Human Glucokinase Gene Research Articles

Review on upregulation of glucokinase expression by selected plants and their phytoconstituents

Glucokinase is, an allosteric enzyme involved in glycolysis, is rapidly regulated in the liver through glucokinase regulatory protein and remains essential for blood glucose homeostasis. The present review provides brief information about the effects of a few selected plants and phytochemicals on human glucokinase activation and gene modulation. A thorough and relevant literature search from several scientific databases, comprising Google Scholar, Web of Science, Scopus and PubMed, was carried out. We highlighted the seven plants (Acorus tatariowii Schott, Allium hirtifolium Boiss, Apache red maize, Mulberry species, Pterocarpus marsupium, Sapium ellepticum, Mangifera indica) and their phytoconstituents ( Tatanans A-C, alliogenin , gitogenin , kaempferol , quercetin and shallomin,1-deoxynojirimycin,cyanidin-3-rutinoside,resveratrol,cyanidin-3-glucoside,oxyresveratrol, lupeol, alpha-tocopherol, uteolin-7-glucoside, amentoflavone, and Mangiferin) on human glucokinase enzyme activation and gene modulation. This review concluded that investigation of glucokinase activators of plant origin is the major research focus in the management of type 2 diabetes.

Complications of Diabetes: An Insight into Genetic Polymorphism and Role of Insulin

Diabetes Mellitus (DM) is an advanced and chronic endocrine disorder characterized by an insufficiency of insulin secretion from pancreatic β-cells and liver, adipose tissues, and skeletal muscles. The main objective of this study is to understand the mechanism and genes which are responsible for the prevalence of diabetes. The study also covers various types of diabetic complications with special reference to insulin role and defects. The scientific literature and patents were reviewed and analyzed based on their suitability and relevance to the theme of the study. The scientific literature was covered from the authentic databases such as Elsevier, Springer, and Bentham Science. The patents were reviewed from http://www.freepatentsonline.com. Glucokinase (ATP: D-glucose-6-phosphotransferase; GCK), initiates glycolysis and acts as a glucose sensor and metabolic signal producer in liver and pancreas. PCR-sequencing showed qualitative differences in diabetic patients in comparison to healthy subjects. Glucokinase is the most important component in glucose detection of pancreatic islet beta cells in diabetes because glucokinase mutations can be one of the most common single gene disorders described. It is known that a genetic variation of a human glucokinase gene, including a point mutation, causes MODY, the concentration of plasma glucose increased and it is supposed to be the cause of diabetes of the present study subjects. Owing to hyperglycemia and individual components of the insulin resistance (metabolic) syndrome, people with Type II DM are prone to the high threat for microvascular complications (including nephropathy, retinopathy, and neuropathy) and macrovascular complications (such as Ischemic Heart Disease). There were also significant differences (P < 0.0001) in glycation levels (0.90, 0.4838mole/mole), random blood sugar (348.8, 105.8mg/dL), cholesterol levels (235.3, 161.8mg/dL), low density lipoprotein in diabetic subjects (155.3, 28.46mg/dL) and in healthy donors. GCK gene mutations were found in 70% of the patients while 30% are non-mutated. In conclusion, lipids, glucose, and protein play an essential role in the initiation of AGE's or diabetic complications (Micro and Macrovascular Complications). The importance of the clinical results should also be recognized in the genetic analysis of heterogeneous disorders as NIDDM/ Type II DM.

Expression of the Human Glucokinase Gene: Important Roles of the 5′ Flanking and Intron 1 Sequences

BackgroundGlucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood.Methodology/Principal FindingsTo better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5′ pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5′ flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5′ flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3′ to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin.Conclusions/SignificanceOur results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.

