Activation of Focal Adhesion Pathway by CIDEA as Key Regulatory Axis in Lipid Deposition in Goat Intramuscular Precursor Adipocytes

Simple Summary

CIDEA, a protein associated with lipid droplets, plays a crucial role in maintaining lipid homeostasis. Our research demonstrates that CIDEA increases the levels of lipid droplets and triglycerides while concurrently repressing cell proliferation. Furthermore, we discovered that CIDEA facilitates lipid accumulation in goat intramuscular adipocytes through the activation of the focal adhesion pathway. This study enhances our understanding of the molecular processes governing intramuscular fat deposition and provides a theoretical framework that could inform molecular breeding efforts aimed at improving the quality of goat meat.

Abstract

Intramuscular fat (IMF) content determines the quality of goat meat and is regulated by the comprehensive effect of the proliferation and adipogenesis of intramuscular preadipocytes. Our previous RNA-seq data revealed that cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) A was upregulated during the development of intramuscular fat in the longissimus dorsi muscle tissue, implying an important role in lipid homeostasis. However, the mechanism by which CIDEA, a member of the CIDE family, regulates intramuscular fat deposition in goat muscle is unknown, so we explored the function and underlying mechanism of CIDEA in goat intramuscular preadipocytes. To address this, we altered CIDEA in intramuscular preadipocytes and resolved the effect and mechanism of CIDEA in adipogenesis through RT-PCR, Western blot, triglyceride and LD determinations, CCK-8, and RNA-seq. It was found that CIDEA increased lipid droplets (LDs) and triglyceride contents and inhibited cell proliferation. Meanwhile, the lipid metabolism-related genes PPARγ, C/EBPα, SREBP1c, PLIN1, TIP47, ADFP, DGAT1, ACC, FASN, ACSL1, and FABP3 were upregulated, while the lipolysis and β-oxidation genes HSL, ACOX1, and CPT1B, as well as the proliferation marker gene CDK1, were all downregulated upon CIDEA overexpression. Differentially expressed genes in CIDEA dysregulation groups through RNA-seq were selected and were enriched in the apelin and focal adhesion signaling pathways. Specifically, the Western blot and rescue assays found that focal adhesion, but not apelin, was the key signaling pathway in CIDEA regulating lipid deposition in goat intramuscular preadipocytes. In summary, this study reveals that CIDEA promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibits cell proliferation. This work clarifies the functional role and downstream signaling pathway of CIDEA in intramuscular fat deposition and provides theoretical support for improving meat quality by targeting key phenotype-related genes.

1. Introduction

Goat meat is favored by consumers as a high-quality meat food with high protein and low cholesterol [1]. Intramuscular fat (IMF), also known as marbling, plays a crucial role in the tenderness, juiciness, and flavor of meat products, determining the quality of meat [2,3]. The IMF content is controlled by adipocytes’ number and volume, which is regulated by the comprehensive effect of proliferation and adipogenesis processes of intramuscular preadipocytes. Thus, understanding the underlying molecular mechanism of preadipocytes’ proliferation and adipogenesis is essential to understand IMF deposition and, thereby, improve the quality of goat meat.

Cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA), a member of the CIDE gene family, is a lipid droplet-associated protein [4,5], mainly localized on the surface of lipid droplets and the endoplasmic reticulum [6,7]. CIDEA plays an important role in lipid droplet expansion and TAG accumulation [8,9]. In mice, CIDEA, induced by a high-fat diet (HFD), was highly expressed in brown adipocytes [10,11] and correlated with the development of hepatic steatosis [12,13]. High expression of CIDEA showed TAG accumulation in hepatocytes and maintained a healthy obese phenotype in the adipose tissue of transgenic mice [14,15]. Its deficiency resulted in the accumulation of smaller LDs and improved insulin sensitivity in brown adipocytes [6,10] and HFD-induced fatty liver [13], as well as increased whole-body energy expenditure in HFD-fed mice. However, the role and underlying molecular mechanism of CIDEA in regulating goat IMF deposition remain to be studied.

Apelin is a peptide hormone identified as the only endogenous ligand for the previously orphaned G protein-coupled APJ receptor [16]. In a previous study, apelin, as an adipokine, inhibited adipogenesis via the mitogen-activated protein kinase (MAPK) pathway [17]. In endothelial cells, apelin promotes cell proliferation by activating the downstream phosphoinositide-3 kinase (PI3K)/Akt signaling pathway [18]. Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes and regulates important cellular processes, including cell migration, proliferation, and survival [19]. By controlling the supply of precursors and the enzyme activity of proteins involved in lipid synthesis, FAK can affect lipid metabolism [20].

