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PubMed2000

Functional glucokinase isoforms are expressed in rat brain.

Recently, the description of glucokinase mRNA in certain neuroendocrine cells has opened new ways to characterize this enzyme in the rat brain. In this study, we found glucokinase mRNA and a similar RNA splicing pattern of the glucokinase gene product in rat hypothalamus and pancreatic islets; the mRNA that codes for B1 isoform was the most abundant, with minor amounts of those coding for the B2, P1, P2, P1/B2, and P2/B2 isoforms. Glucokinase gene expression in rat brain gave rise to a protein of 52 kDa with a high apparent Km for glucose and no product inhibition by glucose 6-phosphate, with a contribution to the total glucose phosphorylating activity of between 40 and 14%; the hypothalamus and cerebral cortex were the regions of maximal activity. Low and high Km hexokinases were characterized by several criteria. Also, using RT-PCR analysis we found a glucokinase regulatory protein mRNA similar to that previously reported in liver. These findings indicate that the glucokinase present in rat brain should facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and the expression of glucokinase and GLUT-2 in the same hypothalamic neurons suggests a role in glucose sensing.

Animals↗

PubMed1997

Acute glucose intolerance in insulinoma cells with unbalanced overexpression of glucokinase.

The INS-r3-GK27 insulinoma cells are endowed with artificially inducible glucokinase under control of the reverse tetracycline-dependent transcriptional activator. Moderate induction of glucokinase has been shown to result in proportionate increases in glycolytic flux and in potentiation of glucose effects on insulin secretion and pyruvate kinase gene expression. In cells with 20-fold overexpression of glucokinase, however, glucose activation of secretion and gene expression was severely impaired. Measurements of the glycolytic flux in cells with 7- and 21-fold increases in glucokinase activity and determination of the flux control coefficient of this enzyme showed that control of glycolysis at the glucokinase step was lost in the cells at the higher level of overexpression. Challenging the cells with glucose above 6 mM resulted in massive accumulation of glucose 6-phosphate and caused a rapid and sustained depletion of cellular ATP, in contrast with the glucose-induced rise in ATP in cells with wild-type glucokinase levels. Loss of cell viability ensued upon prolonged culture in high glucose. In summary, in insulinoma beta cells strongly overexpressing glucokinase, an imbalance between glucose phosphorylation and turnover of glucose 6-phosphate resulted in acute glucose intolerance due to trapping of cellular orthophosphate in dead-end product and severe paralysis of energy metabolism.

Animals↗

PubMed2000

Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation.

Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421).

Catalytic Domain↗

PubMed1996

Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element.

Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.

Animals↗

PubMed1984

New perspectives on pancreatic islet glucokinase.

Control of blood sugar involves the complex interaction of the pancreatic glucose-sensing beta-cells with the liver, which serves as the primary site of glucose disposal after a meal. Glucokinase occupies an important role in controlling glucose phosphorylation and metabolism both in the liver and in pancreatic islets. In the beta-cells, glucokinase functions as pacemaker of glycolysis at physiological glucose levels. It determines the unique characteristics of islet hexose usage, that is, the rate, affinity, cooperativity, and anomeric discrimination of glucose metabolism. Because glycolysis controls hexose-induced insulin release, glucokinase is considered the best-qualified candidate for the elusive glucose sensor of beta-cells. A deficiency of glucokinase would disturb glucose homeostasis. Decreased islet glucokinase would diminish islet glycolysis and would result in a higher set point of beta-cells for glucose-induced insulin release. Decreased liver glucokinase would cause less efficient hepatic glucose disposal. Human maturity-onset diabetes (type II diabetes) has these characteristics. It is thus conceivable that certain forms of type II diabetes are due to a glucokinase deficiency.

Animals↗

PubMed1996

Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets.

