Glucose activates H+-ATPase in kidney epithelial cells

Suguru Nakamura

Division of Nephrology, Hypertension, and Renal Transplant, Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610

Submitted 28 October 2003 ; accepted in final form 25 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The vacuolar H+-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H+-ATPase remain largely unknown. The present study examines the effect of glucose on H+-ATPase activity in the pig kidney epithelial cell line LLC-PK1. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH3/NH4+ prepulse, and rates of intracellular pH (pHi) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K+ free to eliminate Na+/H+ exchange and H+-K+-ATPase activity. After NH3/NH4+-induced acidification, LLC-PK1 cells had a significant pHi recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pHi recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pHi recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H+-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification.

proton secretion; glycolysis; intracellular pH; concanamycin A


THE KIDNEY PLAYS A MAJOR ROLE in the regulation of acid-base balance by controlling extracellular fluid bicarbonate concentration. Approximately 80% of the filtered HCO3 is reabsorbed in the proximal tubule (PT) by proton secretion. Two proton transporters, the Na+/H+ exchanger NHE-3 and H+-ATPase, are involved in the cellular mechanisms in HCO3 reabsorption (6, 29). Previous microperfusion studies by Nakamura and colleagues (36–40) have shown that H+-ATPase mediates HCO3 reabsorption in the collecting duct of NHE-3-deficient and healthy animals.

Metabolic control of ion transport in renal tubular epithelium provides a means to reduce cellular ATP consumption during limited metabolic substrate or oxygen availability but also has an important role in regulating transport during substrate availability. Studies in turtle urinary bladder (3, 52), mammalian proximal tubules (29), and mammalian collecting ducts (21) have demonstrated the importance of metabolic substrates in the control of epithelial H+ transport and have shown that glucose is a preferred substrate.

As a metabolic fuel, glucose is metabolized primarily through glycolysis. Several studies suggest that vacuolar H+-ATPase (V-ATPase)-mediated H+ transport is coupled with glycolysis in urinary epithelia. Steinmetz et al. (55) demonstrated close coupling of proton secretion with anaerobic lactate production in the turtle urinary bladder. Kurtz (29) found that H+-ATPase activity in the rabbit S3 proximal tubule was inhibited by the sulfhydryl reagent iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), suggesting dependence of the H+-ATPase on glycolysis. In the genetic disorder hereditary fructose intolerance, caused by deficiency in the glycolytic enzyme aldolase B, fructose induces rapid, severe proximal renal tubular acidosis (35) thought to be caused by inhibition of residual aldolase activity from fructose metabolites (5).

Recently, studies have shown that H+-ATPase binds directly to the glycolytic enzyme aldolase and colocalizes with the H+-ATPase in two proton-transporting cell types: renal proximal tubule cells and osteoclasts (32). The functional importance of the interaction was demonstrated in Saccharomyces, in which deletion of the aldolase gene resulted in disassembly of V-ATPase (32), similarly to the disassembly observed after glucose removal (25), providing further evidence for direct coupling of V-ATPase with the glycolytic pathway. The present study shows that glucose activates V-ATPase activity in renal epithelial cells through a pathway requiring aerobic glycolysis, providing further evidence for coupling between V-ATPase activity and glycolysis.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Cell culture. LLC-PK1 cells were cultured in medium 199 supplemented with 10% (vol/vol) fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were maintained in culture at 37°C in 5% CO2 in air. The culture medium was replaced every 3 days, and cells were passaged only when confluent. LLC-PK1 cell monolayers were grown on 40-mm-diameter coverslips and plated at a density of 6–8 x 104 cells per dish (60 x 15 mm). Experiments were performed on confluent monolayers 3–6 days after cell passage.

Solutions. The standard HEPES-buffered solutions contained (in mM) 125 NaCl, 5 KCl, 1 CaCl2, 1.2 MgSO4, 2 NaH2PO4, 32 HEPES, and 10.5 glucose, pH adjusted to 7.4 with NaOH. All experiments were performed in the nominal absence of CO2/HCO3. In the Na+- and K+-free solutions, N-methyl-D-glucammonium (NMDG+) was substituted for Na+ and K+, and the solutions were adjusted to pH 7.4 with Tris. In the 20 mM NH3/NH4+ solutions, either 20 mM NaCl or 20 mM NMDG+ for Na+-free solutions was replaced by an equal concentration of NH4+Cl. In the calibration solution, which contained 10 µM of nigericin, NaCl was replaced by 105 mM of KCl and 20 mM of NMDG-Cl.

