Abstract

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.Hormone-sensitive lipase is a broadly expressed enzyme that mediates the hydrolysis of triacylglycerols in adipose cells of animals, leading to the release of fatty acids which are transported in the plasma to supply the energy needs of various tissues (1). This lipolytic process is under strict hormonal and neural regulation, to guarantee an appropriate supply of fatty acids to the peripheral tissues according to the metabolic needs at each moment. Activation of the lipolytic process is mediated by cAMP-dependent protein kinase (PKA), 1 which is acutely stimulated by hormones that elevate cAMP, like catecholamines, corticotropin, glucagon, and others (2).HSL contains three sites for PKA serine phosphorylation (Ser-563, Ser-659, and Ser-660) (3). Phosphorylation of HSL in vitro with exogenous PKA causes a modest (2-fold) activation of HSL, and mututational analysis has revealed that phosphorylation of Ser-659 or Ser-660, but not Ser-563, are required for this modest activation of HSL in vitro (3). However, this meager increase in the specific activity of HSL cannot account for the 30 -100-fold increases in lipolysis observed upon elevation of PKA activity in intact cells. Rather, we have proposed that PKA-mediated translocation of HSL from the cytosol to the surface of lipid droplets more likely explains the large cellular response to lipolytic stimuli (4 -6). Furthermore, while PKAmediated lipolysis has long been thought to be attributed solely to HSL, more recent developments point to a role for the perilipins in lipolytic activation. Perilipin A is an adipocyte protein that coats the lipid storage droplet (7) and contains six sites for PKA phosphorylation (8), most of which are phosphorylated upon lipolytic stimulation. In a separate paper (9), we demonstrate that the interaction of HSL with the intracellular neutral lipid droplets is cri...

No abstract

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.