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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Troglitazone and pioglitazone interactions via PPAR--independent and -dependent pathways in regulating physiological responses in renal tubule-derived cell lines

¹Department of Medicine, Feist-Weiller Cancer Center, ²Gene Therapy Program, and ³Departments of Molecular and Cellular Physiology, Louisiana State University Health Science Center, Shreveport, Louisiana; and ⁴Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 21 July 2006 ; accepted in final form 13 October 2006

	ABSTRACT

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Troglitazone (Tro) and pioglitazone (Pio) activation of peroxisome proliferator-activated receptor (PPAR)- and PPAR--independent pathways was studied in cell lines derived from porcine renal tubules. PPAR--dependent activation of PPAR response element-driven luciferase gene expression was observed with Pio at 1 µM but not Tro at 1 µM. On the other hand, PPAR--independent P-ERK activation was observed with 5 µM Tro but not with Pio (5–20 µM). In addition, Pio (1–10 µM) increased metabolic acid production and activated AMP-activated protein kinase (AMPK) associated with decreased mitochondrial membrane potential, whereas Tro (1–20 µM) did not. These results are consistent with three pathways through which glitazones may act in effecting metabolic processes (ammoniagenesis and gluconeogenesis) as well as cellular growth: 1) PPAR--dependent and PPAR--independent pathways, 2) P-ERK activation, and 3) mitochondrial AMPK activation. The pathways influence cellular acidosis and glucose and glutamine metabolism in a manner favoring reduced plasma glucose in vivo. In addition, significant interactions can be demonstrated that enhance some physiological processes (ammoniagenesis) and suppress others (ligand-mediated PPAR- gene expression). Our findings provide a model both for understanding seemingly opposite biological effects and for enhancing therapeutic potency of these agents.

peroxisome proliferator-activated receptor-; phospho-extracellular signal-regulated kinase; intracellular pH; Na⁺/H⁺ exchanger; AMP-activated protein kinase; mitochondria

TZDs may signal acid production as a consequence of impaired mitochondrial ATP formation (12) and activated AMP-activated protein kinase (AMPK) (16). This in turn accelerates glucose flux via glycolysis (9, 12, 14), lowering extracellular glucose while cellular acidosis limits availability of the gluconeogenic precursors derived from glutamine (8, 29). The combined effect of enhanced glucose utilization and decreased availability of the gluconeogenic precursor alanine would lower blood glucose concentration as a consequence of increased glucose removal (muscle) and decreased production (liver), both processes by which TZDs are known to act in exerting antiglycemic effects (13).

TZDs are also potentially able to activate a third pathway, MAPK and specifically P-ERK1/2 (19, 25), in addition to PPAR- and the mitochondrial pathways. We have shown (19) that Tro activated ERK1/2 within 4 min and that ERK activation was related to the inability to extrude acid and the associated cellular acidosis. Cellular acidosis in turn has as a physiological response the accelerated deamination of glutamine's amino nitrogen (18). A fall in cytoplasm pH was proposed as a basic mechanism for driving glutamate uptake into the mitochondrial matrix space and the deamination reaction producing ammonium (3). Preventing P-ERK activation abrogated these responses and largely prevented the cytosol acidosis (19), restoring the ability to extrude an exogenous acid load and preventing ammoniagenesis from glutamine. Thus, under these conditions, ammonium formed from glutamine's amino nitrogen may serve as a surrogate for the cytosol-to-mitochondrial matrix H⁺ gradient driving glutamate into the matrix site of the deamination reaction.

TZDs may potentially activate all three pathways: PPAR--dependent gene expression/suppression, PPAR--independent ERK activation, and PPAR--independent loss of mitochondrial membrane potential () and acidogenic glycolysis. Nevertheless, little is known about the activation and relative contribution of each of these potential pathways to the physiological responses under investigation. For this reason, we looked for these pathways and their expression in two well-established renal tubule cell lines under the influence of Tro or Pio alone or Tro plus Pio in combination. The results shown here indicate that each TZD alone has unique and clearly discernable effects elicited by differentially activating the above pathways and that in combination their actual contribution to the physiological response depends on an understanding of their subsequent interactions.

