Identifying the specific lipid biomarkers and LYPLA1 as a novel candidate in intramuscular fat deposition of Erhualian pigs

Abstract

Background

Intramuscular fat (IMF) is a key determinant of meat quality enhancement in pigs. However, its correlation with subcutaneous fat (SCF) deposition presents a challenge. The precise regulation of IMF lipogenesis, without impacting SCF deposition, is a critical issue in the pig industry. To investigate this, our study examined the lipid profiles of longissimus dorsi (LD) muscle and subcutaneous adipose tissue in high IMF (IMFH) and low IMF (IMFL) Chinese Erhualian pigs using lipidomics techniques.

Results

We identified 112 differentially abundant lipids (DALs) in the muscle and 101 DALs in the adipose tissue. Notably, 105 specific DALs associated with IMF, including various candidate markers like upregulated lipids of PS (19:2/18:1), TG (14:2/4:0/4:0), PS (17:1/18:2), FA(10:0) + COOH:(s), CL (20:4/18:2/18:2/18:2), and downregulated lipids of FA (20:4), SM (d43:5), TG (38:1/22:6), PC (22:3/20:3), and PC (18:2/24:8), were identified. These specific DALs were implicated in the regulation of linoleic, arachidonic, and alpha-linolenic acid metabolism, and choline metabolism in cancer. We further discovered that the lysophosphlipase 1 (LYPLA1) gene promotes differentiation and lipid deposition of intramuscular pre-adipocytes by affecting the phosphatidylcholine (PC) content.

Conclusions

Our findings identify specific lipids associated with IMF accumulation in both skeletal muscle and subcutaneous adipose depots, thereby offering valuable insights into the lipid composition of porcine IMF and new avenues for targeted IMF deposition.

Background

Pork, as the most demanded meat globally, offers the advantages of excellent flavor, rich nutrients, and reasonable pricing. Consumers typically select pork based on immediate quality indicators such as meat color and odor, while flavor and palatability are crucial in determining repeat purchases. Previously, the Maillard reaction of water-soluble compounds during cooking has been shown to contribute to basic saltiness and meat flavor. However, the degradation of intramuscular lipids imparts a distinctive fat aroma to the meat [1, 2]. Consequently, an increase in intramuscular fat (IMF) content can enhance the flavor and palatability of pork [3,4,5], underscoring the pivotal role of IMF in meat quality development. Among all lipids, triglycerides (TG) and phospholipids are the most prevalent lipid compounds. TG is a major storage lipid, and studies showed that GPAT1 and GPAT2, which are key genes related to TG synthesis, have important effects on the IMF content of pigs [6, 7], whereas phospholipids, structural lipids forming the primary components of cell and organelle membranes [8, 9]. The lipolysis of phospholipids can influence the formation of volatile flavor compounds during cooking [10, 11]. This highlights the significant influence of these lipids on meat flavor.

Meanwhile, there is a noted synergistic effect between IMF and subcutaneous fat (SCF) deposition, indicating that an increase in IMF contents often leads to heightened SCF deposition [12]. In the pig industry, excessive SCF deposition can lower the lean meat yield and rise the breeding costs of pigs [13]. Therefore, understanding the differences between IMF and SCF deposition is crucial for enhancing IMF contents without impacting SCF deposition. Numerous studies have identified genetic variations that specifically influence IMF deposition in pigs. For instance, different genotypes of the SREBF1 gene in the muscle of Sutai pigs have been linked to IMF content, rather than backfat thickness, a measure used for SCF deposition [14]. In a F₂ population crossbred between Korean native pigs and Landrace breeds, two out of nine single nucleotide polymorphisms (SNPs) were found to be exclusively related to IMF and not SCF deposition [15]. Similarly, in Duroc pigs, six SNPs were significantly associated with both IMF contents and backfat thickness, yet four of these SNPs were solely linked to IMF content [16]. However, little is currently known about the specific lipid composition of IMF in pigs.

Chinese Erhualian pigs are renowned for their high fertility and exceptional meat quality. Typically, the IMF content of this breed ranges from 2.5% to 5% at market weight, making it an ideal subject for studying IMF deposition in pigs. Consequently, this study utilized lipidomics to analyze the intramuscular and subcutaneous lipid compositions of high and low IMF content groups in Chinese Erhualian pigs. As this is the first attempt to compare the differences in lipid composition between IMF and SCF tissues, we aim to identify specific lipids and metabolic pathways or genes associated with IMF deposition. These findings offer comprehensive insights into potential lipid metabolites or new genes related to the specific adipogenesis of IMF and their corresponding pathways in pigs.

