<i>LEP</i> Gene Promotes Milk Fat Synthesis via the JAK2-STAT3 and mTOR Signaling Pathways in Buffalo Mammary Epithelial Cells

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 <i>LEP</i> gene and conducted experiments to investigate its function in buffalo mammary epithelial cells (BuMECs). In this study, two transcript variants of <i>LEP</i>, designated as <i>LEP</i>_X1 and <i>LEP</i>_X2, were identified. The coding sequences (CDS) of <i>LEP</i>_X1 and <i>LEP</i>_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 <i>LEP</i> gene expression in lactating BuMECs was significantly higher than in non-lactating cells, with <i>LEP</i>_X2 expression remarkably higher than <i>LEP</i>_X1 in lactating BuMECs. Overexpression of both <i>LEP</i>_X1 and <i>LEP</i>_X2 significantly promoted the expression of genes related to milk fat synthesis in lactating BuMECs, including <i>STAT3</i>, <i>PI3K</i>, <i>mTOR</i>, <i>SCD</i>, and <i>SREBF1</i>, accompanied by an increase in cellular triglycerides (TG). Interestingly, <i>LEP</i>_X2 overexpression significantly suppressed <i>LEP</i>_X1 expression while increasing intracellular TG concentration by 12.10-fold compared to <i>LEP</i>_X1 overexpression, suggesting an antagonistic relationship between the two variants and supposing <i>LEP</i>_X2 plays a dominant role in milk fat synthesis in lactating BuMECs. Additionally, four nucleotide substitutions were identified in the buffalo <i>LEP</i> 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 <i>LEP</i> in milk fat synthesis and can provide a basis for molecular breeding strategies of buffalo.