Abstract
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 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 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 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 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 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 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 beta. 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 beta.
Highlights
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
GCLC Expression Level and IL- Regulation of GCLC Expression, GSH/GSSG Ratios, and Intraislet reactive oxygen species (ROS) Levels—The expression level of GCLC mRNA in pancreatic islets was less than that found in kidney and liver, and greater than that observed in lung, subcutaneous fat, and skeletal muscle (Fig. 1A)
Effects of Adenoviral Overexpression of GCLC on IL-1 Decreases in Insulin Secretion—To determine whether increased GCLC expression causes an increase in GSH levels and prevents the inhibitory effects of oxidative stress on beta cell function, islets were infected with adenovirus encoding the GCLC catalytic subunit
Summary
Vol 279, No 52, Issue of December 24, pp. 53988 –53993, 2004 Printed in U.S.A. Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress*. 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. We have reported that prolonged hyperglycemia or shorter exposure to ribose, a stronger reducing sugar, stimulates the formation of reactive oxygen species (ROS) in pancreatic islets [6] and causes beta cell dysfunction These excessive increases in intraislet ROS levels cause decreases in insulin mRNA levels, insulin content, and glucose-stimulated insulin secretion. IL-1-dependent changes in GCLC on ROS production, intraislet GSH levels, and insulin secretion; 2) whether IL-1 effects on GCLC gene expression depend on p38 MAPK and NF〉 activation; and 3) whether adenoviral overexpression of GCLC increases intraislet levels of GSH and mitigates the adverse effects of IL-1-induced oxidative stress on glucose-induced insulin secretion
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