Buffalo Mammary Epithelial Cells Research Articles

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of β-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Molecular characterization, function, tissue differential expression, and single-nucleotide polymorphism of buffalo TP53 gene

Abstract. TP53 has been shown to be involved in lactation in cattle. However, the role of TP53 in buffalo lactation remains unknown. To this end, we isolated and identified the complete coding sequence (CDS) of the TP53 gene from the buffalo mammary gland and further analyzed its molecular characteristics, function, tissue differential expression, and single-nucleotide polymorphism (SNP). A transcript of this gene was cloned with a CDS length of 1161 bp, encoding a protein consisting of 386 amino acid residues. Bioinformatics analysis showed that buffalo TP53 CDS and the physicochemical characteristics, conserved domains, structure, and function of its encoded protein are highly similar to those of other species in Bovidae. The buffalo TP53 protein contains an N-terminal activation domain, a DNA-binding domain, and a tetrameric domain, and it plays a functional role in the nucleus. TP53 was found to express in all 11 detected buffalo tissues, and its expression in the heart, kidney, brain, muscle, and rumen during lactation was significantly higher than that during non-lactation (p<0.05), while in the liver, lung, and mammary gland, its expression was the opposite (p<0.05). Interference experiments in buffalo mammary epithelial cells (BuMECs) showed that TP53 inhibits the expression of genes related to milk protein and milk fat synthesis through the PI3K–AKT–mTOR pathway. A synonymous nucleotide substitution (c.204C > T) was found in the TP53 CDS of river buffalo, which is the CC homozygote in swamp buffalo. The results indicate that the TP53 gene is involved in buffalo lactation by negatively regulating the synthesis of milk protein and milk fat.

Role of Different Members of the AGPAT Gene Family in Milk Fat Synthesis in Bubalus bubalis.

During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPATs affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1, AGPAT3, and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1, AGPAT3, and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1, FASN, GPAM, DGAT2, and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.

Molecular Characteristics and Polymorphisms of Buffalo (Bubalus bubalis) ABCG2 Gene and Its Role in Milk Fat Synthesis.

The ATP-binding cassette subfamily G member 2 (ABCG2) serves crucial roles in secreting riboflavin and biotin vitamins into the milk of cattle, mice, and humans, as well as in the transportation of xenotoxic and cytostatic drugs across the plasma membrane. However, the specific role of the ABCG2 gene in water buffaloes (Bubalus bubalis), especially its effect on milk fat synthesis in buffalo mammary epithelial cells (BuMECs), remains inadequately understood. In this study, the full-length CDS of the buffalo ABCG2 gene was isolated and identified from the mammary gland in buffaloes. A bioinformatics analysis showed a high degree of similarity in the transcriptional region, motifs, and conservative domains of the buffalo ABCG2 with those observed in other Bovidae species. The functional role of buffalo ABCG2 was associated with the transportation of solutes across lipid bilayers within cell membranes. Among the 11 buffalo tissues detected, the expression levels of ABCG2 were the highest in the liver and brain, followed by the mammary gland, adipose tissue, heart, and kidney. Notably, its expression in the mammary gland was significantly higher during peak lactation than during non-lactation. The ABCG2 gene was identified with five SNPs in river buffaloes, while it was monomorphic in swamp buffaloes. Functional experiments revealed that ABCG2 increased the triglyceride (TAG) content by affecting the expression of liposynthesis-related genes in BuMECs. The results of this study underscore the pivotal role of the ABCG2 gene in influencing the milk fat synthesis in BuMECs.

Pre-treatment but not co-treatment with vitexin alleviates hyperthermia induced oxidative stress and inflammation in buffalo mammary epithelial cells

