Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain

The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.     

Stoichiometry of slow binding of palmitoyl-CoA to liver glucokinase

The interaction of palmitoyl-CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase "bound" up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl-CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839-12845). Sephacryl S-200 gel filtration in the presence of palmitoyl-CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (greater than or equal to 30) of associated palmitoyl-CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl-CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4-20 h), both the rat and pig enzyme were greatly inactivated (greater than 90%) in the presence of palmitoyl-CoA (15 microM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl-CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl-CoA over a long period of time.     

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.     

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K_m values around 8 mM for the substrate d-glucose with a preference for the α-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH₂-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with K_i values in the range of 1 μM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC₅₀ value of 9 μM for alloxan and of 60 μM for ninhydrin. d-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.     

Liberation and reassociation of the microsomal membrane glucokinase in cat liver]

The particulate glucokinase of cat liver is shown to be microsomal. The activity is readily solubilized by glucose-6-phosphate, ATP, pyrophosphate, high salt concentrations and, to a lesser extent, ribonucleoside triphosphates. The solubilization by glucose-6-phosphate is inhibited by Pi. Solubilizations by ATP and glucose-6-phosphate differ in their sensitivity to temperature changes; they are relatively specific for glucokinase as compared to solubilization by detergent (Triton X 100). The enzyme can be bound again to previously eluted microsomal membranes. Treatment of membrane with trypsin, at 0 degrees C, destroys the ability to rebind the enzyme to the membrane. It is suggested that electrostatic forces are of considerable importance for the binding of glucokinase to a possible protein binding site in the membrane.     

Anomeric specificity of human liver and B-cell glucokinase: modulation by the glucokinase regulatory protein

The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of d-glucose as tracers. With d-glucose at anomeric equilibrium and at 30 degrees C, the maximal velocity, Hill number, and K(s) amounted, respectively, to 16 micromol min(-1) mg(-1), 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 micromol min(-1) mg(-1), 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20-22 or 30 degrees C, the maximal velocity, Hill number, and K(m) were significantly lower with alpha-d-glucose than with beta-d-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with alpha-d-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM d-fructose 6-phosphate, the glucokinase regulatory protein caused a concentration-related inhibition of d-glucose phosphorylation, such an effect fading out at high concentrations of either d-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of alpha-d-glucose by liver glucokinase appeared more resistant than that of beta-d-glucose to the inhibitory action of d-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of alpha- and beta-d-glucose to the overall phosphorylation of equilibrated d-glucose by glucokinase.     

Distribution of adenosine 5'-triphosphate (ATP)-dependent hexose kinases in microorganisms.

A systematic study of adenosine triphosphate (ATP)-dependent hexose kinases among microorganisms has been undertaken. Sixteen hexose kinases of five major types were partially purified from 12 microorganisms and characterized with respect to specificity for sugar and nucleotide substrates and Michaelis constants for the sugar substrates. Glucokinase activities that phosphorylate glucose and glucosamine are inhibited by N-acetyl-glucosamine and xylose, were found to be present in the non-sulphur photosynthetic bacteria Rhodospirillum rubrum, the blue-green algae Anacystis montana, and the protists Chlorella pyrenoidosa and Chlamydomonas reinhardtii (green algae), Hypochytrium catenoides (Hypochytridiomycete) and Saprolegnia Iitoralis (Oomycete). The myxobacteria Stigmatella aurantiaca contains a glucokinase activity with a different specificity pattern. Anacystis and Chlorella, besides their glucokinase activities, contain highly specific fructokinases, although that from Anacystis can also phosphorylate fructosamine; fructokinase from Anacystis has a molecular weight of 20 000, and exhibits a sigmoidal saturation curve for ATP when the Mg2+/ATP ratio is 2; this curve is transformed to a Michaelian one when under the same conditions an excess of Mg2+ (5 mM) is added. Saprolegnia however, besides the glucokinase, contains a mannofructokinase activity that phosphorylates mannose (Km 0.06 mM) and fructose (1 mM). On the other hand, hexokinase, a low specificity enzyme, was detected in the protist Allomyces arbuscula (Chytridiomycete) and in fungi Mucor hiemalis and Phycomyces blakesleeanus (Zygomycetes), and Schizophyllum commune (Basidiomycete). Schizophyllum contains a glucomannokinase activity together with hexokinase activity. The pattern of distribution of ATP-dependent hexose kinases among microorganisms seems to parallel that reported for biosynthetic pathways for lysine. The correlation with other biochemical parameters is also considered.     

Bisphenol A Impairs Hepatic Glucose Sensing in C57BL/6 Male Mice

Aims/Hypothesis

Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly.

Methods

To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA’s purported “safe dose” (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1–20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above.

Results

Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002–0.029 at glucose 5–20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA.

Conclusions/Interpretation

Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.     

Purification and properties of adenosine 5′-triphosphate–D-glucose 6-phosphotransferase from rat liver

1. An 870-fold purification of glucokinase from rat liver is described which involves ammonium sulphate fractionation and the use of DEAE-Sephadex, DEAE-cellulose and polyacrylamide columns. 2. The preparation is free of any interfering enzymes and has a specific activity of 8mumoles/min./mg. of protein. 3. Glucokinase catalyses the phosphorylation of glucose, mannose and 2-deoxyglucose. 4. The enzyme is inhibited by high concentrations of glucose 6-phosphate only; ADP is an inhibitor whose effect depends on the Mg(2+) concentration. 5. The properties of glucokinase are compared briefly with those of other phosphotransferases.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

Maintenance of glucokinase activity in primary hepatocyte cultures

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low-K_M isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.     

The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.