Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK
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Updated: Aug 13, 2026
Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice
Published on: May 16, 2021
Nadeeja Wijesekara1, Amanda Tung, Farah Thong
1Programme in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8.
Abstract:
Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.
Insights
Muscle contraction increases glucose transporter type 4 (GLUT4) at the cell surface. Membrane depolarization, independent of PI3K/Akt/AMPK, elevates GLUT4 via reduced endocytosis, potentially involving PKC.
Area of Science:
- Muscle physiology
- Cellular metabolism
- Molecular signaling
Background:
- Skeletal muscle contraction enhances glucose uptake via increased cell surface GLUT4 (glucose transporter type 4).
- The precise mechanisms and signaling pathways regulating GLUT4 translocation during muscle contraction remain largely unknown.
- Muscle contraction involves membrane depolarization, calcium (Ca2+) release, and actomyosin activation.
Purpose of the Study:
- To investigate the role of membrane depolarization in regulating GLUT4 trafficking in skeletal muscle cells.
- To elucidate the signaling pathways involved in depolarization-induced GLUT4 surface expression, distinct from insulin signaling.
Main Methods:
- Utilized L6 myotubes expressing GLUT4myc, a model system lacking sarcomeres and contractility.
- Induced membrane depolarization using potassium (K+) and examined effects on surface GLUT4myc levels.
- Investigated the involvement of Ca2+, PI3K/Akt, AMPK, CaMKII, and PKC signaling pathways using specific inhibitors and genetic manipulation (siRNA).
- Assessed GLUT4myc exocytosis and endocytosis rates.
Main Results:
- K+-induced membrane depolarization increased surface GLUT4myc in a manner additive to insulin.
- This effect was independent of PI3K, Akt, and actin polymerization but dependent on intracellular Ca2+.
- Inhibition of Ca2+ release (dantrolene) or extracellular Ca2+ chelation abolished the depolarization-induced GLUT4myc increase.
- PKC inhibition prevented the surface GLUT4myc gain, while CaMKII and AMPK inhibition did not.
- Depolarization primarily reduced GLUT4myc endocytosis rather than increasing exocytosis.
Conclusions:
- Membrane depolarization triggers GLUT4myc surface accumulation through mechanisms distinct from insulin.
- The primary mechanism involves a reduction in GLUT4 endocytosis, mediated by Ca2+ and potentially involving PKC.
- This pathway is largely independent of PI3K, Akt, AMPK, and CaMKII signaling cascades.
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