This study investigated if in vitro supplementation of vitexin could mitigate the adverse effects of hyperthermia on buffalo mammary epithelial cells (BuMECs). Immortalized BuMECs were divided into seven groups (n = 3): (1) a negative control group at 37 °C; (2) BuMECs exposed to heat stress as a positive control at 42 °C for 1 h; (3−7) heat stressed BuMECs pre-treated or co-treated with different concentrations of vitexin (5 μM, 10 μM, 20 μM, 50 μM, and 100 μM), respectively. Hyperthermia was induced by exposing the cells to 42 ºC for 1 h. For the pre-treatment experiment, BuMECs were treated with vitexin for 2 h before hyperthermia exposure. For co-treatment, vitexin was added simultaneously with hyperthermia for 1 h. Subsequently, the cells were allowed to recover for 12 h at 37 °C. Results showed that pre-treatment with vitexin was more effective than co-treatment in protecting BuMECs from hyperthermia in a dose-dependent manner, with higher concentrations (50 μM and 100 μM) being the most effective. Pre-treatment with vitexin maintained cellular viability and prevented inflammation by inducing the expression of the anti-apoptotic gene (BCL-2) and reducing the expression of the pro-apoptotic gene (Bax) and pro-inflammatory mediators (IL-1β, IL-6) in heat-stressed BuMECs. Pre-treatment with vitexin reduced oxidative stress and induced thermotolerance by increasing the expression of antioxidants mediators such as SOD, GPx and CAT at mRNA and protein levels, and modulating the expression of heat shock proteins. The findings suggest that vitexin has the potential as a therapeutic agent to protect the mammary gland from the negative impact of hyperthermia in dairy cows.

MiR-190a regulates milk protein biosynthesis through the mTOR and JAK2–STAT5 signaling pathways by targeting PTHLH in buffalo mammary epithelial cells

Milk protein is a key quality indicator of milk, so elucidating its synthesis mechanism has been the focus of research in recent years. Studies have shown that both miRNAs and mRNA genes play important roles in the regulation of milk protein synthesis. In this study, a total of 7 and 4073 differentially expressed miRNA and mRNA genes were identified between early and peak lactation, respectively. Overexpression and knockdown experiments of parathyroid hormone like hormone gene (PTHLH) gene showed that it can promote milk protein synthesis in buffalo mammary epithelial cells (BuMECs) through mammalian target of rapamycin (mTOR) and janus kinase 2/signal transducer and activator of transcription 5 (JAK2–STAT5) signaling pathways, and the experiments treated with miR-190a mimic or inhibitor showed that miR-190a inhibited the expression of PTHLH gene. In addition, miR-190a inhibited milk protein synthesis by targeting PTHLH through mTOR and JAK2–STAT5 signaling pathways in BuMECs. Collectively, these findings reveal a novel miR-190a–PTHLH pathway that has a significant role in regulating milk protein synthesis.

CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells

Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2-STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis.

Novel Insight into the Role of Squalene Epoxidase (SQLE) Gene in Determining Milk Production Traits in Buffalo.

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Squalene epoxidase (SQLE) is one of the rate-limiting enzymes for cholesterol biosynthesis and was highly expressed in the buffalo mammary. The objectives of the present study were to detect the polymorphisms within SQLE in buffalo, the genetic effects of these mutations on milk production traits, and to understand the gene regulatory effects on buffalo mammary epithelial cells (BuMECs). A total of five SNPs were identified by sequencing, g.18858G > A loci were significantly associated with fat yield, and g.22834C > T loci were significantly associated with peak milk yield, milk yield, fat yield, and protein yield. Notably, linkage disequilibrium analysis indicated that 2 SNPs (g.18858G > A and g.22834C > T) formed one haplotype block, which was found to be significantly associated with milk fat yield, fat percentage, and protein yield. Furthermore, expression of SQLE was measured in different tissues of buffalo and was found to be higher in the mammary. Knockdown of SQLE gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis, and significantly downregulated the expression of related genes MYC, PCNA, and P21. In addition, knockdown of the SQLE gene significantly reduces triglyceride concentrations and the signal intensity of oil red O staining. In addition, silencing of SQLE was also found to regulate the synthesis and secretion of β-casein and κ-casein negatively. Furthermore, SQLE knockdown is accompanied by the downregulation of critical genes (RPS6KB1, JAK2, eIF4E, and SREBP1) related to milk fat and protein synthesis. The current study showed the potential of the SQLE gene as a candidate for buffalo milk production traits. It provides a new understanding of the physiological mechanisms underlying buffalo milk production regulation.

