Combined Multi-Omics Analysis Reveals the Potential Role of Methionine in Regulating the Proliferation, Differentiation, and Lipid Deposition of Yak Intramuscular Preadipocytes

TL;DR

This study shows that methionine can boost fat deposition in yaks' muscle cells at moderate levels but causes stress at high levels, offering insights for improving meat quality.

Contribution

The study is the first to demonstrate a biphasic effect of methionine on yak intramuscular preadipocytes and identifies the PI3K/AKT pathway as a key regulator.

Findings

Moderate methionine (0.5 mM) promotes yak preadipocyte proliferation and differentiation, while excessive methionine (50 mM) suppresses these processes.

Transcriptomic and proteomic analyses reveal that moderate methionine activates the ECM-receptor interaction and PI3K/AKT pathways.

Excessive methionine induces DNA damage, oxidative stress, and metabolic dysregulation in yak preadipocytes.

Abstract

Methionine (Met) is an essential amino acid that influences intramuscular fat (IMF) deposition, a key determinant of yak meat quality. However, the direct mechanism by which Met regulates yak intramuscular preadipocytes (YIMA) remains unknown. In this study, YIMA were exposed to a range of Met concentrations, and cellular assays combined with transcriptomic and proteomic profiling were used to dissect the dose-dependent effects. We found that moderate Met (0.5 mM) markedly promoted YIMA proliferation and adipogenic differentiation, whereas an excessive dose (50 mM) suppressed these processes. Transcriptomics and proteomics analysis revealed that moderate Met activated the ECM-receptor interaction and PI3K/AKT pathways, while excessive Met induced signatures of DNA damage, oxidative stress, and metabolic dysregulation. Functional validation confirmed that the PI3K/AKT pathway is essential for Met-driven adipogenesis and proliferation. This study provides the first yak-specific evidence of a biphasic Met effect and offers a mechanistic framework for optimizing dietary Met supplementation to improve intramuscular fat deposition without eliciting cellular stress.

This study employed integrated transcriptomics and proteomics analysis to investigate the potential role of methionine (Met) in regulating the proliferation, differentiation, and lipid deposition of yak intramuscular preadipocytes (YIMA). Five Met concentrations (0, 0.05, 0.5, 5, and 50 mM) were used to establish the Met model of YIMA. The results of Bodipy, Oil Red O, EdU staining, and qPCR showed that the appropriate Met (0.05, 0.5, and 5 mM) supplementation significantly promoted the proliferation and adipogenic differentiation of YIMA, whereas excessive Met (50 mM) markedly inhibited these processes. To further evaluate the mechanism of Met on YIMA, cells supplemented with 0 mM (control, CON), 0.5 mM (moderate) and 50 mM (excessive) Met were selected for the transcriptomic and proteomic analyses. The results showed that moderate Met treatment primarily enriched pathways related to extracellular matrix–receptor interaction and the PI3K/AKT signaling pathway, while excessive Met significantly enriched processes involving abnormal methylation, DNA damage, and metabolic stress. Functional validation experiments further confirmed that Met regulates YIMA proliferation and differentiation by upregulating p-Akt protein expression and activating the PI3K/AKT pathway. These findings provide molecular insights that support improving yak meat quality and IMF content through dietary Met supplementation.

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1. Introduction

Yaks (Bos grunniens) are iconic livestock on the Qinghai–Tibet Plateau, supplying meat with high protein, low fat, and rich trace elements [1]. However, compared with taurine cattle, yaks exhibit slower growth, less efficient muscle development, and lower intramuscular fat (IMF) deposition, resulting in tougher meat and reduced economic value in high-altitude production areas [2]. IMF content is positively correlated with meat tenderness, juiciness, and flavor [3]; therefore, strategies to enhance IMF deposition are of great interest for yak industry improvement.

