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Several C-β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(β-d-glucopyranosyl)-imidazoles and 2-aryl-4-(β-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 μM; 13b = 4.58 μM). Ten C-β-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a–c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30–60 μM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.

The mechanisms by which metformin (dimethylbiguanide) inhibits hepatic gluconeogenesis at concentrations relevant for type 2 diabetes therapy remain debated. Two proposed mechanisms are 1) inhibition of mitochondrial Complex 1 with consequent compromised ATP and AMP homeostasis or 2) inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) and thereby attenuated transfer of reducing equivalents from the cytoplasm to mitochondria, resulting in a raised lactate/pyruvate ratio and redox-dependent inhibition of gluconeogenesis from reduced but not oxidized substrates. Here, we show that metformin has a biphasic effect on the mitochondrial NADH/NAD redox state in mouse hepatocytes. A low cell dose of metformin (therapeutic equivalent

Aim To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis. Methods C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge. Results Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-β), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-β mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2. Conclusions Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-β and both catalytic subunits of AMP-activated protein kinase.

Recent environmental sampling around a landfill site in the UK demonstrated that unidentified xenoestrogens were present at higher levels than control sites; that these xenoestrogens were capable of super-activating (resisting ligand-dependent antagonism) the murine variant 2 ERβ and that the ionic liquid 1-octyl-3-methylimidazolium chloride (M8OI) was present in some samples. To determine whether M8OI was a contributor to the xenoestrogen pool in the soils, activation of human estrogen receptors by M8OI was examined. M8OI activated the human ERα in MCF7 cells in a dose-response manner. These effects were inhibited by the ER antagonist ICI182780; occurred in the absence of any metabolism of M8OI and were confirmed on examination of ER-dependent induction of trefoil factor 1 mRNA in MCF7 cells. M8OI also super-activated the murine variant 2 ERβ in a murine hepatopancreatobiliary cell line. The human ERβ was not activated by M8OI when expressed in HEK293 cells. These data demonstrate that M8OI is a xenoestrogen capable of activating the human ERα and super-activating the murine variant 2 ERβ.

Aim Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. Materials and Methods We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. Results Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. Conclusions Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.

International audience; The chronic effects of metformin on liver gluconeogenesisinvolve repression of theG6pcgene, which is regulated by thecarbohydrate-response element–binding protein throughraised cellular intermediates of glucose metabolism. In thisstudy we determined the candidate mechanisms by which met-formin lowers glucose 6-phosphate (G6P) in mouse and rathepatocytes challenged with high glucose or gluconeogenic pre-cursors. Cell metformin loads in the therapeutic range loweredcell G6P but not ATP and decreasedG6pcmRNA at high glu-cose. The G6P lowering by metformin was mimicked by a com-plex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol)and by overexpression of mGPDH, which lowers glycerol3-phosphate and G6P and also mimics theG6pcrepression bymetformin. In contrast, direct allosteric activators of AMPK(A-769662, 991, and C-13) had opposite effects from metforminon glycolysis, gluconeogenesis, and cell G6P. The G6P loweringby metformin, which also occurs in hepatocytes from AMPKknockout mice, is best explained by allosteric regulation ofphosphofructokinase-1 and/or fructose bisphosphatase-1, assupported by increased metabolism of [3-3H]glucose relative to[2-3H]glucose; by an increase in the lactate m2/m1 isotopologratio from [1,2-13C2]glucose; by lowering of glycerol 3-phos-phate an allosteric inhibitor of phosphofructokinase-1; and bymarked G6P elevation by selective inhibition of phosphofruc-tokinase-1; but not by a more reduced cytoplasmic NADH/NADredox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glu-cose by stimulation of glycolysis by an AMP-activated proteinkinase–independent mechanism through changes in allostericeffectors of phosphofructokinase-1 and fructose bisphospha-tase-1, including AMP, Pi, and glycerol 3-phosphate