Methods

Animals

Chinese Erhualian pigs, approximately 270 days old, were sourced from the Changzhou Erhualian Pig Production Cooperation in Changzhou, Jiangsu, China. From a group with similar body weights, 10 and 11 pigs were selected for the high intramuscular fat (IMFH, 4.86% ± 0.57) and low intramuscular fat (IMFL, 2.70% ± 0.39) content groups, respectively. All pigs were reared under identical environmental conditions and fed a standard diet twice a day in compliance with the Chinese feeding standard for pigs (NY/T65-2004). In strict accordance with the Chinese national standard for pig slaughtering (GB/T 17236–2019), the Erhualian pigs were electrically stunned and then exsanguinated at Ligang pig slaughtering center in Jiangyin, Jiangsu, China. Post-slaughter, approximately 20 g of the longissimus dorsi (LD) muscle and 4 g of SCF tissue were collected from each pig. Of these, about 4 g of LD muscle and 4 g of SCF tissues were immediately frozen in liquid nitrogen and subsequently transferred to an ultra-freezer at -80 °C for lipidomics and quantitative real-time RCR (qRT-PCR) analysis. The remaining LD muscle samples were preserved at 4 ℃ for assessing meat quality traits. All animal experiment procedures received approval from the Animal Ethics Committee of Nanjing Agricultural University (Approval No.20220318053).

Measurement of meat quality traits

The IMF contents of each sample was determined through the Soxhlet extraction method. The meat color L* (lightness), a* (redness), and b* (yellowness) values of the longissimus dorsi, were measured at 1 h and 24 h post-slaughter, respectively, using a chroma meter provided by Beijing Kemei Runda Instrument Equipment Co., Ltd, Beijing. Additionally, the shear force, an indicator of meat tenderness, was assessed 48 h after slaughter using a muscle tenderness meter (College of Engineering, Northeast Agricultural University). Backfat of shoulder, thoracolumbar junction, and lumbosacral joint was measured using an electronic vernier caliper. The average value of backfat in these three parts was taken as the average backfat thickness. The fatty acid (FA) composition of the longissimus dorsi was determined by gas chromatograph (GC7890, Shimadzu Corporation of Japan).

Lipid extraction

A total of 100 mg of muscle tissue from each individual was homogenized using a tissue grinder (model TS-24, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai) and transferred to a glass tube with a Teflon-lined cap. To this tube, 0.75 mL of pre-cooled methanol was added and the mixture was vortexed. Subsequently, 2.5 mL of pre-cooled methyl tert-butyl ether (MTBE) was introduced, and the mixture was incubated at room temperature on a shaker for 1 h. Following this, 0.625 mL of mass spectrometry (MS)-grade water was added, and the samples were further incubated at room temperature for 10 min, then centrifuged at 1,000 g for 10 min. The upper organic phase was collected, and the lower phase was subjected to a second extraction using 1 mL of a solvent mixture (MTBE/methanol/water in a 10:3:2.5 ratio, v/v/v). The upper organic phase was collected again, enhancing lipid purity and improving extraction efficiency. The combined organic phases were dried and reconstituted in 100 μL of isopropanol for subsequent UHPLC-MS/MS analysis. An equal volume of supernatant from each processed sample was mixed thoroughly to serve as quality control (QC) samples.

