Departments of 1Molecular and Cellular Physiology and 2Medicine and Feist-Weiller Cancer Center, Louisiana State University Health Science Center, Shreveport, Louisiana 71130; and 3Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Submitted 9 May 2003 ; accepted in final form 12 September 2003
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ABSTRACT |
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intracellular pH; Na+/H+ exchanger; 15N-labeled glutamine; alanine aminotransferase activity; protein kinase C/extracellular regulated kinase
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In the LLC-PK1-F+ cell line, chronic (18-24 h) studies at extracellular pH of 7.1 (32) and 6.9 (20) result in a 20-25% increase in ammonium production (20, 32), adaptive increases in glutamine uptake (31), and phosphate-dependent glutaminase and PEPCK (16, 20). These adaptations in glutamine metabolism occur along with an increased acid extrusion at the apical cell surface (31) consistent with upregulated Na+/H+ exchanger (NHE)3 activity and restoration of the pHi despite the progressively acidic extracellular milieu. The ability of an acute fall in pHi to accelerate glutamine-dependent ammoniagenesis suggests direct effects of cellular acidosis on these pathways (37, 38), whereas adaptive responses are consistent with signaling pathways (2, 10, 40, 48) inducing upregulation of transporters and enzymes involved in the chronic response.
To study the chronic effect of reduced pHi on ammoniagenesis, glutamine metabolism, and dependent cellular processes in culture, a means of inducing a sustained cellular acidosis is important. Recently, the thiazolidinedione troglitazone has been shown to acutely induce a dose-dependent cellular acidosis and to accelerate ammoniagenesis from glutamine in distal tubule-like Madin-Darby canine kidney (MDCK) cells (9) expressing NHE1 (33). However, it is not clear whether this cellular acidosis would develop in the more pH-sensitive LLCPK1-F+ cells also expressing NHE3 (10) and, if so, would this acidosis persist as a chronic cellular acidosis. The present studies therefore were designed to determine in pH-sensitive LLC-PK1-F+ cells whether troglitazone would in fact induce this chronic cellular acidosis and also express effects on glutamine metabolism similar to those observed in metabolic acidosis. Because troglitazone is a well-recognized activator of the peroxisome proliferator-activated receptor (PPAR) signaling system (7), we also included studies using the PPAR
antagonist bisphenol A diglycide ether (BADGE) (47) and the protein kinase C (PKC) inhibitor chelerythrine (23) to assess the relative roles for PPAR
-dependent and putative PPAR
-independent signaling pathways in these physiological responses.
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MATERIALS AND METHODS |
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Cell pHi measurements. The intracellular pH was assayed using the pH-sensitive fluorescent dye (2,7)-biscarboxyethyl-5 (6)-carboxyfluorescein (BCECF) as described previously (9, 45). For acute pHi measurements, the cells were loaded with BCECF-AM (Molecular Probes, Eugene, OR), washed, and equilibrated in HEPES-buffered Krebs-Henseleit (KHH) media (pH = 7.40) containing 10 mM glucose. The chamber was then mounted on an epifluoroscope and the media was then replaced with fresh KHH media; after 4 min of continuous recording at 37°C, new KHH media was added containing either vehicle or 25 µM troglitazone (kindly supplied by Dr. Tagata, Sankyo, Tokyo, Japan), 25 µM troglitazone plus 100 µM BADGE, BADGE alone, or vehicle for a second 4 min of continuous recording. Calibrations of the 490/440-nm ratios were determined on every experiment using the nigericin/high potassium method (45). For determining the chronic effect of troglitazone, monolayers were incubated for 18 h in DMEM minus phenol red and then BCECF-AM loaded with DMEM minus FCS but containing 25 µM troglitazone, 25 µM troglitazone plus 100 µM BADGE, BADGE alone, or vehicle and then pHi monitored over a 30-min time course followed by calibration for each experiment.
Measurement of NHE activity. The activity of NHE was determined as the rate of pHi recovery after an NH4Cl load as modified from the previously described method (9). In the present study, BCECF-acetoxymethyl ester (5 µM) was loaded into cells in KHH media containing 20 mM NH4Cl (substituted for an equal molar NaCl). After being washed with the same media minus BCECF, KHH media was added and the recovery was monitored over an 8-min time course. In the chronic troglitazone experiments, troglitazone was present in both the loading and recovery media; in the acute experiments, troglitazone was added to the recovery media only following the second NH4Cl load. Recovery from a second acid load was not different from the recovery to the first acid load. BCECF fluorescence was measured and the 490/440 ratio was calibrated as described above.
