GLUT4 is the principal insulin-responsive glucose transporter in skeletal muscle. Insulin stimulation leads to exocytosis of intracellular GLUT4-containing vesicles to the cell surface, thereby increasing glucose uptake. Muscle contraction also elevates cell surface GLUT4 by a less understood mechanism. Once at the cell surface, GLUT4 may be subject to additional regulation, such as by modulation of its internalization rate or its intrinsic activity. The objective of this thesis was to identify the mechanism of GLUT4 internalization in muscle cells and to determine whether it is regulated by insulin treatment or by the signals elicited by muscle contraction. Skeletal muscle cells in culture stably expressing myc-tagged GLUT4 were used.
We found that GLUT4 internalizes simultaneously through a clathrin-dependent and a clathrin- and caveolae-independent and cholesterol- and dynamin-dependent pathway. Insulin did not regulate GLUT4 internalization. In contrast, mitochondrial uncoupling, which may mimic the heightened energy demand that occurs during muscle contraction, retarded GLUT4 internalization by inhibiting the clathrin-independent route. Activation of both AMP-dependent kinase (AMPK) and protein kinase C (PKC) was necessary and sufficient for this response.
We further hypothesized that the intrinsic activity of GLUT4 may be regulated under some conditions, based on a discrepancy between the amount of cell surface transporters and the rate of glucose uptake. In particular, inhibitors of p38 mitogen-activated protein kinase (p38MAPK) lowered insulin-dependent glucose uptake without reducing the number of GLUT4 units at the surface. We found that p38MAPK is activated by insulin through TAB1-dependent autophosphorylation, yet p38MAPK was dispensable for insulin-stimulated glucose uptake. Mechanisms other than p38MAPK must be involved in the regulation of GLUT4 intrinsic activity. In conclusion, in addition to its exocytosis, the activity and endocytosis of GLUT4 are regulated by stimuli that increase the rate of glucose uptake into muscle.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/16717 |
| Date | 19 January 2009 |
| Creators | Antonescu, Costin N. |
| Contributors | Klip, Amira |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 30272987 bytes, application/pdf |
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