The effect of insulin and isoproterenol on Rab18 association with LDs was also explored using subcellular fractionation in a sucrose density gradient followed by immunoblotting of the resulting fractions. Under basalSB203580 conditions, the Rab18 immunosignal was mainly localized in the perilipin-enriched and microsomal fractions, although a degree of immunoreactivity was also noticeable in some cytosolic fractions. After insulin treatment, Rab18 immunolabeling was 43% higher in the perilipin-enriched fractions, whereas the amount of protein present in the microsomal fraction remained the same as in control conditions. In addition, insulin provoked a slight decrease in Rab18 immunoreactivity in cytosolic fractions. Regarding isoproterenol, this treatment induced a 48% increase in Rab18 immunoreactivity associated with perilipin-enriched fractions, similar to that previously reported, and a concomitant decrease in the microsomal and cytosolic fractions. In sum, these findings indicate that, as inSAR131675 isoproterenol-induced b-adrenergic receptor activation, the intracellular signaling pathway initiated by insulin involves Rab18 recruitment to the surface of LDs, which further supports the notion of a role for this GTPase in the metabolic response of 3T3-L1 adipocytes to this hormone. We next examined the intracellular signaling pathways that mediate the effect of insulin and isoproterenol in Rab18 association to LDs. In adipocytes, most of the metabolic actions of insulin are mediated by activation of phosphatidilinositol-3- kinase that phosphorylates Akt, the latter being responsible for the activation of various different substrates. We therefore investigated whether the increase in Rab18 at the surface of LDs induced by insulin is a consequence of activation of PI3K/Akt by quantifying the degree of colocalization between Rab18 and perilipin immunolabeling in cells treated with insulin in the presence of the PI3K blocker wortmannin. This showed that in the presence of wortmannin, insulin was prevented from increasing colocalization of Rab18 and perilipin at the surface of LDs. These data indicate that insulin exerts its effect on the intracellular localization of Rab18 via activation of the PI3K/Akt signaling cascade. In the case of isoproterenol, its effect on lipolysis in adipocytes is mediated by activation of b-adrenergic receptors, which initiates the adenylate cyclase /cAMP/ protein kinase A pathway. PKA phosphorylates hormone sensitive lipase, which then translocates to the LD surface and triggers the enzymatic reactions that lead to fatty acid hydrolysis. In the current work, we demonstrate that the isoproterenol-induced effect on Rab18 localization is mediated by activation of the AC/cAMP/PKA pathway, inasmuch as blockade of either AC by treating cells with MDL 12,330A or PKA by using H89 prior to isoproterenol administration significantly decreased colocalization of Rab18 and perilipin immunosignals on the LD surface, reaching levels comparable to those obtained in non-treated cells.