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Am J Physiol Cell Physiol 292: C1874-C1879, 2007. First published January 17, 2007; doi:10.1152/ajpcell.00617.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Inhibition of PI3-kinase signaling by glucocorticoids results in increased branched-chain amino acid degradation in renal epithelial cells

Xiaonan Wang, Junping Hu, and S. Russ Price

Renal Division, Emory University, Atlanta, Georgia

Submitted 12 December 2006 ; accepted in final form 12 January 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phosphatidylinositol 3-kinase(PI3K) is a pivotal enzyme involved in the control of a variety of diverse metabolic functions. Glucocorticoids have been shown to attenuate PI3K signaling in some nonrenal cell types, raising the possibility that some physiological effects of glucocorticoids in renal cells may be achieved by a similar mechanism. Therefore, we tested whether glucocorticoids affect signaling through the insulin receptor substrate (IRS)-1/PI3K/Akt signaling cascade in LLC-PK1-GR101 renal epithelial cells. Treatment of cells with dexamethasone for 24 h: 1) suppressed IRS-1-associated PI3K activity and Akt phosphorylation, 2) increased the level of the PI3K p85 regulatory subunit but not the p110 catalytic subunit, and 3) induced the phosphorylation of IRS-1 on inhibitory Ser307. We have previously reported that glucocorticoids increase branched-chain ketoacid dehydrogenase (BCKD) activity in LLC-PK1-GR101 cells. This response was achieved, in part, by alterations in the transcription of BCKD subunits and BCKD kinase, which inactivates the enzyme complex by phosphorylation. Therefore, we tested whether inhibition of PI3K signaling would mimick glucocorticoids by increasing branched-chain amino acid degradation. Expression of a dominant negative PI3K p85 regulatory subunit (Adp85{Delta}iSH2) increased BCKD activity, and dexamethasone did not further stimulate enzyme activity. Inhibition of PI3K using LY-294002 increased the transcription of the BCKD E2 subunit but not the E1{alpha} subunit or BCKD kinase. Thus, glucocorticoids inhibit signaling through the IRS-1/PI3K/Akt pathway with a consequence of increased branched-chain amino acid catabolism.

phosphatidylinositol 3-kinase; kidney

CLASS IA PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) plays a pivotal role in the control of a variety of diverse metabolic processes. It is a heterodimeric enzyme complex that is typically composed of a p85 regulatory subunit and a p110 catalytic protein. PI3K becomes activated when it interacts with phosphorylated members of the insulin receptor substrate (IRS) family; the latter proteins undergo modification (i.e., phosphorylation) by tyrosine kinase receptors (e.g., growth factor receptors). A major downstream target of PI3K is Akt (also known as protein kinase B), but others also have been identified.

Interestingly, we found that signaling through the IRS-1/PI3K/Akt pathway is impaired in muscles of rats with chronic renal failure (CRF) (2), a condition associated with metabolic acidosis and increased glucocorticoid production (21–23). When the spontaneous metabolic acidosis of CRF was corrected with NaHCO3 treatment, signaling through this pathway was improved but not normalized. The reduced PI3K signaling that persisted after NaHCO3 treatment may have been due to increased glucocorticoid production, because a previous study by May et al. (23) revealed that urinary corticosterone excretion remained high in CRF rats after correction of their acidosis. Others have reported that glucocorticoids inhibit signaling through PI3K in muscle (12, 29, 31).

In patients and experimental rats with CRF, plasma and intracellular levels of the branched-chain amino acids (BCAAs) in skeletal muscle are frequently reduced compared with healthy individuals (4, 5, 14). Feeding CRF rats a high-protein diet did not raise the plasma concentrations of these essential amino acids, suggesting that overriding conditions (i.e., acidosis or a hormonal imbalance) accelerated their degradation (25). We and others have identified glucocorticoids as a physiological signal that increases branched-chain ketoacid dehydrogenase (BCKD) activity in muscle of adrenalectomized rats (1, 14, 21, 28) and in LLC-PK1 renal epithelial cells that had been stably transfected to express glucocorticoid receptors [LLC-PK1-GR101 cells (35)]. The BCKD enzyme complex is responsible for the irreversible degradation of BCAAs. Subsequently, we found that glucocorticoids increased the transcription of two BCKD subunits, E1{alpha} and E2, in LLC-PK1-GR101 cells; they also decreased the expression of BCKD kinase, which inactivates BCKD by phosphorylating the E1{alpha} subunit (34, 36). The intracellular signaling pathway(s) responsible for mediating these responses has not been identified.

