Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study

Rishikesh Mankidy, Pearson Wk Ahiahonu, Hong Ma, Dushmanthi Jayasinghe, Shawn A Ritchie, Mohamed A Khan, Khine K Su-Myat, Paul L Wood, Dayan B Goodenowe, Rishikesh Mankidy, Pearson Wk Ahiahonu, Hong Ma, Dushmanthi Jayasinghe, Shawn A Ritchie, Mohamed A Khan, Khine K Su-Myat, Paul L Wood, Dayan B Goodenowe

Abstract

Background: Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer.

Results: Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition.

Conclusion: The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

Figures

Figure 1
Figure 1
Scheme showing the syntheses of phosphoethanolamine plasmalogen precursors C1-3, C6-10, and diacylglycerols C4 and C5, test compounds for the study. Reagents: (a) i R1Br, NaH/DMF or R1SO4CH3, NaH/THF, reflux; (ii) 10% HCl, reflux; (b) TBDMS-Cl, imidazole/DMF; (c) R2COCl, DMAP, Pyridine, Toluene; (d) TBAF/THF, Imidazole, - 20°C
Figure 2
Figure 2
Relative ethanolamine plasmalogens in DHAPAT-deficient cells (A). Plasmalogen content of CHO (C_V) and NRel-4 (N_V) cell lines. Values are an average of three independent experiments; error bars represent standard deviation. All transitions measured in NRel-4 cells were significantly different from control cells (p < 0.05). Cholesterol profile of DHAPAT-deficient cells (B). Values are an average of independent experiments; error bars represent standard deviation. Total, free and esterified cholesterol of N_V is significantly different from C_V ( p < 0.05).
Figure 3
Figure 3
Plasmalogen biosynthetic pathway showing therapeutic intervention.
Figure 4
Figure 4
Side chain-specific restoration of PlsEtn in NRel-4 cells. Effect of C1, C2, C3, C6-10 treatment of NRel-4 (N_V) cells on sn-1 specific PlsEtn pools.
Figure 5
Figure 5
Sn-2 rearrangement following C1, C2, C3, C6-10 treatment of NRel-4 (N_V) cells. Relative distribution of sn-2 fatty acids within each plasmalogen pool following treatments as above is displayed. All PlsEtn measurements are reported relative to the control CHO cells (C_V). Results are an average of three independent experiments. Error bars represent standard deviation.
Figure 6
Figure 6
Comparison of sn-2 fatty acid substitution (A) and sn-1bond type (B) on total DHA PlsEtn levels in NRel-4 cells. NRel-4 cells were treated with ethanol solvent (N_V), or with test compounds at 20 μM concentration. Compounds C1, C6-10 contain palmityl ether at sn-1 and different fatty acid moieties at sn-2 position; DHA (C1), oleic acid (C7), linoleic acid (C8), linolenic acid (C9), and arachidonic acid (C10), -OH refers to a free hydroxyl group at sn-2 position. Compounds C4 and C5 have acyl linkages at sn-1 and sn-2 positions. Total DHA containing ethanolamine plasmalogens were quantified, and expressed relative to the amount observed in wild-type CHO cells (C_V). Values were an average of three independent experiments. Error bars represent standard deviation.
Figure 7
Figure 7
Concentration response curve of PlsEtn precursor C1 in CHO and NRel-4 cells. Treatments were carried out at concentrations of 1 μM, 5 μM, and 20 μM of C1. (A): Relative restoration/augmentation of DHA PlsEtn; (B): Relative restoration/augmentation of total PlsEtn. Values were normalized to control CHO cells (C_V). Results are an average of three independent experiments. Error bars indicate standard deviation.
Figure 8
Figure 8
Cholesterol profile of CHO/NRel-4 cells. Cholesterol profile of NRel-4 cells following 48 hour treatments with PlsEtn precursors (C1, C6-10) and PtdEtn precursors (C4, C5) compared to control CHO cells. A: total cholesterol; B: free cholesterol; C: esterified cholesterol. Cholesterol is reported as μg per million cells. Results are an average of three independent experiments. Asterisk represents values that are significantly different from those observed in DHAPAT-deficient NRel-4 cells (p < 0.05).
Figure 9
Figure 9
Cholesterol profile of HEK293 cells treated for 48 hr with plasmalogen precursors. Cholesterol (total, free and esterified) content is reported as μg/million cells, and is an average of two independent experiments. Error bars indicate standard deviation. Asterisk indicates significantly lower total or free cholesterol, or significantly higher esterified cholesterol compared with control.
Figure 10
Figure 10
Immunoblots showing SOAT1 protein levels in wild-type HEK293 cells treated with a concentration range of C1 and pravastatin. β-actin was used as a loading control.

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