Long Exposure to High Glucose Concentration Impairs the Responsive Expression of γ-Glutamylcysteine Synthetase by Interleukin-1β and Tumor Necrosis Factor-α in Mouse Endothelial Cells

Urata, Yoshishige; Yamamoto, H.; Goto, Shinji; Tsushima, Hiroyuki; Akazawa, Shouichi; Yamashita, Shunichi; Nagataki, Shigenobu; Kondo, Takahito

doi:10.1074/jbc.271.25.15146

Cited by 134 publications

(100 citation statements)

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“…A decrease in ␥-GCS mRNA expression and increase in DNA damage accompany this reduction. The decrease observed is in agreement with published results; Yoshida et al [15] found GSH levels 77% of controls in erythrocytes from diabetic patients, Urata et al [14] reported GSH levels 59% of normal glucose after 7 d in 22 mM glucose, and Tachi et al [25] found a decrease of 20% in HVSMC exposed to 27.5 mM glucose for 10 d. The depletion of GSH may be brought about through a variety of mechanisms, including an increased breakdown of GSH by ␥-glutamyltranspeptidase, an increased export of GSSG from the cell, or a reduction in de novo synthesis through reduced activity of ␥-GCS or availability of substrate. In human vascular smooth muscle cells, there is no significant difference in the rate of GSH efflux between cells exposed to high glucose compared to those in normal glucose [25].…”

Section: Discussionsupporting

confidence: 92%

“…Induction of ␥-GCS expression by oxidative stress is well established [38 -40], however exposure to high-glucose conditions decreases mRNA expression of both subunits in HVSMC. Urata et al [14] found that high concentrations of glucose in endothelial cells decreased GCS H mRNA expression and inhibited the stimulatory effect of IL-1␤ and TNF-␣ on induction of ␥-GCS. These results would suggest that glucose itself may have a direct effect on transcriptional regulation of both ␥-GCS subunits, but further investigation is required.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have demonstrated the reductions in gene expression of the heavy subunit of ␥-GCS and activity of ␥-GCS in high-glucose conditions [13][14][15]. However, regulation of ␥-GCS activity at the mRNA level with respect to both the light and heavy subunits has not been investigated in high glucose conditions.…”

Section: Introductionmentioning

confidence: 99%

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Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Powell

Nally

McMaster

et al. 2001

Free Radical Biology and Medicine

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Abstract-Hyperglycemia-induced oxidative stress may play a key role in the pathogenesis of diabetic vascular disease. The purpose of this study was to determine the effects of glucose on levels of glutathione (a major intracellular antioxidant), the expression of ␥-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione de novo synthesis), and DNA damage in human vascular smooth muscle cells in vitro. High glucose conditions and buthionine sulphoximine, an inhibitor of ␥-glutamylcysteine synthetase, reduced intracellular glutathione levels in vascular smooth muscle cells. This reduction was accompanied by a decrease in the mRNA expression of both subunits of ␥-glutamylcysteine synthetase as well as an increase in DNA damage. In high glucose conditions, incubation of the vascular smooth muscle cells with ␣-lipoic acid and L-cystine restored glutathione levels. We suggest that the decrease in GSH levels seen in high glucose conditions is mediated by the availability of cysteine (rate-limiting substrate in de novo glutathione synthesis) and the gene expression of the ␥-glutamylcysteine synthetase enzyme. Glutathione depletion is associated with an increase in DNA damage, which can be reduced when glutathione levels are restored.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

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Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Powell

Nally

McMaster

et al. 2001

Free Radical Biology and Medicine

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“…The regulation of GCL expression and activity has been studied in many other cell types, including mesangial and endothelial cells, but not the pancreatic islet. Long term exposure to high glucose levels decreases GCL expression in mesangial, retinal Muller, and endothelial cells, which results in a decrease in GSH levels (3)(4)(5). Long term exposure to high glucose conditions inhibits endothelial cells from responding to cytokine exposure with an increase in GCL expression and activity (3).…”

mentioning

confidence: 99%

Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress

Tran

Parker

Leroy

et al. 2004

Journal of Biological Chemistry

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The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1␤ (IL-1␤). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1␤ up-regulated GCLC expression (10 ng/ml IL-1␤, 3.76 ؎ 0.86; 100 ng/ml IL-1␤, 4.22 ؎ 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF B and also increased reactive oxygen species levels (10 ng/ml IL-1␤, 5.41 ؎ 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 ؎ 0.1; 10 ng/ml IL-1␤, 8.0 ؎ 0.5; 100 ng/ml IL-1␤, 8.2 ؎ 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1␤ on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1␤ significantly decreased glucose-stimulated insulin secretion (control, 123.8 ؎ 17.7; IL-1␤, 40.2 ؎ 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1␤. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1␤.Glutamylcysteine ligase (GCL) 1 is the primary and ratelimiting enzyme responsible for de novo synthesis of intracellular glutathione (GSH). This enzyme catalyzes ATP-dependent ligation of L-glutamate and L-cysteine to form ␥-glutamyl-L-cysteine, which undergoes another ATP-dependent ligation with glycine catalyzed by glutathione synthetase to form the final GSH product (1, 2). GCL is a heterodimer, composed of a light regulatory subunit as well as a heavy catalytic subunit. Although both are required for optimal enzyme activity, overexpression of the catalytic subunit GCLC alone is sufficient to increase enzyme activity significantly over control levels (1). The regulation of GCL expression and activity has been studied in many other cell types, including mesangial and endothelial cells, but not the pancreatic islet. Long term exposure to high glucose levels decreases GCL expression in mesangial, retinal Muller, and endothelial cells, which results in a decrease in GSH levels (3-5). Long term exposure to high glucose conditions inhibits endothelial cells from responding to cytokine exposure with an increase in GCL expression and activity (3). That the pathogenesis of diabetes mellitus involves interactions with IL-1␤ and oxidative stress secondary to chronic hyperglycemia (6, 7) raises the questions of whether GCL is normally expressed in the islet and whe...

