Elucidation of the final stages in coupling of insulin signalling to GLUT4 translocation

Lead Research Organisation: University of Bath
Department Name: Biology and Biochemistry


Maintenance of blood glucose levels is critically dependent on insulin stimulated glucose transport in adipose tissue, heart and skeletal muscle. Insulin combines with its receptor on target tissues and this initiates a cascade of linked reactions that ultimately result in the fusion of vesicles containing the glucose transporter protein GLUT4 with the plasma membrane. This latter process increases the availability of transporter molecules and thereby increases glucose transport into the cell. Although many of the links in the cascade are well studied, many technical difficulties have prevented detailed study of the final steps in the sequence, namely the fusion process. The proposed project will utilise novel approaches to study the fusion reaction and the extent to which small G-proteins of the Rab family link with the fusion machinery. We have identified many similarities between the membrane fusion reactions that occur in the pancreatic beta cell (which lead to secretion of insulin) and in insulin target tissues (which are involved in GLUT4 vesicle fusion). These similarities will guide the research objectives and experimental plans. These plans will be directed at advancing knowledge of how specific and known components of the fusion mechanism are linked together by effector proteins that are downstream of Rab proteins. This work is important as the fundamental mechanism that links signalling to regulated membrane fusion underlies many processes in biology and is relevant to human health. Elucidation of common mechanisms for pancreatic insulin-vesicle traffic and insulin target cell GLUT4-vesicle traffic will allow a unification of our understanding of those key reactions that are critical for control of blood glucose. These may become dysfunctional by a common route in metabolic disease including obesity and type 2 diabetes.

Technical Summary

We plan to study mechanisms involved in regulating the fusion of GLUT4 vesicles with the plasma membrane of insulin target cells. We have identified Rab3 as a target of insulin signalling and using a novel photolabelling procedure have found that the loading of this G-protein is increased by insulin action. This labelling approach has also led to the identification of Rap1 as another regulated G-protein target of insulin action. In the beta cell of the pancreas Rap1 is downstream of trimeric G-proteins that augment glucose-dependent insulin release. Furthermore, it is associated with Rab3 control of vesicle traffic. In addition Munc18 is known to be a critical component of the vesicle fusion in both systems. These similarities have led us to propose that there is a unifying mechanism underlying the membrane fusion roles of these components. We plan to examine whether known Rab3 effectors that link with Munc18 in the pancreas have similar counterparts involved in insulin regulation of GLUT4 vesicle fusion. Therefore, we plan to identify Rab3 effectors in insulin-target tissues and validate their involvement in the fusion reaction. We will then study the interaction of Rab3 and the Rab effectors with Munc18. We will also examine the extent to which Munc18 availability for fusion and interaction with SNARE proteins is dependent of its tyrosine phosphorylation. We will determine whether insulin receptor tyrosine kinase is responsible for this phosphorylation using intact receptor and intact plasma membrane as components in our cell-free fusion system. In addition, we will examine the extent to which Rab3 and its effectors are regulated downstream of trimeric G-proteins and through the intermediate Rap1, as occurs in pancreatic beta cells.

Planned Impact

Economic impact
Potential Beneficiaries: Pharmaceutical companies (improved products and sales); NHS (improved therapies and interventions);
The Holman group has collaborated extensively with UK based Pharmaceutical companies including Glaxo-SmithKline and currently AstraZeneca that have helped translate our research into practice. One example outcome of these studies has been the testing of the potency of an insulin sensitizer that was developed into a highly successful anti-diabetes drug called Rosiglitazone. Information obtained in this collaboration was used to mechanistically validate the drug. Annual sales of this drug peaked at approx $2.5bn in 2006, but declined after reports of adverse effects. Our current work is partially supported AstraZeneca has been supported by two consecutive CASE studentships from 2006-2009 and currently 2009-2013. Both studentships have involved setting up high-throughput cell models for testing drug targets, including insulin sensitive Rab-GAPs. With these continuing links we will be able to translate new output on the proposed project into drug screening programs with an expected eventual impact on new drug sales. Diabetes treatment has economic consequences to the NHS (below).

Social and human health impact
Potential beneficiaries: General public; NHS; Charities; Policy Makers
Type 2 diabetes is a major global problem with more than 250 million people worldwide currently living with this condition and by 2025, this total is expected to increase to over 380 million people. Each year another 7 million people develop diabetes. 2.8m people in the UK are now diagnosed with diabetes and it is estimated that 1 million more people may have pre-diabetes. Diabetes can lead to complications including kidney failure, coronary heart disease, stroke, blindness and foot amputation. It is estimated that Diabetes treatment (mainly type 2) accounts for an estimated 5% of all NHS expenditure. A detailed understanding of the underlying biological system is key for developing effective diagnostic tools, causal intervention strategies and in supporting public health advice (through government departments and charities). Providing a full pathway from insulin receptor to glucose transporter will impact on strategies for the control of glycemia, energy homeostasis and bodyweight. Therefore there is potential to change behaviours with associated improvements in health outcomes.
The targeting of this fundamental research area will reduce animal use by generating more targeted drug discovery. In addition it will provide a focused and directed range of parameters to be monitored in human subjects undergoing clinical drug trials.

