Cholesterol through the looking glass: ability of its enantiomer also to elicit homeostatic responses

Abstract

How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.

FIGURE 1.

Structures and characterization of nat-cholesterol versus ent-cholesterol. A, chemical structures of native cholesterol (nat-cholesterol) and its enantiomer (ent-cholesterol). B, nat-cholesterol and ent-cholesterol (200 ng) measured using the Amplex red cholesterol assay kit. Fluorescence was monitored during incubation with cholesterol oxidase at 1-min intervals for 60 min at 37 °C. Data are the average of two separate experiments (each performed in triplicate), with the plateau given by nat-cholesterol set to 100% relative cholesterol oxidase activity. C, CHO-7 cells pretreated with statin and then incubated with 20 μg/ml nat-cholesterol (nat-C) or ent-cholesterol (ent-C) for 4 h. Nontreated and CD-treated conditions served as controls. CD was delivered at an amount equivalent to the sterol-CD complex. Lysates were analyzed for cholesterol content by HPLC/UV (performed in triplicate, ±S.E. (error bars)). D, CHO-7 cells pretreated with statin and then incubated with 20 μg/ml nat-cholesterol or ent-cholesterol for 4 h in the presence of 1 μCi of [1-¹⁴C]palmitate. Lipids were separated by TLC and detected by phosphorimaging. Cholesteryl [¹⁴C]esters were expressed relative to the maximal condition, which was set to 1 (n = 4, ±S.E.).

FIGURE 2.

Ent-cholesterol suppresses activation of the SREBP-2 pathway. A and B, CHO-7 cells were pretreated with statin and then incubated with 20 μg/ml nat-cholesterol or ent-cholesterol for 4 h. Nontreated and CD-treated conditions served as controls. A, cell lysates were subjected to 7.5% SDS-PAGE and assayed for SREBP-2 by immunoblot analysis with IgG-7D4. P, precursor; M, mature (active) form. Relative SREBP-2 processing was expressed as a ratio of mature to total SREBP (M/P + M) and normalized to the nontreated condition, which was set to 1 (n = 4, ±S.E. (error bars)). *, p ≤ 0.05; and **, p ≤ 0.01 versus nontreated condition. B, relative mRNA levels of three SREBP-2 target genes (LDLR, HMGCR, and SQLE) were determined by quantitative real-time PCR (n = 4, ±S.E., each performed in triplicate). **, p ≤ 0.01 versus nontreated condition, which was set to 1. C, SRD-13A (lacking Scap) and CHO/pGFP-Scap cells were pretreated with statin overnight. After harvesting, microsomes were prepared and subjected to the trypsin cleavage assay of Scap (see “Experimental Procedures”). The densitometric values of the lower (26-kDa) band were normalized relative to nat-cholesterol, which was set to 1 (n = 3, ±S.E.). **, p ≤ 0.01 versus nontreated condition.

FIGURE 3.

Ent-cholesterol causes degradation of squalene monooxygenase. A, SRD-1 cells were pretreated with statin and then incubated with 20 μg/ml nat-cholesterol or ent-cholesterol in medium containing cycloheximide (10 μg/ml) for 8 h. Nontreated and CD-treated conditions served as controls. B, CHO-7 cells were transfected with 1 μg of pTK-SM-V5 for 24 h. Cells were pretreated with statin and then treated as in A. A and B, cell lysates were subjected to 10% SDS-PAGE and immunoblotted with anti-SQLE for endogenous SM (A) or anti-V5 for ectopic SM (B). Densitometric values of SM were expressed relative to the nontreated condition, which was set to 1 (n ≥ 4, ±S.E. (error bars)). *, p ≤ 0.05 for ent-cholesterol versus nontreated condition; **, p ≤ 0.01 for nat-cholesterol versus nontreated condition.

FIGURE 4.

Basal cholesterol esterification in cholesterol-starved CHO-7 cells is undetectable, and loading with ent-cholesterol induces minimal esterification of endogenous cholesterol. A, CHO-7 cells were pretreated overnight with statin (LPDS and nat-C) or with DMEM/F12 supplemented with 5% (v/v) NCS (NCS). They were then incubated in the treatment medium without sterols (LPDS and NCS) or with 20 μg/ml nat-cholesterol (nat-C) for 4 h in the presence of 1 μCi of [1-¹⁴C]palmitate. Lipids were separated by TLC and detected by phosphorimaging. This phosphorimage was exposed for 3 days (top panel) or 2 weeks (bottom panel) and is representative of four separate experiments. Cholesteryl [¹⁴C]esters were expressed relative to the maximal condition, which was set to 1 (n = 4, ±S.E. (error bars)). B, CHO-7 cells were pretreated with statin overnight and then pulse-labeled for 10 min with 10 μCi of [³H]cholesterol-HPCD complex. After washing three times with 0.5 mg/ml BSA/PBS, cells were treated for 4 h with 20 μg/ml nat-cholesterol or ent-cholesterol, or 1 μg/ml 25-HC. Lipids were separated by TLC and detected by phosphorimaging. This phosphorimage is representative of four separate experiments. [³H]Chol esters were expressed relative to the maximal condition, which was set to 1 (n = 4, ±S.E.).

FIGURE 5.

Ent-cholesterol rather than endogenous cholesterol is able to suppress SREBP-2 target genes and induce Scap conformational change. A, CHO-7 cells were pretreated with statin overnight. They were then preincubated with 1% (w/v) HPCD for 1 h or grown in enantiomer medium for 10 days, or preincubated with 4.5 μm U18666A for 2 h before treatment. Cells were then treated with 20 μg/ml nat-cholesterol or ent-cholesterol for 4 h, with U18666A maintained in the last three conditions during this period. The nontreated condition served as a control in each category. Relative mRNA levels of two SREBP-2 target genes (LDLR and HMGCR) were determined by quantitative real-time PCR (n = 3, ±S.E. (error bars), each performed in triplicate). Values were normalized to the nontreated condition, which was set to 1. *, p ≤ 0.05 and **, p ≤ 0.01 versus nontreated condition for each category. B, CHO/pGFP-Scap cells were pretreated with statin overnight and then incubated with 1% (w/v) HPCD for 1 h (left panel) or grown in enantiomer medium for 10 days (right panel). After harvesting, microsomes were prepared and subjected to the trypsin cleavage assay of Scap (see “Experimental Procedures”). The immunoblots are each representative of three separate experiments.

FIGURE 6.

Summary of the estimated nonenantioselective effects on various cholesterol homeostatic parameters. Data from Figs. 1D, 2, and 3 (sharing the same culturing conditions) were used to estimate the nonenantioselective effects, calculated as the difference of ent-cholesterol from the nontreated condition, expressed as a percentage of the difference of nat-cholesterol from the nontreated condition (n ≥ 3, ±S.E. (error bars)). It should be noted that these values assume that equivalent amounts of nat-cholesterol and ent-cholesterol reached the ER (not directly measured).