Role of PFK2/FBPase-2 in regulating hepatic glucokinase

Lead Research Organisation: Newcastle University
Department Name: Institute of Cellular Medicine

Abstract

Type-2 diabetes is a very common metabolic disease that is due to complex genetic and environmental factors. It manifests as an increase in blood sugar (glucose) level which if not adequately corrected leads to chronic morbidity and mortality as a result of long-term damage to blood vessels and nerves with consequent organ failure. Current therapy for Type-2 diabetes does not achieve adequate control of blood sugar levels and there is an urgent need for better oral therapeutics. Drugs that target a protein called glucokinase are currently considered to be very promising candidates for treatment for Type-2 diabetes.

Several mutations in the glucokinase gene have been reported in man which cause a somewhat rare form of Diabetes described as Maturity Onset Diabetes of the Young. Whilst defects in the glucokinase gene are not in themselves a common cause of Type 2 diabetes, nonetheless there are indications that defects in the mechanism(s) that switch ON or OFF the glucokinase gene may be very important. We now know that there are complex networks of ?on? and ?off? mechanisms encoded by different genes that account for the fine tuning of glucokinase function.

Recent work from our laboratory has identified a novel mechanism involving a protein called PFK2/FDP2 that is critical for maintaining glucokinase functional. This proposal is to explore in detail the mechanistic basis by which PFK2/FDP2 regulates glucokinase activity in the liver. This is important on two accounts: first, because this or related mechanisms may be defective in Type-2 diabetes; second, understanding the physiological mechanisms that activate glucokinase is essential for a more complete evaluation of the long-term benefits of drugs targeting glucokinase.

The results of the project will be disseminated through: (i) Research Publications; (ii) Presentations at local, national and international meetings; (iii) Public Lectures and Informal Talks and discussion groups to both Professional and Lay members of Diabetes UK and other organisations.

Technical Summary

Glucokinase (GK) controls blood glucose homeostasis through its dual role as glucose sensor for insulin secretion and as a major determinant of hepatic glucose metabolism. Distinct mechanisms of regulation of GK activity occur in liver and pancreatic b-cells that are in part accounted for by different GK-binding proteins or isoforms thereof.

We previously tested the hypothesis that the bifunctional enzyme phosphofructokinase-2 / fructose bisphosphatase-2 (PFK-2/FDP-2), which catalyses the formation and degradation of the regulator of glycolysis, fructose 2,6-P2, functions as a cytoplasmic receptor for GK in liver (Payne et al. 2005, Diabetes 54:1949). This work showed unexpectedly that overexpression of PFK-2/FDP-2 markedly potentiates GK expression at the mRNA level by a mechanism that is post-transcriptional and independent of fructose 2,6-P2. Since PFK2/FDP2 binds to GK protein through its bisphosphatase domain (Baltrusch et al. 2001, J Biol Chem 276:43915), this suggests a unique role for PFK2/FDP2 as a putative receptor for GK protein but also as regulator of GK mRNA levels.

This proposal is to determine the mechanism(s) by which PFK2/FDP2 potentiates GK-mRNA expression in hepatocytes. To define the role of PFK2/FDP2 as regulator of GK-mRNA expression, endogenous PFK2/FDP2 will be by down-regulated using RNA interference and the induction by insulin of GK and other genes will be determined by RT-PCR.

We will test for association of PFK2/FDP2 with ribosomes, stress granules and ribonucleoprotein complexes. Further work will focus on immunoprecipitates of PFK2/FDP2 from nuclear and cytoplasmic extracts of hepatocytes. We will first test for GK-mRNA and other insulin-induced transcripts by RT-PCR. If specific association is confirmed we will next perform Codelink rat genome microarrays on the immunoprecipitates. However if specific binding of GK-mRNA is not confirmed by RT-PCR we will test the alternative hypothesis that PFK2/FDP2 potentiates GK-mRNA expression by dampening endogenous RNA interference by determining MicroRNA in the immunoprecipitates.

Binding of PFK2/FDP2 to the coding and non-coding (3?-UTR) regions of GK-mRNA will be determined by gel retardation assays. A role for PFK2/FDP2 in stabilising GK-mRNA from degradation will be tested by expression of GK using a tetracycline-regulated (tet-off) vector to enable selective inhibition of GK transcription. The role of the 3?-UTR in mRNA decay will be determined using reporter constructs.

There are as yet no known binding proteins for GK-mRNA. This project will test alternative mechanisms that could explain the potentiation of GK-mRNA by PFK2/FDP2 protein and determine whether PFK2/FDP2 is a pre-requisite for GK expression in liver.

