In our experiment we hypothesized that the size of adipocytes is an important determinant of the adipokine secretion profiles. Accordingly, the large adipocytes released more pro-inflammatory and less anti-inflammatory adipokines than the lean fat cells. Studies have shown that large adipocytes are characteristic for adipose tissue of obese individuals. Moreover, obesity is more common in older people, in whom the ability of new adipocytes to differentiate is significantly decreased. There is a lack of information about inflammatory activity in old fat cells because in most of in vitro studies researchers use young adipocytes, in which the state of inflammation is induced by LPS treatment. In our study we used 3T3-L1 cells on different stages of growth and differentiation. Adipocytes on post-induction day 8 (PID 8) were considered as young, on PID 12 as mature, and on PID 16 as old. We noted time-dependent changes in control cells, which were maintained in standard medium without experimental factors. We observed that age of cultured cells was positively correlated with their fat accumulation. In addition, the presented results revealed significantly increased secretion of IL-6 (pro-inflammatory cytokine) by old adipocytes (PID 16) in comparison with young fat cells (PID 8). Furthermore, secretion of adiponectin (anti-inflammatory cytokine) was significantly decreased on PID 16 in comparison with PID 12. Thus, ageing of adipocytes can cause their large size and higher release of pro-inflammatory adipokines. Presented results are in agreement with a study by Zoico and co-workers, in which the authors also showed similar correlations . These observations suggest that differentiated 3T3-L1 cells on the later stages of maturation can constitute a good experimental model for studying obesity and inflammation in adipose tissue.
Our study also aimed at evaluating the influence of EPA and DHA, which are regarded as PUFA with potential anti-obesity and anti-inflammatory properties, on fat cells secretory activities in relation to their age (days of culture). To our knowledge this is the first study investigating the effect of EPA and DHA during ageing of adipocytes. Our experiment did not reveal any differences in the intensity of lipolysis (glycerol release) and fat accumulation (Oil red O absorbance) between adipocytes treated with EPA, DHA and control cells. These results are contrary to previous findings showing that both EPA and DHA may increase lipolysis in differentiated 3T3-L1 cells [21–23]. However, it is hard to compare our data with the cited studies, in which the doses of evaluated fatty acids were higher, and the time of exposure was shorter. We used higher concentrations of EPA (300 μmol/l) and DHA (100, 200 μmol/l) in our preliminary studies, but these doses caused increased cell death (data not shown). Our observations indicate that the anti-obesity properties of n-3 PUFA are not connected with their influence on lipolysis in adipocytes, but possibly with induction of apoptosis in these cells.
Although we did not observe any effect of EPA and DHA on lipolysis and fat accumulation, we noted their significant role in secretion of adipokines by adipocytes on different stages of maturation. First adipokine investigated in our study was adiponectin, belongs to the anti-inflammatory adipokines secreted by adipose tissue, mainly by adipocytes. In organisms this protein is involved in i.e. glucose metabolism, fatty acids β-oxidation and insulin sensitivity . Numerous studies have shown that in obese humans and animals with insulin resistance, concentration of plasma adiponectin is significantly decreased. Moreover, increased endogenous production of this hormone improves insulin sensitivity . In the last few years, several studies have suggested that n-3 PUFA are able to decrease insulin resistance connected with obesity, possibly through their stimulatory effect on adiponectin secretion. However, the influence of examined fatty acids on synthesis of this protein by adipocytes is still unclear. Recent data from in vivo experiments on rodents seem to confirm the abovementioned thesis. On the other hand, in vitro studies provided contrary information on this subject. Kalupahana and co-workers performed an experiment on mice, as well as 3T3-L1 cell line, and revealed that diet supplementation with EPA improved adiponectin levels in blood of obese mice, but the authors did not observe any influence of EPA (150 μM) treatment on adiponectin release by differentiated 3T3-L1 cells . Findings of another group showed that both EPA and DHA (250 μM) were able to decrease the expression of apM1 gene, which encodes adiponectin . Our study demonstrated that investigated n-3 PUFA (EPA 100 μM, DHA 50 μM) increased adiponectin secretion by 3T3-L1 adipocytes, but only on the early stage of maturation (PID 8). These results are partially in agreement with the study by Oster and co-workers, who observed similar effects of EPA and DHA in young cells . However, our experiment showed that the stimulatory activity of both PUFA on adiponectin secretion by adipocytes was not sustained on the later days of culture (PID 12 and PID 16). Different findings of in vitro experiments performed on young fat cells could be explained by various doses used in both studies. Oster et. al  suggested that high concentrations of EPA and DHA can cause more intense lipid peroxidation in adipocytes, and development of oxidative stress associated with reduction of adiponectin gene expression.
