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

Abstract

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.

Targeting the Circulating MicroRNA Signature of Obesity

“…Moreover, the signature of circulating miRNAs changed concomitantly with extensive weight loss. Interestingly, recent studies have demonstrated that adipocytes secrete microvesicles that may contain oligonucleotides (40 ). Indeed, adipocyte/adipose tissue-specific gene transcripts and microRNAs were found in microvesicles isolated from rat serum (13 ).…”

miR-450a-5p within rat adipose tissue exosome-like vesicles promotes adipogenic differentiation by targeting WISP2

“…The amount of PREF-1, a negative regulator of adipogenesis, decreased in EVs derived from cells during the later stages of differentiation (Connolly et al, 2015). Other adipogenesis-related proteins were also detected in adipocyte-derived exosomes including adenylate kinase 2 (AK2), and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) (Lee et al, 2015;Aoki et al, 2007). Furthermore, mRNAs encoding the adipogenesis-related proteins PPARγ, leptin, C/EBP-α and C/EBP-δ have also been detected in adipocyte-derived EVs and as being delivered to osteoblasts (Martin et al, 2015).…”

Adipocyte fatty acid binding protein: a novel adipokine involved in the pathogenesis of metabolic and vascular disease?

“…As an example, fibroblast growth factor-1 is exported through an unconventional release pathway involving the formation of a specific multiprotein complex [63]. Furthermore, murine adipocytes release various proteins lacking a secretory signal sequence by microvesicles and, most interestingly, AFABP is detected in microvesicles [64]. These findings are supported by an independent study that [73] 98 CAD Associated with MS + TG [74] 479 with carotid IMT Associated with IMT in women + Age, BP, carotid IMT (in women) [75] 234 with coronary angiograms Associated with CAD n.d. [76] 211 CAD ↑ in CAD + CAD 211 controls [77] 125 CAD ↑ in CAD + Plaque volume (CAD) 120 controls [78] 247 CAD ↑ in CAD + Coronary atherosclerosis index 94 controls [79] 18 CAD/CVD ↑ in CVD n.d. 18 controls [80] 87 controls Associated with vascular inflammation + Vascular inflammation [90] 306 acute ischaemic stroke ↑ in ischaemic stroke n.d.…”