Integrated Transcriptomic-Metabolomic Analysis Reveals the Effect of Different Light Intensities on Ovarian Development in Chickens.

Light is a key environmental factor regulating reproduction in avians. However, the mechanism of light intensity regulating ovarian development is still unclear. In this study, 5-week-old (5 wk) partridge broiler breeders were randomly divided into a low-light-intensity group (LL group) and a natural-light-intensity group (NL group) (n = 100). In the rearing period (5 wk to 22 wk), the light intensity of the LL group and NL group were 0.41 ± 0.05 lux and 45.39 ± 1.09 lux, and in the laying period (23 wk to 32 wk) they were 23.92 ± 0.06 lux and 66.93 ± 0.76 lux, respectively. Samples were collected on 22 wk and 32 wk. The results showed that the LL group had a later age at first egg and a longer laying period than the NL group. Serum P4 and LH levels in the LL group were higher than in the NL group on 22 wk (p < 0.05). On 32 wk, P4, E2, LH and FSH levels in the LL group were lower than in the NL group (p < 0.05). Ovarian transcriptomics and metabolomics identified 128 differentially expressed genes (DEGs) and 467 differential metabolites (DMs) on 22 wk; 155 DEGs and 531 DMs on 32 wk between two groups. An enrichment analysis of these DEGs and DMs identified key signaling pathways, including steroid hormone biosynthesis, neuroactive ligand-receptor interaction. In these pathways, genes such as CYP21A1, SSTR2, and NPY may regulate the synthesis of metabolites, including tryptamine, triglycerides, and phenylalanine. These genes and metabolites may play a dominant role in the light-intensity regulation of ovarian development and laying performance in broiler breeders.

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

BACKGROUND: Previous 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. RESULTS: The 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. CONCLUSIONS: In 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.

Exploring the association between fat-related traits in chickens and the RGS16 gene: insights from polymorphism and functional validation analysis.

Introduction: Excessive fat deposition in chickens can lead to reduced feed utilization and meat quality, resulting in significant economic losses for the broiler industry. Therefore, reducing fat deposition has become an important breeding objective in addition to achieving high broiler weight, growth rate, and feed conversion efficiency. In our previous studies, we observed high expression of Regulators of G Protein Signaling 16 Gene (RGS16) in high-fat individuals. This led us to speculate that RGS16 might be involved in the process of fat deposition in chickens. Methods: Thus, we conducted a polymorphism and functional analysis of the RGS16 gene to investigate its association with fat-related phenotypic traits in chickens. Using a mixed linear model (MLM), this study explored the relationship between RGS16 gene polymorphisms and fat-related traits for the first time. We identified 30 SNPs of RGS16 in a population of Wens Sanhuang chickens, among which 8 SNPs were significantly associated with fat-related traits, including sebum thickness (ST), abdominal fat weight (AFW), and abdominal fat weight (AFR). Furthermore, our findings demonstrated that AFW, AFR, and ST showed significant associations with at least two or more out of the eight identified SNPs of RGS16. We also validated the role of RGS16 in ICP-1 cells through various experimental methods, including RT-qPCR, CCK- 8, EdU assays, and oil red O staining. Results: Our functional validation experiments showed that RGS16 was highly expressed in the abdominal adipose tissue of high-fat chickens and played a critical role in the regulation of fat deposition by promoting preadipocyte differentiation and inhibiting their proliferation. Taken together, our findings suggest that RGS16 polymorphisms are associated with fat-related traits in chickens. Moreover, the ectopic expression of RGS16 could inhibit preadipocyte proliferation but promote preadipocyte differentiation. Discussion: Based on our current findings, we propose that the RGS16 gene could serve as a powerful genetic marker for marker-assisted breeding of chicken fat-related traits.

Detection of CD36 gene polymorphism associated with chicken carcass traits and skin yellowness.

Investigations into the association between chicken traits and genetic variations provide helpful breeding information to improve production performance and economic benefits in chickens. The single nucleotide polymorphism technique is an important method in agricultural molecular breeding. In this study, we detected 11 SNPs in the CD36 gene, 2 SNPs (g.-1974 A>G, g.-1888 T>C) located in the 5' flanking regions, 8 SNPs (g.23496 G>A, g.23643 C>T, g.23931 T>C, g.23937 G>A, g.31256 C>A, g.31258 C>T, g.31335 C>T, g.31534 A>C) located in the intron region, 1 SNPs (g.23743 G>T) located in the exon region and it belongs to synonymous mutation. In SNPs g.23743 G>T, the abdominal fat weight and abdominal fat weight rate of the GG genotype were lower than that of the TT genotype. In SNPs g.23931 T>C, the full-bore weight rate and half-bore weight rate of the TT genotype were higher compared with the CC genotype. And the SNPs g.-1888 T>C, g.23496 G>A, g.23643 C>T, g.31335 C>T and g.31534 A>C were significantly associated with skin yellowness traits, the cloacal skin yellowness before slaughter of the TT genotype was higher than that of the TC and CC genotype in SNPs g.-1888 T>C. Furthermore, 3 haplotypes of the above eleven SNPs were calculated and they correlated with heart weight, stomach weight, wing weight, leg skin yellowness and shin skin yellowness before slaughter. Finally, the CD36 expression profile displayed the expression pattern of CD36 mRNA variation in different tissues.

The Role of Chicken Prolactin, Growth Hormone and Their Receptors in the Immune System.

Prolactin (PRL) and growth hormone (GH) exhibit important roles in the immune system maintenance. In poultry, PRL mainly plays its roles in nesting, hatching, and reproduction, while GH is primarily responding to body weight, fat formation and feed conversion. In this review, we attempt to provide a critical overview of the relationship between PRL and GH, PRLR and GHR, and the immune response of poultry. We also propose a hypothesis that PRL, GH and their receptors might be used by viruses as viral receptors. This may provide new insights into the pathogenesis of viral infection and host immune response.

Growth hormone receptor gene influences mitochondrial function and chicken lipid metabolism by AMPK-PGC1α-PPAR signaling pathway.

BACKGROUND: Adipose tissue is an important endocrine and energy-storage organ in organisms, and it plays a crucial role in the energy-metabolism balance. Previous studies have found that sex-linked dwarf (SLD) chickens generally have excessively high abdominal fat deposition during the growing period, which increases feeding costs. However, the underlying mechanism of this fat deposition during the growth of SLD chickens remains unknown. RESULTS: The Oil Red O staining showed that the lipid-droplet area of SLD chickens was larger than that of normal chickens in E15 and 14d. Consistently, TG content in the livers of SLD chickens was higher than that of normal chickens in E15 and 14d. Further, lower ΔΨm and lower ATP levels and higher MDA levels were observed in SLD chickens than normal chickens in both E15 and 14d. We also found that overexpression of GHR reduced the expression of genes related to lipid metabolism (AMPK, PGC1α, PPARγ, FAS, C/EBP) and oxidative phosphorylation (CYTB, CYTC, COX1, ATP), as well as reducing ΔΨm and ATP levels and increasing MDA levels. In addition, overexpression of GHR inhibited fat deposition in CPPAs, as measured by Oil Red O staining. On the contrary, knockdown of GHR had the opposite effects in vitro. CONCLUSIONS: In summary, we demonstrate that GHR promotes mitochondrial function and inhibits lipid peroxidation as well as fat deposition in vivo and in vitro. Therefore, GHR is essential for maintaining the stability of lipid metabolism and regulating mitochondrial function in chicken.

Growth Hormone Receptor Controls Adipogenic Differentiation of Chicken Bone Marrow Mesenchymal Stem Cells by Affecting Mitochondrial Biogenesis and Mitochondrial Function.

Growth hormone receptor (GHR) can activate several signaling pathways after binding to growth hormone (GH) to regulate cell growth and development. Sex-linked dwarf (SLD) chickens, normal protein functions are prevented because of exon mutations in the GHR gene, have more severe fat deposition. However, the specific molecular mechanisms responsible for this phenotype remains unclear. We therefore investigated the effect of the GHR gene on adipogenic differentiation of chicken bone marrow mesenchymal stem cells (BMSCs). We found that bone marrow fat deposition was more severe in SLD chickens compared to normal chickens, and the expression of genes related to adipogenic differentiation was enhanced in SLD chicken BMSCs. We also detected enhanced mitochondrial function of BMSCs in SLD chickens. In vitro, overexpression of GHR in chicken BMSCs increased mitochondrial membrane potential but decreased reactive oxygen and ATP contents, oxidative phosphorylation complex enzyme activity, and mitochondrial number. Expression of genes associated with mitochondrial biogenesis and function was repressed during adipogenic differentiation in chicken BMSCs, the adipogenic differentiation capacity of chicken BMSCs was also repressed. With knockdown of GHR, opposite results were observed. We concluded that GHR inhibited adipogenic differentiation of chicken BMSCs by suppressing mitochondrial biogenesis and mitochondrial function.

gga-miR-200b-3p promotes avian leukosis virus subgroup J replication via targeting dual-specificity phosphatase 1.

MicroRNAs (miRNAs) involved host-virus interaction, affecting the replication or pathogenesis of several viruses. Although avian leukosis virus subgroup J (ALV-J) has been one of the most studied avian viruses, the effects of various host miRNAs on ALV-J infection and its underlying molecular mechanisms are still unclear. Here, we reported that gga-miR-200b-3p acts as a positive host factor enhancing ALV-J replication. We found that gga-miR-200b-3p was increased in response to ALV-J infection in host cells, and that gga-miR-200b-3p effectively enhanced ALV-J replication via targeting host protein dual-specificity phosphatase 1 (DUSP1). Collectively, these findings highlight a crucial role of gga-miR-200b-3p in ALV-J replication.

TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.

BACKGROUND: Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up-regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration. METHODS: RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual-luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182-knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co-immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione-S-transferse pull down, protein purification, and RNA immunoprecipitation assays. RESULTS: TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182-knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387-470) and a conserved region (aa 52-62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK-ERK and FAK-Akt signalling axes during myogenesis. CONCLUSIONS: Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.

MicroRNAs Involved in Oxidative Stress Processes Regulating Physiological and Pathological Responses.

Oxidative stress influences several physiological and pathological cellular events, including cell differentiation, excessive growth, proliferation, apoptosis, and inflammatory response. Therefore, oxidative stress is involved in the pathogenesis of various diseases, including pulmonary fibrosis, epilepsy, hypertension, atherosclerosis, Parkinson's disease, cardiovascular disease, and Alzheimer's disease. Recent studies have shown that several microRNAs (miRNAs) are involved in the development of various diseases caused by oxidative stress and that miRNAs may be useful to determine the inflammatory characteristics of immune responses during infection and disease. In this review, we describe the known effects of miRNAs on reactive oxygen species to induce oxidative stress and miRNA regulatory mechanisms involved in the uncoupling of Keap1-Nrf2 complexes. Finally, we summarized the functions of miRNAs in several antioxidant genes. Understanding the crosstalk between miRNAs and oxidative stress-inducing factors during physiological and pathological cellular events may have implications for the design of more effective treatments for immune diseases.

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.

Characterization of Chicken Skin Yellowness and Exploration of Genes Involved in Skin Yellowness Deposition in Chicken.

Skin color is an important economic trait in meat-type chickens. A uniform bright skin color can increase the sales value of chicken. Chickens with bright yellow skin are more popular in China, especially in the broiler market of South China. However, the skin color of chickens can vary because of differences in breeds, diet, health, and individual genetics. To obtain greater insight into the genetic factors associated with the process of skin pigmentation in chickens, we used a colorimeter and high-resolution skin photographs to measure and analyze the skin color of chickens. By analyzing 534 chickens of the same breed, age, and feed condition, we found that the yellowness values of the chickens varied within this population. A significant positive correlation was found between the cloacal skin yellowness values before and after slaughter, and the cloacal skin yellowness value of live chickens was positively correlated with the overall body skin yellowness value. Additionally, chicken skin yellowness exhibited low heritability, ranging from 0.07 to 0.27. Through RNA sequencing, 882 genes were found to be differentially expressed between the skin with the highest and lowest yellowness values. Some of these differentially expressed genes may play an important role in yellow pigment deposition in chicken skin, which included TLR2B, IYD, SMOC1, ALDH1A3, CYP11A1, FHL2, TECRL, ACACB, TYR, PMEL, and GPR143. In addition, we found that the expression and variations of the BCO2 gene, which is referred to as the yellow skin gene, cannot be used to estimate the skin yellowness value of chickens in this population. These data will help to further our understanding of chicken skin yellowness and might contribute to the selection of specific chicken strains with consistent skin coloration.

High expression of BCL6 inhibits the differentiation and development of hematopoietic stem cells and affects the growth and development of chickens.

BACKGROUND: B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that represses more than 1200 potential target genes. Our previous study showed that a decline in blood production in runting and stunting syndrome (RSS) affected sex-linked dwarf (SLD) chickens compared to SLD chickens. However, the association between BCL6 gene and hematopoietic function remains unknown in chickens. METHODS: In this study, we used RSS affected SLD (RSS-SLD) chickens, SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells (HSCs), to investigate the effect of the BCL6 on differentiation and development of HSCs. RESULTS: The results showed that comparison of RSS-SLD chickens with SLD chickens, the BCL6 was highly expressed in RSS-SLD chickens bone marrow. The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow, bone density was reduced, and the levels of immature erythrocytes in peripheral blood were increased. At the same time, the hematopoietic function of HSCs decreased in RSS-SLD chickens, which was manifested by a decrease in the hematopoietic growth factors (HGFs) EPO, SCF, TPO, and IL-3, as well as hemoglobin α1 and hemoglobin ß expression. Moreover, mitochondrial function in the HSCs of RSS-SLD chickens was damaged, including an increase in ROS production, decrease in ATP concentration, and decrease in mitochondrial membrane potential (ΔΨm). The same results were also observed in SLD chickens compared with normal chickens; however, the symptoms were more serious in RSS-SLD chickens. Additionally, after overexpression of the BCL6 in primary HSCs, the secretion of HGFs (EPO, SCF, TPO and IL-3) was inhibited and the expression of hemoglobin α1 and hemoglobin ß was decreased. However, cell proliferation was accelerated, apoptosis was inhibited, and the HSCs entered a cancerous state. The function of mitochondria was also abnormal, ROS production was decreased, and ATP concentration and ΔΨm were increased, which was related to the inhibition of apoptosis of stem cells. CONCLUSIONS: Taken together, we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function, resulting in impaired growth and development of chickens. Moreover, the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb, pale skin, stunted growth and development, and the tendency to appear RSS in SLD chickens.

gga-miR-200b-3p Promotes Macrophage Activation and Differentiation via Targeting Monocyte to Macrophage Differentiation-Associated in HD11 Cells.

MicroRNAs (miRNAs) play a critical role in various biological processes through regulation of gene expression post-transcriptionally. Although miRNAs are involved in cell proliferation and differentiation in mammals, few reports regarding the effects of host miRNAs on macrophage activation and differentiation are available in birds. Here, we reported that gga-miR-200b-3p acts as a positive regulator, enhancing macrophage activation and differentiation using an avian model. We found that ectopic expression of gga-miR-200b-3p in HD11 cells enhances the amount of MHC-II-positive cells and promotes the expression of pro-inflammatory cytokines and that gga-miR-200b-3p directly targets monocyte to macrophage differentiation-associated (MMD). The inhibition of MMD by gga-miR-200b-3p enhances the activation and differentiation of HD11 cells and increases the expression of pro-inflammatory cytokines. Collectively, these findings highlight a crucial role of gga-miR-200b-3p in macrophage activation and differentiation in birds.

Mutation of TWNK Gene Is One of the Reasons of Runting and Stunting Syndrome Characterized by mtDNA Depletion in Sex-Linked Dwarf Chicken.

Runting and stunting syndrome (RSS), which is characterized by low body weight, generally occurs early in life and leads to considerable economic losses in the commercial broiler industry. Our previous study has suggested that RSS is associated with mitochondria dysfunction in sex-linked dwarf (SLD) chickens. However, the molecular mechanism of RSS remains unknown. Based on the molecular diagnostics of mitochondrial diseases, we identified a recessive mutation c. 409G > A (p. Ala137Thr) of Twinkle mitochondrial DNA helicase (TWNK) gene and mitochondrial DNA (mtDNA) depletion in RSS chickens' livers from strain N301. Bioinformatics investigations supported the pathogenicity of the TWNK mutation that is located on the extended peptide linker of Twinkle primase domain and might further lead to mtDNA depletion in chicken. Furthermore, overexpression of wild-type TWNK increases mtDNA copy number, whereas overexpression of TWNK A137T causes mtDNA depletion in vitro. Additionally, the TWNK c. 409G > A mutation showed significant associations with body weight, daily gain, pectoralis weight, crureus weight, and abdominal fat weight. Taken together, we corroborated that the recessive TWNK c. 409G > A (p. Ala137Thr) mutation is associated with RSS characterized by mtDNA depletion in SLD chicken.

Integrative Analyses of mRNA Expression Profile Reveal SOCS2 and CISH Play Important Roles in GHR Mutation-Induced Excessive Abdominal Fat Deposition in the Sex-Linked Dwarf Chicken.

Sex-linked dwarf (SLD) chicken, which is caused by a recessive mutation of the growth hormone receptor (GHR), has been widely used in the Chinese broiler industry. However, it has been found that the SLD chicken has more abdominal fat deposition than normal chicken. Excessive fat deposition not only reduced the carcass quality of the broilers but also reduced the immunity of broilers to diseases. To find out the key genes and the precise regulatory pathways that were involved in the GHR mutation-induced excessive fat deposition, we used high-fat diet (HFD) and normal diet to feed the SLD chicken and normal chicken and analyzed the differentially expressed genes (DEGs) among the four groups. Results showed that the SLD chicken had more abdominal fat deposition and larger adipocytes size than normal chicken and HFD can promote abdominal fat deposition and induce adipocyte hypertrophy. RNA sequencing results of the livers and abdominal fats from the above chickens revealed that many DEGs between the SLD and normal chickens were enriched in fat metabolic pathways, such as peroxisome proliferator-activated receptor (PPAR) signaling, extracellular matrix (ECM)-receptor pathway, and fatty acid metabolism. Importantly, by constructing and analyzing the GHR-downstream regulatory network, we found that suppressor of cytokine signaling 2 (SOCS2) and cytokine-inducible SH2-containing protein (CISH) may involve in the GHR mutation-induced abdominal fat deposition in chicken. The ectopic expression of SOCS2 and CISH in liver-related cell line leghorn strain M chicken hepatoma (LMH) cell and immortalized chicken preadipocytes (ICP) revealed that these two genes can regulate fatty acid metabolism, adipocyte differentiation, and lipid droplet accumulation. Notably, overexpression of SOCS2 and CISH can rescue the hyperactive lipid metabolism and excessive lipid droplet accumulation of primary liver cell and preadipocytes that were isolated from the SLD chicken. This study found some genes and pathways involved in abdominal fat deposition of the SLD chicken and reveals that SOCS2 and CISH are two key genes involved in the GHR mutation-induced excessive fat deposition of the SLD chicken.

miR-429-3p/LPIN1 Axis Promotes Chicken Abdominal Fat Deposition via PPARγ Pathway.

