PMC:6879884 / 12756-29515 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6879884","sourcedb":"PMC","sourceid":"6879884","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6879884","text":"RESULTS\n\nHexokinase proteins I, II, and III are present in airway epithelial cells.\nAs glucose enters the cell, it is phosphorylated by hexokinases to glucose-6-phosphate reducing the intracellular concentration of free glucose. Western blot of cell extracts from H441 cells grown on plastic (proliferating) or H441 cells and HBECs grown at air-liquid interface indicated the presence of hexokinases I, II, and III in these cells. There was no observed difference in the total cellular abundance (hexokinase/actin) of these proteins in H441 cells after exposure to either 5 or 15 mM glucose (Fig. 1, A and B).\nFig. 1. H441 cells and human bronchial epithelial cells (HBECs) express all three forms of hexokinase. Representative Western blots of cell lysates from H441 cells grown on plastic (A) or H441 cells or HBECs grown at air-liquid interface (B). Lanes indicate cell type and growth conditions of either 5 or 15 mM glucose as indicated. Proteins immunostained for hexokinases I, II, and III are indicated to the right of the blots (all ~100 kDa). The immunostained housekeeping protein β-actin is also indicated (Actin) and serves as a loading control.\n\nHexokinase activity in airway cells is reduced by BrPy.\nAddition of BrPy to H441 cells reduced total hexokinase activity in cell extracts in a dose-dependent manner with an IC50 of 1.2 ± 0.28 mM (Fig. 2A). The data did not follow a classic sigmoid curve, and there was an indication that the inhibition was biphasic. We were unable to unambiguously fit such a curve to the data. However, the IC50 obtained from the initial inhibition of hexokinase activity was lower at 0.04 ± 0.01 mM. As there was no statistical difference in hexokinase activity between pretreatment with 100 µM and pretreatment with 1 mM, it was decided to use the lower concentration of BrPy. At this concentration, total cellular hexokinase activity was reduced by 25.1 ± 11.6% in H441 cells cultured at air-liquid interface (n = 6 experiments; Fig. 2B).\nFig. 2. Hexokinase activity is inhibited by 3-bromopyruvic acid (BrPy). A: effect of BrPy concentration on hexokinase activity in cell extracts from H441 cells exposed to 5 mM glucose. The dose-response data did not follow a classic sigmoid curve, and there was an indication that the inhibition was biphasic. Two curves could be fitted to the data to reflect initial inhibition (left-hand curve) with an IC50 of 0.04 ± 0.01 mM or overall inhibition (right-hand side) with an IC50 of 1.2 ± 0.28 mM (n = 4 experiments). B: total hexokinase activity in cell extracts from control (●) or BrPy (100 μM)-treated cells (○). Individual data points are shown with mean ± SD. ****Significantly different from control, P \u003c 0.0001.\n\nHexokinase activity drives glycolysis in airway cells.\nUsing the Seahorse assay, we previously showed that airway cells produce energy by mitochondrial respiration (OCR) and that elevation of extracellular glucose shifts metabolism to glycolysis (ECAR), which is associated with increased lactic acid secretion (12). We found that BrPy (100 μM) was effective at inhibiting both mitochondrial respiration (Fig. 3A) and glycolysis in these cells (Fig. 3, B and C). We calculated that BrPy inhibited glycolysis with an IC50 of 0.06 ± 0.02 mM (Fig. 3D). Application of 2-DG, an inhibitor of all hexokinase activity, was more effective at inhibiting respiration and glycolysis (Fig. 3, A–C). These data indicate that glycolysis is predominantly driven by hexokinase II activity in these cells.\nFig. 3. 3-Bromopyruvic acid (BrPy) inhibits glycolysis in airway epithelial cells. Seahorse metabolic assay of airway cells exposed to medium or different concentrations of BrPy (1 μM to 1 mM) as indicated on the right-hand side of graphs. A–C: oxygen consumption rate (OCR, A), extracellular acidification rate (ECAR, B), and ECAR/OCR (C) before and after injection of 5 mM glucose, oligomycin, or 2-deoxy-d-glucose (2-DG) at points indicated. D: dose-response data of glycolysis to BrPy were fit with a sigmoidal curve (df = 25, r2 = 0.95) with an IC50 of 0.06 ± 0.02 mM. All n = 5 experiments.