ABSTRACT Metabolic disorders related to obesity are largely dependent on adipose tissue hypertrophy, which involves adipocyte hypertrophy and increased adipogenesis. Adiposize is regulated by lipid accumulation as a result of increased lipogenesis (mainly lipid uptake in mature adipocytes) and reduced lipolysis. Using realtime 2D cell culture analyses of lipid uptake, we show 1) that high glucose concentration (4.5 g/L) was required to accumulate oleic acid increasing lipid droplet size until unilocularisation similar to mature adipocytes in few days, 2) oleic acid reduced Peroxisome-Proliferator Activated Receptor Gamma (PPARG) gene transcription and 3) insulin counteracted oleic acid-induced increase of lipid droplet size. Although the lipolytic activity observed in high versus low glucose (1g/L) conditions was not altered, insulin was found to inhibit oleic acid induced gene transcription required for lipid storage such as CIDEC (Cell Death Inducing DFFA Like Effectors) and G0S2 (G0 switch gene S2), possibly through PPARA activity. Although this signaling pathway requires more detailed investigation, the results point out the differential mechanisms involved in the pro-adipogenic effect of insulin in absence versus its protective effect on adiposity in presence of oleic acid uptake.