CIDEA promotes lipid deposition through focal adhesion pathway in goat intramuscular preadipocytes

CIDEA promotes lipid deposition through focal adhesion pathway in goat intramuscular preadipocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CIDEA promotes lipid deposition through focal adhesion pathway in goat intramuscular preadipocytes Peng Shao, Qi Li, Yu Liao, Yong Wang, Yaqiu Lin, Hua Xiang, Zhanyu Du, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5661803/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Intramuscular fat (IMF) content determines the quality of goat meat, and is strongly associated with the number and volume of adipocytes, which is regulated by the comprehensive effect of proliferation and adipogenesis of intramuscular preadipocytes. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) proteins has emerged as lipid droplets (LDs)-related proteins, implying the important roles in lipid homeostasis. However, the mechanism through which CIDEA , one member of CIDE family, regulates intramuscular fat deposition remains unclear. To address this, we dysregulated CIDEA in intramuscular preadipocytes and resolved the effect and mechanism of CIEDA in adipogenesis through RT-PCR, Western blot, triglyceride and LDs determinations, CCK-8 and RNA-seq. It was found that CIDEA increased LDs and triglyceride contents and inhibited cell proliferation. Lipid metabolism-related genes PPARγ , C/EBPα , SREBP1c , PLIN1 , TIP47 , ADFP , DGAT1 , ACC , FASN , ACSL1 , FABP3 were upregulated after CIDEA overexpression. Moreover, lipolysis and β oxidation genes HSL , ACOX1 , CPT1B and proliferation marker genes CDK1 were upregulated. Differentially expressed genes in RNA-seq results were selected and enriched in the apelin and focal adhesion signaling pathways. Specifically, CIDEA regulated the activation of focal adhesion kinase and AKT signaling proteins, but not p38 signaling. To this end, we did the rescue assay and found that suppressing focal adhesion kinase signaling pathway with PF573228 reversed the lipid droplets and triglyceride contents increase induced by CIDEA overexpressing and further decreased their contents in CIDEA interfering group. In summary, this study reveals that CIDEA promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibiting the 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. Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Molecular biology CIDEA intramuscular fat RNA-seq focal adhesion pathway fat deposition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Goat meat, as a superior health food for its delicate texture, low fat and high-quality protein, have been gaining popularity among modern meat consumers [ 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 the 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 decipher the IMF deposition, and thereby improve the quality of goat meat. Cell death-inducing DNA fragmentation factor alpha-like effector A ( CIDEA ), one member of CIDE family genes, is a lipid droplet-associated protein [ 4 , 5 ], mainly localized on the surface of lipid droplets and on the endoplasmic reticulum [ 6 , 7 ]. CIDEA plays an important role in lipid droplets expanding and TAG accumulation [ 8 , 9 ]. In mice, CIDEA , induced by 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 an increased triacylglycerol (TAG) accumulation in hepatocytes and maintained a healthy obese phenotype in adipose tissue of transgenic mice [ 14 , 15 ]. Its deficiency resulted in the accumulation of smaller LDs and the improvement of 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 ]. In this study, we dysregulated the CIDEA expression and found that CIDEA promoted adipogenesis and inhibited cell proliferation of goat intramuscular preadipocytes. Then, apelin and focal adhesion pathways were enriched through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) after CIDEA overexpression and downregulation. On the basis, Rescue assay toward FAK signaling protein revealed that CIDEA -promoting lipid deposition was partly through FAK signaling pathway. These data reveal the crucial role of CIDEA in lipid deposition of goat intramuscular preadipocytes, which can help to thoroughly interpret the molecular mechanism of intramuscular fat deposition, thus providing the 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, revised in June 2004. Tissue sampling procedures are conducted in strict accordance with 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 of 2 days old in the sterile atmosphere, following by washing with PBS containing 5% penicillin-streptomycin (Boster, PYG0016, CA, USA) for 3 times. the tissues were minced and then transferred into a 50 mL centrifuge tubes. Then, typeⅡ collagenase (Sigma-Aldrich, C2-BIOC, St. Louis, MO, USA) was added for digestion in 37 ℃ for 1.5 h (shaking every 5 min) and termination with an equal volume of complete culture medium. The digested tissue was filtered to remove the large undigested tissue. Then, 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, following by standing for 5 min to remove the red cell. Subsequently, the cell suspension underwent centrifugation at 2,000 revolutions per minute for a duration of 5 minutes, 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 ℃ in 5% CO2. After 2 hours incubation, discarding the cell culture medium and adding a new complete medium to culture the purified goat primary intramuscular preadipocytes. The goat primary intramuscular preadipocytes were cultured with the complete culture medium containing 10% fetal bovine serum (12483012, Gibco, USA) and 90% DMEM/F12 (SH30023 − 01, Hyclone, USA) supplemented with 100 U/mL penicillin/streptomycin (080092569, Harbin Pharmaceutical Group, China). To promote adipogenesis, cells were cultured with an adipogenic medium consisted of complete medium and 50 µM oleic acid (112-80-1, Sigma, USA) for 48 h. 2.3. Goat CIDEA overexpression vector construction and siRNA synthesis According to the CDS region sequence of goat CIDEA on NCBI database. the CDS region of the CIDEA gene was amplified and inserted into thepcDNA3.1 (+) plasmid which was double digested by EcoRІ and HindIII, and named 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 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 (Invitrogen), following the manufacturer's guidelines.. In addition, 1 µg plasmids or 120 µM siRNAs were used for the transfection in each well of 6-wll 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-minute duration. Subsequently, the lipid droplets within the cells were stained with a filtered Oil Red O solution for 20 minutes 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 twice or thrice with PBS and then incubated with 200 µL of lysis buffer for 10 minutes at room temperature. The supernatant was heated at 70°C for 10 minutes, followed by centrifugation at 2,000 rpm for 5 minutes. 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 hours transfection, 10 µL of CCK-8 reagent (AC11L054, Life-iLab, Shanghai, China) was added to each well, then, the cells were incubated for 0.5 h at 37 ℃. At last, the absorbance was measured using microplate reader at 450 nm wavelength. 2.6. Western Blot Cellular proteins were extracted using RIPA buffer (Solarbio Tech Inc., Beijing, China) containing protease inhibitor (04693132001, Roche, Mannheim, Germany) and phosphatase inhibitor. The total proteins were then separated via SDS-PAGE electrophoresis and transferred onto PVDF membranes for abundance analysis. 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 assessed. The RNA was then reverse transcribed using a reverse transcription kit from Vazyme (Cat. No. R32301). qPCR was performed on 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 (listed in Table S1 ). UXT served as the internal reference gene, and the relative expression levels were calculated according to the 2 -ΔΔCT method. 2.8. RNA Sequencing(RNA-seq) Total RNA was extracted using the Trizol (Takara, 9109, Beijing, China) method. Samples were named as NO group (control), AO group ( CIDEA overexpression), AS group ( CIDEA knockdown) and NS group (control). Samples with three replicates were used for high-throughput transcriptome sequencing (Shanghai OE Biotechnology Co, Ltd). DEsq2 was used to screen differentially expressed genes with P < 0.05. DEGs were subjected to the GO and KEGG analysis. 