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.

Leptin (LEP), a protein hormone well-known for its role in metabolic regulation, has recently been linked to lipid metabolism in cattle. However, its function in buffalo mammary glands remains unclear. To address this issue, we isolated and identified the LEP gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of LEP, designated as LEP_X1 and LEP_X2, were identified. The coding sequences (CDS) of LEP_X1 and LEP_X2 are 504 bp and 579 bp in length, encoding 167 and 192 amino acid residues, respectively. Bioinformatics analysis revealed that LEP_X2 is a hydrophobic protein with an isoelectric point below 7 and contains a signal peptide, while LEP_X1 is hydrophilic and lacks a signal peptide. Our study found that LEP gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with LEP_X2 expression remarkably higher than LEP_X1 in lactating BuMECs. Overexpression of both LEP_X1 and LEP_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including STAT3, PI3K, mTOR, SCD, and SREBF1, accompanied by an increase in cellular triglycerides (TG). Interestingly, LEP_X2 overexpression significantly suppressed LEP_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to LEP_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing LEP_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo LEP CDS, including a nonsynonymous substitution c.148C>T (p.Arg50Cys), which was predicted to decrease the stability of the LEP protein without affecting its function. These results collectively underscore the significant role of LEP in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.

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.

Background/Objectives: Sterol regulatory element-binding transcription factor 2 (SREBF2) is a key transcription factor involved in regulating cholesterol homeostasis. However, its role in buffalo mammary gland lipid metabolism remains unclear. Methods: To address this, we isolated and characterized the SREBF2 gene from buffalo mammary glands and performed an in-depth analysis of its molecular characteristics, tissue-specific expression, and functional roles in buffalo mammary epithelial cells (BuMECs). Additionally, we investigated the single nucleotide polymorphisms (SNPs) of SREBF2 in both river and swamp buffalo. Results: The coding sequence (CDS) of buffalo SREBF2 is 3327 bp long and encodes a protein of 1108 amino acid residues. Bioinformatics analysis revealed that the molecular characteristics of buffalo SREBF2 were highly similar across Bovidae species, with collinearity being observed among them. An expression profile analysis revealed that SREBF2 is expressed in all 11 tested tissues of buffalo, with its expression level in the mammary gland being higher during lactation than in the dry period. The knockdown of SREBF2 in BuMECs during lactation led to a significant reduction in the expression of genes involved in triglyceride (TAG) and cholesterol synthesis, including PI3K, AKT, mTOR, SREBF1, PPARG, INSIG1, ACACA, SCD, DGAT1, LPL, CD36, HMGCR, and SQLE. This knockdown led to a 23.53% and 94.56% reduction in TAG and cholesterol levels in BuMECs, respectively. In addition, a total of 22 SNPs were identified in both buffalo types, of which four non-synonymous substitutions (c.301G>C, c.304A>T, c.1240G>A, and c.2944G>A) were found exclusively in the SREBF2 CDS of swamp buffalo, and the assessment revealed that these substitutions had no impact on SREBF2 function. Conclusions: These findings emphasize the critical role of SREBF2 in regulating both triglyceride and cholesterol biosynthesis, providing valuable insights into its functions in buffalo mammary glands.

Objective: Acetyl-CoA synthetase 2 (ACSS2) converts rumen-derived acetate into acetyl-CoA in ruminants. Whether it actively regulates lactation-associated transcriptional networks beyond its catalytic role remains unclear. This study aimed to characterize buffalo ACSS2 and investigate its function within the metabolic-transcriptional network of buffalo mammary epithelial cells (BuMECs).Methods: The buffalo ACSS2 coding sequence was cloned, and its expression across lactation stages was analyzed. Subcellular localization was determined via confocal microscopy. Through siRNA-mediated knockdown in BuMECs, we assessed cell viability, triglyceride (TAG) content, and the expression of core metabolic and regulatory genes.Results: ACSS2 expression was highly enriched in lactating mammary tissue, and the protein localized to both the nucleus and cytoplasm. In BuMECs, ACSS2 knockdown impaired lipogenesis, significantly reducing intracellular TAG and downregulating key lipid metabolism genes (FASN, ACACA, SCD, CD36, LPL, FABP3, DGAT1, DGAT2, and AGPAT6). It also inhibited cell proliferation and downregulated G1/S phase regulators (CCND1, CCNE1, CDK2, and CDK4). Mechanistically, ACSS2 depletion reduced the mRNA levels of master regulators SREBF1 and PPARG, while upregulating the SREBP1-inhibitor INSIG1, suggesting an INSIG1-mediated blockade of the lipogenic program.Conclusion: This study establishes ACSS2 as a critical metabolic checkpoint in the buffalo mammary gland. We propose that ACSS2 maintains a reciprocal positive feedback loop with SREBP1 and PPARG. By ensuring adequate acetyl-CoA to suppress INSIG1 and support nuclear histone acetylation, ACSS2 couples substrate availability to the stability of the lipogenic program and cell cycle progression. These findings reveal a conserved metabolic-epigenetic axis essential for high-efficiency lactation in ruminants.

Research has demonstrated that certain microRNAs (miRNAs) play a pivotal role in governing milk protein synthesis in mice and dairy cows. However, there remains a limited understanding of specific miRNAs that regulate milk protein synthesis in buffalo. While our previous transcriptome data identified miR‐29c and miR‐106b as potential regulators influencing buffalo milk protein synthesis, the precise mechanism by which they modulate this process remains elusive. To address this gap, we conducted overexpression and knockdown experiments in this study. Our findings reveal that miR‐29c and miR‐106b exhibit inhibitory effects on the mRNA levels of CSN2 and CSN3 genes, along with diminishing β ‐casein production, within buffalo mammary epithelial cells (BuMECs). Furthermore, these miRNAs exert regulatory effects on the transcription and phosphorylation states of pivotal components within the mammalian target of rapamycin (mTOR) and JAK2–STAT5 signaling pathways. Employing the luciferase reporter system and qPCR, we confirmed the PTHLH gene as a shared target of miR‐29c and miR‐106b. Notably, they collaboratively regulate PTHLH gene expression. Our previous investigation highlighted the role of the PTHLH gene in augmenting milk protein production within BuMECs, achieved via stimulation of the mTOR and JAK2–STAT5 signaling cascades. Consequently, the inhibitory effect of miR‐29c and miR‐106b on buffalo milk protein synthesis is attributed to their coregulation of PTHLH . These findings emphasize the significance of miR‐29c and miR‐106b as essential regulators of milk protein synthesis, shedding light on the underlying mechanism governing milk production in buffalo.