Milk Fat Metabolism Research Articles

Bta-miR-224 regulates milk fat metabolism by targeting FABP4 in bovine mammary epithelial cells

Milk fat is produced and secreted by the mammary gland, which is mainly regulated by diet and gene-molecule network. Therefore, understanding the molecular mechanism of milk fat synthesis is of practical significance for improving milk quality. Fatty acid-binding protein 4 (FABP4) is a candidate messenger RNA (mRNA) closely linked to milk fat metabolism obtained from transcriptomic analysis of mammary epithelial cells of cows in the pre-existing high- and low-milk-fat groups, and its expression pattern and function are still unclear. The qRT-PCR results depicted that FABP4 was highly expressed in bovine mammary epithelial cells (BMECs) in breast tissues and the high milk fat group. Subsequently, the regulatory effects of FABP4 on BMECs were analyzed by CCK8, EdU, and flow cytometry, and the results demonstrated that FABP4 inhibited the proliferation and viability of BMECs and promoted their apoptosis. Next, the effect of FABP4 on milk lipid metabolism was explored. pEGFP-N1-FABP4 was transfected into BMECs, and FABP4 upregulated the expression levels of the milk lipid marker genes XDH, PPARG, and ACSS2, and promoted the formation of triglycerides (TGs), cholesterol, lipid droplets, and β-casein. Strong interactions between FABP4 and PPARG were identified using STRING prediction. Western blotting revealed that FABP4 interacted with PPARG to promote PPARG expression, while the opposite result was observed after interfering with FABP4. The gene regulation of microRNA (miRNA) is essential for fatty acid metabolism and synthesis. Predicted by website and combined with pre-miRNA transcriptome sequencing results, we hypothesized that FABP4 might be the target gene of bta-miR-224. The results of the dual-luciferase reporter gene and qRT-PCR revealed that bta-miR-224 negatively regulated FABP4 expression by targeting the 3′-UTR of FABP4. By exploring the function of bta-miR-224, we observed that bta-miR-224 mimics downregulated the expression of the milk fat marker genes AGPAT6, ACSS2, and XDH and inhibited TG synthesis and lipid droplet secretion. However, the bta-miR-224 inhibitor depicted the opposite results. In conclusion, FABP4 plays a crucial role in regulating BMEC proliferation and differentiation. Bta-miR-224 targeting FABP4 may promote biological processes such as TG synthesis and lipid droplet formation through PPARG, which lays a solid foundation for further analysis of the functional mechanism of milk lipid metabolism in dairy cows from a miRNA-mRNA perspective.

Overexpression of PER2 Promotes De Novo Fatty Acid Synthesis, Fatty Acid Desaturation, and Triglyceride Accumulation in Bovine Mammary Epithelial Cells.

The core clock gene Period2 (PER2) is associated with mammary gland development and lipid synthesis in rodents and has recently been found to have a diurnal variation in the process of lactation, but has not yet been demonstrated in bovine mammary epithelial cells (BMECs). To explore the regulatory function of PER2 on milk fat synthesis in bovine mammary epithelial cells, we initially assessed the expression of clock genes and milk fat metabolism genes for 24 h using real-time quantitative PCR and fitted the data to a cosine function curve. Subsequently, we overexpressed the PER2 in BMECs using plasmid vector (pcDNA3.1-PER2), with empty vector pcDNA3.1-myc as the control. After transfecting BMECs for 48 h, we assessed the protein abundance related to milk fat synthesis by Western blot, the expression of genes coding for these proteins using real time-quantitative PCR, the production of triacylglycerol, and the fatty acid profile. The findings indicated that a total of nine clock genes (PER1/2, CRY1/2, REV-ERBα, BMAL1, NCOR1, NR2F2, FBXW11), seven fatty acid metabolism genes (CD36, ACSS2, ACACA, SCD, FADS1, DGAT1, ADFP), and six nuclear receptor-related genes (INSIG1, SCAP, SREBF1, C/EBP, PPARG, LXR) exhibited oscillation with a period close to 24 h in non-transfected BMECs (R2 ≥ 0.7). Compared to the control group (transfected with empty pcDNA3.1-myc), the triglyceride content significantly increased in the PER2 overexpression group (p < 0.05). The lipogenic genes for fatty acid transport and triglyceride synthesis (ACACA, SCD, LPIN1, DGAT1, and SREBF1) were upregulated after PER2 overexpression, along with the upregulation of related protein abundance (p < 0.05). The contents and ratios of palmitic acid (C16:0), oleic acid (C18:1n9c), and trans-oleic acid (C18:1n9t) were significantly increased in the overexpression group (p < 0.05). Overall, the data supported that PER2 participated in the process of milk fat metabolism and is potentially involved in the de novo synthesis and desaturation of fatty acid in bovine mammary epithelial cells.

