Studies on the Molecular Mechanism of Insulin Action

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

Diabetes and obesity represent an enormous public health problem that is set to increase substantially over the next decade placing even further strain on a limited health budget. While education will play an important role in reducing their incidence, pharmaceutical intervention will remain a very important method of treatment until methods of beta cell transplantation improve (e.g. stem cells), and/or public attitudes towards diet and exercise change. Indeed, the importance that is placed on diabetes research was exemplified by the ?Department of Health and MRC Review on Diabetes Research? which was published in 2002 as part of the National Service Framework for Diabetes.

One of the most important actions of insulin is to stimulate the uptake of glucose from the blood into muscle and fat tissue. In type I diabetes, insulin is not produced due to auto-immune attack on the beta cells which are responsible for insulin production. In type II diabetes, insulin production is often normal or elevated (until late stage diabetes) but the target tissues for insulin action do not respond effectively (?insulin resistance?). While we have an increasing understanding of how insulin works on glucose uptake, many aspects are still completely unknown. For example, we know that an enzyme called protein kinase B (PKB) is involved but we do not know in exactly what capacity and how this may be affected in diabetes. Our work involves using a unique combination of state-of-the-art cell and molecular biology techniques as well as proteomics to examine the mechanism by which insulin brings about its effects on glucose uptake with particular emphasis on the role of PKB.

We take public engagement and dialogue extremely seriously. Professor Tavare was recently part of a programme broadcast on the BBC called ?Cell City? which examined the inner workings of cells. The programme was part of the Open University?s Open Science series and he discussed our work using green fluorescent protein to follow the dynamics of insulin action inside single living cells. Professor Tavare has also appeared on both HTV and BBC local news programmes and has given a number of lay presentations at Diabetes UK Regional Groups (including Swindon, Bournemouth and London). Two members of the research team are part of the Bristol Teacher-Scientist Partnership Network and have taken part in primary and secondary school science education.

Technical Summary

The stimulation of the translocation of the insulin responsive glucose transporter, GLUT4, from intracellular storage locations to the plasma membrane underlies the ability of insulin to increase glucose uptake. Our principal objective is to define the molecular basis by which insulin stimulates glucose transport in adipocytes, specifically the signalling mechanism employed. A complete molecular explanation of this action of insulin is crucial to uncovering new drug targets for potential therapeutic intervention in both types I and II diabetes.

There is substantial evidence to suggest that the activation of protein kinase B (PKB) is central to the action of insulin on glucose transport, although the substrate proteins involved have not been identified. In the current proposal we plan to delineate the precise role that PKB plays in this action of insulin, and to purify, identify and characterise the critical PKB substrate(s) involved. We have recently identified a phosphoinositide 5-kinase, PIKfyve, as a potentially important PKB substrate in primary adipocytes. We plan to investigate whether this enzyme plays a role in GLUT4 trafficking.

An important aspect of insulin action on glucose uptake is the compartmentalisation of signalling proteins, including PKB. We will develop genetically-encoded fluorescent biosensors for PKB activity, to examine the kinetics of PKB activation in specific intracellular compartments (e.g. on the surface of GLUT4 vesicles and endosomes) such that we can relate this to the stimulation of GLUT4 translocation. Furthermore, we will develop novel fluorescence-based methods for studying the interaction between selected signalling molecules involved in insulin action on GLUT4 translocation (IRS and PI3-kinase, in the first instance), and investigate the intracellular compartments within which these interactions occur.

In parallel, we will take advantage of recent major developments in time-lapse microscopy and GFP technologies to follow the dynamics of GFP-labelled GLUT4 vesicles. Our recent studies suggest that insulin may induce a re-distribution of GLUT4 to the plasma membrane either by initiating a budding event from relatively static peripheral GLUT4 vesicles, or by transit through a tubular vesicular network. The extent to which these events involve PKB will be investigated.

Publications

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Alesutan IS (2010) Regulation of the glutamate transporter EAAT4 by PIKfyve. in Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

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Gehring EM (2009) PIKfyve upregulates CFTR activity. in Biochemical and biophysical research communications

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Gehring EM (2009) Regulation of the glutamate transporter EAAT2 by PIKfyve. in Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

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Hill EV (2010) Regulation of PIKfyve phosphorylation by insulin and osmotic stress. in Biochemical and biophysical research communications

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Klaus F (2009) Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve. in Neurochemistry international

 
Description BHF Project Grant
Amount £240,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2010 
End 07/2013
 
Description Diabetes UK, PhD Studentship
Amount £75,000 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2006 
End 09/2009
 
Description Diabetes UK, Project Grant
Amount £169,906 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2009 
End 07/2012
 
Description MRC Capacity Building Studentship
Amount £501,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2008 
End 09/2013
 
Description MRC Clinical Fellowship
Amount £705,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2006 
End 12/2010
 
Description MRC DPFS Award (devolved portfolio)
Amount £420,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2009 
End 09/2013
 
Description Seeding Drug Discovery Award
Amount £2,814,340 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2006 
End 05/2008
 
Title GFP tagged proteins 
Description Various GFP tagged proteins for use in cell biology research 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Several publications 
 
Description Insulin action in human adipocytes 
Organisation Astellas Pharma
Department Prosidion Ltd
Country United Kingdom 
Sector Private 
PI Contribution We are currently undertaking research into this topic.
Collaborator Contribution Support for a BBSRC/CASE student
Impact Support for a BBSRC/CASE student
Start Year 2008
 
Description Investigations into the efficacy of IGF1R kinase inhibitors towards IGF1 and insulin signalling 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Studies ongoing
Collaborator Contribution Increased our understanding of the contribution of insulin action to tumorigenesis
Impact Studies ongoing but publication in preparation
Start Year 2008
 
Description BBC Radio 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact BBC Radio interview

Public education
Year(s) Of Engagement Activity 2013
 
Description HTV West 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact ITV interview

Public education
Year(s) Of Engagement Activity 2013
 
Description How insulin works 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation to local diabetes fund raising groups and to a schools (year 6)

Public education
Year(s) Of Engagement Activity 2009,2011,2012,2013