Methionine (Met) is the only sulfur-containing essential amino acid in organisms and plays a significant role in polyamine synthesis and protein biosynthesis [4]. As a major limiting amino acid of many animals, it is commonly used as a feed additive in livestock farming. In ruminants, dietary supplementation with rumen-protected methionine (RPM) has been shown to increase IMF deposition efficiency [5], improve nitrogen utilization [6], and enhance growth performance and meat quality in Tan lambs [7]. Moreover, Liu et al. [8] also demonstrated that supplementing a low-protein diet with RPM significantly influences fat deposition by altering the synthesis and breakdown of fat in lamb muscle. However, these studies were conducted in cattle or sheep, and the direct cellular and molecular responses of yak adipogenic progenitors to Met have not been investigated.

Our prior research established that supplementing with RPM can increase IMF content and effectively improve both the fatty acid profile and growth performance of yak longissimus lumborum muscle [9,10]. However, the mechanisms by which it influences IMF in yaks are still unknown. Moreover, in monogastric livestock such as broilers and swine, appropriate Met is known to enhance meat production by promoting protein synthesis and muscle development, while an overdose can be detrimental, causing growth inhibition, impaired synthesis, and even mortality [11,12]. Whether such a biphasic dose-dependent effect exists in yak adipocytes, and the underlying molecular networks, have never been explored. Therefore, this study provides the first evidence of the biphasic dose-dependent effect of methionine in a yak model, elucidates the molecular mechanism by which Met mediates IMF deposition, and offers a scientific basis for precision nutritional interventions in yak husbandry.

2. Materials and Methods

2.1. Isolation and Culture of Intramuscular Preadipocytes

This study was approved by the Animal Ethics Committee of Southwest Minzu University (Approval No.: SMU 202501049). Intramuscular preadipocytes (YIMAs) were isolated from the longissimus dorsi muscle of healthy male Maiwa yaks aged approximately 3 years using the method described in Reference [13], and were cultured in an incubator at 37 °C with 5% CO_2_. Cell proliferation was carried out in a customized medium (Boster, Wuhan, China) containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin-streptomycin dual-antibiotic solution, and varying concentrations of Met (L-Met, Sigma-Aldrich, St. Louis, MO, USA). Differentiation was induced in the same medium supplemented with 100 μM oleic acid.

2.2. Cell Proliferation Assays

EdU-488 Staining: The cells were exposed to EdU reagent with green fluorescence (Beyotime, Shanghai, China) at 37 °C with 5% CO_2_ for a duration of 1 h. Thereafter, the cells were subjected to fixation with 4% paraformaldehyde at room temperature for a period of 30 min. Subsequent steps were then executed in strict accordance with the manufacturer’s guidelines. Finally, 1X Hoechst 33,342 dye with blue fluorescence was added to each well, followed by incubation at room temperature in the dark for 10 min, and then washed three times with washing buffer. The images were captured using an Axio Observer 3 inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). ImageJ software (version 1.54d, National Institutes of Health, Stapelton, NY, USA) was used to quantify the mean fluorescence intensity from at least six random fields per group.

Cell proliferation activity was assessed using the CCK-8 assay: Following the method described in reference [14], YIMA were treated with five concentrations of Met (0, 0.05, 0.5, 5, and 50 mM). Cells were seeded in 96-well plates and, upon reaching approximately 60% confluence, were subjected to treatment with varying concentrations of Met and the PI3K/AKT pathway inhibitor LY294002 (Beyotime, Shanghai, China; concentration gradient: 0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, and 10 µM). After treatment, 10 µL of CCK-8 reagent (Beyotime, Shanghai, China) was added to each well, incubated for 1 h, and absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) to evaluate cell viability. Based on the measurement results, 50 mM Met was determined as the excess treatment concentration. Simultaneously, the effective concentration of LY294002 was preliminarily screened, and the optimal concentration was further confirmed by quantitative real-time polymerase chain reaction (qRT-PCR).

2.3. Bodipy and Oil Red O Staining

Lipid droplet accumulation was visualized using the red dye Oil Red O and the green fluorescent dye Bodipy staining. Following treatment, cells were fixed with 4% paraformaldehyde (Biosharp, Beijing, China) for 30 min and subsequently stained with either Oil Red O (RHAWN, Shanghai, China) or Bodipy (Thermo Fisher Scientific, USA) working solution for 30 min in the dark. Nuclei were counterstained with DAPI (Solarbio, Beijing, China) for 10 min. After washing with PBS, images were captured using a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) for qualitative observation and quantitative analysis of lipid deposition. Specifically, Oil Red O-stained samples were extracted with isopropanol (Cologne Chemical Co., Ltd., Hangzhou, China), and absorbance was measured at 510 nm using a microplate reader to quantify lipid content. Bodipy-stained images were analyzed with Image pro Plus (version 6.0, Media Cybernetics, Rockville, MD, USA) software to assess fluorescence intensity and lipid droplet area, enabling quantitative evaluation of lipid accumulation.

