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

Objective: Acetyl-CoA synthetase 2 (ACSS2) is the obligatory gatekeeper for converting rumen-derived acetate into acetyl-CoA in ruminants.However, whether ACSS2 actively regulates the transcriptional networks governing lactation, beyond its catalytic role, remains unclear.This study aimed to elucidate the molecular characteristics of buffalo ACSS2 and investigate its function as a central node in the metabolic-transcriptional circuitry of buffalo mammary epithelial cells (BuMECs). Methods:The complete coding sequence of buffalo ACSS2 was characterized, and its expression was analyzed across lactation stages.Subcellular localization was determined via high-resolution confocal microscopy.We utilized siRNA-mediated knockdown in BuMECs to assess cell viability, triglyceride (TAG) content, and the expression of core metabolic and regulatory genes to dissect the underlying molecular mechanisms. Results: ACSS2 expression was highly enriched in lactating mammary tissue, and the protein exhibited a dual nucleocytoplasmic distribution.ACSS2 knockdown induced a "dual collapse" of cellular function: it severely impaired lipogenesis (significantly reducing intracellular TAG and downregulating FASN, ACACA, SCD, CD36, LPL, FABP3, DGAT1, DGAT2 and AGPAT6) and arrested cell proliferation (downregulating the G1/S phase regulators CCND1, CCNE1, CDK2 and CDK4).Mechanistically, ACSS2 depletion dismantled the transcriptional machinery itself, suppressing the A c c e p t e d A r t i c l e 4 mRNA levels of master regulators SREBF1 and PPARG.Crucially, this collapse was accompanied by the paradoxical upregulation of the SREBP1-inhibitor INSIG1, suggesting that metabolic stress triggers an INSIG1-mediated blockade of the feedback loop.Conclusion: This study establishes ACSS2 as a critical metabolic checkpoint in the buffalo mammary gland, rather than a passive enzyme.We propose a model where ACSS2 maintains a reciprocal positive feedback loop with SREBP1 and PPARG.By ensuring sufficient acetyl-CoA to suppress INSIG1 and support histone acetylation (implied by nuclear localization), ACSS2 couples substrate availability to the stability of the lipogenic program and cell cycle progression.These findings reveal an evolutionarily conserved metabolic-epigenetic axis essential for high-efficiency lactation in ruminants.