Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle.
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To investigate the effect of exercise on GLUT-4, hexokinase, and glycogenin gene expression in human skeletal muscle, 10 untrained subjects (6 women and 4 men, 21.4 +/- 1.2 yr, 66.3 +/- 5.0 kg, peak oxygen consumption = 2.30 +/- 0.19 l/min) exercised for 60 min on a cycle ergometer at a power output requiring 73 +/- 4% peak oxygen consumption. Muscle samples were obtained by needle biopsy before, immediately after, and 3 h after exercise. Gene expression was quantified, relative to 29S ribosomal protein cDNA, by RT-PCR. GLUT-4 gene expression was increased immediately after exercise (1.7 +/- 0.4 vs. 0.9 +/- 0.3 arbitrary units; P < 0.05) and remained significantly higher than baseline 3 h after the end of exercise (2. 2 +/- 0.4 vs. 0.9 +/- 0.3 arbitrary units; P < 0.05). Hexokinase II gene expression was significantly higher than the resting value 3 h after the end of exercise (2.9 +/- 0.4 vs. 1.3 +/- 0.3 arbitrary units; P < 0.05). Exercise increased glycogenin mRNA more than twofold (2.8 +/- 0.6 vs. 1.2 +/- 0.2 arbitrary units; P < 0.05) 3 h after the end of exercise. For the first time, we report that a single bout of exercise is sufficient to cause upregulation of GLUT-4 and glycogenin gene expression in human skeletal muscle. Whether these increases, together with the associated increase in hexokinase II gene expression, lead to increased expression of these key proteins in skeletal muscle and contribute to the enhanced skeletal muscle glucose uptake, glycogen synthesis, and insulin action observed following exercise remains to be determined. (+info)
Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.
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BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery. (+info)
Hypoxia induces hexokinase II gene expression in human lung cell line A549.
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During adaptation to hypoxic and hyperoxic conditions, the genes involved in glucose metabolism are upregulated. To probe involvement of the transcription factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expression in human pulmonary cells, A549 cells and small-airway epithelial cells (SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and metal ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for enzymatic activity) was observed in A549 cells when exposed to DFO. All stimuli selectively increased the 5.5-kb rather than 4-kb transcript in A549 cells. Cycloheximide and actinomycin D inhibited these responses. In addition, cells were transfected with luciferase reporter constructs driven by the full-length HK-II 5'-regulatory region (4.0 kb) or various deletions of that region. A549 cells transfected with the 4.0-kb construct and exposed to hypoxia or DFO increased their luciferase activity 7- and 10-fold, respectively, indicating that HK-II induction is, at least in part, due to increased gene transcription. Sixty percent of the inducible activity of the 4.0-kb construct was shown to reside within the proximal 0.5 kb. Additionally, cotransfection with a stable HIF-1 mutant and the 4.0-kb promoter construct resulted in increased luciferase activity under normoxic conditions. These results strongly suggest that HK-II is selectively regulated in pulmonary cells by a HIF-1-dependent mechanism. (+info)
Compartmentation protects trypanosomes from the dangerous design of glycolysis.
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Unlike in other organisms, in trypanosomes and other Kinetoplastida the larger part of glycolysis takes place in a specialized organelle, called the glycosome. At present it is impossible to remove the glycosome without changing much of the rest of the cell. It would seem impossible, therefore, to assess the metabolic consequences of this compartmentation. Therefore, we here develop a computer experimentation approach, which we call computational cell biology. A validated molecular kinetic computer replica was built of glycolysis in the parasite Trypanosoma brucei. Removing the glycosome membrane in that replica had little effect on the steady-state flux, which argues against the prevalent speculation that glycosomes serve to increase flux by concentrating the enzymes. Removal of the membrane did cause (i) the sugar phosphates to rise to unphysiologically high levels, which must have pathological effects, and (ii) a failure to recover from glucose deprivation. We explain these effects on the basis of the biochemical organization of the glycosome. We conclude (i) that the glycosome protects trypanosomes from the negative side effects of the "turbo" structure of glycolysis and (ii) that computer experimentation based on solid molecular data is a powerful tool to address questions that are not, or not yet, accessible to experimentation. (+info)
Exposure to glibenclamide increases rat beta cells sensitivity to glucose.
