Moreover, we have identified the intracellular pathways responsible for the Rab18 association with LDs and provided experimental evidence that this process entails rapprochement of LDs to ER membranes. Finally, we have demonstrated, for the first time, the presence of Rab18 in human adipose tissue and showed that the expression of this GTPase is correlated to obesity. Studies in differentiating 3T3-L1 cells, the cell line most commonly used to study adipogenesis, revealed that Rab18 mRNA reached a maximal level on day 3 of differentiation, coinciding with the appearance of Crizotinib late differentiation markers, which are responsible for the maintenance of the adipocyte phenotype in 3T3-L1 cells. In addition, Rab18 protein content progressively increased during differentiation. These data indicate that, as previously suggested for other Rab proteins, Rab18 may play a role in the differentiation of 3T3-L1 fibroblasts to mature adipocytes. Notably, we found that insulin, a key component of the hormonal cocktail employed to induce this process in 3T3-L1 adipocytes, up-regulated Rab18 expression and increased Rab18 protein content in these cells. Furthermore, this hormone also triggered Rab18 association with LDs, a process that seems to be mediated by activation of the key upstream regulator of the metabolic actions induced by insulin in adipocytes, PI3K. Similar to the pattern observed herein for Rab18, previous studies have reported that insulin induces other coating proteins to localize with LDs, including S3–12 and OXPAT. Moreover, it has been shown that insulin, via PI3K, induces the activation and intracellular redistribution in adipocytes of another member of the Rab family, Rab4,PI-103which is involved in GLUT-4 vesicle trafficking. These findings suggest that Rab proteins and, in particular Rab18, may be part of the intracellular machinery transducing the metabolic effects of insulin in this cell type. In line with this notion, the increase in Rab18 association with LDs induced by insulin concurred with the stimulation of intracellular TAG accumulation evoked by this hormone and, in addition, this later effect was inhibited in Rab18-silenced cells. These data support the view that insulin-induced recruitment to LDs may contribute to the lipogenic action of this hormone. A role for Rab18 in promoting lipogenesis is further backed by our observations of the increased lipogenic rate and LD size evoked by the overexpression of this GTPase in 3T3-L1 cells. Intriguingly, the effects of insulin on the expression and intracellular localization of Rab18 were strikingly similar to those induced by b-adrenergic receptor activation which, as is widely known and also shown in this study, has an opposite effect to that of insulin on lipid metabolism.