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【摘要】 Troglitazone was studied in pH-sensitive LLC-PK 1 -F + cells to determine the effect on pH i and glutamine metabolism as well as the role of peroxisome proliferator-activated receptor (PPAR )-dependent and PPAR -independent signaling pathways. Troglitazone induces a dose-dependent cellular acidosis that occurs within 4 min and persists over 18 h as a result of inhibiting Na + /H + exchanger-mediated acid extrusion. Cellular acidosis was associated with glutamine-dependent augmented [ 15 N]ammonium production and decreased [ 15 N]alanine formation from 15 N-labeled glutamine. The shift in glutamine metabolism from alanine to ammoniagenesis appears within 3 h and is associated after 18 h with both a reduction in assayable alanine aminotransferase (ALT) activity as well as cellular acidosis. The relative contribution of troglitazone-induced cellular acidosis vs. the decrease in assayable ALT activity to alanine production could be demonstrated. The PPAR antagonist bisphenol A diglycide ether (BADGE) reversed both the troglitazone-induced cellular acidosis and ammoniagenesis but enhanced the troglitazone reduction of assayable ALT activity; BADGE also blocked troglitazone induction of peroxisome proliferator response element-driven firefly luciferase activity. The protein kinase C (PKC) inhibitor chelerythrine mimics troglitazone effects, whereas phorbol ester reverses the effects on ammoniagenesis consistent with troglitazone negatively regulating the DAG/PKC/ERK pathway. Although functional PPAR signaling occurs in this cell line, the major troglitazone-induced acid-base responses appear to be mediated by pathway(s) involving PKC/ERK.
【关键词】 intracellular pH Na + /H + exchanger Nlabeled glutamine alanine aminotransferase activity protein kinase C/extracellular regulated kinase
ACID - BASE METABOLISM in proximal tubule cells includes regulating intracellular pH (pH i ) and glutamine-dependent ammoniagenesis and gluconeogenesis. The creation of the pH-sensitive LLC-PK 1 -F + cell line ( 18, 19 ) from the continuous LLCPK 1 renal proximal tubule-like cell line provides an in vitro model for studying these responses and the underlying signaling pathways. In vivo renal responses to metabolic acidosis and a sustained fall in pH i ( 1 ) include the upregulation of glutamine transport and transporters ( 28 ), phosphate-dependent glutaminase ( 11 ), and phospho enol pyruvate carboxykinase (PEPCK) ( 10 ). On the other hand, model proximal tubule-like cells and osteoclasts respond to chronic metabolic acidosis with enhanced acid extrusion ( 3, 24, 31, 34 ) and restoration of pH i ( 3, 24, 34 ), thereby limiting the acidosis effects normally observed in vivo. Nevertheless, acute exposure [and before adaptive acid extrusion restores pH i ( 24 )] to an acidic media results in metabolic responses that mimic those observed in vivo ( 32 ). These studies performed in the parental LLC-PK 1 line have shown that within 1 h ammoniagenesis from [2- 15 N]glutamine ( *; Fig. 1 ) increased twofold at a pH of 7.0 while that formed from the [5- 15 N]glutamine did not increase ( 32 ). On the other hand, an earlier study ( 30 ) in the LLC-PK 1 -F + cell line showed that ammonium released from glutamine metabolism is far less than the alanine released suggesting that glutamine's amide nitrogen may be incorporated into alanine. The results from the present study will show that indeed LLC-PK 1 -F + cells incorporate 15N-labeled ammonium formed from glutamine's amide nitrogen into alanine via reductive animation to form glutamate ( Fig. 1, II ) followed by transamination to form alanine. Thus the ability to incorporate both the amide and amino nitrogen of glutamine into alanine at a significant net flux accounts for the ammonium to alanine production ratios previously observed ( 30 ) being far less than unity.
Fig. 1. Potential pathways previously described ( 31 ) by which troglitazone might regulate glutamine metabolism as studied by 15 N-labeled glutamine in pH-sensitive LLC-PK 1 -F + cells. Note that glutamine's amide nitrogen in the form of ammonium can be converted to glutamate by reductive amination and then to alanine by transamination.
In the LLC-PK 1 -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 pH i despite the progressively acidic extracellular milieu. The ability of an acute fall in pH i 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 pH i 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 LLCPK 1 -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-PK 1 -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.
