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Diabetes 52:2943-2950, 2003
© 2003 by the American Diabetes Association, Inc.

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

Martin Köhler1, Sergei V. Zaitsev1,2, Irina I. Zaitseva1,2, Barbara Leibiger1, Ingo B. Leibiger1, Mikael Turunen1, Iouri L. Kapelioukh1,2, Linda Bakkman1, Ioulia B. Appelskog1, Jacques Boutet de Monvel3,4, Gabriela Imreh1, and Per-Olof Berggren1

1 Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden
2 Belozersky Institute of Physico-Chemical Biology and Chemical School, Lomonosov Moscow State University, Moscow, Russia
3 Center for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden
4 Department of Otolaryngology, Karolinska Hospital, Stockholm, Sweden

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 ß-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 ß-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.

Address correspondence and reprint requests to Martin Köhler, the Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm, Sweden. E-mail: martin.kohler{at}molmed.ki.se


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Diabetes Diabetes Care Clinical Diabetes Diabetes Spectrum
Copyright © 2003 by the American Diabetes Association.