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

Identification, Molecular Characterization and Preliminary Functional Analysis of Three SLC27A Full-CDS cDNAs from Water Buffalo (Bubalus bubalis)

Background: Solute carrier family 27 family (SLC27As) have been identified to be involved in long chain fatty acid uptake in various tissues and cells, but their function in buffalo is unclear. Buffalo is an important dairy animal in some parts of the world. Buffalo milk contains more fat, protein, lactose and minerals than cow’s milk. The objective of this study was to identify the buffalo SLC27A1, 2 and 6 genes and to describe their characteristics, structure and basic function. Methods: The full-length coding sequences (CDSs) of SLC27A1, 2 and 6 were isolated from Binglangjiang buffalo and bioinformatics analyses were used to provide insights into the physicochemical characteristics, structure and function of their encoded proteins. Result: The CDSs of buffalo SLC27A1, 2 and 6 were 1941 bp, 1863 bp and 1905 bp in length, respectively, encoding 646, 620 and 634 amino acid residues accordingly. Their proteins FATP1, 2 and 6 have no signal peptide but a transmembrane helix with the N-terminus located outside the membrane and the C-terminus located in cytoplasmic side. All three proteins contain AMP-binding domains that belonged to AFD_class_I superfamily and may play a crucial role in the transport of fatty acids. The amino acid sequences of buffalo FATP1, 2 and 6 showed more than 88.6% identity with other species of Bovidae and more than 76.4% identity with other species of mammals. The phylogenetic tree showed that buffalo FATP1, 2 and 6 clustered in their respective clades and buffalo was more closely genetically related to other species of the Bovidae family. These findings suggest that buffalo FATPs are more similar in function to other species. The present study will provide a theoretical basis for further investigation of the functions of FATP1, 2 and 6 in buffalo lipid metabolism, especially milk fat metabolism.

The Effects of trans-10, cis-12 Conjugated Linoleic Acid on the Production Performance of Dairy Cows and the Expression and Transcription Regulation of Lipid Metabolism-Related Genes in Bovine Mammary Epithelial Cells.

Dietary fatty acids (FAs) determine the quality of dairy products. The trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) is commonly considered an FA factor leading to milk fat depression syndrome (MFDs) in dairy cow. However, its effect on dairy cow performance and involvement in milk fat metabolism have been insufficiently explored. This study administered 136.17 g/day of rumen-protected CLA (RP-CLA) to dairy cows and found a diminution in milk fat percentage and a trend of increasing milk protein percentage on day 21 postpartum. Lactose content, milk yield, and net energy for lactation were unaffected. In the cell experiments, Oil Red O staining showed a notable increase in lipid droplets. Gene and protein expression analysis showed that 300 μM t10c12-CLA upregulated the expression of CD36, DGAT2, and ADRP, while downregulating the expression of ACACA, FASN, SREBP1, FABP3, FATP3, ACSL4, LPIN1, DGAT1, BTN1A1, XDH, SNAP23, and VAMP4. This provides a possible mechanistic pathway for the contradictory phenomenon of t10c12-CLA reducing milk fat while increasing lipid droplets. Overall, t10c12-CLA, as a long-chain fatty acid, can promote lipid droplet synthesis but may reduce milk fat by inhibiting lipid droplet fusion and secretion, FAs de novo synthesis, and triglyceride biosynthesis.

MicroRNA-2285f regulates milk fat metabolism by targeting MAP2K2 in bovine mammary epithelial cells.

