SUN-119 Melatonin Increases 3T3-L1 Adipogenesis and Brown Adipocyte Characteristics

Abstract

Currently, mechanisms that promote the transformation from energy storing white adipose tissue (WAT) to energy expending brown adipose tissue (BAT) are being investigated. Melatonin has been a recent target for the enhancement of BAT in other studies. The hypothesis of our investigation is that melatonin will increase adipogenesis, and white adipocytes exposed to the melatonin treatment, will be induced to develop brown adipocyte characteristics. Melatonin treatment was for 10-11 days from Day 4 of differentiation of 3T3-L1 cells. After the treatment, cells were washed with PBS, fixed with 4% paraformaldehyde and stained with Oil Red O for 1 hour, followed by hematoxylin for 2 minutes for bright field imaging microscopy for adipocytes. Live cells were stained with mitotracker for 20 min at 37°C and viewed under confocal microscopy. At day 5-6, 3T3-L1 cells treated with 1mM of melatonin had slightly increased differentiation at 19% compared to control at 13% and 10µM treated cells at 9%. At day 9-10, 1mM treated cells had 60% differentiation, 10µM treated cells 28% and control 29%. When the results were analyzed, we observed that only the differences in differentiation between control and 1mM treated cells were statistically significant for Day 9-10 (p=0.04). Data obtained also provided a comparison between big lipid (WAT like) and small lipid (BAT like) droplet formation during melatonin treatment. At Day 9-10, the quantitation of small lipid droplet containing (90%) versus big lipid droplet containing (10%) adipocytes, using bright field images for the 1mM melatonin treatment was statistically significant (p=0.002). Data obtained from oil red staining also identified that 85% of the 1mM melatonin treated adipocytes had smaller lipid droplets and 15% big lipid droplets with high statistical significance (p<107). Additionally, 10µM melatonin treated cells showed statistically significant (p<107) smaller lipid droplet containing adipocytes (60%) compared to big lipid droplet containing adipocytes (40%). In contrast, control (0µM) had 51% of the adipocytes with small lipid droplets and 49% adipocytes with big lipid droplets. Preliminary experiments showed a significant increase in adipogenesis between control and melatonin treated 3T3-L1 cells, with more brown adipocyte like characteristics. Preliminary confocal data also suggest that there are more active mitochondria containing adipocytes (Brown adipocyte like) after melatonin treatment. Future experiments will be performed to determine if melatonin is causing significant effects on regulation of mitochondrial gene expression and function during brown adipocyte transformation.