Genome-wide identification and expression profiling analysis of Wnt family genes affecting adipocyte differentiation in cattle

The Wnt family features conserved glycoproteins that play roles in tissue regeneration, animal development and cell proliferation and differentiation. For its functional diversity and importance, this family has been studied in several species, but not in the Bovinae. Herein we identified 19 Wnt genes in cattle, and seven other species of Bovinae, and described their corresponding protein properties. Phylogenetic analysis clustered the 149 Wnt proteins in Bovinae, and 38 Wnt proteins from the human and mouse into 12 major clades. Wnt genes from the same subfamilies shared similar protein motif compositions and exon–intron patterns. Chromosomal distribution and collinearity analysis revealed that they were conservative in cattle and five species of Bovinae. RNA-seq data analysis indicated that Wnt genes exhibited tissue-specific expression in cattle. qPCR analysis revealed a unique expression pattern of each gene during bovine adipocytes differentiation. Finally, the comprehensive analysis indicated that Wnt2B may regulate adipose differentiation by activating FZD5, which is worthy of further study. Our study presents the first genome-wide study of the Wnt gene family in Bovinae, and lays the foundation for further functional characterization of this family in bovine adipocytes differentiation.

Interference With ACSL1 Gene in Bovine Adipocytes: Transcriptome Profiling of mRNA and lncRNA Related to Unsaturated Fatty Acid Synthesis

The enzyme long-chain acyl-CoA synthetase 1 (ACSL1) is essential for lipid metabolism. The ACSL1 gene controls unsaturated fatty acid (UFA) synthesis as well as the formation of lipid droplets in bovine adipocytes. Here, we used RNA-Seq to determine lncRNA and mRNA that regulate UFA synthesis in bovine adipocytes using RNA interference and non-interference with ACSL1. The corresponding target genes of differentially expressed (DE) lncRNAs and the DE mRNAs were found to be enriched in lipid and FA metabolism-related pathways, according to GO and KEGG analyses. The differentially expressed lncRNA- differentially expressed mRNA (DEL-DEM) interaction network indicated that some DELs, such as TCONS_00069661, TCONS_00040771, TCONS_ 00035606, TCONS_00048301, TCONS_001309018, and TCONS_00122946, were critical for UFA synthesis. These findings assist our understanding of the regulation of UFA synthesis by lncRNAs and mRNAs in bovine adipocytes.

Genome-Wide Identification and Expression Profiling Analysis of Wnt Family Genes Affecting Adipocyte Differentiation in Cattle

Wnt is a family of conserved glycoproteins that functions in a variety of crucial biological processes including tissue regeneration, animal development, and cell proliferation and differentiation. For its functional diversity and importance, Wnt gene family has gained considerable research interest in a variety of species. However, comprehensive identification and analysis of Wnt genes in Bovinae is lacking. In this study, we identified the repertoire of Wnt genes in cattle and seven other species of Bovinae and obtained 19 Wnt genes. Protein properties of these Wnt genes were also described. Phylogenetic analysis showed that the 149 Wnt proteins in Bovinae, together with 38 Wnt proteins from human and mouse, were clustered into 12 major clades. The Wnt genes belonging to the same subfamilies shared similar protein motif compositions and exon-intron patterns. Chromosomal distribution and collinearity analysis of Wnt genes among cattle and five species of Bovinae revealed that this gene family was conservative in evolution. RNA-seq data analysis indicated that Wnt genes exhibited tissue-specific expression patterns in cattle. qPCR analysis of Wnt gene family showed that each gene had a unique expression pattern during bovine adipocytes differentiation. And the comprehensive analysis indicated that Wnt2B may regulate adipose differentiation through activation of FZD5, which is worthy of further study. Our study presents the first genome-wide study of Wnt gene family in Bovinae, and lay the foundation for further functional characterization of the Wnt family in bovine adipocytes differentiation.

