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PubMed1989

Effect of thyroid hormones on glucokinase gene transcription in rat liver.

Thyroid hormones contribute to the regulation of blood sugar by accelerating the turnover of glucose. The mechanism by which thyroid hormones stimulate the rate of glucose utilization in the liver was determined by investigating the effect of different thyroid states on the expression of the glucokinase gene, a key enzyme of glycolysis. In euthyroid rats the mass of glucokinase mRNA increased 8-fold during the first 4 h of refeeding a high carbohydrate diet to 48-h starved rats. In hypothyroid rats under the same conditions only a 2-fold induction was observed. In euthyroid rats a 5-fold increase was obtained 1 h after refeeding, while hypothyroid rats displayed no significant response in glucokinase mRNA within this time. Basal levels of glucokinase mRNA in starved rats were the same observed in eu- and hypothyroid rats. Injection of 3,3',5-triiodothyronine (T3) into hypothyroid rats restored the mRNA levels of refed hypothyroid rats to euthyroid levels within 24 h. However, a 3-fold increase over untreated animals was already observed 3 h after T3 administration. Even subphysiological doses of T3 (0.1 microgram/100 g body weight) led to an significant increase in glucokinase mRNA levels (1.5-fold, p less than 0.05) in hypothyroid rats, whereas higher doses (100 micrograms/100 g body weight) restored the mRNA levels to those of euthyroid controls. Parallel increases in the cytosolic mRNA levels and rate of glucokinase gene transcription were detected when hypo- and euthyroid fasted rats were compared after 4 h of refeeding. It is concluded that thyroid hormones are permissive for the induction of glucokinase during refeeding but have no effect during starvation. They rapidly enhance the rate of gene transcription within the range of their physiologically circulating concentrations. The results suggest a major importance of thyroid hormones in regulating glucose utilization.

Animals↗

PubMed1989

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.

Amino Acid Sequence↗

PubMed1983

Discrimination of glucose anomers by glucokinase from liver and transplantable insulinoma.

Phosphorylation of alpha- and beta-D-glucose by glucokinase from rat liver or a radiation-induced, transplantable insulinoma was investigated. Glucokinase partially purified by ion exchange chromatography on DEAE-Cibacron blue F3GA agarose was incubated for brief periods (1 or 3 min) with glucose anomers. Glucokinase from both liver and insulinoma tissue had a higher affinity for alpha-D-glucose (S0.5 = 6-7 mM) than beta-D-glucose (S0.5 = 12-14 mM). The maximum velocity was 15-20% lower for alpha-D-glucose than beta-D-glucose. Cooperative rate dependence with respect to glucose concentration was observed with both anomers (nH = 1.4). These kinetic data imply that both anomers of glucose are phosphorylated by glucokinase, however, at the physiological range of glucose concentrations below 15 mM, the higher affinity of alpha-D-glucose results in higher rates than with beta-D-glucose. At clearly pathological glucose concentrations exceeding 20 mM, the observed velocities are slightly higher with beta- than alpha-D-glucose. Glucokinase is thought to be the glucose sensor of pancreatic beta cells. The present data indicating a preferential phosphorylation of alpha-D-glucose compared to beta-D-glucose by glucokinase, supports the glucokinase-glucose sensor hypothesis, because it parallels the well established greater potency of alpha-D-glucose as a stimulant of insulin release.

Adenoma, Islet Cell↗

PubMed1984

Effects of biotin upon the intracellular level of cGMP and the activity of glucokinase in cultured rat hepatocytes.

The effects of biotin upon the intracellular level of cGMP and the activity of glucokinase were examined in primary cultures of adult rat hepatocytes. The addition of biotin to the culture medium of hepatocytes increased their content of cGMP 3-fold within 1 h. A 4-fold increase in the activity of glucokinase was observed in response to the addition of the vitamin to the culture medium and the maximal response was observed 6 h following the addition to the medium. These maximum effects were noted when biotin was present in the culture medium at a concentration of 10(-6) M. The induction of glucokinase activity by biotin was preceded by an increase in the intracellular level of cGMP. The addition of 8-bromo-cGMP to the culture medium also increased the activity of glucokinase and its effects were not additive with respect to the effects of biotin. The induction of glucokinase by biotin or the cyclic nucleotide analog was not observed in the absence of insulin. The effects of biotin upon the activity of glucokinase could be mimicked by including glucose in the culture medium. When hexose utilization by the hepatocytes was blocked by the addition to the culture medium of N-acetylglucosamine, the induction of glucokinase by biotin was unaffected, whereas the induction brought about by glucose was not observed. The changes in the activity of the enzyme brought about by biotin or 8-bromo-cGMP was shown to arise as the result of changes in the rate of synthesis of the enzyme. In addition, using an in vitro translation assay and immunoprecipitation, it was found that biotin and 8-bromo-cGMP increased the amount of translatable mRNA coding for the enzyme.

