Adipocytes are now recognized as endocrine cells secreting adipocytokines, regulating multiple metabolic pathways. In this study, we addressed secretion of microvesicles by 3T3-L1 adipocytes. We found that MFG-E8, one of the exosomal proteins, was present in the microvesicles and was distributed in the sucrose density fractions with 1.13-1.20 g/ml, which has been reported for exosomes. Several integral, cytosolic, and nuclear proteins such as caveolin-1, c-Src kinase, and heat shock protein 70 were also found to be microvesicle components. Unexpectedly, adiponectin was also substantially distributed in the microvesicle fractions. Furthermore, proteomic analysis of the microvesicles revealed that many other proteins such as extracellular matrix-related proteins were also present. Microvesicles secreted by 3T3-L1 adipocytes exhibited heterogeneity in size and comprised both smaller exosome-like and larger membrane vesicles as revealed by electron microscopy. Milk fat globule-epidermal growth factor 8 (MFG-E8)-associated adiposomes exhibited binding activity toward phosphatidylserine and apoptotic cells. MFG-E8 in the microvesicles was reduced when cultured in the low-glucose medium or cultured in the high-glucose medium with antioxidant N-acetyl cysteine. Insulin and TNF-alpha also up-regulated MFG-E8 in the microvesicles. Moreover, MFG-E8 was strongly up-regulated in the hypertrophic adipose tissue, predominantly in adipocyte fractions, of diet-induced obese C57BL/6 mice, where increased oxidative stress is induced. Thus, it is suggested that microvesicles, especially MFG-E8-associated ones, modulate adipose functions under redox- and hormone-dependent regulation. Based on the above findings, the adipocyte-derived microvesicles were named adiposomes.

We previously reported that 3T3-L1 and rat primary adipocytes secreted microvesicles, known as adipocyte-derived microvesicles (ADMs). In the present study, we further characterized the 3T3-L1 ADMs and found that they exhibited angiogenic activity in vivo. Antibody arrays and gelatin zymography analyses revealed that several angiogenic and antiangiogenic proteins, including leptin, TNFalpha, acidic fibroblast growth factor (FGFa), interferon-gamma, and matrix metalloprotease (MMP)-2 and MMP-9, were present in the ADMs. Gene expression of most of these angiogenic factors was induced in the adipose tissue of diet-induced obese mice. Furthermore, leptin, TNFalpha, and MMP-2 were up-regulated at the protein level in the adipocyte fractions prepared from epididymal adipose tissues of high-fat-diet-induced obese mice. ADMs induced cell migration and tube formation of human umbilical vein endothelial cells, which were partially suppressed by neutralizing antibodies to leptin, TNFalpha, or FGFa but not to interferon-gamma. Supporting these data, a mixture of leptin, TNFalpha, and FGFa induced tube formation. ADMs also promoted cell invasion of human umbilical vein endothelial cells through Matrigel, which was suppressed by the addition of the MMP inhibitor 1,10'-phenanthroline and a neutralizing antibody to MMP-2 but not to MMP-9. These results suggest that ADMs are associated with multiple angiogenic factors and play a role in angiogenesis in adipose tissue.