We also examined the concentration of leptin released by 3T3-L1 adipocytes. White adipose tissue is the main source of this hormone in the organism, but it is also secreted by stomach, brain and bone morrow . This adipokine has a systemic action and is engaged in the regulation of food intake, energy expenditure, body fat storage and insulin signalling . Moreover, high blood concentrations of leptin are strongly correlated with obesity, and it was observed that these concentrations decrease with weight loss. Several reports, based on in vitro and in vivo experiments, revealed that n-3 PUFA are able to modulate leptin secretion by adipocytes. In our study we demonstrated a stimulatory effect of EPA supplementation on this hormone release by young (PID 8) 3T3-L1 adipocytes, but at this stage DHA did not show any impact. In the later period of cell culture we observed that both EPA and DHA significantly decreased secretion of leptin by cells on PID 12, but this effect was not noted on PID 16. The stimulation of leptin secretion by EPA observed on PID 8 was in agreement with other in vitro studies, showing that this fatty acid is able to increase leptin gene expression and leptin secretion in 3T3-L1 cells, as well as primary cultures of rat adipocytes [30, 31]. In contrast, Reseland and co-workers reported that EPA and DHA decreased leptin mRNA expression in vitro and in vivo . However, the results obtained by Reseland group are incomparable with our findings, because they used much higher doses of n-3 PUFA (500 μM). Other in vivo studies also showed that long-term diet supplementation with n-3 PUFA, resulted in decreased plasma leptin concentrations in rats and mice [32, 33]. This inhibitory effect observed in vivo is usually correlated with the anti-obesity influence of n-3 PUFA. To our knowledge, this is the first report in which the influence of EPA and DHA on leptin secretion has been investigated in 3T3-L1 adipocytes on different stages of cellular maturation. Our observations showing the opposite effects of these fatty acids on leptin secretion between PID 8 and PID 12, and no impact on PID 16 indicate that adipocytes reaction and sensitivity to EPA and DHA are age-dependent.
We also examined the influence of n-3 PUFA on secretion of interleukin 6 (IL-6) by 3T3-L1 cells during their aging. IL-6 is a pro-inflammatory cytokine secreted by monocytes, macrophages and fat cells. Studies have shown that the level of blood circulating IL-6 is significantly increased in obese subjects . Adipose tissue is a source of approximately 30% of this cytokine in the organism . IL-6 participates in the development of insulin resistance via different mechanisms. Firstly, this adipokine decreases adiponectin secretion by adipose tissue. Moreover, it directly affects the insulin signal transduction pathway in hepatocytes . The mechanism of this action is not completely understood, but it is believed that SOCS3 protein (suppressor of cytokine signal 3) may be involved. SOCS3 probably inhibits the insulin-dependent autophosphorylation of insulin receptor . Presented results showed that secretion of this pro-inflammatory adipokine by fat cells was strongly connected with their age, and was the highest in old adipocytes (PID 16). n-3 PUFA have been reported to have anti-inflammatory properties. Surprisingly, in our study we found that EPA supplementation increased IL-6 concentration in conditioned medium, while DHA exerted an opposite effect. Similar impact of investigated fatty acids was observed at all stages of cellular maturation. Contrary results were obtained by other research groups working with young adipocytes. Siriwardhana and co-workers observed that EPA treatment (200 μM) increased secretion of IL-6 in differentiated 3T3-L1 cells . On the other hand, Kalupahana and co-workers found that 150 μM concentration of EPA in incubation medium inhibited IL-6 release by adipocytes . These differences are hard to explain because in all experiments the time of treatment was the same, and the reaction of 3T3-L1 cells to n-3 PUFA treatment was not dose-dependent. These discrepancies show that this topic needs further investigation in the future. However, the results of our study indicate that EPA and DHA are able to modulate secretion of IL-6 not only in young cells but also in ageing adipocytes.
In addition, we examined the content of polyunsaturated fatty acids: EPA and DHA in cultured cells. The results obtained revealed that the uptake of investigated fatty acids increased with age. On the other hand, the percentage of EPA and DHA in relation to the total quantity of fatty acids decreased with age in most cases. The second observation correlated with age-dependant differences in secretion of examined adipokines and could at least partially explain the weaker effect of EPA and DHA on endocrine functions of old cells in comparison with young and mature adipocytes.