2.4. RNA Extraction, Reverse Transcription and qPCR

Total RNA was extracted from cells using TRIzol reagent (Tsingke, Beijing, China). RNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples with an A260/A280 ratio between 1.8 and 2.0 were used for subsequent analysis. Subsequently, 1 μg of total RNA was reverse-transcribed into cDNA using HiScript^®^ III RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Real-time quantitative PCR was performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The amplification protocol consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. A melt curve analysis was carried out at the end of each run (95 °C for 30 s, 60 °C for 60 s, and 95 °C for 30 s) to verify reaction specificity. The housekeeping gene GAPDH was used as an internal control for normalization. Relative gene expression was calculated using the 2^−ΔΔCT^ method. All primer sequences are listed in Supplementary Table S1.

2.5. Transcriptome Sequencing

Through preliminary screening of Met concentrations, differentiated YIMA were treated with 0 mM (control, CON), 0.5 mM (moderate Met, MM), and 50 mM (excessive Met, EM) Met for 48 h, with five biological replicates per treatment group, followed by subsequent transcriptomic and proteomic sequencing analyses. Total RNA was extracted from cell samples using TRIzol reagent (Qiagen, Hilden, Germany). The concentration and purity were assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with OD260/280 values required to be between 1.8 and 2.2. Integrity was evaluated by agarose gel electrophoresis, and the RNA Quality Number (RQN) was determined using an Agilent 5300 system (requiring RQN > 6.5). Polyadenylated mRNA was purified using Oligo(dT) magnetic beads, fragmented, and reverse-transcribed into double-stranded cDNA. After end-repair, A-tailing, and ligation of Illumina sequencing adapters, the constructed libraries were purified with AMPure XP beads and amplified by PCR. Transcript quantification was performed using RSEM software (version 1.3.3) to obtain TPM values, which normalize for gene length and sequencing depth. Based on gene read count data, differential expression analysis was conducted using DESeq2 (version 1.42.0) software with screening criteria of FDR < 0.05 and |log2FC| ≥ 1; this software internally employs the median of ratios method to correct for differences in sequencing depth and library composition between samples, ensuring result reliability. Finally, GO functional enrichment and KEGG pathway enrichment analyses of differentially expressed genes (DEGs) were performed using Goatools (version 0.8.9) and the Python scipy package (version 1.11.4), respectively. The raw sequencing data have been deposited in the NCBI SRA under accession number PRJNA1301543.

2.6. Proteomic Sequencing

Protein samples were lysed using 8 M urea lysis buffer, and the supernatant was collected after centrifugation for concentration determination. The protein samples were then sequentially subjected to reduction, alkylation, and buffer exchange. Trypsin was added at an enzyme-to-substrate ratio of 1:50, and digestion was carried out overnight at 37 °C. The resulting peptides were dried under vacuum, reconstituted, and desalted using HLB solid-phase extraction columns, followed by quantification. Finally, the peptides were dissolved in mass spectrometry loading buffer containing iRT internal standards and analyzed via high-resolution mass spectrometry in Data-Independent Acquisition mode. Raw data were processed using Proteome Discoverer software (version 2.2, Thermo Fisher Scientific, USA), and DIA data analysis, including peak extraction, iRT calibration, and protein quantification, was performed using Spectronaut software (version 18.5, Biognosys AG, Schlieren, Switzerland). Differentially expressed proteins (DEPs) were screened with thresholds of |log_2_FC| ≥ 0.585 and FDR ≤ 0.05. Subsequently, GO functional enrichment analysis of the target protein sets was performed using Goatools software (version 0.8.9), and KEGG pathway enrichment analysis was conducted using the in-house analysis pipeline developed by Majorbio (KEGGPATHWAY, version 2.0, Shanghai, China). A corrected p-value (Padjust) < 0.05 was set as the criterion for statistically significant enrichment.

