Glucagon induces translocation of glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2 and the glucokinase regulatory protein

Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25 mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.     

The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.     

Rapid translocation of hepatic glucokinase in response to intraduodenal glucose infusion and changes in plasma glucose and insulin in conscious rats

The rate of liver glucokinase (GK) translocation from the nucleus to the cytoplasm in response to intraduodenal glucose infusion and the effect of physiological rises of plasma glucose and/or insulin on GK translocation were examined in 6-h-fasted conscious rats. Intraduodenal glucose infusion (28 mg.kg(-1).min(-1) after a priming dose at 500 mg/kg) elevated blood glucose levels (mg/dl) in the artery and portal vein from 90 +/- 3 and 87 +/- 3 to 154 +/- 4 and 185 +/- 4, respectively, at 10 min. At 120 min, the levels had decreased to 133 +/- 6 and 156 +/- 5, respectively. Plasma insulin levels (ng/ml) in the artery and the portal vein rose from 0.7 +/- 0.1 and 1.8 +/- 0.3 to 11.8 +/- 1.5 and 20.2 +/- 2.0 at 10 min, respectively, and 12.4 +/- 3.1 and 18.0 +/- 4.8 at 30 min, respectively. GK was rapidly exported from the nucleus as determined by measuring the ratio of the nuclear to the cytoplasmic immunofluorescence (N/C) of GK (2.9 +/- 0.3 at 0 min to 1.7 +/- 0.2 at 10 min, 1.5 +/- 0.1 at 20 min, 1.3 +/- 0.1 at 30 min, and 1.3 +/- 0.1 at 120 min). When plasma glucose (arterial; mg/dl) and insulin (arterial; ng/ml) levels were clamped for 30 min at 93 +/- 7 and 0.7 +/- 0.1, 81 +/- 5 and 8.9 +/- 1.3, 175 +/- 5 and 0.7 +/- 0.1, or 162 +/- 5 and 9.2 +/- 1.5, the N/C of GK was 3.0 +/- 0.5, 1.8 +/- 0.1, 1.5 +/- 0.1, and 1.2 +/- 0.1, respectively. The N/C of GK regulatory protein (GKRP) did not change in response to the intraduodenal glucose infusion or the rise in plasma glucose and/or insulin levels. The results suggest that GK but not GKRP translocates rapidly in a manner that corresponds with changes in the hepatic glucose balance in response to glucose ingestion in vivo. Additionally, the translocation of GK is induced by the postprandial rise in plasma glucose and insulin.     

Activation and translocation of glucokinase in rat primary hepatocytes monitored by high content image analysis

In the liver, glucokinase (GK) regulatory protein (GKRP) negatively modulates the metabolic enzyme GK by locking it in an inactive state in the nucleus. Here, the authors established a high content screening assay in the 384-well microplate format to measure the nucleus-to-cytoplasm translocation of GK by reagents that destabilize the interaction between GK and GKRP. As a cellular model system, primary rat hepatocytes endogenously expressing both GK and GKRP at physiological levels were used. The GK translocation assay was robust, displayed limited day-to-day variability, and delivered good Z' statistics. The increase of the glucose concentration in the extracellular medium from a low glucose situation (2.8 mM) to beyond its physiological set point value of 5 mM was found to drive GK from the nucleus into the cytoplasm. Likewise, both fructose (converted intracellularly into fructose-1-phosphate) and a known allosteric GK activator were found to induce the export of GK from the nucleus and to synergistically enhance the effects of medium or high glucose concentrations with respect to GK translocation. Transfer of the high content screening format to a semiautomated medium throughput screening platform enabled the profiling of large compound numbers with respect to allosteric activation of GK.     

Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone

The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase.     

Transport of macromolecules between the nucleus and the cytoplasm.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.     

Regulation of development of hepatic glucokinase in the neonatal rat by the diet

1. Feeding a high-glucose diet to weanling rats showed that high hepatic glucokinase activities could be induced at 18 days of age, i.e. 2 days after development of the enzyme begins. 2. The normal development of glucokinase activity can be retarded by weaning rats on to carbohydrate-free, high-fat and high-protein diets. 3. Precocious development of the enzyme before 16 days of age cannot be induced by oral glucose administration. 4. It is concluded that the ability to synthesize glucokinase develops very rapidly and that the nature of the diet determines the normal developmental pattern.     

Regulation of hepatic glucose metabolism by leptin in pig and rat primary hepatocyte cultures

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phosphoenolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% (P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease (P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.     

