Differentiation Of Intramuscular Preadipocytes Research Articles

LncBNIP3 Inhibits Bovine Intramuscular Preadipocyte Differentiation via the PI3K-Akt and PPAR Signaling Pathways.

Intramuscular fat (IMF) content is an economic trait in beef cattle that improves the meat quality. Studies have highlighted the correlation between long noncoding RNAs (lncRNAs) and IMF development. In this study, lncBNIP3 knockdown promoted bovine intramuscular preadipocyte differentiation. RNA-seq analysis of intramuscular preadipocytes with lncBNIP3 knockdown identified 230 differentially expressed genes. The PI3K-Akt and PPAR signaling pathways were enriched. lncBNIP3 interference promoted mRNA and protein expression of key genes in PI3K-Akt signaling pathway. LncBNIP3 interference reversed the effects of an AKT-inhibitor MK-2206 on Akt protein expression and lipid droplet accumulation, promoted mRNA and protein expression of essential genes in the PPAR signaling pathway, and ameliorated the inhibitory effects of a PPARg antagonist GW9662 on lipid accumulation. Therefore, lncBNIP3 inhibition of bovine intramuscular preadipocyte differentiation is likely mediated via the PI3K-Akt and PPAR signaling pathways. This study identified a valuable lncRNA with functional roles in IMF accumulation and revealed new strategies to improve beef quality.

Chi-circ_0009659 modulates goat intramuscular adipocyte differentiation through miR-3431-5p/STEAP4 axis.

Circular RNAs (circRNAs) are widely involved in the regulation of lipid deposition in animals, but there are few reports on key circRNAs regulating intramuscular adipocyte differentiation in goats. Therefore, this study took an abundantly expressed in goat adipocytes chi-circ_0009659 as the object. Based on the identification of back splicing site in chi-circ_0009659, its expression level during the goat intramuscular preadipocyte differentiation was detected by qPCR. The chi-circ_0009659 loss-of-function and gain-of-function cell models were obtained by adenovirus and smarter silencer, respectively. and the adipocyte differentiation were explored by Oil Red O staining, Bodipy staining and qPCR. Its major cytoplasmic localization was determined by FISH, nucleocytoplasmic separation and qPCR. The interaction between chi-circ_0009659, miR-3431-5p, and STEAP family member 4 (STEAP4) was verified by bioinformatics, RNA pull down and dual luciferase reporter assay. Silencing chi-circ_0009659 inhibited lipid droplet accumulation and the expression of differentiation-determining genes in goat intramuscular adipocytes, while overexpression of chi-circ_0009659 reversed these results. chi-circ_0009659 was predominantly localized to the cytoplasm and could regulate miR-3431 expression which in turn affects STEAP4. Consistent with expectations, miR-3431-5p acted as a negative regulator of GIMPA differentiation, while STEAP4 promoted differentiation. We demonstrated chi-circ_0009659 positively regulates goat intramuscular preadipocyte differentiation by sponging miR-3431-5p to further regulate the expression of STEAP4. This research provides a new reference for in-depth understanding of the effects of circRNA on adipocyte differentiation.

IGFBP7 promotes the proliferation and differentiation of primary myoblasts and intramuscular preadipocytes in chicken