UHPLC-MS/MS analysis

UHPLC-MS/MS analysis was conducted using the Vanquish UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) and the Orbitrap Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) at Novogene Co., Ltd. (Beijing, China). Samples were injected into the Thermo Accucore C30 chromatographic column with a linear gradient flow rate of 0.35 mL/min. The injection time and column temperature were set at 20 min and 40 ℃, respectively. Mobile phase buffer A consisted of acetonitrile/water (6:4) with 10 mM ammonium acetate and 0.1% formic acid, while buffer B was isopropanol/acetonitrile (9:1) with 10 mM ammonium acetate and 0.1% formic acid. The solvent gradient was adjusted to 30%-70% buffer B over 2–11 min, 99% buffer B for 16 min, and returned to 30% buffer B at 18.1 min. The Q Exactive™ HF mass spectrometer operated with the following settings: sheath gas at 40 psi, sweep gas at 0 L/min, auxiliary gas rate at 10 L/min for positive and 7 L/min for negative ion mode, spray voltage at 3.5 kV, capillary temperature at 320 ℃, heater temperature at 350 ℃, S-lens RF level at 50, scan range from 114–1700 m/z, automatic gain control target at 3e6, normalized collision energy set at 22 eV, 24 eV, and 28 eV for both positive and negative ions, injection time at 100 ms, isolation window at 1 m/z, automatic gain control target at 2e5, and dynamic exclusion at 6 s.

Differential abundant lipid (DAL) identification

Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca). Permutation tests were conducted via OPLS-DA, and variable importance in projection (VIP) scores were derived from both PLS-DA and OPLS-DA results. After base-2 logarithmic transformation of lipid metabolite data, Shapiro–Wilk tests for normality and Levene’s tests for homogeneity of variances were conducted, followed by P-value calculation using the described statistical approaches: Student’s t-test was used for normally distributed data with homogeneous variances; Welch’s t-test for normal but heteroscedastic data; and Mann–Whitney U test for non-normal data. Fold changes (FC) values was calculated as the ratio of the mean value of each lipid compound in the IMFH group to that in the IMFL group. A lipid compound was defined as a DAL if it had a variable importance in projection (VIP) score greater than 1 from both PLS-DA and OPLS-DA models, a p-value < 0.05, and a FC value > 1.2 or < 0.8. Volcano plots were generated using GraphPad Prism 9.0.

Pathway enrichment analysis

The metabolic pathways of the selected DALs were carried out on the NovoMagic platform of Novogene Co., Ltd (https://magic-plus.novogene.com). Specifically, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was employed as the reference. A bioinformatics pipeline was built with Perl 5.18.2, and KEGG enrichment analysis used a hypergeometric test implemented in R 4.0.3. Subsequently, KEGG pathway maps were generated using Python 3.5.0. Pathways exhibiting P-value < 0.05 were designated as statistically significant.

Cell collection and differentiation of intramuscular pre-adipocytes

Three 7-day-old Erhualian piglets were humanely euthanized via intraperitoneal injection of pentobarbital sodium at a dosage of 50 mg/kg body weight, followed by aseptic exsanguination. Subsequently, intramuscular pre-adipocytes were collected from LD muscles isolated from these piglets, following the steps we previously reported [17]. Plasmids harboring LYPLA1 (pcDNA3.1-LYPLA1) and small interfering RNA (siRNA) of LYPLA1 (si-LYPLA1) were constructed separately. When the cells density reached 80%, pcDNA3.1-LYPLA1 or si-LYPLA1 was transfected with Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA) (Table S1). The cells were induced by induction medium for 4 d and fresh induction medium was replaced every other day. The components of induction medium contained 1 μg/mL insulin, 2 μg/mL dexamethasone, 1 μmol/L rosiglitazone, 5 nmol/L triiodothyronine, 100 μmol/L 3-isobutyl-1 methylxanthine, 125 μmol/L indomethacin, 1% penicillin–streptomycin, and 10% fetal bovine serum in Dulbecco’s modified eagle’s medium–high glucose (DMEM-HG) (Servicebio Technology Co., Ltd, Wuhan, China). The cells were then differentiated for 4 d in maintenance medium (1 μg/mL insulin, 1 μmol/L rosiglitazone, 5 nmol/L triiodothyronine, 1% penicillin–streptomycin, 10% fetal bovine serum in DMEM-HG); maintenance medium was replaced every other day.

qRT-qPCR assay

The total RNA of intramuscular pre-adipocytes or LD muscle tissues was extracted by using the Trizol reagent kit. Reverse transcription was conducted using the Hifair®II 1st Strand cDNA Synthesis SuperMix for the qPCR (Yeasen, shanghai, China), followed by qRT-PCR with qPCR SYBR Green Master MIX (Abclonal, Wuhan, Hubei, China) on the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The expression levels of genes were measured using the 2^−ΔΔCt method. GAPDH was selected as an internal reference gene. All primers of genes used in qRT-PCR assay are listed in Table S1.

