AIM To explore the effects of branched-chain amino acids (BCAAs) on the inflammatory response in white adipose tissue (WAT) of obese mice and its downstream mechanisms.

METHODS Eight-week-old male C57BL/6J mice were randomly divided into three groups and fed for 12 weeks with either a normal chow diet (CD), a high-fat diet with added BCAAs (HFD + BCAA), or a calorically matched high-fat diet (HFD paired) corresponding to the HFD + BCAA group. The body weight of the mice was recorded weekly, and glucose tolerance and insulin tolerance tests were conducted after 12 weeks. Hematoxylin-eosin staining was used to evaluate WAT morphology. Bulk RNA sequencing was conducted to assess gene expression in WAT. F4/80 immunofluorescence staining was employed to evaluate macrophage infILtration in WAT. Proteomic analysis was performed to determine the impact of BCAAs on histone modifications in WAT. Western blot analysis was used to assess the levels of histone H3 trimethylation at lysine 36 (H3K36me3) and the expression of its methyltransferase, SET domain containing 2 (Setd2), in primary mouse macrophages incubated with palmitic acid (PA). Small interfering RNA was used to sILence Setd2 gene expression, and real-time quantitative PCR was used to evaluate the expression of inflammatory genes IL1b, IL6, TNF-α, and NOS2 in PA-incubated primary mouse macrophages.

RESULTS Compared to the HFD paired group, the HFD + BCAA group showed no significant change in body weight, but reduced WAT, and further impaired glucose and insulin tolerance (P<0.01). Bulk RNA sequencing revealed significant changes in epididymal WAT gene expression in the HFD + BCAA group, with notable upregulation of inflammatory pathways. BCAA treatment led to increased macrophage infILtration and elevated expression levels of inflammatory factors in WAT (P<0.01). The HFD + BCAA group exhibited significantly higher levels of histone H3K36me3 modification in WAT compared to the HFD paired group (P<0.01), predominantly in infILtrating macrophages. In vitro experiments showed that BCAAs increased H3K36me3 modification levels (P<0.01), upregulated Setd2 methyltransferase, and increased the expression of inflammatory genes in PA-incubated macrophages (P<0.01). SILencing Setd2 expression reversed the pro-inflammatory effects of BCAAs on macrophages (P<0.01).

CONCLUSION BCAAs promote the inflammatory response in WAT of obese mice by increasing macrophage inflammatory gene expression through the Setd2-H3K36me3 pathway.