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Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, Hallström S, Rockenfeller P, Konya V, Heinemann A, Madeo F, Graier WF, Malli R - J. Biol. Chem. (2012)

Bottom Line: Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death.Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA.In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.

ABSTRACT
Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

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PA-induced autophagy and cell death is a Ca2+-dependent process.A, representative Western blot showing LC3 cleavage of BAPTA-AM-loaded cells that were incubated for 8 h with BSA alone or 0.5 mm PA. B, statistical data of LC3 cleavage from Western blots (n = 3 for all conditions). *, p < 0.05 versus control. PA induces release of Ca2+ from ER and results in cytosolic Ca2+ elevation. C, representative Ca2+ signals of Fura-2/AM-loaded cells 16 h after incubation with BSA alone (black continuous line), 0.5 mm OA (gray continuous line), and 0.5 mm PA (black dotted line). The indicated signals were first recorded in Ca2+ containing medium (2 mm) and subsequently Ca2+ was mobilized from the ER by cell stimulation with 100 μm histamine and 15 μm BHQ in a Ca2+-free medium (1 mm EGTA). D, statistics of basal ratio values of Fura-2/AM-loaded cells in the presence of extracellular Ca2+ (2 mm), and E, average δ values of the maximal Ca2+ peaks upon cell stimulation with 100 μm histamine and 15 μm BHQ in the absence of extracellular Ca2+ (1 mm EGTA) at different times after incubation with BSA alone (Control, white columns, n = 9, ×118 cells at 2 h, ×127 cells at 4 h, ×130 cells at 8 h, and ×127 cells at 16 h), with 0.5 mm OA (gray columns, n = 9, ×113 cells at 2 h, ×127 cells at 4 h, ×133 cells at 8 h, and ×132 cells at 16 h), and with 0.5 mm PA (black columns, n = 9, ×115 cells at 2 h, ×124 cells at 4 h, ×118 cells at 8 h and ×107 cells at 16 h). *, p < 0.05 versus BSA. F, effect of chelating cytosolic Ca2+ on cell viability. Cells were pretreated with BAPTA-AM for 20 min and then incubated with 0.5 mm PA or BSA alone for 24 h. Cell viability was measured with the MTT assay and data were normalized to BSA as a control and represented as mean viability (n = 3, for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without BAPTA-AM-loaded cells.
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Figure 6: PA-induced autophagy and cell death is a Ca2+-dependent process.A, representative Western blot showing LC3 cleavage of BAPTA-AM-loaded cells that were incubated for 8 h with BSA alone or 0.5 mm PA. B, statistical data of LC3 cleavage from Western blots (n = 3 for all conditions). *, p < 0.05 versus control. PA induces release of Ca2+ from ER and results in cytosolic Ca2+ elevation. C, representative Ca2+ signals of Fura-2/AM-loaded cells 16 h after incubation with BSA alone (black continuous line), 0.5 mm OA (gray continuous line), and 0.5 mm PA (black dotted line). The indicated signals were first recorded in Ca2+ containing medium (2 mm) and subsequently Ca2+ was mobilized from the ER by cell stimulation with 100 μm histamine and 15 μm BHQ in a Ca2+-free medium (1 mm EGTA). D, statistics of basal ratio values of Fura-2/AM-loaded cells in the presence of extracellular Ca2+ (2 mm), and E, average δ values of the maximal Ca2+ peaks upon cell stimulation with 100 μm histamine and 15 μm BHQ in the absence of extracellular Ca2+ (1 mm EGTA) at different times after incubation with BSA alone (Control, white columns, n = 9, ×118 cells at 2 h, ×127 cells at 4 h, ×130 cells at 8 h, and ×127 cells at 16 h), with 0.5 mm OA (gray columns, n = 9, ×113 cells at 2 h, ×127 cells at 4 h, ×133 cells at 8 h, and ×132 cells at 16 h), and with 0.5 mm PA (black columns, n = 9, ×115 cells at 2 h, ×124 cells at 4 h, ×118 cells at 8 h and ×107 cells at 16 h). *, p < 0.05 versus BSA. F, effect of chelating cytosolic Ca2+ on cell viability. Cells were pretreated with BAPTA-AM for 20 min and then incubated with 0.5 mm PA or BSA alone for 24 h. Cell viability was measured with the MTT assay and data were normalized to BSA as a control and represented as mean viability (n = 3, for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without BAPTA-AM-loaded cells.

Mentions: Recently there are some reports about the role of Ca2+ in autophagy (35). We chelated cytosolic Ca2+ with BAPTA-AM and measured the cleavage of LC3 by Western blotting. It was found that autophagy was inhibited by BAPTA-AM treatment (Fig. 6, A and B). This indicated that the induction of autophagy by PA was dependent on cytosolic Ca2+ elevation.


Inhibition of autophagy rescues palmitic acid-induced necroptosis of endothelial cells.

