Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.1 (hexokinase)
5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2-3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2-3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphorylation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2-3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 microM and a Vmax of 0.1 nmol/min/oocyte. Co-injection of yeast hexokinase increased glucose phosphorylation by five-fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25-30-fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2-3H]glucose were analyzed by a multiple curve-fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate-limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three-fold lower extracellular concentration of 2-deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models.
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PMID:Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: determination of the rate-limiting step. 825 62

Alloxan causes diabetes in experimental animals through its ability to destroy the insulin-secreting B-cells of the pancreas. Alloxan is hydrophilic and chemically unstable; it is reactive toward thiols, undergoing redox cycling in the presence of glutathione and oxidizing protein-bound thiol groups, as reflected by inhibition of the thiol enzymes, hexokinase and glucokinase. It is apparently also selectively taken up by the GLUT-2 glucose transporter in the pancreatic B-cell membrane. In order to investigate which, if any, of these physicochemical properties are important in the toxic action of alloxan, we have examined seven N-alkyl substituted alloxan derivatives of various diabetogenic activity. Hydrophilicity was identified as a factor essential for diabetogenicity. Stability, rate of redox cycling and reactivity toward thiol groups were not correlated with diabetogenicity. Selective uptake by the GLUT-2 glucose transporter is not a prerequisite for the diabetogenicity of alloxan derivatives.
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PMID:The relationship between the physicochemical properties and the biological effects of alloxan and several N-alkyl substituted alloxan derivatives. 825 88

HIT is a hamster-derived beta-cell line which in contrast to normal beta cells that only express the high Km GLUT-2 glucose transporter, also expresses the low Km glucose transporter GLUT 1. In HIT cells the abnormal glucose transport mechanism is associated with a marked shift to the left of the glucose-induced insulin release dose-response curve. We have used this cell model to investigate whether changes in glucose transport affect the glucose-induced insulin release. HIT cells were first incubated with a concentration of cytochalasin B (0.4 mumol/l) that selectively inhibits the GLUT-1 but not the GLUT-2 transporter. The consequences of blocking glucose phosphorylation and insulin release were studied. Exposure to 0.4 mumol/l cytochalasin B for 1 h caused a selective loss of the low Km transport: the calculated Vmax of GLUT 1 was reduced from 1726 +/- 98 to 184 +/- 14 pmol.mg protein-1 5 min-1 (mean +/- SEM, n = 6, p < 0.005), while no major difference in the high Km (GLUT-2) transport was observed. In cytochalasin B exposed HIT cells the glucose phosphorylating activity (due to hexokinase and glucokinase) was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of the high-affinity glucose transporter GLUT 1 affects the sensitivity to glucose in a hamster-derived pancreatic beta cell line (HIT). 827 Jan 37

Sodium butyrate is widely used to differentiate insulinoma cell lines. However, sodium has been shown to decrease glucose phosphorylation in the liver and heart and decrease the expression of glucose transporter. Since these mechanisms are essential for glucose-induced insulin secretion, the ultimate function of the pancreatic beta-cell, we investigated the effect of sodium butyrate on both glucose-phosphorylating enzymes as well as glucose transport in the pancreatic cell line RIN-m5F. Treatment of RIN-m5F cells with 2.5 mM sodium butyrate for 72 h increased by twofold both hexokinase and glucokinase (GK) activities, as well as the gene expression of GK. Sodium butyrate treatment had no effect on GLUT-1 mRNA levels but increased the GLUT-2 mRNA 3.7-fold. Kinetic analysis of 2-deoxyglucose transport displayed a single curve with Km = 1.2 mM and Vmax = 10.9 pmol/micrograms protein/min in the untreated cells, values similar to the low Km glucose transport reported in the pancreatic beta-cells. This low Km transport component markedly decreased with sodium butyrate treatment, and interestingly a second component with a higher Km appeared, consistent with the increase in GLUT-2 mRNA. We conclude that the differentiating action of sodium butyrate involves increases in GK and GLUT-2 gene expression, which characterizes the differentiated state of the pancreatic beta-cell. However, the inhibitory effect of sodium butyrate on low Km glucose transport needs to be considered in the use of this compound to promote differentiation.
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PMID:Effect of sodium butyrate on glucose transport and glucose-phosphorylating enzymes in RIN-m5F cells. 830 95

