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Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans

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Abstract

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.

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Abbreviations

DMSO :

Dimethyl sulfoxide

FBS :

Fetal bovine serum

FDA :

Fluorescein diacetate

IEQ :

Islet equivalent

IL-1β :

Interleukin 1-beta

INF-γ :

Interferon gamma

MTT :

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NG :

Newport green

PARP :

Poly (ADP-ribose) polymerase

PI :

Propidium iodide

TNF-α :

Tumor necrosis factor alpha

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Acknowledgements

This work was supported by the Juvenile Diabetes Research Foundation, the Canadian Institutes for Health Research, and the Canadian Diabetes Association. L. Rosenberg is a National Scientist supported by the Fonds du Recherches Scientifique du Quebec. The authors would like to thank Despina Agapitos, Mark Lipsett, and Ali Hazrati for technical assistance with the islet isolation procedure.

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Authors and Affiliations

  1. Department of Surgery, McGill University, Montreal General Hospital, 1650 Cedar Ave., Montreal, H3G 1A4, Canada

    Reid Aikin, Lawrence Rosenberg & Steven Paraskevas

  2. Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler, McIntyre Medical Science Building, Rm. 1314, Montreal, H3G 1Y6, Canada

    Reid Aikin & Dusica Maysinger

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  1. Reid Aikin
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  2. Lawrence Rosenberg
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Correspondence to Dusica Maysinger.

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Aikin, R., Rosenberg, L., Paraskevas, S. et al. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans. J Mol Med 82, 389–397 (2004). https://doi.org/10.1007/s00109-004-0540-5

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  • DOI: https://doi.org/10.1007/s00109-004-0540-5

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