39 research outputs found
Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells
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
A common trafficking route for GLUT4 in cardiomyocytes in response to insulin, contraction and energy-status signalling
A new mouse model has been developed to study the localisation and
trafficking of the glucose transporter GLUT4 in muscle. The mouse line has
specific expression of a GFP and HA-epitope-tagged version of GLUT4 under the
control of a muscle-specific promoter. The exofacial HA-tag has enabled
fluorescent labelling of only the GLUT4 exposed at the external surface. A
distinction between sarcolemma labelling and transverse-tubule labelling has
also been possible because the former compartment is much more accessible to
intact anti-HA antibody. By contrast, the Fab fragment of the anti-HA antibody
could readily detect GLUT4 at the surface of both the sarcolemma and
transverse tubules. Here, we have used this mouse model to examine the route
taken by cardiomyocyte GLUT4 as it moves to the limiting external membrane
surface of sarcolemma and transverse-tubules in response to insulin,
contraction or activators of energy-status signalling, including hypoxia.
HA-GLUT4-GFP is largely excluded from the sarcolemma and transverse-tubule
membrane of cardiomyocytes under basal conditions, but is similarly trafficked
to these membrane surfaces after stimulation with insulin, contraction or
hypoxia. Internalisation of sarcolemma GLUT4 has been investigated by
pulse-labelling surface GLUT4 with intact anti-HA antibody. At early stages of
internalisation, HA-tagged GLUT4 colocalises with clathrin at puncta at the
sarcolemma, indicating that in cells returning to a basal state, GLUT4 is
removed from external membranes by a clathrin-mediated route. We also observed
colocalisation of GLUT4 with clathrin under basal conditions. At later stages
of internalisation and at steady state, anti-HA antibody labeled-GLUT4
originating from the sarcolemma was predominantly detected in a peri-nuclear
compartment, indistinguishable among the specific initial stimuli. These
results taken together imply a common pathway for internalisation of GLUT4,
independent of the initial stimulus
Communication: Clusters of absorbing disks on a reflecting wall: Competition for diffusing particles
DEPOSITION OF DOUBLE-LAYER COATINGS FOR PREPARING COMPOSITE MEMBRANES WITH SUPERHYDROPHOBIC PROPERTIES
FORMATION OF COMPOSITE MEMBRANES WITH TWO HYDROPHOBIC LAYERS BY THE PLASMA POLYMERIZATION METHOD
Impaired Tethering and Fusion of GLUT4 Vesicles in Insulin-Resistant Human Adipose Cells
Systemic glucose homeostasis is profoundly influenced by adipose cell function. Here we investigated GLUT4 dynamics in living adipose cells from human subjects with varying BMI and insulin sensitivity index (S-i) values. Cells were transfected with hemagglutinin (HA)-GLUT4-green fluorescent protein (GFP)/mCherry (red fluorescence), and were imaged live using total internal reflection fluorescence and confocal microscopy. HA-GLUT4-GFP redistribution to the plasma membrane (PM) was quantified by surface-exposed HA epitope. In the basal state, GLUT4 storage vesicle (GSV) trafficking to and fusion with the PM were invariant with donor subject S-i, as was total cell-surface GLUT4. In cells from insulin-sensitive subjects, insulin augmented GSV tethering and fusion approximately threefold, resulting in a corresponding increase in total PM GLUT4. However, with decreasing S-i, these effects diminished progressively. All insulin-induced effects on GLUT4 redistribution and trafficking correlated strongly with S-i and only weakly with BMI. Thus, while basal GLUT4 dynamics and total cell-surface GLUT4 are intact in human adipose cells, independent of donor S-i, cells from insulin-resistant donors show markedly impaired GSV tethering and fusion responses to insulin, even after overnight culture. This altered insulin responsiveness is consistent with the hypothesis that adipose cellular dysfunction is a primary contributor to systemic metabolic dysfunction