Studies with 15N-labeled glutamine. For analysis of alanine and ammonium formed from [2-15N]glutamine and [5-15N]glutamine, media glutamine was replaced with either [2-15N]- or [5-15N]glutamine (99 atom % excess, Cambridge Isotope Laboratories, Andover MA). After 18 h of incubation in the prescribed media above, media samples and PBS-washed cells were taken and treated with ice-cold 40% perchloric acid. The concentration of ammonium and alanine and their 15N enrichment were determined on the neutralized supernatants. Briefly, the amino acids underwent precolumn derivatization with O-phthaladehyde (FLUKA, Buchs, Switzerland) and separation by HPLC and fluorescence detection (35). Analysis of 15N in the amino acids was done by GC-MS as previously described (6, 32, 43). Ammonium concentration was measured by the microdiffusion method described previously (35).
Formation of [15N]ammonia was determined following conversion of ammonium to norvaline (6). To calculate the conversion of 15N-labeled glutamine to ammonium and alanine, the isotopic enrichment, atom percent excess (a%ex) of 15N in the particular metabolite was multiplied times the amount present and expressed as nanomole per milligram of protein. The results therefore express net fluxes reflecting the balance of underlying unidirectional fluxes depicted in Fig. 1.
Metabolic studies. Studies were performed on confluent LLCPK1-F+ cells grown in the 6- to 12-well plates over 18 h in DMEM media containing DMSO (vehicle) or DMEM plus troglitazone. Media samples were promptly treated with an equal volume of ice-cold 5% trichloroacetic acid, processed, and their amino acid concentration was analyzed by HPLC (35). Utilization or production rates for the respective amino acids were obtained from the concentration differences times the media volume (2 ml). Ammonium concentration was determined by the microdiffusion method (35) and formation rate was determined as above by subtracting the media blank and expressed on the basis of milligrams of protein.
PPAR functional activity assay. LLC-PK1-F+ cells,
80% confluent and fed 1 h before, were transiently transfected with a firefly luciferase reporter plasmid (ARE6.3XTkpGL3, kindly provided by Dr. T. Leff) driven by three copies of the PPAR response element from the aP2 gene inserted (7) into a luciferase vector (Promega, Madison, WI). A thymidine kinase-driven sea pansy luciferase was simultaneously cotransfected using a liposomal cationic vehicle (Lipofectamine, Life Technologies, Gaithersburg, MD) as described previously (17). Briefly, DNA and lipofectamine were separately dissolved in Opti-MEM I (GIBCO, Grand Island, NY) and then mixed and allowed to stand at room temperature for 0.5 h. After the cells were washed two times with Opti-MEM I, the DNA-containing micelles suspended in Opti-MEM I were directly added. After a 6-h incubation, 0.5 ml DMEM were added to the transfection media with overnight incubation. The media was then removed and replaced with fresh media containing either vehicle, DMSO, or increasing concentrations (5-50 µM troglitazone) for 18 h after which the cells were washed with PBS and harvested in passive lysis buffer (Promega) for assayable luciferase and alanine aminotransferase (ALT) activity. Luciferase activity of the monolayers was assayed as described (Dual-Luciferase Reporter Assay, Promega) with the activity of the PPRE-driven firefly luciferase normalized to the constitutively expressed sea pansy luciferase and presented as a ratio.
PPAR expression by immunohistochemistry. The previously studied MDCK cells (9) and LLC-PK1-F+ cells were grown on permanox two-well chamber slides (Nalge-Nunc, Napierville, IL) and fixed with 4% paraformaldehyde/PBS and made permeable using 0.1% saponin in PBS containing 20 mM glycine. Cells were then incubated for 1 h with PPAR
primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 in 0.25% BSA and 0.1% saponin in PBS. After two washes with PBS, the cells were treated for 1 h with the secondary antibody (Alexa Fluor 488 goat anti-mouse antibody, Molecular Probes) diluted 1:500 dilution. The slides were washed as above, and cells were stained with 1 µg Hoechst 33342/ml PBS for 5 min to visualize the nuclei. After being washed, the cells were mounted with Slow Fade anti-fade reagent (Molecular Probes) and viewed on an Olympus Bx60 scope using QuipsPathvision software.