Glucokinase has exclusively high control strength on glucose usage in the pancreatic beta-cell. However, glucokinase also has extraordinarily high control strength on insulin secretion, which is linked to the phosphate potential, [ATP]/([ADP][Pi]) (F.M. Matschinsky, Y.Liang, P. Kesavan, L. Wang, P. Froguel, G. Velho, D. Cohen, M.A. Permutt, Y. Tanizawa, T.L. Jetton, K. Niswender, and M.A. Magnuson. J. Clin. Invest. 92: 2092-2098, 1993). We propose that the ATP produced via the tricarboxylic acid cycle is approximately constant, irrespective of the glucose level. Furthermore, the component of ATP production that is derived from glycolysis and glycolytically derived NADH, which is shuttled into the mitochondria, is a critical signal controlling the ionic events leading to insulin secretion, as suggested previously (M. J. MacDonald. Diabetes 39: 1461-1466, 1990 and I.D. Dukes, M.S. McIntyre, R.J. Mertz, L.H. Philipson, M.W. Roe, B. Spencer, and J.F. Worley III. J. Biol. Chem. 269: 10979-10982, 1994). To test this hypothesis, glucose usage, oxidation, and insulin secretion were measured in cultured rat islets over a wide range of concentrations of glucose and mannoheptulose, an inhibitor of glucokinase. These data were fit to a mathematical model that predicts that glucokinase will govern the rate of glucose usage and ATP production and will also have a strong, but not complete, control over the rate of glucose oxidation, the phosphate potential, and insulin release. Mannoheptulose caused an inhibition of all three fluxes. The estimates of the mechanistic parameters of the model [maximal velocity (Vmax) and Michaelis constant for glucokinase, Vmax for hexokinase and glucose transport, and the inhibition constant of mannoheptulose to glucokinase] were similar to those obtained in vitro. Thus the data are consistent with a model in which the primary importance of glycolysis in transducing the glucose signal into changes of the phosphate potential imparts to glucokinase a high control strength on glucose-induced insulin secretion.

Animals↗

PubMedDate not supplied

Utility of 3-O-methyl-N-acetyl-D-glucosamine, an N-acetylglucosamine kinase inhibitor, for accurate assay of glucokinase in pancreatic islets and liver.

Glucokinase, an enzyme that catalyzes the phosphorylation of glucose, constitutes the key regulatory step in glucose metabolism in pancreatic islets and liver. We found that 3-O-methyl-N-acetyl-D-glucosamine (3-O-methyl-GlcNAc) potently inhibits glucose phosphorylation by N-acetylglucosamine kinase whereas glucokinase is not at all affected by this hexosamine. The addition of 3-O-methyl-GlcNAc to the assay system for glucokinase in rat liver extracts, which contain a high activity of glucokinase (glucose as substrate) relative to N-acetylglucosamine kinase (N-acetyl-D-glucosamine as substrate), affected neither Km nor Vmax values of glucokinase. On the other hand, both Km and Vmax values of glucokinase in rat pancreatic islet extracts, in which N-acetylglucosamine kinase activity is higher than glucokinase activity, were significantly lowered by the use of 3-O-methyl-GlcNAc as an inhibitor of N-acetylglucosamine kinase.

Acetylglucosamine↗

PubMed1994

Regulatory effects of glucose on the catalytic activity and cellular content of glucokinase in the pancreatic beta cell. Study using cultured rat islets.

Glucose regulates the cellular content of glucokinase in the pancreatic beta cell by altering the level of the enzyme. We investigated the existence of a second regulatory pathway, an alteration in the catalytic activity, by comparing Vmax and protein levels of glucokinase in rat islets cultured under high glucose conditions (16.7 mM) for 6, 14, and 24 h. The Vmax was increased by glucose at all time points. In contrast, glucokinase protein levels on Western blots were unchanged from the control value at 6 h but increased 40% at the later time points (P < 0.0002). Further evidence for a dual regulatory system was obtained with a reversal protocol. After a 6-h incubation at high glucose, an additional 3-h incubation at 5.5 mM glucose restored glucokinase Vmax to normal, but failed to change the Vmax after a 24-h incubation at high glucose. Finally, 10 microM cycloheximide partially prevented the increase in glucokinase Vmax induced by 24 h of high glucose, but had no effect at 6 h, suggesting the early increase in enzymatic activity did not require protein synthesis. In summary, glucose regulates both the catalytic activity and cellular content of glucokinase in the beta cell. Glucose-induced increases in glucokinase activity are an important element of the beta cell adaptive response to hyperglycemia.

Animals↗

PubMed1992

Regulation of glucokinase and proinsulin gene expression and insulin secretion in RIN-m5F cells by dexamethasone, retinoic acid, and thyroid hormone.