Oil red phenotype and triglyceride content determination

Intramuscular pre-adipocytes on 8 d of differentiation were washed with PBS and fixed with 4% paraformaldehyde for 30 min. After removing the paraformaldehyde, the cellular specimens were rinsed again with PBS. Subsequently, the accumulation of lipid droplets was visualized through oil red O staining for 30 min. Cell morphology was assessed using a Zeiss inverted microscope (Thornwood, NY, USA). Finally, the cells were decolorized with isopropanolyl and absorbance was measured at 510 nm to obtain the triglyceride content.

Determination of phosphatidylcholine (PC) content

The PC content of intramuscular pre-adipocytes was determined using a phosphatidylcholine ELISA kit (Nanjing Jiancheng Technology Co., Ltd, Nanjing, China) and measured at 450 nm absorbance.

Statistical analysis

Statistical analyses were performed using SPSS 17.0. Data normality and variance homogeneity were assessed via Shapiro–Wilk and Levene's tests, respectively. Two-group comparisons employed Student's t-test, Welch's t-test, or Mann–Whitney U test based on parametric assumptions, consistent with the P-value calculation in prior DAL identification. One-way analysis of variance and Tukey’s test was used when comparing more than two groups. The data represented as mean ± standard deviation (SD). P < 0.05 and P < 0.01 or 0.001 was indicated significant or highly significant differences, respectively.

Results

Phenotypic differences in meat quality between IMFH and IMFL pigs

As presented in Table 1, the IMF contents of the IMFH pigs was significantly higher than that of the IMFL group (P < 0.001). Meanwhile, there was no significant difference in body weight between these two groups (P > 0.05). Furthermore, the IMFH pigs exhibited significantly higher L*24 h and b*24 h values in meat color compared to the IMFL pigs (P < 0.05 or P < 0.01). In contrast, no significant differences were observed in L*1 h, a*1 h, b*1 h, a*24 h, shear force, and average back fat thickness between the IMFH and IMFL pigs (P > 0.05). Compared with IMFL group, IMFH pigs contained more saturated fatty acids (SFA, P = 0.095) and monounsaturated fatty acids (MUFA, P < 0.05), as well as less polyunsaturated fatty acids (PUFA, P < 0.001). Among them, the contents of oleic acid of the IMFH pigs were significantly higher than that of the IMFL pigs (P < 0.05), whereas the contents of pentadecanoic acid, heptadecanoic acid, linoleic acid, α-linolenic acid, cis-11,14-eicosadienoic acid, and arachidonic acid of the IMFH pigs were significantly lower than that of the IMFL pigs (P < 0.05 or P < 0.01/0.001).

Table 1 The phenotype of meat quality between IMFH and IMFL groups

Characteristics of lipid compounds in the longissimus dorsi (LD) muscles of IMFH and IMFL pigs

A total of 1,159 lipid compounds were detected in the LD muscles across all samples, with 688 identified in positive ion mode and 471 in negative ion mode (Table S2). As depicted in Fig. 1A, B, glycerophospholipids (GP, 42.15%) were the most prevalent in positive ion mode, predominantly comprising phosphatidylcholine (PC, 22.67%), phosphatidylethanolamine (PE, 8.28%), phosphatidylserine (PS, 3.20%), lyso-phosphatidylcholine (LPC, 2.33%), phosphatidylinositol (PI, 1.60%), and phosphatidylglycerol (PG, 1.16%). Glycerolipids (GL, 39.40%) were the second most abundant, mainly consisting of triglyceride (TG, 28.20%) and diglyceride (DG, 10.47%). Sphingolipids (SP, 15.26%), including sphingomyelin (SM, 7.27%) and ceramide (Cer, 6.25%), were the third largest lipid class. Additionally, a minor proportion of fatty acids (FA, 2.76%) was also detected (Fig. 1A). In negative ion mode, the primary lipids were glycerophospholipids (GP, 87.47%), sphingolipids (SP, 8.92%), and fatty acids (FA, 3.61%) (Fig. 1E). The most abundant subclasses included PC (25.05%), PE (19.53%), cardiolipin (CL, 8.70%), PS (8.49%), PI (7.64%), Cer (4.88%), and PG (3.82%) (Fig. 1F). The PCA results showed that QC samples clustered together (Fig. 1C and G), demonstrating the stability and high quality of the lipid determination process. Furthermore, the OPLS-DA scatter plot revealed a clear separation between IMFH and IMFL pigs under both positive and negative ion modes (Fig. 1D and H).

