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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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Clustering of GLUT4 vesicles near PM after application of insulin in adipose cells. (A) TIRF image of a cell 5 min after insulin stimulation. (B) Pair-distance correlation functions of vesicle distributions for cells in the basal and insulin-stimulated states. With insulin (red curve) this function exhibits a peak in the range of 1–5 μm, and shows that a significant fraction of vesicles are clustered. The correlation function for cells in the basal state (black) revealed no statistically significant deviation from the random distribution. Green curves represent the 99% confidence interval for spatially random distribution acquired by computer simulation. (C) Sequential images show arrival of new vesicles to a cluster (top) and subsequent redistribution of GLUT4 to adjacent regions outside the cluster (bottom). (D) Time course of fluorescence integrated over (red) and outside (black) the cluster.
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fig4: Clustering of GLUT4 vesicles near PM after application of insulin in adipose cells. (A) TIRF image of a cell 5 min after insulin stimulation. (B) Pair-distance correlation functions of vesicle distributions for cells in the basal and insulin-stimulated states. With insulin (red curve) this function exhibits a peak in the range of 1–5 μm, and shows that a significant fraction of vesicles are clustered. The correlation function for cells in the basal state (black) revealed no statistically significant deviation from the random distribution. Green curves represent the 99% confidence interval for spatially random distribution acquired by computer simulation. (C) Sequential images show arrival of new vesicles to a cluster (top) and subsequent redistribution of GLUT4 to adjacent regions outside the cluster (bottom). (D) Time course of fluorescence integrated over (red) and outside (black) the cluster.

Mentions: Although the initial distribution of GLUT4 vesicles around the cell surface is relatively uniform (Fig. 1, C and D), we observed that insulin stimulates formation of fluorescent domains alternating with dark regions (Fig. 4, A and C). To characterize the degree of nonuniformity of the spatial distribution of GLUT4 vesicles near the PM, we applied a pair-distance correlation analysis. Insulin-stimulated cells showed a distinct peak on the pair-distance correlation curve (Fig. 4 B), demonstrating that a significant percentage of vesicles are clustered. The width of the main peak gives an estimate of average cluster size (∼5 μm). Pair-distance correlations obtained with cells in the basal state exhibited no significant deviation from a randomly scattered point distribution (all points lie inside the 99% confidence interval), as tested by simulation. These observations are consistent with previous studies that insulin targets GLUT4 vesicles to specific places of the PM of 3T3-L1 adipocytes (Patki et al., 2001; Semiz et al., 2003). Moreover, insulin signaling in adipose cells has been proposed to be associated with specific domains of the PM (Saltiel and Pessin, 2003). If insulin promotes tethering of GLUT4 vesicles to the PM, then to complete the insulin response docked vesicles must fuse to release GLUT4 to the PM. We further confirmed that insulin stimulates tethering and fusion of GLUT4 vesicles to the PM within the characteristic time of the appearance of GLUT4 in the PM reported previously (Dawson et al., 2001; Tengholm and Meyer, 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)

Clustering of GLUT4 vesicles near PM after application of insulin in adipose cells. (A) TIRF image of a cell 5 min after insulin stimulation. (B) Pair-distance correlation functions of vesicle distributions for cells in the basal and insulin-stimulated states. With insulin (red curve) this function exhibits a peak in the range of 1–5 μm, and shows that a significant fraction of vesicles are clustered. The correlation function for cells in the basal state (black) revealed no statistically significant deviation from the random distribution. Green curves represent the 99% confidence interval for spatially random distribution acquired by computer simulation. (C) Sequential images show arrival of new vesicles to a cluster (top) and subsequent redistribution of GLUT4 to adjacent regions outside the cluster (bottom). (D) Time course of fluorescence integrated over (red) and outside (black) the cluster.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171949&req=5

fig4: Clustering of GLUT4 vesicles near PM after application of insulin in adipose cells. (A) TIRF image of a cell 5 min after insulin stimulation. (B) Pair-distance correlation functions of vesicle distributions for cells in the basal and insulin-stimulated states. With insulin (red curve) this function exhibits a peak in the range of 1–5 μm, and shows that a significant fraction of vesicles are clustered. The correlation function for cells in the basal state (black) revealed no statistically significant deviation from the random distribution. Green curves represent the 99% confidence interval for spatially random distribution acquired by computer simulation. (C) Sequential images show arrival of new vesicles to a cluster (top) and subsequent redistribution of GLUT4 to adjacent regions outside the cluster (bottom). (D) Time course of fluorescence integrated over (red) and outside (black) the cluster.
Mentions: Although the initial distribution of GLUT4 vesicles around the cell surface is relatively uniform (Fig. 1, C and D), we observed that insulin stimulates formation of fluorescent domains alternating with dark regions (Fig. 4, A and C). To characterize the degree of nonuniformity of the spatial distribution of GLUT4 vesicles near the PM, we applied a pair-distance correlation analysis. Insulin-stimulated cells showed a distinct peak on the pair-distance correlation curve (Fig. 4 B), demonstrating that a significant percentage of vesicles are clustered. The width of the main peak gives an estimate of average cluster size (∼5 μm). Pair-distance correlations obtained with cells in the basal state exhibited no significant deviation from a randomly scattered point distribution (all points lie inside the 99% confidence interval), as tested by simulation. These observations are consistent with previous studies that insulin targets GLUT4 vesicles to specific places of the PM of 3T3-L1 adipocytes (Patki et al., 2001; Semiz et al., 2003). Moreover, insulin signaling in adipose cells has been proposed to be associated with specific domains of the PM (Saltiel and Pessin, 2003). If insulin promotes tethering of GLUT4 vesicles to the PM, then to complete the insulin response docked vesicles must fuse to release GLUT4 to the PM. We further confirmed that insulin stimulates tethering and fusion of GLUT4 vesicles to the PM within the characteristic time of the appearance of GLUT4 in the PM reported previously (Dawson et al., 2001; Tengholm and Meyer, 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