Insulin deficiency (i.e., experimental diabetes) is another condition associated with increased glucocorticoid production and abnormal BCAA metabolism. BCKD activity was increased in the kidney, liver, and muscle of rats rendered insulinopenic by streptozotocin or alloxan treatment (Refs. 1, 20, and 26 and unpublished observations, S. R. Price). Importantly, Lee et al. (19) reported that PI3K/Akt signaling was decreased in muscles of streptozotocin-injected rats compared with controls.

Thus, in two pathological conditions associated with high glucocorticoids, there is an apparent inverse correlation between BCKD and PI3K activities, suggesting that PI3K may be an important regulator of BCKD activity. We now report that glucocorticoids reduce PI3K activity in LLC-PK1-GR101 renal tubular epithelial cells and provide evidence for a mechanism of action. We also used molecular tools to attenuate PI3K signaling and found that BCKD activity was increased.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM, FBS, trypsin-EDTA, and penicillin-streptomycin were from Life Technologies (Grand Island, NY). Anti-p85 PI3K and anti-IRS-1 antibodies were from Upstate Biotechnology (Lake Placid, NY); anti-p110beta PI3K antibody was from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-Akt (Ser473) polyclonal antibodies was from New England Biolabs (Beverly, MA); anti-phosphotyrosine PY20 monoclonal antibody was from Transduction Laboratories (Lexington, KY); anti-phospho-IRS-1 (Ser307) polyclonal antibodies was from Cell Signaling (Beverly, MA); L-[1-14C] leucine was from Amersham (Arlington Heights, IL); phosphatidylinositol was from Avanti Polar Lipids (Alabaster, AL); and Silica H gel thin-layer chromatography (TLC) plates were from Whatman (Kent, UK). All other reagents were obtained from Sigma Chemical (St. Louis, MO).

Cell culture and adenovirus infection. LLC-PK1-GR101 cells were derived from LLC-PK1 porcine renal epithelial cells by stably transfecting the parent line with a plasmid to express the glucocorticoid receptor (35). In this earlier report (35), we confirmed that LLC-PK1 cells do not respond to glucocorticoids and demonstrated (in LLC-PK1-GR101 cells) that dexamethasone-induced activation of BCKD was a glucocorticoid receptor-mediated response. Cells were grown in DMEM containing 10% FBS, 2 mM glutamine, 0.8 mg/ml hygromycin, and 1% penicillin-streptomycin (35). Cells between passages 4 and 15 were grown in 24-well plates for measurement of BCKD activity or in 100-mm dishes for other experiments. For experiments involving glucocorticoids, the normal growth medium was replaced with DMEM containing 10% charcoal-stripped serum plus other normal supplements 24 h before the addition of dexamethasone. This protocol avoids the confounding effects of endogenous glucocorticoid effects. In unpublished studies, the effects of glucocorticoids on BCKD activity were compared in cells supplemented with stripped serum or dialyzed serum. Glucocorticoids activated BCKD to the same extent in each condition. In some experiments, cells were infected with Adp85{Delta}iSH2, an adenovirus encoding a mutant PI3K p85 regulatory subunit in which the inner SH2 domain was deleted plus a second gene for enhanced green fluorescent protein (EGFP) (10); control cells were infected with a control adenovirus encoding EGFP (Ad-EGFP). Both viruses were provided by Dr. J. Du (Baylor Medical College). Cells were infected for 2 h in serum-free DMEM. Afterward, fresh DMEM containing 10% FBS was added. In each experiment, the efficiency of infection was monitored by fluorescence microscopy. Cells were used for experiments when infection efficiencies of >90% were achieved.

BCKD activity. Cells were incubated in DMEM containing charcoal-stripped serum with or without 50 nM dexamethasone for 24 h as previously described (35). To measure BCKD activity, cells were preincubated in assay buffer containing 0.25 µCi/well L-[1-14C] leucine and 0.5 mM cold leucine for 30 min at 37°C (35).

