Chicken Adipogenesis Research Articles

ALKBH5 regulates chicken adipogenesis by mediating LCAT mRNA stability depending on m6A modification

BackgroundPrevious studies have demonstrated the role of N6-methyladenosine (m6A) RNA methylation in various biological processes, our research is the first to elucidate its specific impact on LCAT mRNA stability and adipogenesis in poultry.ResultsThe 6 100-day-old female chickens were categorized into high (n = 3) and low-fat chickens (n = 3) based on their abdominal fat ratios, and their abdominal fat tissues were processed for MeRIP-seq and RNA-seq. An integrated analysis of MeRIP-seq and RNA-seq omics data revealed 16 differentially expressed genes associated with to differential m6A modifications. Among them, ELOVL fatty acid elongase 2 (ELOVL2), pyruvate dehydrogenase kinase 4 (PDK4), fatty acid binding protein 9 (PMP2), fatty acid binding protein 1 (FABP1), lysosomal associated membrane protein 3 (LAMP3), lecithin-cholesterol acyltransferase (LCAT) and solute carrier family 2 member 1 (SLC2A1) have ever been reported to be associated with adipogenesis. Interestingly, LCAT was down-regulated and expressed along with decreased levels of mRNA methylation methylation in the low-fat group. Mechanistically, the highly expressed ALKBH5 gene regulates LCAT RNA demethylation and affects LCAT mRNA stability. In addition, LCAT inhibits preadipocyte proliferation and promotes preadipocyte differentiation, and plays a key role in adipogenesis.ConclusionsIn conclusion, ALKBH5 mediates RNA stability of LCAT through demethylation and affects chicken adipogenesis. This study provides a theoretical basis for further understanding of RNA methylation regulation in chicken adipogenesis.

Long Noncoding RNA 6302 Regulates Chicken Preadipocyte Differentiation by Targeting SLC22A16.

The excessive deposition of abdominal adipocytes in chickens is detrimental to poultry production. However, the regulatory factors that affect abdominal adipogenesis in chickens are still poorly understood. SLC22A16 is differentially expressed in abdominal preadipocytes and 10-day differentiated adipocytes in chickens, but its role in regulating chicken adipogenesis has not been reported. In this study, the function of SLC22A16 in chicken abdominal preadipocytes was investigated. SLC22A16 is significantly upregulated during abdominal adipocyte differentiation. The overexpression of SLC2A16 upregulated the expression of adipogenic marker genes and proliferation-related genes, and promoted the proliferation of adipocytes and the accumulation of triglycerides. The knockdown of SLC22A16 downregulated the expression of adipogenic marker genes and proliferation-related genes, inhibited the proliferation of adipocytes, and impaired the accumulation of triglycerides in adipocytes. In addition, LNC6302 was differentially expressed in abdominal preadipocytes and mature adipocytes, and was significantly positively correlated with the expression of SLC22A16. Interference with LNC6302 inhibits the expression of adipogenic marker genes and proliferation-related genes. The data supported the notion that LNC6302 promotes the differentiation of chicken abdominal adipocytes by cis-regulating the expression of SLC22A16. This study identified the role of SLC22A16 in the differentiation and proliferation of chicken adipocytes, providing a potential target for improving abdominal adipogenesis in chickens.

CircDOCK7 facilitates the proliferation and adipogenic differentiation of chicken abdominal preadipocytes through the gga-miR-301b-3p/ACSL1 axis

BackgroundAbdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes, which is a well-orchestrated multistep process involving many regulatory molecules. Circular RNAs (circRNAs) have emergingly been implicated in mammalian adipogenesis. However, circRNA-mediated regulation in chicken adipogenesis remains unclear. Our previous circRNA sequencing data identified a differentially expressed novel circRNA, 8:27,886,180|27,889,657, during the adipogenic differentiation of chicken abdominal preadipocytes. This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes, and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.ResultsOur results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7 (DOCK7) gene, abbreviated as circDOCK7. CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life. CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens, and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. Functionally, the gain- and loss-of-function experiments showed that circDOCK7 promoted proliferation, G0/G1- to S-phase progression, and glucose uptake capacity of chicken abdominal preadipocytes, in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes. Mechanistically, a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p, which was identified as an inhibitor of chicken abdominal adipogenesis. Moreover, the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p. Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator. Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.ConclusionsCircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene, thus augmenting adipogenesis in chickens. These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.