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Liver glucose metabolism is dependent on glucokinase activity. Glucokinase expression is transcriptionally regulated by hormones and metabolites of glucose, and glucokinase activity is dependent on reversible binding of glucokinase to a specific inhibitor protein, glucokinase regulatory protein (GKRP), and to other binding proteins such as 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK2/FBP2), which functions as an activator. Glucokinase is inhibited in the postabsorptive state by sequestration in the nucleus bound to GKRP, and it is activated postprandially by portal hyperglycemia and fructose through dissociation from GKRP, translocation to the cytoplasm, and binding to PFK2/FBP2. Glucagon dissociates this interaction, promoting translocation back to the nucleus. In humans, changes in glucokinase expression and activity are associated with poorly controlled type 2 diabetes and with nonalcoholic fatty liver disease, and a common variant of GKRP with altered binding affinity for glucokinase is associated with increased blood and liver lipids and other metabolic traits that implicate a role for GKRP in maintaining intrahepatic metabolite homeostasis.

Metformin therapy lowers blood glucose in type 2 diabetes by targeting various pathways including hepatic gluconeogenesis. Despite widespread clinical use of metformin the molecular mechanisms by which it inhibits gluconeogenesis either acutely through allosteric and covalent mechanisms or chronically through changes in gene expression remain debated. Proposed mechanisms include: inhibition of Complex 1; activation of AMPK; and mechanisms independent of both Complex 1 inhibition and AMPK. The activation of AMPK by metformin could be consequent to Complex 1 inhibition and raised AMP through the canonical adenine nucleotide pathway or alternatively by activation of the lysosomal AMPK pool by other mechanisms involving the aldolase substrate fructose 1,6-bisphosphate or perturbations in the lysosomal membrane. Here we review current interpretations of the effects of metformin on hepatic intermediates of the gluconeogenic and glycolytic pathway and the candidate mechanistic links to regulation of gluconeogenesis. In conditions of either glucose excess or gluconeogenic substrate excess, metformin lowers hexose monophosphates by mechanisms that are independent of AMPK-activation and most likely mediated by allosteric activation of phosphofructokinase-1 and/or inhibition of fructose bisphosphatase-1. The metabolite changes caused by metformin may also have a prominent role in counteracting G6pc gene regulation in conditions of compromised intracellular homeostasis.

Liver glycogen is synthesized after a meal in response to an increase in blood glucose concentration in the portal vein and endocrine and neuroendocrine signals, and is degraded to glucose between meals to maintain blood glucose homeostasis. Glycogen degradation and synthesis during the diurnal cycle are mediated by changes in the activities of phosphorylase and glycogen synthase. Phosphorylase is regulated by phosphorylation of serine-14. Only the phosphorylated form of liver phosphorylase (GPa) is catalytically active. Interconversion between GPa and GPb (unphosphorylated) is dependent on the activities of phosphorylase kinase and of phosphorylase phosphatase. The latter comprises protein phosphatase-1 in conjunction with a glycogen-targeting protein (G-subunit) of the PPP1R3 family. At least two of six G-subunits (GL and PTG) expressed in liver are involved in GPa dephosphorylation. GPa to GPb interconversion is dependent on the conformational state of phosphorylase which can be relaxed (R) or tense (T) depending on the concentrations of allosteric effectors such as glucose, glucose 6-phosphate and adenine nucleotides and on the acetylation state of lysine residues. The G-subunit, GL, encoded by PPP1R3B gene is expressed at high levels in liver and can function as a phosphorylase phosphatase and a synthase phosphatase and has an allosteric binding site for GPa at the C-terminus which inhibits synthase phosphatase activity. GPa to GPb conversion is a major upstream event in the regulation of glycogen synthesis by glucose, its downstream metabolites and extracellular signals such as insulin and neurotransmitters.

