Hepatic Lipidomics Reveals the Homeostasis and Profile of Sphingomyelin from Yak Butter in Normal-Fat Diet-Fed Rats


 Background: Dietary sphingomyelin was showed to inhibit the uptake of lipids in mice fed with a high-fat diet, however, the effect of sphingomyelin on normal diet was on reported. The current study aims to examine the effects of sphingomyelin extracts from yak butter on hepatic steatosis and inflammation in C57/B6J mice fed with a normal diet. Methods: A UHPLC-QTOF-MS based lipidomics method was utilized to screen the liver metabolites and predict the dominant potential metabolic pathways after sphingomyelin feeding. Results: The results showed that sphingomyelin extracts reduced the accumulation of lipid droplets, suppressed the expression of pro-inflammatory factors IFN -γ, IL-6 and TNF - α, synchronously, promoted the expression of anti-inflammatory factors IL-10, IL-4 and IL-1Ra. In addition, sphingomyelin extracts exhibited the modulation on liver lipid metabolism when supplement sphingomyelin in normal diet for one month and five months. Specifically, 16, 68 different metabolites and 2, 6 metabolic pathways were identified by quantitative lipidomics, respectively. Six CERs including Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d17:1/22:0), Cer(d17:1/24:1), Cer(d17:1/24:0) and Cer(d17:0/26:1), six SMs including SM(d15:0/24:1), SM(d14:0/26:1), SM(d14:1/24:1), SM(d15:1/22:0), SM(d15:1/24:1) and SM(d19:1/26:1), and PS(18:1/22:6) were identified and can be used as potential biomarkers of steatosis and inflammation.Conclusions: This study highlighted the effects of yak butter sphingomyelin on hepatic steatosis, tissue inflammation and lipid metabolism of mice under a normal diet.


28
Yak butter was a kind of butter like a dairy product, which was the fat extracted from yak milk. It was one of the main dietary 29 manner for herdsmen to eat fat in Qinghai Tibet plateau area. The special plateau climate endowed yak milk with higher 30 nutritional value than ordinary milk [1]. The Tibetan people exhibited adaptive capacity under environment at altitude of 3000 -31 5000 m, which was related to their regular intake of yak butter [2]. It ont only provided sufficient energy for organismal 32 metabolism, but possessed favourable nutritional and health care effect [3].Yak butter contained several vitamins, which 33 showed bright yellow in spring-produced, turned light yellow in winter-produced. Compared with common cream, it has higher 34 nutritional value, higher sodium carbonate and nearly eight times and twice of trans oleic acid and conjugated linoleic acid, 35 respectively. These polyunsaturated fatty acids have the effect of lowering blood lipids and reducing inflammatory reaction [4].

36
Sphingomyelin (SM) consisting of ceramide and phosphorylcholine moieties belonged to the sphingolipid analogue of 37 phosphatidylcholine, which was vital precursor of intracellular mediators [5].SM was also a component of microdomains and 38 lipid rafts, which played the functional role in several immune responses and intercellular signaling [6]. Numerous foods 39 contain different types of SM. In some fruits and vegetables, the content of SM was less than 100 μmol/kg, while it was more 40 than 2000 μmol/kg in eggs and dairy products [7]. In recent years, studies was showed that sphingomyelin was closely related to 41 some diseases caused by fatty acids, cholesterol and mycotoxins in human body. Therefore, sphingomyelin in food has 42 gradually attracted people's attention [8].Dietary sphingomyelin, a sphingolipid found exclusively in animals, was showed to 43 dose-dependently reduce the absorption of cholesterol, triglyceride and fatty acids both in vitro [9,10]and in rat models 44 [11,12].In addition, it effective prevented diet-induced obesity, adipose tissue inflammation and hepatic steatosis [13]. SM from 45 different dietary sources has different amide-linked fatty acids and sphingosine bases [14,15], which may have different 46 biological effects on lipid metabolism in vivo [11,16].Nowadays, few studies have evaluated the effects of chronic dietary SM 47 intake on lipid metabolism in high fat, except for eggs and milk [16][17][18][19].

58
The current study evaluated the effects of dietary SM from yak butter on steatosis, tissue inflammation and lipid 59 metabolism in C57BL/6J mice. The aims of this study was to observe the characteristics of lipid metabolism in mice based on a 60 quantitative lipidomics approach, screen related differential metabolites, analyze the lipid metabolism status and the relationship 61 with diseases through the change of relative content of lipid metabolites, so as to reveal the regulatory mechanism of 62 sphingomyelin on diseases from the metabolic level. Our data may lay a theoretical foundation for further development and Lanzhou, China) were used in this study. All mice were housed in plastic cages (five mice/cage) in the animal facility of 68 Medical College of Qinghai University under a 12 hr light-dark cycle. After acclimatization for one week, mice were 69 randomized into two groups (control group and treatment group, n = 10/group). The control group was given a maintenance 70 diet, while the other group was given a maintenance diet with 1.2% of SM added as previously described [17]. Detailed 71 composition of the diets is listed in Table 1. The analysis was carried out after one month and five months, respectively. All of 72 the animal experiments in this study were approved by The Institutional Animal Care and Use Committee of Qinghai Province.