PNX14 but not PNX20 as a novel regulator of preadipocyte differentiation via activating Epac-ERK signaling pathway in Gallus gallus

Small integral membrane protein 20 (SMIM20) could generate two main peptides, PNX14 and PNX20, which participate in multiple biological roles such as reproduction, inflammation and energy metabolism in mammals. However, little is known about their physiological functions in non-mammalian vertebrates. Using chicken (c-) as an animal model, we found cSMIM20 was moderately expressed in adipose tissues, and its expression was gradually increased during the differentiation of chicken preadipocytes, suggesting that it may play an important role in chicken adipogenesis. Further research showed cPNX14 could facilitate the differentiation of chicken preadipocytes into mature adipocytes by enhancing expression of adipogenic genes including PPARγ, CEBPα and FABP4, and promoting the formation of lipid droplets. This pro-adipogenic effect of cPNX14 was completely attenuated by Epac-specific and ERK inhibitor. Interestingly, cPNX20 failed to regulate the adipogenic genes and lipid droplet content. Collectively, our findings reveal that cPNX14 but not cPNX20 can serve as a novel adipogenesis mediator by activating the Epac-ERK signaling pathway in chickens.

M6A demethylase FTO regulate CTNNB1 to promote adipogenesis of chicken preadipocyte

BackgroundN6-methyladenosine (m6A) is an abundant post-transcriptional RNA modification that affects various biological processes. The fat mass and obesity-associated (FTO) protein, a demethylase encoded by the FTO gene, has been found to regulate adipocyte development in an m6A-dependent manner in multiple species. However, the effects of the m6A methylation and FTO demethylation functions on chicken adipogenesis remain unclear. This study aims to explore the association between m6A modification and chicken adipogenesis and the underlying mechanism by which FTO affects chicken preadipocyte development.ResultsThe association between m6A modification and chicken lipogenesis was assessed by treating chicken preadipocytes with different doses of methyl donor betaine and methylation inhibitor cycloleucine. The results showed that betaine significantly increased methylation levels and inhibited lipogenesis, and the inverse effect was found in preadipocytes after cycloleucine treatment. Overexpression of FTO significantly inhibited m6A levels and promoted proliferation and differentiation of chicken preadipocytes. Silencing FTO showed opposite results. Mechanistically, FTO overexpression increased the expression of catenin beta 1 (CTNNB1) by improving RNA stability in an m6A-dependent manner, and we proved that FTO could directly target CTNNB1. Furthermore, CTNNB1 may be a positive regulator of adipogenesis in chicken preadipocytes.Conclusionsm6A methylation of RNA was negatively associated with adipogenesis of chicken preadipocytes. FTO could regulate CTNNB1 expression in a demethylation manner to promote lipogenesis.

Genome-wide survey identifies TNNI2 as a target of KLF7 that inhibits chicken adipogenesis via downregulating FABP4

Krüppel-like factor 7 (KLF7) negatively regulates adipocyte differentiation; however, the mechanism underlying its activity in mammals and birds remains poorly understood. To identify genome-wide KLF7-binding motifs in preadipocytes, we conducted a chromatin immunoprecipitation-sequencing analysis of immortalized chicken preadipocytes (ICP2), which revealed 11,063 specific binding sites. Intergenic binding site analysis showed that KLF7 regulates several novel factors whose functions in chicken and mammal adipogenesis are underexplored. We identified a novel regulator, troponin I2 (TNNI2), which is positively regulated by KLF7. TNNI2 is downregulated during preadipocyte differentiation and acts as an adipogenic repressor at least in part by repressing FABP4 promoter activity. In conclusion, we demonstrated that KLF7 functions through cis-regulation of TNNI2, which inhibits adipogenesis. Our findings not only provide the first genome-wide picture of KLF7 associations in preadipocytes but also identify a novel function of TNNI2.

