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PubMed1994

Control of glucokinase translocation in rat hepatocytes by sorbitol and the cytosolic redox state.

In rat hepatocytes cultured in 5 mM glucose, glucokinase activity is present predominantly in a bound state, and during permeabilization of the cells with digitonin in the presence of Mg2+ less than 20% of glucokinase activity is released. However, incubation of hepatocytes with a higher [glucose] [concn. giving half-maximal activation (A50) 15 mM] or with fructose (A50 50 microM) causes translocation of glucokinase from its Mg(2+)-dependent binding site to an alternative site [Agius and Peak (1993) Biochem. J. 296, 785-796]. A comparison of various substrates showed that sorbitol (A50 8 microM) was 6-fold more potent than fructose at causing glucokinase translocation, whereas tagatose was as potent and mannitol was > 10-fold less potent (A50 550 microM). These substrates also stimulate glucose conversion into glycogen with a similar relative potency, suggesting that conversion of glucose into glycogen is dependent on the binding and/or location of glucokinase within the hepatocyte. Ethanol and glycerol inhibited the effects of fructose, sorbitol and glucose on glucokinase translocation, whereas dihydroxy-acetone had a small additive effect at sub-maximal substrate stimulation. The converse effects of glycerol and dihydroxy-acetone suggest a role for the cytosolic NADH/NAD+ redox state in controlling glucokinase translocation. Titrations with three competitive inhibitors of glucokinase did not provide evidence for involvement of glucokinase flux in glucose-induced glucokinase translocation: N-acetylglucosamine inhibited glucose conversion into glycogen, but not glucose-induced glucokinase translocation; glucosamine partially suppressed glucose-induced and fructose-induced glucokinase translocation, at concentrations that caused total inhibition of glucose conversion into glycogen; D-mannoheptulose increased glucokinase release and had an additive effect with glucose. 3,3'-Tetramethylene-glutaric acid (5 mM), an inhibitor of aldose reductase, inhibited glucokinase translocation induced by glucose, but not that by sorbitol or fructose, suggesting that glucose may induce glucokinase translocation by conversion into sorbitol. Sorbitol generated from glucose intrahepatically or extrahepatically in hyperglycaemic conditions may be a physiological regulator of hepatic glucokinase translocation.

Animals↗

PubMed1996

Evidence for a role of glucose-induced translocation of glucokinase in the control of hepatic glycogen synthesis.

Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximum binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocyte in situ, by adenovirus-mediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokinase overexpression or in free glucokinase activity. Glucokinase overexpression is associated with an increase in both free and bound activity, with an overall decrease in the proportion of bound activity. In hepatocytes incubated at low [glucose] (0-5 mM), glucokinase binding involves a high-affinity binding site with a Kd of approximately 0.1 microM and a binding capacity of approximately 3 pmol/mg total cell protein and low-affinity binding with a Kd of approximately 1.6 microM. Increasing glucose concentration to 20 mM causes a dose-dependent increase in the Kd of the high- affinity site to approximately 0.6 microM, and this effect was mimicked by 50 microM sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase. Glycogen synthesis determined from the incorporation of [2-3H,U-14C]glucose into glycogen at 5 mM or 10 mM glucose was very sensitive to small increases in total glucokinase activity and correlated more closely with the increase in free glucokinase activity. The relation between glycogenic flux and glucokinase activity is sigmoidal. Expression of the sensitivity of glycogen synthesis to glucokinase activity as the control coefficient reveals that the coefficient is greater for the incorporation of 2-tritium (which occurs exclusively by the direct pathway) than for incorporation of 14C label (which involves direct and indirect pathways) and is greater at 5 mM glucose (when glucokinase is maximally sequestered at its high-affinity site) than at 10 mM glucose. The results support the hypothesis that compartmentation of glucokinase in the hepatocyte increases the sensitivity of glycogen synthesis to small changes in total glucokinase activity and that glucose-induced translocation of glucokinase has a major role in the acute control of glycogen synthesis.