Immunoprecipitation and Western blot analyses. Cells were incubated with dexamethasone or vehicle for 24 h, washed twice with 5 ml of ice-cold PBS, and scraped in 500 µl of RIPA buffer composed of 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 100 µg/ml PMSF. Cleared lysate samples (20 µg protein) were separated by SDS-PAGE. Total IRS-1, phospho-IRS-1 (Ser307), p85 and p110 subunits of PI3K, Akt, and phospho-Akt (Ser473) were evaluated by Western blot analysis using commercial polyclonal antisera. To evaluate the level of IRS-1 tyrosine phosphorylation, anti-IRS-1 antibodies (2 µg/1 mg protein) were added to equal amounts of lysate proteins and mixed at 4°C overnight. Protein A agarose beads were added, and the mixture was incubated for an additional 2 h. Immunoprecipitated IRS-1 protein was subjected to SDS-PAGE and Western blot analysis for tyrosine phosphorylation content using the PY20 anti-phosphotyrosine monoclonal antibody.

PI3K assay. IRS-1-associated PI3K activity was measured using thin-layer chromatography as previously described (9).

Transient transfection and luciferase transcription assays. Cells were transfected with firefly luciferase transgenes containing the following promoter segments: human BCKD E1{alpha} subunit (–710 to +83 bp), mouse BCKD E2 subunit (–800 to +86 bp), or rat BCKD kinase (–3500 to +264 bp) (7, 16, 34, 36). Effectene (Qiagen, Valencia, CA) was used as the transfection reagent according to manufacturer's instructions. For the assessment of transfection efficiency, cells were cotransfected with pRL-TS encoding the Renilla luciferase gene (17). Luciferase activities were measured, and each firefly activity was normalized using the respective Renilla luciferase value (34, 36).

Statistical analysis. All data are reported as means ± SE. Densitometric analysis data are reported in arbitrary densitometric units. BCKD activity was reported as picomoles of CO2 released per microgram of DNA per minute. Normalized firefly luciferase activity (i.e., BCKD E2 promoter activity) was reported in arbitrary units. For pairwise statistical analyses, a Student's t-test was performed. When multiple treatements were compared, ANOVA was performed, followed by pairwise analyses using the Tukey test when appropriate. In all cases, P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
To test the effects of glucocorticoids on PI3K activity in renal cells, we studied LLC-PK1-GR101 renal tubular epithelial cells that had been stably transfected to express a modest level of glucocorticoid receptors (35). To avoid any confounding effects of endogenous glucocorticoids in serum, cells were maintained in growth medium supplemented with charcoal-stripped FBS (i.e., lacking endogenous glucocorticoids). Cells were treated with 50 nM dexamethasone for 24 h before the measurement of PI3K activity associated with IRS-1. Insulin was added to some cells for 10 min before lysis to activate the PI3K system. In the absence of insulin, dexamethasone decreased PI3K activity by 69% (P < 0.05, n = 4; Fig. 1A). The addition of insulin without glucocorticoids increased PI3K activity by 320% (P < 0.05, n = 4). Consistent with the antagonism between insulin and glucocorticoids, dexamethasone treatment blocked the stimulation of PI3K activity by insulin (23% of the untreated control value, P < 0.05, n = 4).


Figure 1
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  		Fig. 1. Glucocorticoids suppress signaling through insulin receptor substrate (IRS)-1/phosphatidylinositol 3-kinase (PI3K)/Akt. LLC-PK1-GR101 cells were incubated in DMEM supplemented with 10% charcoal-stripped FBS; 50 nM dexamethasone (Dex) was added to some cells for 24 h. To stimulate PI3K activity, insulin (100 nM) was added 10 min before cells were harvested. A: anti-IRS-1 antibodies were added to aliquots of the lysate containing equal amounts of protein. PI3K activity associated with the immunocomplexes was assayed by measuring the phosphorylation of phosphatidylinositol using [{gamma}-32P]ATP; the reaction product is phosphatidylinositol (3,4,5)-trisphosphate (PIP). C, vehicle control; D, Dex; I, insulin; D + I, Dex plus insulin; solid bars, no Dex; open bars, with Dex. Results from 4 experiments are shown graphically and are reported as means ± SE. *P < 0.05 vs. untreated controls; **P < 0.05 vs. insulin alone. B: total Akt and phosphorylated Akt (pAkt) were detected by Western blot analysis using pan and phospho-Ser473 (pSer)-specific polyclonal antibodies. Results of 3 experiments are presented graphically and are reported as means ± SE. *P < 0.05 vs. untreated controls; **P < 0.05 vs. insulin alone.