In vitro microperfusion. In vitro microperfusion was performed as described previously (36–40). The kidney from a male Sprague-Dawley rat was decapsulated, cross-sectioned, and placed immediately in a petri dish containing dissecting solution (i.e., glucose-free standard solution). Each section was cut into smaller wedges from the papillary tip to the cortex and transferred into a second petri dish containing dissecting solution maintained at 14°C under a dissecting microscope (Nikon SMZ-645). Proximal tubules were dissected under x50 magnification. They were transferred to a lucite chamber containing bathing solution, which initially was maintained at room temperature. One end of the proximal tubule was pulled into an outer pipette. Once secure, the inner perfusion pipette was advanced, and the proximal tubule was opened with slight positive pressure. The opposite end of the proximal tubule was then pulled into a holding pipette. The proximal tubule was bathed in a low-volume laminar flow chamber. Solutions were continuously bubbled with 100% O2 and delivered at the rate of 6 ml/min to the bathing chamber in water-jacketed lines at 37°C. Perfusion rates were maintained at 10–15 nl/min (microperfusion system; Vestavia Scientific, Birmingham, AL).

Intracellular pH measurements. LLC-PK1 cells were grown to confluence on coverslips and incubated in the presence of 15 µM of BCECF-AM for 20 min at 37°C in culture medium as described above. After BCECF incubation, coverslips were placed in a closed perfusion chamber with a volume of 0.3 ml (Bioptechs FCS2; Bioptechs, Butler, PA) mounted on an inverted fluorescence microscope (Nikon TE-300). Solutions were preheated and delivered to the chamber at 37°C in lines that allowed any combination of four solutions at a flow rate of 5 ml/min. All experiments were started ~5 min after removal of BCECF-AM and were performed with the solutions at 37°C.

Intracellular pH (pHi) in single LLC-PK1 cells was measured by ratiometric fluorescence (50) using excitation at 440 and 490 nm and measurement of light emission at 520 nm (Intracellular Imaging, Cincinnati, OH). Intracellular acid loading was induced by the NH3/NH4+ prepulse method (9, 41). Cells were exposed to 20 mM of NH3/NH4+ for 5 min, and H+-ATPase activity was determined as the initial rate of pH; recovery was measured in the absence of Na+, K+, and HCO3 after NH3/NH4+ removal (41). The solutions also contained 1 µM ethylisopropylamiloride (EIPA) to eliminate Na+/H+ exchange (19).

pHi was calculated on the basis of fluorescence ratios from an intracellular calibration curve constructed at the end of each experiment using the nigericin/high-K+ technique (60) and a linear conversion formula (440-/490-nm ratio = a + b x pHi) (60). Calibration analysis was performed separately for each cell. Rates of pHi recovery (dpHi/dt) were determined on the basis of the slopes of the linear regression lines of measurements taken during the first 3 min of recovery and expressed as change in pH units per second (30, 60).

Determination of buffering capacity. Intracellular buffer capacity ({beta}i) was determined with the formula {beta}i = [NH4+]i/pHi using the technique described by Boyarsky et al. (10) and calculated according to the method of Weintraub and Machen (62). In our system, {beta}i refers to the ability of intrinsic cellular components (excluding HCO3/CO2) to buffer changes in pHi, and thus {beta}i values were estimated with the use of HEPES-buffered solutions.

{beta}i is defined as [base]/pHi and is most precisely estimated in cells whose pHi regulatory mechanisms are blocked. H+-HCO3 membrane transporters were blocked by a 0 mM Na+, 0 mM K+ solution plus 100 nM concanamycin A. At steady-state pHi, addition of 20 mM of NH4+/NH3 (NH4+ replacing NMDG+) caused a rapid initial increase in cell pH due to the influx of NH3 and subsequent generation of NH4+. Extracellular NH4+ concentration ([NH4+]) was then reduced in a stepwise manner to 0 mM (20, 10, 5, 2.5, and 0 mM) in the nominal absence of HCO3/CO2. The rate of transmembrane H+ flux (JH+) was calculated by using the equation JH+ = (dpHi/dt{beta}i, where dpHi/dt is the initial rate of pHi recovery after an acid pulse and {beta}i is the cytosolic buffering capacity averaged for the respective pH interval.