In this study, Holstein dairy cows raised in Ningxia were selected as the research object. Mammary epithelial cells (BMECs) were extracted from the milk of eight Holstein cows with significantly different milk fat expression rates and transcribed for sequencing. Bioinformatics analysis was used to analyse the correlation of fat milk percentage, and the critical miR-2285f regulating milk fat was screened out. The target gene binding sites were predicted, and 293T cells and mammary epithelial cells were used as miRNA and target gene models for functional verification invitro. The tissue difference of miR-2285f Holstein cows was quantitatively analysed by transfecting miR-2285f mimic and inhibitor. Assay (dual luciferase reporter gene assay) and quantitative real-time PCR (quantitative real-time PCR, qRT-PCR), triglyceride (TAG) detection, oil red O detection of lipid droplets, Western Blot assay, Edu and Flow cytometry, The molecular regulatory effects of miR-2285f and target gene MAP2K2 on milk fat metabolism of Holstein dairy cows were studied. The wild-type vector and mutant vector of map2k2-3'utr were constructed, and double luciferase reporting experiments were conducted to verify that MAP2K2 was one of the target genes of miR-2285f. According to qRT-PCR and Western Blot analysis, miR-2285f mainly regulates the expression of MAP2K2 protein in BMECs at the translation level. Bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2. Bta-miR-2285f can promote triglyceride (TAG) and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2. Bta-miR-2285f can regulate protein levels of fat milk marker gene PPARG by targeting MAP2K2. In conclusion, miR-2285f can target the expression of the MAP2K2 gene, promote the proliferation of dairy mammary epithelial cells, inhibit cell apoptosis and regulate the milk fat metabolism in dairy mammary epithelial cells. The results of this study revealed the function of miR-2285f in regulating the differential expression of fat milk in Holstein dairy cows at the cellular level. They provided a theoretical and experimental basis for analysing the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.

The cooperative regulatory effect of the miRNA-130 family on milk fat metabolism in dairy cows.

There is a strong relationship between the content of beneficial fatty acids in milk and milk fat metabolic activity in the mammary gland. To improve milk quality, it is therefore necessary to study fatty acid metabolism in bovine mammary gland tissue. In adipose tissue, peroxisome proliferator-activated receptor gamma (PPARG), the core transcription factor, regulates the fatty acid metabolism gene network and determines fatty acid deposition. However, its regulatory effects on mammary gland fatty acid metabolism during lactation have rarely been reported. Transcriptome sequencing was performed during the prelactation period and the peak lactation period to examine mRNA expression. The significant upregulation of PPARG drew our attention and led us to conduct further research. According to bioinformatics prediction, dual-luciferase reporter system detection, real-time quantitative reverse transcription polymerase chain reaction and Western blotting, miR-130a and miR-130b could directly target PPARG and inhibit its expression. Furthermore, triglyceride and oil red O staining proved that miR-130a and miR-130b inhibited milk fat metabolism in bovine mammary epithelial cells (BMECs), while PPARG promoted this metabolism. In addition, we also found that the coexpression of miR-130a and miR-130b significantly enhanced their ability to regulate milk fat metabolism. In conclusion, our findings indicated that miR-130a and miR-130b could target and repress PPARG and that they also have a functional superposition effect. miR-130a and miR-130b seem to synergistically regulate lipid catabolism via the control of PPARG in BMECs. In the long-term, these findings might be helpful in developing practical means to improve high-quality milk.

Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages

BackgroundCow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals.MethodThe present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA).ResultsA total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = − 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism.ConclusionsOur study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Research Progress on the Mechanism of Milk Fat Synthesis in Cows and the Effect of Conjugated Linoleic Acid on Milk Fat Metabolism and Its Underlying Mechanism: A Review.

