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Decrease in membrane phospholipid unsaturation induces unfolded protein response.

Ariyama H, Kono N, Matsuda S, Inoue T, Arai H - J. Biol. Chem. (2010)

Bottom Line: In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA.Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown.These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

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Polyunsaturated fatty acids suppress UPR induced by SCD1 knockdown. HeLa cells were transfected with the indicated siRNA. 48 h after transfection cells were further incubated for 24 h in media supplemented with the indicated fatty acid (50 μm) and then harvested. A and B, expression of CHOP (A) and GRP78 (B) mRNAs detected by real-time PCR is shown. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells (Tg) were used as a positive control. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01), and the number symbols indicate significant differences compared with siSCD1-transfected cells which were untreated (p < 0.01).
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Figure 4: Polyunsaturated fatty acids suppress UPR induced by SCD1 knockdown. HeLa cells were transfected with the indicated siRNA. 48 h after transfection cells were further incubated for 24 h in media supplemented with the indicated fatty acid (50 μm) and then harvested. A and B, expression of CHOP (A) and GRP78 (B) mRNAs detected by real-time PCR is shown. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells (Tg) were used as a positive control. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01), and the number symbols indicate significant differences compared with siSCD1-transfected cells which were untreated (p < 0.01).

Mentions: We next examined the effect of unsaturated fatty acid administration to the SCD1 knockdown cells on UPR activation. Administration of 18:1n-9, which is a major product of SCD1, completely suppressed the induction of CHOP and GRP78 expression and XBP1 splicing in SCD1 knockdown cells (Fig. 4, A–C). In contrast, the addition of a saturated fatty acid such as 16:0 caused severe cell death in SCD1 knockdown cells (data not shown). The suppression effects were also observed when PUFAs, linoleic acid (18:2), 20:4, or eicosapentaenoic acid (20:5) were administered (Fig. 4, A–C). Treatment with these unsaturated fatty acids also completely rescued SCD1 knockdown-induced cell death but had no effect on thapsigargin-induced alternate splicing of XBP1 (data not shown). Lipid analysis showed that the supplemented unsaturated fatty acids were efficiently incorporated into the phospholipid fraction (supplemental Fig. S2). These results indicate that various unsaturated fatty acids, in addition to 18:1n-9, are able to rescue the SCD1 knockdown phenotype.


Decrease in membrane phospholipid unsaturation induces unfolded protein response.

Ariyama H, Kono N, Matsuda S, Inoue T, Arai H - J. Biol. Chem. (2010)

Polyunsaturated fatty acids suppress UPR induced by SCD1 knockdown. HeLa cells were transfected with the indicated siRNA. 48 h after transfection cells were further incubated for 24 h in media supplemented with the indicated fatty acid (50 μm) and then harvested. A and B, expression of CHOP (A) and GRP78 (B) mRNAs detected by real-time PCR is shown. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells (Tg) were used as a positive control. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01), and the number symbols indicate significant differences compared with siSCD1-transfected cells which were untreated (p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Polyunsaturated fatty acids suppress UPR induced by SCD1 knockdown. HeLa cells were transfected with the indicated siRNA. 48 h after transfection cells were further incubated for 24 h in media supplemented with the indicated fatty acid (50 μm) and then harvested. A and B, expression of CHOP (A) and GRP78 (B) mRNAs detected by real-time PCR is shown. The expression level of each gene was normalized to the GAPDH gene and is represented as -fold induction over siControl. C, semiquantitative reverse transcription-PCR analysis of XBP1 spliced and unspliced mRNA. The positions of the unspliced form (u) and spliced form (s) are indicated. Thapsigargin-treated cells (Tg) were used as a positive control. The asterisks indicate significant differences compared with siControl-transfected cells (p < 0.01), and the number symbols indicate significant differences compared with siSCD1-transfected cells which were untreated (p < 0.01).
Mentions: We next examined the effect of unsaturated fatty acid administration to the SCD1 knockdown cells on UPR activation. Administration of 18:1n-9, which is a major product of SCD1, completely suppressed the induction of CHOP and GRP78 expression and XBP1 splicing in SCD1 knockdown cells (Fig. 4, A–C). In contrast, the addition of a saturated fatty acid such as 16:0 caused severe cell death in SCD1 knockdown cells (data not shown). The suppression effects were also observed when PUFAs, linoleic acid (18:2), 20:4, or eicosapentaenoic acid (20:5) were administered (Fig. 4, A–C). Treatment with these unsaturated fatty acids also completely rescued SCD1 knockdown-induced cell death but had no effect on thapsigargin-induced alternate splicing of XBP1 (data not shown). Lipid analysis showed that the supplemented unsaturated fatty acids were efficiently incorporated into the phospholipid fraction (supplemental Fig. S2). These results indicate that various unsaturated fatty acids, in addition to 18:1n-9, are able to rescue the SCD1 knockdown phenotype.

Bottom Line: In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA.Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown.These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.

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