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The CD44 gene is a critical factor in animal physiological processes and has been shown to affect insulin resistance and fat accumulation in mammals. Nevertheless, little research has been conducted on its precise functions in lipid metabolism and adipogenic differentiation in beef cattle. This study analyzed the expression of CD44 and miR-199a-3p during bovine preadipocyte differentiation. The luciferase reporter assay demonstrated that CD44 was a direct target of miR-199a-3p. Increased accumulation of lipid droplets and triglyceride levels, altered fatty acid metabolism, and accelerated preadipocyte differentiation were all caused by the upregulation of miR-199a-3p or a reduction in CD44 expression. CD44 knockdown upregulated the expression of adipocyte-specific genes ( LPL and FABP4 ) and altered the levels of lipid metabolites (SOPC, l-arginine, and heptadecanoic acid). Multiomics highlights enriched pathways involved in energy metabolism (MAPK, cAMP, and calcium signaling) and shifts in mitochondrial respiration and glycolysis, indicating that CD44 plays a regulatory role in lipid metabolism. The findings show that intracellular lipolysis, glycolysis, mitochondrial respiration, fat deposition, and lipid droplet composition are all impacted by miR-199a-3p, which modulates CD44 in bovine adipocytes.

Simple Summary: According to the FDA, a litre of milk contains about 33 g of total lipids, 95% of which are triacylglycerols made up of varying lengths and saturation of fatty acids. The increase in free fatty acids in milk may lead to changes in the flavour of milk and milk products. Bovine mammary epithelial cells (bMECs) function to synthesize and secrete milk and are good in vitro models for cells in milk quality research. In this research, we report for the first time the effect of the ASIP gene knockout in bovine mammary epithelial cells using CRISPR/Cas9 technology. We found that ASIP knockout could down-regulate the expression of PPARγ, FASN, and SCD, thus affecting the saturation of fatty acids in milk and upregulating the expression of FABP4, ELOVL6, and ACSL1, increasing the synthesis of medium-long chain fatty acids. These results may allow the selection of potential targets in future molecular breeding efforts for dairy cows. Agouti signalling protein (ASIP) is a coat colour-related protein and also is a protein-related to lipid metabolism, which had first been found in agoutis. According to our previous study, ASIP is a candidate gene that affects the lipid metabolism in bovine adipocytes. However, its effect on milk lipid has not been reported yet. This study focused on the effect of the ASIP gene on the lipid metabolism of mammary epithelial cells in cattle. The ASIP gene was knocked out in bMECs by using CRISPR/Cas9 technology. The result of transcriptome sequencing showed that the differentially expressed genes associated with lipid metabolism were mainly enriched in the fatty acids metabolism pathways. Furthermore, the contents of intracellular triglycerides were significantly increased (p < 0.05), and cholesterol tended to rise (p > 0.05) in bMECs with the knockout of the ASIP gene. Fatty acid assays showed a significant alteration in medium and long-chain fatty acid content. Saturated and polyunsaturated fatty acids were significantly up-regulated (p < 0.05), and monounsaturated fatty acids were significantly decreased in the ASIP knockout bMECs (p < 0.05). The Q-PCR analysis showed that knockout of ASIP resulted in a significant reduction of gene expressions like PPARγ, FASN, SCD, and a significant up-regulation of genes like FABP4, ELOVL6, ACSL1, HACD4 prompted increased mid-to long-chain fatty acid synthesis. Overall, ASIP plays a pivotal role in regulating lipid metabolism in bMECs, which could further influence the component of lipid in milk. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Delta-like non-canonical Notch ligand 1 (DLK1) is a candidate gene associated with lipid metabolism. In order to verify the function of the DLK1 gene on lipid metabolism in Chinese Simmental cattle, we identified the effect of DLK1 on lipid metabolism in bovine fetal fibroblast cells (BFFs). At the same time, we also detected the relationship between single-nucleotide polymorphism sites (SNPs) of bovine DLK1 with the economic traits and fatty acids composition in Chinese Simmental cattle, such as the carcass fat coverage rate, loin eye muscle area, and fat color score, etc. In the present study, we precisely constructed and transfected the overexpression and interference vectors of the DLK1 gene in BFFs. the results showed that the overexpression of the DLK1 gene could decrease the contents of intracellular triglycerides (TGs) while interference of the DLK1 gene could increase the TGs contents. Gas chromatography analysis of the fatty acid composition showed that the contents of octanoate acid and γ-linolenate acid were regulated as the expression of DLK1 was altered. We found two SNPs and genes associated with these traits in Chinese Simmental by the restriction fragment length polymorphism (RPLF-PCR) detection method. In summary, we verified the effects of bovine DLK1 on fatty acid metabolism from the cellular level to population genetic polymorphism, which also paved the way for further studying of the effects of the DLK1 gene on lipid metabolism in vivo. 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. [ABSTRACT FROM AUTHOR]