Animal

Total 40 C57BL/6 J male mice (22 ± 2 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd, they were randomly divided into 3 groups. STC group (n = 20), HFD (n = 10), High BCAA group (n = 10). STC group mice were fed with standard chow for 16 weeks. HFD group mice were fed with a high-fat diet for 16 weeks. High BCAA group was fed with a high BCAA diet (L-Amino Acid Rodent Diet Based on Teklad 7002 Rodent Chow With 150% Added BCAA, Research Diet Inc, catalog #A12030801). The housing conditions for the animals were maintained under specific pathogen-free circumstances, with a controlled temperature of 22 ± 2 °C and a relative humidity of 55% (ranging from 45 to 70%), and under a 12-h light–dark cycle. Mice were sacrificed by CO2 asphyxiation after 16 weeks of experimental period, all procedures involving the animals were performed following China's national regulations on the use of experimental animals and were in line with the standards set by the Laboratory Animal Welfare and Ethics Committee of Jinhua Municipal Central Hospital, which reviewed and approved the guidelines (AL-JHYY202415).

Histology and immunohistochemical Study

The collected subcutaneous white adipose tissue (sWAT) was fixed by inflation with a 4% buffered paraformaldehyde solution (dissolve in water, v/v). The tissues were then embedded in paraffin, after dehydration, cut into sections (5 μm) and stained using Hematoxylin & Eosin (H&E), anti-TNF-α, anti-MCP-1 and anti-IL-1β antibody. These stained sections were examined under a light microscope and analyzed using the Image J software. The antibodies used for TNF-α staining were sourced from Abcam (Catalog #ab220210) at a dilution of 1/200, for IL-1β staining from Abcam (Catalog #ab205924) at a dilution of 1/200, and for MCP-1 staining from Abcam (Catalog #ab214819) at a dilution of 1/200.

Measurement of cytokines

Adipose tissue samples were collected and rapidly frozen in liquid nitrogen. Upon experimentation, the tissues were homogenized in 500 µL of 0.2 M sodium acetate solution (pH 4.5) using a Precellys tissue homogenizer. The homogenate was centrifuged at 800g for 10 min at room temperature to remove debris, and the resulting supernatant was used to measure TNF-α (Catalog# DY410, R&D Systems), IL-1β (Catalog# DY401, R&D Systems), and MCP-1 (Catalog# DY479, R&D Systems) by using commercial ELISA kits.

Immunofluorescence staining

For immunofluorescence staining, antigen retrieval was performed on the sections by boiling them in sodium citrate buffer (0.1 mol/L sodium citrate, 0.1% Tween 20, pH 6.0) for 20 min. This was followed by blocking with 5% FBS in PBS for 1 h at room temperature. The sections were then incubated overnight at 4 °C with mouse anti-F4/80 (Abcam, Cat#ab6640, 1/500 dilution) and anti-iNOS (Abcam, Cat#ab178945, 1/500 dilution) antibodies. The next day, fluorescent dye-conjugated secondary antibodies (anti-rabbit 594 nm and anti-mouse 488 nm) were applied at room temperature for 1 h. Finally, the sections were mounted using ProLong™ Glass Antifade Mountant (Catalog# P36980, Invitrogen). The distribution of fluorescence in sWAT was observed using a laser scanning confocal microscope (Leica TCS SP8 CARS, Germany).

Glucose tolerance test and insulin tolerance test

Mice were fasted for 12 h under glucose tolerance test in 15 weeks. After measuring the baseline blood glucose concentration from a tail cut by a Glucometer test strip, mice were injected intraperitoneally with 20% glucose at 1.5 mg/g body weight. Blood glucose concentrations were measured at 15, 30, 45, 60, 90, and 120 min after glucose injection. Mice were fasted for 6 h under insulin tolerance test in 16 weeks. After measuring the baseline blood glucose concentration, mice were injected intraperitoneally with recombinant human insulin at 1.2 mU/g body weight (Catalog# 91,077 C, Sigma–Aldrich). Blood glucose concentrations were then measured at 15, 30, 45, 60, 90, and 120 min after insulin administration.

Isolation of adipose tissue macrophages

To procure visceral adipose tissue, we meticulously excised any apparent blood vessels and connective tissue using sterilized forceps and scissors. We then measured out two grams of this tissue, rinsed it thoroughly with 20 mL of Dulbecco's Phosphate-Buffered Saline (DPBS), and finely chopped it into pieces ranging from 1 to 2 mm in size. Following this, we subjected the chopped tissue to centrifugation at 550g in DPBS at a temperature of 4 °C for 10 min, which served to eliminate any red blood cells. Afterward, we introduced a digestion solution to the tissue and maintained the mixture at 37 °C for an hour to facilitate digestion. We then filtered the digested material through a 70 µm strainer (Catalog #CLS431751, Merck), performed another round of centrifugation, and collected the resultant pellet. This pellet was further processed to extract macrophages, utilizing CD14 + magnetic beads (Invitrogen, Catalog# 11149D) in alignment with the guidelines provided by the bead manufacturer. Once isolated, we quantified the macrophages, evaluated their viability, and prepared them for plating, setting the stage for further experimental investigations.

Flow cytometry

Isolated adipose tissue macrophage was analyzed using FACS assays on an LSRFortessa instrument (BD Biosciences). Subsequent statistical analysis of the data was performed using FlowJo software. For staining, cells were incubated with fluorochrome-conjugated antibodies, specifically anti-F4/80 (Abcam, Catalog #ab6640, at a dilution of 1/500), anti-206 (Abcam, Catalog #ab64693, at a dilution of 1/500), anti-CD86 (Abcam, Catalog #ab239765, at a dilution of 1/500), anti-11b (Abcam, Catalog #ab133357, at a dilution of 1/500).