Animals↗

PubMed1994

Initial expression of glucokinase gene in cultured hepatocytes from suckling rats is linked to the synthesis of an insulin-dependent protein.

The initial accumulation of glucokinase mRNA in response to insulin in cultured hepatocytes from 10-day-old suckling rats was characterized by a delay of 18-24 h with a maximal level reached after 48 h. This delay is not observed in cultured adult rat hepatocytes. When hepatocytes from 10-day-old suckling rats were cultured for 48 h in the presence of insulin (to obtain a maximal accumulation of glucokinase mRNA) and then deprived of insulin for 18 h, glucokinase mRNA returned to very low levels. Reexposure of these cultured hepatocytes to insulin allowed a rapid accumulation of glucokinase mRNA, with a maximal level reached after 8 h, as in adult rat hepatocytes. The aim of the present study was to investigate the factors responsible for the delay in insulin action during first exposure to insulin. The difference in the kinetics of glucokinase mRNA accumulation after the first and secondary exposure to insulin was due to differences in the rate of transcriptional activity of the glucokinase gene, as shown by a run-on assay on isolated nuclei. The half-life of glucokinase mRNA was similar after the first and second exposure to insulin. The delay in the initial accumulation of glucokinase mRNA in response to the first exposure to insulin was not due to elevated levels of cAMP (a potent inhibitor of glucokinase gene expression) or to a defect in insulin signalling (insulin inhibited without delay phosphoenolpyruvate carboxykinase gene expression). In contrast, it was markedly dependent upon whether glucokinase has been already expressed in vivo. Hepatocytes from rats that had already expressed glucokinase in vivo (suckling rats force-fed with glucose or rats weaned to a high-carbohydrate diet) showed no delay in their response to insulin in culture, whereas hepatocytes from rats that have never expressed glucokinase in vivo (suckling rats or rats weaned to a high-fat diet) showed a delay of 24 h. Two different inhibitors of protein synthesis (cycloheximide and puromycin) prevented the initial accumulation of glucokinase mRNA in response to the first exposure to insulin but not to the secondary accumulation of glucokinase mRNA in response to reexposure to insulin. This suggests that the synthesis of one or several insulin-dependent proteins is necessary for the first activation of glucokinase gene transcription in response to the first exposure to insulin.

Aging↗

PubMed1989

An alternate promoter in the glucokinase gene is active in the pancreatic beta cell.

An alternate promoter in the glucokinase gene is active in the beta cell and produces a glucokinase mRNA which is longer and that has a different leader sequence and translation start site than the hepatic glucokinase mRNA. The glucokinase beta cell promoter is located at least 12 kilobases upstream from the glucokinase hepatic promoter. Transcription from the glucokinase beta cell promoter initiates over a region of 62 bases. The absence of a TATA box homology in the proximal promoter region may account for the diffuse transcriptional initiation. Translation of the beta cell glucokinase mRNA predicts a glucokinase isozyme that is different from the hepatic isozyme by 15 amino acids at the N terminus. The use of alternative promoters apparently enables the glucokinase gene to be regulated by insulin in the liver and by glucose in the beta cell, thus possibly constituting an important feedback control loop for maintaining glucose homeostasis. Alternate RNA splicing of the beta cell glucokinase mRNA predicts at least two beta cell glucokinase isoforms. An alternate splice acceptor site in the 4th exon of the glucokinase gene was identified in two glucokinase cDNAs from rat insulinoma tissue. Use of the alternate splice acceptor site results in a 51-nucleotide in frame deletion in the beta cell glucokinase mRNA and removal of 17 amino acids from a region of the protein situated between the putative glucose and ATP binding domains. Analysis of the pattern of RNA splicing in tissues containing beta cells indicates that the splice acceptor site utilized in producing hepatic glucokinase mRNA is also utilized in the beta cell.

Adenoma, Islet Cell↗

PubMed1975

Adaptive character of liver glucokinase.

