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

Deevska GM, Sunkara M, Morris AJ, Nikolova-Karakashian MN - Biosci. Rep. (2012)

Bottom Line: An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Division of Cardiovascular Medicine, University of Kentucky, A. B. Chandler Medical Center, Lexington, KY 40536, U.S.A.

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)- 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.

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

Mentions: The role S-SMase plays in determining the ceramide enrichment of LDL was investigated in sera from asm−/−/ldlr −/− and asm+/+/ldlr −/− mice placed on either modified or standard diet. In a previous study, we reported the generation of this novel composite knockout mouse strain, in which asm deficiency (asm−/−) was introduced into ldlr−/− background [13]. Feeding asm−/−/ldlr−/− mice and their respective controls, asm+/+/ldlr−/−, with atherogenic diet, revealed that the lack of ASMase had no effect on diet-induced hypercholesterolaemia, and mice from both genotypes exhibited similar increases in serum VLDL and LDL cholesterol and similar lipoprotein profiles [13]. The experiments shown in Figure 2(A) confirm that asm-deficient mice and wild-type control develop comparable hypercholesterolaemia when placed on atherogenic diet. The increases in LDL protein were also similar for both genotypes (results not shown). As anticipated, the asm- mice have no detectable S-SMase activity thus confirming that S-SMase is the only SM-hydrolysing enzyme in mouse serum (Figure 2B). Mice deficient in S-SMase consequently have significantly higher LDL-SM content than their wild-type counterparts. This LDL-SM increases similarly for both genotypes when placed on an atherogenic diet (Figure 2C). In contrast, diet-induced increases in LDL-ceramide (and in the ceramide to SM ratio) are substantially attenuated in the asm−/−/ldlr −/− mice (Figures 2D and 2E). The statistical significance of the diet–genotype interaction effect based on two-way ANOVA is P=0.016 for LDL-ceramide (Figure 2D) and P=0.01 for the ceramide/SM ratio (Figure 2E) confirming that asm deletion eliminates diet-induced accumulation of ceramide in the LDL particles. This effect is specific for LDL since the corresponding elevation in ceramide observed in the liver is not reversed by the ASMase deletion [13]. Collectively, these results provide evidence that the activation of serum S-SMase facilitates the conversion of LDL-SM into ceramide in mice on atherogenic diet.


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

Deevska GM, Sunkara M, Morris AJ, Nikolova-Karakashian MN - Biosci. Rep. (2012)

Role of S-SMase in diet-induced changes in serum cholesterol levels and LDL-sphingolipid contentMice 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.
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Related In: Results  -  Collection

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Figure 2: Role of S-SMase in diet-induced changes in serum cholesterol levels and LDL-sphingolipid contentMice 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.
Mentions: The role S-SMase plays in determining the ceramide enrichment of LDL was investigated in sera from asm−/−/ldlr −/− and asm+/+/ldlr −/− mice placed on either modified or standard diet. In a previous study, we reported the generation of this novel composite knockout mouse strain, in which asm deficiency (asm−/−) was introduced into ldlr−/− background [13]. Feeding asm−/−/ldlr−/− mice and their respective controls, asm+/+/ldlr−/−, with atherogenic diet, revealed that the lack of ASMase had no effect on diet-induced hypercholesterolaemia, and mice from both genotypes exhibited similar increases in serum VLDL and LDL cholesterol and similar lipoprotein profiles [13]. The experiments shown in Figure 2(A) confirm that asm-deficient mice and wild-type control develop comparable hypercholesterolaemia when placed on atherogenic diet. The increases in LDL protein were also similar for both genotypes (results not shown). As anticipated, the asm- mice have no detectable S-SMase activity thus confirming that S-SMase is the only SM-hydrolysing enzyme in mouse serum (Figure 2B). Mice deficient in S-SMase consequently have significantly higher LDL-SM content than their wild-type counterparts. This LDL-SM increases similarly for both genotypes when placed on an atherogenic diet (Figure 2C). In contrast, diet-induced increases in LDL-ceramide (and in the ceramide to SM ratio) are substantially attenuated in the asm−/−/ldlr −/− mice (Figures 2D and 2E). The statistical significance of the diet–genotype interaction effect based on two-way ANOVA is P=0.016 for LDL-ceramide (Figure 2D) and P=0.01 for the ceramide/SM ratio (Figure 2E) confirming that asm deletion eliminates diet-induced accumulation of ceramide in the LDL particles. This effect is specific for LDL since the corresponding elevation in ceramide observed in the liver is not reversed by the ASMase deletion [13]. Collectively, these results provide evidence that the activation of serum S-SMase facilitates the conversion of LDL-SM into ceramide in mice on atherogenic diet.

Bottom Line: An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Division of Cardiovascular Medicine, University of Kentucky, A. B. Chandler Medical Center, Lexington, KY 40536, U.S.A.

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)- 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.

Show MeSH
Related in: MedlinePlus