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

Mentions: S-SMase activity under standard diet was high for ABVs, as well as for resident peritoneal macrophages, while it was low for epididymal adipose tissue (Figure 4A). The atherogenic diet had seemingly no stimulatory effect on the S-SMase of ABVs and fat tissue. If anything, the activity of ABVs tended to decline. However, macrophages from mice on modified diet exhibited 2-fold higher SMase activity. As expected, the S-SMase-deficient mice had no detectable secretory activity associated with their macrophages on either diet (Figure 4B). This confirmed that the activity measured in our assays came solely from S-SMase. Importantly, the observed diet-induced enzyme stimulation was S-SMase-specific since the activity of the L-SMases, which is produced by the same gene, was not induced (Figure 4C). The ASMase activity in the cells actually declined, which could indicate re-direction of the intracellular trafficking of the ASMase precursor away from the lysosomes and towards the secretory pathways in macrophages of mice on the atherogenic diet. Such a possibility is supported by published studies showing that atherogenic diet interferes with the mannose 6-phosphate receptor system [41]. The latter is known to regulate the processing of the lysosomal and secretory form of ASMase [3]. It is also possible, however, that the decrease in macrophages ASMase results from diet-induced accumulation of cholesterol, which is an ASMase inhibitor [42]. Whatever the case, it is clear that high-fat diet has an impact on macrophage S-SMase activity and/or secretion.


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 and L-SMase activities in peritoneal macrophages, blood vessels and adipose tissue cultured ex vivoMice 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.
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Related In: Results  -  Collection

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Figure 4: S-SMase and L-SMase activities in peritoneal macrophages, blood vessels and adipose tissue cultured ex vivoMice 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.
Mentions: S-SMase activity under standard diet was high for ABVs, as well as for resident peritoneal macrophages, while it was low for epididymal adipose tissue (Figure 4A). The atherogenic diet had seemingly no stimulatory effect on the S-SMase of ABVs and fat tissue. If anything, the activity of ABVs tended to decline. However, macrophages from mice on modified diet exhibited 2-fold higher SMase activity. As expected, the S-SMase-deficient mice had no detectable secretory activity associated with their macrophages on either diet (Figure 4B). This confirmed that the activity measured in our assays came solely from S-SMase. Importantly, the observed diet-induced enzyme stimulation was S-SMase-specific since the activity of the L-SMases, which is produced by the same gene, was not induced (Figure 4C). The ASMase activity in the cells actually declined, which could indicate re-direction of the intracellular trafficking of the ASMase precursor away from the lysosomes and towards the secretory pathways in macrophages of mice on the atherogenic diet. Such a possibility is supported by published studies showing that atherogenic diet interferes with the mannose 6-phosphate receptor system [41]. The latter is known to regulate the processing of the lysosomal and secretory form of ASMase [3]. It is also possible, however, that the decrease in macrophages ASMase results from diet-induced accumulation of cholesterol, which is an ASMase inhibitor [42]. Whatever the case, it is clear that high-fat diet has an impact on macrophage S-SMase activity and/or secretion.

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