1. Glucokinase is one of four glucose phosphorylating enzymes present in rat liver. Its distinctive features are a high K-m for glucose (high-K-m isozyme) and a rather narrow substrate specificity. In contrast, the other three enzymes, collectively called hexokinases or low-K-m isozymes, exhibit low K-m values for glucose and a wider substrate specificity. 2. Glucokinase is present in the liver os mammals (with some exceptions), amphibians and lower reptiles; It is absent from higher reptiles and birds. The presence or absence of glucokinase may represent an evolutionary adaptation to feeding habits and other physiological peculiarities. Differences in the immunological behavior and in the kinetic parameters of glucokinases from different taxa suggest the operation of divergent evolution. 3. The levels of glucokinase in rat liver depend strictly on the supply of carbohydrate in the diet. Glycogen phosphorylase and glycogen synthetase behave similarly, whereas other carbohydrate-metabolizing enzymes depend on the provision of either protein or protein plus carbohydrate. Glucokinase decays with a half-life of 33 hr when rats are starved or fed a carbohydrate-free diet, and is induced by the administration of glucose. The adaptive character is not exhibited by all mammals, indicating evolutionary discrimination within the same class and even within the same single order Rodentia. Enzyme adaptation in the liver may partially explain the condition known as 'hunger diabetes'. 4. The endocrine system plays a paramount role in glucokinase adaptation, since insulin is essential for glucose-dependent glucokinase induction and, on the other hand, glucagon, catecholamines and cyclic AMP prevent the induction. Glucocorticoids and some pituitary hormones modulate the rate of induction. The mechanisms underlying the hormonal regulation of glucokinase levels are not well known. 5. The variations in liver glucokinase correspond to changes in the amount of enzyme protein as assessed by immunochemical titration. This fact agrees with the effects of inhibitors of protein synthesis on glucokinase induction. 6. An antiserum against rat glucokinase reacts with the enzyme from mammals and turtles but not with the amphibian enzyme. It does not react with low-K-m hexokinases from different sources. 7. The saturation function for glucose is sigmoidal in mammalian and amphibian glucokinases but not in glucokinase from lower reptiles. The Hill's coefficient is very constant with values about 1.6. The K0.5 (concentration for half saturation) values in the different species studied vary between 1.5 and 8 mM. These kinetic parameters may be considered as another adaptive feature aimed to give maximal efficiency to the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.

Adaptation, Physiological↗

PubMed2000

The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte.

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.

Adenoviridae↗

PubMed2001

Cyclic adenosine 3',5'-monophosphate increases pancreatic glucokinase activity and gene expression.

Comparison of the pancreatic and hepatic glucokinase gene transcripts reveals tissue-specific control of expression and the existence of two distinct promoters in a single glucokinase gene. The existence of alternate promoters suggests that separate factors regulate glucokinase transcription in the two tissues. Hepatic glucokinase expression has been shown to be repressed by cAMP; however, in the pancreatic beta-cell it is unlikely that cAMP represses glucokinase activity, as cAMP is known to positively affect glucose-induced insulin secretion, a process that in mature islets requires pancreatic glucokinase activity. In this work we demonstrate that cAMP indeed has a stimulatory effect on pancreatic glucokinase. The cyclic nucleotide stimulates pancreatic glucokinase activity after 3-h incubation, and maximal effects are observed after 6 and 12 h of treatment. Using the bDNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase messenger RNA levels of 40.5 +/- 7.5% after 3-h incubation with cAMP. This stimulatory effect was increased to 106.3 +/- 22% after 6-h incubation and sustained up to 12 h of incubation. Inhibition of gene transcription by actinomycin D abolishes cAMP-induced glucokinase activity. In transfected fetal islets, cAMP increased the activity of the -1000 bp rat glucokinase promoter by 60 +/- 6%. These data demonstrate that cAMP has a stimulatory effect on pancreatic glucokinase gene expression and that the nucleotide has opposite effects on pancreatic and hepatic glucokinase, supporting the concept that glucokinase transcription in the liver and that in the beta-cell differ.

Animals↗

PubMed1988

Characteristics of glucokinase of the Kirkman insulinoma.

