Characterisation and analysis of GLUT4 vesicles in 3T3-L1 adipocytes, and the MUNC18-syntaxin interaction

Mackay, Martin Norman (2006) Characterisation and analysis of GLUT4 vesicles in 3T3-L1 adipocytes, and the MUNC18-syntaxin interaction. MSc(R) thesis, University of Glasgow.

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Abstract

The translocation of GLUT4 from intracellular stores to the plasma membrane in response to insulin results in the large insulin-mediated glucose uptake in fat and muscle tissue. Individuals with insulin resistance and type II diabetes are known to have a defect in this translocation mechanism. As such, a greater understanding of the molecular basis of GLUT4 translocation and recycling is essential in order to design therapies for these diseases. In this study, we have used sucrose gradient analysis to examine the intracellular distribution of GLUT4. This method allowed for the intracellular GLUT4 to be separated into the two pools of GLUT4 containing vesicles. One of these is highly insulin responsive and corresponds to the specialised GLUT4 storage vesicles (GSVs), while the other corresponds to the less insulin responsive endosomal GLUT4 vesicles. It is these specialised GSVs which are believed to allow the large (up to 20- fold) increase in plasma membrane levels of GLUT4, compared to only ~2-fold increase in levels of recycling proteins (such as the transferrin receptor) in response to insulin. The GSVs are small vesicles (~50nm) and exclude the transferrin receptor (and other recycling proteins) found in endosomal vesicles. Here we demonstrated that in response to insulin there is -50% decrease in levels of GLUT4 in GSVs compared to only a 10-20% decrease in levels of GLUT4 in endosomes. We also demonstrate that these sucrose gradients can be used to determine whether other proteins co-localise with GSVs or the endosomal pool. The Sec1/ Munc18 (SM) protein munc 18c is known to interact with syntaxin4 (a t- SNARE present in insulin responsive cells), which is an interaction that appears to inhibit glucose transport by preventing GLUT4 vesicles from undergoing fusion with the plasma membrane. Here we investigated whether a mutated form of the neuronal SM isoform (munc 18a) is capable of functioning in a similar manner to mime 18c (from a sequence alignment of munc 18a and munc 18c we identified specific amino acids to mutate based on the known 3D structure of the munc 18a-syntaxin1 interaction). If indeed the mutated protein is seen to exhibit binding characteristics similar to munc 18c, then it may be possible to establish the importance of the munc 18c-syntaxin4 interaction on GLUT4 integration into the plasma membrane by making recombinant Adenoviral constructs of the munc 18 proteins, and expressing these in mammalian cells. Having made the initial mutations, we demonstrate here that in vitro the mutant munc 18 protein has inherited the syntaxin4 binding capabilities of the wild-type munc 18c. However, we were not able to then get expression of each of the recombinant Adenoviral constructs in mammalian cells, and as such we were unable to establish the effect that these mutations would have upon GLUT4 fusion with the plasma membrane, and ultimately glucose transport.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Additional Information: Adviser: Gwyn W Gould
Keywords: Molecular biology, Physiology
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71382
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71382

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