Wangting
Song‡
a,
Ke
Zhang‡
ab,
Teng
Xue‡
ac,
Jiarui
Han‡
a,
Fangda
Peng‡
d,
Chunguang
Ding‡
d,
Feng
Lin‡
e,
Jiujun
Li‡
fg,
Fat Tin Agassi
Sze‡
ah,
Jianwen
Gan‡
i and
Xianyang
Chen‡
*ac
aBao Feng Key Laboratory of Genetics and Metabolism, Beijing, China. E-mail: cxyibcas@163.com; Fax: +8610 85095705; Tel: +86 10 85095705
bSchool of Grassland Science, Beijing Forestry University, Beijing, 100083, China
cZhong Guan Cun Biological and Medical Big Data Center, Beijing, China
dNational Center for Occupational Safety and Health, NHC, Beijing, 102308, China
eDepartment of Neurology, Sanming First Hospital Affiliated to Fujian Medical University, Sanming, Fujian, China
fDepartment of Pediatrics, Shengjing Hospital of China Medical University, Shenyang, China
gPlateau Medical Research Center of China Medical University, Shenyang, China
hGraduate Institute of Bioresources, National Pingtung University of Science and Technology, Pingtung, Taiwan, China
iMacau University of Science and Technology, Macau, China
First published on 21st January 2022
Acer truncatum Bunge seed oil (ASO) is rich in ω-9 (53.93%) and ω-6 (30.7%) fatty acids (FAs) and characterized by 3–7% nervonic acid (NA, C24:1ω-9). Evidence suggests that ω-9 FAs such as NA participate in processes of cognitive improvement; however, their mechanism remains ambiguous. In this study, we investigated the effect of ASO on rat memory and the change in lipid profiling and underlying metabolism. After ASO was administrated to rats for one, three and seven days, their capacity for learning and memory significantly increased via the MWM test. Lipid profiling showed alterations in a wide range of metabolic features after ASO was administrated to the rats, in which sphingolipids (SP) in the serum and glycerophospholipids (GP) in the brain were regulated significantly. The changes in the fatty acids in the serum and brain showed the synergetic effects of NA, EA, OA and DHA, where NA, EA and OA exhibited similar change trends. The enrichment analysis based on KEGG indicated that ASO supplementation evoked the pathways of neurotrophin signaling, glycerophospholipid metabolism and sphingolipid metabolism, which are related to memory and cognition improvement. Among the metabolites with different molecular forms, the biomarkers with C24:1ω-9 chains exhibited a positive correlation with others both in the serum SP and brain GP. These results suggest the synergistic effects of ω-9 FAs and that their conversion into each other may result in enhanced cognition in rats ingesting Acer truncatum Bunge seed oil.
As a particular Acer species in China, Acer truncatum Bunge is an important widespread prominent species in the hardwood forests from north to west China.8Acer truncatum Bunge seed oil (ASO), as a novel edible oil, possesses a proportion of unsaturated FAs that is up to 92% is richer in LA and OA than other edible oils including sunflower, rapeseed, grape and peanut oil.9,10 ASO is an important woody oil, and its nutritional and economic value largely depend on its FA composition. In 2011, ASO was certified as a new food resource by the Ministry of Health of the People's Republic of China.11 Especially, ASO is the main plant resource to achieve the large-scale production of C24:1ω-9, whose portion is 3–7%.12
C24:1ω-9, a very long-chain FA (VLCFA), plays a vital role in human health, especially in brain development. As the main long-chain unsaturated FA in the branch chain of lipids such as phosphatidylcholine (PC), sphingomyelin (SM) and ceramide (Cer), the synthesis of C24:1ω-9 is the rate-limiting step in the lipid homeostasis of the myelin sheath.13 Nerve fibers consist mainly of myelin sheath and axons, and the myelin sheath is surrounded by a thick lipid layer, which determines the speed of saltatory signal conduction.14,15 The outer lipid membrane of the myelin sheath is in a dynamic equilibrium of shedding and regeneration, and its production requires various outer membrane lipids as raw materials. Therefore, C24:1ω-9 is regarded as one of the structural compounds in the nervous system.16 Substantial evidence has proven that C24:1ω-9 deficiency can trigger a series of neurological diseases. A reduction in C24:1ω-9 has been confirmed to be associated with demyelinating disease, whereas an NA-enriched diet can ameliorate ALD.17 However, the amount of C24:1ω-9 synthesized in humans is relatively low, and thus exogenous supplementation may be considered necessary to ameliorate neuropathy in individuals with a deficiency in C24:1ω-9.13 One way for the production of C24:1ω-9 in vivo is via the conversion of other FAs through a series of biochemical reactions; alternatively, direct ingestion is another way by which C24:1ω-9 is readily available to the body.18
C24:1ω-9 can also be obtained from certain types of oils. Lewkowicz et al. employed a fish oil mixture rich in C24:1ω-9 (13%) and ω-3 PUFAs in a human model of maturating oligodendrocyte precursor cells, and found an enhancement in the synthesis of sphingomyelin and inhibition of proinflammatory cytokines and chemokines.13 Cook et al. supplemented Lunaria oil, which is rich in C24:1ω-9 (6.89%) and erucic acids (C22:1ω-9, 13.8%), in the routine diet of quaking mice pups and observed normalization of the level of C24:1ω-9 in brain sphingomyelin.19 ASO is qualified for providing exogenous C24:1ω-9 as well as other types of essential unsaturated FAs in the human body and represents a new type of plant resource with potential applications for treating human cerebral and neurological problems.20 Nevertheless, the effect of ASO supplementation on cognitive function and molecular alterations in the serum and brain is still unclear.
