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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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Fatty acid composition of the SM and ceramide in liver, VLDL and LDLAcidified 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 C17:0 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.
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Figure 5: Fatty acid composition of the SM and ceramide in liver, VLDL and LDLAcidified 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 C17:0 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.

Mentions: The bioactive properties of sphingolipids seem to depend on the length and saturation of the fatty acid attached to the primary amine group in the sphingoid base [43–45]. Therefore RP-HPLC (reverse-phase HPLC)–ESI–tandem MS analysis was used to determine whether an atherogenic diet affects the levels of sphingolipids with specific fatty-acid content (Figure 5). The major fatty acids found in serum lipoprotein sphingolipids were nervonic (C24:1), lignoceric (C24:0) and palmitic acid (C16:0). The liver sphingolipids exhibited similar fatty-acid distribution, confirming previous studies [46]. The levels of all measured ceramide and SM species were elevated in mice fed on the modified diet. Importantly, the increases in C16:0-containing sphingolipids, however, were larger than those for sphingolipids with unsaturated or very-long-chain saturated fatty acids. As a result, the occurrence of sphingolipids with C16:0 fatty acid in liver and lipoproteins of mice on an atherogenic diet increased, reflecting the high abundance of palmitate in the diet. It should be noted that elevated C16:0 sphingolipid content has been associated with increased rate of apoptosis and fibrosis in liver [46,47]. Thus, the observed increases in the fraction of sphingolipids with C16:0 fatty acid on palmitate-enriched diet could be associated with some of the pathology characteristics of cardiovascular diseases.


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)

Fatty acid composition of the SM and ceramide in liver, VLDL and LDLAcidified 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 C17:0 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.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Fatty acid composition of the SM and ceramide in liver, VLDL and LDLAcidified 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 C17:0 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.
Mentions: The bioactive properties of sphingolipids seem to depend on the length and saturation of the fatty acid attached to the primary amine group in the sphingoid base [43–45]. Therefore RP-HPLC (reverse-phase HPLC)–ESI–tandem MS analysis was used to determine whether an atherogenic diet affects the levels of sphingolipids with specific fatty-acid content (Figure 5). The major fatty acids found in serum lipoprotein sphingolipids were nervonic (C24:1), lignoceric (C24:0) and palmitic acid (C16:0). The liver sphingolipids exhibited similar fatty-acid distribution, confirming previous studies [46]. The levels of all measured ceramide and SM species were elevated in mice fed on the modified diet. Importantly, the increases in C16:0-containing sphingolipids, however, were larger than those for sphingolipids with unsaturated or very-long-chain saturated fatty acids. As a result, the occurrence of sphingolipids with C16:0 fatty acid in liver and lipoproteins of mice on an atherogenic diet increased, reflecting the high abundance of palmitate in the diet. It should be noted that elevated C16:0 sphingolipid content has been associated with increased rate of apoptosis and fibrosis in liver [46,47]. Thus, the observed increases in the fraction of sphingolipids with C16:0 fatty acid on palmitate-enriched diet could be associated with some of the pathology characteristics of cardiovascular diseases.

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