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Protection of pancreatic INS-1 β-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin.

Lablanche S, Cottet-Rousselle C, Lamarche F, Benhamou PY, Halimi S, Leverve X, Fontaine E - Cell Death Dis (2011)

Bottom Line: We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death.As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death.We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1055, Grenoble F-38041, France.

ABSTRACT
Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca²+-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.

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Related in: MedlinePlus

Effect of CsA, rotenone and metformin on the Ca2+ retention capacity of digitonin-permeabilized INS-1 cells. (a) The incubation medium contained 250 mM sucrose, 1 mM Pi, 10 mM Tris-MOPS, 5 mM succinate, 0.25 μM Calcium Green-5N and 50 μg/ml digitonin. The final volume was 1 ml (pH 7.35) at 25°C. Experiments were started by the addition of 3 × 106 INS-1 cells. Where indicated, 12.5 μM Ca2+ pulses were added (arrows). (b) Represents cumulative data of three different experiments performed as described in a, in control cells, in the presence of 1 μM CsA or 1.25 μM rotenone or in cells incubated overnight in the presence of 100 μM metformin. Results are mean±S.E.; *P<0.05 versus control, unpaired Student's t-test
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fig1: Effect of CsA, rotenone and metformin on the Ca2+ retention capacity of digitonin-permeabilized INS-1 cells. (a) The incubation medium contained 250 mM sucrose, 1 mM Pi, 10 mM Tris-MOPS, 5 mM succinate, 0.25 μM Calcium Green-5N and 50 μg/ml digitonin. The final volume was 1 ml (pH 7.35) at 25°C. Experiments were started by the addition of 3 × 106 INS-1 cells. Where indicated, 12.5 μM Ca2+ pulses were added (arrows). (b) Represents cumulative data of three different experiments performed as described in a, in control cells, in the presence of 1 μM CsA or 1.25 μM rotenone or in cells incubated overnight in the presence of 100 μM metformin. Results are mean±S.E.; *P<0.05 versus control, unpaired Student's t-test

Mentions: The Ca2+ retention capacity (CRC) represents the minimum Ca2+ load required to induce PTP opening in an entire population of mitochondria. Therefore, CRC measurement represents a suitable method to quantify and compare the potency of different PTP regulators. CRC is measured by loading mitochondria with a train of Ca2+ pulses until a rapid Ca2+ release occurs as illustrated in Figure 1a.


Protection of pancreatic INS-1 β-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin.

Lablanche S, Cottet-Rousselle C, Lamarche F, Benhamou PY, Halimi S, Leverve X, Fontaine E - Cell Death Dis (2011)

Effect of CsA, rotenone and metformin on the Ca2+ retention capacity of digitonin-permeabilized INS-1 cells. (a) The incubation medium contained 250 mM sucrose, 1 mM Pi, 10 mM Tris-MOPS, 5 mM succinate, 0.25 μM Calcium Green-5N and 50 μg/ml digitonin. The final volume was 1 ml (pH 7.35) at 25°C. Experiments were started by the addition of 3 × 106 INS-1 cells. Where indicated, 12.5 μM Ca2+ pulses were added (arrows). (b) Represents cumulative data of three different experiments performed as described in a, in control cells, in the presence of 1 μM CsA or 1.25 μM rotenone or in cells incubated overnight in the presence of 100 μM metformin. Results are mean±S.E.; *P<0.05 versus control, unpaired Student's t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101812&req=5

fig1: Effect of CsA, rotenone and metformin on the Ca2+ retention capacity of digitonin-permeabilized INS-1 cells. (a) The incubation medium contained 250 mM sucrose, 1 mM Pi, 10 mM Tris-MOPS, 5 mM succinate, 0.25 μM Calcium Green-5N and 50 μg/ml digitonin. The final volume was 1 ml (pH 7.35) at 25°C. Experiments were started by the addition of 3 × 106 INS-1 cells. Where indicated, 12.5 μM Ca2+ pulses were added (arrows). (b) Represents cumulative data of three different experiments performed as described in a, in control cells, in the presence of 1 μM CsA or 1.25 μM rotenone or in cells incubated overnight in the presence of 100 μM metformin. Results are mean±S.E.; *P<0.05 versus control, unpaired Student's t-test
Mentions: The Ca2+ retention capacity (CRC) represents the minimum Ca2+ load required to induce PTP opening in an entire population of mitochondria. Therefore, CRC measurement represents a suitable method to quantify and compare the potency of different PTP regulators. CRC is measured by loading mitochondria with a train of Ca2+ pulses until a rapid Ca2+ release occurs as illustrated in Figure 1a.

Bottom Line: We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death.As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death.We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U1055, Grenoble F-38041, France.

ABSTRACT
Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca²+-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.

Show MeSH
Related in: MedlinePlus