2.9. Statistical analysis Data were showed as mean ± SEM. All experiments were carried out in three biological replicates and repeated three times. GraphPad Prism 9.0 was used for statistical analysis and plotting. The student’s t-test and one-way ANOVA were used to calculate the difference. P < 0.05 was considered as significant, and P < 0.01 was considered as extremely 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 to that of 2-month-old goats (Figure 1 A). To further investigate the influence of CIDEA on intramuscular adipogenesis, we examined the expression of CIDEA during differentiation of intramuscular preadipocytes in goats. The findings indicated a gradual increase in the expression of CIDEA from day 0 to day 8 (Fig. 1 B), implying that CIDEA might have a pivotal function 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 through transfecting the overexpression vector (CIDEA OVER). The results showed that the expression level of CIDEA was increased by about 23-fold ( P < 0.01, Fig. 2 A). Lipid droplets and TAG contents were both increased after CIDEA overexpression ( P < 0.01, Fig. 2 B-D). CCK-8 assay revealed that cell viability was reduced in the CIDEA OVER group ( P < 0.05, Fig. 2 E). Correspondingly, mRNA abundances of transcription factor ( PPARγ , C/EBPα , SREBP1c ), lipid droplet accumulation gene ( PLIN1 , ADFP ), triglyceride synthesis gene ( DGAT1 , DGAT2 ), fatty acid synthesis and transport gene ( ACC , FASN , ACSL1 , FABP3 ) were increased after overexpressing CIDEA in intramuscular preadipocytes (Fig. 2 F-I). However, the mRNA expression of lipolysis and β-oxidation gene ( HSL , ACOX1 , CPT1B ) and proliferation gene ( CCND2 , CDK1 ) were both downregulated (Fig. 2 J-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 (Fig. 3 A). Lipid droplets and TAG contents were both decreased in CIDEA knockdown group ( P < 0.01, Fig. 3 B-D). CCK-8 assay exhibited that siRNA-mediated suppression increased the viability of intramuscular preadipocytes ( P < 0.05, Fig. 3 E). Correspondingly, mRNA expressions of transcription factors ( PPARγ , C/EBPα , SREBP1c ), lipid droplet accumulation genes ( PLIN1 , TIP47 , ADFP ), triglyceride synthesis genes ( GPAM , AGPAT6 , DGAT1 ), fatty acid synthesis and transport genes ( ACC , FASN , ACSL1 , ACSS2 , FABP3 ) were all downregulated after overexpressing CIDEA in intramuscular preadipocytes (Fig. 3 F-I). Moreover, the mRNA abundances of lipolysis and β oxidation genes ( ATGL , HSL , ACOX1 , CPT1A , CPT1B ) and proliferation genes ( CDK1 , PCNA ) were both upregulated (Fig. 3 J, K). 3.4. Screening and analysis of differentially expressed genes (DEGs) with dysregulated CIDEA expression To elucidate the molecular mechanism by which CIDEA regulates lipid deposition in intramuscular preadipocytes, the transcriptional profiles of pcDNA3.1 and CIDEA OVER groups and si-NC and CIDEA-393 groups were identified by RNA-seq. RNA-sequencing data and its analysis were listed in the table S2. After CIDEA overexpression, we identified 134 differentially expressed genes (DEGs) ( P < 0.05), of which 56 genes were up-regulated and 78 genes were down-regulated (Fig. 4 A, Table S3). After CIDEA expression was reduced in goat intramuscular adipocytes, 1493 DEGs were identified ( P < 0.05), of which 977 were up-regulated and 516 were down-regulated (Fig. 4 C, Table S6). The heat map revealed that, despite significant differences among groups, the expression patterns were comparable within groups among samples, suggesting minimal variations between individual samples (Figure S2B, S2D). GO enrichment analysis showed that dysregulated expression of CIDEA DEGs were enriched in biological processes related to lipid metabolism, such as C-terminal protein lipidation, fatty acid omega-oxidation, lipid binding, cellular response to lipid, lipid transport involved in lipid storage and other biological process are associated with lipid metabolism (Figure S2A, 2C, Table S4, S7). KEGG pathway analysis indicated that differential mRNAs were involved in apelin and focal adhesion signaling pathways (Fig. 4 B, 4 D, Table S5, S8). 3.5. CIDEA regulates lipid deposition in goat intramuscular preadipocytes via focal adhesion pathway RNA-seq analysis showed that the apelin and focal adhesion pathway was 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 signaling protein and its phosphorylated form in these two pathways, result exhibited that the abundance of p-FAK and ratio of p-FAK/FAK were both elevated while p-38, one key downstream signaling protein of apelin pathway, was downregulated (Fig. 5 A, B). In contrast, knockdown of CIDEA decreased the abundances of p-FAK and p-AKT, together with ratios of p-FAK/FAK and p-AKT/AKT(Figure S3A-B). On the basis, we detected the mRNA expression of FAK, and found that overexpression of CIDEA promoted FAK mRNA expression, while interference with CIDEA suppressed its expression (Figure S1 A, B). Then, we used PF-573228, a specific FAK inhibitor [ 23 ], to explore whether CIDEA regulating intramuscular adipogenesis was through FAK pathway. Oil red O staining results displayed that inhibition of FAK signaling could recue the lipid droplets content increase induced by CIDEA overexpression (Fig. 5 C, D). Cellular triglyceride content change was consistent with the finding in lipid droplet content (Fig. 5 E). Moreover, we found that inhibiting FAK activity further decreased the lipid droplets and TAG contents induced by CIDEA interference (Figure S2C-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 in regards 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 is significantly elevated in the longissimus dorsi muscle tissue of 24-month-old goats when compared to their 2-month-old counterparts. Consequently, the objective of this study is to explore the impact of CIDEA on lipid deposition within goat intramuscular preadipocytes. Intramuscular fat is mainly determined by adipocyte number and adipocyte volume [ 23 ]. 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 compared to 2-month-old goats, the expression of CIDEA is upregulated in the longissimus dorsi muscle of 24-month-old goats, which corresponds to the increase in IMF content shown in our other 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 homologue of CIDEA , was also increased during the differentiation process of human preadipocytes. [ 24 ]. These findings predict 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 [ 25 , 26 ], and our research has shown 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. [ 27 , 28 ]. Subsequently, the free fatty acids are transported to mitochondria by CPT1s for β-oxidation [ 29 ], or they undergo oxidation by ACOX1 within peroxisome [ 30 ]. Our research also supports the finding that expression of CIDEA leads to 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. Transcription regulatory factors play a significant role in the synthesis of lipids. Previous research has indicated that CIDEA act as an activator of CCAAT/enhancer-binding protein (C/EBP) in the mammary glands of lactating mice [ 31 ]. 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 [ 32 ]. 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 [ 33 ]. 