\n\nElevating extracellular glucose and inhibiting hexokinase activity changed FRET ratio in nonpolarized and polarized H441 cells and HBECs.\nProliferating H441 cells transfected with FLII12Pglu-700µΔ6 and exposed to 5 mM extracellular glucose exhibited a cyclic fluctuation in FRET ratio of eCFP/Citrine over time, with a full cycle taking 3.4 ± 0.2 min (n = 16 experiments; Fig. 4A). This was not observed when the control FRET eCFP/Citrine plasmid was transfected into cells (data not shown). Elevation of extracellular glucose to 15 mM resulted in an increase in FRET ratio from 1.54 ± 0.02 to 1.6 ± 0.02 (P \u003c 0.0001, n = 117 individual cells from n = 16 experiments), indicating a decrease in intracellular glucose. In addition, the cyclic fluctuations slowed to 4.3 ± 0.3 min for a full cycle (n = 16 experiments; P \u003c 0.05; Fig. 4A). Pretreatment with the hexokinase inhibitor BrPy decreased FRET from 1.54 ± 0.02 to 1.41 ± 0.01 (P \u003c 0.0001; n = 117 individual cells from n = 14 experiments) indicating that intracellular glucose was increased (Fig. 4A). Furthermore, BrPy prevented the large cyclic fluctuations in FRET indicating that hexokinase activity was associated with this phenomenon. As an alkylating agent, it is possible that BrPy could directly affect the sensor. However, this would likely reduce glucose binding or stoichiometric changes to the sensor, neither of which would explain these results. Thus, these data indicate that intracellular glucose concentration fluctuated with external glucose concentration and hexokinase activity.\nFig. 4. Förster resonance energy transfer (FRET) ratio [enhanced cyan fluorescent protein (eCFP)/citrine] was measured over a period of 6 min using the glucose FRET sensor FLII12Pglu-700µΔ6. A: H441 cells grown on coverslips were exposed to either osmotically balanced 5 mM glucose (●) or 15 mM glucose (▲), both n = 16 experiments. Cells were also exposed to the same conditions in the presence of the hexokinase inhibitor 3-bromopyruvic acid (BrPy; ○ or △, respectively), both n = 14 experiments. B: FRET ratio (eCFP/Citrine) for H441 cells grown at air-liquid interface and exposed to either 5 mM glucose (●, n = 12 experiments) or 15 mM glucose (▲, n = 6 experiments). Cells were also exposed to 5 mM glucose in the presence of the hexokinase inhibitor BrPy (○, n = 4 experiments). C: FRET ratio (eCFP/Citrine) in human bronchial epithelial cells (HBECs) grown at air-liquid interface, exposed to either osmotically balanced 5 mM glucose (●, n = 12 experiments) or 15 mM glucose (▲, n = 15 experiments). D: FRET ratio (eCFP/Citrine)-glucose dose-response curve for cells shown in A, equilibrated with extracellular glucose as described in results. Data points are shown as means only in A, B, and C for clarity. Data in D are shown as means ± SD. Data were fitted with a sigmoidal one-site binding curve (df = 37, r2 = 0.6). Values shown in A and B are directly comparable, but FRET ratio values in A, B, and C cannot be directly compared because of the different imaging conditions required for the two cell types and their growth substrates. ****Significantly different between groups as indicated, P \u003c 0.0001. H441 cells cultured at air-liquid interface on permeable supports required altered microscope conditions for FRET acquisition, which meant that the measured FRET ratio of eCFP/Citrine was decreased compared with that observed in proliferating cells. Nevertheless, in cells exposed to 5 mM extracellular glucose the pattern of response was similar to that seen in proliferating cells. A cyclic fluctuation in FRET ratio was also observed in these cells with a full cycle taking 4.4 ± 0.6 min, in 5 mM glucose. Elevation of extracellular glucose to 15 mM resulted in an increased FRET ratio from 0.38 ± 0.007 to 0.41 ± 0.005 (P \u003c 0.0001, n = 83 individual cells from n = 6 experiments). Addition of BrPy reduced FRET ratio to 0.34 ± 0.003 and the cycling frequency to 1.3 ± 0.23 min (P ≤ 0.001; n = 16 experiments).\nOptimization of FRET acquisition in HBECs cultured at air-liquid interface also resulted in a change in FRET ratios obtained. However, similar to H441 cells, FRET ratio increased when extracellular glucose was increased from 5 to 15 mM (P \u003c 0.0001, n = 149 individual cells).