This study investigates the effect of CIDEA on the adipogenesis of goat intramuscular precursor adipocytes and explores the molecular mechanism of CIDEA inducing lipid deposition through the FAK signaling pathway. These data reveal the crucial role of CIDEA in the lipid deposition of goat intramuscular preadipocytes, which can help to thoroughly interpret the molecular mechanism of intramuscular fat deposition, thus providing a theoretical basis for the improvement of goat meat quality by molecular breeding.

2. Materials and Methods

2.1. Ethics Statement

All experimental exercises were isolated and approved by the Institutional Animal Care and Use Committee, Southwest Minzu University (Chengdu, China) (permit number: S2020-013). Tissue-sampling procedures were conducted in strict accordance with the state and institutional animal care and ARRIVE guidelines.

2.2. Isolation and Culture of Goat Intramuscular Preadipocytes

Purchased from Sichuan Jianyang Dageda Animal Husbandry Co., Ltd. (Jianyang, China), the 2-day-old Jianzhou big-eared goats were rapidly euthanized via carotid artery bleeding in the laboratory. The longissimus dorsi muscle tissues were collected from 3 male Jianzhou big-eared goats, 2 days old, in a sterile atmosphere, followed by washing with PBS containing 5% penicillin–streptomycin (Boster, PYG0016, Pleasanton, CA, USA) three times. The tissues were minced and then transferred into 50 mL centrifuge tubes. Then, type II collagenase (Sigma-Aldrich, C2-BIOC, St. Louis, MO, USA) was added for digestion at 37 °C for 1.5 h (shaking every 5 min), and digestion was terminated with complete culture medium. The digested tissue was filtered to remove the large undigested tissue. Then, the cell suspension was filtered with a 200-mesh (75 μm) sieve and centrifuged at 2000 rpm for 5 min to obtain the mixed cells. The red blood cell lysate (Boster, AR1118, CA, USA) was used to resuspend the precipitate, followed by standing for 5 min to remove the red cells. Subsequently, the cell suspension underwent centrifugation at 2000 revolutions per minute for a duration of 5 min, followed by resuspension in complete culture medium sourced from Gibco (C11330500BT, Beijing, China). The cells were seeded in a 25 cm² culture plate and cultured at 37 °C in 5% CO₂. After 2 h, the medium was replaced with fresh complete medium, and the attached cells were considered goat primary intramuscular preadipocytes. All subsequent experiments were performed in three biological replicates and three technical replicates.

The goat primary intramuscular preadipocytes were cultured with the complete culture medium containing 10% fetal bovine serum (12483012, Gibco, Carlsbad, CA, USA) and 90% DMEM/F12 (SH30023−01, Hyclone, Logan, UT, USA) supplemented with 100 U/mL penicillin–streptomycin (080092569, Harbin Pharmaceutical Group, Harbin, China). The medium was changed every two days during this process, and there were no significant changes in the adipocyte morphology. To promote adipogenesis, cells were cultured with an adipogenic medium consisting of complete medium and 50 μM oleic acid (112-80-1, Sigma, MO, USA) for 48 h.

In the FAK pathway inhibition assay, the FAK inhibitor was added at a concentration of 100 nM along with the oleic acid induction solution. The control group was an equal volume of DMSO, and the phenotyping examinations were conducted after adipogenic induction for 48 h.

2.3. Goat CIDEA Overexpression Vector Construction and siRNA Synthesis

Based on the CDS sequence of goat CIDEA in the NCBI database, the CDS region of the CIDEA gene was amplified and inserted into the pcDNA3.1 (+) plasmid, which was double-digested by EcoRI and HindIII, and named the CIDEA-OVER vector. Next, the recombinant plasmid was confirmed through enzyme digestion and DNA sequencing, with the empty pcDNA3.1 (+) plasmid serving as the negative control, designated as pcDNA3.1.

The siRNAs for goat CIDEA mRNA were designed and synthesized by Shanghai GenePharma company. siRNA-NC S: UUCUCCGAACGUGUCACGUTT; A: ACGUGACACGUUCGGAGAATT. siRNA-CIDEA393 S: CCACCAUGUACGAGAUGUATT; A: UACAUCUCGUACAUGGUGGTT.