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 [ 34 ]. Furthermore, expression of PPARγ and SREBP1c can directly activate the transcription of FASN and ACC , thereby promoting the formation of lipids [ 35 , 36 ]. On the basis of these results, we hypothesized that CIDEA regulated lipid metabolism through PPARγ and SREBP1c , thereby regulating their downstream genes ( ACC , FASN ) expression. To further unravel the potential molecular mechanism by which CIDEA affects IMF deposition in goat, we performed RNA-seq on intramuscular preadipocytes after overexpressing and interfering CIDEA . Interestingly, KEGG pathway enrichment analysis revealed obvious enrichments of apelin and focal adhesion pathways after CIDEA dysregulation. It is known that 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 [ 37 – 39 ]. PI3K-Akt signaling pathway is also served as the downstream signaling of focal adhesion pathway [ 40 ]. In our study, we found that CIDEA activated the FAK and AKT signaling proteins, two key signaling proteins in focal adhesion pathway, but not the p38 signaling protein, a downstream signaling protein of 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 the basis, we inquired whether CIDEA regulating lipid deposition was through focal adhesion pathway, PF-537228 was used to inhibit the FAK signaling, results suggested that inhibition of FAK rescued the lipid droplets content increase induced by CIDEA overexpressing and ulteriorly decreased the lipid droplets content in CIDEA interfering cells. Unfortunately, due to species limitations, we have not found the antibody matching goat CIDEA protein to characterize CIDEA expression at the protein level. In conclusion, our study provides evidence for the role of CIDEA in intramuscular fat deposition in goats. CIDEA promotes adipogenesis and inhibits cell proliferation in intramuscular preadipocytes and the adipogenesis is achieved through the focal adhesion pathway. These findings contribute to our understanding the functional role of CIDEA in intramuscular fat deposition and lay the theoretical foundation for the developing of goat molecular breeding technology. 5. Conclusions In conclusion, 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 c ontribute to our understanding the comprehensive effect of CIDEA on intramuscular fat deposition and lay the theoretical foundation for the developing of goat molecular breeding technology. Declarations Supplementary Materials : Supplementary Material 1 contains documents Figure S1 (mRNA abundances detection of FAK in CIDEA dysregulated intramuscular preadipocytes), Figure S2 (Screening and analysis of differentially expressed genes (DEGs) with dysregulated CIDEA expression), and Figure S3 ( CIDEA regulates lipid deposition in goat intramuscular preadipocytes via focal adhesion pathway). Supplementary Material 2 contains documents table S1 (Primers for quantitative real-time PCR (RT-qPCR)), table S2 (Sequencing data quality control), table S3 (Overexpression ALL_FPKM), table S4 (Overexpression GO enrichment analysis) ,table S5 (Overexpression KEGG enrichment analysis), table S6 (knockdown ALL_FPKM), table S7 (knockdown GO enrichment analysis),and table S8 (knockdown KEGG enrichment analysis). Author Contributions: “Conceptualization, P.S. and L.H.; methodology, J.Z.; software, Q.L.; validation, P.S. and Y.L.; formal analysis, Y.L.; investigation, Y.W.; resources, L.H.; data curation, Y.L.; writing—original draft preparation, P.S.; writing—review and editing, H.X.; visualization, Z.D.; supervision, C.Z.; project administration, L.H.; funding acquisition, L.H. All authors have read and agreed to the published version of the manuscript.”. Funding : This work was supported by Sichuan Science and Technology Program (Chengdu, China, 2021YFYZ0003, 2024NSFSC0029), National Natural Science Foundation of China (Beijing, China, 32302702), Zhejiang Science and Technology Program (Hangzhou, China, 2022C04017), the Fundamental Research Funds for the Central Universities, Southwest Minzu University (Beijing, China, ZYN2023097) and the Scientific and Technological Innovation Team for Qinghai-Tibetan Plateau Research in Southwest Minzu University（2024CXTD13）. Institutional Review Board Statement: All experimental procedures were approved by the Institutional Animal Care and Use Committee, Southwest Minzu University (Chengdu, China). Permit number: S2020-013, revised in June 2004. Informed Consent Statement: Not applicable. Data Availability Statement : This RNA-seq data are deposited in the NCBI Sequence Read Archive (SRA) and Bio project number PRJNA995405. 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Tang, Y. et al. Expression Variation of CPT1A Induces Lipid Reconstruction in Goat Intramuscular Precursor Adipocytes. International Journal of Molecular Sciences 24, (17). (2023). Li, F. et al. Cell death-inducing DFF45-like effector, a lipid droplet-associated protein, might be involved in the differentiation of human adipocytes. FEBS J. 277 (20), 4173–4183 (2010). Wang, L., Xu, S., Zhou, M., Hu, H. & Li, J. The role of DGAT1 and DGAT2 in tumor progression via fatty acid metabolism: A comprehensive review. Int. J. Biol. Macromol. 278 (Pt 3), 134835 (2024). Yang, C. et al. Diacylglycerol acyltransferase 2 promotes the adipogenesis of intramuscular preadipocytes in goat. Animal Biotechnol. 34 (7), 2376–2383 (2022). Brejchova, K. et al. Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides. Proc. Natl. Acad. Sci. U.S.A. 118 , (2). (2021). Zheng, Y. et al. 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CIDEA Regulates De Novo Fatty Acid Synthesis in Bovine Mammary Epithelial Cells by Targeting the AMPK/PPARγ Axis and Regulating SREBP1. J. Agric. Food Chem. 70 (36), 11324–11335 (2022). Li, J. et al. CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways. J. Hepatol. 63 (6), 1378–1389 (2015). Ho, T. C. et al. C., Effects of In Utero PFOS Exposure on Epigenetics and Metabolism in Mouse Fetal Livers. Environ. Sci. Technol. 57 (40), 14892–14903 (2023). Lee, D. K., George, S. R. & O'Dowd, B. F. Unravelling the roles of the apelin system: prospective therapeutic applications in heart failure and obesity. Trends Pharmacol. Sci. 27 (4), 190–194 (2006). Yao, F. et al. Apelin-13 impedes foam cell formation by activating Class III PI3K/Beclin-1-mediated autophagic pathway. Biochem. Biophys. Res. Commun. 466 (4), 637–643 (2015). Xie, F. et al. Apelin-13 promotes cardiomyocyte hypertrophy via PI3K-Akt-ERK1/2-p70S6K and PI3K-induced autophagy. Acta Biochim. Biophys. Sin. 47 (12), 969–980 (2015). Xiong, Y. et al. LKB1 Regulates Goat Intramuscular Adipogenesis Through Focal Adhesion Pathway. Front. Physiol. 12. (2021). Chen, H. J. et al. High-Fat-Diet-Induced Extracellular Matrix Deposition Regulates Integrin-FAK Signals in Adipose Tissue to Promote Obesity. Mol. Nutr. Food Res. 66 , (7), e2101088. (2022). Lin, X. et al. lncRNA ITGB8-AS1 functions as a ceRNA to promote colorectal cancer growth and migration through integrin-mediated focal adhesion signaling. Mol. therapy: J. Am. Soc. Gene Therapy . 30 (2), 688–702 (2022). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5661803","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":394966225,"identity":"b8cc823c-df89-4fa3-bc3a-b9d0f9b653f1","order_by":0,"name":"Peng Shao","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Shao","suffix":""},{"id":394966226,"identity":"a72b89ed-acbc-4f59-bedd-8a264cc79b64","order_by":1,"name":"Qi Li","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Li","suffix":""},{"id":394966227,"identity":"d41c61dd-ed2a-46cf-845b-b71cdd8b3df8","order_by":2,"name":"Yu Liao","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liao","suffix":""},{"id":394966228,"identity":"644c4fd1-a9d6-44bd-890a-86f52838ca52","order_by":3,"name":"Yong Wang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Wang","suffix":""},{"id":394966229,"identity":"279f97dd-403f-4df4-b6ef-c76f75c3e835","order_by":4,"name":"Yaqiu Lin","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yaqiu","middleName":"","lastName":"Lin","suffix":""},{"id":394966230,"identity":"449c3b70-3ffe-4cd3-90db-7c110ed6a656","order_by":5,"name":"Hua Xiang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Xiang","suffix":""},{"id":394966231,"identity":"190588f8-2469-4847-9f70-83e01bf9234f","order_by":6,"name":"Zhanyu Du","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Zhanyu","middleName":"","lastName":"Du","suffix":""},{"id":394966232,"identity":"f15c543e-517f-409c-89de-b3396caf9cf4","order_by":7,"name":"Changhui Zhang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Changhui","middleName":"","lastName":"Zhang","suffix":""},{"id":394966233,"identity":"20dc5be8-5078-492f-8352-9b63beb64292","order_by":8,"name":"Jiangjiang Zhu","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jiangjiang","middleName":"","lastName":"Zhu","suffix":""},{"id":394966234,"identity":"c3094aa1-9b7e-4b64-b1b2-53791bc5f86a","order_by":9,"name":"Lian Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACfmb2AwcSKmzq+6ECjA2EtEi29yQ++HAmjXFmA7FaDM4cMDac2XaYccMBom2ZkZAmzcPGzGx8/oyZdAGDjeyGA8zPHuDTwi+ReEyah4eNzexGjpn0DIY04w0H2MwNCNsiwcNjdoN3mzQPw+HEDQd42CTw+uVGgpk0j4GEhHH/WZCW/0RoAXl/RoKBgQFDLkjLAcJaIIF8ICFB4kb+Z2seg2TjmYfZzPBqAUdl4r//Cfz9xxJv81TYyfYdb36GVwu6O4GYmQT1o2AUjIJRMAqwAwBekUkTYw5mzQAAAABJRU5ErkJggg==","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Lian","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-12-17 12:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5661803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5661803/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72583942,"identity":"e860ae07-aaca-4746-86f6-1e82e6aa41e1","added_by":"auto","created_at":"2024-12-30 05:58:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217071,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCIDEA \u003c/em\u003eis associated with intramuscular fat deposition.