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 Citations (Scopus)

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

Fingerprint

Insulin-Secreting Cells
Apoptosis
Caspase 3
Fluorescence Resonance Energy Transfer
Staurosporine
Protein C
Photons
Confocal Microscopy
Insulin
Cytophotometry
Amino Acid Sequence
Proteins
Peptide Hydrolases
Cytokines
Glucose

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Köhler, M., Zaitsev, S. V., Zaitseva, I. I., Leibiger, B., Leibiger, I. B., Turunen, M., ... 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

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells. / Köhler, Martin; Zaitsev, Sergel V.; Zaitseva, Irina I.; Leibiger, Barbara; Leibiger, Ingo B.; Turunen, Mikael; Kapelioukh, Iouri L.; Bakkman, Linda; Appelskog, Ioulia B.; De Monvel, Jacques Boutet; Imreh, Gabriela; Berggren, Per Olof.

In: Diabetes, Vol. 52, No. 12, 01.12.2003, p. 2943-2950.

Research output: Contribution to journalArticle

Köhler, M, Zaitsev, SV, Zaitseva, II, Leibiger, B, Leibiger, IB, Turunen, M, Kapelioukh, IL, Bakkman, L, Appelskog, IB, De Monvel, JB, Imreh, G & Berggren, PO 2003, 'On-Line Monitoring of Apoptosis in Insulin-Secreting Cells', Diabetes, vol. 52, no. 12, pp. 2943-2950. https://doi.org/10.2337/diabetes.52.12.2943
Köhler M, Zaitsev SV, Zaitseva II, Leibiger B, Leibiger IB, Turunen M et al. On-Line Monitoring of Apoptosis in Insulin-Secreting Cells. Diabetes. 2003 Dec 1;52(12):2943-2950. https://doi.org/10.2337/diabetes.52.12.2943
Köhler, Martin ; Zaitsev, Sergel V. ; Zaitseva, Irina I. ; Leibiger, Barbara ; Leibiger, Ingo B. ; Turunen, Mikael ; Kapelioukh, Iouri L. ; Bakkman, Linda ; Appelskog, Ioulia B. ; De Monvel, Jacques Boutet ; Imreh, Gabriela ; Berggren, Per Olof. / On-Line Monitoring of Apoptosis in Insulin-Secreting Cells. In: Diabetes. 2003 ; Vol. 52, No. 12. pp. 2943-2950.
@article{77c83df905a544728c396f479efb704c,
title = "On-Line Monitoring of Apoptosis in Insulin-Secreting Cells",
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.",
author = "Martin K{\"o}hler and Zaitsev, {Sergel V.} and Zaitseva, {Irina I.} and Barbara Leibiger and Leibiger, {Ingo B.} and Mikael Turunen and Kapelioukh, {Iouri L.} and Linda Bakkman and Appelskog, {Ioulia B.} and {De Monvel}, {Jacques Boutet} and Gabriela Imreh and Berggren, {Per Olof}",
year = "2003",
month = "12",
day = "1",
doi = "10.2337/diabetes.52.12.2943",
language = "English",
volume = "52",
pages = "2943--2950",
journal = "Diabetes",
issn = "0012-1797",
publisher = "American Diabetes Association Inc.",
number = "12",

}

TY - JOUR

T1 - On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

AU - Köhler, Martin

AU - Zaitsev, Sergel V.

AU - Zaitseva, Irina I.

AU - Leibiger, Barbara

AU - Leibiger, Ingo B.

AU - Turunen, Mikael

AU - Kapelioukh, Iouri L.

AU - Bakkman, Linda

AU - Appelskog, Ioulia B.

AU - De Monvel, Jacques Boutet

AU - Imreh, Gabriela

AU - Berggren, Per Olof

PY - 2003/12/1

Y1 - 2003/12/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=10744224294&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=10744224294&partnerID=8YFLogxK

U2 - 10.2337/diabetes.52.12.2943

DO - 10.2337/diabetes.52.12.2943

M3 - Article

VL - 52

SP - 2943

EP - 2950

JO - Diabetes

JF - Diabetes

SN - 0012-1797

IS - 12

ER -