Effects of 3.beta.-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

Sexton, Russell C.; Panini, Sankhavaram R.; Azran, Florence; Rudney, Harry

doi:10.1021/bi00294a001

Cited by 94 publications

(51 citation statements)

References 28 publications

(30 reference statements)

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“…In CHO cells, GW707 is approximately 10-fold less potent than U18666A with respect to inhibition of LDL-stimulated cholesterol esterification, whereas in HepG2 cells, GW707 is 2-to 3-fold more potent than U18666A in inhibition of cholesteryl ester synthesis (E. Lopez-Perez and M. Issandou, unpublished data). In addition, U18666A, unlike GW707, impairs cholesterol biosynthesis through the inhibition of 2,3-oxidosqualene cyclase (30). In the present study, we found that GW707 does not significantly affect the synthesis of 25-HC in response to LDL cholesterol, whereas U18666A-treated cells exhibit a 40% reduction in the rate of secretion of 25-HC.…”

Section: Downloaded Fromcontrasting

confidence: 45%

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Zhang¹,

Dudley-Rucker²,

Crowley³

et al. 2004

Journal of Lipid Research

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Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.

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Section: Downloaded Fromcontrasting

confidence: 45%

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Zhang¹,

Dudley-Rucker²,

Crowley³

et al. 2004

Journal of Lipid Research

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“…In addition to its effect on the accumulation of cholesterol in late endosomes/lysosomes, U18666A is reported to inhibit oxidosqualene cyclase, an intermediate step in cholesterol synthesis ( 25 ). Inhibition of this enzyme led to the generation of 2,3-monoepoxysqualene and 2,3;22,23-diepoxysqualene, two substrates of oxydosqualene cyclase ( 38,44,45 ). Our results suggest that compounds 5 and 10 inhibit oxidosqualene cyclase.…”

Section: Resultsmentioning

confidence: 69%

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution

Ishitsuka

Saito

Osada

et al. 2011

Journal of Lipid Research

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“…Proteins were solubilized with CHAPS, separated by size exclusion chromatography, and the elution profile of Bax was analyzed by immunoblotting. For each condition, even-numbered fractions were loaded on two separate gels; a short exposure time was chosen for the low molecular weight fractions (30-44), and a longer one for the high molecular weight fractions (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) and its sensitivity to trypsin digestion were analyzed by immunoblotting. The presence of tBid was required for the recruitment of Bax to liposomes and led to the appearance of a Tr-Bax fragment (Figure 1d).…”

Section: Resultsmentioning

confidence: 99%

“…It interferes with the trafficking of cholesterol, causing its increase in late endosomes/lysosomes, 23,24 and inhibits its neosynthesis in the ER. 25 A recent study reported the accumulation of cholesterol in the mitochondria of neurons isolated from an Niemann-Pick Type C1 mouse model, 26 raising the possibility that U18666A might also increase mitochondrial cholesterol content. We therefore incubated HeLa cells with U18666A, isolated mitochondria, and analyzed their lipid composition by thin-layer chromatography (Figure 3a and b).…”

Section: Resultsmentioning

confidence: 99%

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

2007

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Activation of Bax or Bak is essential for the completion of many apoptotic programmes. Under cytotoxic conditions, these proteins undergo a series of conformational rearrangements that end up with their oligomerization. We found that unlike inactive monomeric Bax, active oligomerized Bax is partially resistant to trypsin digestion, providing a convenient read out to monitor Bax activation. Using this assay, we studied how the lipid composition of membranes affects tBid-induced Bax activation in vitro with pure liposomes. We report that Bax activation is inhibited by cholesterol and by decreases in membrane fluidity. This observation was further tested in vivo using the drug U18666A, which we found increases mitochondrial cholesterol levels. When incubated with tBid, mitochondria isolated from U18666A-treated cells showed a delay in the release of Smac/Diablo and Cytochrome c, as well as in Bax oligomerization. Moreover, pre-incubation with U18666A partially protected cells from stressinduced apoptosis. As many tumours display high mitochondrial cholesterol content, inefficient Bax oligomerization might contribute to their resistance to apoptosis-inducing agents. Among the stimuli that lead to apoptosis, a series of stress conditions trigger the permeabilization of the mitochondrial outer membrane (MOM) and the release of pro-apoptotic factors that will activate caspases in the cytosol. This so-called intrinsic apoptotic pathway is tightly regulated by proteins of the Bcl-2 family, among which the pro-apoptotic members Bax and Bak are essential. They seem to be the central players whose activation results in MOM permeabilization.Under resting conditions, whereas Bak essentially resides in the MOMs, Bax is found in loose association with membranes (mainly the MOMs) or as a soluble protein in the cytosol. Following many cytotoxic signals, Bax and Bak undergo a series of conformational rearrangements. 1 Bax translocates to mitochondria and inserts into the MOMs in a form that cannot be detached by alkali treatment. Bax and Bak then uncover N-terminal epitopes and oligomerize. Oligomerization, shown by size exclusion chromatography, 2 crosslinking experiments 3,4 and native gel electrophoresis, 5 appears as the critical step resulting in MOM permeabilization. However, despite many efforts, it remains unclear how each of these conformational rearrangements is regulated, and how Bax/Bak oligomerization leads to the release of mitochondrial apoptogenic factors. Both processes are undoubtedly regulated by other members of the Bcl-2 family, 6,7 but the participation of many unrelated proteins was also proposed. 8 While a BH3-only protein appears to be sufficient for Bax to oligomerize in synthetic liposomes, 9,10 additional proteins could be required for its activation in isolated mitochondria. 11 These might directly interact with Bax, and/or modify the membrane properties to allow its oligomerization. Interaction with mitochondrial lipids is indeed an important parameter to consider as the final conformational rearrange...

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Effects of 3.beta.-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

Cited by 94 publications

References 28 publications

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Fluorescence image screening for chemical compounds modifying cholesterol metabolism and distribution

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes

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