Materials. Medium 199 was purchased from Mediatech (Herndon, VA). LLC-PK1 cells were obtained from the American Type Culture Collection (Manassas, VA). Fetal bovine serum was purchased from GIBCO-BRL (Gaithersburg, MD). BCECF-AM was obtained from Molecular Probes (Eugene, OR). Concanamycin A, nigericin, EIPA, 2-deoxy-D-glucose (2-DG), antimycin A, pyruvate, wortmannin, and other chemicals were purchased from Sigma (St. Louis, MO).

Statistics. Data are expressed as means ± SE where appropriate. Analysis of variance and the t-test were performed as appropriate to determine statistical significance. P < 0.05 was considered statistically significant.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Na-independent concanamycin-sensitive and plasma membrane V-ATPase activity in LLC-PK1 cells. LLC-PK1 cells possess several pH regulatory mechanisms that allow them to restore intracellular pH to baseline levels after acute intracellular acidification (19, 34, 53). To determine whether V-ATPase participates in pH recovery from intracellular acidification, pHi changes were examined in confluent monolayers of LLC-PK1 cells using the pH probe BCECF (50). Cells were maintained in Na+-containing solution, which was replaced by a Na+-free solution 5 min before initiation of pHi measurements. After NH3/NH4+-induced acidification, in the presence of 10.5 mM glucose, Na+- and K+-independent pHi recovery (dpHi/dt) was observed at a rate of 13.8 ± 3.3 x 10–4 pH units/s (n = 23) (Fig. 1A). pHi recovery was inhibited completely by the H+-ATPase inhibitor concanamycin A (100 nM) (Fig. 1B), indicating that Na-independent proton secretion in LLC-PK1 cells is likely due to a plasma membrane V-ATPase. In the presence of concanamycin, Na+ addition caused pHi to recover fully to baseline, likely by Na+/H+ antiport (19, 34), showing that concanamycin does not affect other pH recovery mechanisms.



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Fig. 1. Concanamycin-sensitive vacuolar H+-ATPase (V-ATPase) activity in LLC-PK1 cells. Intracellular pH (pHi) was measured by dual excitation fluorescence using BCECF in sodium-free medium to eliminate Na+-H+ exchange activity, and recovery of pHi was measured after intracellular acidification by NH4Cl prepulse (periods A and B) as described in METHODS. A: Na+-independent pHi recovery in 10 mM glucose (period C). NaCl was added at time indicated (period D) to activate Na+/H+ exchange. B: inhibition of sodium-independent pHi recovery in 10 mM glucose containing V-ATPase inhibitor concanamycin A (CCA; 100 nM) (period C), with partial recovery of pH after addition of NaCl (period D).

 
Glucose activates H+-ATPase activity in LLC-PK1 cells. H+-ATPase activity in LLC-PK1 cells was inhibited markedly by removal of glucose from the medium (Fig. 2). To determine the time course of this effect, the pHi recovery rate in the presence of 10 mM glucose (Fig. 2, period C) was determined and repeated measurements were performed of pHi recovery rate 5 min after removal of glucose from the medium (Fig. 2, period E) and after replacement of glucose in the medium (period F). H+-ATPase-mediated pHi recovery was undetectable 5 min after removal of glucose, and H+-ATPase activity returned rapidly after readdition of glucose. Recovery was detectable at 2.5 ± 0.6 min (n = 6) after readdition of glucose and half-maximal at 6.3 ± 0.7 min (n = 6). These data demonstrate that LLC-PK1 cells contain glucose-activated plasma membrane H+-ATPase, the activity of which is dependent on the continual presence of glucose.



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Fig. 2. Glucose activates V-ATPase activity in LLC-PK1 cells. pHi measurement and intracellular acidification (periods A and B) were performed as in Fig. 1. V-ATPase activity was present in 10 mM glucose (period C). After reacidification (period D), V-ATPase activity was nearly abolished in absence of glucose (period E) but recovered rapidly to initial rate after addition of 10 mM glucose (period F).

