Characterization and visualization of the liposecretion process taking place during ceiling culture of human mature adipocytes

Abstract

Objective To investigate and further characterize the process of mature adipocyte dedifferentiation. Our hypothesis was that dedifferentiation does not involve mitosis but rather a phenomenon of liposecretion. Methods Mature adipocytes were isolated by collagenase digestion of human adipose tissue samples. Ceiling cultures were established using our six‐well plate model. Cells were treated with cytosine β‐d‐arabinofuranoside (AraC) or vincristine (VCR), two agents blocking cell division, and were compared with vehicle. Liposecretion events were visualized by time‐lapse microscopy, with and without AraC in adipocytes transducted with a baculovirus. Microscopic analyses were performed after labeling phosphorylated histone 3 and cyclin B1 in ceiling cultures. Results Treatment with AraC almost entirely prevented the formation of fibroblasts up to 12 days of ceiling culture. Similar results were obtained with VCR. The antimitotic effectiveness of the treatment was confirmed in fibroblast cultures from the adipose tissue stromal‐vascular fraction by proliferation assays and colony‐forming unit experiments. Using time‐lapse microscopy, we visualized liposecretion events in which a large lipid droplet was rapidly secreted from isolated mature adipocytes. The same phenomenon was observed with AraC. This was observed in conjunction with histone 3 phosphorylation and cyclin B1 segregation to the nucleus. Conclusion Our results support the notion that dedifferentiation involves rapid secretion of the lipid droplet by the adipocytes with concomitant generation of fibroblast‐like cells that subsequently proliferate to generate the dedifferentiated adipocyte population during ceiling culture. The presence of mitotic markers suggests that this process involves cell cycle progression, although cell division does not occur.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function

“…The exact mechanisms by which 14-3-3z knockdown in 3T3-L1 cells reduces intracellular lipids is not known, but it is possible that autophagy, which we have previously shown to be up-regulated by 14-3-3z depletion, could be a potential contributor (15,34). Alternatively, dedifferentiation of adipocytes is also associated with a rapid expulsion of lipid droplets from cells via a yet undefined mechanism (11).…”

Ceiling culture chip reveals dynamic lipid droplet transport during adipocyte dedifferentiation via actin remodeling

“…11 The phenomena of LD secretion by adipocytes has recently been documented as a central event of the dedifferentiation process whereby adipocytes transform into a delipidated state through a mechanism distinct from lipolysis. 12,13 Recently, electron micrographs of fat tissue biopsies from obese individuals revealed the presence of in vivo dedifferentiated adipocytes that had also undergone LD secretion to acquire the delipidated phenotype. 14 Despite its physiological importance, however, the molecular and cellular mechanisms of adipocyte dedifferentiation and LD secretion are poorly understood.…”

Insulin negatively regulates dedifferentiation of mouse adipocytes in vitro

“…Dedifferentiation of cells is a reversal of the developmental process, such as in cases of differentiated mature cells with specialized functions that produce undifferentiated progenitor cells [1][2][3] or they undergo a self-repair mechanism [4,5] under certain physiological and/or pathological conditions. Spalding et al reported that approximately 10% of adipocytes in human white adipose tissue are renewed annually during adult age [1].…”