	MATERIALS AND METHODS

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Two renal tubule cell lines, LLC-PK₁ obtained from American Type Culture Collection and the LLC-PK₁-F⁺ strain derived to grow in the absence of glucose (15), were grown to confluence in T150 flasks on DMEM plus 10% FCS containing (in mM) 28 sodium bicarbonate, 10 sodium pyruvate, 5 D-glucose, and 2 L-glutamine at 37°C and 5% CO₂ (pH 7.47). Confluent cells were detached with trypsin-EDTA (Sigma, St. Louis, MO) and reseeded onto 12- or 24-well culture trays for metabolic/transfection studies or onto custom-designed 30-mm glass chambers (Bioptechs, Biological Optical Technologies, Butler, PA) layered with DMEM and allowed to dry for cell pH, NHE activity, and mitochondrial measurements. Cells were allowed to gain near-complete confluence for transfection studies (2–3 days) or complete confluence for metabolic studies (3–4 days). Cells grown on chambers were studied within 1–2 days of seeding. Experiments were designed for a range of concentrations from 1 to 10 µM for Pio and from 5 to 50 µM for Tro; glitazones were obtained from Cayman Chemical (PPAR--PAK; Ann Arbor, MI).

Cell intracellular pH, P-ERK, and NHE activity measurements. Cytosol pH was assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as previously described (8, 19, 29, 30). The cells were loaded with 5 µM BCECF-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 10 min in normal Krebs-Henseleit (KH) medium (pH 7.4, 5 mM glucose), washed, and promptly studied for spontaneous intracellular pH (pH_i) monitored over 4 min, after which the cells were promptly lysed in the previously described (19) lysis buffer containing phosphatase inhibitor buffers 1 and 2 (Sigma). Aliquots of the lysates were analyzed for P-ERK1/2 and total ERK1/2 as previously described with specific antibodies (nos. 9106 and 9102, respectively, obtained from Cell Signaling Technology, Beverly, MA) as well as for tubulin as a control for protein loading. Phospho-AMPK- (Cell Signaling) was assayed with a specific antibody that recognizes phosphorylated threonine 172, with total AMPK- detected with an AMPK- antibody (no. 2532, Cell Signaling) on the reprobed blot. NHE activity was assayed as previously described (29) after an NH₄Cl acid pulse and a 1-min exposure to low Na⁺ medium; NHE activity was then assayed over 0–4 min in either normal or low-Na⁺ (10 mM) medium.

Mitochondrial and metabolic acid production. Cells grown to confluence in chambers were incubated in DMEM containing vehicle, Tro, or Pio for 2 h, after which the medium was harvested for metabolic acid production. The cells were then loaded with the potentimetric dye tetramethylrhodamine ethyl ester (TMRE; Molecular Probes), 50 nM in KH medium plus 1 µg/ml Hoechst 33342 for 20 min at 37°C. TMRE is a nonfixable cationic dye that is actively pumped into energized mitochondria (20). Deenergized mitochondria will transport less of the dye with a predominant extramitochondrial distribution and less mitochondrial fluorescence indicating a decreased . The metabolic acid produced over the 2-h period was determined from the decrease in medium bicarbonate concentration, which in turn reflects the production of lactate accumulating in the medium (19, 29). Cells were then viewed on an Olympus Bx60 scope with excitation and emission settings of 549 and 574 nm, respectively, and QuipsPathvision software.

Metabolic and [2-¹⁵N]glutamine studies. Cells grown to confluence in 12-well trays were studied for effects of Tro and Pio on glutamine metabolism and acid production over 3-h and 18-h time courses as previously described (29). We used [2-¹⁵N]glutamine to determine mitochondrial glutamate uptake and oxidation since both a pH gradient and a respiratory chain complex are integral to the release of ¹⁵NH₄⁺ from the amino nitrogen. Detection of ¹⁵NH₄⁺ and calculation of glutamate conversion were as previously described (29). Metabolic acid production and relation to glucose uptake and lactate production were also described previously (19, 29).

PPAR-, luciferase, and ALT assays. Activation of PPAR- was assessed by measuring firefly luciferase activity in cells transfected with the previously described reporter plasmid (6) driven by three copies of the PPRE from the aP2 gene. Transfections were performed as described previously except that Lipofectamine Plus (Life Technologies, Gaithersburg, MD) was used as the vehicle in place of Lipofectamine. The cells were washed with PBS, harvested in passive lysis buffer (Promega, Madison, WI), and assayed for luciferase activity with the dual luciferase assay (Promega) and ALT (Point Scientific, Canton, MI), and the results were expressed as firefly luciferase activity normalized to sea pansy luciferase activity or as activity per milligram of cell protein.