Enzymatic assays. The assayable ALT and GDH activities were determined as described previously (9) and expressed in units per milligram of protein. For direct effects of troglitazone and pH on assayable activity, either 100 µM troglitazone or 0.2 N HCL was added to the assay media before control sample addition.
Statistical analysis. Differences between control and treated monolayers were analyzed using either the Student's t-test or ANOVA and a corrected t-test (Dunnett's) for multiple groups with differences considered significant at P < 0.05.
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RESULTS |
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The effect of the PPAR antagonist BADGE on the troglitazone-induced cellular acidosis was also studied in both the acute and chronic conditions. As shown in Fig. 2, BADGE (100 µM) modulated the acidifying effect of troglitazone in the acute study (pHi 7.12 ± 0.11 vs. 6.92 ± 0.04 for Tro + BADGE and Tro alone, P < 0.05), which was not different from control (7.12 ± 0.11 vs. 7.22 ± 0.04). In the chronic 18-h treatment, BADGE prevents the acidifying effect of troglitazone (7.18 ± 0.10 vs. 6.91 ± 0.06 and 7.16 ± 0.05 for Tro + BADGE and control alone); BADGE by itself increases the pHi (7.26 ± 0.06 vs. 7.16 ± 0.04, P < 0.05 data not shown).
To determine troglitazone's affect on the pathways of glutamine metabolism in LLC-PK1-F+ cells as depicted in Fig. 1, experiments were carried out over 18 h using [15N]glutamine in either the [5-15N] or [2-15N] position. The isotopic enrichment (a%ex) as well as the ammonium produced from [15N]glutamine are shown in Table 1. Troglitazone (25 µM) increased the ammonium produced from the amide nitrogen of glutamine by 2.45-fold (328 ± 18 to 804 ± 44 nmol/mg, P < 0.0001) and increased by 3-fold (P < 0.001) the ammonium derived from the amino nitrogen of glutamine (174 ± 12 to 524 ± 44 nmol/mg). Note that the increased ammoniagenesis derived from glutamine reflects both an enhanced enrichment of the respective isotope (23 to 30 a%ex for the amide nitrogen and 12 to 21 a%ex for the amino nitrogen, P < 0.001) but as well an increased ammonium production (1,426 ± 76 to 2,671 ± 223 nmol/mg, P < 0.01, for [15N]amide and 1,510 ± 113 to 2,530 ± 257 nmol/mg, P < 0.01, for [15N]amino glutamine study). These results are consistent with an increased net flux through the glutamate dehydrogenase pathway as depicted in Fig. 1.
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The effect of troglitazone on alanine production from labeled glutamine is also presented in Table 1. Troglitazone had no effect on the isotopic enrichment in alanine from either the [5-15N] or [2-15N]glutamine. However, the total alanine production decreased by 52 and 62% (3,812 ± 256 to 1,934 ± 248 nmol/mg and 2,657 ± 143 to 1,049 ± 147 nmol/mg for the amide and amino labeled-glutamine studies, respectively, both P < 0.0001). Consequently, the production of alanine from the amide and amino nitrogen of glutamine decreased proportionately, 801 ± 82 to 388 ± 50 and 732 ± 62 to 284 ± 48 nmol/mg, both decreases P < 0.0001 (Table 1). Note that the isotope enrichments from [15N]amide-labeled glutamine in the cellular glutamate and alanine are similar (17.8 ± 0.2 and 18.1 ± 0.4 a%ex for control and 17.5 ± 0.4 and 17.4 ± 0.2 a%ex for troglitazone, respectively). Therefore, [15N]alanine from [5-15N]glutamine was most likely formed following the sequence of metabolic reactions depicted in Fig. 1: I) formation of via the glutaminase reaction, II) incorporation of [15N]ammonium into
-Kg to form [15N]glutamate via the GDH reaction (reductive amination), and II) transamination of [15N]glutamate to form [15N]alanine. With [2-15N]glutamine, following glutaminase activity, [15N]glutamate and [14N]ammonium (I) was formed and then transamination (III) of [15N]glutamate to form [15N]alanine.