To explore the effect of ASO, a type of novel edible oil, especially on cognitive function, in this study we supplemented ASO to rats for a certain number of days to evaluate its effect on memory enhancement via the Morris water maze (MWM) test. Afterward, we employed lipid profiling to determine the lipid remodeling in the serum and brain. The pathway and multivariate statistical analysis revealed the biological processes and biomarkers related to cognitive function after the supplementation of ASO.
Thirty six-week-old SD rats were randomly and equally divided into two groups, including the control group and ASO group. Before the formal experiment, the rats were adaptively fed for 1–2 weeks. The ASO group was fed ASO by gavage, and the control group received the vehicle. Based on the individual recommended intake of NA of 0.3 g kg−1 d−1, the equivalent dose of the drug was calculated according to the body weight of a human and the animal, where the daily dose of the rat was equivalent to six times that of adults. After continuous administration, the rats from the control (n = 5) and ASO (n = 5) groups were sacrificed via CO2 euthanasia after one, three, and seven days. The weight of the body and brain of each rat were recorded, meanwhile the brain and serum were collected and analyzed by lipidomics (Fig. 1).
According to the general operation of the MWM test, the test in our study included a six-day place navigation test and a spatial probe test on the last day.21,22 The navigation training phase consisted of 24 trials: 4 training trials per day and 6 training days with an intertrial interval of 20 min. The rats were released with their heads facing the pool wall from the four compass locations for each training per day and allowed to swim and search for the platform within 120 s, and the latency to reach the hidden platform (shown as escape latency) was recorded. If the rats did not locate the platform after 120 s, the animals were manually guided to reach the platform, and the escape latency was recorded as 120 s.
On the 7th day (the day after the navigation training phase), the subjects received a probe trial, in which the platform was removed. The rats were released from the NW start point and allowed to swim freely for 120 s. The path that the rat swam was tracked and analyzed for the proportion of swim time in the target quadrant (SE) of the pool.
Ingredient | Description | Content (%) |
---|---|---|
Linoleic acid | C18:2ω-6 | 30.7 |
Oleic acid | C18:1ω-9 | 21.8 |
Erucic acid | C22:1ω-9 | 18.7 |
cis,cis-11-Eicosenoic acid | C20:1ω-9 | 8.54 |
cis-15-Tetracosenoic acid (nervonic acid) | C24:1ω-9 | 6.89 |
Palmitic acid | C16:0 | 4.02 |
Stearic acid | C18:0 | 2.48 |
α-Linolenic acid | C18:3ω-3 | 1.65 |
Behenic acid | C22:0 | 0.96 |
γ-Linolenic acid | C18:3ω-6 | 0.74 |
Tetracosanoic acid | C24:0 | 0.42 |
cis-11,14-Eicosadienoic acid | C20:2ω-6 | 0.34 |
Arachidic acid | C20:0 | 0.28 |
cis,cis,cis-11,14,17-Eicosatrienoic acid | C20:3ω-3 | 0.16 |
Palmitoleic acid | C16:1ω-7 | 0.07 |
Heptadecanoic acid | C17:0 | 0.07 |
cis-10-Heptadecenoic acid | C17:1ω-7 | 0.04 |
Tricosanoic acid | C23:1 | 0.04 |
cis-13,16-Eicosadienoic acid | C22:2ω-6 | 0.02 |
In the navigation training phase (1st day to 6th day), escape latency to reach the platform in the MWM was recorded, and the time for each rat was calibrated against the time on the 1st training day, which served as a background. A continuous decrease in latency was observed for both the ASO and control groups during the six training days. The latency time of the ASO group was significantly less than the latency time of the control group on the 2nd, 3rd and 6th day (Fig. 2A). As the number of days increased, both the control and ASO rats had significantly shorter platform landing times than on day one. The ASO group started significantly lower on day 2 than on day 1, and then continued to decline. In contrast, there was a significant difference in the control group from day 4 onwards (Fig. 2B and C). Although the learning ability of the rats on the 6th and 3rd day was better than that of the control group, there was no difference between them. During the probe trial on the 7th day, the time spent in the target quadrant (SE) by the ASO group was 30.3%, which was significantly longer than the time spent in the target quadrant by the control (24.3%) (Fig. 2D).