Statistical analysis. Differences between control and Tro and Pio versus their combination were analyzed with ANOVA and a corrected t-test (Dunnett), with differences considered significant at P < 0.05.

	RESULTS

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Pio potentiates Tro-induced cellular acidosis: interaction between Tro inhibition of acid extrusion and Pio activation of acidogenic glycolysis. Figures 1 and 2 show typical acute responses to increasing concentrations of Tro and Pio in LLC-PK1-F⁺ cells. At the lowest concentration (5 µM) Tro (Fig. 1A) induces an acute drop in pH_i and elevates P-ERK; in contrast, Pio (Fig. 2A) neither increases P-ERK nor lowers pH_i acutely. At the higher concentrations, 10 and 20 µM, Tro further enhances P-ERK and the associated cellular acidosis (Fig. 1A), whereas Pio does not induce acidosis at any concentration (Fig. 2A) and tends to lower P-ERK (Fig. 2B). Despite not exhibiting an acidifying effect on its own, Pio together with Tro potentiates the cellular acidosis induced by Tro, as can be seen in Fig. 3A. At 10 µM Pio, the acidification induced by 10 µM Tro is enhanced, resulting from a greater rate of decline in pH_i as well as a lower pH_i at the end of the 4-min period; P-ERK levels are not further elevated above the rise with Tro alone. Results for pH_i and P-ERK from four additional LLC-PK₁-F⁺ studies are shown in Fig. 3, B and C. Cytosol pH averaged 7.23 ± 0.02 in the control cells and fell after 4 min to an acidic 6.71 ± 0.08 with 10 µM Tro; Pio at 10 µM tended to slightly lower pH, while combined with Tro (10 µM) it dropped the pH to a markedly acidic 6.58 ± 0.09. P-ERK levels rose threefold with Tro (10 µM) but not with Pio (10 µM), while in combination P-ERK did not further increase above that for Tro alone.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Troglitazone (Tro) decreases intracellular pH (pHi) in association with P-ERK activation. A: typical response of pHi and P-ERK to increasing Tro concentration after 4 min of exposure in 4 separate chambers. pHi was determined by BCECF assay and P-ERK by site-specific antibodies as described previously (19). T-ERK, total ERK. B: Tro elevates P-ERK in a dose-dependent fashion; results are from 4 experiments performed as described in A. CTL, control.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Pioglitazone (Pio) does not decrease pHi or activate P-ERK. A: typical 4-min response of pHi and P-ERK to increasing Pio concentrations in 4 separate chambers. B: Pio fails to increase P-ERK after 4 min; results from 4 experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Pio enhances the cellular acidosis induced by Tro. A: typical responses to 10 µM Pio or Tro alone and in combination; 1007 is the number of this experiment. B: P-ERK activation from 4 experiments identical to those in A. C: pHi measured after exposure to control medium, Pio or Tro alone, or in Pio and Tro in combination; results are from 4 experiments.

To determine whether the combined effect to further enhance the cellular acidosis induced by Tro alone reflects Pio inhibition of the acid extrusion, the rate of acid extrusion in response to an NH₄Cl acid load was determined with Pio, Tro, and Pio plus Tro. As shown in Fig. 4A, Pio at 5 or 20 µM had little effect on acid extrusion; in contrast, Tro at 20 µM markedly blunted acid extrusion (Fig. 4A) and Tro at 10 µM in combination with 10 µM Pio prevented acid extrusion and converted the response to acid loading (Fig. 4B, declining recovery pH_i). The results from three sets of acid load experiments are presented in Fig. 4C, and they show that Pio at 10 µM reduces (P < 0.05) acid extrusion in 10 mM Na⁺ medium by 65% but has no effect on acid extrusion in 140 mM Na⁺ medium. Tro, on the other hand, reduces acid extrusion by >70% in both 140 mM Na⁺ and low-Na⁺ media and in combination with Pio at 140 mM Na⁺ results in acid loading.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Pio has no effect on acid extrusion in normal Krebs-Henseleit (KH) medium after exogenous acid load (20 mM NH4Cl). A: typical recovery responses (0–4 min) to standard acid pulse in the presence of increasing concentrations of Pio vs. 20 µM Tro. B: typical recovery responses in normal KH medium to standard acid pulse with 10 µM Tro alone or in combination with 10 µM Pio. C: acid pulse recovery results from 4 separate experiments measured over 0- to 4-min recovery period in either normal KH medium or low-Na+ (10 mM) KH medium. NHE, Na+/H+ exchanger. *P < 0.05 vs. CTL; **P < 0.01 vs. CTL.