 
To determine if the dexamethasone-induced decrease in PI3K activity was reflected in the activity of downstream effectors, we examined the "activation" status of Akt by measuring phospho-Ser473 by immunoblot analysis (Fig. 1B); Akt becomes activated upon phosphorylation of Ser473 and Thr308. In the absence of insulin, dexamethasone reduced Ser473 phosphorylation by 33% (P < 0.05 vs. no treatment, n = 3). Dexamethasone also blunted the insulin-induced phosphorylation of Akt. Insulin alone increased phosphorylation by 98% (P < 0.05 vs. no treatment, n = 3); in the presence of dexamethasone, insulin did not significantly increase Akt phosphorylation above the level in untreated control cells. Since the hormone did not affect the total amount of Akt, we conclude that the reduction in Akt phosphorylation was due to decreased PI3K activity.

Tyrosine kinase membrane receptors (e.g., insulin receptors) activate IRS-1 through phosphorylation. Conversely, a variety of intracellular serine/threonine kinases inactivate IRS-1 (13). Therefore, we tested whether glucocorticoids affect either tyrosine or serine phosphorylation of IRS-1. As shown in Fig. 2, dexamethasone minimally affected the basal level of IRS-1 tyrosine phosphorylation [P = not significant (NS), n = 3]; however, phosphorylation of IRS-1 on Ser307 was increased by 90% (P < 0.05, n = 3; Fig. 2). The amount of IRS-1 protein was unchanged by dexamethasone (data not shown).


Figure 2
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  		Fig. 2. Effect of Dex on IRS-1 phosphorylation. A: cells were treated with vehicle (C; solid bars) or Dex (50 nM; open bars) for 24 before lysis in RIPA buffer. IRS-1 was immunoprecipitated with anti-IRS-1 antibodies before SDS-PAGE and transfer for Western blot analysis. Phosphotyrosine (pTyr) content was evaluated using the PY20 anti-phosphotyrosine monoclonal antibody. pSer was detected using polyclonal antibodies that only detect phospho-Ser307 in IRS-1. Results of 3 experiments are presented graphically and are expressed as means ± SE. *P < 0.05 vs. control.

 
Changes in the stoichiometry between the p85 regulatory and p110 catalytic PI3K subunits could alter the enzyme's activity in renal epithelial cells (24, 32, 33). Therefore, we evaluated the amounts of each of these subunits (Fig. 3). Dexamethasone increased the amount of the p85 subunit by 185% (P < 0.05, n = 3) but did not change the level of p110 protein (P = NS).


Figure 3
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  		Fig. 3. Dex increases the PI3K p85 regulatory subunit. Cells were treated as described in Fig. 2 before lysis in RIPA buffer and Western blot analysis using polyclonal antibodies against the PI3K p85 regulatory or p110 catalytic subunits. Solid bars, control cells; open bars, Dex-treated cells. Results of 3 experiments are presented graphically and are expressed as means ± SE. *P < 0.05 vs. control.

 
To examine the relationship between PI3K and BCKD, we studied LLC-PK1-GR101 cells that had been infected with a recombinant bicistronic adenovirus that separately encoded EGFP and a dominant negative PI3K p85 regulatory subunit (Adp85{Delta}iSH2) to attenuate PI3K activity. The mutant p85 protein lacks an inner SH2 domain and prevents activation of the p110 catalytic subunit without interfering with the p85-p110 interaction. Fluorescence microscopy indicated that the expression of the transduced EGFP gene was high (Fig. 4A). Western blot analysis for the p85 subunit confirmed a dose-dependent increase in the amount of p85 expressed in infected cells (Fig. 4B).


Figure 4
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  		Fig. 4. Expression of a dominant negative PI3K p85 subunit. Cells were infected with 5 x 107 plaque-forming units (pfu)/ml Adp85{Delta}iSH2 in serum-free medium for 2 h; medium plus charcoal-stripped FBS (10%) was added for the next 24 h. A: expression of the bicistronic gene encoding enhanced green fluorescent protein (EGFP) and p85{Delta}iSH2 was evaluated by fluorescence microscopy. EGFP fluorescence is shown in grayscale. B: expression of p85 in cells infected with different amounts of Adp85{Delta}iSH2 was evaluated by Western blot analysis. The experiment in B was performed 3 times.