2.7. Combinatorial Transcriptome and Proteome Profiling

The thresholds for defining DEGs and DEPs were set at |log_2_ (FC)| ≥ 1.0 and |log_2_ (FC)| ≥ 0.585, respectively, both with a false discovery rate (FDR) ≤ 0.05. Functional enrichment of the overlapping DEGs and DEPs was performed against the GO database, and the KEGG pathway database was used to identify the signaling pathways jointly affected at both the transcript and protein levels.

2.8. Protein Abundance Analysis

YIMA were treated with 0 mM (control, CON + DMSO), 0.5 mM (moderate Met, MM + DMSO), or 0.5 mM Met combined with LY294002 (MM + LY294002) for 48 h, with three biological replicates per group. Subsequently, total proteins were extracted using lysis buffer containing protease and phosphatase inhibitors (Boster, Wuhan, China). Protein concentration of each sample was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Following separation via SDS-PAGE and membrane transfer, the membrane was blocked with 5% BSA for 1 h, then sequentially incubated with primary antibodies against Akt1 (1:5000, Catalog No. 60203-2-Ig; Proteintech, Wuhan, China), p-Akt1 Ser473 (1:2000, Catalog No. 4060T; Cell Signaling Technology, Danvers, MA, USA), and the internal reference GAPDH (1:10,000, Catalog No. 10494-1-AP; Proteintech, Wuhan, China). Subsequently, corresponding species-specific secondary antibodies, goat anti-mouse IgG (1:10,000, Catalog No. KGAAM001) and goat anti-rabbit IgG (1:10,000, Catalog No. RGAR001) (both from Proteintech, Wuhan, China), were used for detection. Finally, protein bands were visualized using a hypersensitive ECL chemiluminescence substrate (Biosharp, Beijing, China) and quantitatively analyzed with ImageJ v1.54d software.

2.9. Statistical Analysis

Statistical analyses were performed using SPSS 26.0, and graphs were generated with GraphPad Prism 8. Differences between two groups were evaluated by an independent-samples Student’s t-test, for differences among multiple groups were assessed by one-way ANOVA followed by Duncan’s multiple-range test. Data are presented as mean ± SEM, with p < 0.05 considered statistically significant. All experiments were independently repeated three times.

3. Results

3.1. Effect of Met on the Proliferation of YIMA

CCK-8 assay demonstrated that compared with the 0 mM Met group, treatment with 0.05, 0.5 and 5 mM Met significantly promoted cell proliferation after 24, 36 and 48 h, whereas 50 mM Met had no significant effect (Figure 1A). RT-qPCR showed that the addition of 0.05, 0.5 and 5 mM Met significantly upregulated the expression of proliferation marker genes (Ki67, CCNB1, and PCNA) compared to the 0 mM group (Figure 1B). Furthermore, the EdU assay confirmed that supplementation with 0.05, 0.5 and 5 mM Met significantly promoted the proliferation of intramuscular preadipocytes (Figure 1C).

3.2. Effect of Met on the Differentiation of YIMA

The effects of Met concentration on the differentiation of YIMA were analyzed using quantitative PCR, Bodipy staining, and Oil Red O staining. The results showed that compared to the 0 mM-treated cells, Met supplementation at 0.05, 0.5 and 5 mM significantly upregulated the expression of key adipogenic genes, including PPARγ, C/EBPα, SREBP1, and FASN (Figure 2A). Bodipy and Oil Red O staining further revealed that the addition of 0.05 and 0.5 mM Met significantly promoted intracellular lipid droplet content compared to the 0 mM-treated cells, with the 0.5 mM-treated cells showing the most pronounced effect (Figure 2B).

3.3. Transcriptomic Profiling

Transcriptome sequencing was conducted on the Illumina Novaseq X Plus system. Principal component analysis and clustering results showed clear distinctions among the CON-, MM-, and EM-treated cells, with good intra-group reproducibility (Figure S1A,B). Analysis of five randomly selected DEGs by qPCR confirmed a high consistency with the RNA-Seq expression trends (Figure S1C).