Acetylglucosamine↗

PubMed1994

[Glucokinase gene abnormalities in maturity-onset diabetes of the young (MODY) and late-onset NIDDM].

Glucokinase is a key enzyme of glucose metabolism that phosphorylates glucose to glucose-6-phosphate (G-6-P). This is the first step of glucose metabolism after the uptake of glucose by glucose transporter 2 (GLUT 2). Glucokinase is one of the hexo-kinases and is expressed only in pancreatic beta cells and hepatocytes. Recently it was reported that glucokinase gene is associated with some families with MODY (maturity-onset diabetes of the young). As MODY is a subtype of diabetes which is inherited autosomal dominantly, the correlation of diabetes with glucokinase gene was vigorously studied in many laboratories. The first mutation in exon 7 of the glucokinase gene was reported in 1992. Since the first report of the glucokinase gene mutation in exon 7, a number of mutations and a deletion were reported to be associated with MODY or late-onset NIDDM. But investigations by many groups revealed that glucokinase gene abnormalities are responsible for less than one per cent of NIDDM which is relatively small compared with diabetes with mitochondrial gene alterations.

Diabetes Mellitus, Type 1↗

PubMed1971

Utilization of glucose by Clostridium thermocellum: presence of glucokinase and other glycolytic enzymes in cell extracts.

Clostridium thermocellum was shown to ferment glucose in a medium containing salts and 0.5% yeast extract. An active glucokinase was obtained with improved conditions for growth, assay, and preparation of cell extracts. Cell extracts appear to contain a glucokinase inhibitor that interferes with the assays at high protein concentrations. Glucokinase activity is stimulated about 60% by pretreatment with dithiothreitol. Little or no fructokinase or mannokinase activity was detected in cell extracts. The absence of glucokinase in mannitol-grown cells, the increase in glucokinase activity upon incubation of cell suspensions with glucose, and the lack of increase in activity when chloramphenical is added are evidence that glucokinase is an inducible enzyme. The following enzymes were detected in cell extracts (the enzyme activities are shown in parentheses are micromoles per minute per milligram or protein at 27 C): glucokinase (0.48), phosphoglucose isomerase (0.73), fructose 6-phosphate kinase (0.24), fructose diphosphate aldolase (0.59), glyceraldehyde 3-phosphate dehydrogenase (0.53), triose phosphate isomerase (0.13), phosphoglycerate kinase (0.20), phosphoglycerate mutase (0.20), enolase (0.28), pyruvic kinase (0.13), and lactic dehydrogenase (0.13). Glucose 6-phosphate dehydrogenase activity was absent or very low (0.0002) and 6-phosphogluconate dehydrogenase activity also was relatively low (0.015). From these data, it is proposed that carbohydrate metabolism in C. thermocellum proceeds by the Embden-Meyerhof pathway.

Alcohol Oxidoreductases↗

PubMed1994

Epidermal growth factor counteracts insulin-induced expression of glucokinase in hepatocytes.

Hepatic glucokinase is induced by insulin and repressed by glucagon. The effects of epidermal growth factor (EGF) on glucokinase expression were investigated in rat hepatocytes. EGF does not affect the decline in glucokinase activity in hepatocytes cultured for 48h in the absence of insulin, but it counteracts the increase in activity induced by insulin. This effect of EGF is greater in cells cultured at low cell density than in confluent cultures. EGF suppressed the insulin-induced increase in glucokinase mRNA levels by 50% indicating that its effect is at least in part at a pretranslational level. However, it potentiated the stimulatory effect of insulin on glucose-6-phosphate dehydrogenase activity and mRNA, indicating that the effect on glucokinase expression is due to a specific post-receptor mechanism. The effect of EGF on glucokinase mRNA expression is mimicked by phospholipase D but not by phosphatidylinositol-specific phospholipase C or by phorbol ester, an activator of protein kinase C, suggesting that it is unlikely to be mediated by activation of protein kinase C.