 
Next, we confirmed that the expression of the dominant negative p85 protein attenuated IRS-1-associated PI3K activity and tested whether the reduction might affect BCKD activity in the absence of glucocorticoids. Insulin stimulated PI3K activity by 244% in control cells infected with Ad-EGFP (P < 0.05, n = 3), and dexamethasone attenuated the response (150% of untreated Ad-EGFP-transfected cells, P < 0.05 vs. control or insulin-treated cells, n = 3). In cells infected with Adp85{Delta}iSH2, basal PI3K activity associated with IRS-1 was unchanged relative to control cells (Fig. 5). Importantly, insulin did not stimulate PI3K activity in cells expressing the mutant p85 subunit (91% of the untreated control cell value, P = NS, n = 3). Moreover, dexamethasone did not significantly suppress PI3K activity in infected cells (85% of untreated EGFP-infected cells, P = NS, n = 3). BCKD activity in Adp85{Delta}iSH2-infected cells was increased by 58% over the activity in uninfected cells and was increased by 44% over the activity measured in cells infected with Ad-EGFP (P < 0.05 vs. either treatment, n = 6; Fig. 6). Dexamethasone did not stimulate BCKD activity in cells infected with Adp85{Delta}iSH2 as it did in control and Ad-EGFP-infected control cells (Fig. 6).


Figure 5
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  		Fig. 5. Expression of dominant negative PI3K p85 subunit inhibits PI3K activity. Cells infected with Ad-EGFP or Adp85{Delta}iSH2 were incubated in media supplemented with 10% dialyzed FBS for 24 h and then treated with or without 50 nM Dex for an additional 24 h; insulin (100 nM) was added to some cells 10 min prior to lysis to activate PI3K. IRS-1-associated PI3K activity was measured as described in Fig. 1. Solid bars, control cells; open bars, Dex treatment; crosshatched bars, insulin treatment; shaded bars, Dex plus insulin treatments. Results of 3 experiments are presented graphically and are expressed as means ± SE. *P < 0.05 vs. control; **P < 0.05 vs. insulin.

 

Figure 6
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  		Fig. 6. Expression of dominant negative PI3K p85 increases branched-chain ketoacid dehydrogenase (BCKD) activity. Cells infected with Adp85{Delta}iSH2 or Ad-EGFP were incubated with vehicle (solid bars) or Dex (50 nM, open bars) for 24 h before measurement of BCKD activity. CTL, control. Results are means ± SE (n = 6) of each condition. The experiment was performed 3 times. *P < 0.05 vs. the same treatment group without Dex; **P < 0.05 vs. the other treatment groups without Dex.

 
Previously, we (34, 36) reported that glucocorticoids increased the transcription of the BCKD E1{alpha} and E2 subunits and inhibited BCKD kinase expression. To determine whether inhibition of PI3K would elicit any of these responses, we transfected cells with plasmids containing proximal segments of the E1{alpha} subunit, E2 subunit, or BCKD kinase promoter DNA sequences linked to a luciferase reporter gene. The PI3K inhibitor LY-294002 (25 µM) was added for 24 h; this concentration of LY-294002 completely inhibited PI3K in renal epithelial cells (30). As previously reported, the addition of dexamethasone for 24 h increased E1{alpha} promoter-controlled luciferase activity by 210 ± 27% (P < 0.05 vs. control, n ≥ 4) and E2-related activity by 156 ± 2% (P < 0.05 vs. control, n ≥ 4); the glucocorticoid suppressed kinase promoter activity by 27 ± 1% (P < 0.05 vs. control, n = 3). LY-294002 alone increased E2-linked reporter activity, but the combination of dexamethasone plus LY-294002 was no more effective than either treatment alone (Fig. 7). Interestingly, LY-294002 alone or in combination with dexamethasone did not affect E1{alpha} and BCKD kinase promoter activities (data not shown).