DEGs in YIMA were analyzed using DESeq2 software. Compared to the CON group, the MM-treated cells had a total of 1193 DEGs (718 upregulated, 475 downregulated), while the EM-treated cells had 2186 DEGs (1163 upregulated, 1023 downregulated). Meanwhile, a comparison between the EM- and MM-treated cells identified 1316 DEGs (511 upregulated, 805 downregulated) (Figure 3A–C).

GO enrichment analysis was performed using Goatools. Compared to the CON group, DEGs in the MM-treated cells were primarily enriched in biological processes including regulation of developmental and multicellular organismal processes, as well as cell surface receptor signaling pathways. For molecular functions, the DEGs showed significant enrichment for protein binding and ion binding, and they were also involved in the composition of the extracellular space. DEGs in the EM-treated cells were mainly enriched in biological processes such as mitotic cell cycle processes, cell cycle processes, chromosome segregation, and developmental processes. Molecular functions were primarily associated with protein binding and ion binding, and the DEGs were also involved in the composition of the extracellular matrix and external encapsulating structures. Compared to the MM-treated cells, DEGs in the EM-treated cells were predominantly enriched in biological processes such as cell cycle processes, mitotic cell cycle processes, chromosome organization, and segregation, with molecular functions mainly related to protein binding (Figure 3D–F). These findings indicate that different concentrations of Met exert distinct biological effects on YIMA: an appropriate concentration of Met tends to regulate cellular developmental processes and signal transduction mechanisms, whereas excessive Met significantly activates processes related to cell cycle progression, mitosis, and chromosome dynamics, potentially inducing cellular stress responses.

KEGG pathway analysis was performed using R scripts. Compared to the CON group, the DEGs in the MM-treated cells were mainly enriched in metabolic pathways such as the PI3K-AKT signaling pathway, ECM-receptor interaction, and steroid biosynthesis and secretion, suggesting that MM-treated cells may regulate adipocyte function by enhancing anabolic metabolism and promoting cell-microenvironment. The DEGs in the EM-treated cells were primarily enriched in cellular senescence, glutathione metabolism, sulfur metabolism, and regulation of inflammatory mediators via TRP channels, indicating that EM-treatment tends to activate cellular stress responses, redox balance regulation, and senescence-related mechanisms. Compared to the MM-treated cells, the DEGs in the EM-treated cells were mainly enriched in DNA replication, the p53 signaling pathway, homologous recombination, mismatch repair, and cellular senescence (Figure 3G–I), demonstrating that excessive Met, relative to moderate methionine, more strongly triggers molecular events associated with DNA damage repair, genomic stability maintenance, and cell cycle surveillance, which may subsequently affect cell proliferation and senescence. These results suggest that 50 mM Met treatment exceeds the metabolic buffering threshold of YIMA and activates genomic stability surveillance systems, providing direct molecular evidence for the empirical observation in production practice that “excessive supplementation is counterproductive.”

3.4. Proteomic Data Analysis

Principal component and clustering analyses revealed clear separations among the CON-, MM-, and EM-treated cells, with strong intra-group biological reproducibility. DEPs exhibited dose-dependent upregulation or downregulation patterns across Met concentrations (Figure S2A,B).

Analysis of DEPs in YIMA showed that compared to the CON group, the MM-treated cells had 575 DEPs (350 upregulated, 225 downregulated), while the EM-treated cells had 1404 DEPs (584 upregulated, 820 downregulated). Compared to the MM-treated cells, the EM-treated cells exhibited 977 DEPs (274 upregulated, 703 downregulated) (Figure 4A–C).

GO enrichment analysis demonstrated that compared to the CON group, DEPs in the MM-treated cells were significantly enriched in biological processes including extracellular structure organization, and external encapsulating structure organization; in cellular components including external encapsulating structures, extracellular matrix, and extracellular regions; and in molecular functions including glycosaminoglycan binding and heparin binding. These results suggest that MM-treated cells may influence cellular functions primarily through the regulation of extracellular microenvironment remodeling and carbohydrate molecular interactions. DEPs in the EM-treated cells were significantly enriched in biological processes including regulation of RNA biosynthesis, DNA-templated transcription, and nitrogen compound metabolic processes; in cellular components including mitochondrial respiratory chain complexes; and in molecular functions including DNA binding. This indicates that EM-treated cells tend to affect gene transcriptional regulation and energy metabolism processes. Compared to the MM-treated cells, DEPs in the EM-treated cells were significantly enriched in biological processes including chromosome organization, DNA replication/repair/metabolism, and negative regulation of nitrogen compound metabolic processes; in cellular components including nucleus and MCM complex; and in molecular functions including DNA and chromatin binding activities (Figure 4D–F). This comparison further confirms that excessive Met, relative to moderate Met treatment, more significantly activates molecular pathways associated with genomic stability maintenance and nucleic acid metabolism regulation.

KEGG pathway analysis indicated that, compared to the CON group, DEPs in the MM-treated cells were significantly enriched in complement and coagulation cascades, ECM-receptor interaction, protein digestion and absorption, the PI3K/AKT signaling pathway, and focal adhesion. These findings suggest that an appropriate concentration of Met may influence cellular functions primarily by modulating immune responses, cell-matrix adhesion, nutrient absorption, and key growth signal transduction. DEPs in the EM-treated cells were mainly enriched in DNA replication, oxidative phosphorylation, base excision repair, and thermogenesis, indicating that excessive Met tends to activate cellular processes related to genetic information integrity maintenance, energy metabolism, and thermogenesis. Compared to the MM-treated cells, DEPs in the EM-treated cells were prominently enriched in DNA replication, base excision repair, nucleotide excision repair, and glutathione metabolism (Figure 4G–I). These results further demonstrate that excessive Met, relative to moderate Met, significantly potentiates metabolic pathways associated with DNA damage repair, genomic stability maintenance, and oxidative stress balance.

3.5. Integrated Analysis of Transcriptomic and Proteomic Data

GO analysis revealed that compared to the CON group, the DEGs and DEPs in the MM-treated cells primarily affected biological processes related to cellular development and extracellular matrix-associated functions, as well as molecular functions such as glycosaminoglycan binding. In contrast, the DEGs and DEPs in the EM-treated cells showed significant enrichment in DNA replication, oxidative stress response, and chromosome-related components. Compared to the MM-treated cells, the DEGs and DEPs in the EM-treated cells were significantly enriched in DNA metabolism, cell cycle, and DNA damage response processes (Figure 5A–C).

KEGG pathway analysis demonstrated that compared to the CON group, the DEGs and DEPs in the MM-treated cells were mainly involved in ECM-receptor interaction, the PI3K-AKT signaling pathway, and cholesterol metabolism. However, the DEGs and DEPs in the EM-treated cells were predominantly enriched in DNA repair pathways and glutathione metabolism. Comparative analysis of the DEGs and DEPs between the EM- and MM-treated cells further confirmed that excessive Met treatment leads to decreased genetic stability and disruption of redox balance (Figure 5D–F). These findings provide key pathway-level evidence for elucidating the metabolic network through which Met regulates IMF deposition from a multi-omics perspective.

3.6. Met Regulates Intramuscular Preadipocyte Proliferation via the PI3K/AKT Signaling Pathway

Integrated transcriptomic and proteomic analyses revealed that the genes associated with the PI3K/AKT signaling pathway were significantly enriched by Met treatment. Results showed that compared to the CON group, the MM-treated cells exhibited significantly upregulated expression of PDK1, AKT1, and AKT3, whereas the EM-treated cells showed no significant changes in PDK1 and AKT1 but a significant downregulation of AKT3 (Figure S3A). CCK-8 and qPCR results demonstrated that 2.5 μM LY294002 significantly inhibited PI3K-AKT pathway activity (Figure S3B,C).

Met may also regulate the proliferation of YIMA through the PI3K/AKT pathway. Results showed that compared to the MM + DMSO group, the MM + LY294002 group significantly downregulated the expression of proliferation marker genes (Ki67, CCNB1, and PCNA) (Figure 6A). EdU staining further confirmed that the combined treatment of Met and LY294002 markedly attenuated the promoting effect of Met on cell proliferation (Figure 6B).