Type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with elevated hepatic glucose production and fatty acid synthesis (de novolipogenesis (DNL)). High carbohydrate diets also increase hepatic glucose production and lipogenesis. The carbohydrate-response element-binding protein (ChREBP, encoded byMLXIPL) is a transcription factor with a major role in the hepatic response to excess dietary carbohydrate. Because its target genes include pyruvate kinase (PKLR) and enzymes of lipogenesis, it is regarded as a key regulator for conversion of dietary carbohydrate to lipid for energy storage. An alternative hypothesis for ChREBP function is to maintain hepatic ATP homeostasis by restraining the elevation of phosphate ester intermediates in response to elevated glucose. This is supported by the following evidence: (i) A key stimulus for ChREBP activation and induction of its target genes is elevation of phosphate esters; (ii) target genes of ChREBP include key negative regulators of the hexose phosphate ester pool (GCKR,G6PC,SLC37A4) and triose phosphate pool (PKLR); (iii) ChREBP knock-down models have elevated hepatic hexose phosphates and triose phosphates and compromised ATP phosphorylation potential; (iv) gene defects inG6PCandSLC37A4and common variants ofMLXIPL,GCKRandPKLRin man are associated with elevated hepatic uric acid production (a marker of ATP depletion) or raised plasma uric acid levels. It is proposed that compromised hepatic phosphate homeostasis is a contributing factor to the elevated hepatic glucose production and lipogenesis that associate with type 2 diabetes, NAFLD and excess carbohydrate in the diet.

Figure S2. Additional animal analysis information. (DOCX 19408 kb)

In population studies hepatic steatosis in subjects with Non-alcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance. This association has encouraged debate whether hepatic steatosis is the cause or the consequence of hepatic insulin resistance? Although genome-wide studies have identified several gene variants associated with either hepatic steatosis or type 2 diabetes, no variants have been identified associated with both hepatic steatosis and insulin resistance. Here, the hypothesis is proposed that high-carbohydrate diets contribute to the association between hepatic steatosis and insulin resistance through activation of the transcription factor ChREBP (Carbohydrate response element binding protein). Postprandial hyperglycaemia raises the hepatic concentrations of phosphorylated intermediates causing activation of ChREBP and induction of its target genes. These include not only enzymes of glycolysis and lipogenesis that predispose to hepatic steatosis but also glucose 6-phosphatase (G6PC) that catalyses the final reaction in glucose production and GCKR, the inhibitor of hepatic glucokinase that curtails hepatic glucose uptake. Induction of G6PC and GCKR manifests as hepatic glucose intolerance or insulin resistance. Induction of these two genes by high glucose serves to safeguard intrahepatic homeostasis of phosphorylated intermediates. The importance of GCKR in this protective mechanism is supported by "less-active" GCKR variants in association not only with hepatic steatosis and hyperuricaemia but also with lower fasting plasma glucose and decreased insulin resistance. This supports a role for GCKR in restricting hepatic glucose phosphorylation to maintain intrahepatic homeostasis. Pharmacological targeting of the glucokinase-GCKR interaction can favour either glucose clearance by the liver or intrahepatic metabolite homeostasis.

Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials.We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D.Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes.Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.

OBJECTIVE The posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells. RESEARCH DESIGN AND METHODS Activity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models. RESULTS Nine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non–β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2. CONCLUSIONS Several GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.

Liver glycogen is synthesized in the postprandial state in response to elevated concentrations of glucose and insulin or by activation of neuroendocrine signals and it is degraded in the postabsorptive state in response to changes in the concentrations of insulin and counter-regulatory hormones. Dysregulation of either glycogen degradation or synthesis through changes in allosteric control or covalent modification of glycogen phosphorylase and glycogen synthase leads to disturbance of blood glucose homeostasis. Liver glycogen phosphorylase has a dual role in the control of glycogen metabolism by regulation of both glycogen degradation and synthesis. The phosphorylated form (GPa) is the active form and determines the rate of degradation of glycogen and it is also a potent allosteric inhibitor of the protein complex, involving the glycogen targeting protein G(L) and protein phosphatase-1, which catalyses dephosphorylation (activation) of glycogen synthase. Drug discovery programmes exploring the validity of glycogen phosphorylase as a therapeutic target for type 2 diabetes have generated a wide array of selective phosphorylase ligands that modulate the catalytic activity and / or the phosphorylation state (interconversion of GPa and GPb) as well as the binding of GPa to the allosteric site of G(L). Glycogen phosphorylase inhibitors that act in hepatocytes either exclusively by dephosphorylating GPa (e.g. indole carboxamides) or by allosteric inhibition of GPa (1,4-dideoxy-1,4-D-arabinitol) are very powerful experimental tools to determine the relative roles of covalent modification of glycogen phosphorylase and/or cycling between glycogen synthesis and degradation in the mechanism(s) by which insulin and neurotransmitters regulate hepatic glycogen metabolism.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 μM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100–140 μM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.