 
The glucose concentration dependence of H+-ATPase activation was determined by stimulating cells with varying (0–30 mM) concentrations of glucose after acute glucose removal (Fig. 3). The maximal effect of glucose was observed at 20 mM, with a Km of 5.1 mM glucose. pH recovery rates were 3.4 ± 0.5 x 10–4 pH units/s for 0 mM glucose (n = 17 cells), 6.2 ± 0.9 x 10–4 pH units/s for 2.5 mM glucose (P < 0.02; n = 18), 9.2 ± 0.4 x 10–4 pH units/s for 5.5 mM glucose (P = 0.005; n = 21), 13.8 ± 0.7 x 10–4 pH units/s for 10.5 mM glucose (P < 0.00001; n = 23), 16.1 ± 1.6 x 10–4 pH units/s for 15 mM glucose [P = 0.082, not significant (NS); 10.5 vs. 15 mM; n = 19], and 17.2 ± 2.0 x 10–4 pH units/s for 20.5 mM glucose (P = NS, 10.5 vs. 20.5 mM; P = NS, 15 vs. 20.5 mM; n = 24). pH recovery rates at 25 and 30 mM glucose decreased from the maximum to 16.9 ± 3.5 x 10–4 pH units/s (P = NS; n = 15) and 16.3 ± 1.2 x 10–4 pH units/s (P = NS; n = 18), respectively. The concentration dependence of glucose-stimulated proton flux [calculated by using the equation JH+ = (dpHi/dt{beta}i, where {beta}i is buffering capacity as described in METHODS] was similar to that for pHi recovery.



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Fig. 3. Glucose concentration dependence of V-ATPase activation. pHi recovery rates were measured as described in METHODS after readdition of glucose (period F in Fig. 2) at varying concentrations as indicated. Values are means ± SE for pHi recovery (dpHi/dt in pH units/s).

 
Amlal et al. (7) reported H+-ATPase activity in LLC-PK1 cells activated by hypotonicity. To determine whether glucose-induced H+-ATPase activation was a result of changing the solution osmolarity in the present study, the effect of 10 mM mannitol was examined as a substitute for glucose, using the procedure shown in Fig. 2. As shown in Fig. 4, mannitol failed to activate H+-ATPase activity, showing that glucose-induced H+-ATPase activation is not due to an increase in solution osmolarity.



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Fig. 4. Glucose-induced V-ATPase activation is not due to change in medium osmolarity. Procedures used were identical to those in Fig. 2, except that 10 mM mannitol replaced glucose (period F), which produced no pHi recovery. Recovery of V-ATPase activity was observed after readdition of 10 mM glucose (period G).

 
Glucose activates H+-ATPase activity in perfused rat proximal tubule. LLC-PK1 cells have several morphological and physiological properties resembling those of proximal tubule cells (14, 43, 49, 58), including Na+-glucose cotransport (49), but they do not possess a complete proximal tubule phenotype. To determine whether glucose activation of H+-ATPase occurs in renal tubular epithelial cells from intact proximal tubules, the effect of glucose on the H+-ATPase activity was examined in isolated perfused rat PT S3 segments. Proximal tubules were perfused with Na+- and K+-free buffers, and the rate of pHi recovery was determined after 5 min of NH3/NH4+ incubation and removal, initially in 2.5 mM glucose, followed by 10.5 mM glucose. As shown in Fig. 5A, changing the glucose concentration from 2.5 to 10.5 mM produced a significant increase in pHi recovery rates, from 6.2 ± 3.4 x 10–4 to 16.9 ± 0.5 x 10–4 pH units/s (P < 0.05, n = 14; Fig. 5B). The results suggest that intact rat renal proximal tubule cells have glucose-activated plasma membrane H+-ATPase activity similar to that observed in LLC-PK1 cells.



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Fig. 5. Glucose activates V-ATPase in isolated rat renal proximal tubule. A: intracellular pH measurements with BCECF were performed on isolated microperfused rat proximal tubules as described in METHODS in Na+-free conditions. After NH4Cl induced pHi acidification (periods A and B), pHi recovery rates were measured in 2.5 mM glucose (period C) and 10 mM glucose (period D). B: pHi recovery rates in 2.5 and 10 mM glucose. Values are means ± SE; n = 14. P < 0.05.