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An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug. (+info)
Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia.
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The critical minimum values of Na,K-ATPase and glycolytic enzyme activities at which the erythrocyte viability is lost were calculated using the mathematical model of the erythrocyte, which included all reactions of glycolysis, adenylate metabolism, ionic balance, and osmotic regulation of erythrocyte volume. The criterion for cell death was an increase in its volume to the level at which it is sequestrated from the circulation or is lysed. In hemolytic anemia associated with hexokinase or pyruvate kinase deficiency, activities of these enzymes measured in patient erythrocytes appeared to be close to the calculated critical values. By contrast, in hemolytic anemia associated with phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, activities of these enzymes measured in patient erythrocytes were significantly greater than the calculated critical values. In this case, if the deficient enzyme were stable, i.e. its activity in the cell were low, but constant in time, the deficiency observed would not account for the erythrocyte destruction observed and the development of hemolytic anemia. It was shown, however, that in phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, hemolytic anemia can arise because of the instability of these enzymes in time. (+info)
Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro.
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Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.(1) Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an approximately 50% increase in GLUT-4 protein and an approximately 80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase. (+info)
The interplay among cardiac ultrastructure, metabolism and the expression of oxygen-binding proteins in Antarctic fishes.
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We examined heart ventricle from three species of Antarctic fishes that vary in their expression of oxygen-binding proteins to investigate how some of these fishes maintain cardiac function despite the loss of hemoglobin (Hb) and/or myoglobin (Mb). We quantified ultrastructural features and enzymatic indices of metabolic capacity in cardiac muscle from Gobionotothen gibberifrons, which expresses both Hb and Mb, Chionodraco rastrospinosus, which lacks Hb but expresses Mb, and Chaenocephalus aceratus, which lacks both Hb and Mb. The most striking difference in cellular architecture of the heart among these species is the percentage of cell volume occupied by mitochondria, V(v)(mit,f), which is greatest in Chaenocephalus aceratus (36.53+/-2.07), intermediate in Chionodraco rastrospinosus (20.10+/-0.74) and lowest in G. gibberifrons (15.87+/-0.74). There are also differences in mitochondrial morphologies among the three species. The surface area of inner mitochondrial membrane per volume of mitochondria, S(v)(imm, mit), varies inversely with mitochondrial volume density so that S(v)(imm,mit) is greatest in G. gibberifrons (29.63+/-1.62 microm(-)(1)), lower in Chionodraco rastrospinosus (21.52+/-0.69 microm(-)(1)) and smallest in Chaenocephalus aceratus (20.04+/-0.79 microm(-)(1)). The surface area of mitochondrial cristae per gram of tissue, however, is greater in Chaenocephalus aceratus than in G. gibberifrons and Chionodraco rastrospinosus, whose surface areas are similar. Despite significant ultrastructural differences, oxidative capacities, estimated from measurements of maximal activities per gram of tissue of enzymes from aerobic metabolic pathways, are similar among the three species. The combination of ultrastructural and enzymatic data indicates that there are differences in the density of electron transport chain proteins within the inner mitochondrial membrane; proteins are less densely packed within the cristae of hearts from Chaenocephalus aceratus than in the other two species. High mitochondrial densities within hearts from species that lack oxygen-binding proteins may help maintain oxygen flux by decreasing the diffusion distance between the ventricular lumen and mitochondrial membrane. Also, high mitochondrial densities result in a high intracellular lipid content, which may enhance oxygen diffusion because of the higher solubility of oxygen in lipid compared with cytoplasm. These results indicate that features of cardiac myocyte architecture in species lacking oxygen-binding proteins may maintain oxygen flux, ensuring that aerobic metabolic capacity is not diminished and that cardiac function is maintained. (+info)