To achieve the overexpression and interference of CIDEA, plasmids and siRNAs were transfected into goat primary intramuscular precursor adipocytes in a 6-well plate once the cells reached 80% confluence. These transfections were carried out using Lipofectamine™ 3000 transfection reagent (L3000015, Invitrogen, Carlsbad, CA, USA), following the manufacturer’s guidelines. In addition, 1 μg of plasmids or 120 μM of siRNAs was used for the transfection in each well of the 6-well plate in our research.

2.4. Oil Red O Staining and Triglyceride Determination

Two days post-transfection, the relative lipid droplet content was assessed using Oil Red O staining (Solarbio, G1262, Beijing, China). Briefly, cells were fixed with formaldehyde for a 30-min duration. Subsequently, the lipid droplets within the cells were stained with a filtered Oil Red O solution for 20 min following a PBS wash. After three additional PBS washes to remove excess stain, the lipid droplets were visualized and imaged under a microscope. The stained lipid droplets were then dissolved in isopropanol, and their absorbance was quantified at a wavelength of 510 nm.

For the detection of triglyceride content, the cells were rinsed three times with PBS and then incubated with 200 µL of lysis buffer for 10 min at room temperature. The supernatant was heated at 70 °C for 10 min, followed by centrifugation at 2000 rpm for 5 min. The resulting supernatant was then used for triglyceride quantification (Applygen, E1014-105, Beijing, China). The optical density (OD) value of triglycerides was measured at a wavelength of 550 nm. To normalize the triglyceride content, the protein concentration of each sample was determined using the BCA method (Thermo Fisher Scientific, 23225, Beijing, China).

2.5. Cell-Counting Kit-8 (CCK-8) Assay

Cells were seeded into a 96-well plate and transfected with CIDEA overexpression vectors or siRNAs and their controls individually. After 0, 24, 36, and 48 h of transfection, 10 μL of the CCK-8 reagent (AC11L054, Life-iLab, Shanghai, China) was added to each well. Then, the cells were incubated for 0.5 h at 37 °C. At last, the absorbance was measured using a microplate reader at a 450 nm wavelength.

2.6. Western Blot

Cellular proteins were extracted using RIPA buffer (Solarbio Tech Inc., Beijing, China) containing a protease inhibitor (04693132001, Roche, Mannheim, Germany) and a phosphatase inhibitor. The total proteins were then separated via SDS-PAGE electrophoresis and transferred onto PVDF membranes for abundance analysis. This was followed by occlusion using 5% BSA (4240GR100, Biofroxx, Einhausen, Germany), incubation of the primary antibody overnight at 4 °C, TBST washing of the PVDF membrane three times, and 1 h of room-temperature incubation of the secondary antibody. The primary antibodies utilized were anti-β-actin (1:6000, BM0627, BOSTER, Wuhan, China), anti-p-p38-MAPK (1:1000, 3285S, Cell Signaling Technology, Danvers, MA, USA), anti-p38-MAPK (1:1000, 8690S, Cell Signaling Technology), anti-p-FAK (1:1000, ab81298, Abcam, Cambridge, UK), anti-FAK (1:1000, #3285, Cell Signaling Technology), anti-p-AKT (1:2000, 4060, Cell Signaling Technology), and anti-AKT (1:1000, ab32505, Abcam). Goat anti-mouse IgG-HRP (1:6000, BA1050, Boster) and goat anti-rabbit IgG-HRP (1:6000, BA1054, Boster) served as the secondary antibodies. The target proteins were visualized using an enhanced chemiluminescence (ECL) detection system from Thermo (Waltham, MA, USA).

2.7. Reverse Transcription–Quantitative PCR (RT-qPCR)

Total RNA was extracted using RNAiso Plus (Takara, Cat. No. 9109), and its concentration and purity were measured. The RNA was then reverse-transcribed using a reverse transcription kit from Vazyme (Cat. No. R32301). RT-qPCR was performed with a Bio-Rad CFX96 PCR System, utilizing the Taq Pro Universal SYBR qPCR Master Mix from Vazyme (Cat. No. Q712-02, Nanjing, China) and gene-specific primers (Table S1). UXT served as the internal reference gene, and the relative expression levels were calculated according to the 2^−ΔΔCT method. To clarify the expression pattern of CIDEA in goat intramuscular precursor adipocytes, cellular RNA was extracted during the adipogenic induction period (days 0, 2, 4, 6, and 8), and the abundance of CIDEA was subsequently detected by RT-qPCR.