Publications

10 25 50
 
Description Daibetes UK PhD Studentship
Amount £84,600 (GBP)
Funding ID BDA/RD07/0003559 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2008 
End 09/2011
 
Description Diabetes UK PhD Studentship
Amount £91,800 (GBP)
Funding ID 13/0004701 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description MRC Industrry Asset Sharing Initiative
Amount £746,509 (GBP)
Funding ID MR/P00285/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 01/2020
 
Description Project Grant
Amount £106,070 (GBP)
Funding ID 11/0004231 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2012 
End 05/2014
 
Title Metabolite analysis by resazurin coupling 
Description Mapping of metabolite control of gene expression was dependent on development of highly sensitive quantitative methods for analysis of intermediary metabolites. We have optimised analytical methods for assay of phosphorylated metabolic intermediates and also of substrate analogs that are selectively metabolised by some but not other metabolic pathways on small samples (<10 ug protein) to enable metabolite mapping of gene expression. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact So far these techniques have been prvided by the PI to the IRB, Barcelona laboratory where the techniques were set up during the sabbatical for generation of adenoviral vectors 
 
Title Metabolite control of gene expression 
Description We showed that contrary to previous predictions glucokinase is regulated by intermediary metabolites at least in part at the transcriptional level. Mapping of the metabolites in the regulation of glucokinase and a selection of other genes showed that this regulation can be explained by a mechanism to restore intracellular metabolite homeostasis. This Model has major implications for understanding the mechanisms that account for develoment of fatty liver (NAFLD). A common condition that predisposes to metabolic syndrome, hepatic steatosis and hepatic insulin resistance. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2008 
Provided To Others? Yes  
Impact The model developed from these findings has been presented by the PI at a UCLA invited seminar, at IRB University of Barcelona and at Diabetes UK, Professional Meeting by Dr Catherine Arden. It has attracted a great deal of Discussion. The Presentation of Dr. Arden was shortlisted for the Nick Hales Award. 
 
Description Beynon Proteomics 
Organisation University of Liverpool
Department Institute of Integrative Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We analysed the direct effects of substrates on expression of genes at mRNA and protein level in hepatocytes. This work cannot be undertaken in vivo. We therefore required information on protein turnover in vivo.
Collaborator Contribution Professor Rob Beynon generated and analysed the data from Proteomic analysis for protein turnover in vivo. This enabled us to interpret the in vitro data.
Impact Joint publication in Diabetes PUBMEDID 22013014
Start Year 2010
 
Description Towle, HC 
Organisation University of Minnesota
Department Department of Biochemistry, Molecular Biology and Biophysics
Country United States 
Sector Academic/University 
PI Contribution Sharing of reagents, ideas, discussion.
Collaborator Contribution Experiments on primary hepatocytes using the reagents provided, interpretation of data, hypotheses generated.
Impact Two publications to date (PUBMED ID: 22214556, 22013014) and one MS currently under review.
Start Year 2008
 
Description Yaqoob In vivo studies 
Organisation Queen Mary University of London
Department Centre for Translational Medicine and Therapeutics
Country United Kingdom 
Sector Academic/University 
PI Contribution During review of one our manuscripts the reviewer asked for complementary ChIP studies in vivo to demonstrating the involvement of a transcription factor in vivo. This required additional in vivo studies.
Collaborator Contribution Our partners who have extensive expertise with in vivo studies performed the in vivo studies and sent us the samples for analysis. This analysis is expected to complete the paper.
Impact One joint paper to date PUBMEDID 22106156; a further paper in revision; and another agreed project to follow.
Start Year 2011
 
Description Diabetes Patient Group-Gateshead Queen Elisabeth Hospital 17/06/2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact This was a mixture of patients with Type 1 and Type 2 diabetes from the Gateshead area. It included patients who are volunteers for ongoing studies. The talk was on the mechanism of action of metformin and various patients with Type 1 diabetes were very interested in the topic and in volunteering for projects on metformin.
Year(s) Of Engagement Activity 2015
 
Description Diabetes UK Lay Committee 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A member of the team gave a talk to the Lay Fund raising committee of Newcastle upon Tyne, to discuss the objectives and outcome of our ongoing research on diabetes.

The Lay Committee is very keen to have talks from labs involved in Diabetes Research to help with fundraising events
Year(s) Of Engagement Activity 2012
URL https://www.diabetes.org.uk/In_Your_Area/Northern__Yorkshire/local-groups/
 
Description Diabetes UK Newcastle Group / Programme for 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact The talk was to members of the Diabetes UK Newcastle Group who included people with Type 1 and Type 2 as well as volunteers involved in fund raising and volunteers for studies. The talk was informal and generated a great deal of Discussion on issues of new and established therapies.

The talk was informal and generated a great deal of Discussion on issues of new and established therapies.
Year(s) Of Engagement Activity 2014
URL http://www.newcastle-diabetes.webeden.co.uk/#/meetings-and-events/4542245209
 
Description Diabetes UK Yorskshire region annual conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 30 Lay members attended and there was discussion afterwards

This led to a further invite for Dr Catherine Arden in October 2013
Year(s) Of Engagement Activity 2013
URL http://www.diabetes.org.uk/In_Your_Area/Northern__Yorkshire/
 
Description Newcastle Biomedicine Research Centre Open Day on Ageing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact This was a full day Conference on Ageing (17th October 2013) for the Local Public; Dr Catherine Arden gave a presentation on "Diabetes in the Ageing Population" and engaged in a Full day of participation and workshops.

Following the talks there was lots of Discussion between the Lay Attendees and the presenters of the talks. It Led it to 2 further invites for Dr, C. Arden and Professor L. Agius at the Local Newcastle Diabetes UK Voluntary support group for 2014.
Year(s) Of Engagement Activity 2013
URL http://www.ncl.ac.uk/biomedicine/news/