Fig. 1

Lipidomic analysis of LD muscles in IMFH and IMFL pigs. A, E Lipid classes in LD muscles under positive and negative ion modes. B, F Lipid subclasses in LD muscles under positive and negative ion modes. C, G Principal component analysis of the confirmed lipids in LD muscles under positive and negative ion modes. D, H Orthogonal partial least squares discriminant analysis of the confirmed lipids in LD muscles under positive and negative ion modes. n = 10–11 for each group

Characteristics of lipid compounds in subcutaneous adipose tissues of IMFH and IMFL pigs

A total of 184 and 378 lipid compounds were identified in the subcutaneous adipose tissues of IMFH and IMFL pigs, respectively, when analyzed in positive and negative ion modes (Table S3). In the positive ion mode, the most abundant lipid was GP (57.07%), followed by GL (25.54%), SP (11.96%), and FA (5.43%) (Fig. S1A). The most abundant subcategories were PC (37.50%) and TG (16.85%) (Fig. S1B). In the negative ion mode, four types of lipids were detected: GP at 80.95%, SP at 11.38%, FA at 7.41%, and sterol (ST) at 0.26% (Fig. S1E). Within these, PC (25.40%), PE (22.49%), PS (11.11%), FA (7.41%), and SM (6.08%) were the subcategories with the highest contents (Fig. S1F). The results of PCA and OPLS-DA indicated that there was no marked separation between these two groups (Fig. S1C-D and S1G-H).

DALs identification between IMFH and IMFL pigs in the LD muscles

A total of 60 DALs were identified in the positive ion mode, including 28 GP, 21 GL, 9 SP, and 2 FA (Fig. 2A and Table S4). In comparison with the IMFL pigs, 6 lipids were significantly upregulated, and 54 were significantly downregulated in the IMFH pigs (Fig. 2B and C). The upregulated lipids included DG (13:0/19:2), PE (38:7), SM (t39:1), TG (16:0/18:0/18:0), TG (10:0/18:0/18:1) and TG (14:2/4:0/4:0), while the downregulated lipids were primarily GP and GL. In the negative ion mode, 52 DALs were identified, with 26 significantly upregulated and 26 downregulated in the IMFH group (Fig. 2D and Table S5). This group consisted of 21 GP, 4 SP, and 1 FA upregulated lipids, and the downregulated lipids included 25 GP and 1 FA (Fig. 2E and F). Further analysis of the peak areas of DALs within the same subclass revealed that the total TG content and PS in IMFH was significantly higher than in IMFL (P < 0.05 or P < 0.01) (Fig. 2G and H), despite the lower number of upregulated TGs compared to the number of downregulated TGs in IMFH (Fig. 2B and C). Inversely, the contents of PC and SM in the IMFH pigs were significantly reduced compared to the IMFL pigs (P < 0.05 or P < 0.01) (Fig. 2I and J).

Fig. 2

Analysis of DALs of IMFH and IMFL in the LD muscles. A, D Volcano plot of DALs in positive and negative ion modes. B, C Upregulated and downregulated DALs in positive ion mode. E, F Upregulated and downregulated DALs in negative ion mode. G-J Summation of peak areas of DALs in different subclasses of IMFH and IMFL pigs. Data are presented as mean ± SD. n = 10–11 for each group. *P < 0.05, **P < 0.01

Identification of specific DALs related to IMF

To explore whether DALs in the LD muscle influence SCF deposition, we compared these DALs in SCF tissues between IMFH and IMFL pigs. We found 40 DALs in positive ion mode and 61 DALs in negative ion mode exhibiting significant differences between these two groups (Tables S6 and S7). Of these, 5 DALs in positive ion mode and 2 in negative ion mode were common to both groups. These 7 lipids displayed a consistent trend of change in both LD and SCF tissues in IMFH and IMFL pigs (Fig. 3A and B). Excluding these lipids, there were 55 specific DALs in LD muscle tissue in positive ion mode and 50 in negative ion mode. The five most significantly upregulated lipids in IMFH pigs were PS (19:2/18:1), TG (14:2/4:0/4:0), PS (17:1/18:2), FA (10:0) + COOH:(s), and CL (20:4/18:2/18:2/18:2) (Fig. 3C-G). In contrast, the five most significantly downregulated lipids in IMFH pigs were FA (20:4), SM (d43:5), TG (38:1/22:6), PC (22:3/20:3), and PC (18:2/24:8) (Fig. 3H-L and Tables S4-S5).