Type-2 diabetes is associated with impaired glucose clearance by the liver in the postprandial state, and with elevated glucose production in the post-absorptive state. New targets within the liver are currently being investigated for development of antihyperglycaemic drugs for type-2 diabetes. They include glucokinase, which catalyses the first step in glucose metabolism, the glucagon receptor, and enzymes of gluconeogenesis and/or glycogenolysis such as glucose 6-phosphatase, fructose 1,6-bisphosphatase and glycogen phosphorylase. Preclinical studies with candidate drugs on animal models or cell-based assays suggest that these targets have the potential for pharmacological glycaemic control. Data from clinical studies is awaited. Further work is required for better understanding of the implications of targeting these sites in terms of possible side-effects or tachyphylaxis. The advantage of combined targeting of two or more sites within the liver for minimizing side-effects and tachyphylaxis caused by single-site targeting is discussed.

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P2) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P2content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P2levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P2. Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.

Parasympathetic (cholinergic) innervation is implicated in the stimulation of hepatic glucose uptake by portal vein hyperglycaemia. We determined the direct effects of acetylcholine on hepatocytes. Acute exposure to acetylcholine mimicked insulin action on inactivation of phosphorylase, stimulation of glycogen synthesis and suppression of phosphoenolpyruvate carboxykinase mRNA levels but with lower efficacy and without synergy. Pre-exposure to acetylcholine had a permissive effect on insulin action similar to glucocorticoids and associated with increased glucokinase activity. It is concluded that acetylcholine has a permissive effect on insulin action but cannot fully account for the rapid stimulation of glucose uptake by the portal signal.

Mutations in the glucokinase (GK) gene cause defects in blood glucose homeostasis. In some cases (V62M and G72R), the phenotype cannot be explained by altered enzyme kinetics or protein instability. We used transient and stable expression of green fluorescent protein (GFP) GK chimaeras in MIN6 β-cells to study the phenotype defect of V62M and G72R. GK activity in lysates of MIN6 cell lines stably expressing wild-type or mutant GFP GK showed the expected affinity for glucose and response to pharmacological activators, indicating the expression of catalytically active enzymes. MIN6 cells stably expressing GFP V62M or GFP G72R had a lower GK activity–to–GK immunoreactivity ratio and GK activity–to–GK mRNA ratio but not GK immunoreactivity–to–GK mRNA ratio than wild-type GFP GK. Heterologous expression of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2/FDP2) in cell lines increased GK activity for wild-type GK and V62M but not for G72R, whereas expression of liver GK regulatory protein (GKRP) increased GK activity for wild type but not V62M or G72R. Lack of interaction of these mutants with GKRP was also evident in hepatocyte transfections from the lack of nuclear accumulation. These results suggest that cellular loss of GK catalytic activity rather than impaired translation or enhanced protein degradation may account for the hyperglycemia in subjects with V62M and G72R mutations.

