Identification and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent induction of milk fat globule-epidermal growth factor 8-associated microvesicles.
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.
Aoki
Naohito
N
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. n-aoki@bio.mie-u.ac.jp
Jin-no
Shinji
S
Nakagawa
Yoshimi
Y
Asai
Noriyuki
N
Arakawa
Erina
E
Tamura
Noriko
N
Tamura
Tomohiro
T
Matsuda
Tsukasa
T
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000954:Antigens, Surface / Q000302:isolation & purification / Q000378:metabolism*; D017209:Apoptosis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D006728:Hormones / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D008894:Milk Proteins / Q000302:isolation & purification / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009765:Obesity / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism; D040901:Proteomics; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013395:Sucrose; D014461:Ultracentrifugation
D000954:Antigens, Surface; D017077:Culture Media, Conditioned; D006728:Hormones; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D010743:Phospholipids; D013395:Sucrose