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1.
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). 相似文献
2.
Saccharomyces Cerevisiae Null Mutants in Glucose Phosphorylation: Metabolism and Invertase Expression 总被引:8,自引:1,他引:7
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A congenic series of Saccharomyces cerevisiae strains has been constructed which carry, in all combinations, null mutations in the three genes for glucose phosphorylation: HXK1, HXK2 and GLK1, coding hexokinase 1 (also called PI or A), hexokinase 2 (PII or B), and glucokinase, respectively: i.e., eight strains, all of which grow on glucose except for the triple mutant. All or several of the strains were characterized in their steady state batch growth with 0.2% or 2% glucose, in aerobic as well as respiration-inhibited conditions, with respect to growth rate, yield, and ethanol formation. Glucose flux values were generally similar for different strains and conditions, provided they contained either hexokinase 1 or hexokinase 2. And their aerobic growth, as known for wild type, was largely fermentative with ca. 1.5 mol ethanol made per mol glucose used. The strain lacking both hexokinases and containing glucokinase was an exception in having reduced flux, a result fitting with its maximal rate of glucose phosphorylation in vitro. Aerobic growth of even the latter strain was largely fermentative (ca. 1 mol ethanol per mol glucose). Invertase expression was determined for a variety of media. All strains with HXK2 showed repression in growth on glucose and the others did not. Derepression in the wild-type strain occurred at ca. 1 mM glucose. The metabolic data do not support- or disprove-a model with HXK2 having only a secondary role in catabolite repression related to more rapid metabolism. 相似文献
3.
Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content. 相似文献
4.
Lindner SN Seibold GM Henrich A Krämer R Wendisch VF 《Applied and environmental microbiology》2011,77(11):3571-3581
Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield. 相似文献
5.
Brevibacterium flavum mutants defective in the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) were selected with high frequency by 2-deoxyglucose-resistance. Most of them (DOGr) still had the fructose-PTS and grew not only on fructose but also on glucose like the wild-type strain. A mutant having 1 /8th the fructose-PTS activity of the wild strain but normal glucose-PTS activity was isolated as a xylitol-resistant mutant. It grew on glucose but not on fructose. The glucose-PTS was active on glucose, glucosamine, 2-deoxyglucose and mannose, and slightly on methyl-a-glucoside and N-acetylglucosamine, but not on fructose or xylitol. The fructose-PTS acted on fructose and xylitol, and to some extent on glucose but not on glucosamine or 2-deoxyglucose. Mutants unable to grow on glucose (DOGrGlc-) derived from a DOGr mutant were all defective in the fructose-PTS. All revertants able to grow on glucose derived from a DOGrGlc“ mutant had the fructose-PTS. The glucokinase activity was about 2/3rds the glucose activity of the fructose-PTS. All the DOGrGlc- mutants had normal levels of glucokinase. One of these mutants grew on maltose and sucrose, which were hydrolyzed to glucose. Thus, glucokinase seems to contribute to the phosphorylation of glucose liberated inside the cell. The fructose-PTS was induced by fructose and repressed by glucose. The glucose repression was not observed in a mutant defective in the glucose-PTS. 相似文献
6.
We studied the role of glucokinase translocation between the nucleus and the cytoplasm in hepatocytes. In cultured hepatocytes, both the translocation of glucokinase from the nucleus to the cytoplasm and the rate of glucose phosphorylation were increased when cells were incubated with high concentrations of glucose. The addition of low concentrations of fructose, which is known to stimulate glucose phosphorylation, stimulated both glucokinase translocation and glucose phosphorylation. There was a good correlation between the increase in cytoplasmic glucokinase induced by fructose and that in the glucose phosphorylation rate induced by fructose. Furthermore, we observed a linear relationship between cytoplasmic glucokinase activity and rate of glucose phosphorylation over various glucose concentrations in the absence or presence of fructose. These results indicate that glucose phosphorylation in hepatocytes depended on glucokinase in the cytoplasmic compartment--that is, the increase in the rate of glucose phosphorylation was due to the increase in translocation of glucokinase out of the nucleus. Also, oral administration of glucose, fructose, or glucose plus fructose to 24-h fasted rats induced translocation of glucokinase in the liver. All of these results indicate that hepatic glucose metabolism is regulated by the translocation of glucokinase. 相似文献
7.