\u003c/p\u003e\n\u003cp\u003eA.The expression levels of \u003cem\u003eCIDEA \u003c/em\u003ein the longissimus dorsi muscle at different developmental stages (2 months and 24 months). B. Expression pattern of \u003cem\u003eCIDEA \u003c/em\u003eduring differentiation of goat preadipocytes. Distinct lowercase letters signify statistically significant differences. (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/f8efd1b65c449f53a4fbd427.png"},{"id":72583943,"identity":"643e725e-bcee-498c-823c-5a6fd50d25a6","added_by":"auto","created_at":"2024-12-30 05:58:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1007142,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eCIDEA \u003c/em\u003epromotes lipid deposition in goat intramuscular preadipocytes.\u003c/p\u003e\n\u003cp\u003eA. 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 \u003cem\u003eCIDEA \u003c/em\u003eoverexpression. D. Relative triglyceride content in \u003cem\u003eCIDEA \u003c/em\u003eoverexpressing cells. E. Cell viability detection after overexpression of \u003cem\u003eCIDEA\u003c/em\u003e. F-K. Effects of \u003cem\u003eCIDEA \u003c/em\u003eoverexpression on the expression levels of genes related to lipid metabolism and cell proliferation. Data are presented as mean ± SEM. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/e29bd87d48cb226189a531f1.png"},{"id":72585665,"identity":"4ff18449-b0d5-440e-951b-ada388a64e0c","added_by":"auto","created_at":"2024-12-30 06:14:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1008614,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of \u003cem\u003eCIDEA \u003c/em\u003einhibits adipogenesis of intramuscular preadipocytes.\u003c/p\u003e\n\u003cp\u003eA.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 \u003cem\u003eCIDEA\u003c/em\u003e. D. Relative triglyceride content detection in \u003cem\u003eCIDEA \u003c/em\u003eknockdown cells. E. Cell viability detection by CCK-8 assay kit after \u003cem\u003eCIDEA \u003c/em\u003eknockdown. F-K. Effect of \u003cem\u003eCIDEA \u003c/em\u003eknockdown on the expression levels of genes related to lipid metabolism and cell proliferation\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/2ee065c19fb432d5af1dd8e4.png"},{"id":72583947,"identity":"9d8249db-2bfa-44d3-ac07-c0079ef312ee","added_by":"auto","created_at":"2024-12-30 05:58:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2146675,"visible":true,"origin":"","legend":"\u003cp\u003eScreening and analysis of differentially expressed genes (DEGs) with dysregulated \u003cem\u003eCIDEA \u003c/em\u003eexpression.\u003c/p\u003e\n\u003cp\u003eA. Volcano plot of DEGs in \u003cem\u003eCIDEA \u003c/em\u003eoverexpressing precursor adipocytes, red dots indicate significant up-regulation of genes, blue dots indicate significant down-regulation of genes. B. KEGG pathway analysis of DEGs in \u003cem\u003eCIDEA \u003c/em\u003eoverexpression group. C. Volcano plot of DEGs in \u003cem\u003eCIDEA \u003c/em\u003eknockdown precursor adipocytes, red dots indicate significant upregulation of genes, blue dots indicate significant downregulation of genes. D. KEGG pathway analysis of DEGs in \u003cem\u003eCIDEA \u003c/em\u003eknockdown group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/48b1b18745cc63357f95c490.png"},{"id":72584574,"identity":"ac9241c4-b7f1-47a2-b656-88dbe4d3cff6","added_by":"auto","created_at":"2024-12-30 06:06:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1446504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCIDEA \u003c/em\u003eregulates lipid deposition in goat intramuscular preadipocytes via focal adhesion pathway.\u003c/p\u003e\n\u003cp\u003eA. Detecting protein levels of p-p38, p38, p-AKT, AKT, p-FAK, and FAK after overexpression of \u003cem\u003eCIDEA \u003c/em\u003eusing Western Blot. B. Determining the ratios of p-FAK/FAK, p-AKT/AKT and p-p38/p38 upon overexpressing \u003cem\u003eCIDEA\u003c/em\u003e. C. Lipid Droplets content detection after co-transfection of PF-573228 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 contents detection after co-transfection of PF-573228 or DMSO and CIDEA OVER or pcDNA3.1. Data are presented as mean ± SEM for three independent experiments. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/73eb9faca69182326446f69a.png"},{"id":81676564,"identity":"d4c78ed1-3ed8-4460-8420-2d280add6d0f","added_by":"auto","created_at":"2025-04-30 07:38:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6870343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/6cb81cdd-79c8-4ab9-bc60-d66e63d27473.pdf"},{"id":72583944,"identity":"00cc2acf-ac25-4f12-91db-2fe7aa62001a","added_by":"auto","created_at":"2024-12-30 05:58:55","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4974477,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.rar","url":"https://assets-eu.researchsquare.com/files/rs-5661803/v1/144046fb2350ac1f6b005cd0.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"CIDEA promotes lipid deposition through focal adhesion pathway in goat intramuscular preadipocytes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGoat meat, as a superior health food for its delicate texture, low fat and high-quality protein, have been gaining popularity among modern meat consumers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. 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[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The IMF content is controlled by the 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 decipher the IMF deposition, and thereby improve the quality of goat meat.\u003c/p\u003e \u003cp\u003eCell death-inducing DNA fragmentation factor alpha-like effector A (\u003cem\u003eCIDEA\u003c/em\u003e), one member of CIDE family genes, is a lipid droplet-associated protein [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], mainly localized on the surface of lipid droplets and on the endoplasmic reticulum [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eCIDEA\u003c/em\u003e plays an important role in lipid droplets expanding and TAG accumulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In mice, \u003cem\u003eCIDEA\u003c/em\u003e, induced by High-fat Diet (HFD), was highly expressed in brown adipocytes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and correlated with the development of hepatic steatosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. High expression of \u003cem\u003eCIDEA\u003c/em\u003e showed an increased triacylglycerol (TAG) accumulation in hepatocytes and maintained a healthy obese phenotype in adipose tissue of transgenic mice [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Its deficiency resulted in the accumulation of smaller LDs and the improvement of insulin sensitivity in brown adipocytes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and HFD-induced fatty liver [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], as well as increased whole-body energy expenditure in HFD-fed mice. However, the role and underlying molecular mechanism of \u003cem\u003eCIDEA\u003c/em\u003e in regulating goat IMF deposition remain to be studied.