The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP+-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P ≤ 0.03). Experiments using equimolar concentrations of both regulatory proteins confirmed these findings (n = 9; P < 0.001). In conclusion, P446L-GKRP has reduced regulation by physiological concentrations of F6P, resulting indirectly in increased GCK activity. Altered GCK regulation in liver is predicted to enhance glycolytic flux, promoting hepatic glucose metabolism and elevating concentrations of malonyl-CoA, a substrate for de novo lipogenesis, providing a mutational mechanism for the reported association of this variant with raised triglycerides and lower glucose levels

Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Aims/hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. Methods Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency

L’Observatoire de la Consommation alimentaire en Région wallonne : pourquoi ? Comment ?

The Observatory of Food Consumption in the Walloon Region (Belgium): why, how ? The behaviour of food products consumers is observed by several organisms. This paper gives some informations about situations abroad and in Belgium. It describes the Observatory of Food Consumption established in the Walloon Region

Prioritising Server Side Reachability via Inter-process Concolic Testing

Context: Most approaches to automated white-box testing consider the client side and the server side of a web application in isolation from each other. Such testers lack a whole-program perspective on the web application under test. Inquiry: We hypothesise that an additional whole-program perspective would enable the tester to discover which server side errors can be triggered by an actual end user accessing the application through the client, and which ones can only be triggered in hypothetical scenarios. Approach: In this paper, we explore the idea of employing such a whole-program perspective in inter-process testing. To this end, we develop StackFul, a novel concolic tester which operates on full-stack JavaScript web applications, where both the client and the server side are JavaScript processes communicating via asynchronous messages-as enabled by e.g., the WebSocket or Socket.IO-libraries. Knowledge: We find that the whole-program perspective enables discerning high-priority errors, which are reachable from a particular client, from low-priority errors, which are not accessible through the tested client. Another benefit of the perspective is that it allows the automated tester to construct practical, step-bystep scenarios for triggering server side errors from the end user’s perspective. Grounding: We apply StackFul on a collection of web applications to evaluate how effective inter-process testing is in distinguishing between high- and low-priority errors. The results show that StackFul correctly classifies the majority of server errors. Importance: This paper demonstrates the feasibility of inter-process testing as a novel approach for automatically testing web applications. Classifying errors as being of high or low importance aids developers in prioritising bugs that might be encountered by users, and postponing the diagnosis of bugs that are less easily reached

Effect of mutations on the sensitivity of human beta-cell glucokinase to liver regulatory protein

Human beta-cell glucokinase and its liver counterpart displayed a half-saturating concentration of glucose (S-0.5) of about 8 mmol/l and a Hill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indicate that the N-terminal region of glucokinase, which differs among these three enzymes, is not implicated in the recognition of the regulatory protein. They also suggest that the regulatory protein, or a related protein, could modulate the affinity of glucokinase for glucose in beta cells. We have also tested the effect of several mutations, many of which are implicated in maturity onset diabetes of the young, The mutations affected the affinity for glucose and for the regulatory protein to different degrees, indicating that the binding site for these molecules is different. An Asp(158)Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affinity for the regulatory protein. The mutations that were found to decrease the affinity for the regulatory protein (Asn(166)Arg, Val(203)Ala, Asn(204)Gln, Lys(414)Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agreement with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme