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CELLULAR AND MOLECULAR

Identification of the Insulin Signaling Cascade in the Regulation of Alpha-Class Glutathione S-Transferase Expression in Primary Cultured Rat Hepatocytes

Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan

Received September 22, 2005; accepted November 15, 2005.

	Abstract

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We reported previously that insulin elevated alpha-class glutathione S-transferase (GSTs) protein levels in primary cultured rat hepatocytes (Kim et al., 2003b). In contrast, glucagon down-regulated alpha- and pi-class GST expression, and mechanistic research implicated cAMP and protein kinase A in this process (Kim et al., 2003b). The present study examines the signaling pathways involved in the regulation of alpha-class GST in response to insulin in primary cultured rat hepatocytes. Protein levels of GSTA1/2 and GSTA3/5 and activity of GST toward 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were increased in an insulin concentration-dependent manner. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or rapamycin, an inhibitor of mammalian target of rapamycin and ribosomal p70 S6 kinase (p70S6K) phosphorylation, or with an adenovirus containing green fluorescent protein and a dominant-negative and kinase-dead Akt, effectively inhibited the insulin-mediated increase in alpha-class GST expression and GST activity toward NBD. In contrast, PD98059 (2'-amino-3'-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of c-Jun N-terminal kinase, SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imadazole], an inhibitor of p38 mitogen-activated protein kinase, or bisindolylmaleimide, a broad spectrum inhibitor of protein kinase C, did not inhibit the insulin-mediated increase in alpha-class GST protein levels in hepatocytes. These results show that PI3K/Akt/p70S6K signaling is active in the insulin-mediated up-regulation of the antioxidant defense system and that low insulin levels, as encountered in diabetes, potentially increase the susceptibility of hepatocytes to xenobiotic-mediated and/or oxidative stress-mediated damage.

It is recognized that the hepatic expression of GSTs is altered in response to development, aging, gender, genetic factors, pregnancy, and pathophysiological conditions such as diabetes, long-term alcohol consumption, inflammation, and protein calorie malnutrition (Thomas et al., 1989; Di Ilio et al., 1995; Egaas et al., 1995; Wormhoudt et al., 1999; Cho et al., 2000; Vanhaecke et al., 2000; McCarver and Hines, 2002; Voss et al., 2002). Endogenous factors, such as hormones, growth factors, and cytokines, may play a critical role in mediating alterations in GST expression in physiological and pathophysiological conditions. Our previous work showed that insulin and glucagon regulate, in an opposing manner, the expression of the alpha-class GSTs, GSTA1/2 and GSTA3/5, suggesting that the development of oxidative stress observed in diabetes may be partially attributed to the decrease in alpha-class GST protein levels (Kim et al., 2003b). Furthermore, this study provided evidence that cAMP and protein kinase A mediated the inhibitory effect of glucagon on GST expression. Glucagon effectively suppressed GSTP1 expression in primary cultured hepatocytes through the cAMP and protein kinase A (Kim et al., 2003b). The signaling pathways responsible for mediating insulin effects on GSTs expression, however, are unknown.

The binding of insulin to the insulin receptor results in a cascade of signaling events that proceed through the insulin receptor substrates 1–4 and result in activation of a variety of downstream effectors, including phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B, ribosomal p70 S6 kinase (p70S6K), and protein kinase C (PKC) (Kim et al., 2004b; Farese et al., 2005). We reported that insulin signaling pathways involving PI3K/Akt/p70S6K are active in the insulin-mediated regulation of microsomal epoxide hydrolase (mEH) expression (Kim et al., 2003a) and in the elevation of GSH synthesis via increased -glutamylcysteine ligase catalytic subunit (GCLC) expression (Kim et al., 2004a). Activation of the insulin receptor also leads to activation of mitogen-activated protein kinase (MAPK) signaling pathways, including the extracellular signal-regulated kinase (ERK), the p38 MAPK, and the c-Jun N-terminal kinase (JNK) (Kim et al., 2004a). Interestingly, the p38 MAPK inhibitors SB203580 or SB202190 abrogated the insulin-mediated increase in microsomal epoxide hydrolase protein, showing that these kinases, in addition to the PI3K, Akt, and p70S6 kinase cascade, also play a role in regulating mEH expression (Kim et al., 2003a).

The objectives of the present study were to determine, using primary cultured rat hepatocytes, the signaling pathways and components involved in insulin-mediated regulation of alpha-class GSTs, with a view of providing information on how low insulin, as encountered in type 2 diabetes, may decrease GST levels and predispose the cell to injury. We show that inhibition of PI3K, Akt, or p70S6K activity prevents the insulin-mediated increase in alpha-class GST protein and activity. In contrast, JNK and MAPK inhibitors were without effect on insulin-mediated alpha-class GST expression. These data implicate PI3K, Akt, and p70S6K in the insulin-mediated regulation of alpha-class GST expression.

	Materials and Methods

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Chemicals. Modified Chee's medium and L-glutamine were obtained from Invitrogen (Carlsbad, CA). Insulin (Novolin R) was purchased from Novo-Nordisk (Princeton, NJ). Collagenase (type I) was purchased from Worthington Biochemicals (Freehold, NJ). Vitrogen (95–98% type I collagen, 2–5% type III collagen) was obtained from Cohesion Technologies (Santa Clara, CA). Class-specific GST antibodies were prepared and characterized previously by our laboratory (Kim et al., 2003b). Horseradish peroxidase-conjugated rabbit anti-goat antibody was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Enhanced chemiluminescence reagents were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Wortmannin, LY294002, rapamycin, bisindolylmaleimide, SB203580, PD98059, and SP600125 were obtained from Calbiochem (San Diego, CA). Mouse Akt1-K179M (dominant-negative and kinase-dead) was obtained from Upstate Biotechnology (Lake Placid, NY). pAdTrack-CMV and pAdEasy were obtained from Dr. B. Vogelstein (The Sidney Kimmel Comprehensive Cancer Center and The Howard Hughes Medical Institute, The Johns Hopkins University Medical Institutions, Baltimore, MD). GSH, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Primary Rat Hepatocyte Cultures. Hepatocytes were isolated from the livers of male Sprague-Dawley rats (200–300 g) using collagenase perfusion as described previously (Woodcroft and Novak, 1997). Hepatocytes were plated onto dishes covalently coated with Vitrogen, and modified Chee's medium was fortified as described previously (Woodcroft and Novak, 1997) and supplemented with 0.1 µM dexamethasone and 1 µM insulin. Cells were plated at a density of 3 x 10⁶ cells/60-mm dish or 1 x 10⁷ cells/100-mm dish. Four hours after plating, cells were washed with insulin-free medium three times and cultured for an additional 2 h in insulin-free medium prior to initiation of treatment. Cells were then treated with various concentrations of insulin (0–100 nM). Kinase inhibitors were dissolved in DMSO and added 1.5 h prior to addition of insulin (10 nM). The final DMSO concentration in the medium was 1 µl/ml (0.1%), and this concentration of DMSO did not affect the alpha-class GST protein level or GST activity. None of the protein kinase inhibitors resulted in increased cell toxicity, compared with untreated cells, at the concentrations used in this study. For adenovirus infection, 4 h after plating, the cells were washed with insulin-free medium three times and adenovirus GFP containing the dominant-negative and kinase-dead mutant of Akt (AdV-Akt; 150 MOI), or control virus (AdV-GFP, 150 MOI) was added to the cells in fresh medium. Following overnight infection, medium was changed, and hepatocytes were treated with insulin (10 nM) for 2 days. Medium was changed every 24 h. Hepatocyte viability was monitored by measuring released lactate dehydrogenase activity as described previously (Woodcroft and Novak, 1997). The Wayne State University Animal Investigation Committee approved all experimental procedures involving animals.

