Characterization of secretory sphingomyelinase activity, lipoprotein sphingolipid content and LDL aggregation in ldlr-/- mice fed on a high-fat diet

Abstract

The propensity of LDLs (low-density lipoproteins) for aggregation and/or oxidation has been linked to their sphingolipid content, specifically the levels of SM (sphingomyelin) and ceramide. To investigate this association in vivo, ldlr (LDL receptor)-null mice (ldlr-/-) were fed on a modified (atherogenic) diet containing saturated fats and cholesterol. The diet led to significantly elevated SM content in all serum lipoproteins. In contrast, ceramide increased only in the LDL particles. MS-based analyses of the lipid acyl chain composition revealed a marked elevation in C16:0 fatty acid in SM and ceramide, consistent with the prevalence of palmitic acid in the modified diet. The diet also led to increased activity of the S-SMase [secretory SMase (sphingomyelinase)], a protein that is generated by ASMase (acid SMase) and acts on serum LDL. An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity. ASMase-deficient mice (asm-/-/ldlr-/-) lacked S-SMase activity and were protected from diet-induced elevation in LDL ceramide. LDL from asm-/-/ldlr-/- mice fed on the modified diet were less aggregated and oxidized than LDL from asm+/+/ldlr-/- mice. When tested in vitro, the propensity for aggregation was dependent on the SM level: only LDL from animals on modified diet that have high SM content aggregated when treated with recombinant S-SMase. In conclusion, LDL-SM content and S-SMase activity are up-regulated in mice fed on an atherogenic diet. S-SMase mediates diet-induced changes in LDL ceramide content and aggregation. S-SMase effectiveness in inducing aggregation is dependent on diet-induced enrichment of LDL with SM, possibly through increased hepatic synthesis.

Figure 1. S-SMase activity in serum

Mice of the indicated genotypes were placed on either a standard or modified diet for 10 weeks. S-SMase activity was measured using C₆-NBD-SM as a substrate. (A) S-SMase activity in C57Bl6 mice measured by using 0.4 nmol of NBD-SM per sample. Results are shown as means±S.D. (n=3) for each mouse. (B–D) Michaelis–Menten kinetics of the S-SMase activity in C57Bl6 and ldlr^−/− mice on either standard or modified diet. The assays are carried out using serum (4–6 animals per assay) and the indicated substrate concentrations. Mean values of triplicates ±S.D. are shown. *P<0.05 and **P<0.01 according to a Student's t test. SM/NBD-SM ratio in (D) was calculated by dividing the measured endogenous LDL-SM concentration (presented in Table 1) and the amount of NBD-SM in each assay.

Figure 2. Role of S-SMase in diet-induced changes in serum cholesterol levels and LDL-sphingolipid content

Mice of the indicated genotype were placed on either a standard or modified diet for 10 weeks (3–5 animals per group). Total serum (A and B) or purified LDL particles (C–E) were analysed for total cholesterol (A), S-SMase activity (B), SM, (C), ceramide (D) or the molar ratio of ceramide to SM (E). Values are means±S.D. (n=3 animals per group). Statistical significance was analysed using two-way ANOVA followed by Bonferonni post-hoc test. The results of the Bonferonni post-hoc test with respect to the diet (*P<0.05, **P<0.01 and ***P<0.001), genotype (^∧∧P<0.01), as well as the interaction effects from two-way ANOVA (^#P<0.05 and ^##P<0.01) are shown.

Figure 3. LDL biophysical properties and susceptibility to modifications

LDL particles were isolated by sequential ultracentrifugation from sera of asm^−/−/ldlr^−/− [labelled with (−/−)] and asm^+/+/ldlr^−/− [labelled with (+/+)] mice placed on either standard or modified diet for 10 weeks (3–5 animals per group). (A) Electrophoretic mobility of LDL particles (10 μg of protein per lane) in 1.8% agarose gel visualized by Coomassie Brilliant Blue staining. (B) Oxidation of LDL assessed by measuring TBARS. Results are means±S.D. of triplicate measurements. Statistical significance was analysed by two-way ANOVA followed by Bonferonni post-hoc test. The results of the Bonferonni post-hoc test with respect to the diet (**P<0.01), genotype (^∧∧P<0.01) as well as the genotype/diet interaction effect from two-way ANOVA (^###P<0.001) are shown. (C) Turbidity assay of LDL aggregation. Changes in absorbance were monitored at 680 nm at the indicated times. (D) TLC/HPLC determination of the increase in LDL ceramide content following treatment with bSMase (0.5 unit/ml). The increases in ceramide content due to bSMase treatment were analysed for statistical significance using two-way ANOVA and Bonferonni post-hoc test with respect to diet and genotype. The results of the Bonferonni test with respect to the diet (**P<0.01 and ***P<0.001) and genotype (^∧P<0.05 and ^∧∧P<0.01) are shown. (E) Effect of bSMase treatment on the electrophoretic properties of LDL particles (10 μg of protein) in 1.8% agarose gel visualized by Coomassie Brilliant Blue staining. Representative results from at least three independent experiments, including three different isolations of LDL, are shown. Std, Standard; Mod, modified. (F) Quantification of the results shown in (E).

Figure 4. S-SMase and L-SMase activities in peritoneal macrophages, blood vessels and adipose tissue cultured ex vivo

Mice were placed either on standard or modified diets for 10 weeks (3–5 animals per group). NBD-SM was used as a substrate to measure Zn²⁺-dependent S-SMase activity in conditioned medium or L-SMase activity in cellular lysates. Results are normalized per mg of cellular protein. Means±S.D. are shown. *P<0.05 compared with mice of same genotype on standard diet. (A) S-SMase activity in various tissues of asm^+/+/ldlr^−/− mice. (B) S-SMase activity in resident peritoneal macrophages from asm^−/−/ldlr^−/− and asm^+/+/ldlr^−/− mice. (C) L-SMase activity in lysates prepared from the same macrophages.

Figure 5. Fatty acid composition of the SM and ceramide in liver, VLDL and LDL

Acidified organic solvents were used to prepare total lipid extracts from liver homogenates, VLDL and LDL from asm^+/+/ldlr^−/− mice (three to five animals per group) fed either standard or modified diet. Ceramide and SM internal standards containing a C_17:0 fatty acid were added at the start to account for any losses during the extraction. Sphingolipid species containing different fatty acids were detected by monitoring species-specific precursor product ion pairs using HPLC–ESI–tandem MS and 4000 Q-Trap hybrid linear ion trap triple-quadrupole mass spectrometer. Levels of the respective dihydro species were sometimes too low for proper quantification and therefore were not included.

Figure 6. Ceramide and SM species in the serum of mice on atherogenic or control diet

Total serum lipid extracts were prepared from asm^−/−/ldlr^−/− and asm^+/+/ldlr^−/− mice placed on either standard (Std) or modified (Md) diet for 10 weeks. Ceramide (A) and SM (B) species were quantified by monitoring precursor product ion pairs using HPLC–ESI/tandem MS. The ceramide/SM molar ratio was calculated in each sample (C). Means±S.D. are shown (n=3 animals per group). Bonferonni post-hoc test analyses comparing the effect of the diet for mice from the same genotype are shown (*P<0.05 and ^#P<0.001). Std, standard diet; Md, modified diet.