The point of interest is that glutamine utilization did not change (1,763 ± 118 vs. 1,834 ± 109 nmol/mg for control and troglitazone, respectively), consistent with unchanged glutaminase flux (I) but with a nearly reciprocal shift of nitrogen from alanine (decrease 861 nmol/mg) to ammonium (
increase 826 nmol/mg). Because of the important role of alanine aminotransferase as well as glutamate dehydrogenase in catalyzing the reactions leading from glutamate to alanine and ammonium (Fig. 1), respectively, the assayable activity of both was measured, whereas GDH activity was unchanged (263 ± 110 vs. 264 ± 26 U/mg) and that of ALT was reduced by 23% (190 ± 16 to 147 ± 10 U/mg, P < 0.001).
Figure 4 shows that the increased ammoniagenesis from glutamine occurs as early as 3 h after troglitazone exposure and was coupled at this early time point as well as throughout the time course to the reduction in alanine production. At 3 h, the ratio of ammonium to alanine produced rose from 0.46 ± 0.02 (consistent with extensive amide as well as with amino nitrogen incorporated into alanine) in the control to 1.24 ± 0.04 with troglitazone as the result of both a rise (P < 0.05) in ammonium and fall (P < 0.05) in alanine (consistent with inhibition of net flux into alanine and enhanced net flux through oxidative deamination and ammonium production); at 6 h, the ratio rose to 1.7 with troglitazone and was maintained at this ratio (1.7) until 18 h as the ratio for control cells declined from 0.46 ± 0.02 after 3 h to 0.29 ± 0.024 (P < 0.05).
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The effect of increasing troglitazone concentration on ammonium and alanine production is shown in Fig. 5. At 5 µM troglitazone, ammonium production increased 1.6-fold (724 ± 86 to 1,131 ± 183 nmol/mg, P < 0.05), whereas alanine production decreased 11% (2,498 ± 169 to 2,227 ± 230 nmol/mg, P < 0.10), or, by roughly similar amounts. This coupling between increased ammonium and decreased alanine production was observed up to 25 µM after which alanine production continued to decrease as increased ammonium production leveled off; this was associated with accumulation of glutamate in the media (314 ± 34 and 610 ± 63 nmol/mg at 50 and 100 µM, respectively, vs. no net accumulation at lower concentrations). Figure 5 also shows that for the control cells the steady-state pHi measured at the end of 18 h increased to 7.30 ± 0.06 consistent with the fall in ammonium to alanine production ratios obtained from Fig. 4 over the 18-h time course; in marked contrast, the pHi fell to 6.50 ± 0.10 at the highest concentration with an ammonium to alanine production ratio of 3.04 ± 0.64. These results are consistent with the measured NHE activity playing a major role in determining pHi and regulating glutamine metabolism as reflected in the production ratio and that changes in this ratio may, in turn, be reflective of changes in the pHi.
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To assess the role that PPAR activation may play on assayable ALT activity, cells were transfected with a PPRE-driven firefly luciferase reporter plasmid as described in MATERIALS AND METHODS and treated for 18 h with increasing concentrations of troglitazone after which both firefly luciferase activity (Fig. 6A) and ALT activity (Fig. 6B) were assayed in the same cell lysate. At the lowest troglitazone concentration used (5 µM), assayable firefly luciferase activity increased (P < 0.01) 2.7-fold while ALT activity decreased 42% (P < 0.05). At the highest concentration of troglitazone (50 µM), luciferase activity had increased 4.9-fold (P < 0.01) and ALT activity had decreased 72% (P < 0.01). These results suggest that PPAR
activation may act to inhibit assayable ALT activity. If so, cells that express the cytosolic ALT isoform containing a PPRE promoter and do not downregulate in response to troglitazone should not express PPAR
. As shown in Fig. 6C, MDCK cells previously shown not to decrease assayable ALT in response to troglitazone (10) do not express PPAR
(A). In sharp contrast, LLC-PK1-F+ cells do express PPAR
predominantly, but not exclusively, in association with the nucleus (C). These findings demonstrate the presence of the PPAR
within the LLC-PK1-F+ cell line and its activation by troglitazone and also suggest that this signaling pathway may be involved in the downregulation of assayable ALT activity. To assess the role that the fall in pHi might have on ALT activity, ALT was assayed at a reduced pH. At pH 6.9 (as opposed to 7.4 normal assay media pH), the ALT activity was reduced 33% (203 ± 10 to 137 ± 4 U/mg, P < 0.01), suggesting that both the total enzyme present as well as the cytosolic pH may contribute to the observed decreased in alanine production. It is noteworthy that adding troglitazone (100 µM) directly to the assay media did not inhibit the activity.
