Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and β-catenin signaling to promote adipocyte dedifferentiation

Background Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming.Methods To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-alpha neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-alpha signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of beta-catenin and applying BML-284, an agonist of beta-catenin.Results Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-alpha signaling that activates Wnt-beta-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-alpha inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-beta-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment.Conclusions Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-alpha as a pro-inflammatory cytokine during the stress response. Importantly, TNF-alpha is essential for the activation of the Wnt/beta-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/beta-catenin signaling using BML-284.

Automated summary

Hypertonicity induces dedifferentiation of adipocytes through the release of mitochondrial EVs and activation of TNF-α and Wnt/β-catenin signaling pathways.