In RIN-5mF cells, treatment with either dexamethasone or retinoic acid resulted in increased glucokinase activity. Treatment with dexamethasone (50 and 500 nM) increased glucokinase activity 140% and 260%, respectively, whereas treatment with retinoic acid (100 and 1000 nM) increased glucokinase activity by 50% and 80%, respectively. An additive effect was observed when 1000 nM retinoic acid was added to either 50 or 500 nM dexamethasone. Treatment with either of these factors resulted in increased glucokinase mRNA levels. In contrast, treatment with thyroid hormone (T3; 0.2 and 10 nM) had no effect on glucokinase activity, but decreased glucokinase mRNA levels. T3 did not affect the response of either activity or mRNA levels to retinoic acid. Binding experiments revealed the presence of a single class of T3 nuclear receptors, with a Kd of 0.46 x 10(-10) and a binding capacity of 3.84 pmol/mg protein in these cells. This suggests that the changes produced by T3 may be due to a physiological effect mediated via nuclear receptors. Proinsulin mRNA levels were increased by retinoic acid (10-100 nM) and dexamethasone (5-500 nM), but decreased by T3 (0.04-10 nM). Insulin secretion in the absence or presence of glucose and potassium was increased about 50% in cells previously treated with 1000 nM retinoic acid. T3 (0.2 nM) did not affect insulin secretion in the absence or presence of glucose or in the presence of secretagogues (potassium and glyceraldehyde). Dexamethasone (50 nM) did not significantly increase insulin secretion in the absence or presence of glucose or potassium. In contrast, dexamethasone decreased glyceraldehyde-induced insulin secretion by 60% (P less than 0.05). We conclude that the glucokinase and proinsulin genes respond in parallel to dexamethasone and retinoic acid (both increased) and to T3 (both decreased).

Animals↗

PubMed1987

Defective regulation of glucokinase in rat pancreatic islet cell tumours.

The role of glucokinase in the regulation of insulin secretion was examined in normal rat pancreatic islets and in chemically- and radiation-induced rat pancreatic B-cell tumours which show an impaired insulin secretory response to glucose. In normal rats glucokinase activity in cytoplasmic fractions of pancreatic islets was decreased with the duration of fasting and increased by refeeding or insulin administration. This observation is consistent with the induction of glucokinase by insulin. Hexokinase activity was only slightly reduced during fasting. Glucokinase activity decreased in cytoplasmic fractions of streptozotocin-nicotinamide-induced rat pancreatic islet cell tumours. Glucokinase activity contributed about 75% to the total glucose phosphorylation capacity in cytoplasmic fractions of normal pancreatic islets and of small (less than 1 mg) streptozotocin-nicotinamide-tumours. This proportion decreased to about 20% in the large streptozotocin-nicotinamide tumours. Glucokinase activity in cytoplasmic fractions of transplantable radiation-induced NEDH (New England Deaconess Hospital) rat B-cell tumours was seven times lower than in normal pancreatic islets and contributed only 15% to the total glucose phosphorylation capacity. In contrast, hexokinase activity of the NEDH tumour B-cells was 2.5 times higher than normal. Decreased glucokinase activity in the chemically- and radiation-induced tumour B-cells appears to result from a loss of the ability of insulin to induce this enzyme and may explain the lack of insulin secretory responsiveness of these tumour B-cells.

Adenoma, Islet Cell↗

PubMed1983

Mannose phosphorylation by glucokinase from liver and transplantable insulinoma. Cooperativity and discrimination of anomers.

Glucokinase from rat liver or transplantable, radiation-induced insulinomas was partially purified by ion exchange chromatography using DEAE-Cibacron Blue F3GA agarose. Phosphorylation of alpha,beta-D-mannose by glucokinase occurred with cooperative rate dependence on mannose concentration (nH: 1.50). Half-maximal phosphorylation rate occurred at 14 mM alpha,beta-D-mannose. The alpha- and beta-anomers of mannose were phosphorylated with sigmoidal kinetics (nH: 1.57 and 1.42, respectively). The affinity of glucokinase for alpha-D-mannose is higher than for beta-D-mannose (S0.5: 12 mM versus 19 mM). The maximum phosphorylation rate is slightly higher, about 10%, with beta-D-mannose than with alpha-D-mannose. Islet glucokinase has previously been shown to be chromatographically and kinetically identical to glucokinase from insulinoma and liver; therefore, evidence that glucokinase from these two tissues phosphorylates mannose with cooperative rate dependence and differentiates mannose anomers supports the glucokinase-glucose sensor hypothesis.

Adenoma, Islet Cell↗

PubMed1986

The glucokinase glucose sensor in human pancreatic islet tissue.