The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte 总被引:6,自引:0,他引:6
de la Iglesia N Mukhtar M Seoane J Guinovart JJ Agius L 《The Journal of biological chemistry》2000,275(14):10597-10603
Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte. 相似文献
8.
Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. 13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb''s central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections. 相似文献
9.
Agrimi G Brambilla L Frascotti G Pisano I Porro D Vai M Palmieri L 《Applied and environmental microbiology》2011,77(7):2239-2246
The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality. 相似文献
10.
Differential regulation of glucokinase activity in pancreatic islets and liver of the rat 总被引:5,自引:0,他引:5
F J Bedoya F M Matschinsky T Shimizu J J O'Neil M C Appel 《The Journal of biological chemistry》1986,261(23):10760-10764
The differential tissue-specific regulation of glucokinase activity in liver and pancreatic islet cells was investigated in the insulinoma-bearing rat. A transplantable insulinoma caused hyperinsulinemia and hypoglycemia in the host by 2-3 months after implantation. Suppression of the pancreatic B-cells by the high insulin and/or low glucose manifested itself by a decrease of insulin in islet tissue. Removal of the tumor initiated transient insulin deficiency and hyperglycemia with extremes of these changes at 24 h after tumor resection. These conditions markedly affected glucose phosphorylation in the islet cells: glucokinase activity was reduced 71% in islet samples from insulinoma-bearing rats, and the enzyme fully recovered within 24 h after tumor resection. Hexokinase activity, by contrast, was not affected by these manipulations. To evaluate the relative contributions of hypoglycemia and hyperinsulinemia in islet glucokinase adaptation, glucose was intravenously infused to insulinoma-bearing rats; glycemia in excess of 150 mg/100 ml combined with excessive hyperinsulinemia resulted in a partial recovery of islet glucokinase activity, first apparent after 9 h of glucose infusion and with doubling of the activity after 24 h after glucose loading. In contrast, liver glucokinase was increased nearly 4-fold at the time of extreme hypoglycemia and hyperinsulinemia and rapidly fell to control rates following tumor removal. Intravenous infusion of glucose for 24 h into the tumor-bearing rat (i.e. hyperglycemia combined with excessive plasma insulin) had no influence on liver glucokinase activity. Liver hexokinase was not influenced by any of these experimental manipulations. The data indicate that the activities of pancreatic islet and liver glucokinase are regulated in a differential manner. Insulin is apparently the primary determinant of liver glucokinase and glucose seems to control islet glucokinase. Biochemical mechanisms for differential organ-specific regulation of glucokinase activity seem to have evolved such that this enzyme may play a dual role in glucose homeostasis, namely to serve as insulin-dependent glucose sensor in the B-cells and as insulin-sensitive determinant of hepatic glucose use. 相似文献
11.
Characterization of sugar uptake in wild-type Streptomyces clavuligerus, which is impaired in glucose uptake, and in a glucose-utilizing mutant. 总被引:1,自引:0,他引:1
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Wild-type Streptomyces clavuligerus NRRL 3585 is unable to utilize glucose. A glucose-utilizing (gut-1) mutant of S. clavuligerus NRRL 3585 has been obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The gut-1 mutant is able to grow on glucose or galactose, while the wild type is unable to catabolize these hexoses. Similar binding affinities of glucose by cells of the wild type and the gut-1 mutant were found, but the wild type was unable to complete glucose transport. A soluble intracellular ATP-dependent (but not phosphoenolpyruvate-dependent) glucokinase activity was found both in the wild type and the gut-1 mutant. The gut-1 mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-1 transport system concentrates glucose inside the cell at least 10- to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus. 相似文献
12.
