The importance of ubiquitin in the trafficking of the insulin responsive glucose transporter GLUT4

Adamson, Iain (2015) The importance of ubiquitin in the trafficking of the insulin responsive glucose transporter GLUT4. MSc(R) thesis, University of Glasgow.

Abstract

The regulation of blood glucose levels in humans, in response to insulin, is essential to survival. This response is mediated through the insulin responsive glucose transporter GLUT4. In response to insulin stimulation GLUT4 is trafficked from intracellular insulin sensitive stores (GSVs GLUT4 storage vesicles) to the plasma membrane of fat and muscle cells allowing uptake of glucose into these cells and lowering of plasma glucose levels. Previous work from our lab has identified that ubiquitination and subsequent deubiquitination of GLUT4 is required for entry and stability in GSVs. This balance of ubiquitination and deubiquitination in mammalian cells is carried out by E3 ligases and deubiqutinating enzymes (DUBs). It appears that E3 ligases allow for targeted entry of GLUT4 into insulin sensitive GSVs and that the DUB USP25 is required for GLUT4 to stably maintained in these GSVs. Using a model developed in our lab my thesis looked at key steps of ubiquitination and deubiquitination to try and identify the E3 ligases and deubiquinases essential for entry and stability of GLUT4 into GLUT4 storage vesicles.
Previous work in our lab identified that knockdown of the ubiquitin specific protease USP25 resulted in a reduction in GLUT4 levels in 3T3-L1 adipocytes. In my thesis I looked at using an inducible retro viral system to identify if the catalytic activity of USP25 was required to maintain GLUT4 expression levels. This method had limited success as I was unable to produce a virus that would always express under induction.
Previous work in our lab had also identified that GLUT4 was ubiquitinated in yeast and in 3T3-L1 adipocytes. βTrCP had previously been identified as a possible E3 ligase that interacts with GLUT4 and so I used this as a candidate ligase. To identify if βTrCP played a role in mediating insulin responsive glucose uptake I utilised siRNA knockdown of βTrCP in 3T3-L1 adipocytes alongside glucose uptake assays. Although knockdown of βTrCP showed no effect on GLUT4 levels in 3T3-L1 adipocytes it did show a decrease in insulin responsive glucose uptake. These data did not provide any further information on the relationship between GLUT4 and USP25. These data do however indicate that βTrCP does interact in some way with insulin responsive glucose uptake in 3T3-L1 adipocytes but it is still unclear if this is due to any interaction with GLUT4.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: GLUT4, ubiquitin, trafficking
• Subjects: Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Bryant, Professor Nia
• Date of Award: 2015
• Depositing User: Mr Iain Adamson
• Unique ID: glathesis:2015-6668
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 Sep 2015 08:37
• Last Modified: 29 Sep 2015 07:45
• URI: https://theses.gla.ac.uk/id/eprint/6668