A role for glutathione peroxidase in protecting pancreatic β cells against oxidative stress in a model of glucose toxicity

Abstract

Antioxidant drugs have been reported to protect pancreatic islets from the adverse effects of chronic exposure to supraphysiological glucose concentrations. However, glucose has not been shown to increase intracellular oxidant load in islets, nor have the effects of increasing or inhibiting glutathione peroxidase (GPx) activity on islet resistance to sugar-induced oxidant stress been studied. We observed that high glucose concentrations increased intracellular peroxide levels in human islets and the pancreatic ␤ cell line, HIT-T15. Inhibition of ␥-glutamylcysteine synthetase (␥GCS) by buthionine sulfoximine augmented the increase in islet peroxide and decrease in insulin mRNA levels, content, and secretion in islets and HIT-T15 cells induced by ribose. Adenoviral overexpression of GPx increased GPx activity and protected islets against adverse effects of ribose. These results demonstrate that glucose and ribose increase islet peroxide accumulation and that the adverse consequences of ribose-induced oxidative stress on insulin mRNA, content, and secretion can be augmented by a glutathione synthesis inhibitor and prevented by increasing islet GPx activity. These observations support the hypothesis that oxidative stress is one mechanism for glucose toxicity in pancreatic islets. T ype 2 diabetes mellitus is polygenic in origin and usually begins in adulthood, although specific genes for subtypes of this disease that occur earlier in life, referred to collectively as maturity-onset diabetes of the young (MODY), have been identified (1). The onset of type 2 diabetes is insidious, thus hyperglycemia develops gradually and often goes untreated for years until symptoms become clinically obvious. Consequent chronic exposure of tissues to supraphysiologic levels of blood glucose can lead to adverse intracellular outcomes, a process known as glucose toxicity (2-5). Possible mechanisms of action for glucose toxicity include the formation of advanced glycosylation end products and glucosamine, increased protein kinase C activity with c-myc induction, autooxidation of glucose, and increased levels of reactive glycolytic intermediates such as glyceraldehyde-3-phosphate or dihydroxyacetone phosphate (6 -13). All these processes usually are accompanied by the formation of reactive oxygen species (ROS), setting up the potential for oxidative stress. Many weeks of exposure to high concentrations of glucose are necessary before glucotoxic effects are expressed by islet ␤ cells in vitro and in vivo (14 -20). Because isolated islets do not reliably survive in culture greater than 2 weeks, the use of short-term exposure to ribose, a sugar that generates ROS more potently than glucose, has become an accepted model for studying islet glucose toxicity (21,22). The use of ribose as a prooxidant sugar is especially valuable in adenoviral overexpression systems, because the overexpression effect is short-lived, lasting only days.In the pancreatic ␤ cell, glucose and ribose toxicity cause decreased insulin mRNA levels (14-22), one mechanism ...

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice

“…Because in previous research only post-transcriptional regulation of Pdx1 expression and function by ROS or antioxidant has been suggested [6,20,23,25], our finding on the hyperacetylation of H3 and H4 histone at the proximal promoter of Pdx1 in the OE mice reveals not only a novel, in vivo epigenetic, but also a potential transcriptional regulation for this key factor by antioxidant enzymes. Preceding transcriptional activation [43], hyperacetylation of H3 and H4 helps remodel the chromatin at Pdx1 promoter to form a more accessible structure for transcription [44].…”

“…Infection of rat islets with adenovirus encoding human GPX1 gene protected against the ribose-induced loss of insulin mRNA, content and secretion. However, a sixfold increase in GPX1 activity and 72 h infection seemed to be insufficient to alter baseline levels of these three parameters [6,24].…”

“…The released insulin (after 60 min incubation) in the medium supernatant fraction was quantified as described above. Intracellular ROS levels were detected using dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR, USA) [6]. Islet Δ= m was determined using a mitochondrial membrane sensor kit (ApoAlert; Clontech Laboratories, Mountain View, CA, USA).…”

“…However, overproduction of antioxidant proteins in animals, either specifically in pancreatic beta cells [5,6] or ubiquitously in various tissues [7,8], has generated highly variable or even negative phenotypes [9]. Most strikingly, we have observed spontaneous development of hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in mice overproducing Se-dependent cellular glutathione peroxidase-1 (GPX1), a major intracellular antioxidant enzyme [10].…”

“…Uncoupling protein 2 (UCP2) serves as a negative regulator of mitochondrial membrane potential (Δ= m ) [18], which positively correlates with glucosestimulated insulin secretion (GSIS) [19]. Levels and function of PDX1 and UCP2 are affected by intracellular ROS status [20,21], glucotoxicity [22] and antioxidants [6,23,24]. However, it is not known whether in vivo global overexpression of Gpx1 could overly diminish intracellular production of hydroperoxides and subsequently dysregulate production of PDX1 and UCP2 and their role in insulin synthesis and secretion.…”