Pio potentiates Tro effect on glutamine metabolism: enhancement of pH_i-dependent glutamate deamination. Tro enhances glutamate flux through the ammoniagenic glutamate dehydrogenase pathway while simultaneously inhibiting glutamate flux through the ALT pathway (29). The actions of Tro (20 µM) to increase ammonium and decrease alanine production are clearly evident after 3 h (Fig. 5A; simultaneously ammonium production increased and alanine production decreased so that the ratio NH₄⁺/Ala increased) and more so after 18 h (Fig. 5B; the ammonium to alanine production rising from 0.36 ± 0.9 to 1.2 ± 0.1 for control and Tro at 3 h and from 0.40 ± 0.08 to 1.65 ± 0.25 at 18 h; P < 0.05); like Tro, 10 µM Pio alone increased ammonium production, but unlike Tro, Pio had no effect on alanine production at either time point. In combination with Tro, Pio enhances (P < 0.05) the Tro effect on ammonium production (4,457 ± 540 to 8,041 ± 915 nmol/mg) without further reducing alanine production.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Tro increases ammonium and decreases alanine production in contrast to Pio, which increases only ammonium production. A: ammonium and alanine (Ala) production responses after 3-h exposure to 20 µM Tro or 10 µM Pio alone and in combination. Results are means ± SE from 4 experiments. B: ammonium and alanine production after 18-h exposure to same concentration of glitazones as in A. *P < 0.05 vs. CTL; **P < 0.01 vs. CTL.

Increased ammoniagenesis from the amino nitrogen of glutamine is the result of oxidative deamination mediated by glutamate dehydrogenase present within the mitochondrial matrix space and accelerated by cytosol acidosis. As shown in Fig. 6, Tro increases ammonium produced from labeled amino nitrogen by 58% (1,358 ± 250 vs. 862 ± 23 nmol/mg; P < 0.05), and for Tro in combination with 10 µM Pio, the ammonium produced from amino-labeled glutamate increased by 111% (1,815 ± 214 vs. 862 ± 23 nmol/mg; P < 0.01). Surprisingly, in view of the failure of Pio to acutely induce a cellular acidosis, Pio alone also increased (P < 0.05) the ammonium produced from the labeled amino nitrogen of glutamine (1,383 ± 201 nmol/mg; Fig. 6). We therefore determined the spontaneous pH_i in cells incubated for 18 h with Pio and compared this pH_i with control and Tro-treated cell pH_i. In line with the increased ammonium formed from the amino nitrogen of glutamine, chronically Pio-treated cells showed a reduction in pH_i (6.68 ± 0.09 vs. 7.02 ± 0.02 for control; P < 0.01) that was not different from Tro alone or Tro plus Pio (6.66 ± 0.08 and 6.69 ± 0.09, respectively; n = 7).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Ammonium production from the amino nitrogen of glutamine by cells incubated in [2-15N]glutamine (N Glu) for 18 h. Results are means ± SE from 3 experiments. Tro and Pio were used at the same concentration as in Fig. 5. *P < 0.05 vs. CTL; **P < 0.01 vs. CTL.

Pio, but not Tro, increases metabolic acid production associated with a fall in mitochondrial .

View larger version (34K): [in this window] [in a new window]   Fig. 7. Pio reduces mitochondrial membrane potential. A: mitochondrial membrane potential (m) measured with tetramethylrhodamine ethyl ester (TMRE; see MATERIALS AND METHODS) after exposure to Tro (20 µM) or Pio (10 µM) for 3 h. CCCP was used as a positive control for depolarized mitochondria. Top: distribution of nuclei in the same field. B: P-AMPK to total (T)-AMPK ratio from typical experiment with metabolic acid production from 6 experiments with cells exposed to 10 µM Pio, 20 µM Tro, or in Pio and Tro in combination. C: P-AMPK and T-AMPK levels in F+ cells measured after 18 h with acid produced over that time: glitazone concentrations are the same as in A and B. D: P-AMPK and T-AMPK in HEK cells measured after 3 h with metabolic acid and ammonium produced over the 3 h; glitazone concentrations the same as in A and B.