3.7. Met Regulates the Differentiation of YIMA via the PI3K/AKT Signaling Pathway

Met may regulate the differentiation of YIMA through the PI3K/AKT signaling pathway. Results demonstrated that compared to the MM + DMSO group, the MM + LY294002 group showed significantly reduced expression of adipogenic marker genes (PPARγ, C/EBPα, SREBP1, and FASN) and downstream pathway genes (PDK1, AKT2, and AKT3) (Figure 7A,B). Bodipy and Oil Red O staining further confirmed that inhibition of the PI3K/AKT pathway significantly attenuated Met-induced lipid droplet accumulation (Figure 7C). Consistent with these findings, western blot analysis revealed that Met treatment increased p-Akt protein levels, whereas LY294002 treatment reduced them (Figure 7D).

4. Discussion

IMF content is a key determinant of meat quality, and its deposition is co-regulated by the proliferation and differentiation of intramuscular preadipocytes [15]. In this study, supplementation with appropriate levels of Met significantly promoted the proliferation and differentiation of YIMA. Research has shown that Met is an essential amino acid in animal diets and plays a crucial regulatory role in regulating growth and lipid accumulation [16]. Therefore, appropriate methionine supplementation can enhance IMF deposition, thereby improving yak meat quality. Meanwhile, Wu et al. [14] demonstrated that Met primarily regulates lipid metabolism in animals by modulating the expression of key genes associated with lipid metabolism. In this study, appropriate Met levels significantly upregulated the expression of PPARγ and C/EBPα, as well as their downstream targets SREBP1 and FASN, thereby promoting adipogenesis and increasing IMF deposition. Furthermore, Met serves as a precursor for methyl donors, and methylation is a key epigenetic mechanism regulating gene expression. The observed upregulation of PPARγ expression may be associated with alterations in the methylation status of its promoter. Research has shown that dietary supplementation with RPM in dairy cows alters the methylation status of PPAR receptor promoter in the liver, modifies the expression of downstream lipid metabolism-related genes, and improves lipid metabolism [17]. Thus, Met may promote fat metabolism and improve IMF deposition through methylation. Additionally, Met regulates cell proliferation via methylation modifications. Appropriate Met supplementation elevates S-adenosylmethionine (SAM) levels, thereby promoting H3K4me3 modifications in the promoter regions of proliferation markers Ki67 and cell cycle regulators including CCNB1 and PCNA, enhancing their transcriptional expression, and consequently driving cell cycle progression and proliferative activity [18]. PCNA, an essential cofactor for DNA replication, is also subject to methylation-dependent regulation [19], whereas CCNB1 orchestrates the G2/M transition and thus governs mitotic entry [20].

Met is an essential substance involved in the synthesis of proteins and nucleic acids, playing a critical role in the growth, development, and metabolic regulation of organisms [21,22]. In this study, DEGs following appropriate Met treatment were significantly enriched in processes such as developmental regulation, the PI3K/AKT signaling pathway, and ECM-receptor interaction. Previous studies have confirmed that dietary supplementation with appropriate levels of RPM significantly promotes growth performance in dairy cows through developmental regulation [23]. The PI3K/AKT signaling pathway, a classical insulin pathway, has been demonstrated to be closely associated with adipogenesis [24], suggesting that Met may regulate IMF deposition via this pathway. Meanwhile, ECM-receptor interactions contribute to maintaining microenvironmental homeostasis and provide support for adipogenesis and normal cellular development [25]. These findings indicate that appropriate Met supplementation may synergistically promote adipogenic differentiation by enhancing ECM structural stability, activating the PI3K/AKT signaling pathway, and improving methylation capacity [26], thereby accelerating IMF deposition and improving meat quality. Furthermore, as a key precursor for glutathione synthesis, Met plays an essential role in the antioxidant defense system [27]. In this study, DEGs following excessive Met treatment were primarily enriched in processes such as glutathione metabolism, the p53 signaling pathway, the mitotic cell cycle process, and the extracellular matrix. As a critical stress-responsive factor, p53 is activated in response to DNA damage, oxidative stress, or metabolic dysregulation, subsequently inducing cell cycle arrest, senescence, or apoptosis [28,29]. Additionally, excessive Met supplementation may lead to DNA and histone hypermethylation, disrupt transcriptional regulation, and impair cell cycle progression and chromosomal stability [30,31]. The extracellular matrix also exerts regulatory functions in the proliferation, differentiation, and migration of preadipocytes [32]. These findings indicate that excessive Met supplementation may induce cellular stress, cell cycle disruption, and senescence-related responses, thereby inhibiting lipid deposition and normal adipogenic processes.