The body weight of the rats increased with time, but there was no significant difference between the control and ASO groups on each day (Fig. 1E). There was no alteration in brain weight during the whole period, and there was no significant difference between the control and ASO groups (Fig. 2E).
The lipid category for classification was based on the LIPIDMAPS database (http://www.lipidmaps.org). These lipids were categorized into eight classes, including prenol lipids, fatty acyls, sterol lipids, polyketides, glycerolipids, glycerophospholipids (GP), sphingolipids (SP) and saccharolipids (SL). There were no marked alterations in the lipids on the 1st day in both the serum and brain. On the 3rd and 7th day, significant and consecutive alterations were observed in the serum SP and brain GP and SL (Fig. S1†). The content of SP in the serum continuously accumulated (Fig. 3A). In the brain, the content of GP increased on the 3rd day and decreased on the 7th day (Fig. 3E), contrary to that of SL (Fig. 3I).
Further analysis of the altered lipids in the serum and brain, the lipids from the subclass of SP in the serum and GP and SL in the brain were investigated. The results showed that in the serum, the content of other SP [SP00] was reduced on the 1st day (Fig. 3B). Sphingoid bases [SP01] and neutral glycosphingolipids [SP05] markedly decreased, whereas ceramides [SP02], phosphosphingolipids [SP03], phosphonosphingolipids [SP04] and acidic glycosphingolipids [SP06] significantly increased on the 3rd day (Fig. 3C). Ceramides [SP02], phosphosphingolipids [SP03] and glycosphingolipids [SP06] were all robustly accumulated on the 7th day (Fig. 3D).
In the brain, of the subclass of GP, CDP-glycerols [GP13] notably decreased and glycerophosphocholine [GP16] increased on the 1st day (Fig. 3F), while no significant alteration was found in the subclass of SL (Fig. 3J). On the 3rd day, of the GP subclass, glycerophosphocholines [GP01], glycerophosphoethanolamines [GP02], glycerophosphoinositols [GP06], glycerophosphates [GP10], glycerophosphocholine [GP16] and glycerophosphonoethanolamines [GP17] were prominently augmented (Fig. 3G). In the SL subclass, acylaminosugars [SL01] significantly increased and acyltrehaloses [SL03] decreased (Fig. 3K). On the 7th day, most of the lipids in the GP subclass were significantly reduced, including glycerophosphoethanolamines [GP02], glycerophosphoinositols [GP06], glycerophosphates [GP10], CDP-glycerols [GP13] and glycerophosphonoethanolamines [GP17] (Fig. 3H). In the subclass of SL, acyltrehaloses [SL03] markedly increased (Fig. 3L).
Further investigation into the changes in NA, OA, EA and DHA due to ASO supplementation, we analyzed the different molecular forms of these fatty acids, which have been reported to help improve memory. Compared to the control group, the serum results showed that in the lipids of NA(C24:1ω-9), mainly PC(24:1) and PC(P-24:1) were significantly up-regulated. In the lipids of OA(C18:1ω-9), mainly acidic glycosphingolipids(18:1), ceramides(18:1), FAHFA(18:1) and PC(18:1) significantly increased. In the lipids of EA(C22:1ω-9) mainly lysoPC(22:1) significantly increased, while in the lipids of DHA(C22:6ω-3) mainly DG(22:6) and TG(22:6) were significantly up-regulated from the 1st day to the 7th day (Fig. 5A–D).