73
Preparation of sphingomyelin: Yak butter was melted by constant temperature oscillation at 45 ℃. Neutral lipids were 74 eluted by adding n-hexane. Oil liquid was obtained by rotating evaporation apparatus (50 ℃). Then, acetone was added, the 75 sample was left overnight in refrigerator at 4 ℃. After vacuum filtration and drying, powdered phospholipids insoluble in 76 acetone were obtained. In the dried phospholipid compound, ether was added (a ratio of material to liquid was 1:10), extracted 77 at 30 ℃ for 25 min, and centrifuged at 3,500 rpm for 10 min to obtain crude sphingomyelin.Chloroform: methanol (2:1, V/V, a 78 ratio of material to liquid was 1:10) was added into the dried crude sphingomyelin, which was dissolved, concentrated and dried 79 in vacuum. The dried solid was purified sphingomyelin. All reagents were provided by Tianjin Fuyu Fine Chemical Co., Ltd.

81
Fresh food was provided daily and mice were allowed free access to the diet and water. Body weights and food intake were 82 recorded on a weekly basis. After one month and five months on their respective diets, mice were fasted for 8-10 h prior to 83 5 blood collection by eyeball. Blood was allowed to clot at room temperature for 30 min before serum was isolated by 84 centrifugation (2,000 rpm for 10 min at 4 ℃) and then stored at -80 ℃. The adipose of abdominal, scapular, perirenal and liver 85 tissues were collected from animals. Then, snap-frozen in liquid nitrogen and stored at -80 ℃.

99
(3) Extraction: add 10 ml of n-hexane, shake it, leave it for 10 min to separate layers, take n-hexane layer and put it into another 100 drying tube.

101
(4) Drying: dry n-hexane with nitrogen to obtain dry free fatty acids.

102
(5) Methyl esterification: take the dried fatty acid and add 1 ml of BF3 whose mass fraction is 15%, seal the tube mouth, and 103 take a water bath at 90 ℃ for 2 h. 104 (6) Extraction and demulsification: after cooling, add 2 ml of n-hexane into the centrifuge tube, shake, add 2 ml of saturated 105 NaCl solution, seal and shake, centrifuge for 2 min at 3,500 rpm, and take n-hexane layer into another centrifuge tube. 106 (7) Drying: add 1 g anhydrous sodium sulfate for dehumidification, shake, centrifugation at 3,500 rpm for 2 min, take n-hexane 107 layer and put it into another drying tube, dry with nitrogen to less than 1 ml. 108 (8) Sample collection: Dissolve 2 times with 300 µl n-hexane, wash and collect the fat on the tube wall and put it into a brown 109 vial for GC-MS analysis.

116
The data processing system of the chemical workstation was used to search the spectrum library, analyze the spectrum and 117 confirm the chemical structure of each fatty acid. The relative content of fatty acids was calculated by peak area normalization.

130
The procedure for metabolite extraction was performed as follows: First, 20 mg of sample was weighed to an EP tube, and 131 homogenized with 400 μl water. Transfer 50 μl of homogenate to a new EP tube (A) and dilute to 400 μl with water. Next, 960 132 μl extraction liquid (MTBE:methanol = 5:1, v/v), including 9 μl of 10 μg/ml IS1, 9 μl of 10 μg/ml IS2, 9 μl of 100 μg/ ml IS3, 133 was added to each sample. The homogeneously mixed sample was vortexed for 30 s, sonicated for 10 min in a 4 ℃ water bath, 134 and then centrifuged for 15 min at 3,000 rpm and 4 ℃. The supernatant (500 μl) was transferred to a new EP tube (B), and 500 135 μl MTBE was added from the original EP tube (A); the sample was then vortexed for 30 s, sonicated for 10 min, and 136 centrifuged for 15 min at 3,000 rpm and 4 ℃. The supernatant (500 μl) was transferred to a new EP tube (C). Next, 500 μl 137 MTBE was added from EP tube A, and the sample was vortexed for 30 s, sonicated for 10 min, and centrifuged for 15 min at   An in-house program, Lipid Analyzer, was developed using R for automatic data analysis. The raw data files (.wiff format) 164 were converted to files in mzXML format using the "msconvert" program from ProteoWizard (version 3.0.6150). The mzxML 165 files were then loaded into LipidAnalyzer for data processing. Peak detection was first applied to the MS1 data. The CentWave 8 algorithm in XCMS was used for peak detection. With the MS/MS spectrum, lipid identification was achieved through a 167 spectral match using an in-house MS/MS spectral library. The absolute content of lipid in samples was calculated according to 168 the peak area, stable isotope-labeled internal standard, and relevant fragment information. for unsupervised data analysis to investigate the clustering of data in each group. In order to maximize the separation between 173 groups, the supervised data analysis was carried out by using orthogonal projects to latent structures-discriminate analysis 174 (OPLS-DA), Finally, a Variable Importance in Projection (VIP) value of greater than 1 was selected as a potential biomarker, 175 and compounds with statistical differences of p-value of less than 0.05 were evaluated as differential metabolites for 176 identification.

178
All statistical analyses were conducted using SPSS Statistics 25.0 software. Data were reported as the mean ± SD. Statistical 179 significance between groups was denoted by *p < 0.05, **p < 0.01, and ***p < 0.001.

182
A month of SM-containing diets, there was no significant difference in body weight compared to control, while after five 183 months, the body weight of mice in the treatment group increased by 4% compared with the control. No differences were 184 observed in food intake between two groups (Figure 1).

190
The fixed tissue is often dehydrated, embedded in paraffin, sectioned and stained with H&E, it was found that the liver tissues 191 and structures of the two groups were normal and intact after one month of adding SM diets. Hepatocytes were arranged radially 192 along the central vein, and no degeneration or necrosis was seen. No inflammatory cell infiltration and fibrosis were found in the 193 stroma. Adipose of abdominal: The structure of the adipose tissue in the control group was complete, the adipose cells were 9 arranged densely, the shape was generally circular, and the size was uniform, no obvious pathological changes were found.

195
While in the treatment group, mild focal/multifocal neutrophil infiltration was seen from abdominal adipose. Perirenal adipose:

196
The adipose tissue structures of the two groups were intact, the adipose cells were arranged densely, the shape was generally 197 circular, and the size was uniform. After five months on diets, there was no obvious pathological change for abdominal adipose 198 in the control group, but the treatment group showed slight infiltration of monocytes and neutrophils; H&E-stained livers 199 showed extensive lipid droplet accumulation in the control group. In contrast, SM extracts in yak butter can reduce the 200 accumulation of lipid droplet in the liver and prevent hepatic steatosis (Figure 2D.).

201
The composition of fatty acids in scapular adipose tissue

202
The composition of fatty acids in the adipose tissue of mouse scapula was analyzed. The results are shown in Table 4

225
Ceramide and sphingosine, the metabolites of sphingomyelin, play an important role in modulating the immune system.

226
After a month of feeding SM diets, the expression levels of pro-inflammatory factors interferon-Y (IFN-Y), interleukin-6 (IL-6) 227 and tumor necrosis factor-α (TNF-α) in the liver were lower than the control group. Anti-inflammatory factor interleukin-1 228 receptor antagonist (IL-1Ra) was highly expressed in the treatment group, the expression of interleukin-10 (IL-10) and 229 interleukin-4 (IL-4) were relatively low. While after five months, the relative expression levels of IL-1Ra, IL-10, and IL-4 were 230 significantly higher than the control group.  Figure 4C&F.

249
The differences in metabolic profiles obtained in the experiment reflect the biological differences between the samples. It can be 250 seen from the picture, noticeable separations were found between the two groups, demonstrating that the PCA and OPLS-DA 251 models could be used to identify the differences between two groups. In addition, exogenous addition of sphingomyelin 252 11 significantly affects liver metabolism in mice.

253
Screening of differential metabolites 254 VIP value of OPLS-DA model (VIP>1) and P value of Student's t test (P<0.05) were used to screen differential metabolites. In 255 the database, the retention time and other conditions are matched with the substances in the database to determine the nature of 256 differential metabolites. The volcano map of differential metabolite screening is shown in Figure 5. Overall, 16 and 68 257 significantly differential lipid species were identified in the control group versus the treatment group at one month and five

392
By means of quantitative lipidomics, we not only identified the significantly different lipids between the two groups, but 393 also studied the lipid metabolism pathways involved in these lipids. Based on the analysis of lipidomics pathway, there are 7 394 metabolic pathways for eleven major lipid classes, glycerophospholipid metabolism was the pathway with the most significantly 395 different lipid involvement. SM and Cer are involved in these seven lipid metabolism pathways. The contents of SM and Cer in 396 the control group were lower than those in the treatment group. Therefore, our results support the hypothesis that the change of 16 sphingomyelin addition may affect glycerolipid metabolism, sphingolipid metabolism, glycerophospholipid metabolism, linoleic 398 acid metabolism, alpha-linoleic acid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis and arachidonic acid 399 metabolism, consequently leading to increase in certain lipids, such as Cers and SMs. Through the analysis, it is helpful for us to 400 further understand the mechanism of sphingomyelin supplementation altered the important differential lipid species in the liver.

402
In the current study, we found that the addition of SM extract of yak butter to mice with a normal diet reduced the 403 accumulation of liver lipid droplets, which could prevent hepatic steatosis and tissue inflammation. This conclusion is