Skills and training impact
Potential beneficiaries: Next generation research students; Pharmaceutical companies; Health coaching
The proposed research on insulin action will involve postgraduate students who will benefit from training in the novel methods and approaches used by our group. We use a combination of cell physiology and biochemistry approaches. We also have chemical biology input with generation of new tools and reagents for analysis of aspects of insulin action. This wide range of skills will be passed on in training of new PhD students. We hope to continue to recruit students who can interact with pharmaceutical companies. Students trained in this way will be able to pass on skills to a range of employment sectors but specifically sectors concerned with treatments for obesity and type 2 diabetes. There are currently two PhD students in the Holman group. One of these is a CASE student with AstraZeneca. Holman has successfully trained 27 previous PhD students. Our interaction with Sports and Exercise Science PhD and MSc students allows us to share expertise in metabolism and signalling with those contemplating careers in human health related areas such as exercise and diet coaching.


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Holman GD (2018) Chemical biology probes of mammalian GLUT structure and function. in The Biochemical journal

Title Isotope tags on GTP photolabels 
Description C13 isotope tags on GTP photolabel. For use inproteomic analysis of GTP binding proteins 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Unpublished data 
Title Photochemical reagent for analysis of GLUT4 translocation 
Description We have developed a photoaffinity labelling reagent which can be used to study glucose transporters at the surface of cells. We have used it to determine the extent to which the glucose transporter GLUT4 is translocated in response to insulin and how this process is impaired in type 2 diabetes 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Used with clinical samples including muscle from type 2 diabetes patients. This was a collaborative study with a group at the Karolinska Institute, Stockholm, Sweden 
Title Photolabel for GTP binding proteins 
Description A GTP analogue that can detect the activation of GTP-binding proteins. This is being used to detect the activation of GTPase (mainly of the Rab family) that are relevant to insulin action in insulin responsive tissues such as adipose, heart and skeletal muscle 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact Patent 
Description Breakfast project 
Organisation University of Bath
Department School of Health Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution Setting up assays for determining insulin sensitivity in adipose tissue
Collaborator Contribution Application of insulin sensitivity assays to human subjects under varying fasting regimes
Impact Publication in Trials
Start Year 2009
Description CASE1 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Setting up of assays for measuring GLUT4 translocation in a muscle cell line
Collaborator Contribution shared expertise
Impact Publication ID 19915010
Start Year 2006
Description Diabetes Institute, Dusseldorf 
Organisation University Hospital Düsseldorf
Department German Diabetes Center
Country Germany 
Sector Academic/University 
PI Contribution Generation of a new reagent for labelling the substrates of TBC1D1 and TBC1D4. These proteins are Rab GAPs
Collaborator Contribution Collaboratively we are studying the GTPase labelling in knockout animals with deletions in TBC1D1 and TBC1D4 that have been generated by the Dr Hadi Al Hasani at the German Diabetes Center
Impact Unpublished data Sabbatical visit to the German Diabetes Centre in 2013 sponsored by DAAD
Start Year 2011
Description Proteomics of insulin action on GLUT4 
Organisation The Garvan Institute for Medical Research
Country Australia 
Sector Hospitals 
PI Contribution Supply of material for analysis
Collaborator Contribution Proteomic analysis
Impact New data
Start Year 2012
Description Research conference organisation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Myself, Dylan Thompson and Javier Gonzalez together with Kei Sakamoto (Nestle Research Institute) were the organisers of a Biochemical Society Focused meeting entitles "Insulin and exercise signalling for glucose homeostasis and metabolic health". This meeting was held at the University of Bath, from the 6th till the 8th of September 2017. 45 participants took part, from UK, Germany, Switzerland, Sweden, Danemark and the USA. At the meeting Prof Geoff Holman delivered the Sir Philip Randle award lecture 2017 from the Biochemical Society. The conference was a great success with a lot of positive feedback from colleagues and post-graduate students. They all appreciated the wide variety of the research programme and the interdisciplinarity of the talks. In the aftermath of the event some of our colleagues expressed interest in transforming this event in a regular event with a 2 year reoccurrence.
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/tabid/379/Filter/64/MeetingNo/SA202/view/Conference/Default.aspx