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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 impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.
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fig4: ER stress impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.

Mentions: To elucidate the functional relevance of impaired ABCA1 expression and localization by ER stress, we analyzed cholesterol efflux to apoA-I. Thapsigargin treatment significantly reduced cholesterol efflux even when cholesterol efflux was induced by a synthetic liver X receptor (LXR)-agonist (Fig. 3A). In contrast, cholesterol efflux to HDL was unaltered (data not shown). However, cholesterol efflux was impaired when experiments were performed in media containing LPDS (Fig. 3B). The finding that ER stress modulates cholesterol efflux to LPDS without the addition of exogenous apoA-I may be explained by the presence of residual acceptors for ABCA1 in the LPDS (e.g., free apoA-I or albumin) or by the reduced efflux to endogenously synthesized and secreted apoA-I. Indeed, thapsigargin treatment reduced apoA-I mRNA (Fig. 4A). Consistently, Western blot and immunofluorescence analyses revealed decreased endogenous apoA-I protein expression (Fig. 4B, C). Our data indicate that ER stress reduces the expression of ABCA1 and apoA-I to coordinately impair cholesterol efflux.


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 impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: ER stress impairs apoA-I expression. HepG2 cells were incubated with increasing concentrations of thapsigargin (A, B) or 0.1 µM thapsigargin (C) in media containing 10% LPDS for 24 h. ApoA-I mRNA was determined by qRT-PCR and normalized to 18s expression (A). ApoA-I protein expression was analyzed by Western blotting (B) and immunofluorescence analyses (C). Green: ABCA1; blue: DAPI. Bar = 10 µm. apoA-I mRNA and protein are decreased by thapsigargin treatment. qRT-PCR: mean ± SD (n = 3). Western blot and immunofluorescence analysis: representative images from two independent experiments are shown.
Mentions: To elucidate the functional relevance of impaired ABCA1 expression and localization by ER stress, we analyzed cholesterol efflux to apoA-I. Thapsigargin treatment significantly reduced cholesterol efflux even when cholesterol efflux was induced by a synthetic liver X receptor (LXR)-agonist (Fig. 3A). In contrast, cholesterol efflux to HDL was unaltered (data not shown). However, cholesterol efflux was impaired when experiments were performed in media containing LPDS (Fig. 3B). The finding that ER stress modulates cholesterol efflux to LPDS without the addition of exogenous apoA-I may be explained by the presence of residual acceptors for ABCA1 in the LPDS (e.g., free apoA-I or albumin) or by the reduced efflux to endogenously synthesized and secreted apoA-I. Indeed, thapsigargin treatment reduced apoA-I mRNA (Fig. 4A). Consistently, Western blot and immunofluorescence analyses revealed decreased endogenous apoA-I protein expression (Fig. 4B, C). Our data indicate that ER stress reduces the expression of ABCA1 and apoA-I to coordinately impair cholesterol efflux.

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