Knockout and Restoration Reveal Differential Functional Roles of PPARγ1 and PPARγ2 in Chicken Adipogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipogenesis and is expressed as two isoforms, PPARγ1 and PPARγ2. Our previous lentiviral overexpression study showed that PPARγ1 and PPARγ2 differentially regulated proliferation, differentiation, and apoptosis of the immortalized chicken preadipocyte cell line (ICP2). However, we cannot rule out the possibility that the endogenous expression of PPARγ isoforms may compromise our findings. In this study, using the dual sgRNA-directed CRISPR/Cas9 system, we generated PPARγ (PPARγ-/-) and PPARγ2-specific knockout (PPARγ2-/-) ICP2 cell lines and investigated the differences in proliferation and differentiation among PPARγ-/-, PPARγ2-/-, and wild-type ICP2 cells. EdU proliferation assay showed that both PPARγ2-specific and PPARγ knockouts significantly increased the proliferation rates. Consistently, real-time RT-PCR analysis showed that both PPARγ2-specific and PPARγ knockouts significantly upregulated the expression of proliferation marker genes PCNA and cyclinD1. FACS analysis revealed that PPARγ knockout significantly increased the number of cells accumulating in the S phase and decreased the number of cells accumulating in the G1/G0 phase. Oil Red O staining and gene expression analysis showed both PPARγ2-specific and PPARγ knockouts dramatically reduced capacity for adipogenic differentiation. To corroborate our previous findings, PPARγ1 and PPARγ2 expression were restored in PPARγ-/- cells by using the lentiviruses expressing chicken PPARγ1 (LV-PPARγ1) and PPARγ2 (LV-PPARγ2), respectively. Subsequent assays showed that restoration of expression of either PPARγ1 or PPARγ2 suppressed proliferation and stimulated differentiation of the PPARγ-/- cells. By comparison, PPARγ2 had stronger anti-proliferative and pro-adipogenic effects than PPARγ1. To understand the molecular mechanism underlying their differential effects on differentiation of the PPARγ-/- cells, we performed RNA-seq in the PPARγ-/- cells in which individual PPARγ isoform expression was restored at 72 h of differentiation. Transcriptomic analysis revealed that restoring PPARγ1 expression caused far more differentially expressed genes (DEGs) than restoring PPARγ2 expression. GO and KEGG pathway enrichment analyses indicated that PPARγ1 and PPARγ2 had distinct and overlapping functions in adipogenesis. Taken together, our results clearly indicate that PPARγ1 and PPARγ2 differentially impact chicken adipogenesis.

Comparative Transcriptome Analysis Reveals Regulatory Mechanism of Long Non-Coding RNAs during Abdominal Preadipocyte Adipogenic Differentiation in Chickens.

Simple SummaryExcessive fat deposition, particularly abdominal fat, negatively affects meat quality, feed utilization, and reproduction performance in chickens. Adipogenesis is a well-orchestrated process that involves a network of regulatory molecules. Emerging evidence has shown that long non-coding RNAs (lncRNAs) participate in mammalian adipogenesis. However, limited information is available about their role in adipogenesis in chickens. Therefore, we investigated the genome-wide identification, expression profiles, and regulatory networks of lncRNAs during adipogenic differentiation of chicken abdominal preadipocytes. We found that lncRNAs were expressed in chicken abdominal adipocytes, characterized by their differentiation stage-specific expression pattern and were involved in multiple lipid metabolism-related pathways. Several lncRNAs were identified as key lncRNAs responsible for adipogenic differentiation. These key lncRNAs could regulate lipid-related protein-encoding genes in cis- and trans-regulation manners, and/or competitively sponge miRNA to sequester it away from lipid-related genes, thereby functioning as regulators of chicken adipogenesis. These findings provide valuable information that improves our understanding of functional mechanisms of lncRNAs underlying adipogenesis and contributes to the genetic improvement of excessive abdominal fat in chickens.Long non-coding RNAs (lncRNAs) are implicated in mammalian adipogenesis and obesity. However, their genome-wide distribution, expression profiles, and regulatory mechanisms during chicken adipogenesis remain rarely understood. In the present study, lncRNAs associated with adipogenesis were identified from chicken abdominal adipocytes at multiple differentiation stages using Ribo-Zero RNA-seq. A total of 15,179 lncRNAs were identified and characterized by stage-specific expression patterns. Of these, 840 differentially expressed lncRNAs were detected, and their cis- and trans-target genes were significantly enriched in multiple lipid-related pathways. Through weighted gene co-expression network analysis (WGCNA) and time-series expression profile clustering analysis, 14 key lncRNAs were identified as candidate regulatory lncRNAs in chicken adipogenic differentiation. The cis- and trans-regulatory interactions of key lncRNAs were constructed based on their differentially expressed cis- and trans-target genes, respectively. We also constructed a competing endogenous RNA (ceRNA) network based on the key lncRNAs, differentially expressed miRNAs, and differentially expressed mRNAs. MSTRG.25116.1 was identified as a potential regulator of chicken abdominal preadipocyte adipogenic differentiation by acting as a transcriptional trans-regulator of fatty acid amide hydrolase (FAAH) gene expression and/or a ceRNA that post-transcriptionally mediates FAAH gene expression by sponging gga-miR-1635.

Transcription Factor 21 Promotes Chicken Adipocyte Differentiation at Least in Part via Activating MAPK/JNK Signaling.

The molecular mechanisms of transcription factor 21 (TCF21) in regulating chicken adipogenesis remain unclear. Thus, the current study was designed to investigate the signaling pathway mediating the effect of TCF21 on chicken adipogenesis. Immortalized chicken preadipocytes cell line (ICP), a preadipocyte cell line stably overexpressing TCF21 (LV-TCF21) and a control preadipocyte cell line (LV-control) were used in the current study. We found that the phosphorylation of c-Jun N-terminal kinases (JNK) was significantly elevated in LV-TCF21 compared to LV-control. After treating ICP cells with a JNK inhibitor SP600125, the differentiation of ICP was inhibited, as evidenced by decreased accumulation of lipid droplets and reduced expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), adipocyte fatty acid binding protein (A-FABP), and lipoprotein lipase (LPL). Moreover, we found that the inhibition of JNK by SP600125 remarkably impaired the ability of TCF21 to drive adipogenesis. Taken together, our results suggest that TCF21 promotes the differentiation of adipocytes at least in part via activating MAPK/JNK pathway.

Genome-wide survey and functional analysis reveal TCF21 promotes chicken preadipocyte differentiation by directly upregulating HTR2A

Background/aimPreviously, we showed that transcription factor 21 (TCF21) promotes chicken preadipocyte differentiation. However, the genome-wide TCF21 binding sites and its downstream target genes in chicken adipogenesis were unknown. MethodsChIP-Seq and RNA-Seq were used to screen candidate targets of TCF21. qPCR and luciferase reporter assay were applied to verify the sequencing results. Western blotting, oil red-O staining and pharmacological treatments were performed to investigate the function of 5-hydroxytryptamine receptor 2A (HTR2A), one of the bonafide direct downstream binding targets of TCF21. ResultsA total of 94 candidate target genes of TCF21 were identified. ChIP-qPCR, RT-qPCR, and luciferase reporter assay demonstrated that HTR2A is one of the bonafide direct downstream binding targets of TCF21. HTR2A expression in adipose tissue was upregulated in fat line broilers. Also, the abundance of HTR2A gradually increased during the adipogenesis process. Interestingly, pharmacological enhancement or inhibition of HTR2A promoted or attenuated the differentiation of preadipocytes, respectively. Furthermore, HTR2A inhibition impaired the TCF21 promoted adipogenesis. ConclusionsWe profiled the genome-wide TCF21 binding sites in chicken differentiated preadipocytes revealing HTR2A as the direct downstream target of TCF21 in adipogenesis.

Dynamic Expression and Regulatory Network of Circular RNA for Abdominal Preadipocytes Differentiation in Chicken (Gallus gallus).

Circular RNA (circRNA), as a novel endogenous biomolecule, has been emergingly demonstrated to play crucial roles in mammalian lipid metabolism and obesity. However, little is known about their genome-wide identification, expression profile, and function in chicken adipogenesis. In present study, the adipogenic differentiation of chicken abdominal preadipocyte was successfully induced, and the regulatory functional circRNAs in chicken adipogenesis were identified from abdominal adipocytes at different differentiation stages using Ribo-Zero RNA-seq. A total of 1,068 circRNA candidates were identified and mostly derived from exons. Of these, 111 differentially expressed circRNAs (DE-circRNAs) were detected, characterized by stage-specific expression, and enriched in several lipid-related pathways, such as Hippo signaling pathway, mTOR signaling pathway. Through weighted gene co-expression network analyses (WGCNA) and K-means clustering analyses, two DE-circRNAs, Z:35565770|35568133 and Z:54674624|54755962, were identified as candidate regulatory circRNAs in chicken adipogenic differentiation. Z:35565770|35568133 might compete splicing with its parental gene, ABHD17B, owing to its strictly negative co-expression. We also constructed competing endogenous RNA (ceRNA) network based on DE-circRNA, DE-miRNA, DE-mRNAs, revealing that Z:54674624|54755962 might function as a ceRNA to regulate chicken adipogenic differentiation through the gga-miR-1635-AHR2/IRF1/MGAT3/ABCA1/AADAC and/or the novel_miR_232-STAT5A axis. Translation activity analysis showed that Z:35565770|35568133 and Z:54674624|54755962 have no protein-coding potential. These findings provide valuable evidence for a better understanding of the specific functions and molecular mechanisms of circRNAs underlying avian adipogenesis.

Construction of miRNA-mRNA network in the differentiation of chicken preadipocytes

ABSTRACT 1. MicroRNAs (miRNAs) play key roles in regulating lipid metabolism, adipogenesis and fat deposition in chicken. To date, there are only a few miRNAs that have been confirmed to be involved in chicken adipogenesis. The detailed mechanisms by which miRNAs regulate chicken adipogenesis remain largely unknown. 2. To identify candidate miRNAs involved in chicken preadipocyte differentiation and explore potential mechanisms behind their functions, the following study analysed and identified miRNA and mRNA expression levels in undifferentiated and differentiated preadipocytes. Hub miRNA-mRNA interactions were identified, and the degree of connectivity of DE miRNAs in the network was established. 3. A total of 145 DE miRNAs and 660 DE mRNAs were identified between undifferentiated and differentiated preadipocytes. An miRNA-mRNA network was constructed, including 29 DE miRNAs and 155 DE mRNAs, forming 470 miRNA-mRNA interactions. Functional enrichment analysis showed that DE mRNAs in the network were significantly enriched in 712 biological processes and 13 KEGG pathways. Based on the connectivity degree, five DE miRNAs with higher degrees miR-195-x, gga-miR-200a-3p, gga-miR-135a-5p, novel-m0067-5p and novel-m0270-5p were identified as hub miRNAs. Fifty-eight DE mRNAs interacted with these five hub miRNAs and formed 70 miRNA-mRNA interactions. 4. This study constructed a miRNA-mRNA network associated with chicken preadipocyte differentiation and identified five hub miRNAs in the network. The findings identified a number of chicken adipogenic miRNAs and laid the foundation for elucidating the miRNA-mediated regulatory mechanism in chicken adipogenesis.

Whole Transcriptome Analysis Reveals a Potential Regulatory Mechanism of LncRNA-FNIP2/miR-24-3p/FNIP2 Axis in Chicken Adipogenesis.

Lipid biosynthesis is a complex process, which is regulated by multiple factors including lncRNA. However, the role of lncRNA in chicken abdominal fat accumulation is still unclear. In this research, we collected liver tissues from six high abdominal fat rate Sanhuang broilers and six low abdominal fat rate Sanhuang broilers to perform lncRNA sequencing and small RNA sequencing. A total of 2,265 lncRNAs, 245 miRNAs, and 5,315 mRNAs were differently expressed. Among of them, 1,136 differently expressed genes were enriched in the metabolic process. A total of 36 differently expressed genes, which were considered as differently expressed lncRNAs’ targets, were enriched in the metabolic process. In addition, we also found out that eight differently expressed miRNAs could target 19 differently expressed genes. FNIP2 and PEX5L were shared in a cis-regulatory network and a differently expressed miRNA target relationship network. LncRNA-FNIP2/miR-24-3p/FNIP2 axis was considered as a potential candidate that may participate in lipid synthesis. Experimentally, the objective reality of lncRNA-FNIP2/miR-24-3p/FNIP2 axis was clarified and the regulation effect of lncRNA-FNIP2/miR-24-3p/FNIP2 axis on synthesis was validated. In brief, our study reveals a potential novel regulatory mechanism that lncRNA-FNIP2/miR-24-3p/FNIP2 axis was considered as being involved in lipid synthesis during chicken adipogenesis in liver.

GATA Binding Protein 3 Is a Direct Target of Kruppel-Like Transcription Factor 7 and Inhibits Chicken Adipogenesis.

Kruppel-like transcription factor 7 (KLF7) is a negative regulator of adipogenesis, however, its precise mechanism is poorly understood. Our previous KLF7 ChIP-seq analysis showed that one of the KLF7 binding peaks was present upstream of GATA binding protein 3 (GATA3) in chicken preadipocytes. In the present study, we identified GATA3 as a target of KLF7. Overexpression analysis showed KLF7 markedly enhanced the endogenous expression of GATA3 in the immortalized chicken preadipcyte cell line (ICP2), and the luciferase reporter assay showed that KLF7 overexpression increased the reporter gene activity of the cloned upstream region (-5285/-4336 relative to the translation initiation codon ATG) of GATA3 in ICP2 and DF1 cells, and mutation of the putative KLF7 binding site abolished the promotive effect of KLF7 overexpression on the reporter gene activity of the cloned GATA3 upstream region. ChIP-qPCR further demonstrated that KLF7 directly bound to the GATA3 upstream region. Gene expression analysis showed that GATA3 mRNA expression in abdominal adipose tissue was significantly higher in lean chicken line than in the fat line at 2, 3, and 6 weeks of age. In addition, GATA3 mRNA expression markedly decreased during the preadipocyte differentiation. Furthermore, a functional study showed that GATA3 overexpression inhibited the differentiation of the ICP2 cells. Taken together, our results demonstrated that KLF7 inhibits chicken adipogenesis, at least in part through direct upregulation of GATA3.

Integrative Analyses of mRNA Expression Profile Reveal the Involvement of IGF2BP1 in Chicken Adipogenesis.

Excessive abdominal fat deposition is an issue with general concern in broiler production, especially for Chinese native chicken breeds. A high-fat diet (HFD) can induce body weight gained and excessive fat deposition, and genes and pathways participate in fat metabolism and adipogenesis would be influenced by HFD. In order to reveal the main genes and pathways involved in chicken abdominal fat deposition, we used HFD and normal diet (ND) to feed a Chinese native chicken breed, respectively. Results showed that HFD can increase abdominal fat deposition and induce adipocyte hypertrophy. Additionally, we used RNA-sequencing to identify the differentially expressed genes (DEGs) between HFD and ND chickens in liver and abdominal fat. By analyzed these DEGs, we found that the many DEGs were enriched in fat metabolism related pathways, such as peroxisome proliferator-activated receptor (PPAR) signaling, fat digestion and absorption, extracellular matrix (ECM)-receptor interaction, and steroid hormone biosynthesis. Notably, the expression of insulin-like growth factor II mRNA binding protein 1 (IGF2BP1), which is a binding protein of IGF2 mRNA, was found to be induced in liver and abdominal fat by HFD. Ectopic expression of IGF2BP1 in chicken liver-related cell line Leghorn strain M chicken hepatoma (LMH) cell revealed that IGF2BP1 can regulate the expression of genes associated with fatty acid metabolism. In chicken preadipocytes (ICP cell line), we found that IGF2BP1 can promote adipocyte proliferation and differentiation, and the lipid droplet content would be increased by overexpression of IGF2BP1. Taken together, this study provides new insights into understanding the genes and pathways involved in abdominal fat deposition of Chinese native broiler, and IGF2BP1 is an important candidate gene for the study of fat metabolism and adipogenesis in chicken.

Immortalization of chicken preadipocytes by retroviral transduction of chicken TERT and TR.

The chicken is an important agricultural animal and model for developmental biology, immunology and virology. Excess fat accumulation continues to be a serious problem for the chicken industry. However, chicken adipogenesis and obesity have not been well investigated, because no chicken preadipocyte cell lines have been generated thus far. Here, we successfully generated two immortalized chicken preadipocyte cell lines through transduction of either chicken telomerase reverse transcriptase (chTERT) alone or in combination with chicken telomerase RNA (chTR). Both of these cell lines have survived >100 population doublings in vitro, display high telomerase activity and have no sign of replicative senescence. Similar to primary chicken preadipocytes, these two cell lines display a fibroblast-like morphology, retain the capacity to differentiate into adipocytes, and do not display any signs of malignant transformation. Isoenzyme analysis and PCR-based analysis confirmed that these two cell lines are of chicken origin and are free from inter-species contamination. To our knowledge, this is the first report demonstrating the generation of immortal chicken cells by introduction of chTERT and chTR. Our established chicken preadipocyte cell lines show great promise as an in vitro model for the investigation of chicken adipogenesis, lipid metabolism, and obesity and its related diseases, and our results also provide clues for immortalizing other avian cell types.

Expression profiling of preadipocyte microRNAs by deep sequencing on chicken lines divergently selected for abdominal fatness.

Through posttranscriptional gene regulation, microRNA (miRNA) is linked to a wide variety of biological processes, including adipogenesis and lipid metabolism. Although miRNAs in mammalian adipogenesis have been worked on extensively, their study in chicken adipogenesis is still very limited. To find miRNAs potentially important for chicken preadipocyte development, we compared the preadipocyte miRNA expression profiles in two broiler lines divergently selected for abdominal fat content, by sequencing two small RNA libraries constructed for primary preadipocytes isolated from abdominal adipose tissues. After bioinformatics analyses, from chicken miRNAs deposited in miRBase 20.0, we identified 225 miRNAs to be expressed in preadipocytes, 185 in the lean line and 200 in the fat line (derived from 208 and 203 miRNA precursors, respectively), which corresponds to 114 miRNA families. The let-7 family miRNAs were the most abundant. Furthermore, we validated the sequencing results of 15 known miRNAs by qRT-PCR, and confirmed that the expression levels of most miRNAs correlated well with those of Solexa sequencing. A total of 33 miRNAs was significantly differentially expressed between the two chicken lines (P<0.05). Gene ontology analysis revealed that they could target genes enriched in the regulation of gene transcription and chromatin function, response to insulin stimulation, and IGF-1 signaling pathways, which could have important roles in preadipocyte development. Therefore, a valuable information and resource of miRNAs on chicken adipogenesis were provided in this study. Future functional investigations on these miRNAs could help explore related genes and molecular networks fundamental to preadipocyte development.

Expression and functional analysis of Krüppel-like factor 2 in chicken adipose tissue.

Studies in mammalian species showed that Krüppel-like factor 2 (KLF2) regulates adipogenesis. However, its role in birds is unclear. The objective of the current study was to explore the expression and function of KLF2 in chicken adipogenesis. Results showed that chicken KLF2 (Gallus gallus KLF2 [gKLF2]) was greatly expressed in abdominal adipose tissue, and its transcripts fluctuated during adipose tissue development. In addition, gKLF2 transcripts in abdominal adipose tissue of lean broilers were greater at 1 wk of age but lower at 3, 5, and 8 wk of age than those in fat broilers (P < 0.05). The gKLF2 was more greatly expressed in preadipocytes than in mature adipocytes (P < 0.05), and its expression level decreased during the preadipocyte differentiation in vitro (P < 0.05). The functional analysis showed that gKLF2 overexpression inhibited chicken preadipocyte differentiation (P < 0.05), accompanied by the reduced expression of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and the elevated expression of GATA binding protein 2 (GATA2). Additionally, the luciferase reporter assays showed that gKLF2 overexpression suppressed the promoter activities of chicken C/EBPα and PPARγ (P < 0.05). In conclusion, our results indicated that gKLF2 inhibits chicken adipogenesis, at least in part, through inhibition of PPARγ and C/EBPα expression.

Characterization of the expression profiles of adipogenesis-related factors, ZNF423, KLFs and FGF10, during preadipocyte differentiation and abdominal adipose tissue development in chickens

Adipogenesis is controlled by a complicated process involving certain transcriptional events. In chicken adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of preadipocyte differentiation and abdominal fat accumulation. However, in a recent study in mammals, some novel factors related to regulation of adipogenesis, including preadipocyte differentiation, were identified in mammals. Therefore, in this study, we aimed to determine the expression profiles of these mammalian adipogenesis-related factors, such as zinc-finger protein 423 (ZNF423), Krüppel-like factor -2, -5, and -15 (KLF-2, -5, -15), and FGF10, in the chicken (Gallus gallus). Specifically, we analyzed their expression in primary preadipocyte differentiation in vitro and also analyzed their tissue distribution and their temporal expression in adipose tissue development in vivo. During chicken adipocyte differentiation, the gene expression of ZNF423, KLF-2, KLF-5 and FGF10 was found to rapidly decrease in the early stage of preadipocyte differentiation. Expression of ZNF423 then increased in the late stage of differentiation. KLF-15 expression increased in a time-dependent manner for 48 h. Protein expressions of these factors were reflected by Western blot analysis. High levels of aP2, PPARγ and FGF10 mRNA were found in adipose tissue. In addition, aP2, PPARγ and ZNF423 mRNA levels in the adipose tissue were elevated at days 10 and 20. These expression profiles of the adipogenesis-related factors in chicken are, in part, different from mammalian adipogenesis but this seems to reflect the differences in the regulation of adipogenesis and in adipose tissue functions between avians and mammals.

Cloning, Expression and Function Analysis of Chicken FBXO38t1

F-box proteins are widely present in eukaryotes,and their biological functions are diverse in animals.In this study,the full-length coding sequence of an unnamed gene LOC416151(GenBank accession XM_414482) was cloned from the chicken abdominal adipose tissue by reverse transcriptase(RT)-PCR,and the sequence analysis showed that the acquired sequence(GenBank accession.JX290204) is one of the transcript variants of chicken FBXO38,which was designated as gFBXO38t1.Real-time RT-PCR analysis showed that gFBXO38t1 was widely expressed in various chicken tissues,with a relatively higher expression level in the pancreas,ileum and abdominal fat tissue.In addition,gFBXO38t1 was expressed in all the chicken abdominal fat tissues used in the present study,and at 3 and 4 weeks of age,the gFBXO38t1 expression in lean males was significantly greater than that in fat males(P 0.05) and no significant difference was observed at the other ages(P 0.05).The gFBXO38t1 expression decreased followed by the differentiation of chicken preadipocytes induced by oleate,and gFBXO38t1 expressed more highly in chicken preadipocytes than in mature adipocytes(P 0.05),suggesting that gFBXO38t1 might play a negative role in the chicken adipogenesis.Additionally,The luciferase reporter assay showed that gFBXO38t1 overexpression inhibited the promoter activities of chicken CCAAT/enhancer-binding protein α(C/EBPα),lipoprotein lipase(LPL),fatty acid synthase(FASN) and fatty acid-binding protein 4(FABP4).The combined overexpression of gKLF7 and gFBXO38t1 did not lead to an enhanced ability for gFBXO38t1 to regulate the promoter activities of chicken LPL,FABP4 and FASN,indicating that gFBXO38t1 may regulate promoter activities of these genes through a KLF7-independent manner.The current study provides evidence that the gFBXO38t1 is involved in chicken adipogenesis.