Animals↗

PubMed1985

Glucokinase is not the pancreatic B-cell glucoreceptor.

A series of recent experimental findings are reviewed to indicate that glucokinase does not represent the pancreatic B-cell glucoreceptor. Whether in liver, pancreatic islet or insulin-producing tumoral cell homogenates, glucokinase fails to yield a higher reaction velocity with alpha-than beta-D-glucose. At a high glucose concentration (40 mmol/l), when the phosphorylation of glucose by glucokinase is indeed higher with beta- than alpha-D-glucose, no preference for beta-D-glucose is observed in intact islets, as judged from the utilization of D-[5-3H]glucose, production of lactic acid, oxidation of D-[U-14C]glucose, net uptake of 45Ca or release of insulin. The glucose 6-phosphate content of intact islets is higher in the presence of beta- than alpha-D-glucose. At a low glucose concentration (3.3 mmol/l), when the participation of glucokinase to hexose phosphorylation is minimal, alpha-D-glucose is still better metabolized and stimulates both 45Ca net uptake and insulin release more efficiently than beta-D-glucose, despite the fact that hexokinase yields a higher reaction velocity with beta- than alpha-D-glucose. In intact islets, beta-D-glucose is used preferentially to alpha-D-glucose in the pentose cycle pathway as judged from the oxidation of alpha- or beta-D-[1-14C]glucose relative to that of alpha- or beta-D-[6-14C]glucose. In islets removed from fasted rats, the rate of glycolysis is more severely decreased than expected from the repression of glucokinase. The metabolism of glucose in tumoral insulin-producing cells differs, in several respects, from that in normal pancreatic islets, although the pattern of hexokinase and glucokinase activities is similar in these two types of cells. All these observations point to the participation of regulatory sites distal to glucose phosphorylation in the control of glucose metabolism in islet cells.

Animals↗

PubMed1993

Positive association in the absence of linkage suggests a minor role for the glucokinase gene in the pathogenesis of type 2 (non-insulin-dependent) diabetes mellitus amongst south Indians.

Mutations of the glucokinase gene have been implicated in the development of glucose intolerance in pedigrees with maturity-onset diabetes of the young. However, the contribution of the glucokinase gene to the aetiology of common Type 2 (non-insulin-dependent) diabetes mellitus is uncertain. We have studied the role of the glucokinase gene in the pathogenesis of Type 2 diabetes in South Indians, using both population-association and linkage methodology. A pair of CA-repeat sequences (GCK(3') and GCK(5')) straddling the glucokinase gene were employed as markers, each subject being typed using the polymerase chain reaction and polyacrylamide gel electrophoresis. Comparisons of allele frequencies at these markers were made between 168 Type 2 diabetic subjects and 70 racially-matched control subjects. No differences in allele frequencies were apparent at the GCK(5') marker; however, there were significant differences in allele frequencies at the GCK(3') marker between the Type 2 diabetic subjects and control subjects (chi 2 = 11.6, df = 3, p = 0.009) with an increase of the z allele (78.0% vs 66.4%) and a decrease of the z + 2 allele (13.7% vs 25.0%) amongst the diabetic subjects. Linkage between glucose intolerance and the glucokinase gene was studied in 53 nuclear pedigrees under a variety of genetic models. Linkage was excluded (lod score < -2) at a recombination fraction of zero under five of the ten models used and highly unlikely (-2 < lod score < -1) under the others. The combination of positive association and negative linkage suggests that glucokinase acts as a minor gene influencing the development of Type 2 diabetes within this population.

Adult↗

PubMed1994

Glucokinase gene in gestational diabetes mellitus: population association study and molecular scanning.

Mutations of the glucokinase gene result in early-onset familial Type 2 (non-insulin-dependent) diabetes mellitus, and several members of the mutant glucokinase kindreds were originally diagnosed as having gestational diabetes. This study examined the glucokinase gene in 270 American Black women, including 94 with gestational diabetes whose diabetes resolved after pregnancy (gestational diabetes only), 77 with gestational diabetes who developed Type 2 diabetes after pregnancy (overt diabetes), and 99 normal control subjects who were recruited during the peripartum period. Two simple sequence repeat polymorphisms flanking either end of the glucokinase gene were evaluated. No association was found between glucokinase alleles and gestational diabetes only or overt diabetes, after adjustment for multiple comparisons. To detect single base changes, all 11 exons and proximal islet and liver promoter regions were examined by polymerase chain reaction plus single-stranded conformational polymorphism analysis in 45 gestational diabetes only patients who had not yet developed Type 2 diabetes. Nine coding region variants were identified: Ala11 (GCC) to Thr11 (ACC) in islet exon 1, and 8 variants either in untranslated regions or in the third base of a codon. Four variant sites were found in introns, but none in splicing consensus sequences. Analysis of the promoter regions revealed two common variants, G-->A at islet -30 (24%), and G-->A at liver -258 (42%). The frequencies of the promoter variants, determined by allele specific polymerase chain reaction analysis, but did not differ among the three groups. Thus, no significant coding sequence glucokinase mutations were found in 90 alleles from 45 patients with gestational diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Human pancreatic beta-cell glucokinase: cDNA sequence and localization of the polymorphic gene to chromosome 7, band p 13.