Preparation of Dominant-Negative Akt Adenoviral GFP Construct. The dominant-negative and kinase-dead form of Akt1 has a point mutation, K179M, removing the ATP-binding site, which results in loss of kinase activity. The coding region of Akt1-K179M containing a Myc-His tag was amplified by PCR using Pfu turbo (Stratagene, La Jolla, CA) and the primers 5'-GCGAGATCTATCCCATGAACGACGTAGCC-3' and 5'-GCGAGATCTAAACTCAATGGTGATGGTGATGAT-3'. The PCR product was A-tailed and ligated into the pGEM-T Easy Vector (Promega, Madison, WI). The kinase-dead Akt was digested from the T Vector with SpeI and NotI and ligated into NotI/XbaI sites in pAdTrack. Homologous recombination of pAdTrack and pAdEasy and infection of 293 cells were performed as described previously (Kim et al., 2004a).

Immunoblot Analysis. Whole-cell lysates were prepared as described previously (Kim et al., 2003b). Protein concentrations were determined using the bicinchoninic acid protein assay (Sigma-Aldrich). For immunoblot analysis, protein samples (5–10 µg of protein/lane) were resolved by SDS-polyacrylamide gel electrophoresis on a 15% gel, transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA), and blocked for 2 h in 5% milk powder in 0.05% Tween 20 in PBS (PBS-T). For immunodetection, blots were incubated overnight with goat anti-rat GSTA1/2, GSTA3/5, and GSTM1/2 antibody (diluted in 5% milk powder in PBS-T) at room temperature, followed by incubation with a secondary antibody conjugated to horseradish peroxidase (diluted 1:10,000 in 5% milk powder in PBS-T) for 3 h at room temperature. Proteins were detected by enhanced chemiluminescence on Kodak X-OMAT film (Sigma-Aldrich) and quantified by densitometry with a Molecular Dynamics scanning laser densitometer and ImageQuant analysis program (GE Healthcare).

Metabolic Assays. The activities of GSTs in whole-cell lysates were measured using NBD as a substrate. The GST activity toward NBD was determined using the method of Ricci et al. (1994).

Statistical Analysis. Significant differences between groups were determined by ANOVA followed by the Newman-Keuls comparison test (p < 0.05). Statistical analysis was performed on cell lysates from a single hepatocyte preparation. Reproducibility of results was confirmed in two to four separate hepatocyte preparations.

	Results

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Effects of Insulin on Alpha-Class GST Protein Levels and GST Activity toward NBD. To examine the insulin concentration dependence of alpha-class GST protein levels and GST activity toward NBD, a selective substrate for the alpha-class GSTs (Ricci et al., 1994; Kim et al., 2003b), hepatocytes were cultured for 24 h in medium supplemented with 0.1 to 100 nM insulin (Fig. 1). GSTA1/2, GSTA3/5 protein, and GST activity were increased in an insulin concentration-dependent manner and were increased maximally 177, 288, and 161%, respectively, relative to cells that were not exposed to insulin (Fig. 1). Significantly increased levels of alpha-class GST protein and GST activity were observed at 1 nM insulin, and a further increase was observed in hepatocytes cultured in the presence of 10 nM insulin. In contrast, insulin failed to affect GSTM1/2, mu-class GSTs, indicating that each class of GSTs is differentially regulated by insulin.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Concentration-dependent effects of insulin on alpha- and mu-class GST protein levels and GST activity in primary cultured rat hepatocytes. Hepatocytes were cultured in the presence of various concentrations of insulin for 24 h. Data are means ± S.D. of three to five preparations of cell lysates from a single hepatocyte preparation. *, **, ***, significantly different from levels monitored in hepatocytes cultured in the absence of insulin, p < 0.05, p < 0.01, or p < 0.001, respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Effects of short-term insulin exposure on alpha- and mu-class GST protein levels and GST activity in primary cultured rat hepatocytes. Hepatocytes were treated with 10 nM insulin for 2, 4, 12, or 24 h; 24 h after initiation of each insulin treatment, cells were harvested. Data are means ± S.D. of three to five preparations of cell lysates from a single hepatocyte preparation. *, **, ***, significantly different from levels monitored in hepatocytes cultured in the absence of insulin, p < 0.05, p < 0.01, or p < 0.001, respectively.

