diethyl-maleate and Hyperglycemia

The deficiency of glutathione (GSH) has been linked to several diseases. The study investigated the role of GSH as a protective factor against hyperglycemia-mediated injury in VL-17A cells treated with 50 mM glucose.. The cell viability and different oxidative stress parameters including glyoxalase I activity were measured.. GSH supplementation with 2 mM N-acetyl cysteine (NAC) or 0.1 mM ursodeoxycholic acid (UDCA) increased the viability, GSH level and the GSH-dependent glyoxalase I activity in 50 mM glucose-treated VL-17A cells. Further, pretreatment of 50 mM glucose-treated VL-17A cells with NAC or UDCA decreased oxidative stress (levels of reactive oxygen species and protein carbonylation), apoptosis (caspase 3 activity and annexin V-propidium iodide positive cells) and glutathionylated protein formation, a measure of oxidative stress. GSH depletion with 0.4 mM buthionine sulfoximine (BSO) or 1 mM diethyl maleate (DEM) potentiated the decrease in viability, glyoxalase I activity and increase in oxidative stress and apoptosis, with decreased GSH levels in 50 mM glucose-treated VL-17A cells.. Thus, changes in GSH levels with exogenous agents such as NAC, UDCA, BSO or DEM modulate hyperglycemia-mediated injury in a cell model of VL-17A liver cells.

Topics: Acetylcysteine; Alcohol Dehydrogenase; Antimetabolites; Antioxidants; Apoptosis; Buthionine Sulfoximine; Cell Survival; Clone Cells; Cytochrome P-450 CYP2E1; Glucose; Glutathione; Hep G2 Cells; Hepatocytes; Humans; Hyperglycemia; Lactoylglutathione Lyase; Maleates; Oxidative Stress; Reactive Oxygen Species; Recombinant Proteins; Ursodeoxycholic Acid

To study regulation of gamma-glutamylcysteine synthetase (GCS) heavy and light subunit gene expression in Müller cells under conditions of oxidative stress.. Experiments were carried out with an SV40 transformed cell line (rMC-1) that exhibits the phenotype of rat retinal Müller cells. Endogenous glutathione levels were modified by treating cells with diethyl maleate (DEM), D,L-buthionine sulfoximine (BSO), or tert-butylhydroquinone (TBH). In other experiments, cells were grown in either high (28 mM) or normal (5.5 mM) glucose medium for 1 week to examine the effects of hyperglycemia. Cells were processed for reduced glutathione (GSH) measurement, RNA extraction, cell count, and, in some cases, lactate dehydrogenase activity. The steady state mRNA levels of GCS heavy and light subunits were measured by northern blot analysis using specific cDNA probes. Changes in mRNA levels were normalized to beta-actin or 18S rRNA.. Treatment with DEM for 30 minutes depleted cell GSH to 20% to 30% of the normal value. GSH content recovered completely 6 hours after returning to normal medium. BSO treatment for 12 hours followed by a medium change for 6 hours resulted in a cell GSH level that was 26% that of untreated cells. If cells were left in BSO for 18 hours, however, GSH levels were reduced to < 1%. Treatment with TBH for 12 hours led to a 77% increase in cellular GSH level. Treatment with DEM, TBH, or BSO for 18 hours led to a significant induction of the mRNA level of the GCS subunits, regardless of glucose concentration in the medium. Shorter BSO treatment exerted no effect. Prolonged hyperglycemia resulted in 30% lower GSH level, 55% lower GCS heavy subunit, and 30% lower GCS light subunit mRNA levels.. Oxidative stress induced the gene expression of GCS heavy and light subunits in Müller cells. The effect of BSO on mRNA levels correlated with the degree of GSH depletion. Prolonged hyperglycemia lowered GCS subunit mRNA and GSH levels.

Topics: Animals; Blotting, Northern; Buthionine Sulfoximine; Cell Line, Transformed; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase; Glutathione; Hydroquinones; Hyperglycemia; Maleates; Neuroglia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Retina; RNA, Messenger; Simian virus 40