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

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

Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets

In the post-microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D-fructose 1-phosphate increased the activity of glucokinase by 20-30% as measured in the presence of D-glucose 6-phosphate and D-fructose 6-phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D-fructose 1-phosphate. In the presence of hexose 6-phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D-fructose-1-phosphate-sensitive manner. In intact islets, D-glyceraldehyde stimulated the generation of 14C-labelled D-fructose 1-phosphate from D-[U-14C]glucose and increased the production of 3H2O from D-[5-3H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D-fructose 6-phosphate and D-fructose 1-phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.     

A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity

A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic beta cells. To search for such a protein in pancreatic beta cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system. We detected a cDNA encoding the precursor of propionyl-CoA carboxylase beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23%. We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.     

The mechanism by which rat liver glucokinase is inhibited by the regulatory protein

The fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer. The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecular-mass inhibitor. Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1-phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.     

Inhibition of glucokinase in hepatocytes by alloxan

The effect of alloxan on glucokinase in isolated rat hepatocytes was studied. Exposure of hepatocytes to alloxan (3 mM) at 30 degrees C for 5 min produced a marked inhibition (77%) of glucokinase activity and altered slightly the phosphofructokinase activity (32% inhibition). Pyruvate kinase and glucose 6-phosphate dehydrogenase, however, were not inhibited at all. Alloxan induced a concentration-dependent inhibition of glucokinase activity with a detectable inhibition at an alloxan concentration of 1 mM. The inhibition of glucokinase activity by alloxan was protected by the simultaneous presence of 15 mM hexose such as D-glucose, 3-O-methylglucose, or D-mannose. D-Galactose showed no protective effect. These results suggest that alloxan may exert its cytotoxic action through the inhibition of glucokinase activity not only in the liver but also in the pancreatic islets, since liver and islet glucokinases are known to be quite similar in various properties.     

Competitive inhibition of liver glucokinase by its regulatory protein

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.     

Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation

In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate.     

Characterization of glucokinase-binding protein epitopes by a phage-displayed peptide library. Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner

The low affinity glucose-phosphorylating enzyme glucokinase shows the phenomenon of intracellular translocation in beta cells of the pancreas and the liver. To identify potential binding partners of glucokinase by a systematic strategy, human beta cell glucokinase was screened by a 12-mer random peptide library displayed by the M13 phage. This panning procedure revealed two consensus motifs with a high binding affinity for glucokinase. The first consensus motif, LSAXXVAG, corresponded to the glucokinase regulatory protein of the liver. The second consensus motif, SLKVWT, showed a complete homology to the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which acts as a key regulator of glucose metabolism. Through yeast two-hybrid analysis it became evident that the binding of glucokinase to PFK-2/FBPase-2 is conferred by the bisphosphatase domain, whereas the kinase domain is responsible for dimerization. 5'-Rapid amplification of cDNA ends analysis and Northern blot analysis revealed that rat pancreatic islets express the brain isoform of PFK-2/FBPase-2. A minor portion of the islet PFK-2/FBPase-2 cDNA clones comprised a novel splice variant with 8 additional amino acids in the kinase domain. The binding of the islet/brain PFK-2/ FBPase-2 isoform to glucokinase was comparable with that of the liver isoform. The interaction between glucokinase and PFK-2/FBPase-2 may provide the rationale for recent observations of a fructose-2,6-bisphosphate level-dependent partial channeling of glycolytic intermediates between glucokinase and glycolytic enzymes. In pancreatic beta cells this interaction may have a regulatory function for the metabolic stimulus-secretion coupling. Changes in fructose-2,6-bisphosphate levels and modulation of PFK-2/FBPase-2 activities may participate in the physiological regulation of glucokinase-mediated glucose-induced insulin secretion.     

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

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

Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis.

To gain better insight into the insulin secretory activity of fetal beta cells in response to glucose, the expression of glucose transporter 2 (GLUT-2), glucokinase and mitochondrial glycerol phosphate dehydrogenase (mGDH) were studied. Expression of GLUT-2 mRNA and protein in pancreatic islets and liver was significantly lower in fetal and suckling rats than in adult rats. The glucokinase content of fetal islets was significantly higher than of suckling and adult rats, and in liver the enzyme appeared for the first time on about day 20 of extrauterine life. The highest content of hexokinase I was found in fetal islets, after which it decreased progressively to the adult values. Glucokinase mRNA was abundantly expressed in the islets of all the experimental groups, whereas in liver it was only present in adults and 20-day-old suckling rats. In fetal islets, GLUT-2 and glucokinase protein and their mRNA increased as a function of increasing glucose concentration, whereas reduced mitochondrial citrate synthase, succinate dehydrogenase and cytochrome c oxidase activities and mGDH expression were observed. These findings, together with those reported by others, may help to explain the decreased insulin secretory activity of fetal beta cells in response to glucose.     

