Adipose triglyceride lipase (ATGL) is the key triacylglycerol hydrolase in 
adipocytes. The precise mechanisms by which ATGL action is regulated by lipid 
droplet (LD) coat proteins and responds to hormonal stimulation are incompletely 
defined. By combining usage of loss- and gain-of-function approaches, we sought 
to determine the respective roles of perilipin 1 and fat-specific protein 27 
(FSP27) in the control of ATGL-mediated lipolysis in adipocytes. Knockdown of 
endogenous perilipin 1 expression resulted in elevated basal lipolysis that was 
less responsive to β-adrenergic agonist isoproterenol. In comparison, depletion 
of FSP27 protein increased both basal and stimulated lipolysis with no 
significant impact on the overall response of cells to isoproterenol. In vitro 
assays showed that perilipin but not FSP27 was able to inhibit the 
triacylglycerol hydrolase activity of ATGL. Perilipin 1 also attenuated 
dose-dependent activation of ATGL by its Coactivator Comparative Gene 
identification-58. Accordingly, depletion of perilipin 1 and CGI-58 in 
adipocytes inversely affected basal lipolysis specifically mediated by 
overexpressed ATGL. Moreover, although depletion of perilipin 1 abolished the LD 
translocation of ATGL stimulated by isoproterenol, absence of FSP27 resulted in 
multilocularization of LDs along with increased LD presence of ATGL under both 
basal and stimulated conditions. Interestingly, knockdown of ATGL expression 
increased LD size and decreased LD number in FSP27-depeleted cells. Together, 
our results demonstrate that although FSP27 acts to constitutively limit the LD 
presence of ATGL, perilipin 1 plays an essential role in mediating the response 
of ATGL action to β-adrenergic hormones.