INTERVENTIONSodium acetate (NaA) at low (0.1 mM) and high (2 mM) concentrations, with or without lipopolysaccharide (LPS) stimulation
COMPARISONLPS-only treated cells (MOD group) and untreated control cells (CON group)
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This study demonstrates that sodium acetate (NaA) bidirectionally regulates macrophage inflammatory activity in a dose-dependent manner: low-dose NaA (0.1 mM) promotes inflammation and M1 polarization, while high-dose NaA (2 mM) suppresses them. The mechanism involves NaA altering intracellular acetate concentration and modulating the PPARγ/UCP2/AMPK/NF-κB signaling pathway independently of GPR43 or HDACs. These findings suggest that high-dose NaA may ameliorate non-alcoholic fatty liver disease (NAFLD) by inhibiting macrophage-driven hepatocyte lipid accumulation, providing a potential therapeutic dose range for NAFLD intervention.
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**Background:** Non-alcoholic fatty liver disease (NAFLD) affects approximately 32.4% of the global population, with non-alcoholic steatohepatitis (NASH) affecting 1.5–6.5%. Short-chain fatty acids (SCFAs), particularly acetate, are gut microbiota metabolites implicated in NAFLD pathogenesis, but their effects on macrophage-mediated inflammation and hepatocyte lipid metabolism remain controversial, with some studies showing anti-inflammatory effects and others showing pro-inflammatory effects. This study aimed to clarify the dose-dependent effects of sodium acetate (NaA) on macrophage activity and its downstream impact on hepatocyte lipid accumulation.
**Methods:** RAW264.7 macrophages and Kupffer cells were treated with LPS (20–100 ng/mL) and varying NaA concentrations (0.01–5 mM). Low-dose (0.1 mM, NaA-L) and high-dose (2 mM, NaA-H) were selected for detailed analysis. Inflammatory markers (TNF-α, IL-6, IL-1β) were measured by qRT-PCR; NF-κB p65 and c-Jun phosphorylation by Western blot; M1/M2 polarization by flow cytometry (CD86/CD206). Intracellular acetate was quantified by GC-MS. AMP/ATP ratio was measured by HPLC. AMPK involvement was tested using Compound C inhibitor. GPR43 involvement was tested via siRNA silencing. Lipid accumulation (TC, TG) and lipid synthesis genes (FAS, Scd1, ACC1, ACSS2, SREBP-1c, FATP2) were assessed in macrophages and AML-12 hepatocytes, including in co-culture and conditioned medium experiments.
**Key Results:** NaA at 0.1 mM increased TNF-α expression by 48.7% in RAW264.7 and 66.4% in Kupffer cells versus LPS-only controls, while 2–5 mM NaA reduced TNF-α by approximately 48% and 53%, respectively. NaA-L increased IL-6, IL-1β, and phosphorylation of NF-κB p65 and c-Jun; NaA-H suppressed these. M1 polarization increased from 54.6% (LPS-only) to 58.1% with NaA-L and decreased to 48.1% with NaA-H. Intracellular acetate fell to approximately 21.3 g/g prot (1/4 of control) with NaA-L but increased significantly with NaA-H. GPR43 siRNA and HDAC expression were unaffected by NaA. Both NaA-L and NaA-H increased TC, TG, ACSS2 protein, and lipid synthesis gene expression in macrophages. The AMP/ATP ratio decreased with NaA-L and was restored with NaA-H; Compound C abolished NaA's effects on AMPK, c-Jun, and NF-κB phosphorylation. NaA regulated PPARγ, UCP2, iNOS, and IκBα phosphorylation in a dose-dependent manner. In direct hepatocyte treatment, NaA (0.1–2 mM) dose-dependently increased lipid accumulation and lipogenic gene expression (e.g., SREBP-1c increased 55.6% at 0.1 mM and 210.0% at 2 mM). However, in co-culture with macrophages, NaA-H reduced hepatocyte lipid deposition while NaA-L exacerbated it.
**Clinical Implications:** This study reveals a critical dose-dependent, bidirectional effect of NaA on macrophage inflammatory activity and subsequent hepatocyte lipid metabolism, mediated through the PPARγ/UCP2/AMPK/NF-κB pathway independent of GPR43 or HDACs. The findings reconcile previously conflicting reports on acetate's role in NAFLD by demonstrating that low concentrations (0.1 mM) are pro-inflammatory and pro-steatotic, while high concentrations (2 mM) are anti-inflammatory and anti-steatotic via macrophage-hepatocyte crosstalk. This suggests that therapeutic acetate supplementation for NAFLD may require achieving sufficient portal or systemic concentrations to activate AMPK-dependent anti-inflammatory pathways. The study provides mechanistic rationale for dose selection in future preclinical and clinical trials of acetate-based interventions for NAFLD/NASH.