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“…8,12,13 Important cis-acting elements implicated in mediating the effect of oxidative stress on the promoter activity of the GCS subunits are antioxidant response element (ARE) and activator protein-1 (AP-1). 8,9,13 Nuclear factor-B (NF-B) may 10,14,15 or may not 9,16,17 regulate the heavy subunit. All studies have used cell lines transfected with recombinant GCS subunit promoterreporter gene constructs.…”

mentioning

confidence: 99%

Effect of ethanol and high-fat feeding on hepatic γ-glutamylcysteine synthetase subunit expression in the rat

Lu¹,

Huang²,

Yang³

et al. 1999

Hepatology

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Glutathione (GSH) is important in antioxidant defense. A major determinant of the rate of GSH synthesis is the activity of the rate-limiting enzyme, ␥-glutamylcysteine synthetase (GCS). A heavy (HS) and light subunit (LS) make up GCS; oxidative stress regulates both transcriptionally. Cis-acting elements important for the oxidative stressinduced transcriptional up-regulation of both subunits are antioxidant response element (ARE) and activator protein-1 (AP-1). Nuclear factor-B (NF-B) may also regulate the heavy subunit. Chronic ethanol ingestion causes oxidative stress, increases AP-1 expression, and depletes hepatic GSH. Data conflict regarding GSH synthesis and are lacking regarding GCS subunit gene expression. We examined the effect of chronic ethanol ingestion on ARE, AP-1, and NF-B activity and GCS subunit expression. Male Wistar rats were fed an ethanol and high-fat (28.7% cal) diet intragastrically for 9 weeks. Liver GSH level fell by 40%, although GCS activity doubled. GCS-HS mRNA level doubled, whereas GCS-LS mRNA level remained unchanged. Electrophoretic mobility shift assay (EMSA) showed that binding to ARE, AP-1, and NF-B probes all increased. In conclusion, chronic ethanol ingestion increased GCS-HS expression and GCS activity by activating cis-acting elements important for transcriptional up-regulation of GCS-HS. GCS-LS mRNA level remained unchanged despite activation of ARE and AP-1, suggesting that negative transcriptional factors may be involved or the mRNA may be unstable. Despite induction in GCS activity, GSH level fell because of alterations in the other factors important in determining the steady-state GSH level. (HEPATOLOGY 1999;30:209-214.) Glutathione (GSH) is a tripeptide, ␥-glutamylcysteinylglycine, which defends against toxins and free radicals. 1 The cellular GSH level is determined by a balance of the rate of its synthesis and the rates of its utilization (conjugation) and loss (export). 2 Synthesizing GSH from its constituent amino acids involves two adenosine triphosphate-requiring enzymatic steps: forming ␥-glutamylcysteine from glutamate and cysteine, and forming GSH from ␥-glutamylcysteine and glycine. The rate of GSH synthesis is determined by the availability of cysteine and the activity of the rate-limiting enzyme, ␥-glutamylcysteine synthetase (GCS), which is subject to feedback-competitive inhibition by GSH (K i ϭ 2.3 mmol/L). 1-3 The GCS enzyme is composed of a heavy (Mr ϳ 73,000) and a light (Mr ϳ 30,000) subunit that are encoded for by different genes and dissociate under reducing conditions. 4,5 The heavy subunit (HS) exhibits all of the catalytic activity of the isolated enzyme as well as the feedback inhibition by GSH. 6 The light subunit (LS) is enzymatically inactive but plays an important regulatory function by lowering the K m of GCS for glutamate and raising the K i for GSH. 5,7 Recent studies have shown that both GCS subunits are up-regulated transcriptionally by oxidative stress. 1,[8][9][10][11][12][13] The 5Ј-flanking region of both human GCS subunits ha...

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Long Exposure to High Glucose Concentration Impairs the Responsive Expression of γ-Glutamylcysteine Synthetase by Interleukin-1β and Tumor Necrosis Factor-α in Mouse Endothelial Cells

Cited by 134 publications

References 43 publications

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress

Effect of ethanol and high-fat feeding on hepatic γ-glutamylcysteine synthetase subunit expression in the rat

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