The results showed that in the brain, lysoPC(24:1) and PE-cer(P-24:1) of NA(C24:1ω-9) were significantly elevated on the 3rd day, but on the 7th day, PC(24:1) significantly increased and lysoPE (24:1) significantly decreased. In the lipids of OA(C18:1ω-9), mainly PE(18:1), PE(O-18:1) and PE(P-18:1) increased significantly on the 1st day and 3rd day, while on the 7th day, acidic lipids(18:1), lysoPC (18:1), LysoPE(18:1) and PC(18:1) significantly decreased, and CDP-DG(18:1), DGTA(18:1), neutral glycosphingolipids(18:1) significantly increased. In the lipids of EA (C22:1ω-9), mainly lysoPA(22:1), lysoPC(22:1), PA(P-22:1) and TG(22:1) increased significantly from the 1st to 7th day, while on the 7th day, lysoPC(22:1) decreased. In the lipids of DHA(C22:6ω-3), mainly DGCC(22:6), PE(22:6), PG(22:6) and TG(22:6) were significantly up-regulated from the 3rd to 7th day, while lysoPE(22:6) and lysoPS(22:6) significantly decreased on the 7th day (Fig. 5E–H). The heat map of clustering showed that the change trends of NA, EA and OA at different time points in the ASO group were similar (Fig. 5A–C and E–G), while the changes in DHA in the ASO group at different time points were similar to the corresponding control group (Fig. 5D and H). We speculated that although supplementation of ASO could cause changes in DHA in the serum and brain, this was likely to be time dependent.
We used a ternary diagram to observe the variation in four fatty acids in three days as three dimensions of their characteristics and investigate their overall variation trends. All the molecular forms of these four fatty acids are shown in a ternary diagram over three days. The closer they are, the more similar they are, and vice versa. Regardless of the serum or brain, the distribution of C22:6ω-3, C18:1ω-9, C22:1ω-9 and C24:1ω-9 was concentrated and close, while the plot showed a loose distribution of C22:6ω-3 (Fig. 5I and J).
The serum and brain pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) is shown in Tables 2 and 3 (with Holm P-value < 0.1). Combined with the results of the Venn diagram (Fig. S3†), it can be seen that the supplementation of ASO interfered with glycerol phospholipid metabolism, glycerol metabolism and fat digestion and absorption in the serum from the 1st to 7th day. With an increase in the time of ASO intake, four more pathways were induced from the 3rd to 7th day, including the neurotrophin signaling pathway, serotonergic synapse, sphingolipid metabolism and steroid biosynthesis. Besides, the phospholipase D signaling pathway on the 1st day and oxidative phosphorylation and arachidonic acid metabolism on the 7th day were mediated in the serum (Table 2). As for the brain, ASO intake persistently mediated eight pathways including neurotrophin signaling pathway, glycerophospholipid metabolism, sphingolipid metabolism, fat digestion and absorption, thermogenesis, phospholipase D signaling pathway, glycerolipid metabolism and serotonergic synapse. Besides, the phosphatidylinositol signaling system was mediated on the 1st day and AMPK signaling pathway on the 7th day (Table 3).
Pathway name | Match status | p | Holm P | FDR | HitsName | Time |
---|---|---|---|---|---|---|
Fat digestion and absorption | 4/13 | 4.09 × 10−10 | 3.84 × 10−8 | 4.8 × 10−9 | C00681|C00422|C00165|C00162 | 1st day |