 
Glucose activation of H+-ATPase requires glycolysis. In principle, glucose could activate the H+-ATPase through metabolism, through signaling pathways, or both. To determine whether activation of H+-ATPase activity by glucose requires metabolism through glycolysis, the effect of 20 mM of 2-DG, a glycolytic inhibitor, was examined. As shown in Fig. 6, pretreatment with 2-DG eliminated glucose-activated H+-ATPase activity, suggesting that the response requires metabolism of glucose through glycolysis. Treatment with the mitochondrial electron transport inhibitor antimycin A (20 µM) partially inhibited activation of H+-ATPase by glucose (Fig. 7), suggesting that activation requires aerobic rather than anaerobic glycolysis. In support of this interpretation, it was found that 10 mM of pyruvate, the mitochondrial substrate for aerobic glycolysis, induced concanamycin-sensitive pH recovery, indicating activation of H+-ATPase activity (Fig. 8). In contrast, sodium acetate produced cytosolic alkalinization that was unaffected by concanamycin A (Fig. 9), probably by metabolism to bicarbonate in mitochondria. Alanine (10 mM) also was unable to activate H+-ATPase activity (Fig. 10).



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Fig. 6. Glycolysis is required for glucose-induced V-ATPase activation. Procedures used were identical to those in Fig. 2, except that glycolytic inhibitor 2-deoxy-D-glucose (2-DG; 20 mM) was added during second glucose treatment (period F), which completely inhibited V-ATPase activation.

 


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Fig. 7. Effect of antimycin A on glucose-induced V-ATPase activation. Procedures used were identical to those in Fig. 2, except that mitochondrial electron transport inhibitor antimycin A (20 µM) was added during second glucose treatment (period F), which partially inhibited V-ATPase activation. Incomplete recovery of V-ATPase activity was observed after antimycin A removal (period G).

 


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Fig. 8. Pyruvate activates V-ATPase in absence of glucose. Cells were treated with NH4Cl in presence of 10 mM glucose to induce pHi acidification (periods A and B) followed by removal of glucose and treatment with 10 mM pyruvate (period C), which produced significant pH recovery. After repeat pHi acidification in presence of glucose (period D), cells were treated with glucose-free solution (period E), which produced no pH recovery, and then with 10 mM pyruvate containing 100 nM CCA (period F), which also produced no recovery. Partial pHi recovery was observed after removal of CCA (period G).

 


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Fig. 9. Sodium acetate (NaAc) alkalinizes intracellular pH but does not activate V-ATPase. Procedures used were identical to those in Fig. 9, except that 10 mM sodium acetate replaced pyruvate. Acetate produced alkalinization of pHi (period C) that was not observed after acetate removal (period E) and was not affected by V-ATPase inhibitor CCA (period F).

 


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Fig. 10. Alanine does not activate V-ATPase. Procedures used were similar to those in Fig. 4, except that 10 mM alanine replaced mannitol. No V-ATPase activity was observed with alanine in the absence of glucose (period F), but activity resumed after replacement of glucose (period G).

 
Glucose activation of H+-ATPase requires phosphatidylinositol 3-kinase activity. In insulin-responsive tissues (11, 20, 59), lymphocytes (48), and other cells (11), signaling pathways involving phosphatidylinositol 3-kinase (PI3K) are involved in controlling glucose entry (59) and metabolism (20). To determine whether a PI3K-dependent signaling pathway contributes to the effect of glucose on H+-ATPase activity, the effect of 500 nM wortmannin, a PI3K inhibitor, was studied. As shown in Fig. 11, H+-ATPase activity induced by 10 mM of glucose was inhibited completely by wortmannin, and activity was restored after wortmannin removal.



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Fig. 11. Glucose-induced V-ATPase activation requires phosphatidylinositol 3-kinase (PI3K) activity. Procedures used were similar to those in Fig. 10, except that 10 mM glucose replaced acetate and PI3K inhibitor wortmannin (500 nM) replaced CCA. Glucose-induced V-ATPase activity was abolished by wortmannin (period F) and partially recovered after removal of wortmannin (period G).