Intraportal infusion of serotonin (5-hydroxytryptamine, 5-HT) or inhibitors of its cellular uptake stimulate hepatic glucose uptake in vivo by either direct or indirect mechanisms. The aims of this study were to determine the direct effects of 5-HT in hepatocytes and to test the hypothesis that atypical antipsychotic drugs that predispose to type 2 diabetes counter-regulate the effects of 5-HT.Rat hepatocytes were studied in short-term primary culture.Serotonin (5-HT) stimulated glycogen synthesis at nanomolar concentrations but inhibited it at micromolar concentrations. The stimulatory effect was mimicked by alpha-methyl-5-HT, a mixed 5-HT1/5-HT2 receptor agonist, whereas the inhibition was counteracted by a 5-HT2B/2C receptor antagonist. alpha-Methyl-5-HT stimulated glycogen synthesis additively with insulin, but unlike insulin, did not stimulate glucose phosphorylation and glycolysis, nor did it cause Akt (protein kinase B) phosphorylation. Stimulation of glycogen synthesis by alpha-methyl-5-HT correlated with depletion of phosphorylase a. This effect could not be explained by elevated levels of glucose 6-phosphate, which causes inactivation of phosphorylase, but was explained, at least in part, by decreased phosphorylase kinase activity in situ. The antipsychotic drugs clozapine and olanzapine, which bind to 5-HT receptors, counteracted the effect of alpha-methyl-5-HT on phosphorylase inactivation.This study provides evidence for both stimulation and inhibition of glycogen synthesis in hepatocytes by serotonergic mechanisms. The former effects are associated with the inactivation of phosphorylase and are counteracted by atypical antipsychotic drugs that cause hepatic insulin resistance. Antagonism of hepatic serotonergic mechanisms may be a component of the hepatic dysregulation caused by antipsychotic drugs that predispose to type 2 diabetes.

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues I and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis. (c) 2006 Elsevier Ltd. All rights reserved. Bioorg Med Chem

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

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.

An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.

Glucokinase and phosphorylase both have a high control strength over hepatocyte glycogen metabolism and are potential therapeutic targets for type 2 diabetes. We tested whether combined phosphorylase inactivation and glucokinase activation is a more effective strategy for controlling hepatic glycogen metabolism than single-site targeting. Activation of glucokinase by enzyme overexpression combined with selective dephosphorylation of phosphorylase-a by an indole carboxamide that favors the T conformation of phosphorylase caused a greater stimulation of glycogen synthesis than the sum of either treatment alone. This result is explained by the complementary roles of elevated glucose-6-phosphate (G6P; a positive modulator) and depleted phosphorylase-a (a negative modulator) in activating glycogen synthase and also by synergistic inactivation of phosphorylase-a by glucokinase activation and the indole carboxamide. Inactivation of phosphorylase-a by the indole carboxamide was counteracted by 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside, which is metabolized to an AMP analog; this effect was reversed by G6P. Our findings provide further evidence for the inverse roles of G6P and AMP in regulating the activation state of hepatic phosphorylase. It is proposed that dual targeting of glucokinase and phosphorylase-a enables improved potency and efficacy in controlling hepatic glucose metabolism.

The inhibition of glucokinase by rat and Xenopus GKRPs (glucokinase regulatory protein) is well documented. We report a comparison of the effects of human and rat GKRPs on glucokinase activity. Human GKRP is a more potent inhibitor of glucokinase than rat GKRP in the absence of fructose 6-phosphate or sorbitol 6-phosphate, and has a higher affinity for these ligands. However, human and rat GKRPs have similar affinities for fructose 1-phosphate and chloride. Residues that are not conserved between the rodent and human proteins affect both the affinity for fructose 6-phosphate and sorbitol 6-phosphate and the inhibitory potency of GKRP on glucokinase in the absence of these ligands.

