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Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells.

Röhrl C, Eigner K, Winter K, Korbelius M, Obrowsky S, Kratky D, Kovacs WJ, Stangl H - J. Lipid Res. (2013)

Bottom Line: Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress.This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced.However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.

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ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.
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fig8: ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.

Mentions: Current literature suggests that ER stress induces SREBP activity. Consistently, ER stress induction by thapsigargin treatment resulted in a dose-dependent upregulation of the established SREBP-2 target genes LDL-receptor and HMG-CoA reductase (Fig. 8A). Moreover, we measured SREBP activity using reporter vectors containing copies of the SREBP binding site. SREBP activity increased as expected when cells were switched from FBS to LPDS (Fig. 8B). This effect was even more pronounced when thapsigargin was added. Therefore, ER stress increases SREBP-2 activity as shown by target gene expression analysis and activity assays. Altogether our data indicate that ER stress impairs cholesterol synthesis despite SREBP-2 activation, suggesting posttranscriptional mechanisms for HMG-CoA reductase inhibition.


Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells.

Röhrl C, Eigner K, Winter K, Korbelius M, Obrowsky S, Kratky D, Kovacs WJ, Stangl H - J. Lipid Res. (2013)

ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927476&req=5

fig8: ER stress increases SREBP-2 activity. A: HepG2 cells were incubated with thapsigargin in media containing 10% LPDS for 24 h. mRNA expression of the established SREBP-2 target genes LDL-receptor and HMGCR was determined by qRT-PCR and normalized to 18s expression. Data show means ± SD from three experiments. B: HepG2 cells were transfected with luciferase reporter vectors containing the SREBP response element. After 4 h, cells were incubated in media containing 10% FBS or 10% LPDS or 10% LPDS plus 0.1 µM thapsigargin for another 24 h. Promoter activity was determined using dual luciferase assay. Withdrawal of exogenous cholesterol by LPDS treatment increases SREBP-2 activity, which is further increased in response to thapsigargin treatment. Data show one representative experiment out of three independently performed experiments.
Mentions: Current literature suggests that ER stress induces SREBP activity. Consistently, ER stress induction by thapsigargin treatment resulted in a dose-dependent upregulation of the established SREBP-2 target genes LDL-receptor and HMG-CoA reductase (Fig. 8A). Moreover, we measured SREBP activity using reporter vectors containing copies of the SREBP binding site. SREBP activity increased as expected when cells were switched from FBS to LPDS (Fig. 8B). This effect was even more pronounced when thapsigargin was added. Therefore, ER stress increases SREBP-2 activity as shown by target gene expression analysis and activity assays. Altogether our data indicate that ER stress impairs cholesterol synthesis despite SREBP-2 activation, suggesting posttranscriptional mechanisms for HMG-CoA reductase inhibition.

Bottom Line: Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress.This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced.However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Vienna, Austria.

ABSTRACT
Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.

Show MeSH
Related in: MedlinePlus