We and others have previously shown that adipocytes secrete significant amounts of prostaglandins [3–7, 30]. Further, it is well established that PGE2 decreases lipolysis in adipocytes [11–14]. Our current study addressed whether PGE2 also induces lipogenic activities in adipocytes, which could further enhance their hypertrophic effects in these cells. We have previously reported responsiveness of the ApcMin/+ mice to changes in prostaglandin levels . In this study we tested whether changes in prostaglandins modulated fatty acid synthesis in adipose tissue of these mice. We demonstrated that inhibition of the COX enzymes by piroxicam (or other COX inhibitors, data not shown) decrease FAS activity (Fig. 1); this effect was reversed by admnistering mice EP receptor agonists. These results demonstrate that a lipogenic and receptor-mediated effect of PGE2, which coupled with its previously reported antilipolytic effects [11–14] likely favor triglyceride storage. To further test direct effects of PGE2 manipulation in adipocytes, we used 3T3-L1 adipocytes and used dietary polyunsaturated fatty acids (AA and EPA) as well as pharmacological means (COX inhibition) to modulate prostaglandin levels. Our goal was to test whether changes in PGE2 levels led to parallel changes in FAS activity or expression.
We report here dose-dependent increases in PGE2 levels with AA and EPA treatments (p < 0.001) and, as expected, AA exhibited a more potent induction of PGE2 secretion versus EPA or control. Very limited information exists in the literature regarding physiological levels of EPA, but they are unlikely to approach those of AA due to large difference in their levels in membrane phospholipids. Studies conducted on postmenopausal women fed fish oil found the EPA levels of plasma lipids was 750 μM [36, 37]. Our dose response studies indicate that despite the clearly powerful effect of AA in increasing PGE2 (compared to EPA), the latter was also able to significantly elevate PGE2 levels especially at the 200 and 500 uM doses. Doses at 500 uM and above visibly impacted cell viability and morphology (data not shown). Based on the manufacturer's information for cross-reactivity (and confirmed in our laboratory), the assay also detects PGE3 (cross reactivity with PGE2 antibody), which may explain at least in part increased PGE2 levels with EPA. Although the binding affinities of AA and EPA for both isoforms of COX are equivalent (Km = 5 μM), COX-1 and -2 oxygenate EPA at ~10% and ~35%, respectively, compared to the rate for AA when added exogenously to cultured cells. Compared to AA, EPA is a poorer substrate for COX-1 in vivo. Complicating this issue further is the fact that very little is known concerning the actions of PGE3 and its subsequent down stream signaling . Studies in NIH 3T3 fibroblasts found that PGE3 activated the same signaling pathways as PGE2, but with much less efficiency . This would imply that high levels of PGE3 might duplicate the actions of PGE2, suggesting there would be a point of diminishing returns with EPA supplementation and thus the importance of using appropriate low doses for experimentation and supplementation. It is also possible that PGE3 may bind to EP receptors with affinities that differ significantly from PGE2 , whereby the type and number of receptors in adipocytes would also influence the impact of EPA supplementation. While it is important to point out these differences in PGE2 vs. PGE3 formation, the main focus of this paper is primarily on the role of PG and COX in modulating fatty acid synthesis.
Based on our dose response data, we chose to use intermediate dosage of 150 μM for the rest of our experiments. This dose is also consistent with the findings of other studies [3, 35], which demonstrated the ability to manipulate secreted PGE2 by using EPA to compete with AA for incorporation into membrane phospholipids and subsequent PG production.
PGE2 levels in the CI treatment group were significantly lower than controls. Although the celecoxib preferentially inhibits COX-2, no studies have shown whether it reduces PGE2 levels in adipocytes. Most published evidence indicates that inducible enzyme COX-2 is not expressed at physiologically relevant levels in mature adipocytes under normal conditions [31, 45]. However, since we used a CI dose (1 μM) similar to that of IC50 (1.2 μM ), it is plausible that constitutively expressed COX-1 activity was also inhibited at this dose. Using FAS as a marker of adipocyte lipogenesis, our data showed decreases in FAS activity with the CI treatment. In agreement with our finding, another study also showed that a non-specific COX inhibitor, aspirin, also decreases lipogenesis or levels of triacylglycerols in adipocytes .
The overall comparison of PGE levels for the AA, EPA, and AA + EPA treatment groups correlated well with previously published data measuring tissue concentrations of these fatty acids in mice fed diets supplemented with these fatty acids, although the end points measured and experimental models were different in our study compared to those reported [33, 40, 47].
