Dietary Sphingomyelin Lowers Hepatic Lipid Levels and Inhibits Intestinal Cholesterol Absorption in High-Fat-Fed Mice

Controlling intestinal lipid absorption is an important strategy for maintaining lipid homeostasis. Accumulation of lipids in the liver is a major risk factor for metabolic syndrome and nonalcoholic fatty liver disease. It is well-known that sphingomyelin (SM) can inhibit intestinal cholesterol absorption. It is, however, unclear if dietary SM also lowers liver lipid levels. In the present study (i) the effect of pure dietary egg SM on hepatic lipid metabolism and intestinal cholesterol absorption was measured with [14C]cholesterol and [3H]sitostanol in male C57BL/6 mice fed a high-fat (HF) diet with or without 0.6% wt/wt SM for 18 days; and (ii) hepatic lipid levels and gene expression were determined in mice given a HF diet with or without egg SM (0.3, 0.6 or 1.2% wt/wt) for 4 weeks. Mice supplemented with SM (0.6% wt/wt) had significantly increased fecal lipid and cholesterol output and reduced hepatic [14C]cholesterol levels after 18 days. Relative to HF-fed mice, SM-supplemented HF-fed mice had significantly lower intestinal cholesterol absorption (−30%). Liver weight was significantly lower in the 1.2% wt/wt SM-supplemented mice (−18%). Total liver lipid (mg/organ) was significantly reduced in the SM-supplemented mice (−33% and −40% in 0.6% wt/wt and 1.2% wt/wt SM, respectively), as were triglyceride and cholesterol levels. The reduction in liver triglycerides was due to inactivation of the LXR-SREBP-1c pathway. In conclusion, dietary egg SM has pronounced hepatic lipid-lowering properties in mice maintained on an obesogenic diet.


Introduction
Controlling lipid absorption in the intestine is an important strategy for maintaining lipid homeostasis. Hepatic steatosis is a significant risk factor for many metabolic and cardiovascular diseases, such as atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Reducing cholesterol absorption is considered to be atheroprotective [1,2], and is associated with reduced risk of NAFLD in mice [3].
This study asks if dietary supplementation with sphingomyelin (SM) reduces cholesterol absorption in mice fed a high-fat (HF) western-type diet. SM is a sphingolipid found in foods such as milk and eggs. Purified SM has been shown to inhibit cholesterol absorption in a number of acute in vitro [4] and in vivo [5][6][7][8] studies. A recent study reported plasma and hepatic lipid lowering effects in Zucker fatty rats fed a low-fat chow diet supplemented with chicken skin-derived SM for 6 weeks [9]. However, the lack of dietary fat in the chow diet used in that study did not address the question as to whether long-term dietary SM supplementation affects cholesterol absorption inhibition. Nor was it able to correlate the inhibition of intestinal cholesterol absorption to lipid-lowering effects [9].
Mice fed an obesogenic diet containing high levels of butter fat and cholesterol develop features similar to those of humans with the metabolic syndrome (increased body weight, hepatic steatosis and insulin resistance) [10]. Previous work from our laboratory has shown that dietary supplementation with phospholipids from different sources such as milk, krill, egg and soy can reduce plasma and liver lipid levels [3,[11][12][13]. In this study, our aim was to determine whether dietary SM supplementation inhibits intestinal cholesterol absorption in mice fed an obesogenic diet, and to determine whether this effect is linked to a beneficial effect on hepatic lipid levels.

Animals and diets
All of the animal studies were approved by the Animal Welfare Committee of the Sydney South West Area Health Service.
Four to five-week-old male C57BL/6 mice were obtained from Monash University, Melbourne, VIC. They were housed in standard cages (5 mice per cage) at a constant temperature of 21uC with a 12 h light/dark cycle and ad libitum access to a normal chow diet and water. After 1 week of acclimatization, the mice were randomly assigned to four groups (n = 10/group). The animals were given a high-fat semi-purified diet with (HFSM) or without (SM) egg SM (99% pure, Lipoid, Ludwigshafen, Germany) for 18 days (Study 1) or for 4 weeks (Study 2).
In Study 1, the high-fat diet was supplemented with SM 0.6% wt/wt. In Study 2, the high-fat diet was supplemented with 0.3%, 0.6% or 1.2% SM (wt/wt). Food consumption was recorded three times per week and body weight was monitored weekly.

