Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets

BACKGROUND Adipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation. METHODS C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation. RESULTS The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β. CONCLUSION This in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.