MATERIALS AND METHODS
LLC-PK 1 -F + cells were grown to confluency in T150 flasks on DMEM media plus 10% FCS containing (in mM) 28 sodium bicarbonate, 10 sodium pyruvate, 5 D -glucose, and 2 L -glutamine at 37°C and 5% CO 2 (pH 7.4). Confluent cells were subcultured by detaching using trypsin-EDTA (GIBCO BRL, Rockville, MD) and reseeded onto 6- to 12-well culture plates (Corning Cell Wells, Corning, NY) for metabolic/transfection studies or onto custom designed 30-mm chambers (Bioptechs, Biological Optical Technologies, Butler, PA) equipped with a heating element and cap port for O 2 :CO 2 aeration. The chambers were placed uncapped inside a 60-mm covered tissue culture dish and incubated at 37°C and 5% CO 2. The cells were allowed to gain confluency, usually 3-4 days for 6- to 12-well plates and 1-2 days for cells in chambers.
Cell pH i 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 pH i 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 pH i 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 pH i recovery after an NH 4 Cl 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 NH 4 Cl (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 NH 4 Cl 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 15 N-labeled glutamine. For analysis of alanine and ammonium formed from [2- 15 N]glutamine and [5- 15 N]glutamine, media glutamine was replaced with either [2- 15 N]- or [5- 15 N]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 15 N 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 15 N 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 [ 15 N]ammonia was determined following conversion of ammonium to norvaline ( 6 ). To calculate the conversion of 15 N-labeled glutamine to ammonium and alanine, the isotopic enrichment, atom percent excess (a%ex) of 15 N 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 LLCPK 1 -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-PK 1 -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-PK 1 -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.
RESULTS
To determine whether troglitazone induces an acute cellular acidosis and whether this acidosis would be sustained over a chronic period, cells were loaded with BCECF and studied during acute 4-min exposure and after longer-term 18-h exposure as described in MATERIALS AND METHODS. In the time controls, [ Fig. 2, Acute study, control (CTL)] pH i for cells incubated in the KHH over the 4-min period remained unchanged (7.20 ± 0.03 and 7.22 ± 0.04); adding 25 µM troglitazone (TRO in figure) to the KHH media results in cellular acidosis that develops within 4 min, 6.92 ± 0.04 vs. 7.22 ± 0.04 for time controls, P < 0.01. These results show for the first time that troglitazone induces a marked cellular acidosis in pH-sensitive LLC-PK 1 -F + proximal tubule-like cells as previously shown for the distal tubule-like MDCK cell line ( 9 ) and glomerular mesangial ( 45 ) cells under conditions in which pH i is determined by NHE activity. In the chronic 18-h study, troglitazone reduced ( P < 0.01) pH i from 7.16 ± 0.05 to 6.91 ± 0.06 in cells incubated in DMEM, indicating that these cells are unable to restore the normal pH i. One possible mechanism for troglitazone to reduce pH i would be if metabolic acid production increased. Because acid production usually represents lactate production coupled to ATPase activity in these cells ( 31 ), lactate formation was measured and found to be unchanged by troglitazone (5,877 ± 314 and 6,032 ± 236 nmol/mg for control and troglitazone treated, respectively), suggesting that increased lactic acid production was not responsible for the cellular acidosis. To confirm that chronic exposure to troglitazone results in inhibition of NHE, acid extrusion following an acid load was measured at this time ( Fig. 3 ). Representative responses to an NH 4 Cl load are shown in Fig. 3, A and B, for 18-h control- and troglitazone-treated monolayers, respectively. Control monolayers ( Fig. 3 A ) respond to the acid load with a rapid initial rate of acid extrusion followed by a slower rate obtaining a pH i of 7.57 after 8 min. In contrast, monolayers exposed to troglitazone for 18 h ( Fig. 3 B ) and then acidified with NH 4 Cl show a much-reduced acid extrusion response in obtaining a pH i of only 6.56 after 8 min. Results from six additional experiments confirm that chronic troglitazone (25 µM) treatment inhibits the initial rapid rise in NHE activity (0.110 ± 0.020 to 0.026 ± 0.012, pH/ t, P < 0.01). The delayed slow rise in pH i was also inhibited (0.070 ± 0.014 to 0.021 ± 0.009, P < 0.01) with the steady-state pH i achieved after 8 min of recovery lower than observed for control (7.20 ± 0.14 to 6.24 ± 0.07, P < 0.01). Acutely adding 25 µM troglitazone ( Fig. 3 A ) following a second NH 4 Cl pulse reduced NHE activity to a rate not different from the chronic exposure (0.029 ± 0.009 vs. 0.038 ± 0.006, pH/ t for chronic vs. acute troglitazone). Note that removing troglitazone from the recovery media in the chronically exposed cells failed to activate the rapid rise in NHE activity observed in the control cells given a similar "trough" pH following the NH 4 Cl pulse ( Fig. 3 A ).
