Early assessment of apoptosis in isolated islets of Langerhans

Transplantation. 2001 Apr 15;71(7):857-62. doi: 10.1097/00007890-200104150-00006.


Background: There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis.

Methods: In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 beta, interferon gamma, and tumor necrosis factor a, cytokines known to induce islet cell damage.

Results: A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8+/-0.3 U/100 islet equivalents and it increased to 1.4+/-0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P<0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%+/-5.8%, and it increased to 27.5%+/-8.1% 6 hr after addition of the cytokine cocktail (P<0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment.

Conclusion: These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Annexin A5 / metabolism
  • Apoptosis*
  • Biomarkers
  • Caspase 3
  • Caspases / metabolism
  • DNA Fragmentation
  • Electrophoresis, Polyacrylamide Gel
  • In Situ Nick-End Labeling
  • In Vitro Techniques
  • Islets of Langerhans / cytology
  • Islets of Langerhans / metabolism
  • Islets of Langerhans / physiology*
  • Rats
  • Rats, Inbred Lew


  • Annexin A5
  • Biomarkers
  • Casp3 protein, rat
  • Caspase 3
  • Caspases