It was investigated whether the well-known transplantable insulinoma of the hamster (the Kirkman tumor) contains glucokinase and if so, what its kinetic characteristics are, and whether its cellular levels might be regulated in a manner typical for islet tissue. The supernatant of tumor homogenates contained a low-affinity component (Km 9.7 mmol/L) of glucose phosphorylating activity, apparently glucokinase. Partially purified insulinoma glucokinase exhibited similar kinetic characteristics to liver glucokinase (Km for glucose 5.0 and 5.3 mmol/L, half-maximal saturation 6.9 and 6.3 mmol/L, Hill coefficient 1.63 and 1.62, Ki for mannoheptulose 0.9 and 0.6 mmol/L in hamster insulinoma glucokinase and hamster liver glucokinase, respectively). Insulinoma glucokinase activity was not affected by the age of the tumor. Tumor-bearing hamsters without further treatment stayed normoglycemic (172 +/- 9.5 mg/dL) for the duration of the experiment. Fasting caused hypoglycemia (49 +/- 5.0 mg/dL), and pretreatment with streptozotocin prior to tumor transplantation caused hyperglycemia (393 +/- 20.6 mg/dL) in the tumor-bearing hamsters. Blood glucose levels of the host hamsters did not affect the content of the insulinoma glucokinase (83 +/- 3.5 mU/g in hypoglycemic group, 88 +/- 9.0 mU/g in hyperglycemic group, and 86 +/- 3.5 mU/g in normoglycemic group). Thus, biosynthesis and degradation of insulinoma glucokinase does not seem to be regulated by glucose as found to be true for islet glucokinase. Since glucokinase is constitutively present, the stable transplantable Kirkman tumor could serve as a useful model for studying the pancreatic B-cell glycolysis system which is characterized by the presence of both hexokinase and glucokinase.

Adenoma, Islet Cell↗

PubMed1987

Regulation of hepatic glucokinase gene expression. Role of carbohydrates, and glucocorticoid and thyroid hormones.

The present study investigates the effect of thyroid and glucocorticoid hormones on the induction of hepatic glucokinase mRNA activity, enzyme synthesis and activity in starved/refed adrenalectomized, thyroidectomized and intact rats. In intact rats glucose refeeding resulted within 2 h in a more than tenfold increase in the functional messenger, followed by a corresponding increase in glucokinase synthesis and, a little later, in enzyme activity. Glucokinase mRNA and synthesis remained elevated at this level for about further 6 h. Then the mRNA activity and enzyme synthesis declined considerably to a new steady state (a factor of about 4 above the starvation level) within a further 8 h, while enzyme activity remained constantly elevated. The half-life of glucokinase mRNA, as determined after administration of cordycepin, was identical during the different refeeding periods. Thus the overshoot phenomenon, provoked by carbohydrate refeeding, in glucokinase mRNA is not explained by alteration of the glucokinase mRNA decay rates. In thyroidectomized or adrenalectomized rats, glucose refeeding resulted in only a small increase in glucokinase mRNA, synthesis and activity. Application of thyroid hormones in thyroidectomized rats, refed a carbohydrate-rich diet, enhanced the specific mRNA considerably within 8-10 h, while it took 20-24 h to enhance glucokinase mRNA by glucocorticoids in adrenalectomized rats refed a carbohydrate-rich diet. The decay in translatable glucokinase mRNA, as determined after administration of cordycepin, was identical in the hypothyroid and euthyroid fed state, while adrenalectomy resulted in a significant decrease in the specific mRNA half-life. We conclude that refeeding a carbohydrate-rich diet rapidly stimulates glucokinase mRNA regeneration showing overshoot kinetics. 3,3',5-Triiodothyronine in its physiological concentration significantly enhances the response in glucokinase mRNA at the nuclear level, while glucocorticoids in their physiological concentration predominantly stabilize the translatable glucokinase mRNA.

Adrenalectomy↗

PubMed2005

Dual role of phosphofructokinase-2/fructose bisphosphatase-2 in regulating the compartmentation and expression of glucokinase in hepatocytes.