\n\nInhibition of glucose transporter-mediated glucose uptake increased FRET ratio in H441 cells grown at air-liquid interface.\nCytochalacin B is a molecule larger than glucose, which binds to the pore of facilitative glucose transporters (GLUTs) and blocks glucose uptake. Cytochalacin B treatment of H441 cells grown at air-liquid interface and exposed to 5 or 15 mM glucose significantly increased FRET ratio (P \u003c 0.0001, n = 24 individual cells, respectively). These data indicate that inhibition of glucose uptake into the cell reduced intracellular glucose (Fig. 5).\nFig. 5. Inhibition of cellular glucose uptake increased Förster resonance energy transfer (FRET) ratio [enhanced cyan fluorescent protein (eCFP)/Citrine] indicating a decrease in intracellular glucose concentration. H441 cells grown at air-liquid interface and exposed to either 5 mM glucose or 15 mM glucose in the absence or presence of the facilitative glucose transport inhibitor cytochalasin B (CytB). Individual data points are shown with mean ± SD; n = 24 individual cells. ****Significantly different between groups as indicated, P \u003c 0.0001.\n\nIntracellular glucose concentration of H441 cells and HBECs.\nA dose-response curve for FRET ratio was generated for the three different cell/growth conditions using the individual imaging conditions used. An exemplar dose-response curve for proliferating H441 cells is shown (Fig. 4B). This was then used to interpolate the data points shown in Fig. 4A to calculate the intracellular concentration of glucose. The mean intracellular glucose concentration for proliferating H441 cells in 5 mM glucose was 0.23 ± 0.05 mM. Raising the glucose concentration to 15 mM glucose resulted in a decrease in intracellular glucose to 0.05 ± 0.04 mM. Pretreatment with BrPy increased intracellular glucose concentration to 0.49 ± 0.01 mM in 5 mM glucose and 0.46 ± 0.03 in 15 mM glucose (P \u003c 0.0001 compared with control, respectively; n = 117 individual cells; Fig. 6A).\nFig. 6. Intracellular glucose concentration calculated from Förster resonance energy transfer ratio dose-response curves. A: calculated intracellular glucose concentration in H441 cells grown on plastic and exposed to 5 mM (●) or 15 mM d-glucose (15 mM; hyperglycemia, ▲) or exposed to the same conditions in the presence of 3-bromopyruvic acid (BrPy; ○ or △, respectively). Values were calculated using the dose-response curve shown in Fig. 4D. Individual data points are shown with mean ± SD; n = 117 individual cells. ****P \u003c 0.0001 between groups as indicated. B: calculated intracellular glucose concentration for H441 cells grown at air-liquid interface in either 5 mM glucose (●), 15 mM glucose (▲), or 5 mM glucose in the presence of BrPy (○). Individual data points are shown with mean ± SD; n = 83 individual cells. ****P \u003c 0.0001 between groups as indicated. C: calculated intracellular glucose for human bronchial epithelial cells (HBECs) cultured at air-liquid interface in either 5 mM glucose (●) or 15 mM glucose (▲). Individual data points are shown with mean ± SD; n = 150 individual cells. ****P \u003c 0.0001 between groups as indicated. Interpolation of data from H441 cells cultured at air-liquid interface indicated that these cells had a mean intracellular glucose concentration of 0.36 ± 0.005 mM in 5 mM basolateral glucose and this decreased to 0.26 ± 0.003 mM when basolateral glucose concentration was increased to 15 mM. Addition of BrPy in the presence of 5 mM basolateral glucose increased intracellular glucose concentration to 0.72 ± 0.003 mM (P ≤ 0.0001; n = 83 individual cells; Fig. 6B).\nA similar pattern was seen in HBECs grown at air-liquid interface. Intracellular glucose concentration was 0.09 ± 0.002 mM in 5 mM glucose, and this decreased to 0.03 ± 0.001 mM when basolateral glucose concentration was raised to 15 mM (n = 150 individual cells; Fig. 6C).\n\nGlucose metabolism.