However, the exact contribution of Rab18 to lipid metabolism and the mechanisms regulating Rab18 function in adipose tissue remain to be elucidated. In the present study, we show that, in addition to mediating b-adrenergic action in adipocytes, Rab18 is a downstream effector of the metabolic changes induced by insulin in this cell type. Our data provide novel experimental evidence supporting the involvement of this GTPase as a key mediator in the bidirectional trafficking of lipids between LDs and the ER. Finally, we explore the regulation of Rab18 GSK1120212expression in human adipose tissue as a function of sex, adipose tissue localization and obesity. In order to ascertain the specific contribution of Rab18 to adipocyte function, we first studied how different extracellular stimuli known to control lipid metabolism affect Rab18 production and its subcellular localization in 3T3-L1 adipocytes. Regarding Rab18 expression, 24-h treatments with either 100 nM insulin or 10 mM isoproterenol significantly increased Rab18 mRNA levels, which accounted for by 183% and 108% above baseline levels, respectively. Other treatments, such as 100 nM dexamethasone and 10 nM GH,ICG-001 company also tended to increase Rab18 gene expression, although these effects were not statistically significant. Finally, exposure of 3T3-L1 adipocytes to 4.8 nM pituitary adenylate-cyclase activating polipeptide-38 or 10 mM rosiglitazone did not alter Rab18 transcript levels. In view of these results, we chose the strongest inductors of Rab18 expression to explore their effects on Rab18 protein content. As depicted in Fig. 2B, 24-h treatment with either 100 nM insulin or 10 mM isoproterenol elicited increases in Rab18 protein content of 38% and 59% as compared to non-stimulated conditions, respectively. These data indicate that Rab18 production is regulated by specific regulatory inputs reaching the adipocytes and suggest that the GTPase may form part of the intracellular machinery activated by such factors. Previously, several studies have reported that Rab18 localizes at the surface of LDs and that isoproterenol treatment increases this association. Inasmuch as in the present work we have found that, like isoproterenol, insulin modulates Rab18 production in adipocytes, we asked whether this hormone could also affect intracellular localization of Rab18. To this end, 3T3-L1 adipocytes were subjected to a 4-h treatment with 100 nM insulin and co-immunostained with antibodies against Rab18 and the LD-surface resident, non-exchangeable protein perilipin, and visualized under a confocal microscope. As a positive control, cells were treated with 10 mM isoproterenol and processed for immunocytochemistry.