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To assess the effect that BADGE would have on troglitazone-induced ammonium production, monolayers were incubated with 25 µM troglitazone with and without 100 µM BADGE. As shown in Fig. 7, 25 µM troglitazone increased the ammonium production by 2.1-fold, whereas the addition of BADGE to the troglitazone media prevented this increase (869 ± 88; 1,859 ± 195; and 781 ± 127 nmol/mg for control; troglitazone alone; and troglitazone plus BADGE); note that BADGE by itself decreased ammonium production below the control level (575 ± 130 nmol/mg). This effect of BADGE to inhibit troglitazone's ammoniagenic action is consistent with BADGE's ability to prevent the cellular acidification as shown in Fig. 2; in addition, the effect of BADGE alone to decrease baseline ammonium production is consistent with the observed rise in pHi (7.26 ± 0.06 vs. 7.16 ± 0.04, P < 0.05).
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In contrast to BADGE's action in completely blocking troglitazone's effect to enhance ammoniagenesis, the reduced alanine production could not be restored to the control level (Fig. 7). Troglitazone suppressed alanine production by 62% (2,620 ± 157 to 986 ± 207 nmol/mg, P < 0.0001) and the addition of BADGE to the troglitazone increased alanine production twofold compared with troglitazone alone (1,971 ± 81 vs. 986 ± 207 nmol/mg, P < 0.01). However, alanine production still remained 25% below control (1,971 ± 81 vs. 2,620 ± 157 nmol/mg, P < 0.05); noteworthy BADGE by itself reduced alanine production by 15% (2,218 ± 160 vs. 2,620 ± 157 nmol/mg, P < 0.05). Because the cellular acidosis induced by troglitazone was corrected by BADGE, the effect of BADGE on the assayable ALT activity was measured in these same monolayers. Assayable ALT activity was reduced by troglitazone (244 ± 22 to 125 ± 24 U/mg, P < 0.01) and further decreased by the combination of troglitazone plus BADGE (125 ± 24 to 72 ± 12 U/mg, P < 0.05) while BADGE alone tended to decrease ALT activity (244 ± 22 to 190 ± 31 U/mg, P < 0.10). These results show that the alanine formation can be largely dissociated from the level of assayable ALT activity under the condition in which the troglitazone-induced cellular acidosis is prevented.
To assess whether BADGE would block the transactivation of the PPRE-driven luciferase reporter plasmid, transiently transfected cells were treated with vehicle, troglitazone, troglitazone plus 100 µM BADGE, or BADGE alone. As shown in Fig. 8, troglitazone at 25 µM increases the PPRE-driven firefly luciferase activity by two- to threefold as expected and BADGE at 100 µM completely blocked the troglitazone induction of firefly luciferase expression; BADGE did not demonstrate any agonist activity (47). Although these results and those in Fig. 6, A-C, indicate that PPAR is a functioning signaling pathway in the LLC-PK1-F+ cell line, the acute and dominant affects of troglitazone and BADGE on pHi and glutamine metabolism are more consistent with a PPAR
-independent pathway rather than through conventional PPAR
signaling.