This study followed changes in the capacities of uptake and phosphorylation of glucose in response to contractile activity in low-frequency stimulated (10Hz, 24 h/d) rat fast-twitch muscle. We investigated the intracellular distribution of GLUT-4, the major glucose transporter isoform in muscle, changes in the amounts of its specific mRNA and total cellular protein, as well as changes in its relative synthesis rate. These analyses were complemented by measurements of total hexokinase activity and hexokinase II (HKII) expression at the levels of mRNA content and protein synthesis. Changes in protein synthesis were determined by in vivo labeling with [35S]methionine. Translocation of GLUT-4 into the sarcolemma was an immediate response to contractile activity, whereas changes in its total amount were observed only with ongoing stimulation (5 d and longer). A twofold increase in GLUT-4 content after 5 d and longer stimulation periods was preceded by elevations of its mRNA and by enhanced [35S]methionine incorporation. Conversely, increases in HKII expression with a rise in total hexokinase activity occurred soon after the onset of stimulation (30-fold elevations of HKII mRNA after 12 h and 20-fold increases in [35S]methionine incorporation after 24 h). With ongoing stimulation, HKII mRNA and synthesis returned to lower levels (fivefold elevations). Nevertheless, hexokinase activity continued to rise, stabilizing at fivefold-elevated levels after 3 d. These observation suggested that posttranscriptional mechanisms contributed to the upregulation of HKII, e.g. stabilization by elevated intracellular glucose and mitochondrial binding of the enzyme. This suggestion was supported by experiments with cessation after 24 h where hexokinase activity continued to increase, although the mRNA content and, especially, the [35S]methionine incorporation decayed steeply. The increase in HKII prior to GLUT-4 suggests that phosphorylation may be rate limiting in glucose utilization of glycolytic fibers under conditions of sustained contractile activity. Taken together, the changes in distribution and content of GLUT-4, as well as in HKII represent early metabolic adaptations. In addition, they are related to the overall process of stimulation-induced fiber type transformation.
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PMID:Low-frequency stimulation of rat fast-twitch muscle enhances the expression of hexokinase II and both the translocation and expression of glucose transporter 4 (GLUT-4). 830 97

A Xenopus oocyte expression-co-injection system was used to study the influence of guanine nucleotides on D-glucose uptake. GTP analogs like GTP gamma S and GppNHp had no effect on 3-O-methylglucose transport determined by zero-trans uptake or equilibrium exchange, but suppressed 2-deoxyglucose uptake into Glut1 glucose transporter-expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTP gamma S being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co-injected with water or GDP beta S (250 and 500 microM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTP gamma S-co-injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down-regulation of 2-deoxyglucose uptake.
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PMID:GTP analogs suppress uptake but not transport of D-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes. 833 1

A line of transgenic mice was constructed in which the human Glut1 glucose transporter is overexpressed in skeletal muscle. Overexpression of Glut1 protein was evident in epitrochlearis, extensor digitorum longus (EDL), and quadriceps muscles, and resulted in 6.6-7.4-fold elevations in basal glucose transport activity as measured in isolated muscles in vitro. The elevated glucose transporter activity in the skeletal muscles of transgenic mice was associated with a 10-fold increase in glycogen concentration in EDL and quadriceps muscles that was not due to an increase in muscle glycogen synthase activity or a decrease in glycogen phosphorylase activity. The increased glucose transport activity also resulted in a 2-fold increase in muscle lactate concentration, with no increase in muscle glucose 6-phosphate. Despite a slight (10%) increase in muscle hexokinase activity, there was a 4-fold increase in total muscle free glucose in transgenic mice, indicating that hexokinase becomes rate-limiting for glucose uptake when the rate of glucose transport is very high. These results demonstrate that the muscle glycogen content can be dramatically elevated by increasing the muscle Glut1 protein level and that glucose transport is a rate-limiting step for muscle glucose disposal in normal, resting mice.
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PMID:Evidence from transgenic mice that glucose transport is rate-limiting for glycogen deposition and glycolysis in skeletal muscle. 834 95