Hepatic glucokinase is regulated by a 68-kDa regulatory protein (GKRP) that is both an inhibitor and nuclear receptor for glucokinase. We tested the role of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) in regulating glucokinase compartmentation in hepatocytes. PFK2 catalyzes formation or degradation of the regulator of glycolysis fructose 2,6-bisphosphate (fructose 2,6-P2), depending on its phosphorylation state (ser-32), and is also a glucokinase-binding protein. Incubation of hepatocytes at 25 mmol/l glucose causes translocation of glucokinase from the nucleus to the cytoplasm and an increase in fructose 2,6-P2. Glucagon caused phosphorylation of PFK2-ser-32, lowered the fructose 2,6-P2 concentration, and inhibited glucose-induced translocation of glucokinase. These effects of glucagon were reversed by expression of a kinase-active PFK2 mutant (S32A/H258A) that overrides the suppression of fructose 2,6-P2 but not by overexpression of wild-type PFK2. Overexpression of PFK2 potentiated glucokinase expression in hepatocytes transduced with an adenoviral vector-encoding glucokinase by a mechanism that does not involve stabilization of glucokinase protein from degradation. It is concluded that PFK2 has a dual role in regulating glucokinase in hepatocytes: it potentiates glucokinase protein expression by posttranscriptional mechanisms and favors its cytoplasmic compartmentation. Thus, it acts in a complementary mechanism to GKRP, which also regulates glucokinase protein expression and compartmentation.

Adenoviridae↗

PubMed2000

Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites.

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.

Animals↗

PubMed1988

Inhibition of glucokinase by alloxan through interaction with SH groups in the sugar-binding site of the enzyme.

Alloxan is known to inhibit pancreatic B cell and liver glucokinase and glucose protects the enzyme against inhibition. The dithiol 1,4-dithiothreitol (1,4-DTT) protected against and reversed the inhibition of glucokinase by alloxan. An investigation into the structure-activity relationship using a variety of different dithiols demonstrated that the ability of the dithiols to protect against and to reverse the inhibition of glucokinase by alloxan was dependent on the spacing between the SH (thiol) groups of the various dithiols. Only 1,3-dimercaptopropane, 1,4-dimercaptobutane, 1,4-dithioerythritol, and 1,4-DTT, with intermediate spacing between the SH groups, reversed the inhibition of glucokinase induced by alloxan. Dithiols with two vicinal SH groups such as 1,2-dimercaptoethane and 2,3-dimercaptopropanol (BAL) were ineffective in the same way as dithiols with more widely spaced SH groups such as 1,5-dimercaptopentane and 1,6-dimercaptohexane. Except for 1,6-dimercaptohexane, all dithiols also protected glucokinase against the inhibition of alloxan. The monothiol cysteine, but not glutathione, a tripeptide monothiol, also protected glucokinase against alloxan inhibition but both were unable to reverse the inhibition. Like alloxan, other dithiol reagents such as ninhydrin, N-ethylmaleimide, and maleimide inhibited glucokinase. Glucose and 1,4-DTT protected glucokinase against this inhibition. 1,4-DTT partially reversed this inhibition. It is concluded, therefore, that the mechanism of inhibition of glucokinase by alloxan is a reaction of alloxan with two adjacent SH groups in the depth of the sugar-binding site of the glucokinase, with formation of a disulfide bond and concomitant inactivation of the enzyme. Because glucokinase can couple changes in the blood glucose concentration to changes in the glycolytic flux rate and corresponding changes in the rate of insulin secretion, it may function as a glucose signal recognition enzyme in the pancreatic B cell. This mechanism of interaction of alloxan with glucokinase may thereby provide an explanation for the ability of alloxan to inhibit glucose-induced insulin secretion.

Alloxan↗

PubMed1992

Tissue-specific regulation of glucokinase gene expression.