In the LLC-PK₁ cell line Pio had the same effect on metabolic acid production as it did in the F⁺ cell line, while Tro had no effect either alone or in combination with Pio. To confirm in another cell line the signal link between mitochondrial depolarization and acidogenesis, P-AMPK was measured with human embryonic kidney-derived HEK cells according to the same protocol as above. Figure 7D shows that after 3-h exposure to Pio (10 µM) there was an elevation in P-AMPK- and metabolic acid production but not in ammoniagenesis; in contrast, Tro did not increase either P-AMPK- or metabolic acid production but, as expected, did increase ammonium production (P-ERK dependent).

Pio activates PPRE-driven gene expression, which is abrogated by Tro: evidence for separate PPAR- pathways. To assess the role of PPAR- activation, PPRE-driven luciferase gene expression was monitored over the concentration range of 1–25 µM for both Pio and Tro. As shown in Fig. 8A, Pio at 1 µM activated luciferase expression 85% (P < 0.05), in contrast to Tro, which did not stimulate gene expression at 1 µM and submaximally (compared with Pio) stimulated luciferase at 10–25 µM. In combination (Fig. 8B), Tro (25 µM) reduced the Pio (10 µM)-elevated gene expression to the level observed with Tro alone. Interestingly, Pio (10 µM) increased assayable ALT activity in these same lysates (219 ± 10 vs. 287 ± 27 U/mg protein; P < 0.05), in contrast to Tro, which reduced assayable ALT activity by 57% (94 ± 26 vs. 219 ± 10 U/mg protein; P < 0.01). In combination (10 µM Pio + 25 µM Tro), ALT activity was expressed at the same level as with Tro alone (83 ± 21 U/mg protein). These results are consistent with Pio and Tro acting via two distinct pathways on the expression of luciferase and ALT activities and the Tro-activated pathway dominating. Similar findings occurred in the LLC-PK₁ cell line, indicating a functional PPAR- signaling pathway and sustained Tro activation of P-ERK after 18 h in both cell lines.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Pio and Tro influence luciferase and alanine aminotransferase (ALT) activities by different pathways. A: peroxisome proliferator-activated receptor (PPAR) response element (PPRE)-driven luciferase activity in cells exposed to either Pio or Tro alone over the same concentration range studied for m and P-ERK activation. B: Tro's (20 µM) submaximal stimulation of luciferase is expressed in the presence of Pio at a maximal stimulatory concentration (10 µM); results are means ± SE from 4 separate experiments. C: Tro's inhibition of ALT activity prevents Pio from increasing ALT activity; dominance of Tro pathway for the alanine producing ALT activity. Results are means ± SE from 6 experiments. *P < 0.05 vs. CTL; **P < 0.01 vs. CTL.

	DISCUSSION

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View larger version (16K): [in this window] [in a new window]   Fig. 9. Summary of results as 3 individual pathways responsive to Pio (I and III) or Tro (II) alone. Dotted arrows indicate connections through multiple steps not listed or resolved. In combination, interaction between pathways is depicted by activating/blocking arrows (see text for details). Pathway I solid bar represents PPAR- as a potential phosphoprotein phosphorylated by pathway II P-ERK (partial agonist) and/or acting to inhibit the ligand-activated pathway I; double helix represents PPRE-containing DNA. Pathway II shows ? at cell surface representing putative receptor and NHE. Pathway III shows putative mitochondrial receptor as ? and connection to glycolysis by broken arrow via phosphorylated AMP-activated protein kinase (P-AMPK) Glc represents glucose, Lac represents lactate, and H+ represents metabolic acid produced and either extruded (a) by NHE or retained in the cytosol (b) with pathway II activated.

Glutamine, glucose, and pyruvate are the major energy substrates available to these cells in vitro, with the F⁺ cell line adapted (15) to grow in the absence of glucose, that is, largely dependent on glutamine metabolism as occurs in the renal proximal tubule in vivo (21). This adaptive shift in fuels underlying, in part, the difference between the LLC-PK₁ and F⁺ cell lines may explain the greater physiological response to the cellular acidosis induced by Tro. Although metabolic acidosis readily activates P-ERK in the F⁺ cells (19) as it does in the kidney cortex in vivo (26), it has not been determined whether acidosis activates P-ERK in the pH-insensitive LLC-PK₁ cell line. In the pH-sensitive F⁺ strain Tro enhances ammoniagenesis more than metabolic acidosis, reflecting the failure to sustain an intracellular acidosis and P-ERK activation with an extracellular acidosis (19), whereas Pio chronically enhances ammoniagenesis associated with a developing intracellular acidosis. It would be of some interest to determine whether P-ERK activation is expressed by Pio after 18 h as well and, if so, by what mechanism.