Though it is well known that insulin-like growth factor (IGF) binding protein 7 (IGFBP7) plays an important role in myogenesis and adipogenesis in mammals, its impact on the proliferation, differentiation, and lipid deposition in chicken primary myoblasts (CPM) and intramuscular preadipocytes remains unexplored. In the present study, we firstly examined the correlation between SNPs within the genomic sequence of the IGFBP7 gene and carcass and blood chemical traits in a F2 resource population by genetic association analysis, and found that a significant correlation between the SNP (4_49499525) located in the intron region of IGFBP7 and serum high-density lipoproteins (HDL). We then examined the expression patterns of IGFBP7 across different stages of proliferation and differentiation in CPMs and intramuscular preadipocytes via qPCR, and explored the biological functions of IGFBP7 through gain- and loss-of-function experiments and a range of techniques including qPCR, CCK-8, EdU, flow cytometry, Western blot, immunofluorescence, and Oil Red O staining to detect the proliferation, differentiation, and lipid deposition in CPMs and intramuscular preadipocytes. We ascertained that the expression levels of the IGFBP7 gene increased as cell differentiation progresses in CPMs and intramuscular preadipocytes, and that IGFBP7 promotes the proliferation and differentiation of these cells, as well as facilitates intracellular lipid deposition. Furthermore, we investigated the regulatory mechanism of IGFBP7 expression by using co-transfection strategy and dual-luciferase reporter assay, and discovered that the myogenic transcription factors (MRF), myoblast determination factor (MyoD) and myogenin (MyoG), along with the adipocyte-specific transcription factor (TF) CCAAT/enhancer-binding protein α (C/EBPα), can bind to the core transcription activation region of the IGFBP7 promoter located 500 bp upstream from the transcription start site, thereby promoting IGFBP7 transcription and expression. Taken together, our study underscores the role of IGFBP7 as a positive regulator for myogenesis and adipogenesis, while also elucidating the functional and transcriptional regulatory mechanisms of IGFBP7 in chicken skeletal muscle development and intramuscular adipogenesis.

MiR-196a Promotes Lipid Deposition in Goat Intramuscular Preadipocytes by Targeting MAP3K1 and Activating PI3K-Akt Pathway.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.

Combined Multi-Omics Analysis Reveals the Potential Role of ACADS in Yak Intramuscular Fat Deposition.

The Yak (Bos grunniens) is a special breed of livestock predominantly distributed in the Qinghai-Tibet Plateau of China. Intramuscular fat (IMF) content in beef cattle is a vital indicator of meat quality. In this study, RNA-Seq and Protein-Seq were respectively employed to sequence the transcriptome and proteome of the longissimus dorsi (LD) tissue from 4-year-old yaks with significant differences in IMF content under the same fattening conditions. Five overlapping genes (MYL3, ACADS, L2HGDH, IGFN1, and ENSBGRG00000000-926) were screened using combined analysis. Functional verification tests demonstrated that the key gene ACADS inhibited yak intramuscular preadipocyte (YIMA) differentiation and proliferation, promoted mitochondrial biogenesis gene expression, and increased the mitochondrial membrane potential (MMP). Furthermore, co-transfection experiments further demonstrated that interfering with ACADS reversed the effect of PPARα agonists in promoting lipid differentiation. In conclusion, ACADS potentially inhibits lipid deposition in YIAMs by regulating the PPARα signalling pathway. These findings offer insights into the molecular mechanisms underlying yak meat quality.

LEP inhibits intramuscular adipogenesis through the AMPK signaling pathway invitro.

Leptin can indirectly regulate fatty-acid metabolism and synthesis in muscle invivo and directly in incubated muscle exvivo. In addition, non-synonymous mutations in the bovine leptin gene (LEP) are associated with carcass intramuscular fat (IMF) content. However, the effects of LEP on lipid synthesis of adipocytes have not been clearly studied at the cellular level. Therefore, this study focused on bovine primary intramuscular preadipocytes to investigate the effects of LEP on the proliferation and differentiation of intramuscular preadipocytes, as well as its regulatory mechanism in lipid synthesis. The results showed that both the LEP and leptin receptor gene (LEPR) were highly expressed in IMF tissues, and their mRNA expression levels were positively correlated at different developmental stages of intramuscular preadipocytes. The overexpression of LEP inhibited the proliferation and differentiation of intramuscular preadipocytes, while interference with LEP had the opposite effect. Additionally, LEP significantly promoted the phosphorylation level of AMPKα by promoting the protein expression of CAMKK2. Meanwhile, rescue experiments showed that the increasing effect of AMPK inhibitors on the number of intramuscular preadipocytes was significantly weakened by the overexpression of LEP. Furthermore, the overexpression of LEP could weaken the promoting effect of AMPK inhibitor on triglyceride content and droplet accumulation, and prevent the upregulation of adipogenic protein expression (SREBF1, FABP4, FASN, and ACCα) caused by AMPK inhibitor. Taken together, LEP acted on the AMPK signaling pathway by regulating the protein expression of CAMKK2, thereby downregulating the expression of proliferation-related and adipogenic-related genes and proteins, ultimately reducing intramuscular adipogenesis.

Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1) Promotes the Adipogenesis of Intramuscular Preadipocytes through PI3K/AKT Pathway in Goats.

As a transcription factor, Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1) binds to downstream target genes to participate in cell proliferation and cell differentiation. We found that the NR4A1 reached the highest expression at 60 h after the differentiation of goat intramuscular preadipocytes. Overexpression of goat NR4A1 increased the number of intracellular lipid droplets and up-regulated the expression of adipocyte-differentiation-related marker genes including AP2, SREBP1, ACC, GPAM, and DGAT2, while the relative expression levels of Pref-1 and HSL were significantly decreased. On the contrary, after NR4A1 was knocked down by siRNA, the number of intracellular lipid droplets and the relative expression levels of LPL, CEBPα, CEBPβ, ACC, and DGAT2 were significantly decreased, and the relative expression levels of Pref-1 and HSL were significantly up-regulated. These results suggest that NR4A1 promotes the differentiation of goat intramuscular preadipocytes. Transcriptome sequencing was carried out after overexpression of goat NR4A1, and the KEGG enrichment analysis result showed that the most differentially expressed genes were related to adipocyte differentiation and were enriched in the PI3K-Akt signaling pathway. LY249002, an inhibitor of the PI3K-Akt signaling pathway, was introduced and decreased the number of intracellular lipid droplets, and the relative expression levels of C/EBPα, SREBP1, AP2, C/EBPβ, GPAM, ACC, DGAT1, DGAT2, and ATGL were decreased accordingly. The above results indicate that overexpression of goat NR4A1 may promote the differentiation of intramuscular preadipocytes through the PI3K-Akt signaling pathway.

Bta-miR-330 promotes bovine intramuscular pre-adipocytes adipogenesis via targeting SESN3 to activate the Akt-mTOR signaling pathway

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3, which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3-Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle.

Profiling of m6A methylation in porcine intramuscular adipocytes and unravelling PHKG1 represses porcine intramuscular lipid deposition in an m6A-dependent manner

Intramuscular fat (IMF) content is mainly determined by intramuscular preadipocyte adipogenesis. Epigenetic modifications are known to have a regulatory effect on IMF. As N6-methyladenosine (m6A) is the most abundant epigenetic modification in eukaryotic RNAs. In the present study, we used m6A methylation and RNA sequencing (seq) to identify the m6A-modified RNAs associated with the adipogenic differentiation of intramuscular preadipocytes. Among them, the expression and m6A level of phosphorylase kinase subunit G1 (PHKG1) were found to be significantly changed during adipogenesis. Further studies revealed that knockdown of the methylase METTL3 decreased the m6A methylation of PHKG1 and led to a reduction in PHKG1. Moreover, knockdown of PHKG1 promoted adipogenic differentiation by upregulating the expression of adipogenic genes. In addition, we found that the IMF content in the longissimus thoracis (LT) of Bamei (BM) pigs was greater than that in Large White (LW) pigs, whereas the m6A and PHKG1 expression levels were lower in BM pigs. These findings indicate that the m6A level and expression of PHKG1 were significantly correlated with IMF content and meat quality. In conclusion, this study sheds light on the mechanism by which m6A modification regulates IMF deposition.

Identification of key differentially methylated genes in regulating muscle development and intramuscular fat deposition in chickens

