Transcriptomic and Proteomic Insights Into Buffalo Milk Fat Synthesis and the Role of IGFBP4 in BMECs

ABSTRACT The content and composition of milk fat are critical determinants influencing milk flavor, nutritional value, and economic significance. Buffalo milk is characterized by its high‐fat content and complex lipid profile, characterized by elevated levels of health‐beneficial fatty acids such as linoleic acid, α‐linolenic acid, and arachidonic acid. However, the molecular regulatory mechanisms governing milk fat synthesis in buffaloes remain incompletely elucidated. This study employed transcriptomic analysis of milk fat globules (MFGs) from buffaloes exhibiting high and low milk fat content, identifying 15 949 annotated genes, including 234 differentially expressed genes (DEGs). Functional enrichment analysis revealed that these DEGs were predominantly associated with cell proliferation and differentiation, glyconeogenesis, and reproductive system development. Notably, the expression of IGFBP4 , AGPAT4 , GPAT3 , GPR84 , and PC exhibited positive correlations with buffalo milk fat content, identifying them as potential candidate genes regulating milk fat synthesis. Proteomic profiling identified 1678 proteins, including 53 differentially expressed proteins (DEPs). Enrichment analysis indicated that DEPs were primarily involved in nucleotide metabolism, the tricarboxylic acid (TCA) cycle, glycerophospholipid metabolism, and TGF‐β signaling. Integrated analysis revealed potential interactions involving the IGFBP4 and PC genes, as well as the ACO1 , TMED7 , and APRT proteins, highlighting IGFBP4 as a pivotal regulator of milk fat synthesis. Functional validation demonstrated that overexpression or knockdown of IGFBP4 in buffalo mammary epithelial cells (BMECs) significantly modulated cell proliferation and altered the expression of key milk fat synthesis‐related genes ( FABP3 , LPL , SCD , ACACA , and FASN ), indicating that IGFBP4 can promote de novo fatty acid synthesis and intracellular lipid storage while inhibiting exogenous fatty acid uptake. Collectively, this study provides novel mechanistic insights into the regulation of milk fat synthesis in buffaloes and establishes a foundation for enhancing lactation traits through targeted genetic breeding strategies.