Abstract

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.

   Keywords: CIDEA, intramuscular fat, RNA-seq, focal adhesion pathway,
   fat deposition

1. Introduction

   Goat meat is favored by consumers as a high-quality meat food with high
   protein and low cholesterol [[42]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
   [[43]2,[44]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 [[45]4,[46]5], mainly localized on the
   surface of lipid droplets and the endoplasmic reticulum [[47]6,[48]7].
   CIDEA plays an important role in lipid droplet expansion and TAG
   accumulation [[49]8,[50]9]. In mice, CIDEA, induced by a high-fat diet
   (HFD), was highly expressed in brown adipocytes [[51]10,[52]11] and
   correlated with the development of hepatic steatosis [[53]12,[54]13].
   High expression of CIDEA showed TAG accumulation in hepatocytes and
   maintained a healthy obese phenotype in the adipose tissue of
   transgenic mice [[55]14,[56]15]. Its deficiency resulted in the
   accumulation of smaller LDs and improved insulin sensitivity in brown
   adipocytes [[57]6,[58]10] and HFD-induced fatty liver [[59]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 [[60]16]. In
   a previous study, apelin, as an adipokine, inhibited adipogenesis via
   the mitogen-activated protein kinase (MAPK) pathway [[61]17]. In
   endothelial cells, apelin promotes cell proliferation by activating the
   downstream phosphoinositide-3 kinase (PI3K)/Akt signaling pathway
   [[62]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 [[63]19]. By controlling the supply of
   precursors and the enzyme activity of proteins involved in lipid
   synthesis, FAK can affect lipid metabolism [[64]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 cm^2 culture plate and cultured at 37 °C in 5% CO[2].
   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. [65]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 ([66]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 [[67]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 ([68]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 ([69]Figure 1B), implying
   that CIDEA may play a pivotal role in the adipogenesis of intramuscular
   preadipocytes.

Figure 1.

   [70]Figure 1
   [71]Open in a new tab

   CIDEA is associated with intramuscular fat deposition. (A) Expression
   levels of CIDEA in longissimus dorsi muscle at different developmental
   stages (2 months and 24 months). (B) Expression pattern of CIDEA during
   differentiation of goat preadipocytes. Distinct lowercase letters
   signify statistically significant differences (p < 0.05).

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;
   [72]Figure 2A). Lipid droplets and TAG contents were both increased
   after CIDEA overexpression (p < 0.01; [73]Figure 2B–D). The CCK-8 assay
   revealed that cell viability was reduced in the CIDEA OVER group (p <
   0.05; [74]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 ([75]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
   ([76]Figure 2J,K).

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   Overexpression of CIDEA promotes lipid deposition in goat intramuscular
   preadipocytes. (A) Overexpression efficiency detection. With UXT as the
   internal reference gene and the negative control as reference. (B,C)
   Oil red O staining and quantification of lipid droplets after CIDEA
   overexpression. (D) Relative triglyceride content in
   CIDEA-overexpressing cells. (E) Cell viability detection after
   overexpression of CIDEA. (F–K) Effects of CIDEA overexpression on the
   expression levels of genes related to lipid metabolism and cell
   proliferation. * p < 0.05; ** p < 0.01.

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 ([79]Figure 3A). Lipid droplets and TAG contents were both
   decreased in the CIDEA knockdown group (p < 0.01; [80]Figure 3B–D). The
   CCK-8 assay showed that siRNA-mediated suppression increased the
   viability of intramuscular preadipocytes (p < 0.05; [81]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 ([82]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
   ([83]Figure 3J,K).

Figure 3.

   [84]Figure 3
   [85]Open in a new tab

   Knockdown of CIDEA inhibits adipogenesis of intramuscular
   preadipocytes. (A) Knockout efficiency detection after transfection of
   CIDEA-393 (siCIDEA) in preadipocytes. (B,C) Oil red O staining and
   quantification of lipid droplets content after knockdown with CIDEA.
   (D) Relative triglyceride content detection in CIDEA knockdown cells.
   (E) Cell viability detection by CCK-8 assay kit after CIDEA knockdown.
   (F–K) Effect of CIDEA knockdown on expression levels of genes related
   to lipid metabolism and cell proliferation. * p < 0.05; ** p < 0.01.

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 [86]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 ([87]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 ([88]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 ([89]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 ([90]Figure
   S2A,C). KEGG pathway analysis indicated that differential mRNAs were
   involved in apelin and focal adhesion signaling pathways ([91]Figure
   4B,D).

Figure 4.

   [92]Figure 4
   [93]Open in a new tab

   Screening and analysis of differentially expressed genes (DEGs) with
   dysregulated CIDEA expression. (A) Volcano plot of DEGs in
   CIDEA-overexpressing precursor adipocytes. Red dots indicate
   significant upregulation of genes; blue dots indicate significant
   downregulation of genes. (B) KEGG pathway analysis of DEGs in CIDEA
   overexpression group. (C) Volcano plot of DEGs in CIDEA knockdown
   precursor adipocytes. Red dots indicate significant upregulation of
   genes; blue dots indicate significant downregulation of genes. (D) KEGG
   pathway analysis of DEGs in CIDEA knockdown group. The red border
   indicates that the focal adhesion signaling pathway was enriched in the
   differential genes analysis of transcriptome sequencing results
   following both overexpression and knockdown of CIDEA. Subsequent
   experiments confirmed the activation of this pathway.

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 [[94]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 ([95]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 ([96]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 ([97]Figure S1A,B). We subsequently
   employed FAK inhibitors to investigate whether CIDEA regulates
   intramuscular adipogenesis via the focal adhesion pathway [[98]23]. Our
   findings demonstrated that FAK inhibitors effectively suppressed the
   focal adhesion pathway ([99]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
   ([100]Figure 5C,D). The cellular triglyceride content change was
   consistent with the finding for the lipid droplet content ([101]Figure
   5E). Moreover, we found that inhibiting FAK activity further decreased
   the lipid droplet and TAG contents induced by CIDEA interference
   ([102]Figure S4C,D).

Figure 5.

   [103]Figure 5
   [104]Open in a new tab

   CIDEA regulates lipid deposition in goat intramuscular preadipocytes
   via focal adhesion pathway. (A) Detecting protein levels of p-p38, p38,
   p-AKT, AKT, p-FAK, and FAK after overexpression of CIDEA using Western
   blot. (B) Determining ratios of p-FAK/FAK, p-AKT/AKT, and p-p38/p38
   upon overexpressing CIDEA. (C) Lipid droplet content detection after
   co-transfection of FAK inhibitor or DMSO and CIDEA OVER or pcDNA3.1 by
   Oil red O staining. (D) Determination of relative OD value of lipid
   droplets extracted after Oil red O staining. (E) Intracellular
   triglyceride content detection after co-transfection of FAK inhibitor
   or DMSO and CIDEA OVER or pcDNA3.1. * p < 0.05.

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 [[105]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
   [[106]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 [[107]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
   [[108]26,[109]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 [[110]28,[111]29]. Subsequently, the free fatty acids
   are transported to mitochondria by CPT1s for β-oxidation [[112]30], or
   they undergo oxidation by ACOX1 within peroxisomes [[113]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 [[114]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 [[115]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 [[116]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 [[117]35]. Furthermore, expression of PPARγ and SREBP1c can
   directly activate the transcription of FASN and ACC, thereby promoting
   the formation of lipids [[118]36,[119]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
   [[120]38,[121]39,[122]40]. The PI3K-Akt signaling pathway also serves
   as the downstream signaling of the focal adhesion pathway [[123]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
   [[124]41,[125]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.

Acknowledgments