Amelioration of hyperglycemia-induced mitochondrial ROS generation of single pancreatic islet β-cells of obese/diabetic mice by chronic N-Acetyl-L-Cysteine

Abstract

BACKGROUND: Mitochondria are the principal source of reactive oxygen species (ROS) in pancreatic islets β-cells and impairment of mitochondrial functions is intrinsically related with diabetes mellitus. Hyperglycemiainduced ROS production by mitochondria is an important aspect in β-cell glucose toxicity. However, most previous studies were performed in either normal islets/single β-cells or insulinoma cells which were bathed in high glucose medium which could not mimic the pathophysiological conditions. OBJECTIVES: To compare and measure hyperglycemia-induced mitochondria ROS generation of primary pancreatic islet β-cells of obese/diabetic (+db/+db) and lean/control (+db/+m) mice, and the effects (acute and chronic) of N-acetyl-L-cysteine (NAC) on ROS generation. METHODS: Collagenase-dissociated single pancreatic islet β-cells of C57BL/KsJ obese/diabetic (+db/+db) mice which exhibit phenotypes of the human T2DM were harvested, and the effects (acute, 10 min; chronic, 24 h) of NAC (20 mM) on high glucose-induced mitochondrial ROS generation were evaluated. Mitochondrial ROS levels were estimated by MitoTracker Red (reduced form) (a selective fluorescence probe for mitochondrial ROS measurement) using con-focal microscope. RESULTS: A trend of, but a non-significant, higher resting/basal ROS level was observed in single pancreatic β-cells of +db/+db mice compared to +db/+m mice. High glucose (15 mM) application gradually caused an increase in ROS levels in single pancreatic β-cells of +db/+db mice whereas no apparent change was observed in +db/+m mice. Chronic (24 h), but not acute (10 min), treatments with NAC (20 mM) ameliorated high-glucose induced ROS generation in single pancreatic β-cells of +db/+db mice. CONCLUSIONS: Hyperglycemia elicited mitochondrial ROS generation only in single pancreatic β-cells of +db/+db mice. Chronic NAC (a well known anti-oxidant) pre-treatment eradicated high glucose-induced ROS generation. Current study is underway to elucidate the underlying mechanism(s) involved in the differential effects of high glucose on ROS generation as well as the identification of the particular mitochondrial ROS generating system.