Phosphorylation of glucose in isolated rat hepatocytes. Sigmoidal kinetics explained by the activity of glucokinase alone
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The conversion of glucose into glucose 6-phosphate in an extract of isolated rat hepatocytes incubated in the presence of MgATP was studied spectrophotometrically at 340nm and also by a radiochemical procedure based on the release of (3)H from [2-(3)H]glucose. Both methods gave similar results. The glucose-saturation curve was sigmoidal and the shape of this curve was not influenced by the ionic composition of the incubation medium. The activity at 0.5mm-glucose was only 1-2% of V(max.), indicating a virtual absence of low-K(m) hexokinase in the preparation. The radiochemical method was also used for the determination of glucose phosphorylation by intact hepatocytes. The glucose-saturation curve was also markedly sigmoidal, but the s(0.5) (substrate concentration at half-maximal velocity) and the Hill coefficient were larger than in extracts of hepatocytes. These two parameters became smaller when cells were incubated in a medium in which Na(+) ions were replaced by K(+) ions. The increased rate of phosphorylation at low glucose concentration in a K(+) medium was accompanied by an increased rate of metabolite recycling between glucose and glucose 6-phosphate and also by an increased uptake of glucose. In both media phosphorylation of glucose was inhibited co-operatively by N-acetylglucosamine. Calculations indicate that this inhibition would reach 100% at saturation of the inhibitor, although at lower concentrations of N-acetylglucosamine it was smaller than expected from the known K(i) of N-acetylglucosamine for glucokinase. The rate of phosphorylation of glucose was proportional to the amount of glucokinase in hepatocytes from newborn rats and in conditions such as starvation and diabetes in which the total amount of glucokinase in the liver is decreased. In the same conditions, glucose 6-phosphatase activity was either normal or increased. It is concluded that the phosphorylation of glucose in isolated hepatocytes follows sigmoidal kinetics, which can be explained by the activity of glucokinase alone with no participation of low-K(m) hexokinase or of glucose 6-phosphatase. 相似文献
13.
Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose. 相似文献
14.
Capillaries phosphorylate glucose in a concentration-dependent manner through a glucokinase-like enzyme: a study in the eel 总被引:1,自引:0,他引:1
F Belfiore S Iannello R Campione G Volpicelli 《Biochemical medicine and metabolic biology》1990,43(3):226-233
Glucose phosphorylation was studied in a pure capillary preparation obtained from the rete mirabile of the eel swimbladder. In the 3000g supernatant of capillary homogenates, the glucose phosphorylating activity did not reach the maximum at low glucose concentration (1 mmole/liter), as it occurs in most tissues, but increased with the increase in glucose concentration and approached the maximum at very high (300 mmole/liter) glucose levels, with values (mean +/- SEM, n = 10) of 5.85 +/- 0.94 nmole.min-1.mg-1 protein and 19.97 +/- 1.89 at 1 and 300 mmole/liter glucose, respectively. The apparent Km value for glucose was about 50 mmole/liter, i.e., at supraphysiological glucose concentration, like the enzyme glucokinase, typically present in the liver but absent from most other tissues. This new enzyme did not phosphorylate fructose (similar to glucokinase from liver, which is rather specific for glucose) but was not inhibited by N-acetyl-glucosamine (in contrast to hepatic glucokinase). Thus, capillaries phosphorylate glucose in a concentration-dependent manner, which suggests that they are equipped with a glucokinase-like enzyme. This may explain the reported increase in glucose uptake during capillary exposure to high glucose concentrations and would suggest that the hyperglycemia of the diabetic state may be associated with increased glucose utilization, which may play a role in the development of microangiopathy. 相似文献
15.
Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes. 总被引:1,自引:0,他引:1
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Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly. 相似文献
16.