The glucose phosphorylating enzyme glucokinase plays an important role in the regulation of glucose homeostasis. Studies in rodents indicate that pancreatic Beta cells and hepatocytes express different isoforms of this protein as a consequence of the presence of tissue-specific promoters and exon 1 sequences which are spliced to a shared group of nine exons which encode most of the mRNA and protein. Here, we report the isolation and characterization of cDNA clones encoding human Beta-cell glucokinase. The sequence of human Beta-cell glucokinase shows 97% amino acid identity with that of the cognate rat protein. We also mapped the human glucokinase gene to the short arm of chromosome 7 by analysing its segregation in a panel of reduced human-mouse somatic cell hybrids. In situ hybridization to metaphase chromosomes confirmed the localization of the human glucokinase gene to chromosome 7 and indicated that it was in band p 13. A microsatellite DNA polymorphism that can be typed using the polymerase chain reaction was identified upstream of exon 1 a, the Beta-cell specific first exon. The glucokinase cDNA clone and highly informative DNA polymorphism will be useful for examining the role of this gene in the pathogenesis of diabetes mellitus.

Amino Acid Sequence↗

PubMed2003

Long-term overexpression of glucokinase in the liver of transgenic mice leads to insulin resistance.

AIMS/HYPOTHESIS: Glucokinase overexpression in the liver increases glucose uptake and utilization, and improves glucose tolerance in young transgenic mice. Here, we examined the long-term effects of hepatic overexpression of glucokinase on glucose homeostasis. Moreover, we determined whether glucokinase overexpression counteracted high-fat diet-induced insulin resistance. METHODS: Transgenic mice overexpressing glucokinase in liver under the control of the phosphoenolpyruvate carboxykinase promoter, fed either a standard diet or a high-fat diet, were studied. We used non-transgenic littermates as controls. RESULTS: Transgenic mice over 6 months old developed impaired glucose tolerance. In addition, at 12 months of age, transgenic mice showed mild hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia. In spite of increased glucokinase activity, the liver of these mice accumulated less glycogen and increased triglyceride deposition. When 2-month-old glucose-tolerant mice were fed a high-fat diet, transgenic mice gained more body weight and became hyperglycaemic and hyperinsulinaemic. This was concomitant to glucose intolerance, liver steatosis and whole-body insulin resistance. CONCLUSION/INTERPRETATION: Long-term overexpression of glucokinase increases hepatic lipogenesis and circulating lipids, which lead to insulin resistance. Our results also suggest that the liver plays a key role in the onset of diabetes.

Animals↗

PubMed1997

Identification of 14 new glucokinase mutations and description of the clinical profile of 42 MODY-2 families.

Mutations in glucokinase are associated with defects in insulin secretion and hepatic glycogen synthesis resulting in mild chronic hyperglycaemia, impaired glucose tolerance or diabetes mellitus. We screened members of 35 families with features of maturity-onset diabetes of the young for mutations in the glucokinase gene and found 16 different mutations. They included 14 new mutations in the glucokinase gene: 9 missense mutations (A53S, G80A, H137R, T168P, M210T, C213R, V226M, S336L and V367M); 2 nonsense mutations (E248X and S360X); a deletion of one nucleotide resulting in a frameshift (V401del1); a substitution of a conserved nucleotide at a splice acceptor site (L122-1G-->T); and a 10 base pair deletion that removed the GT of the splice donor site and the following eight nucleotides (K161 + 2del10). In addition, we found two previously identified mutations: R186X and G261R. Study of 260 subjects with glucokinase-deficient hyperglycaemia from 42 families with 36 different GCK mutations made it possible to define the clinical profile of this subtype of non-insulin-dependent diabetes mellitus (NIDDM). Hyperglycaemia due to glucokinase deficiency is often mild (fewer than 50% of subjects have overt diabetes) and is evident during the early years of life. Despite the long duration of hyperglycaemia, glucokinase-deficient subjects have a low prevalence of micro- and macro-vascular complications of diabetes. Obesity, arterial hypertension and dyslipidaemia are also uncommon in this form of NIDDM.