Inhibition of ERK, p38MAPK, and JNK. To determine whether ERK, p38MAPK, or JNK may play a role in insulin regulation of alpha-class GST expression, hepatocytes were pretreated with the mitogen-activated protein kinase kinase inhibitor PD98059, the p38 MAPK inhibitor SB203580, or the JNK inhibitor SP600125, prior to addition of 10 nM insulin (Fig. 3). MAPK inhibitor concentrations used in this study are sufficient to inhibit each of MAPK signaling pathways (Cuenda et al., 1995; Bennett et al., 2001; Kim et al., 2004a). Each of these inhibitors failed to inhibit the insulin-mediated elevation of GSTA1/2 (Fig. 3A), or GSTA3/5 (Fig. 3B) protein levels, or GST activity toward NBD (Fig. 3C). PD98059 has a small but statistically insignificant effect in inhibiting the insulin-mediated increase in GST3/5, whereas SP600125 had a small but statistically insignificant effect in further elevating GST1/2 and GST3/5. These trends were also marginally reflected in GST activity. These results suggest that MAPK signaling pathways are not primarily involved in the insulin-mediated increase in alpha-class GST protein levels.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Effect of the mitogen-activated protein kinase kinase inhibitor PD98059, the p38 MAPK inhibitor SB203580, or the JNK inhibitor SP600125 on the insulin-mediated increase in GSTA1/2 (A), GSTA3/5 (B), and GST (C) activity. Hepatocytes were treated with PD98059 (PD, 50 µM), SB203580 (SB, 10 µM), or SP600125 (SP, 20 µM) for 1.5 h prior to addition of insulin (10 nM) for 24 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitor. Data are means ± S.D. of three preparations of cell lysates from a single hepatocyte preparation. ##, ###, significantly different from levels monitored in untreated hepatocytes, p < 0.01 or p < 0.001, respectively.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Effect of LY294002 or wortmannin on the insulin-mediated increase in GSTA1/2 (A), GSTA3/5 (B), and GST (C) activity in primary cultured rat hepatocytes. Hepatocytes were treated with LY294002 (0.1, 1, or 10 µM) or wortmannin (25, 100, or 500 nM) for 1.5 h before addition of 10 nM insulin for 24 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitor. Data are means ± S.D. of three to five preparations of cell lysates from a single hepatocyte preparation. #, ##, ###, significantly different from levels monitored in untreated hepatocytes, p < 0.05, p < 0.01, or p < 0.001, respectively. *, **, ***, significantly different from levels monitored in hepatocytes treated with insulin only, p < 0.05, p < 0.01, or p < 0.001, respectively.