TLR4/NFκB-Mediated Anti-Inflammatory and Antioxidative Effect of Hexanic and Ethanolic Extracts of Curcuma longa L. in Buffalo Mammary Epithelial Cells

Mastitis is the commonest disease of bovines imposing a great economic setback and a drastic public health concern worldwide. Antibiotic resistance is the preliminary con of excessive antibiotics use for disease treatment. Studies validate an alarming increase in the antibiotic resistance of both humans and animals. For ages, medicinal plants have been used to treat a number of ailments in humans and animals, including mastitis. Curcuma longa, or turmeric, is the prerequisite in Indian cooking and has been used in traditional medicine for its magical properties. The rhizome of C. longa possesses significant anti-inflammatory, antibacterial, antifungal, and antioxidative properties. In the current study, we evaluated the hexanic and ethanolic extracts of C. longa for their anti-inflammatory and antioxidative potential against LPS-induced inflammation in buffalo mammary epithelial cells (BuMECs). Pretreatment with the extracts downregulated the expression of proinflammatory cytokine TNFα via the TLR4/NFκB-mediated signaling pathway. However, IL-6 was downregulated in only the hexanic C. longa-pretreated group. A significant upregulation of NRF2 mRNA expression was seen in both hexanic- and ethanolic-treated groups. A GC-MS/MS study of the extracts revealed the presence of important sesqueterpenoids and phenolics as the main bioactive phytoconstituents in the extracts. Sesqueterpenoids, such as turmerone, ar-turmerone, curlone, and atlantone, and phenols, such as guaiacol (2-Hydroxyanisole phenol, 2-methoxy), and ethyl ferulate/ethyl 4′-hydroxy-3′methoxycinnamate, were found in C. longa extracts. The protective role of C. longa in BuMECs against LPS-induced inflammation and oxidant insult might be due to the presence of bioactive compounds, such as terpenoid and phenolic compounds. However, we further propose the isolation of these phytoconstituents and their analysis using HPLC and NMR studies.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3–7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 μM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 μM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Identification and characterization of alternative splicing variants of buffalo LXRα expressed in mammary gland

Liver X receptor α (LXRα) is a ligand-dependent transcription factor and plays an important role in the regulation of cholesterol homeostasis, fatty acid biosynthesis and glucose metabolism. In this study, transcripts of LXRα gene were cloned and characterized from buffalo mammary gland, and three alternative splicing transcripts of buffalo LXRα gene were identified, named LXRα1, LXRα2 and LXRα3. The structure of the LXRα transcripts of buffalo and cattle was highly similar. Bioinformatics analysis showed that LXRα1 contains two complete functional domains of LXRα, one is the DNA-binding domain (NR_DBD_LXR) and the other is the ligand-binding domain (NR_LBD_LXR). The reading frame of LXRα2 is altered due to the skipping of exon 9, which truncates its encoding protein prematurely at the 400th amino acid residue, making it contain a complete DNA-binding domain and part of a ligand-binding domain. Due to the deletion of exon 4, the protein encoded by LXRα3 lacks 89 amino acid residues and contains only a complete ligand-binding domain, which makes it lose its transcriptional regulation function. In addition, motifs and conserved domains of three LXRα variants of buffalo were highly consistent with those of corresponding transcripts from other mammal species. Subcellular localization analysis showed that LXRα1 plays a functional role in the nucleus of buffalo mammary epithelial cells, while LXRα2 and LXRα3 are distributed in the nucleus and cytoplasm. Compared with non-lactating period, the mRNA abundance of the three LXRα transcripts in the mammary gland tissue of buffalo increased during lactating period, revealing that they play a key role in the synthesis of buffalo milk fat. Among the three LXRα transcripts, LXRα1 has the highest expression in the mammary gland, indicating that it is the major transcript in the mammary gland and has important regulatory functions, while LXRα2 and LXRα3 may have regulatory effects on the function of LXRα1. This study highlights the key role of LXRα alternative splicing in the post-transcriptional regulation of buffalo lactation.

Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

Functional roles for AGPAT6 in milk fat synthesis of buffalo mammary epithelial cells

AGPAT6 plays a crucial role in the triglyceride (TG) synthesis pathway in mammals. However, its roles in buffalo lactation remain unknown. Therefore, we investigated the functional roles of AGPAT6 in milk fat synthesis by transfecting overexpression and lentivirus interference vectors in buffalo mammary epithelial cells (BuMECs) in vitro. AGPAT6 overexpression in BuMECs significantly enhanced the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, LPIN1, INSIG1, CEBPA and SREBF1 genes, and significantly reduced that of XDH, CPT1A, LIPE, INSIG2 and PPARGC1A, but has no significant influence to the mRNA abundance of FABP3, GPAM, PPARG and SREBF2. However, the interference with AGPAT6, the mRNA expression of FABP4, SLC27A6, ACSL1, DGAT1, DGAT2, INSIG1, CEBPA, SREBF1, XDH, CPT1A, LIPE, INSIG2 and PPARGC1A genes in BuMECs changed contrary to the overexpression experiment, and that of GPAM, PPARG and SREBF2 also did not change significantly, but the expression of FABP3 was significantly decreased. In addition, the overexpression/interference of AGPAT6 gene significantly increased/decreased TG content in BuMECs. The results here indicate that AGPAT6 gene is involved in TG synthesis in BuMECs, and affects the expression of major genes associated with FA transport and activation, TG synthesis and transcription regulation, FA oxidation and TG degradation during the lipogenesis of milk.

Evolutionary and Association Analysis of Buffalo FABP Family Genes Reveal Their Potential Role in Milk Performance.

The fatty acid-binding protein (FABP) family gene encode a group of proteins that affect long-chain fatty acid (LCFAs) trafficking and play a crucial function in the regulation of milk fat synthesis. Nevertheless, little is known regarding the identification of members, theevolutionary background, and functional characteristics of FABP genes in buffalo. In this study, in silico analysis was performed to identify the members of FABPs in buffalo. The results revealed that a total of 17 FABP genes were identified. Based on their phylogenetic relationships, these sequences clustered into five groups with similar motif patterns and gene structures. According to positive selection analyses, all duplicated gene pairs containing FABPs in buffalo had Ka/Ks (nonsynonymous/synonymous) ratios that were less than 1, suggesting that they were under purifying selection. Association analysis showed that one SNP in LOC102401361 was found significantly associated with buffalo milk yield. The expression levels of several FABPs in buffalo mammary epithelial cells were regulated by palmitic and stearic acid treatment. The findings of this study provide valuable information for further research on the role of FABPs in regulating buffalo milk synthesis.

LPIN1 promotes triglycerides synthesis and is transcriptionally regulated by PPARG in buffalo mammary epithelial cells

Studies on 3T3-L1 cells and HepG2 hepatocytes have shown that phosphatidic acid phosphohydrolase1 (LPIN1) plays a key role in adipogenesis, acting as a co-activator of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) to regulate fatty acid metabolism. However, the functional role and regulatory mechanism of LPIN1 gene in milk fat synthesis of buffalo are still unknown. In this study, overexpression of buffalo LPIN1 gene transfected with recombinant fusion expression vector significantly increased the expression of AGPAT6, DGAT1, DGAT2, GPAM and BTN1A1 genes involved in triglyceride (TAG) synthesis and secretion, as well as PPARG and SREBF1 genes regulating fatty acid metabolism in the buffalo mammary epithelial cells (BMECs), while the lentivirus-mediated knockdown of buffalo LPIN1 dramatically decreased the relative mRNA abundance of these genes. Correspondingly, total cellular TAG content in the BMECs increased significantly after LPIN1 overexpression, but decreased significantly after LPIN1 knockdown. In addition, the overexpression or knockdown of PPARG also enhanced or reduced the expression of LPIN1 and the transcriptional activity of its promoter. The core region of buffalo LPIN1 promoter spans from − 666 bp to + 42 bp, and two PPAR response elements (PPREs: PPRE1 and PPRE2) were identified in this region. Site mutagenesis analysis showed that PPARG directly regulated the transcription of buffalo LPIN1 by binding to the PPRE1 and PPRE2 on its core promoter. The results here reveal that the LPIN1 gene is involved in the milk fat synthesis of BMECs, and one of the important pathways is to participate in this process through direct transcriptional regulation of PPARG, which in turn significantly affects the content of TAG in BMECs.

Negative effect of insulin-induced gene 2 on milk fat synthesis in buffalo mammary epithelial cells.