\u003c/p\u003e \u003cp\u003eApelin is a peptide hormone identified as the only endogenous ligand for the previously orphaned G protein-coupled APJ receptor [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In a previous study, Apelin as an adipokine inhibited adipogenesis via the mitogen-activated protein kinase (MAPK) pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In endothelial cells, Apelin promotes cell proliferation by activating the downstream phosphoinositide-3 kinase (PI3K)/Akt signaling pathway [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. By controlling the supply of precursors and the enzyme activity of proteins involved in lipid synthesis, FAK can affect lipid metabolism [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we dysregulated the \u003cem\u003eCIDEA\u003c/em\u003e expression and found that \u003cem\u003eCIDEA\u003c/em\u003e promoted adipogenesis and inhibited cell proliferation of goat intramuscular preadipocytes. Then, apelin and focal adhesion pathways were enriched through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) after \u003cem\u003eCIDEA\u003c/em\u003e overexpression and downregulation. On the basis, Rescue assay toward FAK signaling protein revealed that \u003cem\u003eCIDEA\u003c/em\u003e-promoting lipid deposition was partly through FAK signaling pathway. These data reveal the crucial role of \u003cem\u003eCIDEA\u003c/em\u003e in lipid deposition of goat intramuscular preadipocytes, which can help to thoroughly interpret the molecular mechanism of intramuscular fat deposition, thus providing the theoretical basis for the improvement of goat meat quality by molecular breeding.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics Statement\u003c/h2\u003e \u003cp\u003e All experimental exercises were isolated and approved by the Institutional Animal Care and Use Committee, Southwest Minzu University (Chengdu, China). Permit number: S2020-013, revised in June 2004. Tissue sampling procedures are conducted in strict accordance with state and institutional animal care and ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation and culture of goat intramuscular preadipocytes\u003c/h2\u003e \u003cp\u003ePurchased 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 of 2 days old in the sterile atmosphere, following by washing with PBS containing 5% penicillin-streptomycin (Boster, PYG0016, CA, USA) for 3 times. the tissues were minced and then transferred into a 50 mL centrifuge tubes. Then, typeⅡ collagenase (Sigma-Aldrich, C2-BIOC, St. Louis, MO, USA) was added for digestion in 37 ℃ for 1.5 h (shaking every 5 min) and termination with an equal volume of complete culture medium. The digested tissue was filtered to remove the large undigested tissue. Then, cell suspension was filtered with a 200-mesh (75 \u0026micro;m) 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, following by standing for 5 min to remove the red cell. Subsequently, the cell suspension underwent centrifugation at 2,000 revolutions per minute for a duration of 5 minutes, 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 ℃ in 5% CO2. After 2 hours incubation, discarding the cell culture medium and adding a new complete medium to culture the purified goat primary intramuscular preadipocytes.\u003c/p\u003e \u003cp\u003eThe goat primary intramuscular preadipocytes were cultured with the complete culture medium containing 10% fetal bovine serum (12483012, Gibco, USA) and 90% DMEM/F12 (SH30023\u0026thinsp;\u0026minus;\u0026thinsp;01, Hyclone, USA) supplemented with 100 U/mL penicillin/streptomycin (080092569, Harbin Pharmaceutical Group, China). To promote adipogenesis, cells were cultured with an adipogenic medium consisted of complete medium and 50 \u0026micro;M oleic acid (112-80-1, Sigma, USA) for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Goat \u003cem\u003eCIDEA\u003c/em\u003e overexpression vector construction and siRNA synthesis\u003c/h2\u003e \u003cp\u003eAccording to the CDS region sequence of goat \u003cem\u003eCIDEA\u003c/em\u003e on NCBI database. the CDS region of the \u003cem\u003eCIDEA\u003c/em\u003e gene was amplified and inserted into thepcDNA3.1 (+) plasmid which was double digested by EcoRІ and HindIII, and named 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.\u003c/p\u003e \u003cp\u003eThe siRNAs for goat \u003cem\u003eCIDEA\u003c/em\u003e mRNA were designed and synthesized by Shanghai GenePharma company. siRNA-NC S: UUCUCCGAACGUGUCACGUTT, A: ACGUGACACGUUCGGAGAATT. siRNA-CIDEA393 S: CCACCAUGUACGAGAUGUATT, A: UACAUCUCGUACAUGGUGGTT.\u003c/p\u003e \u003cp\u003eTo achieve overexpression and interference of \u003cem\u003eCIDEA\u003c/em\u003e, 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\u0026trade; 3000 transfection reagent (Invitrogen), following the manufacturer's guidelines.. In addition, 1 \u0026micro;g plasmids or 120 \u0026micro;M siRNAs were used for the transfection in each well of 6-wll plate in our research.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Oil red O staining and triglyceride determination\u003c/h2\u003e \u003cp\u003eTwo 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-minute duration. Subsequently, the lipid droplets within the cells were stained with a filtered Oil Red O solution for 20 minutes 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.\u003c/p\u003e \u003cp\u003eFor the detection of triglyceride content, the cells were rinsed twice or thrice with PBS and then incubated with 200 \u0026micro;L of lysis buffer for 10 minutes at room temperature. The supernatant was heated at 70\u0026deg;C for 10 minutes, followed by centrifugation at 2,000 rpm for 5 minutes. 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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell Counting Kit-8 (CCK-8) Assay\u003c/h2\u003e \u003cp\u003eCells were seeded into a 96-well plate and transfected with \u003cem\u003eCIDEA\u003c/em\u003e overexpression vectors or siRNAs, and their controls individually. after 0, 24, 36 and 48 hours transfection, 10 \u0026micro;L of CCK-8 reagent (AC11L054, Life-iLab, Shanghai, China) was added to each well, then, the cells were incubated for 0.5 h at 37 ℃. At last, the absorbance was measured using microplate reader at 450 nm wavelength.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western Blot\u003c/h2\u003e \u003cp\u003eCellular proteins were extracted using RIPA buffer (Solarbio Tech Inc., Beijing, China) containing protease inhibitor (04693132001, Roche, Mannheim, Germany) and phosphatase inhibitor. The total proteins were then separated via SDS-PAGE electrophoresis and transferred onto PVDF membranes for abundance analysis. 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)..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Reverse Transcription Quantitative PCR (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using RNAiso Plus (Takara, Cat. No. 9109), and its concentration and purity were assessed. The RNA was then reverse transcribed using a reverse transcription kit from Vazyme (Cat. No. R32301). qPCR was performed on 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 (listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). UXT served as the internal reference gene, and the relative expression levels were calculated according to the 2\u003csup\u003e-ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. RNA Sequencing(RNA-seq)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the Trizol (Takara, 9109, Beijing, China) method. Samples were named as NO group (control), AO group (\u003cem\u003eCIDEA\u003c/em\u003e overexpression), AS group (\u003cem\u003eCIDEA\u003c/em\u003e knockdown) and NS group (control). Samples with three replicates were used for high-throughput transcriptome sequencing (Shanghai OE Biotechnology Co, Ltd). DEsq2 was used to screen differentially expressed genes with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. DEGs were subjected to the GO and KEGG analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were showed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. All experiments were carried out in three biological replicates and repeated three times. GraphPad Prism 9.0 was used for statistical analysis and plotting. The student\u0026rsquo;s t-test and one-way ANOVA were used to calculate the difference. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant, and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered as extremely significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cem\u003eCIDEA\u003c/em\u003e is associated with intramuscular fat deposition\u003c/h2\u003e \u003cp\u003eOur previous RNA-seq data [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and RNA abundance detected by RT-qPCR showed that the expression of \u003cem\u003eCIDEA\u003c/em\u003e was upregulated in the longissimus dorsi muscle tissue of 24-month-old goats compared to that of 2-month-old goats (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further investigate the influence of \u003cem\u003eCIDEA\u003c/em\u003e on intramuscular adipogenesis, we examined the expression of \u003cem\u003eCIDEA\u003c/em\u003e during differentiation of intramuscular preadipocytes in goats. The findings indicated a gradual increase in the expression of \u003cem\u003eCIDEA\u003c/em\u003e from day 0 to day 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), implying that \u003cem\u003eCIDEA\u003c/em\u003e might have a pivotal function in the adipogenesis of intramuscular preadipocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Overexpression of \u003cem\u003eCIDEA\u003c/em\u003e promotes lipid deposition in goat primary intramuscular preadipocytes\u003c/h2\u003e \u003cp\u003eTo elucidate the role of \u003cem\u003eCIDEA\u003c/em\u003e in lipid deposition in goat intramuscular preadipocytes, we overexpressed \u003cem\u003eCIDEA\u003c/em\u003e through transfecting the overexpression vector (CIDEA OVER). The results showed that the expression level of \u003cem\u003eCIDEA\u003c/em\u003e was increased by about 23-fold (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Lipid droplets and TAG contents were both increased after \u003cem\u003eCIDEA\u003c/em\u003e overexpression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). CCK-8 assay revealed that cell viability was reduced in the CIDEA OVER group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Correspondingly, mRNA abundances of transcription factor (\u003cem\u003ePPARγ\u003c/em\u003e, \u003cem\u003eC/EBPα\u003c/em\u003e, \u003cem\u003eSREBP1c\u003c/em\u003e), lipid droplet accumulation gene (\u003cem\u003ePLIN1\u003c/em\u003e, \u003cem\u003eADFP\u003c/em\u003e), triglyceride synthesis gene (\u003cem\u003eDGAT1\u003c/em\u003e, \u003cem\u003eDGAT2\u003c/em\u003e), fatty acid synthesis and transport gene (\u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e, \u003cem\u003eACSL1\u003c/em\u003e, \u003cem\u003eFABP3\u003c/em\u003e) were increased after overexpressing \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular preadipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-I). However, the mRNA expression of lipolysis and β-oxidation gene (\u003cem\u003eHSL\u003c/em\u003e, \u003cem\u003eACOX1\u003c/em\u003e, \u003cem\u003eCPT1B\u003c/em\u003e) and proliferation gene (\u003cem\u003eCCND2\u003c/em\u003e, \u003cem\u003eCDK1\u003c/em\u003e) were both downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Knockdown of \u003cem\u003eCIDEA\u003c/em\u003e inhibits adipogenesis in intramuscular preadipocytes\u003c/h2\u003e \u003cp\u003eThen, we performed siRNA knockdown of \u003cem\u003eCIDEA\u003c/em\u003e in goat intramuscular preadipocytes, which resulted in a reduction of up to 82% in transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Lipid droplets and TAG contents were both decreased in \u003cem\u003eCIDEA\u003c/em\u003e knockdown group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). CCK-8 assay exhibited that siRNA-mediated suppression increased the viability of intramuscular preadipocytes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Correspondingly, mRNA expressions of transcription factors (\u003cem\u003ePPARγ\u003c/em\u003e, \u003cem\u003eC/EBPα\u003c/em\u003e, \u003cem\u003eSREBP1c\u003c/em\u003e), lipid droplet accumulation genes (\u003cem\u003ePLIN1\u003c/em\u003e, \u003cem\u003eTIP47\u003c/em\u003e, \u003cem\u003eADFP\u003c/em\u003e), triglyceride synthesis genes (\u003cem\u003eGPAM\u003c/em\u003e, \u003cem\u003eAGPAT6\u003c/em\u003e, \u003cem\u003eDGAT1\u003c/em\u003e), fatty acid synthesis and transport genes (\u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e, \u003cem\u003eACSL1\u003c/em\u003e, \u003cem\u003eACSS2\u003c/em\u003e, \u003cem\u003eFABP3\u003c/em\u003e) were all downregulated after overexpressing \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular preadipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-I). Moreover, the mRNA abundances of lipolysis and β oxidation genes (\u003cem\u003eATGL\u003c/em\u003e, \u003cem\u003eHSL\u003c/em\u003e, \u003cem\u003eACOX1\u003c/em\u003e, \u003cem\u003eCPT1A\u003c/em\u003e, \u003cem\u003eCPT1B\u003c/em\u003e) and proliferation genes (\u003cem\u003eCDK1\u003c/em\u003e, \u003cem\u003ePCNA\u003c/em\u003e) were both upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, K).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Screening and analysis of differentially expressed genes (DEGs) with dysregulated \u003cem\u003eCIDEA\u003c/em\u003e expression\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism by which \u003cem\u003eCIDEA\u003c/em\u003e regulates lipid deposition in intramuscular preadipocytes, the transcriptional profiles of pcDNA3.1 and CIDEA OVER groups and si-NC and CIDEA-393 groups were identified by RNA-seq.\u0026nbsp;RNA-sequencing data and its analysis were listed in the table S2. After \u003cem\u003eCIDEA\u003c/em\u003e overexpression, we identified 134 differentially expressed genes (DEGs) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), of which 56 genes were up-regulated and 78 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table S3). After \u003cem\u003eCIDEA\u003c/em\u003e expression was reduced in goat intramuscular adipocytes, 1493 DEGs were identified (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), of which 977 were up-regulated and 516 were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Table S6). The heat map revealed that, despite significant differences among groups, the expression patterns were comparable within groups among samples, suggesting minimal variations between individual samples (Figure S2B, S2D). GO enrichment analysis showed that dysregulated expression of \u003cem\u003eCIDEA\u003c/em\u003e DEGs were enriched in biological processes related to lipid metabolism, such as C-terminal protein lipidation, fatty acid omega-oxidation, lipid binding, cellular response to lipid, lipid transport involved in lipid storage and other biological process are associated with lipid metabolism (Figure S2A, 2C, Table S4, S7). KEGG pathway analysis indicated that differential mRNAs were involved in apelin and focal adhesion signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Table S5, S8).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. \u003cem\u003eCIDEA\u003c/em\u003e regulates lipid deposition in goat intramuscular preadipocytes via focal adhesion pathway\u003c/h2\u003e \u003cp\u003eRNA-seq analysis showed that the apelin and focal adhesion pathway was enriched in both \u003cem\u003eCIDEA\u003c/em\u003e overexpression and interference cells. This suggests that the effect of \u003cem\u003eCIDEA\u003c/em\u003e on lipid deposition in goat preadipocytes may be mediated via these two pathways [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, we examined the abundances of signaling protein and its phosphorylated form in these two pathways, result exhibited that the abundance of p-FAK and ratio of p-FAK/FAK were both elevated while p-38, one key downstream signaling protein of apelin pathway, was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). In contrast, knockdown of \u003cem\u003eCIDEA\u003c/em\u003e decreased the abundances of p-FAK and p-AKT, together with ratios of p-FAK/FAK and p-AKT/AKT(Figure S3A-B). On the basis, we detected the mRNA expression of FAK, and found that overexpression of \u003cem\u003eCIDEA\u003c/em\u003e promoted FAK mRNA expression, while interference with CIDEA suppressed its expression (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). Then, we used PF-573228, a specific FAK inhibitor [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], to explore whether \u003cem\u003eCIDEA\u003c/em\u003e regulating intramuscular adipogenesis was through FAK pathway. Oil red O staining results displayed that inhibition of FAK signaling could recue the lipid droplets content increase induced by \u003cem\u003eCIDEA\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Cellular triglyceride content change was consistent with the finding in lipid droplet content (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Moreover, we found that inhibiting FAK activity further decreased the lipid droplets and TAG contents induced by \u003cem\u003eCIDEA\u003c/em\u003e interference (Figure S2C-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePrevious research indicates that \u003cem\u003eCIDEA\u003c/em\u003e is a pivotal player in both disease pathogenesis and lipid metabolism. Nonetheless, the precise function and regulatory mechanisms of \u003cem\u003eCIDEA\u003c/em\u003e in regards 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 \u003cem\u003eCIDEA\u003c/em\u003e is significantly elevated in the longissimus dorsi muscle tissue of 24-month-old goats when compared to their 2-month-old counterparts. Consequently, the objective of this study is to explore the impact of \u003cem\u003eCIDEA\u003c/em\u003e on lipid deposition within goat intramuscular preadipocytes.