Muscle development and intramuscular fat (IMF) deposition are intricate physiological processes characterized by multiple gene expressions and interactions. In this research, the phenotypic variations in the breast muscle of Jingyuan chickens were examined at three different time points: 42, 126, and 180 days old. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify differentially methylated genes (DMGs) responsible for regulating muscle development and IMF deposition. The findings indicate a significant increase in breast muscle weight (BMW), myofiber diameter, and cross-sectional area, as well as IMF content, in correlation with the progressive number of growing days in Jingyuan chickens. The findings also revealed that 380 hypo-methylated and 253 hyper-methylated DMGs were identified between the three groups of breast muscle. Module gene and DMG association analysis identified m6A methylation-mediated multiple DMGs associated with muscle development and fat metabolism. In vitro cell modeling analysis reveals stage-specific differences in the expression of CUBN, MEGF10, BOP1, and BMPR2 during the differentiation of myoblasts and intramuscular preadipocytes. Cycloleucine treatment significantly inhibited the expression levels of CUBN, BOP1, and BMPR2, and promoted the expression of MEGF10. These results suggest that m6A methylation-mediated CUBN, MEGF10, BOP1, and BMPR2 can serve as potential candidate genes for regulating muscle development and IMF deposition, and provide an important theoretical basis for further investigation of the functional mechanism of m6A modification involved in adipogenesis.

MiR-129 Regulates Yak Intramuscular Preadipocyte Proliferation and Differentiation through the PI3K/AKT Pathway.

miR-129 plays a crucial role in regulating various cellular processes, including adipogenesis; however, its downstream molecular mechanisms remain unclear. In this study, we demonstrated that miR-129 promotes yak adipogenesis in vitro via the PI3K/AKT pathway. Overexpression and interference of miR-129 in yak intramuscular preadipocytes (YIMAs) enhanced and inhibited cell differentiation, respectively, with corresponding changes in cell proliferation. Further investigation revealed that miR-129 enhances AKT and p-AKT activity in the AKT pathway without affecting cell apoptosis, and a specific inhibitor (LY294002) was used to confirm that miR-129 regulates YIMAs proliferation and differentiation through the PI3K/AKT pathway. Our findings suggest that miR-129 promotes yak adipogenesis by enhancing PI3K/AKT pathway activity. This study provides the foundation to precisely elucidate the molecular mechanism of miR-129 in YIMAs adipogenesis and develop advanced miRNA-based strategies to improve meat nutrition and obesity-related ailments in beef production.

The role of lncFABP4 in modulating adipogenic differentiation in buffalo intramuscular preadipocytes.

Intramuscular fat (IMF) is a crucial determinant of meat quality and is influenced by various regulatory factors. Despite the growing recognition of the important role of long noncoding RNAs (lncRNAs) in IMF deposition, the mechanisms underlying buffalo IMF deposition remain poorly understood. In this study, we identified and characterized a lncRNA, lncFABP4, which is transcribed from the antisense strand of fatty acid-binding protein 4 (FABP4). lncFABP4 inhibited cell proliferation in buffalo intramuscular preadipocytes. Moreover, lncFABP4 significantly increased intramuscular preadipocyte differentiation, as indicated by an increase in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG), CCAAT enhancer binding protein alpha (C/EBPα), and FABP4. Mechanistically, lncFABP4 was found to have the potential to regulate downstream gene expression by participating in protein-protein interaction pathways. These findings contribute to further understanding of the intricate mechanisms through which lncRNAs modulate intramuscular adipogenesis in buffaloes.

Goat miR-92a-3p Targets APOL6 Gene to Regulate the Differentiation of Intramuscular Precursor Adipocytes.

The quality of lamb meat is positively correlated with intramuscular fat content. In recent years, a large number of studies have shown that miRNAs play an important role in the proliferation and differentiation of adipocytes. In this study, we aimed to explore the effect of miR-92a-3p on the differentiation of goat intramuscular preadipocytes. The results showed that the expression level of miR-92a-3p was low in the early stage of differentiation, reached the highest level on the third day of differentiation, and then decreased. And miR-92a-3p can inhibit the accumulation of lipid droplets and down-regulate the determinants of adipogenic differentiation. Mechanistically, by predicting target genes, we found that miR-92a-3p affects the differentiation of goat intramuscular preadipocytes and the accumulation of lipid droplets by regulating the expression of goat gene APOL6. This study provides important new information to better understand the relationship between miRNAs and the differentiation of goat intramuscular preadipocytes, thus providing a new reference for goat intramuscular adipogenesis.

Effect of DHCR7 on adipocyte differentiation in goats

Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/β, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/β, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.