Opposite Clinical Phenotypes of Glucokinase Disease: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase (GCK) Gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Opposite Clinical Phenotypes of Glucokinase Disease: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase (GCK) Gene

Opposite Clinical Phenotypes of Glucokinase Disease: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase (<i>GCK</i>) Gene

Opposite Clinical Phenotypes of “Glucokinase Disease”: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase Gene

ABSTRACT Glucokinase (GK) is essential for glucose-stimulated insulin release from pancreatic β-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of “Glucokinase Disease”, i.e. Monogenic Diabetes of Youth (GCK-MDY), Permanent Neonatal Diabetes (inactivating mutations) and Congenital Hyperinsulinism, respectively. Here we present a novel GCK activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/l) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to GCK-MDY. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually “heterogeneous and progressive” diabetic phenotype, a feature not typical of MDY. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Opposite Clinical Phenotypes of “Glucokinase Disease”: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase Gene

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic β-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE...

Activating Mutations in the Human Glucokinase Gene Revealed by Genetic Selection

We describe the discovery of 11 new activating mutations in the human glk gene associated with the disease persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Three of the newly identified substitutions colocalize to a region of the glucokinase polypeptide where a synthetic allosteric activator binds. Of these substitutions, I211F is the most active variant identified to date, with a k(cat)/K(0.5,glucose) value (6.6 x 10(4) M(-1) s(-1)) that is 12-fold higher than that of wild-type glucokinase. The stimulatory mutations described herein represent surreptitious genetic determinants of PHHI. They also identify novel features of the glucokinase scaffold that could be targeted during the development of diabetes therapeutics.

Analysis of upstream promoter region and corresponding 5’ UTR of glucokinase (GCK) gene in horse breeds

A region of glucokinase (GCK) gene was sequenced in 14 horses of 14 different breeds. The resulting GCK nucleotide sequence (GenBank number EF136885) showed 77% homology with human GCK gene portion containing the upstream promoter region and the corresponding 5’ UTR of the exon 1. Conserved regulatory sequences near the putative transcriptional start site were identified. The obtained sequences were aligned to detect polymorphism. A new C>T transition within the 5’ UTR of exon 1 was found. Allele frequencies of this polymorphism were studied by PCR-RFLP in 193 horses of 14 breeds (Bardigiano, 21; Esperia Pony, 5; Haflinger, 10; Italian Heavy Draught Horse, 28; Italian Saddle, 25; Italian Trotter, 16; Lipizzan, 12; Maremmano, 15; Murgese, 14; Norico, 10; Salernitano, 12; Thoroughbred, 10; Tolfetano, 7 and Ventasso Horse, 8). The polymorphism was found in all breeds and differences in allelic frequencies among the breeds were observed. The new SNP identified within a regulative region of GCK gene, which plays an important role in insulin secretion and feeding behaviour, could be used for association studies with performance traits of the horses.

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

Induction of glycation suppresses glucokinase gene expression in HIT-T15 cells.

Chronic hyperglycaemia in patients with Type II (non-insulin-dependent) diabetes mellitus often leads to a decline in glucose-responsive insulin secretion from pancreatic beta cells, a phenomenon called glucose toxicity. Upon hyperglycaemia, glycation reaction occurs in the beta cells and induces oxidative stress. To understand the molecular basis of the beta-cell glucose toxicity, we investigated the possible effects of glycation on the expression and enzymatic activity of glucokinase, which plays a crucial part in glucose-responsive insulin secretion. Glycation and reactive oxygen species were induced in HIT-T15 cells by treatment with D-ribose and effects on glucokinase gene transcription, glucokinase protein amount, glucose phosphorylation activity, and DNA-binding activities of putative glucokinase gene transcription factors were evaluated. When glycation was induced in HIT-T15 cells, the activity of the human glucokinase gene beta-cell-type promoter was suppressed substantially (83% reduction at 60 mmol/l D-ribose). Also, similar reductions in mRNA and protein amounts of glucokinase and in the Vmax of its enzymatic activity were observed. In agreement with the reduction in the promoter activity, the two major transcription factors of the glucokinase gene, the Pal-binding factor and PDX-1, reduced their binding to their target sequences in the glucokinase gene promoter in glycation-induced HIT cells. Because these effects of D-ribose were counteracted by aminoguanidine or N-acetylcysteine, reactive oxygen species, generated by the glycation reaction, appears to be involved in the phenomena. The induction of the glycation reaction, which is known to occur in pancreatic beta cells in chronic hyperglycaemia, suppresses the glucokinase gene transcription and its enzymatic activity. Thus, hyperglycaemia-dependent inhibition of glucokinase activity could in part explain beta-cell glucose toxicity.