Intestinal cholesterol absorption
In order to investigate the ability of SM to inhibit intestinal cholesterol absorption, two groups of mice (n = 10/group) were fed a high-fat diet with or without SM (0.6% wt/wt) for 18 days (Study 1). At 14 days after commencing the diet, olive oil (200 ml) containing 1 mCi [ 14 C]cholesterol and 1 mCi [ 3 H]sitostanol, a sterol that is minimally absorbed by the intestine [14], was administered by intragastric gavage under light methoxyflurane anaesthesia. One animal from the SM-supplemented group did not survive the gavage and was excluded from the analysis. The mice were then transferred into individual cages with perforatedmetal floors. Feces was collected daily for 4 days and stored at 280uC until analysis. Mice were given ad libitum access to their respective diets during this collection period so that the total length of the dietary intervention was 18 days. At the end of the fecal collection period, the mice were anaesthetized with methoxyflurane and exsanguinated by heart puncture.
At sacrifice, livers were immediately excised, weighed and divided into 100-150 mg sections for storage at 280uC until analysis for lipid and radiolabel content. Fecal samples were combined, dried at 60uC for 24 hours then ground to a fine powder. Total lipid content in feces and the pre-gavage radioactive cholesterol-sitostanol-olive oil mixture was analyzed by extracting lipids from 100 mg of the fecal powder or 10 ml aliquots of the olive oil mixture with 1.2 ml of chloroform:methanol (2:1, v/v) [15]. The lipid extracts were dried under nitrogen, saponified in 1 ml of 2 M sodium hydroxide:methanol (1:1, v/v) and incubated at 60uC for 1 hour. The labelled sterols were then extracted into diethyl ether (2 ml). Duplicate aliquots (500 ml) of the organic phase were dried by evaporation, scintillation fluid (5 ml) was added and the samples were counted. The percentage of cholesterol absorption was calculated using the ratio of 14 C and 3 H counts in the olive oil mixture and feces according to the fecal dual-isotope method [16].
% Cholesterol absorption1 The total cholesterol and triglyceride levels in the liver and feces were measured biochemically as described below.

Hepatic lipid analysis
To investigate the effect of SM supplementation on hepatic lipid profile, four groups of mice (n = 10/group) were given high-fat diet without or with 0.3%, 0.6% and 1.2% (wt/wt) SM for 4 weeks (Study 2). The animals were fasted for 12 hours before sacrifice. Two high-fat fed animals were subsequently excluded from the study because they failed to thrive. Livers were excised immediately after sacrifice, weighed and sectioned (100-150 mg/section) for storage at 280uC for subsequent lipid analysis, or added to RNAlaterH solution (Albion, Austin, TX) for gene expression analysis.
Total liver and fecal lipids were determined gravimetrically after extraction with cholororm:methanol (2:1, v/v) [15]. Liver and fecal total cholesterol and triglyceride levels were quantified enzymatically after solubilization in isopropanol using CHOD-PAP and GPO-PAP kits (Roche Diagnostics), respectively. Isopropanol-solubilized total liver phospholipids were measured using a Wako Phospholipids C kit (Wako Pure Chemicals, Osaka, Japan). . The samples were vortexed, rotated for 10 minutes, sonicated for 30 minutes and left to stand at room temperature for a further 20 minutes. The extracts were then centrifuged at 13,0006g for 10 minutes, and supernatants were transferred to a 96-well plate and dried under nitrogen at 40uC. Lipids were redissolved in water-saturated butanol (50 ml) with sonication (10 min) followed by the addition of methanol (50 ml) containing 10 mM NH 4 COOH. Samples were analysed by electrospray ionisation-tandem mass spectrometry using a PE Sciex API 4000 Q/TRAP mass spectrometer with a turboionspray source and Analyst 1.5 data system. Prior liquid chromatographic separation was performed on a Zorbax C18, 1.8 um, 5062.1 mm column at 300 ml/min using the following gradient conditions; 100% solvent A to 0% solvent A over 8.0 min followed by 2.5 min at 0% solvent A, a return to 100% solvent A over 0.5 min then 3.0 min at 100% solvent A prior to the next injection. Diglycerides and triglycerides were separated on an isocratic (85% B) system over 6 minutes. Solvents A and B consisted of tetrahydrofuran:methanol:water in the ratios (30:20:50, v/v/v) and (75:20:5, v/v/v), respectively, plus 10 mM NH 4 COOH.

