On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

Martin Köhler, Sergel V. Zaitsev, Irina I. Zaitseva, Barbara Leibiger, Ingo B. Leibiger, Mikael Turunen, Iouri L. Kapelioukh, Linda Bakkman, Ioulia B. Appelskog, Jacques Boutet De Monvel, Gabriela Imreh, Per Olof Berggren

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate β-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose- and cytokine-induced apoptosis in the β-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 ± 23 min (n = 9) and 257 ± 59 min (n = 4; mean ± SE) after activation of apoptosis with staurosporine (6 μmol/l), showing that this method worked in insulin-producing cells.

Original languageEnglish (US)
Pages (from-to)2943-2950
Number of pages8
JournalDiabetes
Volume52
Issue number12
DOIs
StatePublished - Dec 1 2003

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

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    Köhler, M., Zaitsev, S. V., Zaitseva, I. I., Leibiger, B., Leibiger, I. B., Turunen, M., Kapelioukh, I. L., Bakkman, L., Appelskog, I. B., De Monvel, J. B., Imreh, G., & Berggren, P. O. (2003). On-Line Monitoring of Apoptosis in Insulin-Secreting Cells. Diabetes, 52(12), 2943-2950. https://doi.org/10.2337/diabetes.52.12.2943