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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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LDL biophysical properties and susceptibility to modificationsLDL 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).
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Figure 3: LDL biophysical properties and susceptibility to modificationsLDL 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).

Mentions: To assess the physiological significance of the observed differences in SM and ceramide content, LDL particles from asm+/+/ldlr −/− and asm−/−/ldlr−/− on either standard or atherogenic diets were analysed for oxidation and aggregation as described previously [22]. When run on agarose gel (Figure 3A), LDL from asm+/+/ldlr −/− and asm−/−/ldlr −/− mice fed on the standard diet run as single bands. LDL particles from asm+/+/ldlr −/− mice fed on the high-fat diet exhibited different electrophoretic mobility, with some migrating very slowly (aggregated LDL) and others migrating at a higher rate and forming a secondary band (oxidized LDL) (Figure 3A). These indications for aggregation and oxidation were less pronounced in the LDL from asm−/−/ldlr−/− mice. LDL oxidation and self-aggregation were further assessed by measuring the levels of TBARS and by assaying changes in turbidity. As shown in Figures 3(B) and 3(C), LDL particles from asm+/+/ldlr −/− mice on the modified diet exhibited higher TBARS values and marked increases in the turbidity of the LDL solution, as compared with LDL from asm+/+/ldlr−/− mice on a standard diet. In contrast, there was little increase in the turbidity and, in fact, a decrease in TBARS levels for LDL from asm−/−/ldlr−/− mice on a high-fat diet, as compared with asm−/−/ldlr−/− mice on a standard diet. Such a decrease is likely to reflect the higher SM content of the former. As mentioned before, SM has been shown to protect against LDL oxidation [25]. These results suggest that S-SMase activity contributes to the increased LDL oxidation and aggregation in mice on an 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)

LDL biophysical properties and susceptibility to modificationsLDL 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).
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Figure 3: LDL biophysical properties and susceptibility to modificationsLDL 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).
Mentions: To assess the physiological significance of the observed differences in SM and ceramide content, LDL particles from asm+/+/ldlr −/− and asm−/−/ldlr−/− on either standard or atherogenic diets were analysed for oxidation and aggregation as described previously [22]. When run on agarose gel (Figure 3A), LDL from asm+/+/ldlr −/− and asm−/−/ldlr −/− mice fed on the standard diet run as single bands. LDL particles from asm+/+/ldlr −/− mice fed on the high-fat diet exhibited different electrophoretic mobility, with some migrating very slowly (aggregated LDL) and others migrating at a higher rate and forming a secondary band (oxidized LDL) (Figure 3A). These indications for aggregation and oxidation were less pronounced in the LDL from asm−/−/ldlr−/− mice. LDL oxidation and self-aggregation were further assessed by measuring the levels of TBARS and by assaying changes in turbidity. As shown in Figures 3(B) and 3(C), LDL particles from asm+/+/ldlr −/− mice on the modified diet exhibited higher TBARS values and marked increases in the turbidity of the LDL solution, as compared with LDL from asm+/+/ldlr−/− mice on a standard diet. In contrast, there was little increase in the turbidity and, in fact, a decrease in TBARS levels for LDL from asm−/−/ldlr−/− mice on a high-fat diet, as compared with asm−/−/ldlr−/− mice on a standard diet. Such a decrease is likely to reflect the higher SM content of the former. As mentioned before, SM has been shown to protect against LDL oxidation [25]. These results suggest that S-SMase activity contributes to the increased LDL oxidation and aggregation in mice on an 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