Lipids and ceramide analysis by ESI-MS/MS
Individual lipid species were quantified using scheduled multiple-reaction monitoring (MRM) in positive ion mode. Individual lipid species monitored were the major species (greater than 1% of total) identified in human plasma. MRM experiments were based on product ion of m/z 264 [sphingosine-H 2 O] + for Cer, GC and DHC, m/z 184 [phosphocholine] + for SM, m/z 369 [cholesterol-H 2 O] + for cholesterol and CE. Each ion pair was monitored for 10-50 ms with a resolution of 0.7 amu at half-peak height and averaged from continuous scans over the elution period. Lipid concentrations were calculated by relating the peak area of each species to the peak area of the corresponding internal standard. Total lipids of each class were calculated by summing the individual lipid species.

Gene expression analysis
Total RNA was isolated from liver samples using an RNeasy kit (Qiagen, Melbourne, Australia). RNA (100 ng) was reverse transcribed into cDNA using random primers provided with the iScript cDNA Synthesis Kit (Bio-Rad, Sydney, Australia). mRNA levels were measured by real time PCR. Selected genes were amplified using iQ SYBR Green Supermix (Bio-Rad) in an iCycler system (Bio-Rad) with 20 pmol of both forward and reverse primers (Table S1). PCR conditions were as follows: 1 cycle of 95uC for 3 min, 40 cycles of 95uC for 30 sec, 55-60uC for 3 sec and 72uC for 30 sec, followed by 1 cycle of 95uC for 1 min. Purity of the PCR products was assessed by melt curve analysis. Relative gene expression was calculated by normalizing cycle threshold (Ct) values for genes of interest with Ct values for cyclophilin using the DDCt method.

Statistical analysis
The results are reported as means6SEM. Graphpad Prism (version 4.0c, GraphPad Software, Inc.) was used for statistical analyses. Significant differences between the HF and HFSM groups were assessed by either one-way ANOVA or Student's ttest (homoscedastic, two-tailed). Pearson correlation coefficients (r) were determined to assess the relationship between parameters, with P,0.05 considered to be statistically significant. The p-values were corrected for multiple comparisons (P corrected ) using the Benjamini-Hochberg [17] approach for genes, lipids and lipid classes separately, P corrected ,0.05 was considered to be statistically significant.
Hierarchical clustering based on correlation coefficients derived from all significant genes and lipids between the HF and HFSM groups was performed using the MATLAB 2012b Bioinformatics Toolbox.
A relevance network was created using Cytoscape [18] (version 2.2.2) where the nodes are genes and lipids and an edge is defined between genes (green) or genes and lipids (blue) for |r|.0.7 and pvalue,0.05.

Study 1: Inhibition of intestinal cholesterol absorption and reduction in hepatic lipids by dietary SM
Dietary supplementation with SM for 18 days did not affect total body weight (data not shown). Mice supplemented with SM had slightly smaller livers than the control animals (1.1360.05 v 1.2160.05 g) and their liver/body weight ratio was also less (4.4960.23 v 4.9560.18). These differences did not reach statistical significance. The livers of the high fat-fed mice supplemented with SM contained significantly less total lipid (237%, P,0.05), cholesterol (255% P,0.001) and triglyceride (249%, P,0.05) than the livers of the high fat-fed control mice (Table 1). They also contained significantly lower levels of intestinally-derived [ 14 C]cholesterol. There was no difference in hepatic [ 3 H]sitostanol levels between the two groups.
SM supplementation was associated with a 30% increase in fecal [ 14 C]cholesterol content (P,0.05) ( Table 1). Similar levels of fecal [ 3 H]sitostanol in both groups of animals indicated that comparable amounts of radioactive sterol were administered on day 14. Using these data, cholesterol absorption was found to be significantly less in mice supplemented with SM than in the mice fed a HF diet without SM supplementation (230%, P,0.01). This was also associated with higher amounts of cholesterol and total lipid in the feces of the SM-fed animals. Linear regression analysis showed that liver cholesterol in the HF-and HFSM-fed animals was significantly correlated with percentage of intestinal cholesterol absorption (r 2 = 0.38, P,0.01) (Fig. 1A), and with levels of intestinally-derived cholesterol in the liver (r 2 = 0.86, P,0.001) (Fig. 1B). Liver cholesterol levels were also correlated with liver triglyceride levels (r 2 = 0.70, P,0.001) (Fig. 1C). Separate correlation analyses for the control and SM-treated groups were also performed. Liver cholesterol levels correlated significantly with % cholesterol absorption, liver [ 14 C]cholesterol content and liver triglyceride levels in the control group. The same correlations for the SM-treated group were all poor because cholesterol absorption was very low regardless of liver cholesterol levels. This is consistent with dietary SM being highly efficacious in terms of lowering hepatic lipid levels and inhibiting intestinal cholesterol absorption, and probably reflects maximal inhibition of cholesterol absorption by the SM dose used in the study. In order to display the relationships of interest over a wider range, the combined data sets are shown in Figure 1.

