794 Effect of potential glutathione precursors on human islet survival in hypoxia
Wednesday November 18, 2015 from 15:30 to 17:00
Plenary Room 1

Paul R.V. Johnson, United Kingdom

Director of the Oxford Islet Transplant Programme and Professor of Paediatric Surgery




Effect of potential glutathione precursors on human islet survival in hypoxia

Heide Brandhorst PhD1, Niamh Mullooly PhD1, Samuel Acreman BSc1, Daniel Brandhorst PhD1, Paul RV Johnson PhD1.

1Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom

Objectives: Hypoxia is the main determinant of morphological and functional integrity of isolated pancreatic islets and occurs during organ procurement, islet isolation, culture/shipment and after transplantation affecting islet viability and survival. Radical oxygen species (ROS) are the main contributors to hypoxia-induced islet cell death. The present study assessed the effect of the potential glutathione precursors N-acetyl-cysteine (NAC), taurine (TAU) and L-glutamine (GLN) on survival of human islets exposed to significant hypoxia.

Methods: Isolated human islets were incubated with 1-80 mM NAC, 1.25-40 mM TAU and 1-8 mM of GLN and cultured in hypoxic conditions for 2-3 days (n=6). Hypoxia-exposed untreated islets were used as controls. Post-culture quality assessment included islet recovery, fragmentation, viability and ROS production. Islet in vitro function was assessed in subsequent studies using the most efficient concentration for islet treatment. All data were normalised to hypoxia-exposed control islets and are expressed as mean ± SEM.

Results: Compared to hypoxic controls (100%) TAU was most efficient at 10 mM to reduce islet fragmentation (70±6.9%,p<0.05) and to increase islet recovery after culture at 1.5% oxygen (168±25%, p<0.01). Four mM of GLN was similarly effective to prevent islet fragmentation (59±6.2%, p<0.001) and to enhance post-culture islet recovery (185±23, p<0.001). The most effective concentration of 5 mM NAC reduced fragmentation (71±5.6, p<0.001) but did not improve post-culture islet recovery (135±5.6%, NS). Although 5 mM NAC was effective to reduce ROS generation (54±2.9%, p<0.05) relative viability was not significantly increased (106±1.8%, NS). In contrast, reduced ROS generation (60±5.6%, p<0.05; 51±6.4%, p<0.01) inversely correlated with viability (114±3.5%, p<0.05; 120±3.0%, p<0.05) when 10 mM TAU or 4 mM GLN was used for islet treatment, respectively. Because of the relatively low overall survival of islets which considers both recovery as well as viability (143±5.7%, NS vs. controls) NAC was excluded from further studies.

Islet exposure to severe hypoxia at 0.75% oxygen revealed that 10 mM TAU is significantly more effective than GLN to increase islet recovery (182±17.5% vs. 146±10.4%, p<0.001) and islet overall survival (258±26.3% vs. 220±23.6%, p<0.001) when compared to hypoxic controls. In addition, islet insulin secretory response to glucose was best preserved when hypoxic islets were treated with 10 mM TAU.

Conclusions: This study demonstrates that glutathione precursors can significantly improve islet survival during hypoxia. The protective features of GLN and TAU may be considered for supplementation of media used during isolation, culture and shipment of islets.

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