Insulin-induced gene 2 (INSIG2) is a recently identified gene that is implicated in the regulation of cholesterol metabolism and lipogenesis in mammals. Although the data in goats emphasizes a role for INSIG2 in milk fat synthesis, the regulatory mechanism in buffalo is not clear. In this study, we analyzed the protein abundance of INSIG2 at peak lactation and dry-off period in buffalo mammary tissue. The results indicated that, relative to the peak lactation, the protein abundance of INSIG2 in the dry-off period was higher. To determine the function of INSIG2 in milk fat synthesis, INSIG2 was overexpressed and knocked down by lentiviral transfection in buffalo mammary epithelial cells (BuMECs). The response to overexpressing INSIG2 included down-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 coupled with a decrease in content of triacylglycerol (TAG). However, in response to knockdown of INSIG2, the significant increase in content of TAG along with marked up-regulation of SREBP, PPARG, FASN, ELOVL6, SCD, APGAT6 and TIP47 suggests that INSIG2 negatively affects milk fat synthesis in BuMECs. No significant difference in mRNA abundance of GPAM and DGAT2 in response to overexpression or interference of INSIG2 indicates that they might also be influenced by other regulatory factors. Taken together, our results provide strong support for the negative effect of INSIG2 on milk fat synthesis in BuMECs.

Liver X receptor α promotes milk fat synthesis in buffalo mammary epithelial cells by regulating the expression of FASN

Liver X receptor α (LXRα; NR1H3) is an important transcription factor that can facilitate milk fat synthesis by regulating the transcription of FASN in mice and goats. Nevertheless, the lipid synthesis related to LXRα and its regulation on FASN in the buffalo mammary gland remain elusive. Here, we demonstrated that the mRNA and protein expression of LXRα in buffalo mammary tissue increased in lactation compared with that in the dry-off period. Overexpression of NR1H3 enhanced the lipid droplet formation and triacylglycerol concentration in buffalo mammary epithelial cells (BuMEC), whereas the knockdown of NR1H3 resulted in a decrease in the number of lipid droplets. At the same time, NR1H3 also affected the expression of regulatory factors (INSIG1, INSIG2, SREBF1, and PPARG) related to milk fat synthesis and that of genes involved in de novo synthesis (FASN, ACACA, and SCD), and uptake and transport (LPL, CD36, and FABP3) of fatty acids as well as triacylglycerol synthesis (GPAM, APGAT6, and DGAT1). Luciferase reporter assays indicated that overexpression of NR1H3 resulted in an increase in the activity of FASN promoter, whereas the knockdown of NR1H3 had an opposite effect. When NR1H3 was overexpressed, mutations in LXRE or SRE could decrease the promoter activity of FASN. Furthermore, mutagenesis of both LXRE and SRE within the FASN promoter completely eliminated the induced activity of LXRα. Our results reveal that buffalo LXRα promotes milk fat synthesis through regulating the expression of FASN by directly interacting with FASN promoter and affecting the SREBF1 expression. This study underscores a crucial role of LXRα in regulating lipid synthesis of the buffalo mammary gland.

TMT based deep proteome analysis of buffalo mammary epithelial cellsand identification of novel protein signatures during lactogenic differentiation.

The lactating mammary gland harbours numerous matured alveoli with their lumen surrounded by differentiated mammary epithelial cells (MECs), which are exclusively involved in milk synthesis and secretion. Buffalo (Bubalus bubalis) is the second major milk-producing animal, and its physiology is different from cattle. The complete protein machinery involved in MECs differentiation is still not defined in ruminants, in particular, buffalo. Therefore, we have studied the differential expression of regulated proteins in the in vitro grown buffalo MECs (BuMECs) at different time points (on 3, 6, 12, and 15 days) of their differentiation in the presence of lactogenic hormones. TMT-based MS analysis identified 4,934 proteins; of them, 681 were differentially expressed proteins (DEPs). The principal component analysis suggested a highly heterogeneous expression of DEPs at the four-time points of hormone treatment, with most of them (307) attained the highest expression on 12 days. Bioinformatics analysis revealed the association of DEPs with 24 KEGG pathways. We observed few new proteins, namely ABCA13, IVL, VPS37, CZIB, RFX7, Rab5, TTLL12, SMEK1, GDI2, and TMEM131 in BuMECs. The function of one of the highly upregulated proteins, namely involucrin in the differentiation of BuMECs was confirmed based on biochemical inhibition assay. The results further conclude that the proteins with higher abundance can be considered as the potential biomarkers for differentiation, and they may have a significant association with the lactation process in buffalo too. The proteome dataset obtained can be used to understand the species-specific variations among other lactating animals.