Fig. 3

Analysis of specific DALs in LD muscles. A, B DALs shared by IMF and SCF tissues. C-G Five most significantly upregulated DALs in IMFH pigs compared with IMFL pigs. H–L Five most significantly downregulated DALs in IMFH pigs compared with IMFL pigs. M, N KEGG enrichment analysis of DALs under positive and negative ion modes. The color represents -log10 p-value, while the size represents the number of DALs in the corresponding pathway. Data are presented as mean ± SD. n = 10–11 for each group. **P < 0.01, ***P < 0.001

Pathway analysis of specific DALs related to IMF

KEGG enrichment analysis revealed that the specific DALs associated with IMF primarily engage in metabolic pathways in positive ion mode (Fig. 3M, Table S8). Conversely, DALs in negative ion mode were significantly involved in linoleic acid metabolism, arachidonic acid metabolism, alpha-linolenic acid metabolism, and choline metabolism in cancer (Fig. 3N, Table S9).

Expression levels of LYPLA1 associated with IMF deposition

Because 24 of the 26 (92%) unique DALs enriched in these above four metabolic pathways of negative ion mode belong to PC (Table S9), five genes related to PC metabolism, including CHKA, LYPLA1, PEMT, PLA2G16, and PTDDS1, were selected using qRT-PCR analysis between IMFH and IMFL pigs. As shown in Fig. 4A, only the expression of LYPLA1 was significantly higher in IMFH pigs than in IMFL pigs (P < 0.01). During the differentiation process of intramuscular pre-adipocytes, the level of LAPLA1 began to rise on day 2 of induction differentiation, reached its peak on day 4 and was significantly higher than that on day 0 (P < 0.05) (Fig. 4B).

Fig. 4

The mRNA expression of genes related to PC metabolism between IMFH and IMFL pigs or the differentiation process of intramuscular pre-adipocytes. A The mRNA expression of CHKA, LYPLA1, PEMT, PLA2G16, and PTDDS1 between IMFH and IMFL pigs. n = 10–11 for each group. B The mRNA expression of LYPLA1 at different stages of differentiation in intramuscular pre-adipocytes. Data are presented as mean ± SD. *P < 0.05, **P < 0.01

Overexpression of LYPLA1 promotes lipid deposition while reducing the PC content in intramuscular pre-adipocytes

When treating pcDNA3.1-LYPLA1 for 48 h, the data demonstrated that the overexpression efficiency of LYPLA1 was highly significant (P < 0.001) (Fig. 5A). After inducing differentiation for 8 d, overexpression of LYPLA1 significantly up-regulated the levels of adipogenic markers PPARG and FABP4 (P < 0.01; Fig. 5B and C) and triglycerides (P < 0.01; Fig. 5D and E), and down-regulated the levels of PC (P < 0.05; Fig. 5F).

Fig.5

Overexpression of LYPLA1 promotes lipid deposition in intramuscular pre-adipocytes. A The mRNA levels of LYPLA1 in pre-adipocytes treated with pcDNA3.1 or pcDNA3.1-LYPLA1 for 48 h. B, C The mRNA levels of PPARG and FABP4 in pre-adipocytes treated with pcDNA3.1 or pcDNA3.1-LYPLA1 for 8 d. D, E Oil Red phenotype (D) and triglyceride content (E) in pre-adipocytes treated with pcDNA3.1 or pcDNA3.1-LYPLA1 for 8 d. F Phosphatidylcholine (PC) content in pre-adipocytes treated with pcDNA3.1 or pcDNA3.1-LYPLA1 for 8 d. Data are presented as mean ± SD. n = 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001

Interference of LYPLA1 inhibits lipid deposition while enhancing the PC content in intramuscular pre-adipocytes

When treating si-LYPLA1 for 48 h, the interference efficiency of LYPLA1 was significant (P < 0.001; Fig. 6A). After inducing differentiation for 8 d, interference of LYPLA1 significantly down-regulated the levels of adipogenic markers PPARG and FABP4 (P < 0.05 or P < 0.01; Fig. 6B and C) and triglycerides (P < 0.01; Fig. 6D and E), and up-regulated the levels of PC (P < 0.01; Fig. 6F).