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One possibility for PPAR-independent signaling, but by no means only possibility, is troglitazone action that results in the inhibition of PKC (25). If so, an inhibitor of PKC might be expected to mirror the effect of troglitazone on glutamine metabolism. To test this possibility, chelerythrine (10 µM), a specific PKC inhibitor (23), was added to the media and the effect on ammonium and alanine production was measured over an 18-h time course and compared with 25 µM troglitazone. As shown in Fig. 9, chelerythrine alone increased ammonium production by 1.4-fold (P < 0.05) compared with a 1.7-fold (P < 0.05) increase with troglitazone, whereas in combination ammonium production increased further to 1.92-fold (P < 0.01 vs. control); alanine production was inhibited 33% (P < 0.05) by chelerythrine vs. 50% for troglitazone (P < 0.01) and 78% (P < 0.01) in combination. Note that assayable ALT activity was not reduced with chelerythrine (193 ± 10 vs. 221 ± 12 U/mg for chelerythrine vs. control, n = 3), whereas alanine production decreases again showing the dependence on both the assayable activity and pHi. Because NHE activity is activated by PKC-dependent phosphorylation (42), we assessed whether chelerythrine has an affect on pHi by adding 10 µM chelerythrine to the media alone or in the presence of troglitazone (25 µM) for 18 h after which the pHi was determined. Chelerythrine alone decreased pHi (P < 0.01) to 6.98 ± 0.02 vs. 7.51 ± 0.03 for control; in combination with troglitazone, chelerythrine further reduced pHi (P < 0.01) to 6.54 ± 0.02. These results show that chelerythrine alone induces a cellular acidosis similar to that induced by troglitazone and that in combination with troglitazone chelerythrine further decreases pHi. To test whether the enhancement of ammoniagenesis by troglitazone could be prevented by activation of PKC, monolayers were treated with phorbol ester (500 nM) and troglitazone (25 µM) alone or in combination for 18 h, and the glutamine-dependent ammonium production was determined. As shown in Fig. 9, troglitazone alone nearly doubled ammonium production (1,240 ± 91 to 2,171 ± 236 nmol/mg, P < 0.01), whereas phorbol ester in combination with troglitazone prevented this increase (1,415 ± 212 vs. 2,171 ± 236 nmol/mg). In contrast to the ability of phorbol ester to prevent the troglitazone-induced ammoniagenesis, the suppression of alanine production was only partially restored by phorbol ester (1,223 ± 199 vs. 1,830 ± 282 nmol/mg for troglitazone plus phorbol ester vs. control, P < 0.05) but still increased above the troglitazone treated (1,223 ± 199 vs. 715 ± 170 nmol/mg, P < 0.01). The troglitazone reduction in assayable ALT activity (P < 0.01) was not increased by phorbol ester treatment (195 ± 12, 95 ± 9, and 105 ± 10 U/mg for control, troglitazone, and troglitazone plus phorbol ester, respectively), again demonstrating the contribution of both assayable ALT activity and pHi to alanine production (Fig. 10).
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DISCUSSION |
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The results (Fig. 2) clearly show that troglitazone induces a severe cellular acidosis in this proximal tubule-like cell line just as it did in the glomerular mesangial (45) and distal tubule-like MDCK (9) cell lines. In addition, we now show that this cellular acidosis is sustained for at least 18 h (Fig. 2). A sustained cellular acidosis of this degree of severity might be caused by enhanced acid production or inhibition of acid extrusion. However, lactate production, the main source of metabolic acid in these cells, did not increase, pointing to inhibition of acid extrusion. Indeed, NHE activity measured after chronic treatment with troglitazone was markedly reduced (Fig. 3B) and similar to that observed with acute troglitazone treatment in this (Fig. 3A) and in our previous studies (9, 45) in cell lines expressing only NHE1. Because LLC-PK1-F+ cells express both the NHE1 and 3 isoforms (10), and because only the NHE3-expressing apical cell surface was exposed to the media, it may be concluded that troglitazone's effectiveness in inducing cellular acidosis extends to cells expressing either one, e.g., NHE1, NHE3, or, now, both acid extruders. Perhaps the simplest explanation for the mechanism of troglitazone's effect to inhibit NHE activity would be binding at the external sodium binding site, but, if this was the sole site, then removal of troglitazone from the media (Fig. 3B) should have resulted in NHE activation and a prompt return to the control pHi. Further studies are required to determine the site(s) and pathways involved in troglitazone's unique action in inhibiting both NHE isoforms and to induce the cellular acidosis observed.