Glucokinase contributes to the maintenance of blood glucose homeostasis by catalyzing the high Km phosphorylation of glucose in the liver and the pancreatic beta cell, the only two tissues known to express this enzyme. Molecular biological studies of the glucokinase gene and its products have advanced our understanding of how this gene is differentially regulated in the liver and beta cell. The production of an active glucokinase isoform is determined by both transcriptional and post-transcriptional events. Two different promoter regions that are widely separated in a single glucokinase gene are used to produce glucokinase mRNAs in the liver, pancreatic beta cell, and pituitary. The different transcription control regions are tissue-specific in their expression and are differentially regulated. In liver, glucokinase gene expression is regulated by insulin and cAMP, whereas in the beta cell it is regulated by glucose. The upstream glucokinase promoter region, which gives rise to the glucokinase mRNA in pituitary and pancreas, is structurally and functionally different from the downstream promoter region, which gives rise to the glucokinase mRNA in liver. The use of distinct promoter regions in a single glucokinase gene enables a different set of transcription factors to be utilized in the liver and islet, thus allowing a functionally similar gene product to be regulated in a manner consistent with the different functions of these two tissues. In addition, the splicing of the glucokinase pre-mRNA is regulated in a tissue-specific manner and can affect the activity of the gene product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase.

The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.

Alloxan↗

PubMed2002

Liver glucokinase gene expression is controlled by the onecut transcription factor hepatocyte nuclear factor-6.

AIMS/HYPOTHESIS: Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter. METHODS: We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration. RESULTS: HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value. CONCLUSIONS/INTERPRETATION: Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.

Animals↗

PubMed1997

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 alpha-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 Ki values in the range of 1 microM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC50 value of 9 microM for alloxan and of 50 microM 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.

Alloxan↗

PubMed1965

The development of hepatic glucokinase in the neonatal rat.

1. Glucokinase and hexokinase activities have been determined in the livers of newborn rats and attempts made to influence in vivo the development of the glucokinase. 2. Glucokinase first appears in rat liver about 16 days after birth and adult activities are reached 10-12 days later. Evidence is presented which indicates that this represents synthesis of new protein. Hexokinase activities remain constant throughout the period of glucokinase development. 3. Both exogenous glucose and insulin are necessary for the natural development of glucokinase, for this is retarded in starved and alloxan-diabetic neonatal rats. 4. The absence of glucokinase during the first 2 weeks of extrauterine life in the rat is not due to lack of insulin. 5. Attempts to advance the time at which glucokinase first appears by infusions of glucose, insulin and chlorpropamide alone and in various combinations have resulted in marginal effects only. 6. When rats are starved for 3 days during the period of glucokinase development and then re-fed, glucokinase is more rapidly synthesized, indicating that the potential ability to synthesize glucokinase continues to develop throughout the period of starvation. 7. Some possible reasons for the comparatively late development of glucokinase are discussed.

Animals↗

PubMed1999

Metabolic regulation, activity state, and intracellular binding of glucokinase in insulin-secreting cells.

Regulation of glucose-induced insulin secretion is crucially dependent on glucokinase function in pancreatic beta-cells. Glucokinase mRNA expression was metabolically regulated allowing continuous translation into enzyme protein. Glucokinase enzyme activity in the beta-cell was exclusively regulated by glucose. Using a selective permeabilization technique, different intracellular activity states of the glucokinase enzyme in bioengineered glucokinase-overexpressing RINm5F tissue culture cells were observed. These results could be confirmed in analogous experiments with dispersed islet cells. A diffusible glucokinase fraction with high enzyme activity could be distinguished from an intracellularly bound fraction with low activity. Glucose induced a significant long-term increase of the active glucokinase fraction. This effect was accomplished through the release of glucokinase enzyme protein from an intracellular binding site of protein character. The inhibitory function of this protein factor was abolished through proteolytic digestion or heat inactivation. Northern blot analyses revealed that this binding protein was not identical to the well-known liver glucokinase regulatory protein. This hitherto unknown new protein factor may have the function of a glucokinase regulatory protein in the pancreatic beta-cell, which may regulate glucokinase enzyme activity in a glucose-dependent manner.

Animals↗