Cis-9, Trans-11 CLA Alleviates Lipopolysaccharide-Induced Depression of Fatty Acid Synthesis by Inhibiting Oxidative Stress and Autophagy in Bovine Mammary Epithelial Cells

Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H2O2 inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.

Regulation of Key Genes for Milk Fat Synthesis in Ruminants

Milk fat is the most important and energy-rich substance in milk and plays an important role in the metabolism of nutrients during human growth and development. It is mainly used in the production of butter and yogurt. Milk fat not only affects the flavor and nutritional value of milk, but also is the main target trait of ruminant breeding. There are many key genes involve in ruminant milk fat synthesis, including ACSS2, FASN, ACACA, CD36, ACSL, SLC27A, FABP3, SCD, GPAM, AGPAT, LPIN, DGAT1, PLIN2, XDH, and BTN1A1. Taking the de novo synthesis of fatty acids (FA) and intaking of long-chain fatty acids (LCFA) in blood to the end of lipid droplet secretion as the mainline, this manuscript elucidates the complex regulation model of key genes in mammary epithelial cells (MECs) in ruminant milk fat synthesis, and constructs the whole regulatory network of milk fat synthesis, to provide valuable theoretical basis and research ideas for the study of milk fat regulation mechanism of ruminants.

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.

Bta-miR-34b controls milk fat biosynthesis via the Akt/mTOR signaling pathway by targeting RAI14 in bovine mammary epithelial cells

Background The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes, including milk fat synthesis. miR-34b is a member of the miR-34 miRNA cluster, which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods. Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis; therefore, it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells (BMECs). Results In this study, elevated miR-34b levels reduced milk fat synthesis, upregulated 1,999 genes, and downregulated 2,009 genes in BMECs. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway by reducing phosphorylation levels. Notably, the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b. Furthermore, we demonstrated that retinoic acid-induced protein 14 (RAI14) is a target of miR-34b via TargetScan and immunofluorescence assays. RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor. Moreover, the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway. Conclusions Overall, our results identified a miR-34b-RAI14-Akt/mTOR regulatory network, while also providing a theoretical basis for the molecular breeding of dairy cows.

Screening and Joint Analysis of Key lncRNAs for Milk Fat Metabolism in Dairy Cows

Background: Long noncoding RNAs (lncRNAs) play an important regulatory role in various biological processes as a key regulatory factor. However, there are largely unknown for the function and expression profile of lncRNAs in milk fat synthesis of dairy cows. Results: In this study, RNA sequencing (RNA-seq) was used to research the whole genome expression of lncRNAs and mRNA transcripts in bovine mammary epithelial cells (BMECs) of dairy cows with high and low milk fat percentage (MFP), and joint analysis was carried out. We identified a total of 47 differentially expressed genes (DEGs) and 38 differentially expressed lncRNAs (DELs, Padj < 0.05), 11 candidate DEGs that may regulate milk fat metabolism were screened by enrichment analysis. Downregulated differential gene ENPP2 and upregulated differential gene BCAT1 are more likely to participate in the milk fat metabolism, and its function needs further experiments verification. The enrichment analysis of target genes predicted by DELs identified 7 cis (co-localization) and 10 trans (co-expression) candidate target genes related to milk lipid metabolism, corresponding to a total of 18 DELs. Among them, the targeting relationship between long intervening/intergenic noncoding RNA (lincRNA) TCONS_00082721 and FABP4 gene that predicts milk fat metabolism by co-localization and co-expression is worthy of attention. Based on the expression information of DELs, differential microRNAs (miRNAs), and lipid metabolism-related target genes, 156 competing endogenous RNAs (ceRNAs) interaction regulation networks related to milk fat metabolism were constructed. The regulatory network centered on miR-145 will be the focus of subsequent experimental research. The ceRNAs regulatory network related to TCONS_00082721 and TCONS_00172817 are more likely to be involved in milk fat synthesis. Conclusions: These results will provide new ways to understand the complex biology of dairy cow milk fat synthesis and provide valuable information for the breed improvement of Chinese Holstein cattle.

MiR-485 targets the DTX4 gene to regulate milk fat synthesis in bovine mammary epithelial cells

MicroRNAs (miRNAs) are mRNA suppressors that regulate a variety of cellular and physiological processes, including cell proliferation, apoptosis, triglyceride synthesis, fat formation, and lipolysis, by post-transcriptional processing. In previous studies, we isolated and sequenced miRNAs from mammary epithelial cells from Chinese Holstein cows with high and low milk fat percentages. MiR-485 was one of the significantly differentially expressed miRNAs that were identified. In the present study, the relationship between the candidate target gene DTX4 and miR-485 was validated by bioinformatics and real-time fluorescent quantitative PCR (qRT-PCR) and Western blot (WB) analyses in bovine mammary epithelial cells (bMECs). The results indicated that miR-485 negatively regulated the mRNA expression of the target gene DTX4. Furthermore, an shRNA interference vector for the target gene DTX4 was constructed successfully, and it increased the triglyceride content and reduced the cholesterol content of transfected cells. These results suggest that miR-485 may affect the contents of triglycerides (TGs) and cholesterol (CHOL) by targeting the DTX4 gene. This study indicates that miR-485 has a role in regulating milk fat synthesis and that miR-485 targets the DTX4 gene to regulate lipid metabolism in bMECs. These findings contribute to the understanding of the functional significance of miR-485 in milk fat synthesis.

TATA Element Modulatory Factor 1 Negatively Regulates Sodium Acetate Activated Milk Fat Synthesis Through SREBP1 Pathway in Bovine Mammary Epithelial Cells

Background: Sodium acetate is one of the important nutrients that regulate milk fat synthesis in bovine mammary epithelial cells (BMECs), and it regulates milk fat synthesis mainly through the SREBP1 pathway. Our previous study has showed that TATA element modulatory factor 1 (TMF1) may be interacts with SREBP1 and regulates the sodium acetate-dependent milk synthesis in BMECs, but the underlying mechanism is unclear. In the current study, the effect of TMF1 on sodium acetate activated milk fat synthesis in BMECs was assessed. Results: Overexpressing or inhibiting TMF1 demonstrated that TMF1 negatively regulated sodium acetate activated sterol regulatory element-binding protein 1 (SREBP1) pathway and milk fat synthesis; Overexpressing or inhibiting SREBP1 showed that TMF1 inhibited sodium acetate activated milk fat synthesis through SREBP1 pathway; Co-immunoprecipitation analysis showed that TMF1 interacted with SREBP1; Nuclear localization of SREBP1 analysis showed that sodium acetate activated nuclear localization of SREBP1 was inhibited by TMF1; Depletion or supply sodium acetate demonstrated that sodium acetate negatively regulated expression of TMF1 and the interaction between TMF1 and SREBP1. Conclusions: Together, these results indicate that TMF1 is a negative regulatory factor for sodium acetate activated milk fat synthesis, it induced expression by sodium acetate depletion, and interacts with SREBP1 in cytoplasmic, prevents the nuclear localization of SREBP1 and then suppresses the expression of SREBP1 target gene and subsequent milk fat synthesis in BMECs.

MicroRNA-432 inhibits milk fat synthesis by targeting SCD and LPL in ovine mammary epithelial cells

In our previous studies, microRNA-432 (miR-432) was found to be one of differentially expressed miRNAs in ovine mammary gland between the two breeds of lactating sheep with different milk production...