Gestational diabetes mellitus and gene mutations which affect insulin secretion

We investigated whether genetic mutations known to impair insulin secretion and glucose tolerance are operative in a group of American women with gestational diabetes mellitus. Study groups were comprised of elderly non-diabetic controls ( n=55) with normal glucose tolerance and patients with gestational diabetes ( n=50), together with one family with maturity-onset diabetes of the young (three controls and three affected). No mutations were detected in any exon of the human glucokinase gene or the mitochondrial tRNA Leu(UUR) gene by single strand conformational analysis and direct exon sequencing. Also, X 2 analysis showed no significant association with gestational diabetes for a polymorphism at position −30 (G→A) of the β-cell-specific glucokinase gene promoter. We have determined that glucokinase and mitochondrial tRNA Leu(UUR) gene mutations, which are known to impair insulin secretion are relatively uncommon and do not constitute a large component of genetic risk for gestational diabetes in the study population.

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells

The human glucokinase gene beta-cell-type promoter: an essential role of insulin promoter factor 1/PDX-1 in its activation in HIT-T15 cells

Liver-specific Enhancer of the Glucokinase Gene

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

Variable effects of maturity-onset-diabetes-of-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme.

Mutations in the human glucokinase (GK) gene are thought to cause maturity-onset diabetes of youth (MODY) by leading to the production of enzymes with reduced catalytic activities and increased glucose Km values. However, in some cases the diabetic phenotype is more severe than might be predicted from these apparent kinetic effects alone. To determine whether these mutations might also effect other characteristics of the enzyme, nine MODY-associated mutants were expressed as fusion proteins with Schistosoma japonicum glutathione S-transferase (GST) and compared with three wild-type human GK isoforms that were also expressed in the same manner. Three GST-GK isoforms (liver 1, liver 2 and islet) were kinetically indistinguishable from each other and from purified rat liver GK. Noteworthy is a glucose-induced fit effect for the interaction of trinitrophenyl (TNP)-ATP with GST-GK, whereby glucose significantly increased the affinity of TNP-ATP binding to GST-GK without changing the stoichiometry of binding. The nine MODY-associated mutations studied either showed diminished catalytic activity, substrate affinities, allosteric regulation, or stability of the fusion enzyme. We conclude that: (1) Gly261 and Lys414 are important for ATP binding; (2) Val203 may be essential for a glucose-induced fit effect; and (3) the stability of fusion protein may be significantly reduced when Glu300 is replaced by Lys. These results suggest that, in addition to effects on the Km and Vmax. of GK, a decrease in the ATP-binding affinity or stability of the mutated enzyme may also contribute to a reduction of GK activity in individuals with GK-MODY. In the B-cell this would have the effect of blunting glucose-stimulated insulin release, thereby contributing to the diabetic phenotype.

Polymorphic Microsatellite Repeat Markers at the Glucokinase Gene Locus Are Positively Associated With NIDDM in Japanese

