Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms

Abstract

We have shown that nitric oxide treatment for 30-90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24-72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.

Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

“…We have also found that NO donors decrease glucose oxidation (25) and redox function in insulin-containing cell lines and rat islets (6). NO donors have been reported to inhibit total protein synthesis in hepatocytes (27), vascular smooth muscle cells (28), and the insulin-containing cell line HIT-T15 (6). Furthermore, these donors caused necrotic cell death in rat islets in as little as 4 h when used at high doses (29).…”

“…Exposure of insulin-containing cell lines or islets of Langerhans to NOreleasing compounds, such as S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine (SIN-1), or Roussin's black salt, results in inhibition of glucose-induced insulin secretion, which is dose-and time-dependent (11,(24)(25)(26). We have also found that NO donors decrease glucose oxidation (25) and redox function in insulin-containing cell lines and rat islets (6). NO donors have been reported to inhibit total protein synthesis in hepatocytes (27), vascular smooth muscle cells (28), and the insulin-containing cell line HIT-T15 (6).…”

“…Furthermore, these donors caused necrotic cell death in rat islets in as little as 4 h when used at high doses (29). Low-dose NO donors induce apoptotic cell death in HIT-T15 cells and rat islets after as little as 24 h of exposure (5,6).…”

“…The calcium-independent inducible NOS (iNOS) or NOS2 is induced by IL-1␤ (2,(13)(14)(15)(16) and other cytokines (17) in insulin-containing cell lines and rat islets. The use of arginine analogs such as N G -monomethyl-L-arginine (NMMA) or N G -nitro-L-arginine (L-NAME) has shown that NO from iNOS participates in the following actions of IL-1␤: inhibition of insulin secretion (2,3,18), decreased activity of aconitase and glucose oxidation (9,10), induction of DNA damage (19,20), and necrotic (21) and apoptotic cell death (5,6,22,23) in insulin-containing cell lines and rat islets.…”

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

“…Therefore, following exposure to nitric oxide, IRE1 may also signal to TSC2 through AMPK to reduce mTOR activity. One consequence of this signaling would be the inhibition of protein synthesis, and it is well known that nitric oxide is an effective inhibitor of protein synthesis in many cell types, including ␤ cells (10,11). Additional studies will clarify the role for each of these pathways in response to nitric oxide and the role of this regulation in cellular recovery and survival following nitric oxide-mediated damage.…”