scholarly journals GLUT4 Is Sorted to Vesicles Whose Accumulation Beneath and Insertion into the Plasma Membrane Are Differentially Regulated by Insulin and Selectively Affected by Insulin Resistance

2010 ◽  
Vol 21 (8) ◽  
pp. 1375-1386 ◽  
Author(s):  
Wenyong Xiong ◽  
Ingrid Jordens ◽  
Eva Gonzalez ◽  
Timothy E. McGraw
Keyword(s):  
Insulin Resistance ◽  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Glucose Transport ◽  
Total Internal Reflection ◽  
Glucose Transporter ◽  
Internal Reflection ◽  
General Mechanism ◽  
Recycling Endosomes ◽  
Degree Of Redistribution

Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.

scholarly journals ELKS, a Protein Structurally Related to the Active Zone-associated Protein CAST, Is Expressed in Pancreatic β Cells and Functions in Insulin Exocytosis: Interaction of ELKS with Exocytotic Machinery Analyzed by Total Internal Reflection Fluorescence Microscopy

2005 ◽  
Vol 16 (7) ◽  
pp. 3289-3300 ◽  
Author(s):  
Mica Ohara-Imaizumi ◽  
Toshihisa Ohtsuka ◽  
Satsuki Matsushima ◽  
Yoshihiro Akimoto ◽  
Chiyono Nishiwaki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Active Zone ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic β cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic β cells.

scholarly journals Imaging Constitutive Exocytosis with Total Internal Reflection Fluorescence Microscopy

2000 ◽  
Vol 149 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Jan Schmoranzer ◽  
Mark Goulian ◽  
Dan Axelrod ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Fluorescence Microscopy ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Flattening Process

Total internal reflection fluorescence microscopy has been applied to image the final stage of constitutive exocytosis, which is the fusion of single post-Golgi carriers with the plasma membrane. The use of a membrane protein tagged with green fluorescent protein allowed the kinetics of fusion to be followed with a time resolution of 30 frames/s. Quantitative analysis allowed carriers undergoing fusion to be easily distinguished from carriers moving perpendicularly to the plasma membrane. The flattening of the carriers into the plasma membrane is seen as a simultaneous rise in the total, peak, and width of the fluorescence intensity. The duration of this flattening process depends on the size of the carriers, distinguishing small spherical from large tubular carriers. The spread of the membrane protein into the plasma membrane upon fusion is diffusive. Mapping many fusion sites of a single cell reveals that there are no preferred sites for constitutive exocytosis in this system.

scholarly journals Role of Microtubules in Fusion of Post-Golgi Vesicles to the Plasma Membrane

2003 ◽  
Vol 14 (4) ◽  
pp. 1558-1569 ◽  
Author(s):  
Jan Schmoranzer ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Cell Periphery ◽  
Golgi Vesicles ◽  
Cytoskeletal Elements

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.

scholarly journals Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

2002 ◽  
Vol 159 (4) ◽  
pp. 625-635 ◽  
Author(s):  
Jyoti K. Jaiswal ◽  
Norma W. Andrews ◽  
Sanford M. Simon
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Cell Surface ◽  
Total Internal Reflection ◽  
Cytosolic Calcium ◽  
Internal Reflection ◽  
Secretory Cells ◽  
Calcium Dependent ◽  
Late Endosomes ◽  
Early Endosomes

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.

High-fat diet reduces glucose transporter responses to both insulin and exercise

1994 ◽  
Vol 266 (1) ◽  
pp. R95-R101 ◽  
Author(s):  
M. N. Rosholt ◽  
P. A. King ◽  
E. S. Horton
Keyword(s):  
Insulin Resistance ◽  
Plasma Membrane ◽  
Glucose Transport ◽  
High Fat Diet ◽  
Glucose Transporter ◽  
Membrane Vesicles ◽  
Intrinsic Activity ◽  
Sprague Dawley ◽  
High Fat ◽  
Plasma Membrane Vesicles

High-fat diet (HFD) induces skeletal muscle insulin resistance. To investigate associated changes in the plasma membrane glucose transporter, male Sprague-Dawley rats were fed either chow [high-carbohydrate diet (HCD)] or HFD for 3 wk. Plasma membrane vesicles were prepared from hindlimb muscle of control, insulin-stimulated (Ins), and acutely exercised (Ex) rats. Maximal vesicle glucose transport activity (Vmax) increased threefold with Ins and Ex treatment compared with controls in HCD rats; in HFD rats, increases were less than twofold. Transporter numbers (measured by cytochalasin B binding, CB) approximately doubled with Ins and Ex in both diet groups. Intrinsic activity (carrier turnover, Vmax/CB) increased significantly with stimulation in HCD but not HFD rats. Therefore, vesicles from HFD rats showed resistance to both exercise and insulin stimulation of muscle glucose transport. Transporter number increased normally, but intrinsic activity in HFD rats did not respond. Two conclusions are discussed: 1) translocation and activation are distinct, separable steps in transporter stimulation and 2) HFD produces effects that resemble the insulin resistance of starvation.

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