Figure 7
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  		Fig. 7. BCKD E2 promoter activity is increased by Dex. Cells were transfected with a mouse BCKD E2 0.8-kb promoter-firefly luciferase (Luc) reporter plasmid plus pRL-TS to assess transfection efficiencies. Some cells were treated with 50 nM Dex and/or 25 µM LY-294002 (LY), a PI3K inhibitor, for 24 h before measurement of Luc activities. Normalized Luc values were calculated; data (means ± SE) are reported as percentages of the control cell value. *P < 0.05 vs. control cells without Dex.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In renal cells, the PI3K/Akt pathway participates in a number of diverse physiological processes including lysosomal proteolysis in tubular epithelial cells, oxidative stress responses in mesangial cells, and epithelial Na+ channel activity in cortical collecting duct principal cells (15, 18, 30). In some nonrenal cells (e.g., muscle), glucocorticoids can attenuate PI3K-mediated cell signaling (11, 12), suggesting that some metabolic responses to glucocorticoids in renal cells could result from inhibition of this pathway. Therefore, we tested whether glucocorticoids reduce signaling through the IRS-1/PI3K/Akt pathway in LLC-PK1-GR101 cells. We identified two distinct responses to glucocorticoids that contribute to PI3K inhibition. First, dexamethasone increased the phosphorylation of IRS-1 on Ser307. In contrast, tyrosine phosphorylation of IRS-1 was unchanged. In adipocytes and muscle cells, insulin initially increases phosphorylation of IRS-1 on tyrosine residues but subsequently induces phosphorylation of Ser307 (13). Phosphorylation of Ser307 results in an uncoupling of IRS-1 from the insulin receptor and decreases its tyrosine phosphorylation. The finding that dexamethasone increased Ser307 phosphorylation without affecting tyrosine modification suggests that the glucocorticoid-activated signaling events leading to phosphorylation of Ser307 are distinct from those events that are activated by insulin. In this case, it would seem that glucocorticoids must be acting indirectly by activating a signaling pathway that phosphorylates IRS-1 Ser307. Second, dexamethasone selectively increased the level of the PI3K p85 regulatory subunit. This is notable because the ratio of p85 and p110 subunits may be a critical determinant of PI3K activity (24, 32, 33). According to some models, excess free p85 subunit competes with the p85/p110 holoenzyme complex for the binding site on IRS-1, leading to a reduction in PI3K activity (3, 24, 32). Thus, we believe the selective increase in p85 subunit in the presence of glucocorticoids contributes to the reduction in PI3K signaling in LLC-PK1-GR101 cells.

Our study also demonstrated that one physiological repercussion of PI3K inhibition in renal epithelial cells is an increase in the irreversible degradation of BCAAs. This is notable because we (34, 35) previously reported that glucocorticoids stimulated BCKD activity in these same cells. Thus, our results identify a mechanism that contributes to the reduction of plasma BCAAs often seen in patients with CRF. Interestingly, signaling through the IRS1/PI3K/Akt pathway was impaired in muscles of rats with CRF (2). Feeding NaHCO3 to CRF rats to correct their metabolic acidosis did not completely normalize PI3K signaling in muscle to levels measured in pair-fed control rats. May et al. (23) reported that corticosterone excretion remained high in CRF rats after correction of their acidosis. Our present results would suggest that glucocorticoids are partially responsible for the observed signaling defects in multiple cell types.

Nellis et al. (27) also examined the relationship between PI3K and BCKD activities. They reported that LY-294002 did not increase the basal level of BCKD activity in rat clone 9 hepatic cells but prevented the suppression of BCKD activity by insulin. In contrast, inhibition of PI3K activity in LLC-PK1-GR101 cells by glucococorticoids or expression of a dominant negative PI3K regulatory subunit resulted in higher BCKD activity. Moreover, dexamethasone did not produce an additional induction of BCKD activity in Adp85{Delta}iSH2-infected cells. It is unclear why our results do not concur with those of Nellis et al., but the contrasting results could be due to differences in cell types.

Based on our previous studies, it is unlikely that inhibition of PI3K is the only mechanism by which glucocorticoids enhance BCKD activity. Previously, we (34, 36) found that dexamethasone increased the transcription of both E1{alpha} and E2 subunits and suppressed the expression of BCKD kinase, which phosphorylates (i.e., inactivates) E1{alpha}. Presently, inhibition of PI3K with LY-294002 increased E2 transcription only. This result is consistent with our earlier report (34) showing that glucocorticoids activate E2 and E1{alpha} promoters by different mechanisms. Dexamethasone activated the E2 promoter by reducing NF-{kappa}B binding, whereas the E1{alpha} promoter lacks an NF-{kappa}B binding site.

In summary, we have demonstrated that glucocorticoids attenuate signaling through the IRS-1/PI3K signaling pathway in LLC-PK1-GR101 renal cells. This response results in increased activity of BCKD. Other effectors (e.g., hormones and cytokines) that regulate BCKD activity also are frequently associated with insulin resistance and, presumably, a reduction in PI3K activity (6, 8). Therefore, our findings may represent a general mechanism for controlling BCAA degradation and other physiological functions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50740 (to S. R. Price) and DK-62796 (to X. Wang).


   ACKNOWLEDGMENTS
 
We thank Hong-Yan Qu for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Price, Renal Div., Emory Univ., Rm. 338, Woodruff Memorial Bldg., 1639 Pierce Dr., Atlanta, GA 30322 (e-mail: russ.price{at}emory.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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 DISCUSSION
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