I Miwa H Fukatsu Y Toyoda J Okuda 《Biochemical and biophysical research communications》1990,173(1):201-207
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. 相似文献
17.
Mukhtar MH Payne VA Arden C Harbottle A Khan S Lange AJ Agius L 《American journal of physiology. Regulatory, integrative and comparative physiology》2008,294(3):R766-R774
The rate of glucose phosphorylation in hepatocytes is determined by the subcellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g., sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study, we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity, suggesting altered interaction of glucokinase and GKRP. In summary, mechanisms downstream of AMPK activation, involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes. 相似文献
18.
Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c 总被引:1,自引:0,他引:1
Hagiwara A Cornu M Cybulski N Polak P Betz C Trapani F Terracciano L Heim MH Rüegg MA Hall MN 《Cell metabolism》2012,15(5):725-738
Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2. 相似文献
19.
Glucose-dehydrogenase-deficient (Gcd–) strains ofPseudomonas cepacia 249 compensated for loss of operation of the direct oxidative pathway by expanding the phosphorylative pathway. When grown on glucose, they had between two- and fourfold higher than normal levels of glucokinase and NAD-linked glucose-6-phosphate dehydrogenase activity and a comparable increase in capacity to transport glucose. Similar expansion of the phosphorylative pathway was noted when the wild type was grown on cellobiose or trehalose. Gcd– strains grew normally on cellobiose and trehalose, but not if also deficient in glucokinase; this indicates that the disaccharides were converted to glucose and metabolized via the phosphorylative pathway. The expansion of the phosphorylative pathway during growth of the wild type on disaccharides or of Gcd– mutants on glucose was a consequence of hyperinduction of pathway enzymes. Other compounds that promoted such hyperinduction included aromatic conjugates of glucose such as arbutin and salicin, and mannose. Under conditions leading to expansion of the phosphorylative pathway, enzymes related to the direct oxidative pathway, such as gluconate dehydrogenase and the 6-phosphogluconate dehydrogenase active with NAD, were not formed. The results indicate that intracellular glucose and extracellular glucose are metabolized to 6-phosphogluconate via different routes. 相似文献
20.
Martin Tobias Kaminski Julia Schultz Rica Waterstradt Markus Tiedge Sigurd Lenzen Simone Baltrusch 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
The glucose phosphorylating enzyme glucokinase regulates glucose metabolism in the liver. Glucokinase activity is modulated by a liver-specific competitive inhibitor, the glucokinase regulatory protein (GRP), which mediates sequestration of glucokinase to the nucleus at low glucose concentrations. However, the mechanism of glucokinase nuclear export is not fully understood. In this study we investigated the dynamics of glucose-dependent interaction and translocation of glucokinase and GRP in primary hepatocytes using fluorescence resonance energy transfer, selective photoconversion and fluorescence recovery after photobleaching. The formation of the glucokinase:GRP complex in the nucleus of primary hepatocytes at 5 mmol/l glucose was significantly reduced after a 2 h incubation at 20 mmol/l glucose. The GRP was predominantly localized in the nucleus, but a mobile fraction moved between the nucleus and the cytoplasm. The glucose concentration only marginally affected GRP shuttling. In contrast, the nuclear export rate of glucokinase was significantly higher at 20 than at 5 mmol/l glucose. Thus, glucose was proven to be the driving-force for nuclear export of glucokinase in hepatocytes. Using the FLII12Pglu-700μ-δ6 glucose nanosensor it could be shown that in hepatocytes the kinetics of nuclear glucose influx, metabolism or efflux were significantly faster compared to insulin-secreting cells. The rapid equilibration kinetics of glucose flux into the nucleus facilitates dissociation of the glucokinase:GRP complex and also nuclear glucose metabolism by free glucokinase enzyme. In conclusion, we could show that a rise of glucose in the nucleus of hepatocytes releases active glucokinase from the glucokinase:GRP complex and promotes the subsequent nuclear export of glucokinase. 相似文献