T1D is characterized by a loss of insulin-producing β-cells in pancreatic islets. The immune destruction of β-cells is partly mediated by high levels of Th1 cytokines. Previously we have shown that cytokines alter islet function via protein kinase C δ (PKCδ) dependent mechanisms. Previous studies have suggested a role for PKCδ in mediating β-cell death, however little is known about the pro-apoptotic role of PKCδ in T1D. I hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via translocation to the nucleus and cleavage into a constitutively active fragment.

To test this hypothesis, we utilized mice with an inducible β-cell specific knockout of PKCδ (PKCδ-βKO). Isolated WT and PKCδ-βKO islets were treated with 10ng/ml TNF-α, 5ng/ml IL-1β, and 100ng/ml IFN-γ for 24h. Human islets were treated with 10μM of the PKCδ inhibitor δV1-1 for 24h with or without cytokines. PKCδ-βKO islets were virally transduced with GFP tagged PKCδ and incubated with or without cytokines for 3 or 24h. MIN6 cells were transfected with a FRET-based sensor for PKCδ and were incubated with or without cytokines.

Cytokine treatment increased β-cell apoptosis in mouse (p=0.003) and human islets (p=0.007). Isolated PKCδ-βKO islets showed significantly reduced cytokine-induced apoptosis compared to WT controls (p=0.003). Human islets treated with δV1-1 had reduced cytokine-induced apoptosis (p=0.020) compared to cytokine treatment alone. After 24h cytokine treatment PKCδ translocated to the nucleus (p=0.003) where it showed increased activity as measured by FRET, and an increase in a ~40kDa cleavage product of PKCδ as measured by western blot.

In conclusion, our results implicate a role for PKCδ in mediating cytokine-induced β-cell apoptosis via translocation to the nucleus and cleavage into a constitutively active fragment in both mouse and human islets. The role of PKCδ in mediating cytokine-induced death presents a novel β-cell specific mechanism which may be targeted for therapies to preserve β-cell mass in T1D.

Disclosure

N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None.

Funding

JDRF (5-CDA-2014-198-A-NJDRF, 3-APF-2019-749-A-N)

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