Gene Profiling of Human Adipose Tissue During Evoked Inflammation In Vivo

OBJECTIVE

Adipose inflammation plays a central role in obesity-related metabolic and cardiovascular complications. However, few human adipose-secreted proteins are known to mediate these processes. We hypothesized that microarray mRNA profiling of human adipose during evoked inflammation could identify novel adipocytokines.

RESEARCH DESIGN AND METHODS

Healthy human volunteers (n = 14) were treated with intravenous endotoxin (3 ng/kg lipopolysaccharide [LPS]) and underwent subcutaneous adipose biopsies before and after LPS. On Affymetrix U133Plus 2.0 arrays, adipose mRNAs modulated >1.5-fold (with P < 0.00001) were selected. SignalP 3.0 and SecretomeP 2.0 identified genes predicted to encode secreted proteins. Of these, 86 candidates were chosen for validation in adipose from an independent human endotoxemia protocol (N = 7, with 0.6 ng/kg LPS) and for exploration of cellular origin in primary human adipocytes and macrophages in vitro.

RESULTS

Microarray identified 776 adipose genes modulated by LPS; 298 were predicted to be secreted. Of detectable prioritized genes, 82 of 85 (96% [95% CI 90–99]) were upregulated (fold changes >1.0) during the lower-dose (LPS 0.6 ng/kg) validation study and 51 of 85 (59% [49–70]) were induced greater than 1.5-fold. Treatment of primary adipocytes with LPS and macrophage polarization to M1 proinflammatory phenotype increased expression by 1.5-fold for 58 and 73% of detectable genes, respectively.

CONCLUSIONS

We demonstrate that evoked inflammation of human adipose in vivo modulated expression of multiple genes likely secreted by adipocytes and monocytes. These included established adipocytokines and chemokines implicated in recruitment and activation of lymphocytes, adhesion molecules, antioxidants, and several novel genes with unknown function. Such candidates may represent biomarkers and therapeutic targets for obesity-related complications.Activation of innate and adaptive immunity is a crucial link between adiposity and its metabolic complications (1–4). In rodents, modulation of toll-like receptor-4 (5), tumor necrosis factor (TNF) receptors (6), chemokines, and downstream kinases (7) attenuate diet-induced obesity and insulin resistance. Further, cross talk between immune cells and adipocytes promotes an inflammatory, insulin-resistant state in obesity. A key initiating event in adipose inflammation is recruitment of T-lymphocytes (8,9) and monocyte/macrophages (10,11) with elaboration of inflammatory adipocytokines that modulate metabolic signaling (12–15). Despite experimental evidence in rodent models, most evidence supporting these concepts in humans derives from observational and correlative studies (16–18). Indeed, validated adipokines that mediate, or serve as biomarkers for, complications of human adiposity remain limited.Expression of inflammatory, insulin-signaling, and lipid genes are perturbed in adipose of obese humans (19–21). Recently, the in vitro secretome of subcutaneous and visceral primary human adipocytes was described and includes many unexplored proteins modulated during adipogenesis (1,22). Remarkably, less than half of genes found in the human subcutaneous adipocyte secretome were previously found in the murine 3T3-L1 preadipocyte secretome (22), underscoring the importance of studies in human tissue.Experimental human endotoxemia can provide unique insights into the relationship of inflammation to metabolic disturbance in man (23,24). Others and we have shown that endotoxemia induces acute metabolic, lipoprotein, and oxidant responses that resemble the chronic changes in insulin resistance and metabolic syndrome (25,26). Notably, endotoxemia induces adipose inflammation (27) with activation of several adipose inflammatory cascades, including cytokines, chemokines, and suppressor of cytokine signaling (SOCS) molecules (26) that attenuate insulin signaling and are implicated in obesity and type 2 diabetes (28).We applied microarray mRNA profiling of human adipose during endotoxemia to identify novel inflammation-induced adipose genes. We focused on genes predicted to be secreted and validated our findings in vivo through independent experiments of low-grade human inflammation. Finally, we identified in vitro the likely human adipose cellular source of these top candidates.