The role of the downstream target kinase, Akt, on the insulin-mediated increase in alpha-class GSTs was examined through infection of primary cultured hepatocytes with an adenoviral GFP construct containing a dominant-negative and kinase-dead mutant of Akt1 (Fig. 5). To examine adenoviral infection of hepatocytes, GFP expression was monitored using a fluorescence microscope. The adenovirally introduced Akt was expressed 2- or 7-fold greater than the endogenous Akt 24 h after 30 or 150 MOI AdV GFP-Akt viral particle infection, respectively (Fig. 5A). We have reported previously that overexpression of a dominant-negative and kinase-dead mutant of Akt1 resulted in 65% inhibition of Akt kinase activity induced by 10 nM insulin, as monitored by phosphorylation of glycogen synthase kinase-3, a downstream target of Akt (Kim et al., 2004a). Control AdV-GFP-infected hepatocytes were comparable with uninfected cells in their elevated alpha-class GST protein and GST activity toward NBD in response to insulin (Fig. 5, B–D). Dominant-negative Akt expression resulted in a 70 to 90% inhibition of the insulin-mediated increase in alpha-class GST protein and GST activity. These results suggest that Akt, a downstream effector of PI3K, is involved in the insulin-mediated increase in alpha-class GST expression.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effect of dominant-negative Akt expression on insulin-mediated increased in GSTA1/2, GSTA3/5, and GST activity in primary cultured rat hepatocytes. A, hepatocytes were infected with 30 or 150 MOI AdV-Akt, and 24 h later, cells were harvested for determination of Akt protein level. B, C, and D, 24 h after infection with 150 MOI AdV-Akt or AdV-GFP, hepatocytes were treated with 10 nM insulin for 2 days. Data are means ± S.D. of three to four preparations of cell lysates from a single hepatocyte preparation. **, ***, significantly different from levels monitored in corresponding control hepatocytes, p < 0.01 or p < 0.001, respectively.

The possible involvement of p70S6K or PKC in the insulin-mediated elevation of alpha-class GST expression was examined using rapamycin or bisindolylmaleimide, respectively (Fig. 6). Pretreatment of cells with the mammalian target of rapamycin inhibitor, rapamycin, partially inhibited the insulin-mediated increase in GSTA1/2 and GSTA3/5 protein levels and GST activity toward NBD (Fig. 6), suggesting that p70S6K also plays a role in the insulin-mediated increase in alpha-class GST expression. The broad spectrum PKC inhibitor bisindolylmaleimide, at concentrations up to 10 µM, failed to inhibit the insulin-mediated increase in alpha-class GST expression (Fig. 6). These results suggest that PKC does not contribute to the insulin-mediated increase in alpha-class GST protein expression.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Effect of rapamycin and bisindolylmaleimide on the insulin-mediated increase in GSTA1/2 (A), GSTA3/5 (B), and GST (C) activity in primary cultured rat hepatocytes. Hepatocytes were treated with rapamycin (10, 100, or 1000 nM) or bisindolylmaleimide (10 µM) for 1.5 h before addition of 10 nM insulin for 24 h. Untreated (UT) hepatocytes were cultured in the absence of insulin and inhibitor. Data are means ± S.D. of three to six preparations of cell lysates from a single hepatocyte preparation. #, ##, ###, significantly different from levels monitored in untreated hepatocytes, p < 0.05, p < 0.01, or p < 0.001, respectively. *, **, significantly different from levels monitored in hepatocytes treated with insulin only, p < 0.05 or p < 0.01, respectively.

	Discussion

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GSTs not only play a protective role against electrophilic substrate- and reactive oxygen species-induced damage in cells but also serve as regulators of cell signaling pathways (Eaton and Bammler, 1999; Townsend and Tew, 2003). Therefore, understanding the insulin-mediated regulation of GST expression is critical given the role that GSTs play in cell physiology in response to oxidative stress, xenobiotics, and hormones. It was reported that hepatic GST activity was decreased in chemical-induced diabetic rats and restored by insulin administration (Thomas et al., 1989). The i.v. injection of insulin plus glucose increased GST activity toward 1-chloro-2,4-dinitrobenzene, and the injection of glucagon decreased this activity in rats (Carrillo et al., 1995). We reported previously that insulin and glucagon regulate the expression of alpha- and pi-class GSTs in an opposing manner in primary cultured rat hepatocytes and that glucagon effectively suppressed GSTP1 expression (Kim et al., 2003b). The present study provides evidence that PI3K signaling pathways, but not MAPK signaling pathways, are primarily responsible for the insulin-mediated regulation of alpha-class GST expression in primary cultured rat hepatocytes.

