1717-P: Protein Kinase C δ Activation by Nitric Oxide Mediates Pancreatic ß-Cell Apoptosis

In type 1 diabetes (T1D), autoreactive immune cells secrete cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ), that initiate β-cell apoptosis in pancreatic islets and contribute to disease progression. Protein kinase C delta (PKCδ) activation plays a pivotal role in mediating cytokine-induced β-cell apoptosis via nuclear translocation and activation of pro-apoptotic signaling. In other cell types, nitric oxide (NO) produced in response to cytokines can also activate PKCδ via nitrosylation of tyrosine; however, the exact modification sites and effects on apoptotic signaling have not been investigated in β-cells. We hypothesize cytokine-induced production of NO activates PKCδ leading to nuclear translocation and upregulation of apoptotic signaling.

Islets from WT mice, mice with a β-cell specific knockout of PKCδ (PKCδβ-KO), or human islets were cultured for 24hr with a NO donor (SNAP, 1mM), cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFN-γ), cytokines with an inducible nitric oxide synthase inhibitor (iNOSinh, 5mM), or an adenovirus to produce GFP-tagged PKCδ. Translocation was determined by GFP colocalization with a nuclear stain. Apoptosis was determined by fluorescent live/dead staining. PKCδ activity was determined with a FRET-based sensor in MIN6 cells. Nitrosylation of PKCδ was determined via a biotin switch assay and western blot.

Increases in PKCδ activity were observed in NO treated islets (p=0.05). Inhibiting PKCδ activation protected against β-cell apoptosis in cytokine and NO treated islets (p=0.004). Overall, our results support a role for cytokine induced NO in activating PKCδ and mediating downstream apoptotic β-cell death. Uncovering mechanisms to inhibit PKCδ activation that are associated with T1D can improve targeted protection of β-cells and reduce off target affects. Identifying novel mechanisms of cytokine-mediated apoptosis will provide insight into novel targets to prevent or delay β-cell death in T1D.