The novel adipokine acylation stimulating protein (ASP) is involved in lipid metabolism and obesity-related disorders. Adipophilin and perilipin, two members of the lipid droplet protein family, participate not only in fat storage within adipocytes, but also in ectopic lipid deposition in the form of cytoplasmic triglyceride (TG) droplets within many types of mammalian cells. During differentiation to mature adipocytes, mechanisms controlling the synthesis and turnover of these lipid droplet proteins are only partially understood, the mechanisms regulating gene/protein expression as yet unidentified. In our previous study, ASP has been shown to regulate adipophilin and perilipin expression to facilitate TG synthesis during 3T3-L1 cell differentiation. Our aim in this study was to provide insight into the physiological importance of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in ASP-triggered alteration of adipophilin and perilipin expression. We found that acute (2.5 h) inhibition of PLC or PI3K results in a decrease in mRNA and protein of perilipin and adipophilin at any time during differentiation. The fact that there is such a rapid change even with mRNA levels suggests a rapid turnover of both mRNA and protein independent of a direct ASP effect. Also, the presence of these inhibitors blocked the ASP stimulatory effects with a maximal decrease in gene and protein expression of adipophilin (-45% and -60%, respectively, P < 0.01) and perilipin (-96% and -63%, respectively, P < 0.01 and P < 0.05). These findings provide further understanding of the adipogenic properties of ASP in adipocytes. J. Cell. Biochem. 112: 1622-1629, 2011. (C) 2011 Wiley-Liss, Inc.