Milk fat synthesis in cows mainly includes the synthesis of short- and medium-chain fatty acids, the uptake, transport, and activation of long-chain fatty acids (LCFAs), the synthesis of triglycerides, and the synthesis of the genes, transcription factors, and signaling pathways involved. Although the various stages of milk fat synthesis have been outlined in previous research, only partial processes have been revealed. CLA consists of an aggregation of positional and geometric isomers of linoleic fatty acid, and the accumulated evidence suggests that the two isomers of the active forms of CLA (cis-9, trans-11 conjugated linoleic acid and trans-10, cis-12 conjugated linoleic acid, abbreviated as c9, t11-CLA and t10, c12-CLA) can reduce the fat content in milk by regulating lipogenesis, fatty acid (FA) uptake, oxidation, and fat synthesis. However, the mechanism through which CLA inhibits milk fat synthesis is unique, with most studies focusing only on the effects of CLA on one of the genes, transcription factors, or signaling pathways involved. In this study, we summarized the structure and function of classic genes and pathways (mTOR, SREBP, AMPK, and PPARG) and new genes or pathways (THRSP, METTL3, ELOVL, and LPIN1) involved in each stage of milk fat synthesis and demonstrated the interactions between genes and pathways. We also examined the effects of other substances (melanin, nicotinic acid, SA, etc.). Furthermore, we evaluated the influence of β-sitosterol, sodium butyrate, Met arginine, and Camellia oleifera Abel on milk fat synthesis to improve the mechanism of milk fat synthesis in cows and provide a mechanistic reference for the use of CLA in inhibiting milk fat biosynthesis.

Non-targeted metabolomics identifies biomarkers in milk with high and low milk fat percentage

Milk is widely recognized as an important food source with health benefits. Different consumer groups have different requirements for the content and proportion of milk fat; therefore, it is necessary to investigate the differential metabolites and their regulatory mechanisms in milk with high and low milk fat percentages (MFP). In this study, untargeted metabolomics was performed on milk samples from 13 cows with high milk fat percentage (HF) and 13 cows with low milk fat percentage (LF) using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS). Forty-eight potential differentially labeled compounds were screened using the orthogonal partial least squares-discriminant analysis (OPLS-DA) combined with the weighted gene co-expression network analysis (WGCNA) method. Amino acid metabolism was the key metabolic pathway with significant enrichment of L-histidine, 5-oxoproline, L-aspartic acid, and L-glutamic acid. The negative correlation with MFP differentiated the HF and LF groups. To further determine the potential regulatory role of these amino acids on milk fat metabolism, the expression levels of marker genes in the milk fat synthesis pathway were explored. It was noticed that L-histidine reduced milk fat concentration primarily by inhibiting the triglycerides (TAG) synthesis pathway. L-aspartic acid and L-glutamic acid inhibited milk fat synthesis through the fatty acid de novo and TAG synthesis pathways. This study provides new insights into the mechanism underlying milk fat synthesis and milk quality improvement.

Progress of Conjugated Linoleic Acid on Milk Fat Metabolism in Ruminants and Humans.

As a valuable nutrient in milk, fat accounts for a significant proportion of the energy requirements of ruminants and is largely responsible for determining milk quality. Fatty acids (FAs) are a pivotal component of milk fat. Conjugated linoleic acid (CLA) is one of the naturally occurring FAs prevalent in ruminant dairy products and meat. Increasing attention has been given to CLA because of its anti-cancer, anti-inflammatory, immune regulation, and lipid metabolism regulation properties, and these benefits potentially contribute to the growth and health of infants. In breast milk, CLA is present in trace amounts, mainly in the form of cis-9, trans-11 CLA. Notably, cis-9, trans-11 CLA improves the milk fat rate while trans-10, cis-12 CLA inhibits it. Apart from having multiple physiological functions, CLA is also a pivotal factor in determining the milk quality of ruminants, especially milk fat rate. In response to growing interest in green and healthy functional foods, more and more researchers are exploring the potential of CLA to improve the production performance of animals and the nutritional value of livestock products. Taken together, it is novel and worthwhile to investigate how CLA regulates milk fat synthesis. It is the purpose of this review to clarify the necessity for studying CLA in ruminant milk fat and breast milk fat.

MicroRNA-19a regulates milk fat metabolism by targeting SYT1 in bovine mammary epithelial cells

MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3′-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows.

