Role of PFK2/FBPase-2 in regulating hepatic glucokinase
Lead Research Organisation:
Newcastle University
Department Name: Clinical Medical Sciences
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.
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.
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.