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.

BackgroundPolychlorinated biphenyls (PCBs) are persistent organic pollutants. Due to their lipophilic character, they are preferentially stored within the adipose tissue. During the mobilisation of lipids, PCBs might be released from adipocytes into the bloodstream. However, the mechanisms associated with the release of PCBs have been poorly studied. Several in vivo studies followed their dynamics of release but the complexity of the in vivo situation, which is characterised by a large range of pollutants, does not allow understanding precisely the behaviour of individual congeners. The present in vitro experiment studied the impact of (i) the number and position of chlorine atoms of PCBs on their release from adipocytes and (ii) the presence of other PCB congeners on the mobilisation rate of such molecules.Methodology/Principal FindingsDifferentiated rat adipocytes were used to compare the behaviour of PCB-28, -118 and -153. Cells were contaminated with the three congeners, alone or in cocktail, and a lipolysis was then induced with isoproterenol during 12 hours. Our data indicate that the three congeners were efficiently released from adipocytes and accumulated in the medium during the lipolysis. Interestingly, for a same level of cell lipids, PCB-153, a hexa-CB with two chlorine atoms in ortho-position, was mobilised slower than PCB-28, a tri-CB, and PCB-118, a penta-CB, which are both characterised by one chlorine atom in ortho-position. It suggests an impact of the chemical properties of pollutants on their mobilisation during periods of negative energy balance. Moreover, the mobilisation of PCB congeners, taken individually, did not seem to be influenced by the presence of other congeners within adipocytes.Conclusion/SignificanceThese results not only highlight the obvious mobilisation of PCBs from adipocytes during lipolysis, in parallel to lipids, but also demonstrate that the structure of congeners defines their rate of release from adipocytes.

Adipose tissue plays key roles in energy homeostasis. Understanding its metabolism and regulation is essential to predict the impact of environmental changes on wildlife health, especially in fasting-adapted species. However, in vivo experimental work in wild vertebrates can be challenging. We have developed a novel in vitro approach of precision-cut adipose tissue slices from northern elephant seal (Mirounga angustirostris) as a complementary approach to whole animal models. Blubber biopsies were collected from 14 pups during early and late post-weaning fast (Año Nuevo, CA, United States), precision-cut into 1 mm thick slices and maintained in culture at 37°C for at least 63 h. The slices exhibited an efficient response to ß-adrenergic stimulation, even after 2 days of culture, revealing good in vitro tissue function. The response to lipolytic stimulus did not vary between regions of outer and inner blubber, but was higher at early than at late fast for inner blubber slices. At early fast, lipolysis significantly reduced leptin production. At this stage, inner blubber slices were also more efficient at producing leptin than outer blubber slices, especially in the non-lipolytic condition. This model will aid the study of adipose tissue metabolism and its response to environmental stressors in marine mammals.

h i g h l i g h t s MeHg induced a dose-dependent accumulation of neutral lipids in preadipocytes. Accumulation of lipids persisted beyond the cellular exposure to MeHg. MeHg increased n-3 PUFA levels in lipid droplets and dropped AA levels in membranes. Expression of adipocyte-and lipid-specific genes was modulated by MeHg. Total mercury accumulated in cells was released when MeHg exposure stopped.

AbstractRecent studies suggest that environmental pollutants play a role in the continuous increase of the worldwide prevalence of obesity. To our knowledge, there is only one study investigating the impacts of methylmercury, a common and more toxic form of mercury, mainly present in fish and shellfish, on adipose tissue. It shows that methylmercury can cause changes in the structure and function of 3T3-L1 preadipocytes. In this context, we decided to study the effects of methylmercury on primary cultured adipocytes of rainbow trout. This species is a well-known model to study the impact of pollution. In addition, fish adipose tissue metabolism is close to the one of humans. Using an in vitro model allows us to focus on adipocyte differentiation and lipid metabolism.Preadipocytes were obtained after isolating precursor cells from perivisceral adipose tissue by enzymatic digestion. Cells were cultured at 19°C until confluence before being incubated for 6 days in growth medium supplemented or not with a hormonal cocktail (insulin, dexamethasone, ciglitizone) enriched with 0, 0.3, 1.7 or 3.8 mM of methylmercury and lipid mixture. Cytotoxicity was evaluated every two days via LDH assay. At day 6, quantification of total mercury in cells was performed with Direct Mercury Analyser-80. Phospholipid and neutral lipid fractions were analyzed by gas chromatography and expression of adipocyte-related genes was evaluated by RT-PCR. Three biological replicates were carried out.No cytotoxicity was observed during the methylmercury treatment. Methylmercury was partially accumulated but in a dose-dependent manner. At day 6, all fatty acids (FA) from neutral lipid fraction significantly increased at the highest methylmercury concentration. However, the rate of increase was different for each FA. In particular, n-3 polyunsaturated FA were preferentially accumulated. The total amount of phospholipids was not impacted by methylmercury but a decrease of n-6 polyunsaturated FA was observed. Surprisingly, methylmercury decreased the expression of three adipocyte markers, FA transport protein 1 and glycerol-3-phosphate dehydrogenase in presence or absence of hormonal cocktail, and CCAAT/enhancer binding protein delta in presence of hormonal cocktail. On the other hand, methylmercury increased the expression of FA synthase and perilipin.As a conclusion, methylmercury appears to be a candidate obesogen as it increased the cellular lipid content in primary cultured trout adipocytes. Additional genes involved in adipogenesis, FA de novo synthesis and FA intracellular transport are being investigated. Further experiments currently conducted in our lab should allow us to better clarify the mechanisms involved.