n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

Abstract

A dramatic rise in the incidence of obesity in the U.S. has accelerated the search for interventions that may impact this epidemic. One recently recognized target for such intervention is adipose tissue, which secretes a variety of bioactive substances including prostaglandins. Prostaglandin E₂ (PGE₂) has been shown to decrease lipolysis in adipocytes, but limited studies have explored alternative mechanisms by which PGE₂ might impact obesity, such as adipogenesis or lipogenesis. Studies conducted on ApcMin/+ mice indicated that selective inhibition of the cyclooxygenase (COX)-2 enzyme led to significant reductions in fatty acid synthase (FAS) activity in adipose tissue suggesting lipogenic effects of PGE₂. To further investigate whether these lipid mediators directly regulate lipogenesis, we used 3T3-L1 adipocytes to determine the impact of eicosapentaenoic acid (EPA) and celecoxib on PGE₂ formation and FAS used as a lipogenic marker. Both arachidonic acid (AA) and EPA dose-dependently increased PGE secretion from adipocytes. AA was expectedly more potent and exhibiting at 150 uM dose a 5-fold increase in PGE₂ secretion over EPA. Despite higher secretion of PGE by EPA and AA compared to control, neither PUFA significantly altered FAS activity. By contrast both AA and EPA significantly decreased FAS mRNA levels. Addition of celecoxib, a selective COX-2 inhibitor, significantly decreased PGE₂ secretion (p < 0.05) versus control, and also significantly decreased FAS activity (p < 0.05). Unexpectedly, the combination of exogenous PGE₂ and celecoxib further decreased the FAS activity compared to PGE₂ alone or untreated controls. In conclusion, EPA-mediated inhibition of AA metabolism did not significantly alter FAS activity while both AA and EPA significantly decreased FAS mRNA expression. COX-2 inhibition significantly decreased PGE₂ production resulting in a decrease in FAS activity and expression that was not reversed with the addition of exogenous PGE₂, suggesting an additional mechanism that is independent of COX-2.

Figures

Figure 1

Effects of piroxicam and PGE2 receptor agonists onadipose FAS activity in ApcMin/+ mice. Male C57BL/6J ApcMin/+ mice were maintained on the AIN-93G diet until approximately 80 days of age at which time they were randomly assigned to treatment groups. Treatments consisted of control, piroxicam (0.5 mg/mouse/day), EPR-A (16,16-dimethyl-PGE2 and 17-phenyl-trinor PGE2 -10 μg each), or piroxicam + EPR-A. Mice were sacrificed after 6 days of treatment and epididymal adipose tissue was harvested and snap frozen in liquid nitrogen. Tissue was homogenized in sucrose buffer and cytosolic extracts were analyzed for FAS activity using an activity assay as described in Materials and Methods. For treatments C and P + EPR-A n = 6; for P and EPR-A n = 5. Results represent the mean ± SEM. Values labeled with different letters are significantly different (p < 0.05). Values with the same letters do not differ significantly.

Figure 2

Dose-response effects of AA and EPA on PGE2 levels in 3T3-L1 adipocytes. 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of AA and EPA at 25, 50, 100, 200, and 500 μM concentrations. After 48 hours, media was removed and stored at -80°C until PGE2 levels were measured as described in the Materials and Methods. For treatments AA/EPA 500 n = 5; control (C) and AA/EPA 25 n = 10; AA/EPA 50, 100, 200 n = 15. Values labeled with different letters are significantly different (p < 0.05). Results represent the mean ± SEM. Values with the same letters do not differ significantly.

Figure 3

Effects of EPA and COX-2 inhibition on secreted PGE2 levels from 3T3-L1 adipocytes. 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of EPA (50 and 150 μM), EPA (150 μM) + CI (5 μM), CI (5 μM) and EPA (150 μM) in media without cells. After 48 hours, 2 mL of media was removed and stored at -80°C. Media PGE2 levels were analyzed using EIA as described in the Materials and Methods. Results represent the mean ± SEM with a number of treatments n = 3–4. Values labeled with different letters are significantly different (p < 0.05). Values with the same letters do not differ significantly. * Denotes culture media exposed to cell culture conditions without cells.

Figure 4

Effects of COX-2 inhibition and EPA addition on PGE2 levels (4A) and FAS activity (4B). 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of CI (1 μM), OA, EPA, AA (150 μM each treatment), and AA + EPA (75 μM each FA). After 48 hours, 2 mL of media was removed, and cells were scraped using 350 μL of sucrose buffer, sonicated, and centrifuged for 1 hour. Media and cytosolic extracts were stored at -80°C. Media PGE2 levels and FAS activity were measured as described in the Materials and Methods. For all treatments n = 10. Results represent the mean ± SEM. Values labeled with different letters are significantly different (p < 0.05). Values with the same letters do not differ significantly.

Figure 5

Effects of n3 and n6 PUFA and COX-2 inhibition on FAS mRNA expression. 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum-free media containing FA-free BSA. Fatty acids were incubated in media containing FA-free BSA for two hours at 37°C in a shaking water bath prior to treatment. Treatment consisted of CI (1 μM), EPA and AA (150 μM each treatment), and AA + EPA (75 μM each FA). After 48 hours, cells were scraped using 350 μL of Qiazol lysis reagent, and total RNA was extracted using the RNeasy™ lipid tissue midi kit (Qiagen) following the manufacturer's protocol. RNA was stored at -80° for further analysis. Real time RT-PCR analysis was performed as described in the materials and methods. For all treatments n = 6. Results represent the mean ± SEM. All values were significantly different than control, and values labeled with different letters are significantly different (p < 0.05) from each other. Values with the same letters do not differ significantly.

Figure 6

Effect of exogenous PGE2 and COX inhibition on adipocyte PGE2 secretion (6A) and FAS activity (6B). 3T3 L-1 adipocytes were grown 6–7 days post-confluence, and starved for 24 h in serum free media containing FA free BSA. Treatment consisted of CI (1 μM), PGE2 (300 pM), and CI + PGE2. After 48 hours, 2 mL of media was removed and cells were scraped using 350 μL of sucrose buffer, sonicated, and centrifuged for 1 hour. Media and cytosolic extract were stored at -80° Media PGE2 levels and FAS activity were measured as described in the Materials and Methods. For all treatments n = 8. Results represent the mean ± SEM. Values labeled with different letters are significantly different (p < 0.05). Values with the same letters do not differ significantly.