\u003c/p\u003e \u003cp\u003eIntramuscular fat is mainly determined by adipocyte number and adipocyte volume [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. 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 compared to 2-month-old goats, the expression of \u003cem\u003eCIDEA\u003c/em\u003e is upregulated in the longissimus dorsi muscle of 24-month-old goats, which corresponds to the increase in IMF content shown in our other study [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], despite the different detection methods, the accuracy and depth of the results vary accordingly. Moreover, our study observed a gradual upregulation of \u003cem\u003eCIDEA\u003c/em\u003e expression from day 0 to day 8 during adipogenic differentiation in goat intramuscular tissue. Analogously, the expression level of \u003cem\u003eCIDEC\u003c/em\u003e, a homologue of \u003cem\u003eCIDEA\u003c/em\u003e, was also increased during the differentiation process of human preadipocytes. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These findings predict that \u003cem\u003eCIDEA\u003c/em\u003e may play a beneficial role in the adipogenesis of intramuscular preadipocytes in goats.\u003c/p\u003e \u003cp\u003eIn our study, we observed that the expression levels of several lipid metabolism-related genes were closely associated with \u003cem\u003eCIDEA\u003c/em\u003e. \u003cem\u003eDGAT1\u003c/em\u003e and \u003cem\u003eDGAT2\u003c/em\u003e are rate-limiting enzymes required for triglyceride synthesis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and our research has shown that \u003cem\u003eCIDEA\u003c/em\u003e promotes the expression of \u003cem\u003eDGAT1\u003c/em\u003e and \u003cem\u003eDGAT2\u003c/em\u003e in goat intramuscular adipocytes. \u003cem\u003eATGL\u003c/em\u003e and \u003cem\u003eHSL\u003c/em\u003e are enzymes that facilitate the breakdown of triglycerides into free fatty acids. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Subsequently, the free fatty acids are transported to mitochondria by CPT1s for β-oxidation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], or they undergo oxidation by \u003cem\u003eACOX1\u003c/em\u003e within peroxisome [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our research also supports the finding that expression of \u003cem\u003eCIDEA\u003c/em\u003e leads to downregulation of a series of lipolysis (\u003cem\u003eATGL\u003c/em\u003e, \u003cem\u003eHSL\u003c/em\u003e, and \u003cem\u003eACOX1\u003c/em\u003e)and β-oxidation (\u003cem\u003eCPT1A\u003c/em\u003e and \u003cem\u003eCPT1B\u003c/em\u003e) related genes. \u003cem\u003eFASN\u003c/em\u003e and \u003cem\u003eACC\u003c/em\u003e, as the key rate-limiting enzymes in de novo fatty acid synthesis, play a crucial role in lipid production. The upregulation of \u003cem\u003eCIDEA\u003c/em\u003e expression enhances the expression of these enzymes. Consequently, \u003cem\u003eCIDEA\u003c/em\u003e promotes lipid deposition by facilitating fatty acid uptake and triglyceride synthesis.\u003c/p\u003e \u003cp\u003eTranscription regulatory factors play a significant role in the synthesis of lipids. Previous research has indicated that \u003cem\u003eCIDEA\u003c/em\u003e act as an activator of CCAAT/enhancer-binding protein (C/EBP) in the mammary glands of lactating mice [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Additionally, the promoter region of \u003cem\u003eCIDEA\u003c/em\u003e contains a sterol regulatory element (SRE) and peroxisome proliferator-activated receptor (PPAR) elements that can be bound by \u003cem\u003eSREBP1c\u003c/em\u003e and \u003cem\u003ePPARγ\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Interestingly, we observed that the expressions of \u003cem\u003eSREBP1c\u003c/em\u003e and \u003cem\u003ePPARγ\u003c/em\u003e were affected by \u003cem\u003eCIDEA\u003c/em\u003e in goat intramuscular preadipocytes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting a potential interaction between \u003cem\u003eSREBP1c\u003c/em\u003e, \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eCIDEA\u003c/em\u003e. In dairy goats, \u003cem\u003eC/EBPα\u003c/em\u003e enhances triacylglycerol synthesis by modulating the activity of the \u003cem\u003ePPARG\u003c/em\u003e promoter [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our observations are in good agreement with the known effects of \u003cem\u003eC/EBPα\u003c/em\u003e on \u003cem\u003ePPARγ\u003c/em\u003e. Other studies have shown that \u003cem\u003eCIDEA\u003c/em\u003e expression inhibits AMP-activated protein kinase (AMPK) activity, which enhances \u003cem\u003ePPARγ\u003c/em\u003e expression, thereby increasing triglyceride content, and that \u003cem\u003eCIDEA\u003c/em\u003e expression promotes the nuclear translocation of \u003cem\u003eSREBP1c\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, expression of \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eSREBP1c\u003c/em\u003e can directly activate the transcription of \u003cem\u003eFASN\u003c/em\u003e and \u003cem\u003eACC\u003c/em\u003e, thereby promoting the formation of lipids [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. On the basis of these results, we hypothesized that \u003cem\u003eCIDEA\u003c/em\u003e regulated lipid metabolism through \u003cem\u003ePPARγ\u003c/em\u003e and \u003cem\u003eSREBP1c\u003c/em\u003e, thereby regulating their downstream genes (\u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e) expression.\u003c/p\u003e \u003cp\u003eTo further unravel the potential molecular mechanism by which \u003cem\u003eCIDEA\u003c/em\u003e affects IMF deposition in goat, we performed RNA-seq on intramuscular preadipocytes after overexpressing and interfering \u003cem\u003eCIDEA\u003c/em\u003e. Interestingly, KEGG pathway enrichment analysis revealed obvious enrichments of apelin and focal adhesion pathways after \u003cem\u003eCIDEA\u003c/em\u003e dysregulation. It is known that 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 [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. PI3K-Akt signaling pathway is also served as the downstream signaling of focal adhesion pathway [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In our study, we found that \u003cem\u003eCIDEA\u003c/em\u003e activated the FAK and AKT signaling proteins, two key signaling proteins in focal adhesion pathway, but not the p38 signaling protein, a downstream signaling protein of MAPK pathway, which is consistent with previous findings in mice [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Intriguingly, our data revealed that interfering with \u003cem\u003eCIDEA\u003c/em\u003e inhibited the activation of AKT, while overexpression of \u003cem\u003eCIDEA\u003c/em\u003e did not influence the signaling protein, which needed further exploration. On the basis, we inquired whether \u003cem\u003eCIDEA\u003c/em\u003e regulating lipid deposition was through focal adhesion pathway, PF-537228 was used to inhibit the FAK signaling, results suggested that inhibition of FAK rescued the lipid droplets content increase induced by \u003cem\u003eCIDEA\u003c/em\u003e overexpressing and ulteriorly decreased the lipid droplets content in \u003cem\u003eCIDEA\u003c/em\u003e interfering cells. Unfortunately, due to species limitations, we have not found the antibody matching goat \u003cem\u003eCIDEA\u003c/em\u003e protein to characterize \u003cem\u003eCIDEA\u003c/em\u003e expression at the protein level.