Association of DGAT1 with goat milk and meat production traits

Genetic factors can influence the composition of milk fat and its genetic variation. The purpose of our work was to familiarize and study the structure, role, and influence of the DGAT1 (or diacylglycerol-O-acyltransferase 1) gene on goat milk production, its structure, and relationship with milk fat for further research in improving the goat selection system. The selection of goats, aimed at increasing the frequency of alleles with a positive effect on this trait, was initiated by geneticists. In general, identifying and evaluating genetic markers for milk performance traits are the initial and crucial steps for establishing a marker-assisted selection system (MAS). Thus, increasing productivity through genetic selection is a common goal for many animal breeding programs worldwide. The amount of milk, milk fat, and proteins are essential features of dairy farming. In cheese production, milk fatty acids perform a crucial technical function, as they are the main building blocks of milk fat, giving the cheese its unique taste and textural properties. Milk fatty acids are necessary for producing many dairy products, while intramuscular fat is associated with meat quality. Triacylglycerols (TAG) are the main components of intramuscular fat and milk fat. Therefore, understanding polymorphisms and genes related to fat synthesis is essential for animal husbandry. The identification of quantitative trait loci (QTL) and genes associated with milk production traits has been supported by various studies over the past decade. It was established that QTL genes on chromosomes 14, 15, and 9 are related to the properties of milk and meat production in goats. The presence of the diacylglycerol-O-acyltransferase 1 (DGAT1) gene in goat chromosomes 14, 15, and 9 has been reported. Having a key role in fat metabolism and TAG synthesis, DGAT1 genes have attracted considerable attention, especially in animal milk production. Several polymorphisms have been documented in DGAT1 in different animal species, including many cattle and small cattle, for their association with milk production traits. The critical role of the DGAT1 gene in milk fat metabolism makes it an exciting candidate for genetic variation in milk characteristics in dairy goats.

MiR-497 Regulates LATS1 through the PPARG Pathway to Participate in Fatty Acid Synthesis in Bovine Mammary Epithelial Cells.

Nutrient metabolism is required to maintain energy balance in animal organisms, and fatty acids play an irreplaceable role in fat metabolism. In this study, microRNA sequencing was performed on mammary gland tissues collected from cows during early, peak, and late lactation to determine miRNA expression profiles. Differentially expressed miRNA (miR-497) was selected for functional studies of fatty acid substitution. Simulants of miR-497 impaired fat metabolism [triacylglycerol (TAG) and cholesterol], whereas knockdown of miR-497 promoted fat metabolism in bovine mammary epithelial cells (BMECs) in vitro. In addition, in vitro experiments on BMECs showed that miR-497 could down-regulate C16:1, C17:1, C18:1, and C20:1 as well as long-chain polyunsaturated fats. Thus, these data expand the discovery of a critical role for miR-497 in mediating adipocyte differentiation. Through bioinformatics analysis and further validation, we identified large tumor suppressor kinase 1 (LATS1) as a target of miR-497. siRNA-LATS1 increased concentrations of fatty acids, TAG, and cholesterol in cells, indicating an active role of LATS1 in milk fat metabolism. In summary, miR-497/LATS1 can regulate the biological processes associated with TAG, cholesterol, and unsaturated fatty acid synthesis in cells, providing an experimental basis for further elucidating the mechanistic regulation of lipid metabolism in BMECs.

Docosahexaenoic Acid Alters Lipid Metabolism Processes via H3K9ac Epigenetic Modification in Dairy Goat.

Goat milk is increasingly recognized by consumers due to its high nutritional value, richness in short- and medium-chain fatty acids, and richness in polyunsaturated fatty acids (PUFA). Exogenous supplementation of docosahexaenoic acid (DHA) is an important approach to increasing the content of PUFA in goat milk. Several studies have reported benefits of dietary DHA in terms of human health, including potential against chronic diseases and tumors. However, the mechanisms whereby an increased supply of DHA regulates mammary cell function is unknown. In this study, we investigated the effect of DHA on lipid metabolism processes in goat mammary epithelial cells (GMEC) and the function of H3K9ac epigenetic modifications in this process. Supplementation of DHA promoted lipid droplet accumulation increased the DHA content and altered fatty acid composition in GMEC. Lipid metabolism processes were altered by DHA supplementation through transcriptional programs in GMEC. ChIP-seq analysis revealed that DHA induced genome-wide H3K9ac epigenetic changes in GMEC. Multiomics analyses (H3K9ac genome-wide screening and RNA-seq) revealed that DHA-induced expression of lipid metabolism genes (FASN, SCD1, FADS1, FADS2, LPIN1, DGAT1, MBOAT2), which were closely related with changes in lipid metabolism processes and fatty acid profiles, were regulated by modification of H3K9ac. In particular, DHA increased the enrichment of H3K9ac in the promoter region of PDK4 and promoted its transcription, while PDK4 inhibited lipid synthesis and activated AMPK signaling in GMEC. The activation of the expression of fatty acid metabolism-related genes FASN, FADS2, and SCD1 and their upstream transcription factor SREBP1 by the AMPK inhibitor was attenuated in PDK4-overexpressing GMEC. In conclusion, DHA alters lipid metabolism processes via H3K9ac modifications and the PDK4-AMPK-SREBP1 signaling axis in goat mammary epithelial cells, providing new insights into the mechanism through which DHA affects mammary cell function and regulates milk fat metabolism.