To assess the possible role of glucokinase defects contributing to a genetic susceptibility to NIDDM in Japanese, allelic frequencies of two microsatellite repeat polymorphisms, one in the 3'-flanking region (GCK1) and the other in the 5'-flanking region (GCK2) of the human glucokinase gene, were analyzed in subjects with NIDDM and in nondiabetic control subjects. After typing 107 diabetic and 74 nondiabetic subjects, we found four GCK1 alleles (Z, Z2, Z4, Z6) and six GCK2 alleles (0, -4, -2, 2, 4, 8). The frequency distribution of GCK1 alleles was different between the two groups (P = 0.005), although not significant after correction for multiple comparisons. The Z4 allele was found more frequently in diabetic than in nondiabetic subjects (23 vs. 10%, P = 0.002). This was still significant after correction for multiple comparisons (P < 0.05). The frequency distribution of GCK2 alleles was not different between the two groups. However, the -2 allele was more common in diabetic than in nondiabetic subjects (P = 0.044), although not significant after adjusting for multiple comparisons. Clinical characteristics were compared between the diabetic subjects with Z4 and/or -2 allele and those without either of these two alleles. No differences were found in the age of diagnosis, positive family history, mode of therapy, current HbA1c, or daily urinary C-peptide immunoreactivity excretion between the two groups. We demonstrated a significant association between GCK1 and GCK2 alleles and NIDDM. The results indicate that the polymorphic alleles GCK1 and GCK2 could be genetic markers in NIDDM in Japanese, suggesting a relationship between glucokinase defects and the susceptibility to NIDDM in this population.

Two microsatellite repeat polymorphisms flanking opposite ends of the human glucokinase gene: use in haplotype analysis of Welsh Caucasians with type 2 (non-insulin-dependent) diabetes mellitus.

The purpose of this study was to evaluate the role of potential glucokinase defects contributing to susceptibility to Type 2 (non-insulin-dependent) diabetes mellitus in Welsh Caucasians. For this analysis, two microsatellite repeat polymorphisms flanking opposite ends of the gene were employed. For a recently described microsatellite (GCK2), located 6 kilobases upstream of islet exon 1, six different sized alleles were observed, with heterozygosity of 0.50 and polymorphism information content 0.44. Combined heterozygosity with another microsatellite repeat (GCK1) was 0.72. Significant linkage disequilibrium was noted between GCK2 and GCK1, suggesting that haplotypes may be a better predictor of Type 2 diabetes than analysis with either microsatellite alone. Using these two markers, the association with Type 2 diabetes was examined. The frequencies of alleles and genotypes at GCK1 did not differ between the patients with Type 2 diabetes (n = 157) and control subjects (n = 73). Similarly no differences were observed in GCK2 alleles or genotypes. The frequencies of haplotypes, derived from the two markers, also did not differ between the two groups. To investigate the possibility of minor metabolic effects of glucokinase defects, we also studied the association between the GCK alleles or haplotypes and the response profiles to meal tolerance tests. No association was observed between plasma glucose or insulin responses to meal tolerance tests with GCK haplotypes or alleles. These results suggest that glucokinase mutations in Welsh Caucasians are not major determinants of susceptibility to the common type of Type 2 diabetes.

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.

Characterization of a third simple tandem repeat polymorphism in the human glucokinase gene.

Recent studies have implicated mutations in the glucokinase gene as a cause of early-onset Type 2 (non-insulin-dependent) diabetes mellitus. Two simple tandem repeat DNA polymorphisms have been identified in this gene and used for genetic studies. However, their heterozygosity is relatively low and not all families are informative for linkage with these markers. Here we report the characterization of a third simple tandem repeat polymorphism that can be used for genetic studies.

Structure of the human glucokinase gene and identification of a missense mutation in a Japanese patient with early-onset non-insulin-dependent diabetes mellitus.

Glucokinase is thought to play a glucose-sensor role in the pancreas, and abnormalities in its structure, function, and regulation can induce diabetes. We isolated the human glucokinase gene, and determined its genomic structure including exon-intron boundaries. Structure of the glucokinase gene in human was very similar to that in rat. Then, by screening Japanese diabetic patients using polymerase chain reaction--single strand conformation polymorphism (PCR-SSCP) and direct-sequencing strategies, we identified a missense mutation substituting arginine (AGG) for glycine (GGG) at position 261 in exon 7 of the glucokinase gene in a patient with early-onset non-insulin-dependent diabetes (NIDDM).