The triglycerides (TGs) stored in the white adipose tissue are mobilized during periods of negative energy balance. To date, there is no in vitro model of adipocytes imitating a long period of negative energy balance in which triglycerides are highly mobilized. Such model would allow studying the mobilization of TGs and lipophilic compounds trapped within the adipose tissue (e.g., pollutants and vitamins). The present study aims at developing a performing long-term in vitro lipolysis in adipocytes, resulting in a significant decrease of TG stores. Lipolysis was induced on differentiated rat adipocytes by a lipolytic medium with or without isoproterenol for 12 h. The condition with isoproterenol was duplicated, once with medium renewal every 3 h and once without medium renewal. Adding isoproterenol efficiently triggered lipolysis in a short time (3 h). However, a single stimulation by isoproterenol, without medium renewal, was not sufficient to reduce the TG content during a longer term (12 h). A reesterification of fatty acids occurred after a few hours of lipolysis, resulting in a novel increase of cellular lipids. Regular medium renewal combined with repeated isoproterenol stimulations led to almost emptied cells after 12 h. However, medium renewal without isoproterenol stimulation for 12 h was as efficient in terms of lipid mobilization. Our study demonstrates that, over a short-term period, isoproterenol is required to exert a significant lipolytic effect on adipocytes. During a long-term period, the presence of isoproterenol is no longer essential. Instead, medium renewal becomes the main factor involved in cell emptying. The efficiency of this protocol was demonstrated by visual tracking of the cells and by monitoring the dynamics of release of a lipophilic compound, PCB-153, from adipocytes during lipolysis.

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BackgroundPolychlorinated biphenyls (PCBs) are persistent environmental pollutants that preferentially accumulate in lipid-rich tissues of contaminated organisms. Although the adipose tissue constitutes a major intern reservoir of PCBs and recent epidemiological studies associate PCBs to the development of obesity and its related disorders, little is known about the mechanisms involved in their uptake by the adipose tissue and their intracellular localization in fat cells.Methodology/Principal FindingsWe have examined the intracellular distribution of PCBs in mouse cultured adipocytes and tested the potential involvement of caveolin-1, an abundant adipocyte membrane protein, in the uptake of these compounds by fat cells. We show that 2,4,4′-trichlorobiphenyl (PCB-28), 2,3′,4,4′,5-pentachlorobiphenyl (PCB-118) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) congeners rapidly and extensively accumulate in 3T3-L1 or mouse embryonic fibroblast (MEF) derived cultured adipocytes. The dynamics of accumulation differed between the 3 congeners tested. By subcellular fractionation of primary adipocytes, we demonstrate that these pollutants were almost exclusively recovered within the lipid droplet fraction and practically not associated to cell membranes. The absence of caveolin-1 expression in primary adipocytes from cav-1 deficient mice did not modify lipid droplet selective targeting of PCBs. In cav-1 KO MEF differentiated adipocytes, PCB accumulation was decreased, which correlated with reduced cell triglyceride content. Conversely, adenoviral mediated cav-1 overexpressing in 3T3-L1 cells, which had no impact on total cell lipid content, did not change PCB accumulation.Conclusion/SignificanceOur data indicate that caveolin-1 per se is not required for selective PCB accumulation, but rather point out a primary dependence on adipocyte triglyceride content. If the crucial role of lipid droplets in energy homeostasis is considered, the almost exclusive accumulation of PCBs in these organelles warrants future attention as the impairment of their function could be linked to the worldwide obesity epidemic.