Study 2: Effect of dietary SM on hepatic lipid metabolism
In order to further investigate the effect of dietary SM on liver lipid levels, a second study was carried out in which high-fat fed mice received dietary supplementation with 0.3%, 0.6% and 1.2% (wt/wt) SM for 4 weeks (Study 2). The high fat-fed control mice, and the mice fed a high fat diet supplemented with SM, all had similar body weight gain and food intake throughout the study ( Table 2). The liver weights of the animals supplemented with SM decreased in a dose-dependent manner. Mice fed 0.6% and 1.2% (wt/wt) SM had liver weights that were 12% (P,0.05) and 18% (P,0.001) lower, respectively, than what was observed for the HF animals ( Table 2). Their liver/body weight ratios were also lower, but this reached statistical significance only in the animals that received supplementation with 1.2% (wt/wt) SM (P,0.05). Total hepatic lipid, cholesterol, triglyceride and phospholipid levels were also reduced in a concentration-dependent manner in the SM-supplemented animals (Fig. 2). Total hepatic lipid levels (mg/organ) were decreased by 20%, 33% and 40% in the 0.3%, 0.6% and 1.2% (wt/wt) SM-supplemented groups, respectively ( Fig. 2A). Hepatic cholesterol levels were decreased by 36%, 48% and 67% (Fig. 2B); while hepatic triglyceride levels were decreased by 31%, 46% and 56%, respectively (Fig. 2C). Hepatic phospholipid levels were also lower in the SM-supplemented animals, but not to the same extent (14%, 21% and 24%, respectively, Fig. 2D).
Individual and total lipid concentrations in the livers of the high fat-fed control mice, and the mice fed a high fat diet supplemented with SM were also determined using a p-value corrected for multiple comparisons as described in the Methods. These results are shown in Tables 3 and 4 (Table S2 for complete results).
Total liver cholesterol levels were significantly lower (255%) in the SM-supplemented mice (Table 3). Fifteen lipids of the CE class were detected by ESI-MS/MS (Table S2), ten of which were significantly lower in the livers of the animals that received SM (Table 4). Total hepatic CE was reduced by 59% in the SMsupplemented mice (Table 3). Unesterified cholesterol, levels were also lower in SM-supplemented animals (219%), but this did not reach statistical significance.
Dietary supplementation with SM did not significantly affect hepatic SM levels (Table 3). Total ceramide (Cer) levels were also unchanged. However, Cer 24:1 was significantly lower in the SMsupplemented (Table 4) group and Cer 16:0 also showed a nonsignificant negative trend after correction for multiple comparisons (Table S2). Similar trends were observed in both monohexosylceramides and dihexosylceramides, with the 24:1 and 16:0 species showing negative trends in the SM-supplemented group (Table 3,  Table S2).
Messenger RNA levels of genes involved in liver fatty acid and cholesterol metabolism are shown in Table 5. The hierarchical clustering of correlations of genes and lipids which were significantly different in the two groups is shown in Figure 3. The relevance network of these genes and lipids is shown in Figure 4. Strikingly, mRNA levels for all four ATP-binding cassette transporter genes, ABCA1, ABCG1, ABCG5 and ABCG8, that were measured were significantly lower in the SMsupplemented mice (228%, 257%, 244% and 232% respectively, P,0.001 for all). These genes were also positively correlated with several CE and TG lipids (Figure 3). The ABCG5/8 and SREBP-2 genes were also positively correlated, with the significantly altered CEs (Figure 3).
We also hypothesized that genes controlling fatty acid oxidation may have been increased by SM supplementation, but no evidence to this effect was obtained. In fact mRNA levels of the fatty acid oxidation genes (acetyl-CoA oxidase (ACO), cytochrome P450 4A10 (CYP4A10), carnitine palmitoyltransferase 1a (CPT1a), medium-chain acyl-coenzyme A dehydrogenase (MCAD) and very long-chain acyl-coenzyme A dehydrogenase (VLCAD) tended to be lower in the mRNA levels mice than in the control, high fat-fed mice, as was the mRNA level of their transcription factor, peroxisome proliferator-activated receptor a (PPARa).
Hepatic mRNA levels of enzymes involved in de novo ceramide synthesis, as well as SM catabolism, were also quantified. Serine palmitoyltransferase, long chain base subunit 2 (SPTLC2) and ceramide synthase 5 (CERS5) were decreased by 29% (P,0.001) and 49% (P,0.01) respectively (Table 5), and were both strongly correlated to Cer 24:1 ( Figure 3). Dietary SM supplementation also down-regulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), which hydrolyses SM to phosphocholine and ceramide, by 34% (P,0.01). However, while this was strongly correlated to both ABCG5 and ABCG8, it did not show the same strength of correlation to any ceramide, sphingomyelin or other lipid species (Figures 3 and 4).