Glycerolipid metabolism | 4/38 | 1.52 × 10−7 | 1.2 × 10−5 | 6.2 × 10−7 | C00681|C00422|C00162|C02133 | 1st day |
Glycerophospholipid metabolism | 4/56 | 1.11 × 10−6 | 7.43 × 10−5 | 2.76 × 10−6 | C00681|C00670|C01233|C03819 | 1st day |
Phospholipase D signaling pathway | 2/11 | 6.85 × 10−6 | 0.000384 | 1.19 × 10−5 | C00681|C00165 | 1st day |
Fat digestion and absorption | 4/13 | 9.5 × 10−10 | 8.74 × 10−8 | 6.78 × 10−8 | C00162|C01885|C00165|C00422 | 3rd day |
Neurotrophin signaling pathway | 2/5 | 6.69 × 10−7 | 5.75 × 10−5 | 8.79 × 10−6 | C00165|C00195 | 3rd day |
Glycerolipid metabolism | 3/38 | 1.59 × 10−5 | 0.001303 | 0.000117 | C00162|C01885|C00422 | 3rd day |
Glycerophospholipid metabolism | 3/56 | 7.55 × 10−5 | 0.00566 | 0.000375 | C00670|C03819|C01233 | 3rd day |
Sphingolipid metabolism | 1/25 | 0.005031 | 0.055341 | 0.005577 | C00195 | 3rd day |
Serotonergic synapse | 1/42 | 0.013834 | 0.055341 | 0.014142 | C00165 | 3rd day |
Steroid biosynthesis | 1/58 | 0.025513 | 0.055341 | 0.025513 | C01694 | 3rd day |
Neurotrophin signaling pathway | 2/5 | 2.43 × 10−7 | 2.16 × 10−5 | 1.19 × 10−5 | C00165|C00195 | 7th day |
Fat digestion and absorption | 2/13 | 6.85 × 10−6 | 0.000576 | 0.000102 | C00422|C00165 | 7th day |
Glycerophospholipid metabolism | 3/56 | 1.92 × 10−5 | 0.001551 | 0.000189 | C00670|C03819|C01233 | 7th day |
Serotonergic synapse | 2/42 | 0.000261 | 0.013413 | 0.000541 | C00165|C00909 | 7th day |
Oxidative phosphorylation | 1/16 | 0.00109 | 0.019909 | 0.001311 | C00399 | 7th day |
Sphingolipid metabolism | 1/25 | 0.002683 | 0.02951 | 0.002984 | C00195 | 7th day |
Glycerolipid metabolism | 1/38 | 0.006145 | 0.02951 | 0.00636 | C00422 | 7th day |
Steroid biosynthesis | 1/58 | 0.013953 | 0.02951 | 0.014112 | C01694 | 7th day |
Arachidonic acid metabolism | 1/75 | 0.022739 | 0.02951 | 0.022739 | C00909 | 7th day |
Pathway name | Match status | p | Holm P | FDR | HitsName | Time |
---|---|---|---|---|---|---|
Neurotrophin signaling pathway | 2/5 | 4.21 × 10−7 | 3.62 × 10−5 | 1.67 × 10−5 | C00165|C00195 | 1st day |
Glycerophospholipid metabolism | 4/56 | 1.11 × 10−6 | 9.37 × 10−5 | 2.16 × 10−5 | C00670|C01233|C03819|C00269 | 1st day |
Sphingolipid metabolism | 3/25 | 1.49 × 10−6 | 0.000122 | 2.16 × 10−5 | C12145|C00195|C12126 | 1st day |
Fat digestion and absorption | 2/13 | 1.18 × 10−5 | 0.000934 | 0.000114 | C00165|C00422 | 1st day |
Thermogenesis | 2/23 | 7.16 × 10−5 | 0.005374 | 0.000327 | C00165|C00422 | 1st day |
Phospholipase D signaling pathway | 1/11 | 0.000712 | 0.024218 | 0.001068 | C00165 | 1st day |
Phosphatidylinositol signaling system | 1/29 | 0.00506 | 0.037708 | 0.005435 | C00269 | 1st day |
Glycerolipid metabolism | 1/38 | 0.008595 | 0.037708 | 0.008797 | C00422 | 1st day |
Serotonergic synapse | 1/42 | 0.010437 | 0.037708 | 0.010437 | C00165 | 1st day |
Fat digestion and absorption | 3/13 | 1.64 × 10−7 | 1.41 × 10−5 | 1.41 × 10−5 | C01558|C00165|C00422 | 3rd day |
Neurotrophin signaling pathway | 2/5 | 6.69 × 10−7 | 5.69 × 10−5 | 2.88 × 10−5 | C00195|C00165 | 3rd day |
Glycerolipid metabolism | 3/38 | 1.59 × 10−5 | 0.001271 | 0.000195 | C02133|C00422|C06041 | 3rd day |
Glycerophospholipid metabolism | 3/56 | 7.55 × 10−5 | 0.00566 | 0.000456 | C01233|C00670|C03819 | 3rd day |
Thermogenesis | 2/23 | 0.000113 | 0.007851 | 0.000463 | C00165|C00422 | 3rd day |
Sphingolipid metabolism | 2/25 | 0.000146 | 0.009488 | 0.000542 | C06062|C00195 | 3rd day |