The enzyme glucokinase controls glucose metabolism in islets and is proposed to be the glucose sensor in pancreatic beta-cells. This concept was developed from studies with rodents and it remained to be explored whether it also applies to man. Studies in man were hampered, however, by the difficulty in obtaining well-preserved pancreatic islet tissue and also because the high activity of hexokinase made it difficult to measure glucokinase. To overcome these obstacles, quantitative histochemical sampling techniques were developed allowing precise dissection of pure human islet tissue and a newly designed radiometric microassay was used, avoiding hexokinase interference, and providing the sensitivity necessary to measure the relatively low glucokinase activity in small samples of tissue obtained from brain-dead tissue donors. The present data indicate that glucokinase is present in human pancreatic islet tissue and is not found in the exocrine pancreas. The enzyme's Vmax with D-glucose as substrate was similar to the Vmax for glucose utilization reported previously for intact, isolated human islets and the enzyme's Km for D-glucose was about 5 mM. Since glucokinase was also present in islet tissue of hamster, mouse, and rat, it is suggested that the glucokinase-glucose sensor concept has general applicability and that it could explain many aspects of the physiology and pathology of glucose homeostasis. This well-defined pancreatic islet glucokinase-glucose sensor should, therefore, be incorporated in any comprehensive model of glucose homeostasis.

Adolescent↗

PubMed1988

Control of glucose metabolism in pancreatic beta-cells by glucokinase, hexokinase, and phosphofructokinase. Model study with cell lines derived from beta-cells.

Glucose usage by soluble fractions of cell extracts from two insulin-producing cell lines, RINm5F and HIT, was investigated. Analysis of enzyme activities indicated that glucose phosphorylation and phosphofructokinase are likely to be the rate-limiting steps of glycolysis in both RINm5F and HIT cell extracts. RINm5F extracts, which lack glucokinase, exhibited relatively flat concentration-dependency curves of glucose usage and showed substantial inhibition of hexokinase. HIT cell extracts, which contain glucokinase but lack hexokinase, exhibited sigmoidal concentration-dependency curves of glucose usage, reflecting almost fully expressed glucokinase activity. A reconstituted system prepared from RINm5F and HIT cell extracts exhibited a composite concentration-dependency curve of glucose usage and showed substantial inhibition of hexokinase and almost fully expressed glucokinase. However, conditions that activate phosphofructokinase, such as addition of ammonium sulfate or fructose 2,6-bisphosphate or alkalization, removed the inhibition of hexokinase without noticeably affecting the glucokinase component of usage. Results obtained with a reconstituted system containing RINm5F cell extract and purified glucokinase were consistent with these findings. The data presented here indicate that this reconstituted cell-free system serves as a valid model for the study of aspects of glycolytic control in the islet. This model illustrates the preeminent role of glucokinase in the control of glycolysis, consistent with its glucose-sensor function in the islet. In addition, these studies help to define the contribution of phosphofructokinase to the control of glycolysis and the mechanism whereby changes in phosphofructokinase activity could modulate, via changes in the glucose 6-phosphate concentration, the activity of hexokinase and hence the net glycolytic flux.

Ammonium Sulfate↗

PubMed1992

Human islet glucokinase gene. Isolation and sequence analysis of full-length cDNA.

Pancreatic islet glucokinase (ATP:D-hexose-6-phosphotransferase) cDNAs were isolated from a human islet cDNA library in lambda-gt11. One clone (hlGLK2), 2723 bp plus additional poly(A) residues, appeared to be full length because its size was consistent with a single 2.9-kb glucokinase mRNA on Northern-blot analysis of islet RNA. This cDNA contained an open reading frame of 1395 bp from an ATG codon at position 459, encoding a predicted protein of 465 amino acids (52,000 M(r)). Comparison of the nucleotide sequences of the human islet glucokinase cDNA with that of the recently isolated human liver glucokinase cDNA revealed that the two cDNAs differed completely on their 5'-ends, followed by an identical 2204-bp overlap extending to the 3'-ends. The 5'-ends of islet and liver glucokinase cDNAs predicted proteins that differ by 15 NH2-terminal residues. The overall sequence identity (70%) between the first exons of the human islet and rat islet cDNAs suggested that the islet promoter regions, like the liver promoter regions, have been conserved through evolution. Thus, NH2-terminal differences for human liver and islet enzymes might be explained by use of alternate promoters between the two tissues, analogous to the NH2-terminal differences of the rat liver and rat islet enzymes. If so, this relationship predicts important tissue-specific regulatory functions of these regions. Variations in the glucokinase gene are likely to occur in humans. Isolation of a human islet glucokinase cDNA has provided the sequence necessary to determine whether these variants are important determinants in the genetic predisposition for diabetes mellitus.

Amino Acid Sequence↗

PubMed1997

Abnormal regulation of hepatic glucose output in maturity-onset diabetes of the young caused by a specific mutation of the glucokinase gene.