Discussion
The results of the present study demonstrate that dietary SM potently decreases total hepatic cholesterol and triglyceride levels and reduces intestinal cholesterol absorption. This suggests that dietary SM may lower hepatic lipid levels by inhibiting intestinal cholesterol absorption, and is consistent with our previously published studies demonstrating the ability of dietary phospholipid extracts (containing significant amounts of SM) to reduce hepatic lipid levels and intestinal cholesterol absorption in high-fat fed mice [3,13].
At present, the mechanism for the inhibitory effect of dietary SM on cholesterol absorption is not well understood. The ability of dietary SM to inhibit cholesterol absorption has been shown previously [6][7][8], and it has been proposed that the strong interaction between cholesterol and SM [26,27] reduces the micellar solubility of intestinal cholesterol, resulting in reduced intestinal cholesterol uptake [14,16].
It has also been suggested that ceramide, the hydrolytic product of SM, inhibits intestinal cholesterol uptake [4]. Since the degradation of ceramide along the gastrointestinal tract is relatively slow and inefficient, it is possible that the inhibitory effect of ceramide on cholesterol absorption could be quite significant [19]. In addition, SM has been shown to inhibit secretory (type II) phospho]lipase A2 (sPLA2), which is structurally similar to pancreatic (type I) PLA2 (PPLA2) [20,21]. PPLA2 activity is important in the hydrolysis of TG in the intestinal tract. It has been shown that the hydrolysis of PC to lysoPC facilitates the binding of lipase-colipase to TG (33-36) and that minimal TG hydrolysis is essential to stimulate intestinal cholesterol uptake [22]. It has thus been proposed that SM-mediated inhibition of pancreatic lipase may limit PC bioavailability, which reduces mixed micelle formation, and inhibits SM-mediated intestinal cholesterol absorption [7,23]. It is interesting to note in the present study that total lipids, and not just cholesterol, were increased in the feces of the SM-supplemented mice (Table 1). Triglyceride itself was not significantly increased, however, and there was no evidence of severe fat malabsorption (diarrhoea or a marked reduction in body weight). The effects of dietary SM on the intestinal absorption of other lipids thus deserves further investigation.
The present study shows that lower hepatic cholesterol levels were strongly associated with reduced intestinal cholesterol absorption. Decreased flux of intestinal cholesterol to the liver is supported by the analysis of hepatic gene expression, whereby LXRa-regulated genes involved in hepatic cholesterol export, such as ABCA1, ABCG1, ABCG5 and ABCG8 [24], were down-regulated in the SM-supplemented mice. Genes responsible for hepatic cholesterol uptake, such as LDLr and SR-B1, and genes involved in cholesterol synthesis, such as HMGCoA reductase and HMGCoA synthase, by contrast, tended to be lower or were unchanged.
The observed reduction in hepatic cholesterol levels was strongly associated with lower levels of hepatic triglyceride in the present study. We hypothesize that reduced hepatic triglyceride levels in SM-supplemented mice was a consequence of reduced LXRa activity through ATP-binding cassette transporter genes, ABCA1, ABCG1, ABCG5 and ABCG8. We postulate that this  may have been due to reduced availability of oxysterols, which are known to activate LXRa expression. Reduced LXRa activity has been reported to significantly decrease the expression of SREBP-1c, FAS, ACC and SCD-1 in mice [25,26] and in hepatoma cells [27]. This is in line with our gene expression analysis showing down-regulation of SREBP-1c and the fatty acid synthesis genes FAS, ACC, ELOVL5, ELOVL6 