Transcriptional Repression of MFG-E8 Causes Disturbance in the Homeostasis of Cell Cycle Through DOCK/ZP4/STAT Signaling in Buffalo Mammary Epithelial Cells.

The mammary gland is a unique apocrine gland made up of a branching network of ducts that end in alveoli. It is an ideal system to study the molecular mechanisms associated with cell proliferation, differentiation, and oncogenesis. MFG-E8, also known as Lactadherin, is a vital glycoprotein related to the milk fat globule membrane and initially identified to get secreted in bovine milk. Our previous report suggests that a high level of MFG-E8 is indicative of high milk yield in dairy animals. Here, we showed that MFG-E8 controls the cell growth and morphology of epithelial cells through a network of regulatory transcription factors. To understand the comprehensive action, we downregulated its expression in MECs by MFG-E8 specific shRNA. We generated a knockdown proteome profile of differentially expressed proteins through a quantitative iTRAQ experiment on a high-resolution mass spectrometer (Q-TOF). The downregulation of MFG-E8 resulted in reduced phagocytosis and cell migration ability, whereas it also leads to more lifespan to knockdown vis-a-vis healthy cells, which is confirmed through BrdU, MTT, and Caspase 3/7. The bioinformatics analysis revealed that MFG-E8 knockdown perturbs a large number of intracellular signaling, eventually leading to cessation in cell growth. Based on the directed network analysis, we found that MFG-E8 is activated by CX3CL1, TP63, and CSF2 and leads to the activation of SOCS3 and CCL2 for the regulation of cell proliferation. We further proved that the depletion of MFG-E8 resulted in activated cytoskeletal remodeling by MFG-E8 knockdown, which results in the activation of three independent pathways ZP4/JAK-STAT5, DOCK1/STAT3, and PIP3/AKT/mTOR. Overall, this study suggests that MFG-E8 expression in mammary epithelial cells is an indication of intracellular deterioration in cell health. To date, to the best of our knowledge, this is the first study that explores the downstream targets of MFG-E8 involved in the regulation of mammary epithelial cell health.

Elongase of very long chain fatty acids 6 (ELOVL6) promotes lipid synthesis in buffalo mammary epithelial cells.

Recent studies have shown elongase of very-long-chain fatty acids 6 (ELOVL6) is a vital protein for endogenous synthesis of saturated and monounsaturated long-chain fatty acids in some mammals. Nevertheless, its role in lipid synthesis in buffalo mammary gland is still unclear. In this work, the full-length coding sequence (CDS) of ELOVL6 was cloned and identified from buffalo mammary gland. As a result, the CDS of this gene is 795bp, which encodes a polypeptide of 264 amino acid residues. The buffalo ELOVL6 contains an ELO domain which belongs to the ELO superfamily. Among the 10 tissues of buffalo in peak lactation detected by RT-qPCR, the expression level of ELOVL6 was the highest in the brain, followed by the spleen, and then decreased in the mammary gland, muscle, kidney, heart, liver, rumen, intestine and lung. However, only the expression in the brain and spleen was statistically different from that in other tissues (p<0.05). Compared with that of the dry-off period, the mRNA abundance of ELOVL6 in the mammary gland was significantly increased in peak lactation. The experiments based on lentivirus transfection in buffalo mammary epithelial cells (BuMECs) displayed that the overexpression of ELOVL6 markedly promoted the expression of INSIG1, INSIG2, SREBP, PPARG, FASN, GPAM, DGAT2 and APGAT6 genes, and the knockdown of ELOVL6 significantly decreased the mRNA abundance of INSIG2, SREBP, FASN, SCD, GPAM, APGAT6 and TIP47 genes. In addition, the increase or decrease of ELOVL6 expression level also caused the corresponding change of total triglyceride content in the BuMECs. The results here suggest that the ELOVL6 can catalyse the synthesis of long-chain fatty acids in the BuMECs, and it can indirectly affect the expression of genes related to milk fat synthesis through its catalytic products to promote the lipid biosynthesis of BuMECs.