Treatment of adipocytes with either EPA or AA elicited significant reductions in FAS mRNA compared to control, consistent with previously reported inhibition of FAS message by PUFA (36) in a degree of unsaturation and chain length-dependent manner [17, 48]. Therefore, regulation of FAS expression in adipocytes by PUFA is independent of changes in prostaglandin levels. Alternatively, PG could be directly controlling FAS gene expression via a receptor-mediated mechanism. Such opposing effects may be explained by direct transcriptional effects of EPA and AA on the FAS gene. Indeed, this theory is in line with results from Deng et al. showing that degree of unsaturation correlated highly with suppression of the SREBP-1c promoter , the main transcription factor regulating FAS and other lipogenic genes. However, it is worth noting that since mRNA stability was not measured in these experiments, it is also possible that increased stability was responsible for the discrepancy between decreased FAS mRNA expression and small changes in FAS activity in response to PUFA treatments. It is also possible that this discrepancy is due to a longer half-life of the FAS protein such that we were unable to detect changes in enzyme activity within our treatment times (24–48 hours). Indeed, previous studies have shown that changes in FAS mRNA half-life depend on cell culture treatments and state of differentiation [49, 50]. Another possibility is that PGE2 treatments stimulate leptin secretion (as previously documented Fain et al ) and elevated leptin levels may subsequently decrease lipogenesis . Thus, decreased FAS expression may indirectly reflect effects of PGE2 mediated by leptin [52, 53]. Unfortunately, due to the very low levels of leptin secretion in 3T3-L1, we were not able to assess regulation of leptin in these cells. Additional possible mechanisms may involve the antithetic actions of the EP receptors. Long et al. investigated the role of COX mediated products of AA metabolism on regulation of glucose transporter 4 (GLUT 4). They found that a 50-fold increase in endogenous PGE2 or exposure to 10 μM exogenous PGE2 resulted in an increase in cAMP concentrations, consistent with activation of the EP2/EP4 receptor . Additionally, studies using the specific COX-2 inhibitor NS-398 on cortical collecting duct cells found that NS-398 treatment increased EP3 and EP4 receptor expression 3-fold . Although the concentration of exogenous PGE2 added in our treatments was much lower, preliminary gene expression studies demonstrate that celecoxib also influenced the expression of EP receptors (specifically EP4, data not shown), which may differ from effects of aspirin or other non-specific COX inhibitors. An increase in EP4 receptors and the resulting increase in cAMP would activate a pathway that would oppose the decrease in cAMP responsible for the PGE2 mediated decrease in lipolysis. Further work with EP receptor concentration and mechanism of action is necessary to delineate the exact role of each receptor in regulating adipocyte metabolism.
One surprising finding is that PGE2 recapitulated the decrease of FAS activity and expression by COX inhibition but that combined PGE2 and celecoxib treatments further decreased FAS. These results may be due to and complicated by changes in EP receptor expression and signaling with PGE2 addition. It is also possible that the pathways mediating PGE2 effects via its receptors are different from those affected by COX inhibition. Further, COX inhibition was more potent than EPA in reducing FAS, possibly due to higher levels of the two PGE isoforms in the presence of EPA compared to control.
Overall, our studies demonstrated the ability to pharmacologically decrease production of PGE2 using the selective COX-2 inhibitor celecoxib, which resulted in a significant reduction in lipogenic enzyme activity. The use of EPA was also shown to result in lower production of PGE2 when compared to AA treatment. FAS mRNA expression was decreased by FA treatment in a manner similar to the PUFA effects seen in the liver [16, 27, 28]. Given that SREBP1c, an insulin responsive transcription factor, mediates PUFA regulation of hepatic lipogenic genes, it is possible that PUFA regulation of adipocyte metabolism modulates insulin sensitivity. Indeed, in the absence of insulin, our cells expressed significantly higher PGE2 levels than in the presence of insulin (data not shown).
Low PGE2 levels led to decreased lipogenesis and thus a reduction in PGE2 levels would result in less inhibition of lipolysis, which coupled with reported antilipogenic effects of EPA and other polyunsaturated fatty acids would still favorably impact adipose tissue levels and result in decreased adiposity.
Further experimentation is required to determine the mechanism of action of celecoxib and EPA versus PGE2 in adipose tissue. Additional data on the receptor affinities and actions of PGE3, although difficult to approach at this time given the lack of data in this area, are necessary in order to understand the mechanism mediating EPA and PGE3 effects on lipid metabolism.
This study also brings to light the need for further studies on the function and impact of PGE3 in adipocytes to determine if it does in fact elicit the same responses as PGE2. If this is the case, then it may indicate a point of diminishing benefit and the need to specify a consumption range instead of recommending minimum consumption levels. Understanding the mechanisms involved with EPA metabolism takes on further importance with the FDA move to allow qualified health claims for omega-3 fatty acids , as this will most certainly bring more attention to these fatty acids and increase their consumption even further.