and SCD-1 in SM-supplemented animals. Somewhat surprisingly, PPARa expression was not affected by SM-supplementation, and the expression of various PPARa-regulated genes involved in fatty acid oxidation such as ACO, CPT1a and VLCAD, were reduced, rather than increased. The current results are therefore consistent with a dietary SMinduced reduction in hepatic TG levels being associated with suppression of the LXR-SREBP-1c regulated fatty acid synthesis pathway, but not increased fatty acid oxidation. This is also supported by significant correlations between hepatic TG levels and the LXR-SREBP-1c regulated genes.
A recent study published by Yunoki et al. has demonstrated that feeding leptin-resistent Zucker rats a normal chow diet supplemented with sphingolipids of different origins reduced total hepatic and plasma lipids through improvement of adiponectin signaling and insulin sensitivity [9]. In contrast to the present findings, hepatic cholesterol levels did not change when sphingolipids were administered to those animals. Although total hepatic fatty acids were significantly reduced by sphingolipid supplementation in that study, it was unclear whether this was associated with a reduction in hepatic triglyceride levels. Overall, the effects of sphingolipid supplementation on individual hepatic fatty acids in the prior study were not as significant as the effects observed in the current study.
Gene expression analyses have also shown that dietary sphingolipids suppressed SCD1 expression, which in turn increases insulin sensitivity and activation of acetyl-CoA synthesis (Pdk4), resulting in increased fat catabolism [13]. These results are in contrast to the present study in which we found no consistent effect of increasing doses of SM on plasma glucose, insulin or adiponectin levels (data not shown). It is possible that a longer feeding period might result in subsequent effects on insulin sensitivity. However it is unlikely that the primary effect of dietary SM in the current model is due to an effect on glucose and/or insulin metabolism.
Dietary sphingolipids have previously been reported by Jiang et al. to have adverse effects on plasma lipids and atherosclerosis [28]. In their study, LDLr knockout female mice fed a sphingolipid-rich normal chow diet for 3 months had increased levels of plasma cholesterol, SM and non-HDL lipids. These adverse effects clearly contrast with the benefit that was observed in the present study, and could be due to different animal models and/or different diets. We found no change in plasma cholesterol and triglyceride levels in the present study (data not shown), which contrasts with our previous work, where 6 weeks of feeding a SM-containing phospholipid-rich diet significantly reduced plasma cholesterol, triglyceride and phospholipid levels in the same model [13]. We postulate that a longer period of dietary egg SM supplementation may reduce plasma lipid levels in a similar manner. Interestingly, SM supplementation in the present study was associated with a down-regulation of de novo ceramide synthesis that correlated with the reduced levels of hepatic ceramide, MHC and DHC species.
In conclusion, the present results demonstrate that dietary SM has potentially beneficial effects on diet-induced hepatic steatosis by reducing intestinal cholesterol absorption. More specifically, these experiments provide evidence that dietary SM, when added to a high-fat diet, potently lowers hepatic cholesterol and triglyceride levels in a dose-dependent manner, most likely through inactivation of LXRa expression and reduced expression of down-stream target genes. Whether dietary SM has the therapeutic potential to ameliorate hepatic steatosis in humans remains to be determined.  Author Contributions