Cyclooxygenase 2 Regulates Isoprenaline Induced Adipolysis In Brown Adipocytes

Abstract

PURPOSE: By converting fat into heat, brown adipocytes (BAC) play as an energy expenditure in mammalian. Cyclooxygenase 2 (COX2) is a key factor of Uncoupling protein 1 (UCP1) synthesis which is essential for dissipates energy into heat via adipolysis. Isoprenaline (ISO) can induce adipolysis in brown adipocytes (BAC); however, whether COX2 takes part in ISO induced adipolysis remains unclear. METHODS: C3H10T1/2 cells were differentiated into brown adipocytes. BACs were treated with 10μM ISO or 10μM ISO and 100μM NS-398 (COX2 inhibitor) for 0 (as control), 1, 3, 6, 12 or 24 hours. For lipid droplet (LP) size analysis, cells were fixed and stained with Oil Red O. Images were captured with a Leica CTR 4000 microscope with 10x objectives. All images were thresholded for LPs signal and watershed for individual LP size analysis using Image J. Data were loaded into GraphPad Prism 8 for LP size analysis and size frequency distribution. BACs were collected for UCP1 expression detection after 6-hour-incubation (beta tubulin as loading control). All results are presented as means ± std. error of mean. Statistics were performed in SPSS using Student's t test. RESULTS: Average LP size decreased as ISO incubation time prolonged (113.67 ± 2.40, 115.69 ± 2.21, 83.61 ± 1.69, 35.00 ± 0.88, 31.60 ± 0.69, 34.28 ± 0.77 μm2 after 0, 1, 3, 6, 12, 24 hours incubation, respectively). Incubation of ISO and NS-398 slowed down the rate of LP size reduction (95.08 ± 1.65, 107.73 ± 2.26, 85.22 ± 1.87, 56.97 ± 1.49, 41.32 ± 0.87, 37.43 ± 0.65 μm2 after 0, 1, 3, 6, 12, 24 hours incubation, respectively; slope (-53.04 ± 1.21 vs. -65.45 ± 1.33) LP size frequency distribution showed a shift of LP size towards smaller LPs following incubation with ISO. Inhibition of COX2 activity can delay the occurrence of left shift (became smaller) of LP size frequency distribution (12h vs. 3h). UCP1 expression were lower following ISO and NS-398 incubation than those treated with ISO only (0.74 ± 0.08 vs. 1.00 ± 0.00, p<0.05). CONCLUSION: COX2 inhibition can repress ISO induced adipolysis in BACs and may related to a decrease in UCP1 expression. Funded by FRF for the Central Universities of China (2018GJ017)