A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate

At a concentration of 1 mM, fructose 1-phosphate stimulated about twofold, and glucose 6-phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high-speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion-exchange chromatography of liver extracts separated glucokinase from a fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho-Ultrogel. It was destroyed by trypsin and was heat-labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6-phosphate although not by glucose 6-phosphate. Fructose 1-phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6-phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).     

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.     

Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Contributions of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 to the elevated glycolysis in hepatocytes from Zucker fa/fa rats

The insulin-resistant Zucker fa/fa rat has elevated hepatic glycolysis and activities of glucokinase and phosphofructokinase-2/fructose bisphosphatase-2 (PFK2). The latter catalyzes the formation and degradation of fructose-2,6-bisphosphate (fructose-2,6-P(2)) and is a glucokinase-binding protein. The contributions of glucokinase and PFK2 to the elevated glycolysis in fa/fa hepatocytes were determined by overexpressing these enzymes individually or in combination. Metabolic control analysis was used to determine enzyme coefficients on glycolysis and metabolite concentrations. Glucokinase had a high control coefficient on glycolysis in all hormonal conditions tested, whereas PFK2 had significant control only in the presence of glucagon, which phosphorylates PFK2 and suppresses glycolysis. Despite the high control strength of glucokinase, the elevated glycolysis in fa/fa hepatocytes could not be explained by the elevated glucokinase activity alone. In hepatocytes from fa/fa rats, glucokinase translocation between the nucleus and the cytoplasm was refractory to glucose but responsive to glucagon. Expression of a kinase-active PFK2 variant reversed the glucagon effect on glucokinase translocation and glucose phosphorylation, confirming the role for PFK2 in sequestering glucokinase in the cytoplasm. Glucokinase had a high control on glucose-6-phosphate content; however, like PFK2, it had a relative modest effect on the fructose-2,6-P(2) content. However, combined overexpression of glucokinase and PFK2 had a synergistic effect on fructose-2,6-P(2) levels, suggesting that interaction of these enzymes may be a prerequisite for formation of fructose-2,6-P(2). Cumulatively, this study provides support for coordinate roles for glucokinase and PFK2 in the elevated hepatic glycolysis in fa/fa rats.     

Long-chain fatty acyl-coenzyme A-induced inhibition of glucokinase in pancreatic islets from rats depleted in long-chain polyunsaturated omega3 fatty acids

The metabolism of D-glucose was recently reported to be impaired in pancreatic islets from second generation rats depleted in long-chain polyunsaturated omega3 fatty acids. Considering the increased clearance of circulating non-esterified fatty acids prevailing in these rats, a possible inhibition of glucokinase in insulin-producing cells by endogenous long-chain fatty acyl-CoA was considered. The present study was mainly aimed at assessing the validity of the latter proposal. The activity of glucokinase in islet homogenates, as judged from the increase in D-glucose phosphorylation rate in response to a rise in the concentration of the hexose represented, in the omega3-depleted rats, was only 81.8 +/- 4.8% (n = 11; p < 0.005) of the paired value recorded in control animals. This coincided with the fact that the inclusion of D-glucose 6-phosphate (3.0 mM) and D-fructose 1-phosphate (1.0 mM) in the assay medium resulted in a lesser fractional decrease of D-glucose phosphorylation in omega3-depleted rats than in control animals. Moreover, whereas palmitoyl-CoA (50 microM) decreased the activity of glucokinase by 38.0 +/- 6.0% (n = 4; p < 0.01) in islet homogenates from normal rats, the CoA ester failed to affect significantly the activity of glucokinase in islet homogenates from omega3-depleted rats. These findings afford direct support for the view that glucokinase is indeed inhibited by endogenous long-chain fatty acyl-CoA in islets from omega3-depleted rats, such an inhibition probably participating to the alteration of D-glucose catabolism prevailing in these islets.