Antioxidant Resveratrol Increases Lipolytic and Reduces Lipogenic Gene Expression under In Vitro Heat Stress Conditions in Dedifferentiated Adipocyte-Derived Progeny Cells from Dairy Cows

Heat stress (HS) induces oxidative stress by increasing reactive oxygen species (ROS), and the polyphenol resveratrol (RSV) has been shown to have antioxidant properties by reducing ROS. Hence, we aimed to examine the effects of RSV, HS and their interaction on bovine adipocytes. We generated bovine dedifferentiated adipocyte-derived progeny (DFAT) cells from subcutaneous adipose tissue and examined the effects of RSV (100 µM), heat conditions: isothermal (ISO-37 °C), short heat (SH-41.2 °C for 1 h) and long HS (LH-41.2 °C for 16 h), and their interaction on gene expression in DFAT-cells. In medium of DFAT-cells treated with RSV, malondialdehyde levels were reduced and oxygen-radical absorbance-capacity levels were increased compared to control. Treating DFAT-cells with RSV increased the relative mRNA expression of stress-induced-phosphoprotein-1 (STIP1) and the expression of hormone-sensitive-lipase (LIPE) and perilipin-1 (PLIN1), whereas it reduced the expressions of fatty-acid-synthase (FASN) and of pro-inflammatory chemotactic-C-C-motif-chemokine-ligand-2 (CCL2) also under HS. Moreover, reduced protein abundance of FASN was found in RSV-treated DFAT-cells compared to controls. Molecular docking of RSV with FASN confirmed its possible binding to FASN active site. This work demonstrates that RSV has an antioxidant effect on bovine DFAT cells and may induce adipose lipolysis and reduce lipogenesis also under in vitro HS conditions.

The Combined Influence of Magnesium and Insulin on Central Metabolic Functions and Expression of Genes Involved in Magnesium Homeostasis of Cultured Bovine Adipocytes

At the onset of lactation, dairy cows suffer from insulin resistance, insulin deficiency or both, similar to human diabetes, resulting in lipolysis, ketosis and fatty liver. This work explored the combined effects of different levels of magnesium (0.1, 0.3, 1 and 3 mM) and insulin (25, 250 and 25,000 pM) on metabolic pathways and the expression of magnesium-responsive genes in a bovine adipocyte model. Magnesium starvation (0.1 mM) and low insulin (25 pM) independently decreased or tended to decrease the accumulation of non-polar lipids and uptake of the glucose analog 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG). Activity of glycerol 3-phosphate dehydrogenase (GPDH) was highest at 25 pM insulin and 3 mM magnesium. Expression of SLC41A1 and SLC41A3 was reduced at 0.1 mM magnesium either across insulin concentrations (SLC41A1) or at 250 pM insulin (SLC41A3). MAGT1 expression was reduced at 3 mM magnesium. NIPA1 expression was reduced at 3 mM and 0.1 mM magnesium at 25 and 250 pM insulin, respectively. Expression of SLC41A2, CNNM2, TRPM6 and TRPM7 was not affected. We conclude that magnesium promotes lipogenesis in adipocytes and inversely regulates the transcription of genes that increase vs. decrease cytosolic magnesium concentration. The induction of GAPDH activity by surplus magnesium at low insulin concentration can counteract excessive lipomobilization.

RNA-Seq Reveals Function of Bta-miR-149-5p in the Regulation of Bovine Adipocyte Differentiation

Intramuscular fat is a real challenge for the experts of animal science to improve meat quality traits. Research on the mechanism of adipogenesis provides invaluable information for the improvement of meat quality traits. This study investigated the effect of bta-miR-149-5p and its underlying mechanism on lipid metabolism in bovine adipocytes. Bovine adipocytes were differentiated and transfected with bta-miR-149-5p mimics or its negative control (NC). A total of 115 DEGs including 72 upregulated and 43 downregulated genes were identified in bovine adipocytes. The unigenes and GO term biological processes were the most annotated unigene contributor parts at 80.08%, followed by cellular component at 13.4% and molecular function at 6.7%. The KEGG pathways regulated by the DEGs were PI3K-Akt signaling pathway, calcium signaling pathway, pathways in cancer, MAPK signaling pathway, lipid metabolism/metabolic pathway, PPAR signaling pathway, AMPK signaling pathway, TGF-beta signaling pathway, cAMP signaling pathway, cholesterol metabolism, Wnt signaling pathway, and FoxO signaling pathway. In addition to this, the most important reactome enrichment pathways were R−BTA−373813 receptor CXCR2 binding ligands CXCL1 to 7, R−BTA−373791 receptor CXCR1 binding CXCL6 and CXCL8 ligands, R−BTA−210991 basigin interactions, R−BTA−380108 chemokine receptors binding chemokines, R−BTA−445704 calcium binding caldesmon, and R−BTA−5669034 TNFs binding their physiological receptors. Furthermore, the expression trend of the DEGs in these pathways were also exploited. Moreover, the bta-miR-149-5p significantly (p < 0.01) downregulated the mRNA levels of adipogenic marker genes such as CCND2, KLF6, ACSL1, Cdk2, SCD, SIK2, and ZEB1 in bovine adipocytes. In conclusion, our results suggest that bta-miR-149-5p regulates lipid metabolism in bovine adipocytes. The results of this study provide a basis for studying the function and molecular mechanism of the bta-miR-149-5p in regulating bovine adipogenesis.