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
These studies demonstrate that glucose is a potent and rapid activator of H+-ATPase-mediated proton secretion in both LLC-PK1 cells and isolated renal proximal tubules. The glucose effect was concentration dependent, with a half-maximal effect at 5.1 mM of glucose and a maximal effect at 20 mM. Glucose-induced V-ATPase activation was rapidly reversible. V-ATPase activity was lost after 5–10 min of glucose removal and was restored rapidly by the readdition of glucose, with mean times of 2–3 min to initial recovery and 5.5–7 min to half-maximal recovery. The effect was not due to changes in extracellular fluid osmolarity, because mannitol produced no significant effect. Glucose-induced V-ATPase activation required metabolism of glucose through the glycolytic pathway, because it was inhibited entirely by 2-DG. Metabolism likely occurs through aerobic glycolysis because the mitochondrial complex III inhibitor antimycin partially inhibited V-ATPase activation. Pyruvate, an end product of the glycolytic pathway, activated V-ATPase activity in the absence of glucose, but alanine and acetate did not activate V-ATPase activity. These results extend those of the recent studies of the interaction of the V-ATPase with aldolase (32) and provide further evidence for coupling between V-ATPase activity and glycolysis.

The brewer's yeast Saccharomyces cerevisiae has V-ATPase on the vacuolar membrane that is similar in structure and properties to mammalian V-ATPases (15, 17, 42, 56, 63). Yeast use glucose preferentially as a substrate for anaerobic glycolysis in ethanol production (23). Glucose removal induced rapid disassembly of yeast V-ATPase. Rapid reassembly occurred in a concentration-dependent manner with glucose retreatment (25, 45) and required metabolism of glucose beyond the formation of glucose 6-phosphate (45). A recent study (32) showed that V-ATPase binds directly to aldolase and that V-ATPase disassembly occurs in yeast strains that are deficient in aldolase and other glycolytic enzymes. Collectively, these experiments demonstrate that glycolysis is essential for assembly and function of the Saccharomyces V-ATPase.

Several studies have examined metabolic pathways supporting proton transport by V-ATPases in urinary epithelia (3, 29). In the turtle urinary bladder, a model epithelium resembling the mammalian kidney cortical collecting duct (2, 54) with an electrogenic plasma membrane V-ATPase (16, 65), glucose stimulated electrogenic V-ATPase-mediated proton transport (1, 3, 27, 55). Under standard conditions, transport was inhibited by 2-DG and deoxygenation (1) and was not coupled with lactate production (27). In bladders treated with aldosterone to activate H+ transport, active proton secretion showed a greater response to glucose addition and was stimulated by pyruvate (4). These studies suggest coupling of H+ transport with aerobic glycolysis under these conditions. Under anaerobic conditions, H+ transport was tightly coupled with lactate production through anaerobic glycolysis (55).

H+-ATPase activity was identified in rabbit renal proximal tubules that was inhibited by the glycolytic inhibitor iodoacetate, suggesting coupling of activity with glycolysis (29). Proximal tubules have a low rate of lactate production (8), indicating that glucose metabolism in this segment occurs primarily by aerobic glycolysis. The present study shows that H+-ATPase activity in rat proximal tubules is stimulated by increasing the extracellular glucose concentration, a response similar to the glucose-induced V-ATPase activation observed in LLC-PK1 cells.

Nakamura et al. (39) previously examined the effect of glucose on the levels of ATP, ADP, and ATP-to-ADP ratio in serum- and/or glucose-starved LLCPK cells. Their study showed that incubation in glucose-free medium for 16 h reduced cell ATP content by 37% and increased ADP content slightly, changing the ATP-to-ADP ratio significantly from 2.53 to 1.22. However, stimulation with 10 mM glucose for periods ranging from 2.5 to 30 min produced no significant changes in ATP or ADP content. These results indicate that stimulation of V-ATPase activity by glucose, at least within short time intervals, likely does not occur by increasing ATP availability.

Recently, other studies have shown that ATP level remained practically unchanged when yeast cells were grown in either 2% or 0.025% glucose (44). Krauss et al. (28) demonstrated that under physiological conditions, hyperglycemia-induced mitochondrial superoxide production activates uncoupling protein 2, which decreases the ATP-to-ADP ratio.