Fig. 6

Interference of LYPLA1 inhibits lipid deposition in intramuscular pre-adipocytes. A The mRNA level of LYPLA1 in pre-adipocytes treated with si-NC or si-LYPLA1 for 48 h. B, C The mRNA levels of PPARG and FABP4 in pre-adipocytes treated with si-NC or si-LYPLA1 for 8 d. D, E Oil Red phenotype (D) and triglyceride content (E) in pre-adipocytes treated with si-NC or si-LYPLA1 for 8 d. F PC content in pre-adipocytes treated with si-NC or si-LYPLA1 for 8 d. Data are presented as mean ± SD. n = 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In this study, our data indicated that only the IMF, L*24 h, and b*24 h values were significantly higher in the IMFH group compared to the IMFL group (Table 1). This aligns with findings from a previous study [18], which observed that Ningxiang pigs with high IMF content at 24 h post-slaughter exhibited increased brightness and yellowness in the LD muscle. Since the L and b values represent meat color brightness and yellowness respectively, our results support the notion that higher IMF content may be associated with brighter, more yellow meat colors. Additionally, a previous study has shown that IMF is positively correlated with oleic acid content and negatively correlated with linoleic acid and PUFA content [19], which is similar to our data, suggesting that these FA might play an important role in IMF deposition of pigs (Table 1). Actually, the thermal oxidation of oleic acid can promote the formation of aldehydes, which have a pleasant fatty and oily odor, whereas the thermal oxidation of linoleic acid forms substances that may produce unpleasant odors at higher concentrations [20]. Compared with IMFL pigs, IMFH pigs had higher levels of oleic acid and simultaneously possessed lower levels of linoleic acid, indicating that the meat flavor of individuals from the IMFH group are better than that of IMFL pigs.

To identify specific DALs related to IMF, we conducted a lipidomics analysis on LD muscle and SCF tissues from both IMFH and IMFL groups. Our analysis revealed that IMF contained more TG, CL, and PI and less PC, SM, and FA than that of SCF tissues in positive and negative ion mode (Fig. 1B and 1F; Fig.S1B and S1F). Notably, only 7 DALs were common to both LD and SCF tissues across the two groups (Fig. 3A-B), indicating significant differences in lipid composition between IMF and SCF. Upon excluding these common DALs, we found that specific DALs related to IMF predominantly included TG, PC, PE, PI, PS, and SP (Fig. 2B-F; Tables S4-S5). Furthermore, the total contents of these differential TGs were significantly higher in the IMFH group than in the IMFL group (Fig. 2G). This is consistent with previous studies on Hu sheep and Beijing Heiliu pigs, where individuals with higher IMF had greater TG and DG contents compared to those with lower IMF [21, 22]. These parallels suggest that variations in TG content might be a key factor influencing differences in IMF deposition in pigs.

PC and PE stand out as the most predominant phospholipids within mammalian cell and organelle membranes [23, 24]. On one hand, the increase of PE on the surface of lipid droplets promotes the merging of small lipid droplets to form large ones. On the other hand, the addition of PC to lipid droplets reduces the relative content of PE, thereby influencing droplet fusion [25, 26]. In this study, we identified 44 types of PC among the DALs, with 39 showing a downward trend in the IMFH group (Table S4-S5). Notably, two of the five most significantly downregulated lipids were PC (22:3/20:3) and PC (18:2/24:8) (Fig. 3K-L). This finding aligns with another study [27], which reported that Laiwu pigs, known for higher IMF, had more downregulated PCs compared to Yorkshire pigs. In contrast, of the 9 types of PE identified as DALs, 6 exhibited an upward trend in the IMFH group (Tables S4-S5), supporting the idea that IMF deposition may be inversely proportional to PC content and directly proportional to PE content. In animal adipose tissue, compared to PC and PE, the content of PS is lower, but it is crucial for the activation of many enzymes [28, 29]. A total of 24 differential PS were found in Xidu black pigs, all of which were significantly upregulated in the high IMF group [30]. In our study, 7 different types of PS were found in IMFH and IMFL (Fig. 2C-F), among which PS (19:2/18:1), PS (17:1/18:2), PS (17:1/20:3), and PS (18:0/19:2) were significantly upregulated in IMFH (Tables S5). And in the statistical analysis of the peak areas of these 7 PS, it was found that the content of PS in IMFH was significantly higher than that in IMFL (Fig. 2H). This suggests that PS may promote the deposition of IMF in Erhualian pigs. Additionally, in animal cells, CL is almost exclusively found in the inner membrane of mitochondria and plays a crucial role in regulating mitochondrial function [31,32,33]. In our study, CL (20:4/18:2/18:2/18:2) and CL (22:6/16:1/16:1/18:2) were found to be significantly upregulated in the IMFH group (Table S5), suggesting that muscles of IMFH individuals may have higher levels of mitochondrial respiratory function. However, further research is needed to investigate the specific mechanisms of CL in mitochondrial metabolism in pigs.