Under these conditions, troglitazone induces a large (more than 2-fold) increase in ammoniagenesis from glutamine (Table 1) consistent with the chronic cellular acidosis actually measured, e.g., 6.9 and previous reports of the effect of acute studies at a similar extracellular pH in the parent LLC-PK1 line (32, 37, 38). This increase in ammonium production reflected a two- to threefold increase in the contributions to ammonium production from both the amino and amide nitrogen of glutamine (Table 1). Surprisingly, the large increase in ammonium release to the media represents the inhibition of net flux through transamination and alanine formation and enhancement of net flux through the oxidative deamination pathway rather than an increased glutaminase flux. The dose-dependent increase in ammoniagenesis (Fig. 5) and the dose-dependent effect of troglitazone in lowering pHi (Fig. 5) are consistent with pHi (38) being an important signal by which troglitazone enhances the net flux through the glutamate dehydrogenase pathway. In support of H+ being the primary signal, the effect of troglitazone to enhance ammoniagenesis could be completely abrogated by BADGE (Fig. 8) associated with restoration of the pHi (Fig. 2). The ability of BADGE to acutely null the troglitazone-induced cellular acidosis is in line with its effect as an ionophore in equilibrating ion gradients across cell membranes (15) rather than its effect to block PPAR transactivation (44). Nevertheless, this is the first report of an effect of BADGE on cell pH.
Unlike ammoniagenesis, the troglitazone-induced fall in alanine production likely reflects both the reduction in assayable ALT and the cellular acidosis acting together to reduce in situ ALT activity. The relative contribution of each factor to the alanine production decrease can be assessed from the dose-dependent decrease in assayable activity and pHi. At the lowest troglitazone dose (5 µM), there is a large reduction in assayable ALT (42%; Fig. 6A) with only a small reduction in alanine production (11%, P < 0.05); at 25 µM troglitazone, but with the pHi restored to control values with BADGE, alanine production was still reduced below the control level (25%; Fig. 7), as was the assayable ALT activity (71% reduced) consistent with the flux determined by the amount of enzyme. Conversely, the cell pH was reduced using chelerythrine, which decreased the alanine flux 33%, but without decreasing the assayable ALT activity. Therefore, the dual effect of troglitazone in inducing a marked cellular acidosis and decreasing assayable ALT activity may account for the extraordinary potency of this compound to inhibit a major metabolic pathway sustaining growth in normal (18) and specifically in malignant cells (3, 12) as well as to contribute to its potential hepatotoxicity.
In the parental LLC-PK1 cell line, aminoxyacetate inhibited in situ ALT activity as judged by a decrease in alanine flux and also reduced assayable ALT activity (21). In the present study, we observed an inverse relationship between the fall in alanine production and rise in ammonium production reflecting the shift of glutamine's nitrogen from transamination into the deamination pathway as shown using 15N. Although this may (21) or may not (36) occur by inhibiting ALT with AOA, it is clear from the present and previous study (9) that inhibiting assayable ALT is not necessary for troglitazone to affect this shift. Rather, pHi appears to be the more important factor in the LLC-PK1-F+ cell line and the sole determining factor in the MDCK cell line (7). These findings with troglitazone as a probe to induce a severe cellular acidosis may reveal important roles that cellular acidosis potentially plays in regulating cellular processes dependent on cell pHi and glutamine metabolism.
Previous studies (21, 31, 37, 38) have not shown that acidosis in LLC-PK1 cells reduces alanine production and, in fact, even increased the alanine production along with ammonium. The 15N studies showed that acidosis increased the amino nitrogen incorporation into both alanine and ammonium in parallel suggesting that the same mechanism responsible for enhanced flux through GDH was also driving the alanine formation. One possibility for this discrepancy between the present study and those in the parental cell line could be the bimodal distribution of the ALT activity between the cytosol and mitochondrial compartments (13). If the dominant ALT activity is within the cytosol in the LLC-PK1-F+ cell line, then a reduction in cytosolic pH would reduce overall alanine production; conversely, if the mitochondrial ALT was dominant in the LLC-PK1 cell line, then alanine formation would be enhanced with increased glutamate availability in parallel with the mitochondrial GDH flux as observed. Noteworthy is the cytosolic ALT gene, which contains the PPRE promoter (14) and presumably would be affected by PPAR agonists/partial agonists, is also pH sensitive (13).