A subtype of maturity-onset diabetes of the young (MODY) is caused by mutations of the glucokinase gene, an enzyme expressed in pancreatic beta-cells and the liver. To assess the consequences of a functional alteration of glucokinase at the level of the liver, endogenous (hepatic) glucose production and glucose cycling (an indirect assessment of hepatic glucokinase activity) were measured with 2-2H glucose and 6,6-2H glucose in patients who developed MODY because of the V203A mutation of glucokinase, and in control subjects at similar levels of glycemia. Measurements were performed in the postabsorptive state and after ingestion of 13C-labeled glucose. In the postabsorptive state, MODY patients had normal glucose production (10.9 +/- 1.3 vs. 11.3 +/- 0.6 micromol x kg(-1) x min(-1)) but decreased glucose cycling (0.6 +/- 0.3 vs. 1.5 +/- 0.3 micromol x kg(-1) x min(-1); P < 0.05) when compared with control subjects. However, at plasma glucose and insulin levels similar to those observed in MODY patients, control subjects' glucose production was markedly lower (3.2 +/- 1.5 micromol x kg(-1) x min(-1). After glucose ingestion, endogenous glucose production was reduced by only 29% in MODY patients compared with 80% in control subjects at a similar level of hyperglycemia (P < 0.05). This suggests that the V203A mutation of glucokinase results in decreased activity of glucokinase in liver cells. Thus endogenous glucose production is inadequately inhibited by hyperglycemia in MODY patients, possibly as a result of impaired hepatic glucokinase activity. These alterations contribute to the pathogenesis of hyperglycemia.

Adult↗

PubMed1999

Structural model of human glucokinase in complex with glucose and ATP: implications for the mutants that cause hypo- and hyperglycemia.

Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.

Adenosine Triphosphate↗

PubMed2004

Establishment of liver specific glucokinase gene knockout mice: a new animal model for screening anti-diabetic drugs.

AIM: To characterize the liver-specific role of glucokinase in maintaining glucose homeostasis and to create an animal model for diabetes. METHODS: We performed hepatocyte-specific gene knockout of glucokinase in mice using Cre-loxP gene targeting strategy. First, two directly repeated loxP sequences were inserted to flank the exon 9 and exon 10 of glucokinase in genomic DNA. To achieve this, linearized targeting vector was electroporated into ES cells. Then G418- and Gancyclovir-double-resistant clones were picked and screened by PCR analysis and the positives identified by PCR were confirmed by Southern blot. A targeted clone was selected for microinjection into C57BL/6J blastocysts and implanted into pseudopregnant FVB recipient. Chimeric mice and their offspring were analyzed by Southern blot. Then by intercrossing the Alb-Cre transgenic mice with mice containing a conditional gk allele, we obtained mice with liver-specific glucokinase gene knockout. RESULTS: Among 161 double resistant clones 4 were positive to PCR and Southern blot and only one was used for further experiments. Eventually we generated the liver specific glucokinase knockout mice. These mice showed increased glucose level with age and at the age of 6 weeks fasting blood glucose level was significantly higher than control and they also displayed impaired glucose tolerance. CONCLUSION: Our studies indicate that hepatic glucokinase plays an important role in glucose homeostasis and its deficiencies contribute to the development of diabetes. The liver glucokinase knockout mouse is an ideal animal model for MODY2, and it also can be applied for screening anti-diabetic drugs.

Animals↗

PubMed1989

Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system.

Primary cultures of rat hepatocytes were used to investigate the regulation of glucokinase gene expression by insulin and glucagon. Insulin added in physiological concentrations to the culture medium causes de novo induction of glucokinase mRNA. The induced plateau is reached 4 to 8 h after insulin addition, and the mRNA level remains high as long as insulin is present. Comparison of the potencies of insulin, proinsulin, and insulin-like growth factor I in this system indicates that induction by insulin is mediated via the insulin receptor. The magnitude of the insulin effect is independent of the extracellular glucose concentration. Run-on transcription assays with isolated nuclei show that the mRNA build up depends primarily on a specific stimulation of glucokinase gene transcription. Glucagon added to hepatocytes together with a supramaximal concentration of insulin prevents induction of glucokinase mRNA in a dose-dependent manner. The inhibitory effect of glucagon is mimicked by 8-(4-chlorophenylthio)-cAMP. The effect of this agent has also been tested in hepatocytes first induced for maximal glucokinase gene transcription by culture with insulin alone for 12 h. The transcriptional activity of the gene as measured by run-on assay was completely turned off within 30 min after addition of the cyclic nucleotide. Under these conditions, glucokinase mRNA decays rapidly, with an apparent half-life of 45 min. The mRNA degradation rate was similarly rapid after insulin withdrawal from induced cells. Thus, a cAMP-mediated repression mechanism is a key aspect in the regulation of glucokinase gene transcription in the hepatocyte. Insulin may act by relieving the gene from repression.

Animals↗