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides evidence for the role of \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular fat deposition in goats. \u003cem\u003eCIDEA\u003c/em\u003e promotes adipogenesis and inhibits cell proliferation in intramuscular preadipocytes and the adipogenesis is achieved through the focal adhesion pathway. These findings contribute to our understanding the functional role of \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular fat deposition and lay the theoretical foundation for the developing of goat molecular breeding technology.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, our study provides evidence for the role of \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular fat deposition in goats. \u003cem\u003eCIDEA\u003c/em\u003e promotes adipogenesis through the focal adhesion pathway, and inhibits cell proliferation in goat intramuscular preadipocytes. These findings c ontribute to our understanding the comprehensive effect of \u003cem\u003eCIDEA\u003c/em\u003e on intramuscular fat deposition and lay the theoretical foundation for the developing of goat molecular breeding technology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eSupplementary Material 1 contains documents Figure S1 (mRNA abundances detection of FAK in \u003cem\u003eCIDEA\u0026nbsp;\u003c/em\u003edysregulated intramuscular preadipocytes), Figure S2 (Screening and analysis of differentially expressed genes (DEGs) with dysregulated \u003cem\u003eCIDEA\u0026nbsp;\u003c/em\u003eexpression), and Figure S3 (\u003cem\u003eCIDEA\u0026nbsp;\u003c/em\u003eregulates lipid deposition in goat intramuscular preadipocytes via focal adhesion pathway). Supplementary Material 2 contains documents table S1 (Primers for quantitative real-time PCR (RT-qPCR)), table S2 (Sequencing data quality control), table S3 (Overexpression ALL_FPKM), table S4 (Overexpression GO enrichment analysis) ,table S5 (Overexpression KEGG enrichment analysis), table S6 (knockdown ALL_FPKM), table S7 (knockdown GO enrichment analysis),and table S8 (knockdown KEGG enrichment analysis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026ldquo;Conceptualization, P.S. and L.H.; methodology, J.Z.; software, Q.L.; validation, P.S. and Y.L.; formal analysis, Y.L.; investigation, Y.W.; resources, L.H.; data curation, Y.L.; writing\u0026mdash;original draft preparation, P.S.; writing\u0026mdash;review and editing, H.X.; visualization, Z.D.; supervision, C.Z.; project administration, L.H.; funding acquisition, L.H. All authors have read and agreed to the published version of the manuscript.\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e This work was supported by Sichuan Science and Technology Program (Chengdu, China, 2021YFYZ0003, 2024NSFSC0029), National Natural Science Foundation of China (Beijing, China, 32302702), Zhejiang Science and Technology Program (Hangzhou, China, 2022C04017), the Fundamental Research Funds for the Central Universities, Southwest Minzu University (Beijing, China, ZYN2023097) and the Scientific and Technological Innovation Team for Qinghai-Tibetan Plateau Research in Southwest Minzu University（2024CXTD13）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eAll experimental procedures were approved by the Institutional Animal Care and Use Committee, Southwest Minzu University (Chengdu, China). Permit number: S2020-013, revised in June 2004.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis RNA-seq data are deposited in the NCBI Sequence Read Archive (SRA) and Bio project number PRJNA995405.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Thanks to Shanghai OE Biotechnology Co, Ltd for providing sequencing service.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKumar, P. et al. In-vitro meat: a promising solution for sustainability of meat sector. \u003cem\u003eJ. Anim. 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Gene Therapy\u003c/em\u003e. \u003cb\u003e30\u003c/b\u003e (2), 688\u0026ndash;702 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CIDEA, intramuscular fat, RNA-seq, focal adhesion pathway, fat deposition","lastPublishedDoi":"10.21203/rs.3.rs-5661803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5661803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntramuscular fat (IMF) content determines the quality of goat meat, and is strongly associated with the number and volume of adipocytes, which is regulated by the comprehensive effect of proliferation and adipogenesis of intramuscular preadipocytes. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) proteins has emerged as lipid droplets (LDs)-related proteins, implying the important roles in lipid homeostasis. However, the mechanism through which \u003cem\u003eCIDEA\u003c/em\u003e, one member of CIDE family, regulates intramuscular fat deposition remains unclear. To address this, we dysregulated \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular preadipocytes and resolved the effect and mechanism of \u003cem\u003eCIEDA\u003c/em\u003e in adipogenesis through RT-PCR, Western blot, triglyceride and LDs determinations, CCK-8 and RNA-seq.\u0026nbsp;It was found that \u003cem\u003eCIDEA\u003c/em\u003e increased LDs and triglyceride contents and inhibited cell proliferation. Lipid metabolism-related genes \u003cem\u003ePPARγ\u003c/em\u003e, \u003cem\u003eC/EBPα\u003c/em\u003e, \u003cem\u003eSREBP1c\u003c/em\u003e, \u003cem\u003ePLIN1\u003c/em\u003e, \u003cem\u003eTIP47\u003c/em\u003e, \u003cem\u003eADFP\u003c/em\u003e, \u003cem\u003eDGAT1\u003c/em\u003e, \u003cem\u003eACC\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e, \u003cem\u003eACSL1\u003c/em\u003e, \u003cem\u003eFABP3\u003c/em\u003e were upregulated after \u003cem\u003eCIDEA\u003c/em\u003e overexpression. Moreover, lipolysis and β oxidation genes \u003cem\u003eHSL\u003c/em\u003e, \u003cem\u003eACOX1\u003c/em\u003e, \u003cem\u003eCPT1B\u003c/em\u003e and proliferation marker genes \u003cem\u003eCDK1\u003c/em\u003e were upregulated. Differentially expressed genes in RNA-seq results were selected and enriched in the apelin and focal adhesion signaling pathways. Specifically, \u003cem\u003eCIDEA\u003c/em\u003e regulated the activation of focal adhesion kinase and AKT signaling proteins, but not p38 signaling. To this end, we did the rescue assay and found that suppressing focal adhesion kinase signaling pathway with PF573228 reversed the lipid droplets and triglyceride contents increase induced by \u003cem\u003eCIDEA\u003c/em\u003e overexpressing and further decreased their contents in \u003cem\u003eCIDEA\u003c/em\u003e interfering group. In summary, this study reveals that \u003cem\u003eCIDEA\u003c/em\u003e promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibiting the cell proliferation. These works clarify the functional role and downstream signaling pathway of \u003cem\u003eCIDEA\u003c/em\u003e in intramuscular fat deposition and provide theoretical support for improving meat quality through manipulating phenotype-related key genes.\u003c/p\u003e","manuscriptTitle":"CIDEA promotes lipid deposition through focal adhesion pathway in goat intramuscular preadipocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-30 05:58:50","doi":"10.21203/rs.3.rs-5661803/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53e24c35-9256-43e5-bfec-6ede8e533fc7","owner":[],"postedDate":"December 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42087997,"name":"Biological sciences/Cell biology"},{"id":42087998,"name":"Biological sciences/Genetics"},{"id":42087999,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-04-30T07:38:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-30 05:58:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5661803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5661803","identity":"rs-5661803","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}