2.8. RNA Sequencing (RNA-seq)

After 48 h of oleic acid induction, total RNA was extracted using TRIzol (Takara, 9109, Beijing, China) reagent for RNA-seq analysis. The samples were named as the NO group (negative control for overexpression), the AO group (CIDEA overexpression), the AS group (CIDEA knockdown), and the NS group (CIDEA knockdown control). Samples with three replicates were used for high-throughput transcriptome sequencing (Shanghai OE Biotechnology Co, Ltd. Shanghai, China). The Illumina Novaseq 6000 sequencing platform was used to sequence the library and generate 150 bp paired-end reads with 100-300 cycles. DEsq2 was used to screen differentially expressed genes with p < 0.05. DEGs were subjected to GO and KEGG analysis.

2.9. Statistical Analysis

All experiments were carried out in three biological replicates and repeated three times. GraphPad Prism 9.0 was used for statistical analysis and plotting. Student’s t-test and one-way ANOVA were used to calculate the difference. p < 0.05 was considered significant, and p < 0.01 was highly significant.

3. Results

3.1. CIDEA Is Associated with Intramuscular Fat Deposition

Our previous RNA-seq data [21] and RNA abundance detected by RT-qPCR showed that the expression of CIDEA was upregulated in the longissimus dorsi muscle tissue of 24-month-old goats compared with that of 2-month-old goats (Figure 1A). To further investigate the influence of CIDEA on intramuscular adipogenesis, we examined the expression of CIDEA during the differentiation of intramuscular preadipocytes in goats. The findings indicated a gradual increase in the expression of CIDEA from day 0 to day 8 (Figure 1B), implying that CIDEA may play a pivotal role in the adipogenesis of intramuscular preadipocytes.

3.2. Overexpression of CIDEA Promotes Lipid Deposition in Goat Primary Intramuscular Preadipocytes

To elucidate the role of CIDEA in lipid deposition in goat intramuscular preadipocytes, we overexpressed CIDEA by transfecting the overexpression vector (CIDEA OVER). The results showed that the expression level of CIDEA was increased by about 23-fold (p < 0.01; Figure 2A). Lipid droplets and TAG contents were both increased after CIDEA overexpression (p < 0.01; Figure 2B–D). The CCK-8 assay revealed that cell viability was reduced in the CIDEA OVER group (p < 0.05; Figure 2E). Correspondingly, the mRNA abundances of transcription factors (PPARγ, C/EBPα, and SREBP1c), lipid droplet accumulation genes (PLIN1 and ADFP), triglyceride synthesis genes (DGAT1 and DGAT2), and fatty acid synthesis and transport genes (ACC, FASN, ACSL1, and FABP3) were increased after overexpressing CIDEA in intramuscular preadipocytes (Figure 2F–I). However, the mRNA expressions of lipolysis and β-oxidation genes (HSL, ACOX1, and CPT1B) and proliferation genes (CCND2 and CDK1) were both downregulated (Figure 2J,K).

3.3. Knockdown of CIDEA Inhibits Adipogenesis in Intramuscular Preadipocytes

Then, we performed siRNA knockdown of CIDEA in goat intramuscular preadipocytes, which resulted in a reduction of up to 82% in transcript levels (Figure 3A). Lipid droplets and TAG contents were both decreased in the CIDEA knockdown group (p < 0.01; Figure 3B–D). The CCK-8 assay showed that siRNA-mediated suppression increased the viability of intramuscular preadipocytes (p < 0.05; Figure 3E). Correspondingly, mRNA expressions of transcription factors (PPARγ, C/EBPα, and SREBP1c), lipid droplet accumulation genes (PLIN1, TIP47, and ADFP), triglyceride synthesis genes (GPAM, AGPAT6, and DGAT1), and fatty acid synthesis and transport genes (ACC, FASN, ACSL1, ACSS2, and FABP3) were all downregulated after knockdown of CIDEA in intramuscular preadipocytes (Figure 3F–I). Moreover, the mRNA abundances of lipolysis and β-oxidation genes (ATGL, HSL, ACOX1, CPT1A, and CPT1B) and proliferation genes (CDK1 and PCNA) were both upregulated (Figure 3J,K).