Phospholipase D signaling pathway | 1/11 | 0.000955 | 0.02962 | 0.00137 | C00165 | 3rd day |
Serotonergic synapse | 1/42 | 0.013834 | 0.035217 | 0.013834 | C00165 | 3rd day |
Neurotrophin signaling pathway | 2/5 | 6.69 × 10−7 | 5.89 × 10−5 | 2.01 × 10−5 | C00195|C00165 | 7th day |
Glycerolipid metabolism | 3/38 | 1.59 × 10−5 | 0.001335 | 0.000169 | C02133|C00162|C06041 | 7th day |
Fat digestion and absorption | 2/13 | 1.87 × 10−5 | 0.001517 | 0.000169 | C00165|C00162 | 7th day |
Glycerophospholipid metabolism | 3/56 | 7.55 × 10−5 | 0.005735 | 0.000415 | C00670|C03819|C01233 | 7th day |
Thermogenesis | 2/23 | 0.000113 | 0.007957 | 0.000424 | C00165|C00162 | 7th day |
Phospholipase D signaling pathway | 1/11 | 0.000955 | 0.032487 | 0.00141 | C00165 | 7th day |
AMPK signaling pathway | 1/23 | 0.004264 | 0.051171 | 0.004858 | C00162 | 7th day |
Sphingolipid metabolism | 1/25 | 0.005031 | 0.055341 | 0.00559 | C00195 | 7th day |
Serotonergic synapse | 1/42 | 0.013834 | 0.055341 | 0.01399 | C00165 | 7th day |
On the 3rd day, SM(d17:1/24:1) and Cer(d18:1/24:1(15Z)) were detected in the serum SP lipids, all concluded to be C24:1ω-9. There were 41 candidates in the brain GP lipids, which were concluded to be C18:3ω-3 (PC, PE), C20:5ω-3 (PE), C22:5ω-3 (PC, PE), C22:6ω-3 (PE, PI), C18:2ω-6 (PC, PE, PS), C20:3ω-6 (PE, PS, PA), C20:4ω-6 (PE), C18:1ω-9 (PC, PE, PS, PG, PI, PA), C20:1ω-9 (PC, PE) and C24:1ω-9 (PC) (Tables S2 and S3†). Most of the candidates in were brain positively correlated with serum SM(d17:1/24:1) or Cer(d18:1/24:1(15Z)), especially the significant correlation between cer (d18:1/24:1 (15z)) in the serum and PC (24:1 (15z)/18:1 (9z)) in the brain (Fig. 7A).
On the 7th day, SM(d17:1/24:1), Cer(d18:1/24:1(15Z)) and GalCer(d18:1/24:1(15Z)) were detected in the serum SP lipids, all concluded to be C24:1ω-9. There were 38 candidates in the brain GP lipids, which were concluded to be C18:3ω-3 (PC), C20:5ω-3 (PE, PI), C22:5ω-3 (PC), C22:6ω-3 (PC, PE, PS, PI), C18:2ω-6 (PC, PE, PS, PI), C20:3ω-6 (PE, PI), C20:4ω-6 (PC, PE, PS, PG, PI, Glc-GP), C18:1ω-9 (PC, PE, PS, PG, PI, PA), C20:1ω-9 (PE, PA) and C24:1ω-9 (PC) (Tables S2 and S3†). The candidates in the brain mainly showed a negative correlation with that in the serum on the 7th day. However, PC(24:1(15Z)/18:3(9Z,12Z,15Z)), PI(22:4(7Z,10Z,13Z,16Z)/18:1(9Z)) and PS(P-20:0/20:4(5Z,8Z,11Z,14Z)) in the brain were positively correlated with the serum candidates, notably, the correlation between PC(24:1(15Z)/18:3(9Z,12Z,15Z)) in the brain and SM(d17:1/24:1) or GalCer(d18:1/24:1(15Z)) in the serum presented a statistical significance (Fig. 7B).
A total of six screened candidates with C24:1ω-9 chains in the serum and brain maintained a positive correlation on the 3rd and 7th day. We arranged these biomarkers and displayed the contents from the 1st to 7th day. The contents of the six biomarkers in the serum and brain were all accumulated. In the serum, the contents of SM(d17:1/24:1) and Cer(d18:1/24:1(15Z)) were dramatically enhanced from the 3rd to 7th day (Fig. 7A and B), and GalCer(d18:1/24:1(15Z)) robustly increased on the 7th day (Fig. 7C). In the brain, the content of PC(24:1(15Z)/18:1(9Z)) and PC(18:0/24:1(15Z)) markedly accumulated on the 3rd day (Fig. 7D and E), and PC(24:1(15Z)/18:3(9Z,12Z,15Z)) on the 7th day (Fig. 7F).
