Elevated oxidative stress–NF-κB–p62 pathway in adipose tissues from T2DM patients. (A) The heatmap displayed DEGs among isolated primary human adipocytes from the lean, obesity, and T2DM groups using hierarchical clustering analysis (n = 6/group). (B and C) Statistical analysis of RNA-sequencing gene expression for obesity (B) and T2DM (C) compared with lean as volcano plots. (D) KEGG pathway analysis of DEGs indicated that ROS pathway, NF-κB signaling, and fatty acid metabolism were affected pathway in T2DM adipocytes (arrow). (E) The mRNA level of representative genes related to ROS regulation, inflammation, and fatty acid metabolism in adipocytes (n = 6/group). (F) Heatmap showing the expression of key inflammatory activation related genes in adipocytes between the lean and T2DM groups. (G) GSEA demonstrating enrichment score (ES) of gene sets in RNA-sequencing data of adipocytes. Genes in each gene set were ranked by signal-to-noise ratio according to their differential expression between the lean group and the T2DM group. FDR, false discovery rate. (H–N) Trx2–NF-kB analyses in ND subjects (n = 10) and patients with T2DM-HS (n = 12). (H and I) Relative levels of Trx2 and TNFA mRNA in visceral adipose tissues. (J) Representative Western blot analysis of Trx2, phospho-P65, and P65 in individual visceral adipose tissues of ND and T2DM-HS. Protein levels were quantified and presented as fold changes by taking WT as 1.0. n = 4 for each group. (K) Representative immunofluorescence staining of Trx2 and P65 in visceral adipose tissues from ND and T2DM-HS. (L–N) Gene expression of PINK-1, PRKN, and SQSTM1 in isolated visceral adipose tissues from ND and T2DM-HS patients. Quantitative data are presented as mean ± SEM. ns, not significant, *, P < 0.05; **, P < 0.01 versus corresponding control. Significance was assessed by two-tailed Student’s t test. Scale bars, 50 µm (K). AGE-RAGE, advanced glycation end product–receptor for advanced glycation end product.