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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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Inhibition of autophagy rescued endothelial cells from PA-induced death.A, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone, 0.5 mm OA, or 0.5 mm PA in the absence (−) and presence (+) of 10 μm wortmannin. B, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. C, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone or 0.5 mm PA in the absence (−) and presence (+) of 10 mm 3-MA. D, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. E, columns represent average cell viabilities that were determined using the MTT assay of cells not pretreated with wortmannin (left pair of columns, −Wortmannin) that were incubated for 24 h with BSA alone (left white column, n = 3) or with 0.5 mm PA complexed to BSA (left black column, n = 3), and cells pretreated with 10 μm wortmannin (right pair of columns, +Wortmannin) for 20 min prior to an incubation with either BSA alone (right white column, n = 3) or with 0.5 mm PA complexed to BSA (right black column, n = 3). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without wortmannin. F, cells were treated with 10 mm 3-MA, another specific inhibitor of PI3K III and autophagy, and cell death was analyzed by the MTT assay (n = 3 for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without 3-MA.
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Figure 4: Inhibition of autophagy rescued endothelial cells from PA-induced death.A, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone, 0.5 mm OA, or 0.5 mm PA in the absence (−) and presence (+) of 10 μm wortmannin. B, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. C, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone or 0.5 mm PA in the absence (−) and presence (+) of 10 mm 3-MA. D, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. E, columns represent average cell viabilities that were determined using the MTT assay of cells not pretreated with wortmannin (left pair of columns, −Wortmannin) that were incubated for 24 h with BSA alone (left white column, n = 3) or with 0.5 mm PA complexed to BSA (left black column, n = 3), and cells pretreated with 10 μm wortmannin (right pair of columns, +Wortmannin) for 20 min prior to an incubation with either BSA alone (right white column, n = 3) or with 0.5 mm PA complexed to BSA (right black column, n = 3). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without wortmannin. F, cells were treated with 10 mm 3-MA, another specific inhibitor of PI3K III and autophagy, and cell death was analyzed by the MTT assay (n = 3 for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without 3-MA.

Mentions: To investigate the role of autophagy induction on cell death we inhibited autophagy with wortmannin or 3-MA and measured the effect on lipotoxic cell death. Wortmannin and 3-MA are known to prevent autophagy by their inhibitory effect on VPS34 (33, 34). As expected, inhibition of VPS34 by either wortmannin prevented PA-induced autophagy indicated by a lack of LC3 cleavage in cells pretreated with this inhibitor (Fig. 4, A–D).


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)

Inhibition of autophagy rescued endothelial cells from PA-induced death.A, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone, 0.5 mm OA, or 0.5 mm PA in the absence (−) and presence (+) of 10 μm wortmannin. B, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. C, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone or 0.5 mm PA in the absence (−) and presence (+) of 10 mm 3-MA. D, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. E, columns represent average cell viabilities that were determined using the MTT assay of cells not pretreated with wortmannin (left pair of columns, −Wortmannin) that were incubated for 24 h with BSA alone (left white column, n = 3) or with 0.5 mm PA complexed to BSA (left black column, n = 3), and cells pretreated with 10 μm wortmannin (right pair of columns, +Wortmannin) for 20 min prior to an incubation with either BSA alone (right white column, n = 3) or with 0.5 mm PA complexed to BSA (right black column, n = 3). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without wortmannin. F, cells were treated with 10 mm 3-MA, another specific inhibitor of PI3K III and autophagy, and cell death was analyzed by the MTT assay (n = 3 for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without 3-MA.
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Related In: Results  -  Collection

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Figure 4: Inhibition of autophagy rescued endothelial cells from PA-induced death.A, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone, 0.5 mm OA, or 0.5 mm PA in the absence (−) and presence (+) of 10 μm wortmannin. B, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. C, representative Western blot showing LC3 cleavage of cells that were incubated for 8 h with BSA alone or 0.5 mm PA in the absence (−) and presence (+) of 10 mm 3-MA. D, statistical data of LC3 cleavage from Western blots shown in panel f (n = 3 for all conditions). *, p < 0.05 versus control. E, columns represent average cell viabilities that were determined using the MTT assay of cells not pretreated with wortmannin (left pair of columns, −Wortmannin) that were incubated for 24 h with BSA alone (left white column, n = 3) or with 0.5 mm PA complexed to BSA (left black column, n = 3), and cells pretreated with 10 μm wortmannin (right pair of columns, +Wortmannin) for 20 min prior to an incubation with either BSA alone (right white column, n = 3) or with 0.5 mm PA complexed to BSA (right black column, n = 3). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without wortmannin. F, cells were treated with 10 mm 3-MA, another specific inhibitor of PI3K III and autophagy, and cell death was analyzed by the MTT assay (n = 3 for all conditions). *, p < 0.05 versus BSA, and #, p < 0.05 versus PA without 3-MA.
Mentions: To investigate the role of autophagy induction on cell death we inhibited autophagy with wortmannin or 3-MA and measured the effect on lipotoxic cell death. Wortmannin and 3-MA are known to prevent autophagy by their inhibitory effect on VPS34 (33, 34). As expected, inhibition of VPS34 by either wortmannin prevented PA-induced autophagy indicated by a lack of LC3 cleavage in cells pretreated with this inhibitor (Fig. 4, A–D).

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