Increased levels of calprotectin in obesity are related to macrophage content: impact on inflammation and effect of weight loss

Abstract

Calprotectin has been recently described as a novel marker of obesity. The aim of this study was to determine the circulating concentrations and expression levels of calprotectin subunits (S100A8 and S100A9) in visceral adipose tissue (VAT), exploring its impact on insulin resistance and inflammation and the effect of weight loss. We included 53 subjects in the study. Gene expression levels of the S100A8/A9 complex were analyzed in VAT as well as in both adipocytes and stromovascular fraction cells (SVFCs). In addition, circulating calprotectin and soluble receptor for the advanced glycation end product (sRAGE) concentrations were measured before and after weight loss achieved by Roux-en-Y gastric bypass (RYGB) (n = 26). Circulating concentrations and VAT expression of S100A8/A9 complex were increased in normoglycemic and type 2 diabetic obese patients (P < 0.01) and associated with markers of inflammation (P < 0.01). Oppositely, concentrations of sRAGE were significantly lower (P < 0.001) in both obese groups compared to lean volunteers. Elevated calprotectin levels in obese patients decreased (P < 0.00001) after RYGB, whereas sRAGE concentrations tended to increase. Calprotectin was mainly expressed by SVFCs, and its expression was significantly correlated (P < 0.01) with mRNA levels of the monocyte-macrophage-related molecules macrophage-specific antigen CD68 (CD68), monocyte chemotactic protein 1 (MCP1), integrin α-M (CD11B), and NADPH oxidase 2 (NOX2). Tumor necrosis factor-α treatment significantly enhanced (P < 0.05) the mRNA levels of S100 calcium-binding protein A8 (S100A8) of human visceral adipocytes. The increased levels of calprotectin in obesity and obesity-associated type 2 diabetes, its positive association with inflammation as well as the higher expression levels in the SVFCs in VAT suggests a potential role of this protein as a chemotactic factor in the recruitment of macrophages to VAT, increasing inflammation and the development of obesity-associated comorbidities.

Figures

Figure 1

Circulating levels of calprotectin and sRAGE of LN, obese NG and obese T2D volunteers as well as the effect of weight loss in obese patients achieved by RYGB. Bars represent the mean ± SEM of calprotectin and sRAGE plasma concentrations under basal conditions (LN: n = 16; NG: n = 20; T2D: n = 17) (A, B) and 13 months after RYGB (n = 26) (C, D). Differences between groups were analyzed by one-way ANOVA followed by Tukey tests or by two-tailed paired Student t tests. **P < 0.01 versus LN and versus presurgery values.

Figure 2

Analysis of the calprotectin subunits S100A8 and S100A9 in VAT. (A, B) Gene expression levels of S100A8 and S100A9 in VAT of LN, obese NG and obese T2D volunteers. Bars represent the mean ± SEM of the ratio between the gene expression to 18S rRNA. The expression level in LN subjects was assumed to be 1 (LN: n = 9; NG: n = 10; T2D: n = 10). Differences between groups were analyzed by one-way ANOVA followed by Tukey tests. *P < 0.05, **P < 0.01, versus LN, and †P < 0.05 versus NG. (C, D) Comparison of S100A8 and S100A9 gene expression in adipocytes and SVFCs isolated from VAT of obese patients. Bars represent the mean ± SEM of the ratio between the gene expression to 18S rRNA. The expression level in adipocytes was assumed to be 1 (adipocytes: n = 9; SVFCs: n = 11). Statistical differences were assessed by two-tailed unpaired Student t tests. ***P < 0.001 versus adipocytes.

Figure 3

Gene expression levels of CD68 (A), CD11B (B), MCP1 (C) and NOX2 (D) in VAT of LN, obese NG and obese T2D volunteers. Bars represent the mean ± SEM of the ratio between the gene expression to 18S rRNA. The expression level in LN subjects was assumed to be 1 (LN: n = 9; NG: n = 10; T2D: n = 10). Differences between groups were analyzed by one-way ANOVA followed by Tukey tests. *P < 0.05, **P < 0.01, versus LN, and †P < 0.05 versus NG.

Figure 4

Effect of TNF-α on the expression of both calprotectin subunits S100A8 and S100A9 in human omental adipocytes. Gene expression levels of TNFA (A) in VAT of LN, obese NG and obese T2D volunteers. Bars represent the mean ± SEM of the ratio between the gene expression to 18S rRNA. The expression level in LN subjects was assumed to be 1 (LN: n = 9; NG: n = 10; T2D: n = 10). Differences between groups were analyzed by one-way ANOVA followed by Tukey tests. **P < 0.01 versus LN. Bar graphs show the effect of coincubation for 24 h of TNF-α with omental adipocytes on the transcript levels of S100A8 (B) and S100A9 (C). The gene expression levels in unstimulated cells was assumed to be 1. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Tukey tests. *P < 0.05 versus unstimulated cells.