RNA isolation

Total RNA was isolated using TRIzol reagent (Catalog# 15,596,026, Invitrogen), and reverse transcription of 1 μg of this RNA was conducted utilizing the GoScript™ Reverse Transcription Mix (Catalog# A2801, Promega). Subsequent real-time quantitative PCR (QPCR) analyses were carried out on an Applied Biosystems ViiA 7 Real-Time PCR System, employing QuantiNova SYBR® Green PCR Kit (Catalog# 208,056, QIAGEN) alongside specific primers for various genes as listed in Supplementary Table 1.

RNA‑sequencing

Total RNA was isolated using the RNeasy mini kit (Qiagen). Strand-specific RNA-seq libraries were prepared with the TruSeq stranded total RNA sample preparation kit (Illumina), quantified by the Qubit 2.0 Fluorometer (Life Technologies), and assessed for insert size with the 2100 bioanalyzer (Agilent). Clusters were generated on the cBot at a concentration of 10 pM and sequenced on the NovaSeq 6000 system (Illumina). Library preparation and sequencing were carried out by the Meiji Biotechnology Corporation.

KEGG enrichment analysis and gene set enrichment analysis (GSEA)

Differentially expressed genes (DEGs) were identified using DESeq2 from RNA-seq data. The gene list was filtered based on an adjusted P-value < 0.05 and a log2 fold change > 1. The resulting gene symbols were converted to Entrez Gene IDs using the org.Hs.eg.db package in R. KEGG Pathway Enrichment: KEGG pathway enrichment analysis was performed using the clusterProfiler package in R. The Entrez Gene IDs were input into the enrichKEGG function, specifying the organism as Homo sapiens (hsa). The significance of pathway enrichment was determined using a hypergeometric test, and P-values were adjusted for multiple testing using the Benjamini–Hochberg method. Pathways with an adjusted P-value < 0.05 were considered significantly enriched. Visualization of enriched pathways was conducted using the dotplot function.

GSEA was performed using the fgsea package in R. The ranked gene list and gene sets were input into the fgsea function. The analysis was run with a minimum gene set size of 15 and a maximum size of 500. Enrichment scores were calculated, and P-values were adjusted using the Benjamini–Hochberg method. Gene sets with an adjusted P-value < 0.05 were considered significantly enriched. Enrichment plots were generated for visualization.

Protein analysis by immunoblotting

Cellular or tissue samples were lysed in RIPA buffer (comprising 150 mM NaCl, 50 mM Tris–HCl at pH 7.4, 2 mM EDTA, 0.1% SDS, and 1% NP-40), which was fortified with a protease inhibitor mix (Catalog# HY-K0010, MedChemExpress) and a phosphatase inhibitor cocktail (Catalog# B15001, Bimake). The lysates were then subjected to protein separation by SDS-PAGE and the proteins were subsequently transferred to a PVDF membrane (Catalog# 1,620,177, BIO-RAD) with 300 mA for 2 h. Blocking of the membrane was performed using 10% nonfat milk for one hour, after which it was incubated with the appropriate primary antibody at 4 °C overnight. Following primary antibody incubation, the membrane was washed with TBST (containing 2.7 mM Tris base, 137 mM NaCl, and 0.1% Tween 20) and incubated with the relevant HRP-conjugated secondary antibody from Cell Signaling Technology for one hour at ambient temperature. Post-secondary antibody incubation, the membrane was washed again with TBST, and the protein bands were detected using ECL reagents (BIO-RAD), with band intensity quantification performed via ImageJ software. Anti-PP2CM antibody (catalog #ab135286, abcam), Anti-BCAT1 antibody (catalog # ab232700, abcam), Anti-BCAT2 antibody (catalog # ab307833, abcam), Anti-BCKDH-A antibody (catalog # ab305168, abcam), Anti-BDK antibody (catalog # ab128935, abcam), Anti-phospho-BCKDH-A antibody (catalog # ab302504, abcam), Anti-beta Actin antibody (catalog #ab8226, abcam), Anti-iNOS antibody (catalog # ab283655, abcam), Anti-JAK1 antibody (catalog # ab133666, abcam), Anti-phospho-JAK1 antibody (catalog # ab138005, abcam), Anti-SIRT1 antibody (catalog # ab189494, abcam), Anti-phospho-SIRT1 antibody (catalog # ab76029, abcam), Anti-IFGR1 antibody (catalog #12–5945-82, Invitrogen), Anti-IFGR2 antibody (catalog #PA5-109,847, Invitrogen).

Small interfering RNA (siRNA) transfection

siRNAs (GenePharma) were mixed with DharmaFECT 3 transfection reagent (Catalog# T-2003–03, Dharmacon) followed the manufacturer’s instruction and added to the macrophages at a final concentration of 20 nM for 48 h. The sequence of si-IFNGR1 is 5’-CCGGGCCAGAGTTAAAGCTAAGGTTCTCGAGAACCTTAGCTTTAAC.

TCTGGCTTTTTG-3’ (SHCLNG-NM_010511), IFNGR1 siRNA-Sigma.

Statistical analysis

Data were processed using GraphPad Prism 8.0.1 software. The Kaplan–Meier survival analysis with a log-rank test was used for the horizontal grid test comparisons. Histological data were analyzed using the Mann–Whitney test. Two-way ANOVA with Tukey’s multiple comparison test was applied for other comparisons. P-value < 0.05 was considered as statistical significance.