Respiratory chain activity and mitochondrial DNA content of nonpurified and purified pancreatic islet cells

Abstract

Considerable interest has recently focused on the possible role of alterations in mitochondrial activity and mutations in the mitochondrial genome for the development of non—insulin-dependent diabetes. Our study aimed at investigating the normal mitochondrial respiratory chain activity of nonpurified and purified islet cells to further explore whether some diabetic states are associated with alterations of mitochondrial oxidative processes. For this purpose, pancreatic islets were isolated from Wistar rats. Unpurified islet cells were obtained in the presence of trypsin and DNAse, and purified β and non- β cells were prepared by autofluorescence-activated sorting using a flowcytometer. Intact cell respiration and substrate oxidation in digitonin-permeabilized cells were measured polarographically with a Clark oxygen electrode in a micro—water-jacketed cell. Specific activity of the individual complexes of the respiratory chain was determined spectrophotometrically in unpurified islet cells. The relative amount of mitochondrial (mtDNA) and nuclear (nDNA) DNA in all three cell populations and in rat brain and skeletal muscle was estimated by dot blotting. The intact cell respiration of unpurified islet cells corresponds to the mean of values obtained for β and non- β islet cells. Oxidation rates of different substrates by permeabilized β cells were lower than those for unpurified and non- β cells. The amount of mtDNA relative to nDNA was similar in all three groups of cells, and was also similar to that obtained from brain and skeletal muscle. In summary, we have described mitochondrial respiratory chain activity in unpurified, β, and non- β islet cells. Our results represent an initial step in investigating the potential pathogenic role that alterations in oxidative phosphorylation could play in some diabetic states.