Khan MJ, Rizwan Alam M, Waldeck-Weiermair M, Karsten F, Groschner L, Riederer M, Hallström S, Rockenfeller P, Konya V, Heinemann A, Madeo F, Graier WF, Malli R - J. Biol. Chem. (2012)

PA-induced autophagy and cell death is a Ca2+-dependent process.A, representative Western blot showing LC3 cleavage of BAPTA-AM-loaded cells that were incubated for 8 h with BSA alone or 0.5 mm PA. B, statistical data of LC3 cleavage from Western blots (n = 3 for all conditions). *, p < 0.05 versus control. PA induces release of Ca2+ from ER and results in cytosolic Ca2+ elevation. C, representative Ca2+ signals of Fura-2/AM-loaded cells 16 h after incubation with BSA alone (black continuous line), 0.5 mm OA (gray continuous line), and 0.5 mm PA (black dotted line). The indicated signals were first recorded in Ca2+ containing medium (2 mm) and subsequently Ca2+ was mobilized from the ER by cell stimulation with 100 μm histamine and 15 μm BHQ in a Ca2+-free medium (1 mm EGTA). D, statistics of basal ratio values of Fura-2/AM-loaded cells in the presence of extracellular Ca2+ (2 mm), and E, average δ values of the maximal Ca2+ peaks upon cell stimulation with 100 μm histamine and 15 μm BHQ in the absence of extracellular Ca2+ (1 mm EGTA) at different times after incubation with BSA alone (Control, white columns, n = 9, ×118 cells at 2 h, ×127 cells at 4 h, ×130 cells at 8 h, and ×127 cells at 16 h), with 0.5 mm OA (gray columns, n = 9, ×113 cells at 2 h, ×127 cells at 4 h, ×133 cells at 8 h, and ×132 cells at 16 h), and with 0.5 mm PA (black columns, n = 9, ×115 cells at 2 h, ×124 cells at 4 h, ×118 cells at 8 h and ×107 cells at 16 h). *, p < 0.05 versus BSA. F, effect of chelating cytosolic Ca2+ on cell viability. Cells were pretreated with BAPTA-AM for 20 min and then incubated with 0.5 mm PA or BSA alone for 24 h. Cell viability was measured with the MTT assay and data were normalized to BSA as a control and represented as mean viability (n = 3, for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without BAPTA-AM-loaded cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375534&req=5

Figure 6: PA-induced autophagy and cell death is a Ca2+-dependent process.A, representative Western blot showing LC3 cleavage of BAPTA-AM-loaded cells that were incubated for 8 h with BSA alone or 0.5 mm PA. B, statistical data of LC3 cleavage from Western blots (n = 3 for all conditions). *, p < 0.05 versus control. PA induces release of Ca2+ from ER and results in cytosolic Ca2+ elevation. C, representative Ca2+ signals of Fura-2/AM-loaded cells 16 h after incubation with BSA alone (black continuous line), 0.5 mm OA (gray continuous line), and 0.5 mm PA (black dotted line). The indicated signals were first recorded in Ca2+ containing medium (2 mm) and subsequently Ca2+ was mobilized from the ER by cell stimulation with 100 μm histamine and 15 μm BHQ in a Ca2+-free medium (1 mm EGTA). D, statistics of basal ratio values of Fura-2/AM-loaded cells in the presence of extracellular Ca2+ (2 mm), and E, average δ values of the maximal Ca2+ peaks upon cell stimulation with 100 μm histamine and 15 μm BHQ in the absence of extracellular Ca2+ (1 mm EGTA) at different times after incubation with BSA alone (Control, white columns, n = 9, ×118 cells at 2 h, ×127 cells at 4 h, ×130 cells at 8 h, and ×127 cells at 16 h), with 0.5 mm OA (gray columns, n = 9, ×113 cells at 2 h, ×127 cells at 4 h, ×133 cells at 8 h, and ×132 cells at 16 h), and with 0.5 mm PA (black columns, n = 9, ×115 cells at 2 h, ×124 cells at 4 h, ×118 cells at 8 h and ×107 cells at 16 h). *, p < 0.05 versus BSA. F, effect of chelating cytosolic Ca2+ on cell viability. Cells were pretreated with BAPTA-AM for 20 min and then incubated with 0.5 mm PA or BSA alone for 24 h. Cell viability was measured with the MTT assay and data were normalized to BSA as a control and represented as mean viability (n = 3, for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without BAPTA-AM-loaded cells.
Mentions: Recently there are some reports about the role of Ca2+ in autophagy (35). We chelated cytosolic Ca2+ with BAPTA-AM and measured the cleavage of LC3 by Western blotting. It was found that autophagy was inhibited by BAPTA-AM treatment (Fig. 6, A and B). This indicated that the induction of autophagy by PA was dependent on cytosolic Ca2+ elevation.

Bottom Line: Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death.Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA.In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.

ABSTRACT
Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

Show MeSH
Related in: MedlinePlus