We have demonstrated that treatment of hepatocytes with insulin increased expression of antioxidant enzymes, including alpha-class GSTs, mEH, and GCLC (Kim et al., 2003a,b, 2004). Thus, insulin plays an important role in the regulation of these protective enzymes. Oxidative stress, defined as an imbalance between the production of highly reactive molecular species and antioxidant defenses, is increased in diabetic conditions and is a major factor contributing to the chronic complication of diabetes (Baynes and Thorpe, 1999). It has been reported that oxidative stress observed in the diabetic condition is ameliorated with insulin administration (Sano et al., 1998; Otsyula et al., 2003). The present results, in conjunction with our previous studies, suggest that impairment of the antioxidant defense system, vis-à-vis lower insulin, may be a contributing factor to the increased oxidative stress and incidence of hepatic disease that may occur during diabetes.

The PI3K inhibitors LY294002 and wortmannin completely inhibited the insulin-mediated increase in alpha-class GST protein expression and GST activity, implicating PI3K as an obligatory kinase component in the regulation of alpha-class GST expression by insulin. Moreover, overexpression of the dominant-negative and kinase-dead Akt in hepatocytes and treatment of hepatocytes with rapamycin, an inhibitor of mammalian target of rapamycin, resulted in a decline in the insulin-mediated increase in alpha-class GST protein, suggesting that Akt and the downstream p70S6K are involved in the insulin-mediated increase in alpha-class GST expression. Kang et al. (2001, 2002) reported that 0.1 µM insulin induced rGSTA2 at 12 to 24 h in H4IIE rat hepatoma cell line and suggested that PI3K and Akt served as an essential pathway for the induction of alpha-class GSTA2 in response to oxidative stress induced by tert-butylhydroquinone. These results suggest that PI3K signaling pathways may be responsible for regulation of alpha-class GST expression in pathophysiological conditions, resulting in alteration of insulin secretion/response as well as oxidative stress.

Our previous studies implicated insulin signaling pathways involving PI3K/Akt/p70S6K in the insulin-mediated regulation of mEH expression and GSH synthesis through induction of GCLC (Kim et al., 2003a, 2004). These results, in conjunction with the present study, suggest that the insulin-mediated PI3K signaling pathways play a critical role in protection of hepatocytes against oxidative stress through induction of antioxidant defense system, including GSH and antioxidant enzymes.

The activation of PI3K results in the production of 3-phosphorylated phosphatidylinositides, which then serve to participate in the activation of a variety of downstream effectors including Akt, a serine/threonine protein kinase (Cantley, 2002). The Akt signaling pathway is recognized as one of the most critical pathways in regulating cell survival triggered by insulin and growth factors. Akt activity protects against apoptosis through its phosphorylation and inhibition of proapoptotic mediators such as Bad, a member of the Bcl-2 family (del Peso et al., 1997), forkhead family of transcription factors (Biggs et al., 1999), transcription factor Yes-associated protein (Basu et al., 2003), and apoptosis signal-regulating kinase1, a mitogen-activated protein kinase kinase kinase (Kim et al., 2001). Oxidative stress is one of the major factors effecting cell survival. Impairment of antioxidant defense systems, including production of GSH and antioxidant enzymes, increases cell vulnerability to oxidative stress-induced cell death (Sharma et al., 2004). The present results, in conjunction with our previous studies (Kim et al., 2003a, 2004), show that insulin signaling pathways involving PI3K/Akt are active in the insulin-mediated up-regulation of antioxidant defense system, raising the possibility that increased expression of antioxidant defense system may be involved in Akt-induced cell survival.

In the present study, rapamycin produced a partial inhibition of the insulin-mediated increase in alpha-class GST protein, suggesting that p70S6K, a downstream effector of PI3K, may also play a role in the insulin-mediated increase in alpha-class GST protein expression. The p70S6K catalyzes the phosphorylation of the S6 protein, a component of the 40S subunit of eukaryotic ribosomes and, thus, plays a role in protein synthesis in response to nutrients and hormones (Shah et al., 2000). These results raise the possibility that the insulin effects on alpha-class GST levels may be related to increased translational capacity through activation of p70S6K.

