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  • A key limiting factor in the

    2022-05-25

    A key limiting factor in the semi-quantitative analysis of changes in the nuclear-to-cytoplasmic distribution of glucokinase is the large intercellular heterogeneity of GSK461364 synthesis of glucokinase in both isolated hepatocytes in vitro and also in liver in vivo[13], [23], [24], [26], [36]. This necessitates the analysis of large numbers of cells. To date, two methods have been used for semi-quantitative analysis of glucokinase translocation: either based on the determination of the ratio of cellular nuclear-to-cytoplasmic (N/C) mean pixel intensity [23], [24], [25] or from the automated nuclear or cytoplasmic intensity multiplied by the total cytoplasmic or nuclear area estimated with an independent stain [13], [26], [37]. We show in this study from the analysis of N/C ratio normalised per cell that glucagon antagonises the effect of elevated glucose (10, 15 and 25mM) on glucokinase translocation but has no significant effect at basal glucose concentration (5mM). We also confirm that the N/C ratio is a more accurate assessment of translocation than the mean nuclear or cytoplasmic intensity based on cell outline methods consistent with a previous study [24]. The failure to detect the effects of glucagon on glucokinase translocation in recent studies [13] could be explained by the low glucose concentration used, as well as the exclusive analysis of global nuclear or cytoplasmic intensity. We show in this study that the counter-regulatory effect of glucagon on glucose-induced translocation of glucokinase is abolished by glucagon receptor antagonists and mimicked by non-metabolisable cyclic-AMP analogues. We used the proximity ligation assay (PLA) for the quantification of the protein–protein interaction of glucokinase with either GKRP or PFK2/FBPase2. This assay enables the visualisation of two endogenously expressed proteins that are in close proximity such that the antibody-linked oligonucleotide tag can be ligated [18]. Using this assay we show that at low glucose concentration the interaction between glucokinase and GKRP occurred predominately (though not exclusively) in the nucleus, consistent with the high nuclear to cytoplasmic ratio of GKRP [24], [40]. We also show that elevated glucose decreases the glucokinase–GKRP interaction, with concomitant translocation of glucokinase from the nuclear to cytoplasmic compartment and increased binding between glucokinase and PFK2/FBPase2, an interaction that occurs predominately (though not exclusively) in the cytoplasm. Glucokinase has a bilobal structure linked by a flexible hinge [41] and exists as an equilibrium of conformational states ranging from a wide-open state at low glucose concentration to a closed conformation at high glucose [42], [43]. Glucokinase binds to GKRP in the wide-open state [44], [45] and it binds to PFK2/FBPase2 most likely in the closed conformation [46]. The converse effects of elevated glucose on the interaction of glucokinase with GKRP and PFK2/FBPase2 are therefore consistent with the expected binding to these two proteins. Interestingly, glucagon caused complete reversal of the effect of elevated glucose on both the glucokinase–GKRP and glucokinase–PFK2/FBPase2 interactions (Fig. 5) despite causing only partial reversal of the effect of glucose on the distribution of glucokinase between the cytoplasm and nucleus (Fig. 1). It is noteworthy that whilst immunostaining provides a global measure of the distribution of glucokinase between the nuclear and cytoplasmic compartments, the PLA assay is a measure of two proteins in close proximity and in an orientation that allows the antibody tags to form a complex. Therefore, the signal detected by the PLA assay most likely represents a subset of the glucokinase molecules. In principle, glucokinase may bind simultaneously to more than one protein. For example, shuttling between the cytoplasm and nucleus may involve interaction with additional shuttling proteins, which may mask the epitopes to PFK2/FBPase2 or GKRP or alternatively force the complex into a more or less favourable orientation for ligation of the complementary tags. The marked effect of glucagon in reversing the effects of elevated glucose on both the glucokinase–PFK2/FBPase2 interaction and the glucokinase–GKRP interaction is compelling evidence for hormonal control of glucokinase shuttling through interactions with these partners.