Abstract
Decreased glutathione (GSH) levels and γ-glutamylcysteine ligase (GCL) activity have been observed in diabetic patients, and insulin reportedly increases GSH synthesis via increased GCL catalytic subunit (GCLC) gene expression. The signaling pathways responsible for mediating insulin effects on GCLC expression and GSH levels, however, are unknown. The signaling pathways involved in the regulation of GSH synthesis in response to insulin were examined in primary cultured rat hepatocytes. GSH levels, GCL activity, GCLC protein, and mRNA levels were increased to 140, 160, 600, and 340% of that monitored in untreated cells, respectively, in hepatocytes cultured with 100 nM insulin. The phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002 [2-(4-morpholinyl)-9-phenyl-4H-1-benzopyran-4-one], dominant-negative Akt, or rapamycin, an inhibitor of mTOR (mammalian target of rapamycin) and ribosomal p70 S6 kinase (p70S6K) phosphorylation, inhibited the insulin-mediated increase in GCLC protein and GSH levels. Although the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase, p38 MAPK, and JNK (c-Jun N-terminal kinase) were activated in response to insulin, PD98059 (2′-amino-3′-methoxyflavone), an inhibitor of mitogen-activated protein kinase kinase, SP600125 (1,9-pyrazoloanthrone), an inhibitor of JNK, and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], an inhibitor of p38 MAPK, failed to inhibit the insulin-mediated increase in GCLC protein levels. In conclusion, these data show that insulin signaling pathways involving PI3K/Akt/p70S6K, but not MAPKs, are active in the insulin-mediated regulation of GSH synthesis via increased GCLC expression.
Footnotes
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This work was supported by the National Institutes of Health Grant ES03656 to R.F.N. and by the Cell Culture, Imaging and Cytometry, and Microarray and Bioinformatics Facility Cores of Environmental Health Sciences Center Grant P30 ES06639 from the National Institute of Environmental Health Sciences.
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doi:10.1124/jpet.104.070375.
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ABBREVIATIONS: GSH, glutathione; GCL, γ-glutamylcysteine ligase; GCLC, GCL catalytic subunit; GCLR, GCL regulatory subunit; PI3K, phosphatidylinositol 3-kinase; p70S6K, ribosomal p70 S6 kinase; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; PD98059, 2′-amino-3′-methoxyflavone; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene; SP600125, 1,9-pyrazoloanthrone; DMSO, dimethyl sulfoxide; AdV-GFP, GFP-expressing adenovirus; AdV-Akt, adenovirus containing a dominant-negative kinase-dead mutant of Akt; MOI, multiplicity of infection; MEK, mitogen-activated protein kinase kinase; PCR, polymerase chain reaction; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.
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↵1 Present address: College of Pharmacy and Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon, 305-764, South Korea.
- Received April 20, 2004.
- Accepted May 27, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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