3.4. Screening and Analysis of Differentially Expressed Genes (DEGs) in Intramuscular Preadipocytes with Dysregulated CIDEA Expression

To elucidate the molecular mechanism by which CIDEA regulates lipid deposition in intramuscular preadipocytes, the transcriptional profiles of CIDEA OVER CIDEA-393 groups, together with their controls, were identified by RNA-seq. The RNA-sequencing data and their analysis are listed in Table S2. After CIDEA overexpression, we identified 134 differentially expressed genes (DEGs) (p < 0.05), of which 56 genes were upregulated and 78 genes were downregulated (Figure 4A). After CIDEA expression was reduced in goat intramuscular adipocytes, 1493 DEGs were identified (p < 0.05), of which 977 were upregulated and 516 were downregulated (Figure 4C). The heat map revealed that, despite significant differences between groups, the expression patterns were comparable within groups among samples, suggesting minimal variations between individual samples (Figure S2B,D). GO enrichment analysis showed that dysregulated expression of CIDEA DEGs was enriched in biological processes related to lipid metabolism, such as C-terminal protein lipidation, fatty acid omega-oxidation, lipid binding, cellular response to lipids, lipid transport involved in lipid storage, and other biological processes associated with lipid metabolism (Figure S2A,C). KEGG pathway analysis indicated that differential mRNAs were involved in apelin and focal adhesion signaling pathways (Figure 4B,D).

3.5. CIDEA Regulates Lipid Deposition in Goat Intramuscular Preadipocytes via Focal Adhesion Pathway

RNA-seq analysis showed that the apelin and focal adhesion pathways were enriched in both CIDEA overexpression and interference cells. This suggests that the effect of CIDEA on lipid deposition in goat preadipocytes may be mediated via these two pathways [22]. Therefore, we examined the abundances of the signaling proteins and their phosphorylated forms in these two pathways. The results showed that the abundance of p-FAK and the ratio of p-FAK/FAK were both elevated, while p-38, one key downstream signaling protein of the apelin pathway, exhibited no change (Figure 5A,B). In contrast, knockdown of CIDEA decreased the abundances of p-FAK and p-AKT, together with the ratios of p-FAK/FAK and p-AKT/AKT (Figure S4A,B). On this basis, we detected the mRNA expression of FAK and found that overexpression of CIDEA promoted FAK mRNA expression, while knockdown of CIDEA suppressed its expression (Figure S1A,B). We subsequently employed FAK inhibitors to investigate whether CIDEA regulates intramuscular adipogenesis via the focal adhesion pathway [23]. Our findings demonstrated that FAK inhibitors effectively suppressed the focal adhesion pathway (Figure S3A,B). The Oil red O staining results displayed that inhibition of FAK signaling could rescue the lipid droplet content increase induced by CIDEA overexpression (Figure 5C,D). The cellular triglyceride content change was consistent with the finding for the lipid droplet content (Figure 5E). Moreover, we found that inhibiting FAK activity further decreased the lipid droplet and TAG contents induced by CIDEA interference (Figure S4C,D).

4. Discussion

Previous research indicates that CIDEA is a pivotal player in both disease pathogenesis and lipid metabolism. Nonetheless, the precise function and regulatory mechanisms of CIDEA with regard to intramuscular fat accumulation in goats have yet to be fully understood. Based on our preliminary RNA-seq data, it was observed that the expression level of CIDEA was significantly elevated in the longissimus dorsi muscle tissue of 24-month-old goats compared with their 2-month-old counterparts. Consequently, the objective of this study was to explore the impact of CIDEA on lipid deposition within goat intramuscular preadipocytes.

Intramuscular fat is mainly determined by adipocyte number and adipocyte volume [24]. Mature adipocytes are unable to divide and differentiate, so IMF deposition is due to the comprehensive effect of progenitor adipocyte cell proliferation and differentiation. Our research shows that the expression of CIDEA is upregulated in the longissimus dorsi muscle of 24-month-old goats compared with 2-month-old goats. Since 2-month-old goats are in the juvenile stage, the accumulation of IMF has not yet begun. In contrast, 24-month-old goats have reached adulthood, during which IMF gradually accumulates, leading to a significant increase in its content. This finding aligns with the increase in IMF content reported in our previous study [21]. Despite the different detection methods, the accuracy and depth of the results vary accordingly. Moreover, our study observed a gradual upregulation of CIDEA expression from day 0 to day 8 during adipogenic differentiation in goat intramuscular tissue. Analogously, the expression level of CIDEC, a homolog of CIDEA, was also increased during the differentiation process of human preadipocytes [25]. These findings suggest that CIDEA may play a beneficial role in the adipogenesis of intramuscular preadipocytes in goats.