A number of investigations have indicated that an individual's GST genotype and expression may be associated with altered susceptibility to various diseases, such as acute xenobiotic toxicity, cardiovascular and respiratory disease, and cancer (Eaton and Bammler, 1999; Hayes et al., 2005). Alpha-class GSTs, one of the major classes of GSTs expressed in adult liver, can effectively reduce lipid peroxides through their peroxidase activity (Yang et al., 2001). 4-Hydroxynonenal, the most abundant 4-hydroxyalkenal formed in cells, is a toxic end product of lipid peroxidation contributing to the deleterious effects of oxidative stress (Leonarduzzi et al., 2004). Awasthi et al. (2004) have suggested that 4-hydroxynonenal is involved in the mechanisms of stress-mediated signaling and that it can be modulated by alpha-class GSTs through the regulation of the intracellular concentrations of 4-hydroxynonenal. These results, in conjunction with the results of the present study, warrant further examination for determining the role of alpha-class GST induction in insulin-mediated inhibition of cell death induced by oxidative stress through regulation of 4-hydroxynonenal levels.

In summary, the results of the present study indicate that insulin treatment for 4 h is sufficient to initiate increased expression of alpha-class GST proteins in primary cultured rat hepatocytes. Although transcription factors/coactivators may be involved, these data suggest that phosphorylation of these factors may play a role in the regulatory machinery for governing GST expression. The results of the study support the concept that signaling pathways and components play an important role in the regulation of basal GST expression. Moreover, this study implicates insulin signaling pathways involving PI3K/Akt/p70S6K in the insulin-mediated regulation of alpha-class GST expression. To our knowledge, this is the first report of the identification of the signaling pathways involved in mediating the effect of insulin on alpha-class GST expression in hepatocytes. These results, especially in conjunction with our previous studies demonstrating the signaling pathways involved in insulin-mediated elevation of mEH and GCLC expression (Kim et al., 2003a, 2004), suggest that PI3K/Akt/p70S6K is active in the insulin-mediated up-regulation of antioxidant defense system and that diabetic insulin levels, which elevate CYP2E1 and oxidative stress, also result in lower levels of alpha-class GSTs, GCLC, and GSH, all of which collectively conspire to progressively increase the risk of cellular damage in diabetes.

	Footnotes

 
This work was supported by National Institutes of Health Grant ES03656 and by the Cell Culture and Gene Transfer Technologies and Imaging and Cytometry Facility Cores of the Environmental Health Sciences Center Grant P30 ES06639 from the National Institute of Environmental Health Sciences.

doi:10.1124/jpet.105.096065.

ABBREVIATIONS: GST, glutathione S-transferase; GSH, glutathione; PI3K, phosphatidylinositol 3-kinase; p70S6K, ribosomal p70 S6 kinase; PKC, protein kinase C; GCLC, -glutamylcysteine ligase catalytic subunit; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SB202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; mEH, microsomal epoxide hydrolase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PD98059, 2'-amino-3'-methoxyflavone; SP600125, 1,9-pyrazoloanthrone; NBD, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; AdV-Akt, adenovirus containing a dominant-negative and kinase-dead mutant of Akt; MOI, multiplicity of infection; AdV-GFP, GFP-expressing adenovirus; PBS-T, 0.05% Tween 20 in phosphate-buffered saline.

¹ Current affiliation: College of Pharmacy and Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon, South Korea.

Address correspondence to: Dr. Raymond F. Novak, Institute of Environmental Health Sciences, Wayne State University, 2727 South Avenue, Room 4000, Detroit, MI 48201. E-mail: r.novak{at}wayne.edu

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