The ammoniagenic response generated by troglitazone greatly exceeds that expressed by LLC-PK1-F+ exposed to an extracellular acidosis. In our previous study in LLC-PK1-F+ cells exposed to an extracellular acidosis (31) equal to the intracellular acidosis induced by 25 µM troglitazone, ammonium production increased only 20% compared with the more than 200% increase with troglitazone. This discrepancy in the ammoniagenic responses presumably reflects a mild and transient cellular acidosis as a consequence of the acute buffering response (26) as well as an adaptive increase in apical surface acid extrusion (31). Noteworthy, LLC-PK1 (24, 26) cells as well as OKP (2) cells exposed to a marked reduction in the extracellular pH acutely exhibit a far smaller fall in their pHi and chronically respond by returning their pHi to normal, both processes reflecting upregulation of the NHE activity. In fact, recent studies (22) in the parental LLC-PK1 cell line show that a sustained intracellular acidosis of 6.8 for only 4 min is sufficient to enhance NHE3 activity by more than twofold, restoring pHi to the normal range. In the present study, over the 18-h incubation period, control cells generated a significant amount of acid to reduce their media bicarbonate by 6 mM and to create a mild extracellular acidosis, yet intracellular pHi rose to 7.30 and the ammonium to alanine production ratio fell from 0.5 to 0.3 consistent with an important role for adaptive NHE activity in modulating acid-base metabolism. In contrast, troglitazone acutely induces a profound and persistent cellular acidosis associated with a twofold increase in ammonium production and a 50% decrease, and more, of the glutaminedependent alanine production.
The signaling pathway(s) involved in the effect of troglitazone to induce the marked cellular acidosis is unclear. However, it is clear that the rapidity of the developing acidosis rules out the PPAR pathway acting via transcriptional regulation, pointing to PPAR
-independent pathways. In this regard, it has been shown that troglitazone exerts inhibitory effects on cholesterol biosynthesis through an apparent PPAR
-independent action (41). Our previous study (9) showed that both rosiglitazone and ciglitazone induced a cellular acidosis in PPAR
-deficient MDCK cells, although of a lesser magnitude than troglitazone in contrast to their relative potency in activating PPAR
(40) and their reported tubular distribution (39). We chose to consider PKC because a previous study showed that both troglitazone and pioglitazone negatively regulated this pathway in rat mesangial cells (25), and we observed that troglitazone induces a marked cellular acidosis in these cells (45). To see whether inhibiting PKC mimics the troglitazone effect on pHi and ammoniagenesis, we used chelerythrine as a potent and noncompetitive PKC inhibitor (22). Indeed, the observed cellular acidosis occurring with chelerythrine is consistent with PKC playing an important role in maintaining the steady-state pHi in LLC-PK1-F+ cells, whereas the increase in ammonium and decreased alanine production, respectively, are in line with the cellular acidosis and troglitazone's effect on acid-base metabolism. Note that a previous study demonstrated that PKC plays an important role in ammoniagenesis by LLCPK1 cells both in normal and acute metabolic acidosis and that this effect was mediated through NHE activity and apparently steady-state pHi (37, 38). In fact, phorbol ester reduces the acute acidosis (media pH = 6.8) increase in ammonium production back to the control (media pH = 7.4) level. In the present study, phorbol ester suppresses the troglitazone-enhanced ammonium production consistent with a return to normal pHi. Note, however, that phorbol ester activates ERK as well as PKC (5). How troglitazone might act to reduce the PKC/ERK activity is unclear, but the combination of troglitazone and chelerythrine effects suggests that they may act at different sites, e.g., PKC and ERK, or on different PKC isoforms to exert their additive effects. For example, if chelerythrine acts directly to inhibit PKC (23), troglitazone might act upstream, for example, through inhibition of DAG formation (25), which might also explain why phorbol ester reverses the troglitazone effect. Alternatively, more than one PKC isoform might be involved (27, 46) so that by inhibiting different isoforms, an additive effect is the result. Studies focusing on these and other possibilities (46) while monitoring phosphorylated substrates will be required to gain a further understanding of the signaling pathways through which troglitazone acts in modulating cellular acid-base metabolism and related cellular processes (43).
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institutes of Health Grants DK-53761 and CA-79495 (to I. Nissim); the Southern Arizona Foundation (to T. Welbourne); and the Louisiana Gene Therapy Research Consortium (to F. Turturro).
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FOOTNOTES |
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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.
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REFERENCES |
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