ACSS2 governs milk fat synthesis in buffalo via a reciprocal positive feedback loop with SREBP1 and PPARG

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).

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.

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.

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.