The role of mVps45 in regulating GLUT4 trafficking in 3T3L1 adipocytes

Insulin stimulates glucose transport in fat cells by inducing the movement of glucose transporters (Glucose transporter-4) from specialised storage vesicles to the plasma membrane. Insulin resistant individuals and those with Type II Diabetes exhibit impairment in the ability of insulin to stimulate glucose transport. The molecular mechanisms of glucose transporter-4 trafficking in adipocytes are an important focus in understanding the underlying etiology of this disease. Glucose transporter-4 (GLUT4) recycles between the plasma membrane and intracellular stores in the absence of insulin using a complex intracellular pathway. This involves two intracellular cycles: one is the prototypical endosomal system, the other a specialised cycle involving the trans-Golgi network and a sub-set of intracellular vesicles called GSVs (the slow cycle). Understanding the control of the entry into this second cycle is the subject of this thesis. In particular, the work in this thesis will examine the role of Syntaxin 16 and its cognate Sec1/Munc18 protein mammalian Vps45 (mVps45). The regulation of Syntaxin 16 has not been fully elucidated and understanding the role of Syntaxin 16 in SNARE complex regulation and subsequent control of GLUT4 traffic into the slow cycle requires an understanding of its cognate binding partner Sec1/Munc18 (SM) protein, mammalian Vps45 (mVps45). The absolute levels of both Syntaxin 16 and mVps45 were quantified and found to be present in 3T3-L1 adipocytes in roughly stoichiomeric amounts. IP experiments also showed the ability of mVps45 to interact with Syntaxin 16 in the absence of insulin. Using the model eukaryote Saccharomyces cerevisiae, we found that mVps45 could complement for the deletion of Vps45p. Assays for CPY secretion showed that mVps45 is able to complement for the loss of Vps45p function in the trafficking of carboxypeptidase Y (CPY). Additionally, mVps45 mutants were made that correspond to yeast mutants made previously in the lab and were tested for homology of function. Depleting 3T3-L1 adipocytes of mVps45 showed alterations in the levels of GLUT4 protein as well as the protein levels of Syntaxin 16, IRAP, and Rabenosyn. Insulin-stimulated deoxyglucose uptake was also profoundly decreased upon depletion of mVps45. Further experiments using mVps45 depleted cells show that these cells lose their sensitivity to insulin and that the loss of mVps45 in these cells causes GLUT4 to have the inability to enter the slow cycle. Taken together, these findings demonstrate that mVps45 has a role in allowing GLUT4 entry into the slow cycle

Can a Multi-Student Research Project Deliver a High Quality Learning Experience at Masters Level?

No abstract available

mVps45 knockdown selectively modulates VAMP expression in 3T3-L1 adipocytes

Insulin stimulates the delivery of glucose transporter-4 (GLUT4)-containing vesicles to the surface of adipocytes. Depletion of the Sec1/Munc18 protein mVps45 significantly abrogates insulin-stimulated glucose transport and GLUT4 translocation. Here we show that depletion of mVps45 selectively reduced expression of VAMPs 2 and 4, but not other VAMP isoforms. Although we did not observe direct interaction of mVps45 with any VAMP isoform; we found that the cognate binding partner of mVps45, Syntaxin 16 associates with VAMPs 2, 4, 7 and 8 in vitro. Co-immunoprecipitation experiments in 3T3-L1 adipocytes revealed an interaction between Syntaxin 16 and only VAMP4. We suggest GLUT4 trafficking is controlled by the coordinated expression of mVps45/Syntaxin 16/VAMP4, and that depletion of mVps45 regulates VAMP2 levels indirectly, perhaps via reduced trafficking into specialized subcellular compartments

Curriculum Mapping and Pathology Teaching: A Novel Low Cost Mapping Method

No abstract available

Curriculum Mapping and Pathology Teaching: A Novel Low Cost Mapping Method

No abstract available

Creation of a novel simple heat mapping method for curriculum mapping, using pathology teaching as the exemplar

Background The undergraduate five-year MBChB programme at the University of Glasgow has a high volume of pathology teaching integrated into the course. The ability to better understand what pathology is taught and when, so as to build a picture of the types and depth of pathology topics covered across the programme stages is crucial, especially in a spiral curriculum. A novel method of curriculum mapping, known as curriculum heat mapping, was developed as a way to visualise where and when topics are taught, in an easier to understand format. Methods This method involved comparing the Glasgow curriculum to a pre-determined standard of what should be taught. In this case, The Royal College of Pathologists’ ‘Pathology Undergraduate Curriculum’ was used as a comparison of what a graduating doctor should know about pathology. Results Following the developed template, heat maps showcasing the range of pathology topics covered, and where they are covered, were developed for local use. These heat maps provided a clear visual representation of where and when topics are taught, and how they cluster. Conclusions Heat mapping is a novel low-cost, high-input method of curriculum mapping. It requires a person to input the data which can take a long time for large curricula. There are no other upfront financial costs. It can be used in any area with a curriculum and an external or internal comparator. Examples of gold standard external comparators include validated national or international curricula. Heat mapping can help integrated, spiral curriculum programmes to identify where core topics are taught throughout their course. The heat maps themselves successfully demonstrate the required information and are easy to interpret. The process of mapping, as well as the final heat map, can yield important information. This includes information about trends within the curriculum, areas for potential improvement in sessional design and a clearer understanding of the depth to which each topic is covered in each lecture. Overall, it is a viable novel method, which has been successful locally and is easily transferable to other areas such as pharmacology

Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the ‘insulin responsive vesicles’ are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro. Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes

Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub-cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment