Methionine Promotes Milk Protein Synthesis via the PI3K-mTOR Signaling Pathway in Human Mammary Epithelial Cells.

Breast milk is widely considered to be the most natural, safe, and complete food for infants. However, current breastfeeding rates fall short of the recommendations established by the World Health Organization. Despite this, there are few studies that have focused on the promotion of human lactation through nutrient supplementation. Therefore, the aim of this study was to investigate the effect of methionine on milk synthesis in human mammary epithelial cells (MCF-10A cells) and to explore the underlying mechanisms. To achieve this, MCF-10A cells were cultured with varying concentrations of methionine, ranging from 0 to 1.2 mM. Our results indicated that 0.6 mM of methionine significantly promoted the synthesis of milk protein. An RNA-seq analysis revealed that methionine acted through the PI3K pathway. This finding was validated through real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. In addition, PI3K inhibition assays confirmed that methionine upregulated the expression of both mTOR and p-mTOR through activation of PI3K. Taken together, these findings suggest that methionine positively regulates milk protein synthesis in MCF-10A cells through the PI3K-mTOR signaling pathway.

Syndecan-3 Coregulates Milk Fat Metabolism and Inflammatory Reactions in Bovine Mammary Epithelial Cells through AMPK/SIRT1 Signaling Pathway.

Transcriptome sequencing showed that syndecan-3 (SDC3) was differentially expressed in high-fat and low-fat mammary epithelial cells of Chinese Holstein cows. Previous studies found that SDC3 plays an important role in inflammatory diseases and virus infection. However, those studies did not confirm whether or not the functional gene SDC3, which plays an important role in regulating milk fat metabolism, has an effect on susceptibility to breast tissue diseases. Therefore, we studied the effects of SDC3 on milk lipid metabolism and inflammation in bovine mammary epithelial cells (BMECs) and further explored the common regulatory pathway of SDC3 in both. The overexpression of SDC3 increased the contents of triglycerides and cholesterol, reduced the content of non-esterified fatty acids, inhibited the expression of inflammatory factors (IL-6, IL-1ß, TNF-α and COX-2), and reduced the production of ROS in BMECs. However, silenced SDC3 had the opposite effect. Further exploring the mechanisms of SDC3, we found that SDC3 upregulated the expression of peroxisome proliferator-activated receptor gamma (PPARG) through the AMPK/SIRT1 signal pathway to promote milk fat synthesis. It also regulated the activation of the NF-κB pathway through the AMPK/SIRT1 signal pathway, reducing the expression of inflammatory factors and ROS production, thus inhibiting the inflammatory response of BMECs. Nuclear factor kappa B subunit 1 (NF-κB p50) was an important target of SDC3 in this process. To sum up, our results showed that SDC3 coregulated milk fat metabolism and inflammation through the AMPK/SIRT1 signaling pathway. This study laid a foundation for the comprehensive evaluation of breeding value based on multi-effect functional genes in dairy cow molecular breeding.

Effects of Adipose Tissue-Specific Knockout of Delta-like Non-Canonical Notch Ligand 1 on Lipid Metabolism in Mice.

Delta-like non-canonical Notch ligand 1 (DLK1), which inhibits the differentiation of precursor adipocytes, is a recognized marker gene for precursor adipocytes. Lipids play a crucial role in energy storage and metabolism as a vital determinant of beef quality. In this study, we investigated the mechanism of the DLK1 gene in lipid metabolism by constructing adipose tissue-specific knockout mice. We examined some phenotypic traits, including body weight, liver coefficient, fat index, the content of triglyceride (TG) and cholesterol (CHOL) in abdominal white adipose tissue (WAT) and blood. Subsequently, the fatty acid content and genes related to lipid metabolism expression were detected in DLK1-/- and wild-type mice via GC-MS/MS analysis and quantitative real-time PCR (qRT-PCR), respectively. The results illustrated that DLK1-/- mice exhibited significant abdominal fat deposition compared to wild-type mice. HE staining and immunohistochemistry (IHC) results showed that the white adipocytes of DLK1-/- mice were larger, and the protein expression level of DLK1-/- was significantly lower. Regarding the blood biochemical parameters of female mice, DLK1-/- mice had a strikingly higher triglyceride content (p < 0.001). The fatty acid content in DLK1-/- mice was generally reduced. There was a significant reduction in the expression levels of the majority of genes that play a crucial role in lipid metabolism. This study reveals the molecular regulatory mechanism of fat metabolism in mice and provides a molecular basis and reference for the future application of the DLK1 gene in the breeding of beef cattle with an excellent meat quality traits. It also provides a molecular basis for unravelling the complex and subtle relationship between adipose tissue and health.

Chitosan Oligosaccharide Inhibits the Synthesis of Milk Fat in Bovine Mammary Epithelial Cells through AMPK-Mediated Downstream Signaling Pathway.

Chitosan oligosaccharide (COS) is a variety of oligosaccharides, and it is also the only abundant basic amino oligosaccharide in natural polysaccharides. Chitosan oligosaccharide is a low molecular weight product of chitosan after enzymatic degradation. It has many biological effects, such as lipid-lowering, antioxidant and immune regulation. Previous studies have shown that chitosan oligosaccharide has a certain effect on fat synthesis, but the effect of chitosan oligosaccharide on milk fat synthesis of bovine mammary epithelial cells (BMECs) has not been studied. Therefore, this study aimed to investigate chitosan oligosaccharide's effect on milk fat synthesis in bovine mammary epithelial cells and explore the underlying mechanism. We treated bovine mammary epithelial cells with different concentrations of chitosan oligosaccharide (0, 100, 150, 200, 400 and 800 µg/mL) for 24 h, 36 h and 48 h respectively. To assess the effect of chitosan oligosaccharide on bovine mammary epithelial cells and determine the concentration and time for chitosan oligosaccharide treatment on cells, several in vitro cellular experiments, including on cell viability, cycle and proliferation were carried out. The results highlighted that chitosan oligosaccharide (100, 150 µg/mL) significantly promoted cell viability, cycle and proliferation, increased intracellular cholesterol content, and reduced intracellular triglyceride and non-esterified fatty acids content. Under the stimulation of chitosan oligosaccharide, the expression of genes downstream of Phosphorylated AMP-activated protein kinase (P-AMPK) and AMP-activated protein kinase (AMPK) signaling pathway changed, increasing the expression of peroxisome proliferator-activated receptor alpha (PPARα) and hormone-sensitive lipase (HSL), but the expression of sterol regulatory element-binding protein 1c (SREBP1) and its downstream target gene stearoyl-CoA desaturase (SCD1) decreased. In conclusion, these results suggest that chitosan oligosaccharide may inhibit milk fat synthesis in bovine mammary epithelial cells by activating the AMP-activated protein kinase signaling pathway, promoting the oxidative decomposition of fatty acids and inhibiting fatty acid synthesis.

Transcriptome analysis of CRISPR/Cas9-mediated GPAM-/- in bovine mammary epithelial cell-line unravelled the effects of GPAM gene on lipid metabolism.

Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is an enzyme in animal lipid metabolism pathways that catalyzes the initial and most committed step of glycerolipid biosynthesis. The present study mainly focused on exploring the relationship between the GPAM gene and the lipid metabolism of mammary epithelial cells and the effect of GPAM on the related pathways of lipid metabolism. The GPAM gene was knocked out entirely in bovine mammary epithelial cells(BMECs) using CRISPR/Cas9 technology, and the mechanism by which the GPAM gene regulates lipid metabolism in BMECs was confirmed. Furthermore, after the complete loss of GPAM, BMECs' triglycerides (TGs) and cholesterol (CHOL) levels were significantly decreased (p < 0.05). Concurrently, the content of octanoic acid, a medium-chain saturated fatty acid, increased substantially in BMECs. RNA-seq of GPAM-/- BMECs revealed that GPAM could affect the expression of genes related to lipid metabolism, downregulated the expression of Acyl-CoA synthetase long-chain family member 5 (ACSL5), Fatty Acid Binding Protein 3 (FABP3), Hormone-sensitive lipase (HSL), Protease, serine-2 (PRSS2), 1-Acylglycerol-3-Phosphate O Acyltransferase 4 (AGPAT4), and regulated the milk synthesis metabolism pathway.The findings revealed that a number of genes were expressed, a number of genes were differentially expressed genes (DEGs), and a number of GO terms were enriched, with a number of GO terms considerably increased. Further, the differentially expressed genes (DEGs) were significantly enriched in Fat digestion and absorption pathway, Fatty acid metabolic pathway, Biosynthesis of unsaturated fatty acids, Biosynthesis of unsaturated fatty acids and steroids, NF-kappa B signalling pathway, MAPK signalling pathway. In conclusion, the current research results show that GPAM is a crucial regulator of BMEC lipid metabolism. GPAM-/- BMEC may also become useful genetic materials and tools for future research on gene functions related to lipid and fatty acid metabolism. This study will contribute to the discovery of gene regulation and molecular mechanisms in milk fat synthesis.

RNA-Seq Analysis Identifies Differentially Expressed Genes in the Longissimus dorsi of Wagyu and Chinese Red Steppe Cattle.

Meat quality has a close relationship with fat and connective tissue; therefore, screening and identifying functional genes related to lipid metabolism is essential for the production of high-grade beef. The transcriptomes of the Longissimus dorsi muscle in Wagyu and Chinese Red Steppe cattle, breeds with significant differences in meat quality and intramuscular fat deposition, were analyzed using RNA-seq to screen for candidate genes associated with beef quality traits. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the 388 differentially expressed genes (DEGs) were involved in biological processes such as short-chain fatty acid metabolism, regulation of fatty acid transport and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In addition, crystallin alpha B (CRYAB), ankyrin repeat domain 2 (ANKRD2), aldehyde dehydrogenase 9 family member A1 (ALDH9A1) and enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) were investigated for their effects on intracellular triglyceride and fatty acid content and their regulatory effects on genes in lipogenesis and fatty acid metabolism pathways. This study generated a dataset from transcriptome profiling of two cattle breeds, with differing capacities for fat-deposition in the muscle, and revealed molecular evidence that CRYAB, ANKRD2, ALDH9A1 and EHHADH are related to fat metabolism in bovine fetal fibroblasts (BFFs). The results provide potential functional genes for maker-assisted selection and molecular breeding to improve meat quality traits in beef cattle.

The effect of CPT1B gene on lipid metabolism and its polymorphism analysis in Chinese Simmental cattle.

Carnitine palmitoyltransferase 1B (CPT1B) is a candidate gene that regulates livestock animal lipid metabolism and encodes the rate-limiting enzyme in fatty acid ß-oxidation. To explore the effect of this gene on lipid metabolism in cattle, this study examined CPT1B gene polymorphism in Chinese Simmental cattle and the effect of CPT1B on lipid metabolism. The results showed that the triglyceride content increased significantly with increasing CPT1B gene expression in bovine fetal fibroblasts (BFFs) (p < 0.05), while CPT1B knockout led to decreased CPT1B expression and a downward trend in triglyceride levels. Correlation analysis showed a significant association between the g.119896238 G > C locus and Chinese Simmental cattle backfat thickness (p < 0.05). Backfat thickness was significantly greater in individuals with the GC genotype (0.93 ± 0.67 cm) than in those with the CC genotype (0.84 ± 0.60 cm). The g.119889302 T > C locus was significantly correlated with arachidonic acid content in Chinese Simmental cattle (p < 0.05). The arachidonic acid content in the longissimus muscle was significantly higher in CC genotype beef cattle (0.054 g/100 g) than in those with the other two genotypes (0.046 g/100 g, 0.049 g/100 g). These molecular markers can be effectively used for marker-assisted selection in cattle breeding.

Comprehensive Analysis of miRNAs and Target mRNAs between Immature and Mature Testis Tissue in Chinese Red Steppes Cattle.

This study aims to screen potential regulators and regulate fecundity networks between microRNAs (miRNAs) and target genes. The bovine testes of immature and mature Chinese Red Steppes were performed by genome-wide analysis of mRNAs and miRNAs. Compared with testicular tissues of newborns, 6051 upregulated genes and 7104 downregulated genes in adult cattle were identified as differentially expressed genes (DEGs). The DEGs were significantly enriched in 808 GO terms (p < 0.05) including male gonad development, male genitalia development, spermatogenesis, and sperm motility. Moreover, DEGs were also significantly enriched in 105 KEGG pathways (p < 0.05), including cGMP-PKG signaling pathway and calcium signaling pathway. To explore the expression of miRNA-regulated gene expression, 896 differentially expressed target genes negatively regulated with the expression levels of 31 differentially expressed miRNAs (DERs) were predicted and analyzed, and a network-integrated analysis was constructed. Furthermore, real-time PCR was performed to verify the expression levels of DEGs and DERs. Our results identified novel candidate DEGs and DERs correlated with male reproduction and intricate regulating networks between miRNAs and genes, which will be valuable for future genetic and epigenetic studies of sperm development and maturity, as well as providing valuable insights into the molecular mechanisms of male fertility and spermatogenesis in cattle.

Transcriptomic Analysis of Short/Branched-Chain Acyl-Coenzyme a Dehydrogenase Knocked Out bMECs Revealed Its Regulatory Effect on Lipid Metabolism.

The acyl-CoA dehydrogenase family of enzymes includes short/branched-chain acyl-CoA dehydrogenase (ACADSB), which catalyzes the dehydrogenation of acyl-CoA derivatives in fatty acid metabolism. Our previous findings suggested that ACADSB was a critical candidate gene affecting milk fat synthesis by comparing the transcriptome in bovine mammary epithelial cells (bMECs) from Chinese Holstein dairy cows producing high-fat and low-fat milk as well as gene functional validation studies on the cellular level. In the present study, ACADSB in bMECs was knocked out (KO) using a CRISPR/Cas9 system, and mRNA transcriptome was further sequenced to verify the function of the ACADSB gene and analyze its correlation with lipid metabolism. The findings revealed that 15,693 genes were expressed, 1,548 genes were differentially expressed genes (DEGs), and 6,098 GO terms were enriched, of which 637 GO terms were greatly enhanced, such as phospholipid-translocation ATPase activity (GO:0004012), lipoprotein lipase activity (GO:0004465), acyl-CoA desaturase activity (GO:0016215), and so on. The analysis by KEGG showed that DEGs were distributed over 247 pathogens, of which 49 were significantly enriched, including the metabolism of fatty acids (PATH: 01212), metabolism of glycerolipid (PATH: 00561), and signaling of adipocytokines (PATH: 04920). The CHOL, TGs and FFA contents in bMECs were reduced when the ACADSB gene was knocked out. The RT2 Profiler PCR array also revealed that the loss of the ACADSB gene changed the expression levels of functional genes involved in lipid metabolism, including ACADL, ACOX2, ACAT2, and FABP3. In conclusion, the current findings show that ACADSB is a key regulator of lipid metabolism in bMECs. The ACADSB-/- bMECs could also be useful genetic material and tools for future research into gene functions related to lipid and fatty acid metabolism. It will be valuable for revealing the gene regulatory roles and molecular mechanisms in milk fat synthesis.

MiRNA-34c Regulates Bovine Sertoli Cell Proliferation, Gene Expression, and Apoptosis by Targeting the AXL Gene.

MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase (AXL) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3'-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis.

Effects of polymorphism of the GPAM gene on milk quality traits and its relation to triglyceride metabolism in bovine mammary epithelial cells of dairy cattle.

Mitochondrial glycerol-3-phosphate acyltransferase (GPAM) catalyses the initial and rate-regulated first-stage pathway of glycerol lipid synthesis and helps to allocate acyl-CoA (acyl-coenzyme A) to triglyceride (TG) synthesis and away from degradation pathways in animal lipometabolism-related pathways. In this study, RNA interference (RNAi) and GPAM gene overexpression were used to examine the correlation between the expression of GPAM and adipogenesis in bovine mammary epithelial cells (bMECs). Additionally, three novel polymorphisms were identified within the bovine key functional domain of GPAM with Sanger sequencing. The relationship between variants of the GPAM gene and milk quality traits of Chinese Holstein cows was then analysed using statistical methods. The results showed that knockdown of the GPAM gene significantly reduced the synthesis of triglycerides in the bMECs ( p   <  0.05), whereas the overexpression of the GPAM gene significantly increased the synthesis of TG ( p   <  0.05). In Chinese Holstein dairy cattle, the polymorphic locus of the GPAM gene E20-3386G  >  A was significantly correlated with fat, protein and somatic cell count ( p   <  0.05); I18-652A  >  G was significantly correlated with fat, total fat content, protein, dry matter and somatic cell count ( p   <  0.05); and I18-726A  >  G was significantly correlated with protein, milk yield, dry matter and somatic cell count ( p   <  0.05). Specifically, individuals with the AA genotype of the I18-652A  >  G and E20-3386G  >  A polymorphic loci had a higher milk fat percentage ( p   <  0.05). In summary, GPAM plays a pivotal role in the intracellular regulation of triglyceride, and its mutations could work as a competent molecular marker for selective breeding in dairy cattle.

Integrative analysis of miRNAs and mRNAs revealed regulation of lipid metabolism in dairy cattle.

Lipid metabolism in bovine mammary epithelial cells has been the primary focus of the research of milk fat percentage of dairy cattle. Functional microRNAs can affect lipid metabolism by regulating the expression of candidate genes. The purpose of the study was to screen and identify differentially expressed miRNAs, candidate genes, and co-regulatory pathways related to the metabolism of milk fat. To achieve this aim, we used miRNA and transcriptome data from the mammary epithelial cells of dairy cattle with high (H, 4.85%) and low milk fat percentages (L, 3.41%) during mid-lactation. One hundred ninety differentially expressed genes and 33 differentially expressed miRNAs were significantly enriched in related regulatory networks, of which 27 candidate genes regulated by 18 differentially expressed miRNAs significantly enriched in pathways related to lipid metabolism (p < 0.05). Target relationships between PDE4D and bta-miR-148a, PEG10 and bta-miR-877, SOD3 and bta-miR-2382-5p, and ADAMTS1 and bta-miR-2425-5p were verified using luciferase reporter assays and quantitative RT-PCR. The detection of triglyceride production in BMECs showed that bta-miR-21-3p and bta-miR-148a promote triglyceride synthesis, whereas bta-miR-124a, bta-miR-877, bta-miR-2382-5p, and bta-miR-2425-5p inhibit triglyceride synthesis. The conjoint analysis could identify functional miRNAs and regulatory candidate genes involved in lipid metabolism within the co-expression networks of the dairy cattle mammary system, which contributes to the understanding of potential regulatory mechanisms of genetic element and gene signaling networks involved in milk fat metabolism.

Comparative Genome-Wide Alternative Splicing Analysis of Longissimus Dorsi Muscles Between Japanese Black (Wagyu) and Chinese Red Steppes Cattle.

Alternative splicing is a ubiquitous regulatory mechanism in gene expression that allows a single gene generating multiple messenger RNAs (mRNAs). Significant differences in fat deposition ability and meat quality traits have been reported between Japanese black cattle (Wagyu) and Chinese Red Steppes, which presented a unique model for analyzing the effects of transcriptional level on marbling fat in livestock. In previous studies, the differentially expressed genes (DGEs) in longissimus dorsi muscle (LDM) samples between Wagyu and other breeds of beef cattle have been reported. In this study, we further investigated the differences in alternative splicing in LDM between Wagyu and Chinese Red Steppes cattle. We identified several alternative splicing types including cassette exon, mutually exclusive exons, alternative 5' splice site, alternative 3' splice site, alternative start exon, and intron retention. In total, 115 differentially expressed alternatively spliced genes were obtained, of which 17 genes were enriched in the metabolic pathway. Among the 17 genes, 5 genes, including MCAT, CPT1B, HADHB, SIRT2, and DGAT1, appeared to be the novel spliced candidates that affect the lipid metabolism in cattle. Additionally, another 17 genes were enriched in the Gene Ontology (GO) terms related to muscle development, such as NR4A1, UQCC2, YBX3/CSDA, ITGA7, etc. Overall, altered splicing and expression levels of these novel candidates between Japanese black cattle and Chinese Red Steppes revealed by RNA-seq suggest their potential involvement in the muscle development and fat deposition of beef cattle.

Function Identification of Bovine ACSF3 Gene and Its Association With Lipid Metabolism Traits in Beef Cattle.

Acyl-CoA synthetase family member 3 (ACSF3) carries out the first step of mitochondrial fatty acid synthesis II, which is the linkage of malonate and, to a lesser extent, methylmalonate onto CoA. Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry that is generated and utilized in the cytoplasm. In this research, we verified the relationship between expression of the ACSF3 and the production of triglycerides (TGs) at the cellular level by silencing and over-expressing ACSF3. Subsequently, through Sanger sequencing, five polymorphisms were found in the functional domain of the bovine ACSF3, and the relationship between ACSF3 polymorphism and the economic traits and fatty acid composition of Chinese Simmental cattle was analyzed by a means of variance analysis and multiple comparison. The results illustrated that the expression of ACSF3 promoted triglyceride synthesis in bovine mammary epithelial cells and bovine fetal fibroblast cells. Further association analysis also indicated that individuals with the AG genotype (g.14211090 G > A) of ACSF3 were significantly associated with the fatty acid composition of intramuscular fat (higher content of linoleic acid, α-linolenic acid, and arachidonic acid), and that CTCAG haplotype individuals were significantly related to the fatty acid composition of intramuscular fat (higher linoleic acid content). Individuals with the AA genotypes of g.14211055 A > G and g.14211090 G > A were substantially associated with a larger eye muscle area in the Chinese Simmental cattle population. ACSF3 played a pivotal role in the regulation of cellular triacylglycerol and long-chain polyunsaturated fatty acid levels, and polymorphism could serve as a useful molecular marker for future marker-assisted selection in the breeding of intramuscular fat deposition traits in beef cattle.

miR-2382-5p Regulates Lipid Metabolism by Targeting NDRG2 in Mammary Epithelial Cells of Dairy Cattle.

microRNA is a class of single-stranded RNA molecules of about 22-24 nucleotides in length, which regulate a variety of biological processes, including lipid metabolism and triglyceride synthesis at transcriptional and translational levels by degrading target mRNAs or interfering with the protein production. In this study, the effect of miR-2382-5p on triglyceride levels was examined in bovine mammary epithelial cells (BMECs), and the results showed that miR-2382-5p could decrease the content of triglyceride. Furthermore, miR-2382-5p regulated the expression of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma co-activator 1beta (PPARGC1B), hormone-sensitive lipase (HSL), and peroxisome proliferator-activated receptor gamma (PPARγ), which are known to increase triglyceride decomposition in lipid metabolism. Luciferase reporter assay and quantitative real-time PCR (qPCR) validated that miR-2382-5p downregulated the mRNA expression of target gene N-myc downstream-regulated gene 2 (NDRG2) by specifically recognizing and binding to its 3'-untranslated region (UTR). Meanwhile, overexpression of NDRG2 led to increased triglyceride and cholesterol production in BMECs. In summary, this study suggested that miR-2382-5p regulated lipid metabolism by targeting NDRG2, which might be a potential target for molecular manipulation of milk fat composition to produce healthy milk. This study also provided basic data for further understanding lipid metabolism in dairy cattle.

Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis.

The elucidation of the mechanisms of preadipocyte differentiation and fat accumulation in adipocytes is a major work in beef cattle breeding. As important post-transcriptional regulators, microRNAs (miRNAs) take part in cell proliferation, differentiation, apoptosis, and fat metabolism through binding seed sites of targeting mRNAs. The aim of this study was to isolate and identify bovine preadipocytes and screen miRNAs associated with adipogenesis. Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. Verification of preadipocytes and adipocytes was performed by qRT-PCR (real-time quantitative reverse transcription PCR), Oil Red O staining, and immunofluorescence staining. Total RNA was extracted for small RNA sequencing. The sequencing data showed that 131 miRNAs were highly expressed in adipocytes, and 119 miRNAs were highly expressed in preadipocytes. Stem-loop qPCR (stem-loop quantitative real-time PCR) results showed that the expression patterns of 11 miRNAs were consistent with the sequencing results (miR-149-5p, miR-24-3p, miR-199a-5p, miR-33a, etc.). According to KEGG pathway and Gene Ontology (GO) analyses, multiple predicted target genes were associated with lipid metabolism. In summary, this study provides a protocol of isolating bovine preadipocytes and screening various differently expressed miRNAs during preadipocyte differentiation.

New function of the CD44 gene: Lipid metabolism regulation in bovine mammary epithelial cells.

The CD44 gene encodes a cell-surface glycoprotein that participates in a variety of biological processes such as cell interactions, adhesion, hematopoiesis, and tumor metastasis. We compared the transcriptome in bovine mammary epithelial cells (bMEC) of Chinese Holstein dairy cows producing milk of high and low fat contents. Our results suggest that CD44 might be a candidate gene affecting milk fat synthesis. In the present study, the overexpression of the CD44 gene increased the contents of intracellular triglycerides (TG) and cholesterol (CHOL), whereas knockdown of the CD44 gene decreased bMEC CHOL and TG contents. Gas chromatography analysis of fatty acid composition showed that the contents of α-linolenic acid, palmitic acid, and cis-8,11,14-eicosatrienoic acid were altered due to changes in the level of expression of the CD44 gene. Additionally, elaidic acid, palmitoleic acid, tridecanoic acid, and oleic acid were markedly reduced in the CD44 gene overexpression group compared with the control group. On the contrary, cis-5,8,11,14-eicosatetraenoic acid and stearic acid were markedly increased in the CD44 knockdown group compared with the control group. And RT2 Profiler PCR array (Qiagen, CLAB24070A Frankfurt, Germany) further suggested that overexpression or knockdown of the CD44 gene altered expression levels of functional genes associated with lipid metabolism. The present data indicate that CD44 plays a key regulatory role in lipid metabolism in bMEC.

Analysis of the Bovine DLK1 Gene Polymorphism and Its Relation to Lipid Metabolism in Chinese Simmental.

In this study, we precisely constructed and transfected the overexpression and interference vectors in BFFs to evaluate the role of DLK1 gene on lipid metabolism in vitro. The expression of of DLK1 in the mRNA and protein level tended to reduce, and TGs were significantly increased in the pGPU6-shDLK1 group compared to the control group (p < 0.05). The expression of DLK1 in the mRNA and protein level were increased in the pBI-CMV3-DLK1 group compared to the control group, and the TGs content showed a significant decrease in the pBI-CMV3-DLK1 group (p < 0.05). Meanwhile, we used the restriction fragment length polymorphism (RFLP-PCR) detection method to screen SNPs further to explore and analyze the relationship between the gene and the economic traits of 28-month-old Chinese Simmental and the fatty acids composition of cattle longissimus muscle. The result showed that two SNPs, IVS3 + 478 C>T and IVS3 + 609 T>G, were identified as being significantly associated with carcass and meat quality traits in Chinese Simmental, such as the carcass fat coverage rate, loin eye muscle area, and fat color score. In summary, our results indicated that DLK1 can affect lipid metabolism in bovine and these two SNPs might be applied as genetic markers of meat quality traits for beef cattle breeding.

Polymorphisms of the ASIP gene and the haplotype are associated with fat deposition traits and fatty acid composition in Chinese Simmental steers.

Unlike specific expression in the skin of wild mice, the agouti signaling protein (ASIP) is expressed widely in the tissue of cattle, including adipose and muscle tissue. Hence, it has been suggested that ASIP plays a role in bovine fat metabolism. An inserted L1-BT element was recently identified upstream of the ASIP locus which led to an ectopic expression of ASIP mRNA in cattle. In this study, we detected the indel of the L1-BT element at g.  - 14 643  nt and three SNPs in introns of the ASIP gene (g.  - 568 A  >  G, g.  - 554 A  >  T, and g. 4805A  >  T) in a Chinese Simmental steer population. The association analysis between variants of ASIP and economic traits showed that the homozygous genotype of L1-BT element insertion, AA genotype of g.  - 568 A  >  G, and AT genotype of g. 4805A  >  T were significantly correlated with carcass and fat-related traits, such as live weight and back fat thickness. Moreover, three haplotypes (H1: AT; H2: AA; H3: GT) were identified by linkage disequilibrium analysis and formed six combined genotypes. Results indicated that Chinese Simmental steers with an H1H2 combined genotype had a higher measured value of fat-deposition-related traits ( p < 0.05 ), including thickness of back fat and percentage of carcass fat coverage, but a lower content of linoleic acid and α -linolenic acid ( p < 0.05 ). Individuals of an H3H3 combination had a lower marbling score, perirenal fat weight, and carcass weight ( p < 0.05 ). This suggests that these three SNPs and two combined haplotypes might be molecular markers for beef cattle breeding selection.

The effect of acyl-CoA synthetase long-chain family member 5 on triglyceride synthesis in bovine preadipocytes.

Acyl-CoA synthetase long-chain family member 5 (ACSL5) is a member of the acyl coenzyme A (CoA) long-chain synthase families (ACSLs), and it plays a key role in fatty acid metabolism. In this study, we proved an association between the ACSL5 gene and triglyceride metabolism at the cellular level in cattle. pBI-CMV3-ACSL5 and pGPU6/GFP/Neo-ACSL5 plasmids were constructed and transfected into bovine preadipocytes by electroporation. The expression level of ACSL5 was detected by real-time quantitative PCR and western blot. The triglyceride content was detected by a triglyceride kit. The results indicated that the expression level of ACSL5 mRNA and protein in the pBI-CMV3-ACSL5-transfected group was significantly increased compared with those in the control group. Furthermore, the pGPU6/GFP/Neo-ACSL5-transfected group was significantly decreased compared with those in the control group. A cell triglyceride test showed that overexpression or silencing of the ACSL5 gene could affect synthesis of cellular triglycerides. This study investigated the mechanism of ACSL on bovine fat deposition, and also provides a new candidate gene for meat quality traits in beef cattle.