Bovine Pre-adipocyte Adipogenesis Is Regulated by bta-miR-150 Through mTOR Signaling

Micro RNA (miR) are recognized for their important roles in biological processes, particularly in regulatory componentization. Among the miR, miR-150 has been the focus of intense scrutiny, mostly due to its role in malignant tumors. A comparison between steer and bull adipose tissues identified bta-miR-150 as one of the nine downregulated miRNAs, although its function remains unknown (GEO:GSE75063). The present study aimed to further characterize the role of bta-miR-150 in cattle. bta-miR-150 has a negative regulatory effect on the differentiation of bovine adipocytes and promotes proliferation. Overexpression of bta-miR-150 can promote mRNA and protein expression of the marker genes CDK1, CDK2, and PCNA, increase the number of EdU-stained cells, promote adipocyte proliferation, inhibit adipocyte differentiation, and reduce lipid droplet formation. Results of RNA-seq and WGCNA analyses showed that the mammalian target of the rapamycin signaling pathway, which plays a major regulatory role, is dysregulated by the overexpression and inhibition of miR-150. We found that the target gene of bta-miR-150 is AKT1 and that bta-miR-150 affects AKT1 phosphorylation levels. These results showed that bta-miR-150 plays a role in adipogenic differentiation and might therefore have applications in the beef industry.

Bta-miR-376a Targeting KLF15 Interferes with Adipogenesis Signaling Pathway to Promote Differentiation of Qinchuan Beef Cattle Preadipocytes

Intramuscular fat (IMF) is a quality index associated with the taste and juiciness of meat. The deposition of IMF is affected by genetic and non-genetic factors, such as age, slaughter location, gender of the animal, and diet. Micro-ribonucleic acids (miRNA) are transcriptional regulators involved in adipogenesis, but the specific role of miR-376a in regulation of bovine adipocytes remains unknown. Our findings indicated that miR-376a was a potential negative regulator of bovine adipocyte differentiation. A bta-miR-376a mimic inhibited mRNA and protein expression of the marker genes, CDK1, CDK2, PCNA, C/EBPα, FAS, and PPAR γ, and significantly reduced ratios (%) of S-phase cells, the number of cells stained with 5-ethynyl-2′-deoxyuridine, and adipocyte proliferation. Oil red O staining and triglyceride content analysis also confirmed that bta-miR-376a was involved in adipocyte differentiation. Luciferase activities confirmed that Krüppel-like transcription factor 15 (KLF15) was a direct target gene of bta-miR-376a, and that KLF15 was a key transcription factor in adipogenesis. Therefore, bta-miR-376a might be a target for increasing beef IMF.

Characterization and Transcriptome Analysis of Exosomal and Nonexosomal RNAs in Bovine Adipocytes

Exosomes are endosome-derived extracellular vesicles that allow intercellular communication. However, the biological significance of adipocyte exosomal RNAs remains unclear. To determine the role of RNAs from bovine adipocytes and exosomes in bovine adipogenesis, exosomal and nonexosomal RNAs were extracted from three bovine primary white adipocyte samples and then profiles were generated using DNBSEQ/BGISEQ-500 technology. The RNAome of adipocytes consisted of 12,082 mRNAs, 8589 lncRNAs, and 378 miRNAs for a higher complexity that that detected in exosomes, with 1083 mRNAs, 105 lncRNAs, and 48 miRNAs. Exosomal miRNA-mRNA and lncRNA–miRNA–mRNA networks were constructed and enrichment analysis was performed to predict functional roles and regulatory mechanisms. Our study provides the first characterization of RNAs from bovine adipocyte and exosomes. The findings reveal that some RNAs are specifically packaged in adipocyte-derived exosomes, potentially enabling crosstalk between adipocytes and/or other cells that is mediated by exosomes. Our results greatly expand our understanding of exosomal RNAs from bovine adipocytes, and provide a reference for future functional investigations of adipocyte exosomal RNAs under normal physiological conditions.