Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs

Abstract

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.

FIGURE 1:

Chemical structures of trans and cis fatty acids. Modeling of elaidic acid or 9-trans-octadecenoic acid (top) and oleic acid or 9-cis-octadecenoic (bottom). The trans isomer adopts a linear configuration, while the cis isomer is in a bent configuration.

FIGURE 2:

Lipid dependence of membrane cholesterol partitioning. Equilibrium cholesterol partition coefficient was determined between LUV that contains either cis (open bars) or trans (hatched bars) fatty acid-derived phospholipids and methyl-β-cyclodextrin (CD) (A). Cholesterol partition coefficient (K_cis^trans) between trans and its corresponding cis-phospholipid (B) was derived from measurements similar to those shown in panel A.

FIGURE 3:

DSC scans of reconstituted rhodopsin-containing vesicles. Shown in panel A are thermal transitions of rhodopsin in di-18:1n9(c) PC vesicles (⋯) and in di-18:1n9(t) PC vesicles (---). The thermal transitions were analyzed according to a two-state transition model, shown as (—) for both lipid vesicles. Shown in panel B are phospholipid phase transitions in rhodopsin-containing di-18:1n9(c)PC vesicles (⋯) and rhodopsin-containing di-18:1n9-(t)PC vesicles before (---) and after (—) rhodopsin is thermally denatured. Also shown in panel B is the di-18:1n9(t)PC phase transition in pure di-18:1n9(t)PC vesicles without rhodopsin (-··-). It was scaled down by a factor of 2 for clarity.

FIGURE 4:

Metarhodopsin I (MI) and metarhodopsin II (MII) equilibrium difference spectra in reconstituted vesicles. Spectra were acquired in rhodopsin-containing vesicles consisting of di-18:1n9-(c)PC (O) and di-18:1n9(t)PC (·) in pH 7.0 PBS buffer at 37°C. The dashed and solid lines were the corresponding deconvoluted spectra of MI (480 nm) and MII (385 nm) in di-18:1n9(c)PC and di-18:1n9(t)PC, respectively. The equilibrium constants for MI-MII were derived from these spectra.

FIGURE 5:

Trans fatty acid-derived phospholipids reduced rhodopsin activation. MI-MII equilibrium constants (K_eq) were derived from equilibrium spectra as shown in Figure 4. Shown here are K_eq values from reconstituted rhodopsin-containing vesicles consisting of di-18:1n9(c)PC (open bar) and di-18:1n9(t)PC (hatched bar). The top panel was measured at 37°C, where both di-18:1n9(c)PC and di-18:1n9(t)PC are in the liquid crystalline phase, while the bottom panel was acquired at 5°C, where di-18:1n9(c)PC is in the liquid crystalline phase and di-18:1n9(t)PC is in the gel phase.

FIGURE 6:

van’t Hoff plot of the MI-MII equilibrium constants. Plot of ln K_eq from reconstituted rhodopsin vesicles consisting of di-18:1n9(c)PC (O) and di-18:1n9(t)PC (▲)vs1/T. The dashed and solid lines were the corresponding linear fits of the data. The open triangle data point was excluded in the linear fit since di-18:1n9-(t)PC was below its phase transition at 5°C.