It is noteworthy that Tro did not increase glycolysis, as suggested by the failure of acid production to increase, although it certainly does in other renal cell lines, e.g., mesangial cells (30) and Madin-Darby canine kidney cells (8). In contrast, Pio exerts a striking enhancement of acid production as a reflection of glucose utilization coupled to glycolysis (9, 12). Mechanistically, Pio's ability to increase metabolic acid production may reflect glitazone's inhibitory effect on the mitochondrial respiratory chain (23) or more specifically pyruvate dehydrogenase activity (9, 12), since downstream glutamate oxidation was not impaired. Elimination of pyruvate's oxidative contribution to mitochondrial membrane polarization and hence ATP formation would be associated with activation of AMPK (Fig. 7, B and C) and glycolysis resulting in the enhanced acid production (Fig. 7, B and C) as mitochondrial falls (Fig. 7A). Tro did not increase acid production, activate AMPK, or prevent mitochondrial dye uptake, all indications that pathway III is not activated by Tro over these concentrations in the three renal-derived cell lines.

Glitazones are antihyperglycemic agents that both increase glucose uptake (insulin sensitizers) and reduce glucose production (13). The present study provides a model for both of these actions supported by the present as well as previous findings in renal cells (8, 19, 29). Accordingly, Pio enhances glucose uptake, reflecting a fall in the cellular energy charge leading to compensatory glycolysis; this may contribute to the antihyperglycemic activity as suggested previously (4, 12) and as observed in skeletal muscle (14). Tro, on the other hand, may decrease glucose production by shifting glutamine metabolism to releasing ammonium rather than alanine (gluconeogenic substrate) at sites upstream to the liver, effectively limiting substrate availability for hepatic glucose production (29). It is noteworthy that Tro acts in vivo to both enhance peripheral glucose uptake and decrease hepatic glucose production in lowering blood glucose levels (13).

The receptor(s) apparently present in the plasma membrane responsible for Tro's activation of P-ERK (pathway II) is not clear. Our previous study (29) showed that the PPAR- receptor antagonist bisphenol A diglycide ether (BADGE) abrogated the Tro-induced fall in cellular pH and Tro-induced increased ammoniagenesis. It should be noted that a previous study had shown a PPAR--independent pathway for BADGE (11). Whether BADGE also prevents P-ERK activation in our cell lines requires further study, but, if it does, the possibility of a PPAR-like receptor present in the plasma membrane would gain further support (25). Another well-established PPAR- inhibitor, GW9662, reportedly (24) acts via a PPAR--independent pathway, since by itself it has antiproliferative effects, an observation consistent with our previous study (27) of pathway II exerting an antiproliferative effect through a cellular acidosis. In addition, the present model provides a perspective for explaining how activators of P-ERK, e.g., metabolic acidosis, growth factors, might contribute to the well-known physiological response of enhanced ammoniagenesis and kidney growth (17).

Others (4) have suggested that a PPAR-like mitochondrial receptor mediates the mitochondrial effects of TZDs. It is noteworthy that we previously reported (28) that a nonthiazolidinedione dual PPAR-/PPAR- agonist, KT6–207, both inhibits acid extrusion and enhances acid production in limiting growth of tumorigenic cell lines, in other words, acts similar to the Tro plus Pio combination shown here. If this is so, then dual agonists that are not TZDs and that activate all three of these pathways would provide evidence in support of receptor-mediated signaling initiating all three pathways shown in Fig. 9. Clearly, further studies are warranted to determine whether these interactive pathways controlling critical metabolic events underlying physiological processes are indeed PPAR-like receptors positioned in nontraditional sites.

	GRANTS

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This work was supported by the Southern Arizona Foundation (to T. Welbourne) and the Feist-Weiller Research Fund and Louisiana Gene Therapy Research Consortium, Inc. (to F. Turturro)

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Welbourne, Dept. of Molecular and Cellular Physiology, LSUHSC, Shreveport, LA 71130 (e-mail: twelbo{at}lsuhsc.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.

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