Fig. 2. Troglitazone induces cellular acidosis within 4 min (acute) and maintains cells acidotic for 18 h (chronic). CTL, TRO, and B are control, troglitazone (25 µM), and bisphenol A diglycide ether (BADGE; 100 µM), respectively. Results are means ± SE from 5 chambers per group; * different from control, P < 0.05.
Fig. 3. A : representative recovery response to NH 4 Cl acid load in controls. Monolayers incubated for 18 h in DMEM were (2,7)-biscarboxyethyl-5 (6)carboxyfluorescein (BCECF) loaded in Krebs-Henseleit HEPES (KHH) 20 mM NH 4 Cl,. Recovery response initiated by adding of KHH. A second NH 4 Cl acid load was followed by KHH plus 25 µM troglitazone. B : representative recovery response to NH 4 Cl acid load in chronic troglitazone-treated cells. Monolayers incubated for 18 h in DMEM plus 25 µM troglitazone were BCECF loaded in KHH plus 20 mM NH 4 Cl and then exposed to KHH plus 25 µM troglitazone for 12 min followed by KHH minus troglitazone for another 12 min.
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 (pH i 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 pH i (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-PK 1 -F + cells as depicted in Fig. 1, experiments were carried out over 18 h using [ 15 N]glutamine in either the [5- 15 N] or [2- 15 N] position. The isotopic enrichment (a%ex) as well as the ammonium produced from [ 15 N]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 [ 15 N]amide and 1,510 ± 113 to 2,530 ± 257 nmol/mg, P < 0.01, for [ 15 N]amino glutamine study). These results are consistent with an increased net flux through the glutamate dehydrogenase pathway as depicted in Fig. 1.
Table 1. Effect of troglitazone on 15 N enrichment in ammonia and alanine and production of 15 N-labeled ammonia and alanine following incubation with 15 N-labeled glutamine
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- 15 N] or [2- 15 N]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 [ 15 N]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, [ 15 N]alanine from [5- 15 N]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 [ 15 N]ammonium into -Kg to form [ 15 N]glutamate via the GDH reaction (reductive amination), and II ) transamination of [ 15 N]glutamate to form [ 15 N]alanine. With [2- 15 N]glutamine, following glutaminase activity, [ 15 N]glutamate and [ 14 N]ammonium ( I ) was formed and then transamination ( III ) of [ 15 N]glutamate to form [ 15 N]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).
Fig. 4. Time course showing that troglitazone (25 µM) increases ammonium and decreases alanine production within 3 h and then maintains these fluxes over the 18-h time course; in contrast, control monolayers show a progressive decline in ammonium and rise in alanine production over the 18-h time course. Results are means ± SE from 3 wells per time point.
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 pH i 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 pH i 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 pH i 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 pH i.
Fig. 5. Ammonium and alanine production from glutamine over the troglitazone concentration range 5-100 µM. Numbers in parentheses are pH i measured after 18 h as described in MATERIALS AND METHODS. Results are means ± SE from 4- to 6-well trays for ammonium/alanine and 3 chambers per pH i measure. * Significant difference from control, P < 0.05 by ANOVA and corrected Student's t -test.
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. 6 A ) and ALT activity ( Fig. 6 B ) 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. 6 C, MDCK cells previously shown not to decrease assayable ALT in response to troglitazone ( 10 ) do not express PPAR ( A ). In sharp contrast, LLC-PK 1 -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-PK 1 -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 pH i 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.
Fig. 6. A : assayable firefly luciferase activity driven by PPRE-promoter and normalized to constitutively expressed sea pansy luciferase as a function of troglitazone concentration. Results are means ± SE ( n = 3). B : assayable ALT activity (U/mg protein) as a function of troglitazone concentration from same cells as assayed for luciferase in A. Results are means ± SE ( n = 3). C : absence of peroxisome proliferator-activated receptor- antibody staining in Madin-Darby canine kidney (MDCK; A ) cells, which do not respond to troglitazone with reduced assayable ALT activity ( 9 ), and the presence of staining in LLC-PK 1 -F + cells ( C ), which do respond to troglitazone; visualization of their nuclei are presented in B and D for MDCK and LLC-PK 1 -F +, respectively.
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 pH i (7.26 ± 0.06 vs. 7.16 ± 0.04, P < 0.05).
Fig. 7. BADGE prevents troglitazone-induced ammoniagenesis and partially restores alanine production. Monolayers in 6-well trays were incubated for 18 h with vehicle (CTL, DMSO), troglitazone (25 µM), and 100 µM BADGE. Results are means ± SE from 6 trays. * Different from control or ** different from troglitazone ( P < 0.05).
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-PK 1 -F + cell line, the acute and dominant affects of troglitazone and BADGE on pH i and glutamine metabolism are more consistent with a PPAR -independent pathway rather than through conventional PPAR signaling.