Other previous studies have shown that glucose stimulates glycolysis and increases the activity of several glycolytic enzymes in LLC-PK1 cells (18). The present study revealed that the glycolytic inhibitor 2-DG prevented glucose-induced V-ATPase activation in LLC-PK1 cells. Taken together, these results suggest that glucose activates H+-ATPase by stimulating glycolysis.

The present study shows that pyruvate also activated H+-ATPase activity in the absence of glucose. It is possible that pyruvate could be converted to glucose through the gluconeogenic pathway and subsequently metabolized by glycolysis (51). In the presence of the H+-ATPase inhibitor concanamycin A, it was observed that pyruvate-induced H+-ATPase activity was inhibited (Fig. 8; pyruvate + CCA, period F) and that H+-ATPase activity was restored by pyruvate after CCA removal (Fig. 8; pyruvate only, period G). As shown in Fig. 9, however, the H+-ATPase inhibitor (i.e., CCA) had no effect on acetate-induced pH recovery (Fig. 9; NaAc + CCA, period F), likely due to a mechanism other than H+-ATPase activity involvement for the regulation of intracellular pH. The alkalinization observed with acetate might be due to metabolic generation of alkali. Ishikawa et al. (22) showed that acetate induced cytosolic alkalinization that was not affected by H+-ATPase inhibitors and probably was a result of mitochondrial metabolism of acetate to bicarbonate. Studies in the kidney (26, 51) have demonstrated the importance of such "futile" cycles of glycolysis and gluconeogenesis, which provide the capacity for rapid changes in glycolytic flux (24, 46). Net ATP consumption in futile cycles may be reduced by separation and compartmentalization of the glycolytic and gluconeogenic pathways (24).

Both pyruvate and acetate are metabolized by the citric acid cycle. The metabolism of acetate through acetyl coenzyme A-synthetase consumes one ATP and generates one less NADH and CO2 (22) than does metabolism of pyruvate through pyruvate dehydrogenase. Acetate also inhibits some of the pathways for pyruvate metabolism (13). The observed differences between these two agents on V-ATPase activation strongly implicate mitochondrial metabolism in the activation pathway, consistent with the partial inhibition of activation observed with antimycin.

It is also significant that alanine was unable to activate V-ATPase. Although alanine can be converted to pyruvate by transamination (61, 64) and is a potential substrate for gluconeogenesis (33, 61), studies in both isolated renal proximal tubules (31, 47) and human volunteers (12, 57) have shown that alanine is a poor substrate for renal gluconeogenesis compared with lactate. These studies support the interpretation that substrate flux through the glycolytic pathway is required for V-ATPase activation. The results observed are not specific for V-ATPase, however, because Hering-Smith and Hamm (21) found that alanine was not an effective metabolic substrate for collecting duct Na+ transport in the absence of glucose.

The results of the present study indicate that PI3K activity is required for glucose activation of V-ATPase (Fig. 11). PI3K activity is required for several signaling pathways involved in glucose control, including glucose entry and glycogen metabolism (11, 20, 59). The signaling pathways downstream from PI3K and targets leading to V-ATPase activation remain to be determined in future studies.

In conclusion, glucose activates V-ATPase activity in renal epithelial cells through a pathway requiring aerobic glycolysis and PI3K activity, providing further evidence for coupling between V-ATPase activity and glycolysis.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported by an American Heart Association grant and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54362.


    ACKNOWLEDGMENTS
 
We thank Dr. S. L. Gluck and Dr. M. Lu for important suggestions and valuable discussions and Alexia Lundberg for technical help.

Current address of S. Nakamura: Dept. of Biological Sciences, Murray State Univ., 334 Blackburn Science Bldg., Murray, KY 42071-3346.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Nakamura, Dept. of Biological Sciences, Murray State Univ., 334 Blackburn Science Bldg., Murray, KY 42071-3346 (E-mail: suguru.nakamura{at}murraystate.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Al-Awqati Q. Effect of aldosterone on the coupling between H+ transport and glucose oxidation. J Clin Invest 60: 1240–1247, 1977.[ISI][Medline]

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3. Al-Awqati Q, Mueller A, and Steinmetz PR. Transport of H+ against electrochemical gradients in turtle urinary bladder. Am J Physiol Renal Fluid Electrolyte Physiol 233: F502–F508, 1977.[Abstract/Free Full Text]

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