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-1β, 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.

Excavation and characterization of key circRNAs for milk fat percentage in Holstein cattle.

Milk fat percentage is one of the significant indicators governing the price and quality of milk and is regulated by a variety of non-coding RNAs. We used RNA sequencing (RNA-seq) techniques and bioinformatics approaches to explore potential candidate circular RNAs (circRNAs) regulating milk fat metabolism. After analysis, compared with low milk fat percentage (LMF) cows, 309 circRNAs were significantly differentially expressed in high milk fat percentage (HMF) cows. Functional enrichment and pathway analysis revealed that the main functions of the parental genes of differentially expressed circRNAs (DE-circRNAs) were related to lipid metabolism. We selected four circRNAs (Novel_circ_0000856, Novel_circ_0011157, novel_circ_0011944, and Novel_circ_0018279) derived from parental genes related to lipid metabolism as key candidate DE-circRNAs. Their head-to-tail splicing was demonstrated by linear RNase R digestion experiments and Sanger sequencing. However, the tissue expression profiles showed that only Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 were expressed with high abundance in breast tissue. Based on the subcellular localization found that Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 mainly function as competitive endogenous RNAs (ceRNAs) in the cytoplasm. Therefore, we constructed their ceRNA regulatory networks, and the five hub target genes (CSF1, TET2, VDR, CD34, and MECP2) in ceRNAs were obtained by CytoHubba and MCODE plugins in Cytoscape, as well as tissue expression profiles analysis of target genes. These genes play a key role as important target genes in lipid metabolism, energy metabolism, and cellular autophagy. The Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 regulate the expression of hub target genes through interaction with miRNAs and constitute key regulatory networks that may be involved in milk fat metabolism. The circRNAs obtained in this study may act as miRNA sponges and thus influence mammary gland development and lipid metabolism in cows, which improves our understanding of the role of circRNAs in cow lactation.

Milk fatty acids as covariates in multiple regression analysis is a robust approach to model the decrease in milk fat concentration and yield in small ruminants.

Milk fat depression (MFD) syndrome has been associated with the antilipogenic effects of trans-10 fatty acids (FA), such as t10, c12-CLA (CLA) and t10-18:1 (T10). However, these FA alone cannot completely explain the changes in milk fat in small ruminants. Thus, the aim of this study was to use multiple regression analysis to evaluate other FA that may be related to shifts in milk fat, as well as to improve model accuracy when different milk FA are used as covariates in the models. Previously published data were used in multiple regression analysis for goats (n = 106) and ewes (n = 68). Body weight (BW), vaccenic acid (t11-18:1), both trans-10 FA and the major milk FA were tested as covariates to model four response variables associated with MFD: fat concentration (FC), percentage change in milk fat concentration (CFC; %), fat yield (FY; g/d) and percentage change in milk fat yield (CFY; %). All four multiple regression models were significant for both species. When compared with simple regression models, all multiple regression models improved accuracy when estimating MFD. The improvements in model accuracy (lower RMSE) for FC, CFC, FY and CFY were 60.6%, 43.3%, 35.6% and 44.4% for ewes, and 52.1%, 60.1%, 33.6% and 14.9% for goats respectively. Linolenic acid and t11-18:1 were covariates in all models for goats, and palmitic acid and CLA were covariates in all ewe models. These FA should be investigated regarding their direct effect on gene expression associated with milk fat metabolism in the mammary gland of small ruminants. Multiple regression analysis is the most robust approach to account for the variation of milk fat and yield in goats and ewes.