Fig. 8. BADGE blocks troglitazone's transactivation of PPRE-driven firefly luciferase. Results are from 2 sets of 4 experiments using 25 µM troglitazone alone or troglitazone (25 µM) plus either 100 µM BADGE or BADGE alone; * different from control, P < 0.05.
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 pH i. Because NHE activity is activated by PKC-dependent phosphorylation ( 42 ), we assessed whether chelerythrine has an affect on pH i by adding 10 µM chelerythrine to the media alone or in the presence of troglitazone (25 µM) for 18 h after which the pH i was determined. Chelerythrine alone decreased pH i ( P < 0.01) to 6.98 ± 0.02 vs. 7.51 ± 0.03 for control; in combination with troglitazone, chelerythrine further reduced pH i ( 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 pH i. 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 pH i to alanine production ( Fig. 10 ).
Fig. 9. Chelerythrine (Chel) mimicks the action of troglitazone on ammonium and alanine production. Results are means ± SE from 5- to 6-well trays incubated for 18 h in vehicle, control, troglitazone (25 µM), Chel (10 µM), and troglitazone (25 µM) plus Chel (10 µM). * Different from control; ** different from troglitazone, P < 0.05.
Fig. 10. Phorbol ester (500 nM TPA) reverses the effect of troglitazone on ammoniagenesis but does not completely restore alanine production. Results are means ± SE from 5 experiments. * Difference from control, P < 0.05.
DISCUSSION
The LLC-PK 1 -F + cell line was selected ( 19 ) to grow on glutamine in place of glucose, generating a proximal tubulelike cell model designed for studying acid-base metabolism. In addition to expressing a high level of the ammoniagenic enzymes phosphate-dependent glutaminase and glutamate dehydrogenase ( 18, 19 ), these cells also express both NHE1 and NHE3 ( 10 ) acid extrusion systems similar to the in situ proximal tubule. In addition, these cells have been shown to be pH responsive in responding to cellular acidosis induced by either lowering the media pH ( 20, 31 ) or potassium ( 20 ) with an increased ammoniagenesis and upregulated expressions of both PDG ( 20 ) and PEPCK ( 20 ) activities as well as enhanced apical surface acid extrusion ( 31 ). We now show that these cells also express a functional PPAR signaling pathway. Given their high fidelity with the physiological responses to an acid challenge, we asked whether troglitazone would induce a cellular acidosis in this cell line and, if so, whether there would be a significant glutamine-dependent ammoniagenic response. Furthermore, we wished to obtain some insight into the potential signaling pathway(s) affected by troglitazone and specifi-cally the relative contribution of PPAR as opposed to PPAR -independent pathways.
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. 3 B ) and similar to that observed with acute troglitazone treatment in this ( Fig. 3 A ) and in our previous studies ( 9, 45 ) in cell lines expressing only NHE1. Because LLC-PK 1 -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. 3 B ) should have resulted in NHE activation and a prompt return to the control pH i. 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-PK 1 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 pH i ( Fig. 5 ) are consistent with pH i ( 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 pH i ( 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 pH i. At the lowest troglitazone dose (5 µM), there is a large reduction in assayable ALT (42%; Fig. 6 A ) with only a small reduction in alanine production (11%, P < 0.05); at 25 µM troglitazone, but with the pH i 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-PK 1 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 15 N. 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, pH i appears to be the more important factor in the LLC-PK 1 -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 pH i and glutamine metabolism.
Previous studies ( 21, 31, 37, 38 ) have not shown that acidosis in LLC-PK 1 cells reduces alanine production and, in fact, even increased the alanine production along with ammonium. The 15 N 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-PK 1 -F + cell line, then a reduction in cytosolic pH would reduce overall alanine production; conversely, if the mitochondrial ALT was dominant in the LLC-PK 1 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-PK 1 -F + exposed to an extracellular acidosis. In our previous study in LLC-PK 1 -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-PK 1 ( 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 pH i and chronically respond by returning their pH i to normal, both processes reflecting upregulation of the NHE activity. In fact, recent studies ( 22 ) in the parental LLC-PK 1 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 pH i 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 pH i 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 pH i 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 pH i in LLC-PK 1 -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 LLCPK 1 cells both in normal and acute metabolic acidosis and that this effect was mediated through NHE activity and apparently steady-state pH i ( 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 pH i. 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 ).
ACKNOWLEDGMENTS
We thank R. Oliver III for outstanding technical assistance.
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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作者单位：Departments of 1 Molecular and Cellular Physiology and 2 Medicine and Feist-Weiller Cancer Center, Louisiana State University Health Science Center, Shreveport, Louisiana 71130; and 3 Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Med