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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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S-SMase activity in serumMice 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.
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Figure 1: S-SMase activity in serumMice 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.

Mentions: Eight-week-old male mice (C57Bl6 and ldlr−/−) were placed for 10 weeks either on standard chow diet (18% protein diet, containing 1% saturated fats and no cholesterol) or a modified (atherogenic) one (42% fat, calorie-adjusted diet, containing 13.8% saturated fats and 1.5% cholesterol). S-SMase activity was measured in serum using NBD-SM as a substrate. Significant increases in S-SMase activity were observed in C57Bl6 mice fed on the atherogenic diet, as compared with C57Bl6 on standard chow (Figure 1A). Judging by Michaelis–Menten analyses, both the Km and Vmax of the enzyme reaction were affected (Figure 1B). These results indicate that S-SMase activity is elevated in mice fed on a modified 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)

S-SMase activity in serumMice 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475451&req=5

Figure 1: S-SMase activity in serumMice 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.
Mentions: Eight-week-old male mice (C57Bl6 and ldlr−/−) were placed for 10 weeks either on standard chow diet (18% protein diet, containing 1% saturated fats and no cholesterol) or a modified (atherogenic) one (42% fat, calorie-adjusted diet, containing 13.8% saturated fats and 1.5% cholesterol). S-SMase activity was measured in serum using NBD-SM as a substrate. Significant increases in S-SMase activity were observed in C57Bl6 mice fed on the atherogenic diet, as compared with C57Bl6 on standard chow (Figure 1A). Judging by Michaelis–Menten analyses, both the Km and Vmax of the enzyme reaction were affected (Figure 1B). These results indicate that S-SMase activity is elevated in mice fed on a modified 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