The role of glucose 6-P (glucose 6-phosphate) in regulating the activation state of glycogen synthase and its translocation is well documented. In the present study, we investigated the effects of glucose 6-P on the activation state and compartmentation of phosphorylase in hepatocytes. Glucose 6-P levels were modulated in hepatocytes by glucokinase overexpression or inhibition with 5-thioglucose and the effects of AMP were tested using AICAR (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside), which is metabolized to an AMP analogue. Inhibition of glucokinase partially counteracted the effect of glucose both on the inactivation of phosphorylase and on the translocation of phosphorylase a from a soluble to a particulate fraction. The increase in glucose 6-P caused by glucokinase overexpression caused translocation of phosphorylase a to the pellet and had additive effects with glucose on inactivation of phosphorylase. It decreased the glucose concentration that caused half-maximal inactivation from 20 to 11 mM, indicating that it acts synergistically with glucose. AICAR activated phosphorylase and counteracted the effect of glucose 6-P on phosphorylase inactivation. However, it did not counteract translocation of phosphorylase by glucose 6-P. Glucose 6-P and AICAR had opposite effects on the activation state of glycogen synthase, but they had additive effects on translocation of the enzyme to the pellet. There was a direct correlation between the translocation of phosphorylase a and of glycogen synthase to the pellet, suggesting that these enzymes translocate in tandem. In conclusion, glucose 6-P causes both translocation of phosphorylase and inactivation, indicating a more complex role in the regulation of glycogen metabolism than can be explained from regulation of glycogen synthase alone.

High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase.

The concept of activation of glucokinase (encoded by the Gck gene) as a potential therapy for type 2 diabetes has been explored by several pharmaceutical companies. Small-molecule Gck activators (GKAs) were found to be effective at increasing glucose disposal by hepatocytes and lowering blood glucose in animal models of diabetes during acute or chronic exposure and in human type 2 diabetes during short-term exposure. However, several clinical trials of GKAs were discontinued because of declining efficacy during chronic exposure or other issues. In some cases, declining efficacy was associated with an increase in plasma triglycerides. Accordingly, increased hepatic triglyceride production or steatosis was inferred as the likely cause for declining efficacy. However, other mechanisms of tachyphylaxis need to be considered. For example, elevated glucose concentration causes induction of glucose 6-phosphatase (G6pc) and repression of Gck in hepatocytes. This is best explained as an adaptative mechanism to maintain intracellular phosphometabolite homeostasis. Enhancement of G6pc induction and Gck repression by GKAs because of perturbed phosphometabolite homeostasis could explain the decline in GKA efficacy during chronic exposure. Progress in understanding the mechanisms of intracellular phosphometabolite homeostasis is crucial for development of better drug therapies and appropriate dietary intervention for type 2 diabetes.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

We investigated whether hepatocytes permeabilized with alpha-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2, 6-bisphosphate [Fru(2,6)P(2)], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P(2), rates of detritiation of [2-(3)H]glucose and [3-(3)H]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1, 6)P(2), decreased the rate of detritiation of [3-(3)H]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-(3)H]glucose or [3-(3)H]Glc6P as labelled substrate, there was dilution of metabolism of [3-(3)H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P(2). They provide an alternative explanation for the concept that there is more than one Glc6P pool.

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.

Glucokinase (hexokinase IV) has a major role in the control of blood glucose homeostasis because it is the predominant hexokinase expressed in the liver, has a very high control strength on hepatic glucose disposal (1), and is the glucose sensor for insulin secretion in pancreatic β-cells (2). Glucokinase is currently considered a strong candidate target for antihyperglycemic drugs for type 2 diabetes (2–4). This is supported by the impact of mutations in the glucokinase gene on blood glucose concentration in humans. Inactivating mutations that lower the enzyme affinity for glucose or compromise glucokinase expression cause diabetes (maturity onset diabetes of the young type 2), whereas activating mutations lower blood glucose (2). Pharmacological activators of glucokinase (GKAs) that mimic the effect of activating mutations represent a potential novel strategy for antihyperglycemic therapy (2–4). Type 2 diabetes is associated with defective regulation of hepatic glucose metabolism, involving elevated glucose production in euglycemic conditions and subnormal clearance of glucose by the liver after a meal because of delayed suppression of hepatic glucose production and impaired conversion of glucose to glycogen (5,6). This inefficient clearance of glucose by the liver is due at least in part to impaired regulation of glucose production and usage by hyperglycemia, a process sometimes described as decreased “autoregulation” or “glucose effectiveness” (7). Whether the defect in diabetes in humans involves decreased glucokinase activity is not established. Hepatic glucokinase activity was shown to be either elevated in newly diagnosed type 2 diabetic patients (8) or decreased in obese subjects with diabetes (9). Hepatic glucokinase is regulated by an inhibitory protein (glucokinase regulatory protein [GKRP]) that binds glucokinase with high affinity at basal glucose concentrations (5 mmol/l) and sequesters glucokinase in the nucleus in an inactive state (1,10). In the postprandial state, …

