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Updated: Oct 5, 2026
Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry
Published on: November 7, 2010
GLUT-4 translocation in skeletal muscle studied with a cell-free assay: involvement of phospholipase D
S Kristiansen1, J N Nielsen, S Bourgoin
1Copenhagen Muscle Research Center, Department of Human Physiology, University of Copenhagen, 13 Universitetsparken, DK-2100 Copenhagen, Denmark.
Abstract:
GLUT-4-containing membranes immunoprecipitated from insulin-stimulated rat skeletal muscle produce the phospholipase D (PLD) product phosphatidic acid. In vitro stimulation of PLD in crude membrane with ammonium sulfate (5 mM) resulted in transfer of GLUT-4 (3.0-fold vs. control) as well as transferrin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomycin also reduced insulin-stimulated glucose transport in intact incubated soleus muscles. Furthermore, protein kinase B(beta) (PKB(beta)) was found to associate with the GLUT-4 protein and was transferred to small vesicles in response to ammonium sulfate in vitro. Finally, addition of cytosolic proteins, prepared from basal skeletal muscle, and GTP nucleotides to an enriched GLUT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small membranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-induced GLUT-4 transfer could be blocked by neomycin and N-ethylmaleimide. In conclusion, we have developed a cell-free assay that demonstrates in vitro GLUT-4 transfer. This transfer may suggest release of GLUT-4-containing vesicles from donor GLUT-4 membranes involving PLD activity and binding of PKB(beta) to GLUT-4.
Insights
This study shows that phospholipase D (PLD) activity facilitates the transfer of glucose transporter 4 (GLUT-4) to smaller vesicles in skeletal muscle. This process, involving protein kinase B(beta) (PKB(beta)), is crucial for insulin-stimulated glucose uptake.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Insulin stimulates glucose uptake in skeletal muscle primarily via the translocation of glucose transporter 4 (GLUT-4).
- The precise mechanisms regulating GLUT-4 vesicle trafficking remain incompletely understood.
Purpose of the Study:
- To investigate the role of phospholipase D (PLD) in GLUT-4 membrane trafficking.
- To develop a cell-free system to study GLUT-4 transfer.
Main Methods:
- Immunoprecipitation of GLUT-4-containing membranes from insulin-stimulated rat skeletal muscle.
- In vitro stimulation of PLD activity using ammonium sulfate.
- Assessment of protein transfer between membrane fractions using inhibitors like neomycin.
- Analysis of protein kinase B(beta) (PKB(beta)) association with GLUT-4.
Main Results:
- PLD activation in vitro induced the transfer of GLUT-4 and transferrin receptor proteins to smaller membrane structures.
- Neomycin, a PLD inhibitor, blocked in vitro GLUT-4 transfer and reduced insulin-stimulated glucose transport in intact muscles.
- PKB(beta) associated with GLUT-4 and was transferred to small vesicles upon PLD stimulation.
- Cytosolic proteins and GTP nucleotides promoted in vitro GLUT-4 transfer, which was inhibited by neomycin and N-ethylmaleimide.
Conclusions:
- A cell-free assay was established to demonstrate in vitro GLUT-4 transfer.
- PLD activity and PKB(beta) binding are implicated in the release of GLUT-4-containing vesicles from donor membranes.
