Insulin-Producing Cells, Iron, Oxidative Stress, and Lysosomal Pathology

Abstract: Accumulating evidence suggests that injnries caused by oxygen-derived radicals contribute to the destruction of pancreatic islet ß-cells in autoinnnune diabetes mellitus (diabetes type I, or IDDM). Oxidative stress may be caused by an enhanced production of oxygen-derived radicals, or by a decreased scavenging of such molecules. It was recently suggested that iron-mediated intralysosomal oxidative reactions result in the destabilization of lysosomal membranes, leakage of lysosomal contents to the cytosol, cellular destruction and, moreover, that such mechanisms may operate in IDDM.In the present study, we have investigated the mechanisms by which hydrogen peroxide induces cell damage, and its possible relationship to intralysosomal iron. The work was done on three insulin-producing insulinoma cell lines: HIT-TI5, NIT-1, and RINmF cells, on mouse pancreatic islets ß-cells, and the macrophage-like J-774 cells. In particular, we studied the influence of induced autophagocytosis (by glucose- and amino acid starvation) on the sensitivity to oxidative stress; the influence of high-glucose growth media on hydrogen peroxide cytotoxicity; the protective effects by starvation-stimulated intracellular fertitin synthesis against oxidative stress; the possible relationship between oxidative stress, lysosomal destabilization and apoptotic/necrotic cell death; and the impact of iron chelation on lysosomal stability, and insulinoma- and ß-cell survival.A higb susceptibility to oxidative stress was demonstrated for all the insulin-producing cells. Starvation-induced autophagocytosis increased the concentration of desfertioxarnine-available lowmolecular- weight iron in HIT-T15 cells, as assayed by HPLC. Tbe iron was mainly found in secondary lysosomes, as shown by the autometallography technique when applied at electron nticroscopical level. Starvation enhanced oxidative stress-induced damage of the IDT-T15, RINm5F and J-774 cells, as assayed by the trypan blue dye exclusion test and tests for lysosomal stability (the actidine orange relocation/uptake tests). In contrast, the pronounced starvationinduced autophagocytosis that was shown by the most vulnerable insulinoma cell line (NIT -1) was paralleled by enhanced resistance to oxidative stress, and by increased lysosomal stability as well. A rapid NIT -1 fertitin synthesis was demonstrated by inununocytochentistry under conditions of starvation. It is believed that autophagocytotic lysosomal uptake of non-iron-saturated fertitin will allow such fertitin to act as an iron chelator and stabilize lysosomes against oxidative stress. NIT -1 and B-cells which were subjected to a low level of oxidative stress (30 J.LM H20 2 for 15 min) were still largely intact at the light nticroscopical level ,but 10-20% of the cells exhibited nuclear chromatin condensation as an early sign of apoptosis when examined by the Ho334/PI staining technique, or by TEM, 0.5-1 h after the insult. At the same point of time, a decrease in the number of intact lysosomes was also observed. The rate of oxidative stress-induced lysosomal destabilization progressed with time, and a widespread apoptotic/necrotic-type degeneration/fragmentation ensued, as demonstrated by SEM, TEM, and the TUNEL-reaction. The ntitochondria revealed a mixture of lamelliform and swollen cristae, indicating altered properties of the mitochondrial membranes. Pre-treatment with the iron chelator desfenioxamine attenuated the lysosomal destabilization, and increased cell viability, following exposure to oxidative stress.At high-glucose conditions, the ~02-sensitivity of HIT-T15, NIT-I, and B-cells was reduced which, was consistent with a moderately enhanced stability of their lysosomes, as measured by the acridine orange-relocation test, and with reduced amounts of desfenioxamine-available iron.We conclude that the decisive role of free lysosomal iron in oxidative stress is strongly supported by the following lines of evidence, provided by the present study (a) glucose- and amino acid-starvation promotes autophagic/crinophagic activity of the cells, resulting in enrichment of intracellular (intralysosomal) desfenioxamine-available iron; (b) high-glucose conditions depress autophagic/crinophagic activity and, consequently, the occurrence of intralysosomal iron; (c) starvation-stimulated fenitin synthesis enhances lysosomal stability during oxidative stress by limiting lysosomal redox-active iron; (d) lysosomal destabilization and related apoptotic cell death are associated with the amounts of intralysosomal iron in redox -active form,