s-nitro-n-acetylpenicillamine and Diabetes-Mellitus--Type-1

Several reports propose that apoptosis of pancreatic beta cells may play a central role in the pathogenesis of both spontaneous and induced insulin-dependent diabetes mellitus (IDDM) in animal models. Whether apoptosis is a major cell death pathway during diabetes development, however, is highly controversial. The aim of this study was to examine the mode of islet cell death in prediabetic diabetes-prone (dp) BB rats, which spontaneously develop diabetes and serve as an animal model for human IDDM. In addition we investigated the cell death pathway of islet cells treated with the widely used diabetogenic compound streptozotocin or with nitric oxide (NO), which during IDDM development has been found to be present in inflamed islets in high concentrations because of the expression of inducible NO synthase. Islets of prediabetic BBdp rats were analyzed for DNA strand breaks and screened by electron microscopy. The mode of islet cell death in vitro after treatment with cytotoxic concentrations of streptozotocin or of NO was investigated using different methods including morphologic analysis by electron microscopy, detection of DNA strand breaks, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Although cells with DNA stand breaks-often accepted as a proof for apoptosis-could be identified, we did not find apoptosis-specific features during islet cell death. Instead we observed massive necrosis as evidenced by disrupted plasma membranes and spilled-out cellular constituents in vitro as well as during disease manifestation in BBdp rats. These results may have serious consequences with regard to the treatment of humans to prevent the development of IDDM.

Topics: Animals; Annexin A5; Apoptosis; Blotting, Western; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; DNA Fragmentation; Flow Cytometry; In Situ Nick-End Labeling; Islets of Langerhans; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Necrosis; Nitric Oxide; Penicillamine; Poly(ADP-ribose) Polymerases; Rats; Rats, Inbred BB; Rats, Wistar; Streptozocin

An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. In the present study, we show evidence that supports a tight link between NO, Ca2+, protease and apoptosis in beta-cells. Three different NO donors, SNAP, NOR3 and NOC7, induced apoptosis in a beta-cell line, MIN6 cells, in a concentration-dependent manner. SNAP at 200 microM increased cytosolic Ca2+ concentration ([Ca2+]i) and induced apoptosis. The SNAP-induced apoptosis was blocked by a Ca2+ chelator, BAPTA-AM, and by an inhibitor of a Ca2+-dependent protease, calpain. In conclusion, an excessive NO production induces apoptosis, wherein an increase in [Ca2+]i and resultant activation of calpain play a key role.

Topics: Animals; Apoptosis; Calcium; Cell Survival; Chelating Agents; Diabetes Mellitus, Type 1; Egtazic Acid; Insulinoma; Islets of Langerhans; Mice; Nitric Oxide; Nitric Oxide Donors; Pancreas; Pancreatic Neoplasms; Penicillamine; Tumor Cells, Cultured