CircRNA screening and ceRNA network construction for milk fat metabolism in dairy cows.

Milk fat is one of the main reference elements for evaluating milk quality and is a primary objective trait in dairy cattle breeding. In recent years, circular RNAs (circRNAs) have been found to play crucial roles in many biological processes. However, the function and expression profiles of circRNAs in milk fat synthesis in cows are not completely understood. We performed RNA sequencing to analyze the genome-wide expression of circRNA transcripts in bovine mammary epithelial cells (BMECs) from cows with extreme differences in milk fat percentage. We identified candidate differential circRNAs associated with milk fat metabolism using functional enrichment analysis and constructed a lipid metabolism-related competing endogenous RNA (ceRNA) interactive regulatory network. A total of 290 circRNAs were significantly differentially expressed (DE-circRNAs) in high milk fat percentage (HMF) cows compared to that in low milk fat percentage (LMF) cows. Of the 290 circRNAs, 142 were significantly upregulated and 148 were significantly downregulated. Enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified four DE-circRNAs (circ_0001122, circ_0007367, circ_0018269, and circ_0015179) that potentially regulate milk fat metabolism. Among them, circ_0001122, circ_0007367, and circ_0015179 had relatively high expression levels in cow mammary gland tissue compared to other tissues (heart, liver, kidney, uterus, ovaries, and small intestine) of cows. The regulatory networks circ_0001122:miR-12043:LIPG, circ_0007367:miR-331-3p:CIDEA/PML, and circ_0018269:miR-11989:RORC/HPX are potential networks to explore the mechanism of milk fat regulation. These results reveal the possible role of circRNAs in milk fat metabolism in dairy cows. Several important circRNAs and ceRNAs affecting milk fat synthesis were identified, providing insights into the complex biology of milk fat synthesis as well as a novel theoretical perspective for future research on lactation, milk quality, and breed improvement in dairy cows.

Deciphering the Key Regulatory Roles of KLF6 and Bta-miR-148a on Milk Fat Metabolism in Bovine Mammary Epithelial Cells.

MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of their target genes involved in many cellular functions at the post-transcriptional level. Previously, bta-miR-148a showed significantly high expression in bovine mammary epithelial cells (BMECs) of Chinese Holstein cows producing high milk fat compared to those with low milk fat content. Here, we investigated the role of bta-miR-148a through targeting Krüppel-like factor 6 (KLF6) and further analyzed the role of KLF6 in regulating fat metabolism through targeting PPARA, AMPK/mTOR/PPARG, and other fat marker genes in BMECs of Chinese Holstein. The bioinformatics analysis showed that the 3’ UTR of KLF6 mRNA possesses the binding sites for bta-miR-148a, which was further verified through dual-luciferase reporter assay. The BMECs were transfected with bta-miR-148a-mimic, inhibitor, and shNC, and the expression of KLF6 was found to be negatively regulated by bta-miR-148a. Moreover, the contents of triglyceride (TG), and cholesterol (CHO) in BMECs transfected with bta-miR-148a-mimic were significantly lower than the contents in BMECs transfected with bta-miR-148a-shNC. Meanwhile, the TG and CHO contents were significantly increased in BMECs transfected with bta-miR-148a-inhibitor than in BMECs transfected with bta-miR-148a-shNC. In addition, the TG and CHO contents were significantly decreased in BMECs upon the down-regulation of KLF6 through transfection with pb7sk-KLF6-siRNA1 compared to the control group. Contrarily, when KLF6 was overexpressed in BMECs through transfection with pBI-CMV3-KLF6, the TG and CHO contents were significantly increased compared to the control group. Whereas, the qPCR and Western blot evaluation of PPARA, AMPK/mTOR/PPARG, and other fat marker genes revealed that all of the genes were considerably down-regulated in the KLF6-KO-BMECs compared to the normal BMECs. Taking advantage of deploying new molecular markers and regulators for increasing the production of better-quality milk with tailored fat contents would be the hallmark in dairy sector. Hence, bta-miR-148a and KLF6 are potential candidates for increased milk synthesis and the production of valuable milk components in dairy cattle through marker-assisted selection in molecular breeding. Furthermore, this study hints at the extrapolation of a myriad of functions of other KLF family members in milk fat synthesis.