ASO supplementation resulted in an improvement in the cognitive function of the rats, coupled with lipid remodeling in both their serum and brain. ASO supplementation stimulated the accumulation of SP in the serum (Fig. 3A) and GP and SL in the brain (Fig. 3E and I). GP, SP and SL are commonly used as components of cellular membranes; moreover the GP and SP classes play signaling roles in diverse cellular processes.33 Thus, the aberrant metabolism of GP and SP is associated with various neurodegenerative diseases, such as Parkinson's disease,34 and also induces depression- and anxiety-related behaviors.35 Much evidence suggests that dietary oil, especially long-chain FAs, can synergistically influence brain health and function. C18:1ω-9 and C22:1ω-9 act as major ingredients of Lorenzo's oil for dietary therapy of X-ALD.36 As a neuroactive molecule, C18:1ω-9 was applied for the treatment of neuropathic pain after neurotrauma,37 and C22:1ω-9 supplementation enhanced memory in a cognitive impairment mouse model.38 C24:1ω-9 is naturally involved as the major constituent of sphingolipids in the myelin membrane5 and essential for the growth and maintenance of brain physiology and peripheral nervous tissue.16 Multiple studies have demonstrated C24:1ω-9 deficiency in individuals with neurodegeneration,3,39 and C24:1ω-9 supplementation as a type of nutraceutical is considered to improve brain development and cognition. C18:2ω-6 is an essential precursor of AA (C20:4ω-6), which is one of the major components of cellular membranes and of special importance to the brain and blood vessels.40 C20:4ω-6 serves as an intercellular messenger in the central nervous system once released from membrane phospholipids.40 C18:2ω-6 deficiency is associated with reduced tissue accumulation of C20:4ω-6 and lower growth.40 Evidence has established that C22:1ω-9 and C18:1ω-9 can be converted to C24:1ω-9 in the brain via elongation of their chain in rats.41,42 We speculated that the enhanced cognitive ability of the ASO-supplemented rats was attributed to the synergistic effects of NA and essential FAs.
Besides the top 5 unsaturated fatty acids in ASO content, we also observed the well-known DHA, although ASO does not contain it. We found that on the third day, all the substances showed a significant upward trend (Fig. 4), while on the 7th day, all the substances decreased, including DHA and NA (Fig. 5). The changes in the FAs in the ASO group and the control group suggest that ASO regulates lipid remodeling with a faster response, possibly to improve cognitive performance in rats (Fig. 4). According to previous reports, the synthesis of ω-9 fatty acids in the human body is lower than that of other species,43 and the intake of exogenous NA can support the remyelination process by improving the sphingolipid synthesis of oligodendrocytes (OLs).44 One possibility for the dynamic change in FAs is that the loss of NA may result from its binding via an amide bond to a sphingosine base45 during sphingolipid synthesis by finally differentiated OLs.46 The other possibility is the downregulation of SCD or FA elongase in oligodendrocytes and/or astrocytes by mediator signaling. Both of these would deplete the supply of DHA (C22:6ω-3) and NA (C24:1ω-9) for sphingolipid synthesis. The most abundant ingredients in ASO are ω-6 and ω-9 unsaturated FAs, whose content reaches 86.63% (Table 1). We found that lysoPE(22:6) and lysoPS(22:6) were significantly down-regulated on the 7th day (Fig. 5). According to the unsaturated FA biosynthesis pathway, the excessive intake of ω-6 and ω-9 unsaturated FAs may simultaneously inhibit the biosynthesis of ω-3 unsaturated FAs (Fig. 8A). We inferred that DHA (C22:6ω-3) biosynthesis was affected and lipid metabolism was shifted to NA(C24:1ω-3) production (Fig. 8A).
