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Characterization of the role of myosin II during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Shelly Woody (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Yashomati Patel

Abstract: Insulin-stimulated glucose uptake requires the activation of the nonmuscle motor protein myosin II. Our previous studies using pharmacological inhibitors suggest that insulin signaling results in the phosphorylation of myosin IIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Since pharmacological inhibitors are not specific, we wanted to use a siRNA approach to complement our previous studies. In this report we demonstrate that knockdown of myosin IIA using a myosin IIA-specific siRNA resulted in impaired insulin-stimulated glucose uptake. To delineate the signaling pathway involved we asked if siRNA specific to myosin light chain kinase (MLCK), an upstream regulator of myosin IIA, would have the same effect. While we did not observe a reduction in MLCK or impairment of insulin-stimulated glucose uptake, we were able to observe that MLCK is phosphorylated upon insulin stimulation suggesting a role for MLCK in insulin-stimulated glucose uptake. Next, we used siRNA specific to extracellular-signal regulated kinase 2 (ERK2) to establish a role for this kinase in insulin-stimulated glucose uptake. Our results revealed that knockdown of ERK2 resulted in reduced phosphorylation of MLCK. Knockdown of ERK2 also impaired insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Lastly, we used siRNA specific to the calcium/calmodulin kinase II delta isoform to explore its role as a potential upstream activator of ERK2. Only a slight decrease in CaMKIIδ expression was achieved, but this did not result in a significant change in insulin-stimulated glucose uptake. Taken together, our results suggest that myosin IIA is involved in insulin-stimulated glucose uptake and that it is regulated via MLCK phosphorylation by ERK2 resulting in regulation of glucose uptake upon insulin stimulation in 3T3-L1 adipocytes.

Additional Information

Publication
Thesis
Language: English
Date: 2010
Keywords
Adipocytes, Glucose uptake, Insulin, Myosin II
Subjects
Fat cells $x Physiology.
Glucose $x Metabolism.
Phosphorylation.
Insulin.
Myosin.