Regulation of hepatic lipase activity by sphingomyelin in plasma lipoproteins

Abstract

Hepatic lipase (HL) is an important enzyme in the clearance of triacylglycerol (TAG) from the circulation, and has been proposed to have pro-atherogenic as well as anti-atherogenic properties. It hydrolyzes both phospholipids and TAG of lipoproteins, and its activity is negatively correlated with HDL levels. Although it is known that HL acts preferentially on HDL lipids, the basis for this specificity is not known, since it does not require any specific apoprotein for activity. In this study, we tested the hypothesis that sphingomyelin (SM), whose concentration is much higher in VLDL and LDL compared to HDL, is an inhibitor of HL, and that this could explain the lipoprotein specificity of the enzyme. The results presented show that the depletion of SM from normal lipoproteins activated the HL roughly in proportion to their SM content. SM depletion stimulated the hydrolysis of both phosphatidylcholine (PC) and TAG, although the PC hydrolysis was stimulated more. In the native lipoproteins, HL showed specificity for PC species containing polyunsaturated fatty acids at sn-2 position, and produced more unsaturated lyso PC species. The enzyme also showed preferential hydrolysis of certain TAG species over others. SM depletion affected the specificity of the enzyme towards PC and TAG species modestly. These results show that SM is a physiological inhibitor of HL activity in lipoproteins and that the specificity of the enzyme towards HDL is at least partly due to its low SM content.

Keywords: Enzyme inhibition; Hepatic lipase; PC species; Sphingomyelin; Substrate specificity; TAG species.

Fig. 1. Effect of SM depletion on the hydrolysis of labeled PC incorporated into lipoproteins

The lipoproteins were pre-labeled with dipalmitoyl [¹⁴C] PC, and an aliquot of the labeled lipoprotein was subjected to SMase treatment as described in the text. The SMase-treated and untreated lipoproteins were then incubated with recombinant HL (500 μl, 32 μg protein) for the indicated periods of time at 37 °C. The lipids were extracted, and the radioactivity in LPC, PC, and FFA was determined following their TLC separation. The results show the decrease in PC counts (%) compared to the control (incubated with no HL), and are mean ± SEM of 4 experiments. * p < 0.01 HL vs HL + SMase.

Fig. 2. Effect of SM depletion on the hydrolysis of labeled TAG incorporated into lipoproteins

The lipoproteins were labeled with ³H triolein, and subjected to SMase treatment as described in the text. The SMase-treated and untreated lipoproteins were reacted with recombinant HL (32 μg protein) at 37 °C for the indicated periods of time. The lipids were extracted and the radioactivity in TAG and FFA was determined after TLC separation. The results shown are % decrease in TAG radioactivity from the control (no HL), and are mean ± SEM of 4 experiments. * p,< 0.05 SMase-treated vs untreated samples.

Fig. 3. SM/PC ratios of the lipoproteins, and the effect of SM depletion on the hydrolysis of labeled PC and TAG incorporated into lipoproteins

SM and PC values were determined from the estimation of lipid phosphorus following the separation of the lipids by TLC. The stimulation of PC or TAG hydrolysis by SM depletion was calculated from the samples of 6 h incubation with HL (Fig 1 and Fig 2). The values shown are mean ± SEM of 4 experiments.

Fig. 4. Hydrolysis of PC in unlabeled lipoproteins

Unlabeled lipoproteins (400 μg each) were first treated with SMase, followed by heat-inactivation of SMase, as described in the text. The SM-depleted and intact lipoproteins were then incubated with recombinant HL (32 μg protein) for 4 h at 37 °C.The lipids were extracted after adding an internal standard (17:0–17:0 PC) and the PC composition was analyzed by LC/MS/MS as described in the text, by MRM. The decrease in PC amount was calculated from the difference in the total PC between control (no HL) and experimental (with HL) samples. Values shown are mean ±SEM of 6 analyses (HDL) or 4 analyses (for VLDL and LDL), but were all obtained from the same batch of the enzyme.. * p< 0.05 compared to HDL; # p< 0.05 HL vs SMase + HL.

Fig. 5. Hydrolysis of individual molecular species of PC in the lipoproteins

The SMase-treated and native lipoproteins (400 μg protein) were incubated with 32 μg recombinant human HL for 4 h at 37 °C (Fig. 4), and the composition of molecular species of PC was analyzed by LC/MS/MS, with 17:0 PC as the internal standard. The decrease in individual PC species from the control (no HL) values was calculated. Values shown are mean ± SEM of 4 separate analyses, performed with a single batch of enzyme and 2 different batches of lipoproteins. * p< 0.05 compared to the control (no HL) values (not shown).

Fig. 6. Selectivity of HL for the molecular species of PC in native lipoproteins

The relative specificity values were calculated by dividing the % contribution of each PC species to the total decrease in PC by the % concentration of the species in the native lipoprotein (incubated with no HL). The PC species with a value of > 1.0 are those preferred by the enzyme, whereas the PC species with values < 1.0 are poorer substrates than “average” PC.

Fig. 7. LPC species generated by HL in lipoproteins

The SM-depleted and native lipoproteins were reacted with HL for 4 h (Fig. 5) and the molecular species of LPC were analyzed by LC/MS/MS. The values shown are mean ± SEM of 4 experiments, performed with a single batch of the enzyme and 2 different batches of lipoproteins. * p < 0.05 HL vs SMase + HL

Fig. 8. Effect of SM depletion on TAG hydrolysis in lipoproteins

Unlabeled lipoproteins (400 μg protein) were first depleted of SM by SMase treatment as described in the text. The SMase-treated and native lipoproteins were incubated with recombinant human HL (32 μg) for 4 h, and the TAG composition was analyzed by MRM using 15:0/15:0/15:0 TAG as internal standard. The decrease in TAG from the controls (incubated with no HL) was calculated, and expressed either as % decrease (top) or as decrease in mass (bottom). The values shown are mean ± SEM of 4 analyses (two analyses performed in duplicate, with a single batch of enzyme). # p<0.05 compared to HDL; * p< 0.05 HL alone vs SMase+ HL.

Fig. 9. Hydrolysis of molecular species of TAG by HL

Unlabeled lipoproteins were first depleted of SM by SMase treatment as described in the text. The SM-depleted and native lipoproteins (400 μg) were then incubated with recombinant HL (32 μg) for 4 h and the TAG species were analyzed by MRM, using 15:0/15:0/15:0 TAG as the internal standard. The decrease in each species compared to the control (samples incubated without HL) was calculated. The values shown are mean ± SEM of 4 analyses, as in Fig. 8.. * p< 0.05 compared to control.