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.

In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)1 have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.

The compartmentation of glucokinase in the hepatocyte is regulated by the extracellular glucose concentration and by substrates that alter the concentration of fructose 1-phosphate in the hepatocyte. At low glucose concentrations, that mimic the fasted state, glucokinase is sequestered in an inactive state bound to the 68 kDa regulatory protein in the nucleus. In these conditions the rate of glucose phosphorylation is less than 15% of the total glucokinase activity. An increase in extracellular glucose concentration, within the range occurring in the portal vein in the absorptive state, or low concentrations of fructose or sorbitol (precursors of fructose 1-phosphate), cause the translocation of glucokinase from the nucleus to the cytoplasm and this is associated with a corresponding increase in glucose phosphorylation. The effect of glucose on translocation is mimicked by mannose which is also phosphorylated by glucokinase as well as by competitive inhibitors of glucokinase (mannoheptulose and 5-thioglucose) which are not phosphorylated. Various lines of evidence suggest that the action of these analogues is most likely due to binding to an allosteric or non-catalytic site. The saturation curve of glucose phosphorylation in intact hepatocytes is sigmoidal with an S0.5 of approximately 20 mM and a Hill coefficient approximately 2. This saturation curve can be explained by the activity of glucokinase in the cytoplasmic compartment. Translocation of glucokinase from the nucleus to the cytoplasm in response to precursors of fructose 1-phosphate (which cause dissociation of glucokinase from the regulatory protein) is associated with stimulation of glucose phosphorylation, glycolysis and glycogen synthesis. Using Metabolic Control Analysis to determine the Control Coefficient (Control Strength) of cytoplasmic (free) glucokinase on glucose metabolism it can be shown that the free glucokinase activity has a very high control strength on glycogen synthesis (CFGKJ > 1), indicating a major role of translocation of glucokinase in the control of hepatic glycogen synthesis. Overexpression of glucokinase in hepatocytes by adenovirus-mediated glucokinase overexpression is associated with a marked increase in glycogen synthesis. The relation between glycogen synthesis and enzyme overexpression is sigmoidal with an enzyme concentration causing half-saturation (S0.5) in the physiological range. The high Control Coefficient of glucokinase on hepatic glycogen synthesis explains the abnormalities of hepatic glycogen synthesis in patients with a single mutant allele of the glucokinase gene (Maturity Onset Diabetes of the Young, type 2).

Two important factors that determine the flux of hepatic β-oxidation of long-chain fatty acids are the availability of fatty acid and the activity of carnitine palmitoyltransferase I (CPT I). Using Metabolic Control Analysis, the flux control coefficient of CPT I in rat hepatocyte monolayers was determined by titration with 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir), which is converted to Etomoxir-CoA, an irreversible inhibitor of CPT I. We measured CPT I activity and flux through β-oxidation at 0.2 mM and 1.0 mM palmitate to simulate substrate concentrations in fed and fasted states. Rates of β-oxidation were 4.5-fold higher at 1.0 mM palmitate compared with 0.2 mM palmitate. Flux control coefficients of CPT I, estimated by two independent methods, were similar: 0.67 and 0.79 for 0.2 mM palmitate, and 0.68 and 0.77 for 1 mM palmitate. It is concluded that the regulatory potential of CPT I is similar at low and high physiological concentrations of palmitate.