We recently reported that exposing white adipose cells to 4mM sodium acetate reduces the rate and total release of non-esterified fatty acids (NEFA) [1]. It was unclear from this study whether NEFA release was suppressed or whether acetate inhibited lipolysis. The aim of the current study was to examine whether acetate administration reduces the phosphorylation of hormone sensitive lipase (pHSL) in 3T3-L1 cells that were stimulated with the β-adrenergic receptor agonist, isoproterenol (ISO). The 3T3-L1 cell line was used throughout this study at day 9 post-differentiation. Cells were treated with a combination of the following: ISO (5µM), insulin (1µg/ml) and sodium acetate (4mM) for 3 hours. Under sodium acetate conditions cells were pre-treated (4mM) for 30 minutes. Samples of media were collected every 60 minute for a total duration of 180 minutes and analysed for NEFA and glycerol concentration. Cells for western blot analysis were lysed after 120 minutes. All samples were normalised to total protein content and statistical analysis was by non-parametric Friedman and Mann-Whitney tests. Over the 3 hour period, ISO stimulation of the cells resulted in a >2 fold increase in both NEFA (Fig. 1) and glycerol release and the greatest relative volume of pHSL (Fig. 2) at 120 minutes. Insulin with ISO resulted in a reduction in NEFA release at 120 and 180 minutes (P=0.010; (P=0.016, respectively) (Fig. 1). Incubation with acetate and ISO also reduced NEFA release at 120 and 180 minutes, compared with ISO ((P=0.004; (P=0.004, respectively). Glycerol release was found not to differ between treatment groups (data not shown). Under ISO stimulated lipolysis, the relative quantity of pHSL at serine residue 563 was lower in the presence of acetate, insulin and acetate plus insulin compared with ISO treatment alone (Fig. 2). These data support our previous findings that acetate reduces the appearance of NEFA under ISO stimulated lipolysis. The reduced accumulation of NEFA appears to be due to a down regulation of pHSL and thus lipolysis. The acute metabolic effects of increased acetate availability on lipolysis closely resemble those of insulin however; it is unlikely that the mechanism of reducing pHSL under these two conditions is the same. Future studies to identify the effects of an increase in acetate availability on other lipolytic proteins and secondary messengers upstream of pHSL are warranted.