Transport between the cytoplasm and the nucleus

Summary Active transport of proteins and RNAs across the nuclear-pore complex (NPC) is mediated by a family of related transport receptors which shuttle between the cytoplasm and the nucleoplasm. A number of import and export pathways have been described. Some transport substrates require adapters which mediate association with certain transporters. The transport receptors specifically bind to a recognition signal within the transport substrate or adapter, pass the NPC in one direction, and deliver their cargo to the other side of the nuclear envelope. The Ran GTPase is the crucial regulator of bidirectional transport. Ran-modulating proteins establish an asymmetric intracellular distribution of Ran. As a result, Ran is mainly bound to GTP in the nucleus and to GDP in the cytoplasm. Evidently, RanGTP regulates binding and release of the transport substrates by binding to the transport receptors in the nucleus as well as the transport direction across the NPC. However, little is known about the molecular mechanism of translocation through the NPC.     

An allosteric activator of glucokinase impairs the interaction of glucokinase and glucokinase regulatory protein and regulates glucose metabolism

Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V(max)) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.     

Transport pathways of macromolecules between the nucleus and the cytoplasm.

Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.     

Inhibition of glucokinase in hepatocytes by alloxan

The effect of alloxan on glucokinase in isolated rat hepatocytes was studied. Exposure of hepatocytes to alloxan (3 mM) at 30 degrees C for 5 min produced a marked inhibition (77%) of glucokinase activity and altered slightly the phosphofructokinase activity (32% inhibition). Pyruvate kinase and glucose 6-phosphate dehydrogenase, however, were not inhibited at all. Alloxan induced a concentration-dependent inhibition of glucokinase activity with a detectable inhibition at an alloxan concentration of 1 mM. The inhibition of glucokinase activity by alloxan was protected by the simultaneous presence of 15 mM hexose such as D-glucose, 3-O-methylglucose, or D-mannose. D-Galactose showed no protective effect. These results suggest that alloxan may exert its cytotoxic action through the inhibition of glucokinase activity not only in the liver but also in the pancreatic islets, since liver and islet glucokinases are known to be quite similar in various properties.     

Evidence for the presence of rat liver glucokinase in the nucleus as well as in the cytoplasm

Subcellular distribution of glucokinase was studied in rat liver. With an immunohistochemical procedure, glucokinase immunoreactivity was clearly shown in the nucleus of parenchymal cells of rat liver, but faintly in the cytoplasm. Nuclei, cytosol (extranuclear fraction in the strict sense), and homogenate prepared in nonaqueous medium, i.e. glycerol, were analyzed for glucokinase by both immunoblotting and activity measurement. Such analyses demonstrated that glucokinase concentration was far higher in the nuclei than in the cytosol when compared on the basis of total protein content and that total glucokinase activity in the cytosolic fraction was about 1.8 times that in the nuclear fraction. These results indicate that hepatocyte glucokinase is present in the nucleus as well as in the cytoplasm in contradiction to the widespread belief of its exclusive localization in the cytoplasm.     

Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus.

The Mx2 protein of rats is a cytoplasmic GTPase that protects cells against vesicular stomatitis virus but not against influenza virus. Since vesicular stomatitis virus replicates in the cytoplasm and influenza virus replicates in the nucleus, it was possible that the antiviral specificity of rat Mx2 protein was determined solely by the protein's subcellular localization. Here, we found that, indeed, rat Mx2 protein lost its anti-vesicular stomatitis virus activity and gained anti-influenza virus activity when it was directed to the nucleus by way of a foreign nuclear-transport signal appended to its amino terminus. These data show that rat Mx2 protein possesses an antiviral activity that is revealed only when the protein is shuttled to the nucleus.     

Molecular coordination of hepatic glucose metabolism by the 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase:glucokinase complex

Glucokinase (GK) and 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBP-2) are each powerful regulators of hepatic carbohydrate metabolism that have been reported to influence each other's expression, activities, and cellular location. Here we present the first physical evidence for saturable and reversible binding of GK to the FBP-2 domain of PFK-2/FBP-2 in a 1:1 stoichiometric complex. We confirmed complex formation and stoichiometry by independent methods including affinity resin pull-down assays and fluorescent resonance energy transfer. All suggest that the binding of GK to PFK-2/FBP-2 is weak. Enzymatic assays of the GK:PFK-2/FBP-2 complex suggest a concomitant increase of the kinase-to-bisphosphatase ratio of bifunctional enzyme and activation of GK upon binding. The kinase-to-bisphosphatase ratio is increased by activation of the PFK-2 activity whereas FBP-2 activity is unchanged. This means that the GK-bound PFK-2/FBP-2 produces more of the biofactor fructose-2,6-bisphosphate, a potent activator of 6-phosphofructo-1-kinase, the committing step to glycolysis. Therefore, we conclude that the binding of GK to PFK-2/FBP-2 promotes a coordinated up-regulation of glucose phosphorylation and glycolysis in the liver, i.e. hepatic glucose disposal. The GK:PFK-2/FBP-2 interaction may also serve as a metabolic signal transduction pathway for the glucose sensor, GK, in the liver. Demonstration of molecular coordination of hepatic carbohydrate metabolism has fundamental relevance to understanding the function of the liver in maintaining fuel homeostasis, particularly in managing excursions in glycemia produced by meal consumption.