\nGlycolysis was increased in HBECs in response to elevation of extracellular glucose concentration from 5 to 15 mM consistent with our previous observations in H441 cells (Fig. 7A; 12). In addition, the amount of glycogen per culture was increased twofold after exposure to 15 mM glucose (from 9.1 ± 1.3 to 20.2 ± 1.5 mg/mL, P \u003c 0.0001, n = 6 experiments). Inhibition of hexokinase with BrPy (100 µM) reduced glycogen in H441 cells exposed to 15 mM (P \u003c 0.001, n = 6 experiments) but not 5 mM glucose (Fig. 7B). Thus, elevation of extracellular glucose increased hexokinase-driven glycolysis and glycogen synthesis.\nFig. 7. Glycolysis, glycogen, and sorbitol are increased by elevation of extracellular glucose concentration. A: glycolysis measured in airway cells as extracellular acidification rate (ECAR) after injection of 5 mM glucose (●) or 15 mM glucose (▲); n = 34 experiments. ****P \u003c 0.0001. B: glycogen measured in airway cells after exposure to 5 mM glucose (●) or 15 mM glucose (▲) and 3-bromopyruvic acid (BrPy; ○ or ▽, respectively). Individual data points are shown with mean ± SD; n = 6 experiments. ***P \u003c 0.001, ****P \u003c 0.0001. C: sorbitol measured in airway cells after exposure to 5 mM glucose (●) or 15 mM glucose (▲) and BrPy (○ or △, respectively) or epalrestat (EP; half-shaded symbols). Individual data points are shown with mean ± SD; n = 8 experiments. *P \u003c 0.05, **P \u003c 0.01. Hexokinase-independent pathways are also present in airway cells, such as the polyol pathway, which utilizes aldose reductase to convert glucose to sorbitol. Such a pathway could also contribute to maintaining low intracellular glucose in the face of increased extracellular glucose. There was no significant difference in mean intracellular sorbitol between cells grown in 5 or 15 mM glucose. However, inhibition of hexokinase activity with BrPy in the presence of 15 mM glucose caused a small but significant elevation of sorbitol (from 0.04 ± 0.001 to 0.05 ± 0.002, P \u003c 0.01, n = 8 experiments). This elevation was inhibited by the aldose reductase inhibitor epalrestat (30 μM; n = 8 experiments; Fig. 7C). These data indicate that under circumstances when intracellular glucose rises, the sorbitol pathway can contribute to glucose utilization in these cells.\n\nAirway surface liquid glucose.\nGlucose concentration in washes from the ASL of cell cultures grown at air-liquid interface was increased from 3.6 ± 0.7 to 45.2 ± 1.7 μM, P \u003c 0.001, n = 4 and 7 experiments, respectively) when basolateral glucose was raised from 5 to 15 mM for 6 h. Taking into account the original volume of ASL, these values approximate to 0.5 and 6 mM, respectively, similar to previously published values (12). Treatment with BrPy had no further effect on ASL glucose concentrations. Transepithelial electrical resistance (TEER) was unaffected by treatments (Fig. 8).\nFig. 8. Paracellular diffusion drives airway surface liquid (ASL) glucose concentration. A and B: transepithelial electrical resistance (TEER, A) and glucose concentration (B) in ASL washes after exposure to 5 mM glucose (●) or 15 mM glucose (▲) and 3-bromopyruvic acid (BrPy; ○ or ▽, respectively). Individual data points are shown with mean ± SD; n = 6 experiments. ****P \u003c 0.0001. C and D: proposed mechanism for the role of hexokinase II (HKII) in maintaining low intracellular glucose in normoglycemia (C) and hyperglycemia (D). There is a diffusion gradient for paracellular movement of glucose from the blood/interstitium to the ASL. Glucose uptake via glucose transporters (GLUTs) is maintained by metabolism, which generates low intracellular glucose. We propose that this occurs predominantly by HKII-driven conversion of glucose to glucose-6-phosphate (G-6-P) and glycolysis. When blood glucose levels are raised to 15 mM (hyperglycemia), there is increased paracellular movement of glucose into the ASL. Increased glucose uptake elevates HKII activity at the mitochondria, increasing G-6-P, glycolysis, and glycogen synthesis. This effectively reduces intracellular glucose concentration, which maintains a glucose gradient for clearance of glucose from the ASL and prevents transcellular efflux into the ASL. Inhibition of HKII with BrPy elevates intracellular glucose, but concentrations remain low compared with external glucose concentration indicating additional contribution of HKI/III and the HK-independent polyol pathway to glucose metabolism. 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