To explore the regulatory mechanism of abdominal fat deposition in broilers, 100-day-old Sanhuang chickens (n = 12) were divided into high-fat and low-fat groups, according to the abdominal fat ratio size. Total RNA isolated from the 12 abdominal fat tissues was used for miRNA and mRNA sequencing. Results of miRNA and mRNA sequencing revealed that miR-429-3p was highly expressed in high-fat chicken whereas LPIN1 expression was downregulated. Further, we determined that miR-429-3p promoted preadipocyte proliferation and differentiation, whereas LPIN1 exerted an opposite effect. Notably, we found that the miR-429-3p/LPIN1 axis facilitated PPARγ pathway activation, which is closely associated with the progression of adipogenesis. In conclusion, our results provide evidence that a novel miR-429-3p/LPIN1 axis is involved in the regulation of adipogenesis, which may have a guiding role in the improvement of breeding for abdominal fat traits in broiler chickens.

[The cell cycle pathway regulates chicken abdominal fat deposition as revealed by transcriptome sequencing].

With the improvement of growth traits and feed conversion rate, the abdominal fat rate of Chinese local breeds of broilers has been increasing. Excessive abdominal fat deposition not only reduces the slaughter rate and disease resistance of broiler chickens, but also produces waste due to the difficulty of fat treatment. In order to study the regulatory genes and pathways involved in abdominal fat deposition of broilers, we used high-fat diets to feed the Xinghua Chicken, which is a Chinese local breed. Two weeks after feeding, we found that the abdominal fat weight and rate of broilers in the high-fat diet group increased significantly, and the diameter and area of abdominal fat cells also increased significantly. Transcriptome sequencing of abdominal fat and livers showed that the differentially expressed genes in the abdominal fat were mainly enriched in the cell cycle, peroxisome proliferator- activated receptor (PPAR) and extracellular matrix (ECM) receptor signaling pathways. The differentially expressed genes in livers were also significantly enriched in the cell cycle pathway, as well as in the steroid biosynthesis and PPAR signaling pathway. By analyzing the common differentially expressed genes in abdominal fat and liver tissues, we found that these genes were also enriched in cell cycle. Finally, we used the chicken LMH (chicken hepatoma cell) cell line and chicken ICP (immortalized chicken preadipocytes) cell line to do the in vitro validation assays. We used high-fat and common medium to culture the cells. The results showed that after 48 hours, the high-fat medium could significantly promote cell cycle and increase the number of cells in S phase. Additionally, qRT-PCR results showed that the high-fat medium could significantly promote the expression of genes related to cell cycle. In conclusion, we found that high-fat diets activate the cell cycle progression of chicken hepatocytes and preadipocytes, promote cell proliferation, and then increase abdominal fat deposition.

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.

Growth Hormone Receptor Gene is Essential for Chicken Mitochondrial Function In Vivo and In Vitro.

The growth hormone receptor (GHR) gene is correlated with many phenotypic and physiological alternations in chicken, such as shorter shanks, lower body weight and muscle mass loss. However, the role of the GHR gene in mitochondrial function remains unknown in poultry. In this study, we assessed the function of mitochondria in sex-linked dwarf (SLD) chicken skeletal muscle and interfered with the expression of GHR in DF-1 cells to investigate the role of the GHR gene in chicken mitochondrial function both in vivo and in vitro. We found that the expression of key regulators of mitochondrial biogenesis and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (OXPHOS) genes were downregulated and accompanied by reduced enzymatic activity of OXPHOS complexes in SLD chicken skeletal muscle and GHR knockdown cells. Then, we assessed mitochondrial function by measuring mitochondrial membrane potential (ΔΨm), mitochondrial swelling, reactive oxygen species (ROS) production, malondialdehyde (MDA) levels, ATP levels and the mitochondrial respiratory control ratio (RCR), and found that mitochondrial function was impaired in SLD chicken skeletal muscle and GHR knockdown cells. In addition, we also studied the morphology and structure of mitochondria in GHR knockdown cells by transmission electron microscopy (TEM) and MitoTracker staining. We found that knockdown of GHR could reduce mitochondrial number and alter mitochondrial structure in DF-1 cells. Above all, we demonstrated for the first time that the GHR gene is essential for chicken mitochondrial function in vivo and in vitro.