A clearer understanding of biochemical properties of oocytes and embryos and their changes in oocyte maturation and embryonic development may have significant clinical implications, especially for in vitro fertilization techniques. Microtechniques and highly sensitive methods such as enzymatic cycling, micro-Western analysis, reverse transcription polymerase chain reaction and so on were employed to study these processes. Low hexokinase activity and high activities of enzymes in the phosphate pathway were characteristic of immature oocytes. During maturation, the activities of hexokinase and phosphofructokinase increased significantly. These changes were used to analyze involvement of epidermal growth factor (EGF) and prostaglandins (PG) in oocyte maturation. EGF is shown to stimulate maturation by increasing PG production in granulosa cells. Electrophysiologically, the sensitivity of oocyte to inositol triphosphate increased and Ca2+ release system developed during maturation. Progesterone production of oocyte and embryos are shown by enzymatic cycling and other methods using radiometry. This hormone produced by embryos themselves may play a role in embryonic development in intracrine fasion. There is 100-fold increase in glucose uptake from oocyte to blastocyst in mice. A switch in substrate preference of the embryo from pyruvate to glucose during preimplantation development may be explained by increases in the activity of hexokinase and expression of glucose transporter, GLUT1. Hexokinase activities determined by NADP cycling increased 20-fold while expression of GLUT1 assessed by micro-Western method 10-fold. GLUT1 expression was also analyzed by RT-PCR, which indicated that the expression is regulated at transcription level. There is a delay in the developmental changes in glucose uptake, hexokinase activity and GLUT1 expression when the embryos are developed in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Studies in oocyte maturation and embryonic development]. 837 Oct 11

Feeding rats beta-guanidinopropionic acid (beta-GPA), a creatine analogue, results in depletion of creatine and phosphocreatine and induces increases in mitochondrial oxidative enzymes and hexokinase in skeletal muscle. Comparisons of different muscle types and studies of the adaptation to exercise suggest that 1) the levels of the insulin-responsive glucose transporter (GLUT-4), mitochondrial oxidative enzymes, and hexokinase may be coregulated and 2) GLUT-4 content can determine maximal glucose transport activity in muscle. To further evaluate these possibilities, we examined the effects of feeding rats 1% beta-GPA in their diet for 6 wk on muscle GLUT-4 expression and glucose transport activity. beta-GPA feeding induced 40-50% increases in cytochrome c concentration, citrate synthase activity, and hexokinase activity in plantaris muscle. GLUT-4 protein concentration was increased approximately 50% in plantaris and epitrochlearis muscles, while GLUT-4 mRNA was increased approximately 40% in plantaris muscles of beta-GPA-fed rats. Glucose transport activity maximally stimulated by insulin was increased in parallel with GLUT-4 protein concentration in the epitrochlearis. These results provide evidence that chronic creatine depletion increases GLUT-4 expression by pretranslational mechanisms. They support the hypothesis that the levels of mitochondrial enzymes, hexokinase, and GLUT-4 protein are coregulated in striated muscles. They also support the concept that the GLUT-4 content of a muscle determines its maximal glucose transport activity when the signaling pathways for glucose transport activation are intact.
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PMID:Adaptation of muscle to creatine depletion: effect on GLUT-4 glucose transporter expression. 843 Jul 63

We examined the effects of voluntary exercise on glucose transporter concentration in skeletal muscle from young adult and old female Long-Evans rats. Rats had free access to voluntary running wheels beginning at 4 months of age or remained sedentary. Exercising rats ran approximately 7.5, 6.2, 5.6 and 5.3 km/day during their 6th, 8th, 9th and 10th month of age, respectively. During the 23rd, 24th and 25th month of age running distance averaged 3.0, 2.8 and 2.4 km/day, respectively. At 10 and 25 months of age, glucose transporter protein concentration was assessed in epitrochlearis and flexor digitorum brevis muscles with a polyclonal antibody directed against the GLUT4 transporter isoform. GLUT4 protein concentration was not altered by the aging process (i.e., comparing 10- and 25-month-old rats) in either muscle type. Wheel running increased GLUT4 protein concentration by 45% in epitrochlearis muscles of 10-month-old rats relative to age-matched sedentary controls. The training-induced adaptation in GLUT4 protein was no longer present at age 25 months, probably because the running distance had declined by 50%. In the flexor digitorum brevis, exercise did not alter GLUT4 concentration at either 10 or 25 months, presumably due to insufficient recruitment of this muscle during wheel running as assessed by measurement of citrate synthase and hexokinase enzyme activities. Wheel running induced cardiac and soleus muscle hypertrophy in 10- and 25-month-old rats. In summary, voluntary wheel running can induce an increase in skeletal muscle GLUT4 protein concentration in adult rats. Older rats that run less exhibit cardiac and soleus muscle hypertrophy, but do not maintain an elevated GLUT4 protein concentration in the epitrochlearis muscle. Aging does not alter GLUT4 protein concentration in the epitrochlearis or FDB muscles.
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PMID:Effects of wheel running on glucose transporter (GLUT4) concentration in skeletal muscle of young adult and old rats. 846 30


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