The extracellular matrix (ECM) is a three-dimensional network structure composed of macromolecules such as glycosaminoglycans, collagen, heparin, and fibronectin, playing a core role in tissue morphogenesis, structural remodeling, and the regulation of cellular behavior [33]. In this study, DEPs in cells treated with an appropriate level of Met were primarily enriched in processes including ECM organization, glycosaminoglycan binding, and protein digestion and absorption. Research suggests that appropriate Met supplementation may promote the synthesis and post-translational modification of ECM-related proteins, thereby supporting the maintenance of normal structure and functional development of adipose tissue [34]. Furthermore, appropriate levels of Met can enhance the efficiency and capacity of protein synthesis and utilization in the body, thus improving muscle tissue development [35]. Notably, in this study, DEPs in cells subjected to excessive Met treatment were mainly enriched in processes such as DNA binding, DNA-templated transcriptional regulation, and respiratory chain complexes. As a key component of the one-carbon metabolism pathway, excessive Met intake can significantly elevate SAM levels and disturb the SAM/S-adenosylhomocysteine (SAH) ratio, thereby enhancing global methylation potential [36]. However, hypermethylation may impede the effective binding of transcription factors to DNA recognition elements, subsequently inducing global transcriptional repression [37]. Meanwhile, excessive Met may also inhibit methyltransferase activity through a negative feedback mechanism, leading to the accumulation of DNA damage and aberrant methylation patterns [38]. Furthermore, high-dose Met intake may induce mitochondrial dysfunction, accompanied by increased generation of reactive oxygen species (ROS) [39], and promote the activation of inflammatory signaling pathways, which in severe cases may even lead to systemic metabolic disorders [40].

The PI3K/AKT signaling pathway serves as a critical regulatory hub during adipogenic differentiation and is extensively involved in cell proliferation and lipid synthesis [41]. In this study, both DEGs and DEPs in cells treated with an appropriate concentration of Met were significantly enriched in the PI3K/AKT signaling pathway. To investigate whether Met regulates the proliferation and differentiation of YIMA via the PI3K/AKT pathway, our results demonstrated that appropriate methionine supplementation significantly promoted YIMA proliferation, lipid droplet formation, and p-Akt protein expression, whereas these effects were markedly inhibited in the group cotreated with appropriate Met and LY294002. These findings are consistent with previous studies, which have reported that Met enhances the differentiation potential of adipose-derived stem cells via the PI3K/AKT pathway [42], activates the Nrf2-HO-1 signaling axis to improve antioxidant capacity [43], and participates in the regulation of milk protein and milk fat synthesis [44]. The present study provides the first evidence elucidating the molecular mechanism by which Met regulates YIMA proliferation, differentiation, and IMF deposition through the PI3K/AKT signaling pathway. These findings fill a research gap in the field of yak lipid metabolism and offer a theoretical foundation for precision yak husbandry.

5. Conclusions

This study revealed that Met supplementation exerts a dose-dependent effect on YIMA, with appropriate levels significantly promoting their proliferation and differentiation. Integrated transcriptomics and proteomics analysis demonstrated that optimal Met supplementation enhances protein synthesis and lipid metabolism in YIMA through enrichment of pathways such as ECM organization and PI3K/AKT signaling. Functional validation further confirmed that Met regulates YIMA proliferation, differentiation, and intramuscular fat deposition via activation of the PI3K/AKT signaling pathway. These findings provide the first molecular-level evidence elucidating the mechanism by which dietary Met supplementation improves yak meat quality and IMF content, thereby offering a theoretical foundation for its application.