mentions: In stimulated cells we observed a drastic reduction in the traffic (Video 4, available at http://www.jcb.org/cgi/content/full.200412069/DC1). Only 1.4 ± 1.4 traces/100 m2/min (18 cells, SD) were detected 10 min after the insulin treatment, approximately eightfold less compared with the basal state (Fig. 2, C and D). Our analysis indicates that the reduction of the traffic is due to immobilization of GLUT4 vesicles at the PM. First, upon insulin stimulation, GLUT4 vesicles attach to the PM much tighter. Fig. 3 A shows two GLUT4 vesicles that attached to the PM shortly after appearance in the TIRF zone. After attachment, the vesicles do not “wiggle” near the point of attachment (Li et al., 2004) as much in the presence of insulin as they do in the basal state; the amplitude of wiggling has two peaks (42 ± 11 and 88 ± 26 nm, n = 15, SD) in the basal state, whereas in the insulin-stimulated state, only the first peak (49 ± 20 nm, n = 15, SD, statistically indistinguishable from the first “basal” peak) is present (Fig. 3, B and C). Thus, insulin stimulation almost completely eliminates the long-range “wiggling” of the attached vesicles. Moreover, these vesicles rarely move again and so the mobile vesicles all remain in the TIRF zone. Second, insulin diminished the distance traveled by the vesicles in the TIRF zone (from 15 ± 6 to 5 ± 2 μm, n = 70, SD; Fig. 3 D). Thus, insulin stimulates tighter and quicker attachment of mobile GLUT4 vesicles to the PM of adipose cells (see Fig. S4, available at http://www.jcb.org/cgi/content/full.200412069/DC1, and the section Kinetic analysis of GLUT4 recycling in primary adipose cells in the online supplemental material).


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)

Tethering of mobile GLUT4 vesicles to the PM of primary adipose cells. (A) Sequential frames show two GLUT4 vesicles (arrows) approaching and quickly tethering to the PM. (B) Examples of trajectories (projected onto the x-y “coverslip” plane) of GLUT4 vesicles tethering to the PM in the basal (blue) and insulin-stimulated (red) states; each point corresponds to the center of the vesicle ROI. (C) Histograms of SD of vesicle position from the point of tethering in the x-y plane (amplitude of “wiggling” = \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\sqrt{ \left \left(SD_{x}+SD_{y}\right) \right }\end{equation*}\end{document}); ∼100 consecutive frames were used for the SD calculation for each vesicles; blue, vesicles in the basal state; red, vesicles in the insulin-stimulated state (10 min after insulin treatment). (D) Histograms of the distances a GLUT4 vesicle travels until the first stop in the basal (blue) and insulin-stimulated (red) states.
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fig3: Tethering of mobile GLUT4 vesicles to the PM of primary adipose cells. (A) Sequential frames show two GLUT4 vesicles (arrows) approaching and quickly tethering to the PM. (B) Examples of trajectories (projected onto the x-y “coverslip” plane) of GLUT4 vesicles tethering to the PM in the basal (blue) and insulin-stimulated (red) states; each point corresponds to the center of the vesicle ROI. (C) Histograms of SD of vesicle position from the point of tethering in the x-y plane (amplitude of “wiggling” = \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}\sqrt{ \left \left(SD_{x}+SD_{y}\right) \right }\end{equation*}\end{document}); ∼100 consecutive frames were used for the SD calculation for each vesicles; blue, vesicles in the basal state; red, vesicles in the insulin-stimulated state (10 min after insulin treatment). (D) Histograms of the distances a GLUT4 vesicle travels until the first stop in the basal (blue) and insulin-stimulated (red) states.
Mentions: In stimulated cells we observed a drastic reduction in the traffic (Video 4, available at http://www.jcb.org/cgi/content/full.200412069/DC1). Only 1.4 ± 1.4 traces/100 m2/min (18 cells, SD) were detected 10 min after the insulin treatment, approximately eightfold less compared with the basal state (Fig. 2, C and D). Our analysis indicates that the reduction of the traffic is due to immobilization of GLUT4 vesicles at the PM. First, upon insulin stimulation, GLUT4 vesicles attach to the PM much tighter. Fig. 3 A shows two GLUT4 vesicles that attached to the PM shortly after appearance in the TIRF zone. After attachment, the vesicles do not “wiggle” near the point of attachment (Li et al., 2004) as much in the presence of insulin as they do in the basal state; the amplitude of wiggling has two peaks (42 ± 11 and 88 ± 26 nm, n = 15, SD) in the basal state, whereas in the insulin-stimulated state, only the first peak (49 ± 20 nm, n = 15, SD, statistically indistinguishable from the first “basal” peak) is present (Fig. 3, B and C). Thus, insulin stimulation almost completely eliminates the long-range “wiggling” of the attached vesicles. Moreover, these vesicles rarely move again and so the mobile vesicles all remain in the TIRF zone. Second, insulin diminished the distance traveled by the vesicles in the TIRF zone (from 15 ± 6 to 5 ± 2 μm, n = 70, SD; Fig. 3 D). Thus, insulin stimulates tighter and quicker attachment of mobile GLUT4 vesicles to the PM of adipose cells (see Fig. S4, available at http://www.jcb.org/cgi/content/full.200412069/DC1, and the section Kinetic analysis of GLUT4 recycling in primary adipose cells in the online supplemental material).

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