Gga-let-7a-3p inhibits the proliferation and differentiation of chicken intramuscular preadipocytes

ABSTRACT 1. Intramuscular fat (IMF) is a key parameter for chicken meat quality. IMF deposition is driven by genetic, nutritional and management factors, with genetics being the determining factor. Previous whole transcriptome sequencing revealed that microRNA gga-let-7a-3p was related to lipid metabolism in breast muscle. This study further investigated the potential role of gga-let-7a-3p in IMF deposition. 2. The mimic and inhibitor of gga-let-7a-3p were individually transfected into chicken intramuscular preadipocytes. Subsequently, the proliferation and differentiation states of the cells were detected. Transcriptome sequencing was performed on cells transfected with gga-let-7a-3p mimic. 3. The results indicated that gga-let-7a-3p suppressed the mRNA levels of proliferation and differentiation-related genes, as well as the protein levels. EdU and Oil Red O assays revealed that gga-let-7a-3p restrained preadipocyte proliferation and differentiation. In addition, a total of 333 up-regulated genes and 807 down-regulated genes were identified in cells transfected with gga-let-7a-3p mimic. Using Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis, differential genes were found to be enriched in processes such as the peroxisome proliferator activated receptor (PPAR) signalling pathway and oxidative phosphorylation. 4. The study demonstrated that gga-let-7a-3p inhibits the proliferation and differentiation of chicken intramuscular preadipocytes, which provides new understanding to further unravel the function of gga-let-7a-3p.

Comparative alternative polyadenylation profiles in differentiated adipocytes of subcutaneous and intramuscular fat tissue in cattle

Alternative polyadenylation (APA) is a key molecular mechanism involved in the post-transcriptional regulation of gene expression, which has been proven to play a critical role in cell differentiation. In the present study, we performed IVT-SAPAS sequencing to profile the dynamic changes of APA sites in bovine subcutaneous preadipocytes and intramuscular preadipocytes during adipogenesis. A total of 52621 high quality APA sites were identified in preadipocytes and adipocytes. Compared with preadipocytes, the increased usage of canonical AATAAA was observed in the cell-biased APA sites of adipocytes. Furthermore, 1933 and 2140 differentially expressed APA (DE-APA) sites, as well as 341 and 337 untranslated region-APA (UTR-APA) switching genes were identified in subcutaneous preadipocytes and intramuscular preadipocytes during adipogenesis, respectively. The UTR-APA switching genes showed divergent trends in preadipocytes, among which UTR-APA switching genes in intramuscular preadipocytes tended to use shorter 3′UTR for differentiation into mature adipocytes. APA events mediated by UTR-APA switching in intramuscular adipocytes were enriched in lipid synthesis and adipocyte differentiation. TRIB3, WWTR1, and INSIG1 played important roles in the differentiation of intramuscular preadipocytes. Briefly, our results provided new insights into understanding the mechanisms of bovine adipocyte differentiation.

LncHLEF promotes hepatic lipid synthesis through miR-2188-3p/GATA6 axis and encoding peptides and enhances intramuscular fat deposition via exosome

Long noncoding RNAs (lncRNAs) have emergingly been implicated in mammalian lipid metabolism. However, their biological functions and regulatory mechanisms underlying adipogenesis remain largely elusive in chicken. Here, we systematically characterized the genome-wide full-length lncRNAs in the livers of pre- and peak-laying hens, and identified a novel intergenic lncRNA, lncHLEF, an RNA macromolecule with a calculated molecular weight of 433 kDa. lncHLEF was primarily distributed in cytoplasm of chicken hepatocyte and significantly up-regulated in livers of peak-laying hens. Functionally, lncHLEF could promote hepatocyte lipid droplet formation, triglycerides and total cholesterol contents. Mechanistically, lncHLEF could not only serve as a competitive endogenous RNA to modulate miR-2188-3p/GATA6 axis, but also encode three small functional polypeptides that directly interact with ACLY protein to enable its stabilization. Importantly, adeno-associated virus-mediated liver-specific lncHLEF overexpression resulted in increased hepatic lipid synthesis and intramuscular fat (IMF) deposition, but did not alter abdominal fat (AbF) deposition. Furthermore, hepatocyte lncHLEF could be delivered into intramuscular and abdominal preadipocytes via hepatocyte-secreted exosome to enhance intramuscular preadipocytes differentiation without altering abdominal preadipocytes differentiation. In conclusion, this study revealed that the lncHLEF could promote hepatic lipid synthesis through two independent regulatory mechanisms, and could enhance IMF deposition via hepatocyte-adipocyte communications mediated by exosome.

Epigenomics Analysis of the Suppression Role of SIRT1 via H3K9 Deacetylation in Preadipocyte Differentiation.

Sirtuin 1 (SIRT1) overexpression significantly inhibits lipid deposition during yak intramuscular preadipocyte (YIMA) differentiation; however, the regulatory mechanism remains unknown. We elucidated the role of SIRT1 in YIMA differentiation using lentivirus-mediated downregulation technology and conducted mRNA-seq and ChIP-seq assays using H3K9ac antibodies after SIRT1 overexpression in order to reveal SIRT1 targets during YIMA adipogenesis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed in order to identify the functional annotation of common genes. In addition, a potential target of SIRT1 was selected to verify its effects on the differentiation and proliferation of YIMAs. SIRT1 interfered with lipid deposition and promoted YIMA differentiation. In total, 143,518 specific peaks were identified after SIRT1 overexpression, where genes associated with downregulation peaks were enriched in transcription, gene expression, lipid-related processes, and classical lipid-related pathways. The H3K9ac signal in the whole genome promoter region (2 kb upstream and downstream of the transcription start site (TSS)) was weakened, and the peaks were distributed across all gene functional regions. Genes that lost signals in their TSS region or gene body region were enriched in both biological processes and pathways associated with lipogenesis. The ChIP-seq results revealed 714 common differential genes in mRNA-seq, which were enriched in "MAPK signaling", "lipid and atherosclerosis", "mTOR signaling", and "FoxO signaling" pathways. A total of 445 genes were downregulated in both their H3K9ac signals and mRNA expression, and one of their most significantly enriched pathways was FoxO signaling. Nine genes (FBP2, FPGT, HSD17B11, KCNJ15, MAP3K20, SLC5A3, TRIM23, ZCCHC10, and ZMYM1) lost the H3K9ac signal in their TSS regions and had low mRNA expression, and three genes (KCNJ15, TGM3, and TRIM54) had low expression but lost their H3K9ac signal in the gene body region. The interference of TRIM23 significantly inhibited fat deposition during preadipocyte differentiation and promoted cell proliferation by increasing S-phase cell numbers. The present study provides new insights into the molecular mechanism of intramuscular fat content deposition and the epigenetic role of SIRT1 in adipocyte differentiation.

Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m6A-YTHDF1–dependent manner

BackgroundIntramuscular fat (IMF) content is a critical indicator of pork quality, and abnormal IMF is also relevant to human disease as well as aging. Although N6-methyladenosine (m6A) RNA modification was recently found to regulate adipogenesis in porcine intramuscular fat, however, the underlying molecular mechanisms was still unclear.ResultsIn this work, we collected 20 longissimus dorsi muscle samples with high (average 3.95%) or low IMF content (average 1.22%) from a unique heterogenous swine population for m6A sequencing (m6A-seq). We discovered 70 genes show both differential RNA expression and m6A modification from high and low IMF group, including ADIPOQ and SFRP1, two hub genes inferred through gene co-expression analysis. Particularly, we observed ADIPOQ, which contains three m6A modification sites within 3′ untranslated and protein coding region, could promote porcine intramuscular preadipocyte differentiation in an m6A-dependent manner. Furthermore, we found the YT521‑B homology domain family protein 1 (YTHDF1) could target and promote ADIPOQ mRNA translation.ConclusionsOur study provided a comprehensive profiling of m6A methylation in porcine longissimus dorsi muscle and characterized the involvement of m6A epigenetic modification in the regulation of ADIPOQ mRNA on IMF deposition through an m6A-YTHDF1-dependent manner.