Expression of the receptor of advanced glycation end-products (RAGE) and membranal location in peripheral blood mononuclear cells (PBMC) in obesity and insulin resistance

Abstract

Objectives: The present study aimed to evaluate the receptor of advanced glycation end-products (RAGE), NF-kB, NRF2 gene expression, and RAGE cell distribution in peripheral blood mononuclear cells (PBMC) in subjects with obesity and IR compared with healthy subjects.

Materials and methods: The mRNA expression levels of RAGE, NF-kB, NRF2, and GAPDH were determined in PBMC by qPCR in 20 obese (OB), 17 obese with insulin resistance (OB-IR), and 20 healthy subjects (HS), matched by age and sex. RAGE protein expression and its localization were determined by Western Blot and immunocytochemistry (ICC) analysis, total soluble RAGE (sRAGE) and MCP-1 plasma levels by ELISA.

Results: RAGE, NF-kB, and NRF2 genes mRNA expression in PBMCs did not show variation between groups. RAGE protein was lower in OB and OB-IR groups; RAGE was located predominantly on the cell-surface in the OB-IR group compared to the HS group (22% vs 9.5%, P<0.001). OB-IR group showed lower sRAGE plasma levels, and correlated negatively with HOMA-IR, ALT parameters (r= -0.374, r= -0.429, respectively), and positively with NFE2L2 mRNA (r= 0.540) P<0.05.

Conclusion: In this study, OB-IR subjects did not reflect significant differences in gene expression; however, correlations detected between sRAGE, biochemical parameters, and NRF2, besides the predominant RAGE distribution on the cell membrane in PBMC could be evidence of the early phase of the inflammatory cascade and the subsequent damage in specific tissues in subjects with OB-IR.

Keywords: AGER protein human; Insulin resistance; Obesity; Oxidative stress; Receptor for advanced - glycation end products.

Figure 1

mRNA and protein expression in PBMC. The AGER, RELA, NFE2L2 and GAPDH gene expression of total sampling was evaluated by RT-qPCR of 1 µg of total RNA isolated from PBMC. Lean, healthy subjects (HS, n= 20), obese individuals (OB, n=20), and obese subjects with IR (OB-IR, n=17). Expression levels were normalized with the TBP gene. Mean±SEM corresponds to a.u., arbitrary units. NS= No Significance

Figure 2

Comparisons of total protein lysates were analyzed in PBMC using Western blot. A) The bar graphs show decreased densitometric signal of 50 kDa RAGE protein and increased GAPDH protein expression in OB-IR and OB as compared to the HS group. B) Representative and complete blot of RAGE and GAPDH are shown. Samples (n=9 for each group) were normalized with respective densities of β-actin bands. Results are shown as fold change representing a relative expression according to the levels of expression in the HS group. Mean±SEM corresponds to a.u., relative arbitrary units

Figure 3

Distribution of RAGE protein expression in PBMC. PBMC slides were stained with anti-human RAGE. 40X Original magnification. Samples were: A) Negative control, B) Lean healthy subjects (HS), C) Obese subjects (OB) and D) obese and insulin resistant individuals (OB-IR). The distribution of RAGE was intracellular and on cell-surface, and these are marked with thin and thick arrows, respectively. E) Positive RAGE quantification of subcellular distribution. Data are expressed as percentage of 200 cells count by preparation (n=6 for each group). Data were analyzed using X2, P<0.001. The localization of RAGE is identified predominantly on the membrane in OB-IR group