In our study, we observed that the expression levels of several lipid metabolism-related genes were closely associated with CIDEA. DGAT1 and DGAT2 are rate-limiting enzymes required for triglyceride synthesis [26,27], and our research showed that CIDEA promotes the expression of DGAT1 and DGAT2 in goat intramuscular adipocytes. ATGL and HSL are enzymes that facilitate the breakdown of triglycerides into free fatty acids [28,29]. Subsequently, the free fatty acids are transported to mitochondria by CPT1s for β-oxidation [30], or they undergo oxidation by ACOX1 within peroxisomes [31]. Our research also supports the finding that expression of CIDEA leads to the downregulation of a series of lipolysis (ATGL, HSL, and ACOX1) and β-oxidation (CPT1A and CPT1B) related genes. FASN and ACC, as the key rate-limiting enzymes in de novo fatty acid synthesis, play a crucial role in lipid production. The upregulation of CIDEA expression enhances the expression of these enzymes. Consequently, CIDEA promotes lipid deposition by facilitating fatty acid uptake and triglyceride synthesis.

Transcriptional regulatory factors play a significant role in the synthesis of lipids. Previous research has indicated that CIDEA acts as an activator of CCAAT/enhancer-binding protein (C/EBP) in the mammary glands of lactating mice [32]. Additionally, the promoter region of CIDEA contains a sterol regulatory element (SRE) and peroxisome proliferator-activated receptor (PPAR) elements that can be bound by SREBP1c and PPARγ [33]. Interestingly, we observed that the expressions of SREBP1c and PPARγ were affected by CIDEA in goat intramuscular preadipocytes (p < 0.01), suggesting a potential interaction between SREBP1c, PPARγ, and CIDEA. In dairy goats, C/EBPα enhances triacylglycerol synthesis by modulating the activity of the PPARG promoter [34]. Our observations are in good agreement with the known effects of C/EBPα on PPARγ. Other studies have shown that CIDEA expression inhibits AMP-activated protein kinase (AMPK) activity, which enhances PPARγ expression, thereby increasing triglyceride content, and that CIDEA expression promotes the nuclear translocation of SREBP1c [35]. Furthermore, expression of PPARγ and SREBP1c can directly activate the transcription of FASN and ACC, thereby promoting the formation of lipids [36,37]. On the basis of these results, we hypothesized that CIDEA regulated lipid metabolism through PPARγ and SREBP1c, thereby regulating their downstream genes (ACC and FASN) expression.

To further unravel the potential molecular mechanism by which CIDEA affects IMF deposition in goats, we performed RNA-seq on intramuscular preadipocytes after overexpression and knockdown of CIDEA. Interestingly, KEGG pathway enrichment analysis revealed obvious enrichments of apelin and focal adhesion pathways after CIDEA dysregulation. It is known that the apelin pathway enhances insulin sensitivity, promotes glucose uptake and utilization, inhibits fatty acid synthesis, and stimulates fatty acid oxidation by activating downstream signaling pathways such as PI3K/Akt and MAPK [38,39,40]. The PI3K-Akt signaling pathway also serves as the downstream signaling of the focal adhesion pathway [23]. In our study, we found that CIDEA activated the FAK and AKT signaling proteins, two key signaling proteins in the focal adhesion pathway, but not the p38 signaling protein, a downstream signaling protein of the MAPK pathway, which is consistent with previous findings in mice [41,42]. Intriguingly, our data revealed that interfering with CIDEA inhibited the activation of AKT, while overexpression of CIDEA did not influence the signaling protein, which needed further exploration. On this basis, we inquired into whether CIDEA regulates lipid deposition through the focal adhesion pathway. The FAK inhibitor was used to inhibit FAK signaling. The results suggested that the inhibition of FAK rescued the lipid droplet content increase induced by CIDEA overexpression and further decreased the lipid droplet content in CIDEA-interfering cells. Unfortunately, due to species limitations, we did not find the antibody matching the goat CIDEA protein to characterize CIDEA expression at the protein level.

In summary, this study reveals that CIDEA promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibits cell proliferation. These works clarify the functional role and downstream signaling pathway of CIDEA in intramuscular fat deposition and provide theoretical support for improving meat quality through manipulating phenotype-related key genes.

5. Conclusions

Our study provides evidence for the role of CIDEA in intramuscular fat deposition in goats. CIDEA promotes adipogenesis through the focal adhesion pathway and inhibits cell proliferation in goat intramuscular preadipocytes. These findings contribute to our understanding of the comprehensive effect of CIDEA on intramuscular fat deposition and lay the theoretical foundation for the development of goat molecular breeding technology.