RAGE−/− mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All protocols for animal use were reviewed and approved by the Animal Care Committee of Southwest Medical University following Institutional Animal Care and Use Committee guidelines.

Eight-week-old male and female RAGE−/−mice were fed a high-fat diet (HFD) (TP2330055A; calories fat 60%, carbohydrate 25%, and protein 15%; Trophic Animal Feed High-tech Co. Ltd, China) for 16 weeks, as described previously [10]. Age-matched male mice that were fed a standard chow diet (ND; TP2330055AC; calories fat 10%, carbohydrate 75%, and protein 15%; Trophic Animal Feed High-tech Co. Ltd, China) were used as controls. Group animal size was n = 6–8 per group. The exact group size is specially described in the figure legends.

Following a 4-h fast, glucose (GTT) and insulin (ITT) tolerance tests were performed in response to intraperitoneal (IP) injection of D-glucose (Roth, Karlsruhe, Germany) (2 g of glucose/kg body mass) or insulin (0.75 U insulin/kg body mass, respectively. Blood samples were obtained from the tail vein, and whole blood glucose levels were measured at 0, 30, 60, and 120 min using a One Touch® Vita® glucometer (Lifescan, Zug, Switzerland).

Subcutaneous adipose tissue (sAT) was collected and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were pre-treated with deoxyribonuclease I (Invitrogen Life Technologies, Carlsbad, CA, USA), and a SuperScript kit (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to synthesize cDNA according to the manufacturer’s recommendations. qRT-PCR was analyzed using miScript SYBR Green PCR Kits (Qiagen). Levels of macrophage polarization and oxidative stress markers mRNAs were determined using an ABI PRISM 7700 cycler (Applied Biosystems, Foster City, CA). Fold changes in gene expression were determined using the 2 − ΔΔCT method. The values are presented as the mean ± SEM. All primers are listed in Additional file 2: Table S1.

sAT and visceral adipose tissues (epididymal adipose tissues; eAT) lysates were prepared, and equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. After blocking, the membranes were incubated with antibodies directed against phospho-Akt (Ser473), total AKT (Cell Signaling Technology, Massachusetts, USA). Secondary antibody was horseradish-peroxidase (HRP)-conjugated goat IgG raised against IgG (Santa Cruz Biotechnology). Blots were developed with ECL substrate (Pierce).

sAT was isolated, lysed, and the total amount of ROS was determined using the dihydroethidium (DHE) probe according to the manufacturer’s instructions (bjbalb Inc. Beijing, China). All values were normalized to total cellular protein, determined using a BCA assay, and expressed as intensity/mg protein.

sAT was isolated, fixed, embedded in paraffin, and serially sectioned (6 µm). Cross-sections were stained with hematoxylin–eosin. The images were captured using a microscope (Nikon). The percentage of positive cells/total adipocytes was quantified in five microscopic fields in each of the three cross sections of each tissue using ImagePro Plus software.

Data are presented as the mean ± SEM of triplicate experiments. The significance of the differences among groups was analyzed by one-way analysis of variance with a post hoc test to determine group differences in the study parameters. All analyses were performed with SPSS software (version 24.0 for Windows; Armonk, NY, USA), and a level of p < 0.05 was defined as indicative of statistical significance.