Notably, C24:1ω-9 was the unique FA chain screened in the serum SP, and simultaneously correlated positively between the serum and brain, and regarded as biomarkers, including Cer(d18:1/24:1(15Z)) SM(d17:1/24:1) and GalCer(d18:1/24:1(15Z)) in the serum, and PC(24:1(15Z)/18:1), PC(18:0/24:1(15Z)) and PC(24:1(15Z)/18:3(9Z,12Z,15Z)) in the brain (Fig. 7). Ceramide is a key sphingolipid metabolite in both the biosynthesis and degradation of complex sphingolipids.47 SM and PC serve as structural constituents of cell membranes and plasma lipoproteins.48 Galactosylceramide is integral to neural function, performing different roles in the brain. The former is a component of myelin and plays a critical role in myelin-related functions,49 and the latter is involved in axonal growth.50,51 Evidence revealed that these lipids can participate in certain metabolic procedures in brain cells, including structure,52 growth, differentiation53 and survival,54 and hence they affect memory and behavior.53,55,56 The previous studies and KEGG pathway analysis of C24:1ω-9 in the serum and brain revealed that the conversion of C24:1ω-9 biomarkers can result in sphingolipid metabolism and glycerophospholipid metabolism. C24:1ω-9 intake together with ASO was accessed firstly in the serum and detected as Cer(d18:1/24:1) and SM(d17:1/24:1), and then converted and transported to the brain as PC(18:0/24:1), PC(24:1/18:1), GlcCer(d18:0/24:1) and GalCer(d18:1/24:1). The probable conversion pathways according to the previous studies and KEGG pathway analysis of C24:1ω-9 in the serum and brain are illustrated in Fig. 8B. Previous reports showed that sphingomyelin biosynthesis in mammalian cells is catalyzed by sphingomyelin synthase, which occurs primarily through the transfer of the phosphorylcholine group from phosphatidylcholine to ceramide.57 Consequently, sphingomyelin can be degraded to ceramide under the catalysis of acid sphingomyelinase.58 Ceramide can galactosylate with the effect of GalCerase to produce galactosylceramide.59 These pathways result in sphingolipid metabolism.58 Besides, ceramide can be converted into sphingosine due to the effect of ceramidase, and subsequently sphingosine into sphingosine 1-phosphate (S1P) by catalysis with sphingosine kinase.60 S1P is catalyzed by S1P lyase to form hexadecenal and phosphoethanolamine, which is also included in glycerophospholipid metabolism in the KEGG pathway analysis. Phosphoethanolamine can undergo catalysis by phosphoethanolamine cytidyltransferase and phosphatidylethanolamine N-methyltransferase, and eventually convert into phosphatidylcholine.61 These pathways may contribute to the conversion of C24:1ω-9 in the serum and brain and provide the learning and improvement in cognitive function in rats.
Understanding metabolite changes after ASO supplementation can contribute to determining the metabolites related to the improvement in cognitive function and discovering the molecular pathways regulated by ASO. The KEGG pathway analysis revealed that the neurotrophin signaling pathway, glycerophospholipid metabolism, sphingolipid metabolism, fat digestion and absorption, glycerolipid metabolism and serotonergic synapse were consecutively and collectively perturbed in the serum and brain (Tables 2 and 3, and Fig. 4). Neurotrophins are a family of trophic factors and considered to play a wide range of roles in the development and function of the nervous system. Certain researches have confirmed that the neurotrophin signaling pathway plays an important role in neural development and additional higher-order activities such as learning and memory.62 Glycerophospholipids consist of neuron membranes and play both structural and functional roles.63 The stage of well-pronounced Alzheimer's disease-like pathology in OXYS rats is also related to abnormalities in glycerophospholipid metabolism.64 Sphingolipids are particularly abundant in the brain and are essential for the development and maintenance of the functional integrity of the nervous system,65 and dysregulation of sphingolipid metabolism has been associated with a vast number of neurological diseases via disturbances in membrane organization66 such as Alzheimer's disease, Parkinson's disease, several types of epilepsy and Huntington's disease.67 Meanwhile, glycerolipid metabolism is associated with neuronal axon regeneration.68 Serotonin is a monoamine neurotransmitter and plays an important role. Serotonergic synapse exerts a profound impact on physiological functions including learning and memory, emotion, sleep, pain and motor function, as well as on pathological states such as abnormal mood and cognition.69 Theses perturbed pathways indicate that the supplementation of ASO mediated the pathways in FAs metabolism and cognitive improvement.
We found that the improvement in cognitive function was the result of the overall effect of essential fatty acids including NA. Considering that lipid changes and metabolic biomarkers associated with ω-9 fatty acid supplementation have not been reported, our original research revealed overall lipid remodeling and cognitive improvement associated with ω-9 fatty acid supplementation, especially NA supplementation, through serum and whole brain analyses in healthy mice. The contribution of this study is to provide the basis of metabolic theory and methodology for the future application of ω-9 fatty acid or compositions. Based on the results of the metabolic characteristics of the ω-9 fatty acid and lipid remodeling, we can design accurate disease model rat experiments in the future and conduct more detailed brain segmentation studies (such as white matter in hippocampus). The half-life of NA in the serum and RBC of NA is an important experiment for the clinical application of single lipids, and we will use separate compounds to supplement and conduct follow-up studies in the future.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1fo03671h |
‡ All authors contributed equally to this work. |
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