Objective: To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level.Methods: Adipose tissue from diet‐induced obese mice was digested by collagenase treatment and fractionated by density centrifugation to obtain an adipocyte floating layer and a pellet of stromal vascular cells. The cellular architecture of the adipocyte‐macrophage interaction in both intact white adipose tissue (WAT) and the separated density gradient floating layer fraction was analyzed by confocal immunohistochemistry. Cytokine expression was detected by semi‐quantitative real time PCR and immunohistochemical analysis.Results: Three dimensional image analysis of WAT and the separated “adipocyte” floating layer revealed lipid‐engorged macrophages, macrophages in contact with lipid droplets and sheath‐like assemblies of macrophages surrounding adipocytes. The macrophages immunostained for TNFα and to a lesser extent for the immunoregulatory cytokine IL‐10. TNFα staining was associated only with macrophages indicating that macrophages and not adipocytes are the source of TNFα expression in the adipocyte floating layer.Conclusion: Macrophages form assemblies that tightly adhere to and cover adipocytes and lipid droplets. TNFα found in low density adipocyte preparations is due to contamination with macrophages.

CEACAM1 regulates endothelial barrier integrity. Because insulin signaling in extrahepatic target tissues is regulated by insulin transport through the endothelium, we aimed at investigating the metabolic role of endothelial CEACAM1. To this end, we generated endothelial cell-specific Ceacam1 null mice (VECadCre+Cc1^fl/fl) and carried out their metabolic phenotyping and mechanistic analysis by comparison to littermate controls. Hyperinsulinemic-euglycemic clamp analysis showed intact insulin sensitivity in VECadCre+Cc1^fl/fl mice. This was associated with the absence of visceral obesity and lipolysis and normal levels of circulating non-esterified fatty acids, leptin, and adiponectin. Whereas the loss of endothelial Ceacam1 did not affect insulin-stimulated receptor phosphorylation, it reduced IRS-1/Akt/eNOS activation to lower nitric oxide production resulting from limited SHP2 sequestration. It also reduced Shc sequestration to activate NF-κB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFα levels. Loss of endothelial Ceacam1 also induced the expression of the anti-inflammatory CEACAM1-4L variant in M2 macrophages in white adipose tissue. Together, this could cause endothelial barrier dysfunction and facilitate insulin transport, sustaining normal glucose homeostasis and retaining fat accumulation in adipocytes. The data assign a significant role for endothelial cell CEACAM1 in maintaining insulin sensitivity in peripheral extrahepatic target tissues.