Adolescent↗

PubMed1992

A comparison of hepatic glucokinase gene expression in high- and low-activity strains of mice.

1. Compared with the rat, mouse liver glucokinase activities show a different sensitivity to changes in insulin concentrations. When animals from a high glucokinase-high insulin level strain C3H/He are crossed with those from a low glucokinase-low insulin strain C58, individuals from recombinant inbred lines show non-parental phenotypes with high glucokinase activity but low insulin and vice versa. 2. Messenger RNA levels are greater in high-enzyme-activity mice strains than in low-activity animals, suggesting that differences in either transcription of the glucokinase gene or in mRNA stability occur between the two strains. 3. There is no evidence of a different number of glucokinase genes in the high- and low-activity strains. Differences in activity therefore suggest that dissimilarities in the regulation of the expression of these genes may well occur.

Animals↗

PubMed2005

Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels.

Several studies have shown that organophosphate pesticides affect carbohydrate metabolism and produce hyperglycemia. It has been reported that exposure to the organophosphate pesticide dichlorvos affects glucose homeostasis and decreases liver glycogen content. Glucokinase (EC 2.7.1.1) is a tissue-specific enzyme expressed in liver and in pancreatic beta cells that plays a crucial role in glycogen synthesis and glucose homeostasis. In the present study we analyzed the effect of one or three days of dichlorvos administration [20 mg/kg body weight] on the activity and mRNA levels of hepatic and pancreatic glucokinase as well as on insulin mRNA abundance in the rat. We found that the pesticide affects pancreatic and hepatic glucokinase activity and expression differently. In the liver the pesticide decreased the enzyme activity; on the contrary glucokinase mRNA levels were increased. In contrast, pancreatic glucokinase activity as well as mRNA levels were not affected by the treatment. Insulin mRNA levels were not modified by dichlorvos administration. Our results suggest that the decreased activity of hepatic glucokinase may account for the adverse effects of dichlorvos on glucose metabolism.

Animals↗

PubMed1990

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase.

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.

Animals↗

PubMed1997

Co-localization of glucokinase with actin filaments.

A portion of glucokinase appeared to be co-localized with actin filaments in the cytoplasm of cultured rat hepatocytes incubated with 25 mM glucose. When liver- or islet-type glucokinase was transiently expressed in COS-7 cells, the expressed glucokinase was also co-localized with actin filaments in the cytoplasm of these transfected cells. Although co-localization of glucokinase with actin filaments was not clearly demonstrated in the pancreatic beta-cell line MIN6, islet glucokinase was found to be present in both the nucleus and the cytoplasm, though predominantly in the nucleus. These findings suggest that subcellular localization of glucokinase, including co-localization with actin filaments, may have an important physiological role in metabolic regulation.

Actins↗

PubMed2001

Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes.

It has been shown that all-trans retinoic acid induces prematurely hepatic glucokinase mRNA in ten days-old neonatal rat hepatocytes, however, this effect could be related to the capacity of the retinoid to promote a more differentiated state of the hepatocyte. In this report we demonstrate that physiological concentrations of all-trans retinoic acid stimulate glucokinase activity in both mature fully differentiated hepatocytes and at the onset of the induction of the enzyme in 15 to 17 days-old neonatal hepatocytes. The effects produced by the retinoid were similar both in magnitude and in time, to those elicited by insulin, a well-known stimulator of hepatic glucokinase expression. No additive effect was observed when insulin and retinoic acid were tested together. Using the branched DNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of about 70% at 3 and 24 h after the treatment with 10(-6) M all-trans retinoic acid, in both neonatal and adult hepatocytes. These data show that retinoic acid exerts a stimulatory effect on hepatic glucokinase independent of the hepatocyte stage of maturity and suggest a physiological role of retinoic acid on glucose metabolism. The action of retinoic acid on hepatic glucokinase might explain previous observations on the relationship between vitamin A status and liver glycogen synthesis. These findings may serve as basis for further investigations on the biological functions of retinoic acid derivatives on hepatic glucose metabolism.

Animals↗