As the third largest category of lipids in the LD muscles of pigs, SP have been demonstrated to play a significant role in cellular functions and pathology, including cell growth, senescence, immune responses, and inflammatory processes [5, 34]. In our study, we found that the contents of SM, a subclass of SP, were significantly lower in the IMFH group compared to the IMFL group (Fig. 2J). Nine types of SM among the DALs were identified between IMFH and IMFL groups, among which SM (d43:5), SM (t40:3), SM (t40:6), SM (t38:1), SM (t42:6), and SM (d42:3) were significantly downregulated in the IMFH group (Tables S4-S5). This finding aligns with results from studies on Laiwu pigs, where individuals with higher IMF also exhibited notably lower SM levels compared to those with lower IMF [5]. Additionally, comparisons between different pig breeds revealed higher SM levels in Large White pigs, which typically have lower IMF content, as opposed to Jianhe White Xiang pigs with higher IMF content [35]. Thus, our data contribute new evidence suggesting that SM may influence the deposition of IMF in pigs.

KEGG enrichment analysis revealed that the DALs were primarily involved in linoleic acid metabolism, arachidonic acid metabolism, alpha-linolenic acid metabolism, and choline metabolism in cancer in negative ion mode (Fig. 3N). Interestingly, PC was found to be the main lipid involved in these four metabolic pathways (Table S9). Therefore, we further detected the expression of PC metabolism-related genes between IMFH and IMFL pigs and discovered that only LYPLA1 showed a significant difference between these two groups. As a key regulatory factor in the decomposition of PC, LYPLA1 plays an important role in maintaining lipid homeostasis and membrane lipid composition in cells. A genome-wide association analysis in beef cattle found that LYPLA1 was closely related to carcass weight and related characteristics [36]. In this study, we found that levels of LYPLA1 in IMFH pigs were significantly higher than those in IMFL pigs. Overexpression of LYPLA1 promoted the intracellular lipid accumulation while reducing PC content, whereas interference with LYPLA1 decreased the intracellular lipid accumulation and simultaneously enhanced PC content, indicating that LYPLA1 positively regulates IMF deposition. The negative correlation between PC content and IMF deposition was observed in Fig. 2I; these data suggested that LYPLA1 may affect IMF deposition by altering intracellular PC content. So far, little is known about the exact function of LYPLA1 in IMF deposition. Here our data provides first-hand evidence that LYPLA1 can regulate the adipogenesis of porcine IMF.

Conclusion

In summary, this study offers a comprehensive overview of the lipid metabolite composition of IMF and SCF in pigs. Crucially, we identified specific lipids associated with IMF deposition in both skeletal muscle and subcutaneous adipose depots and their corresponding metabolic pathways. Besides, we further discovered that LYPLA1 can serve as a novel candidate for affecting IMF adipogenesis. Our findings not only enhance the understanding of lipid composition in different body parts, but also propose potential biomarkers and gene for the targeted deposition of IMF in pigs. However, the limitations inherent in this study, primarily stemming from a restricted sample size, may compromise key parametric assumptions (e.g., normality and variance homogeneity) and thereby potentially undermine the statistical power of the analysis. Future investigations employing expanded sample sizes would allow for enhanced statistical power to enable more precise detection of lipidomic alterations.