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AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its phys- iological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (iso- proterenol, forskolin, and isobutylinethyixanthine), which stimu- late lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activa- tion by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stim- ulated lipolysis and AMPK activation but not that of PKA. Forsko- lin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isopro- terenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activa- tion of AMPK in adipocytes by cAMP-inducing agents is a conse- quence of lipolysis and not of PICA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acy- lation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis. [ABSTRACT FROM AUTHOR]

A decrease in the lipid droplet-associated protein perilipin may constitute a mechanism for enhanced adipocyte lipolysis under nonstimulated (basal) conditions, and increased basal lipolysis has been linked to whole body metabolic dysregulation. Here we investigated whether the lipolytic actions of the human immunodeficiency virus protease inhibitor, nelfinavir, are mediated by decreased perilipin protein content and studied the mechanisms by which it occurs. Time course analysis revealed that the decrease in perilipin protein content preceded the increase in lipolysis. A causative relationship was suggested by demonstrating that nelfinavir potently increased lipolysis in adipocytes derived from mouse embryonal fibroblasts expressing perilipin but not in mouse embryonal fibroblast adipocytes devoid of perilipin and that adenoviral mediated overexpression of perilipin in 3T3-L1 adipocytes blocked the lipolytic actions of nelfinavir. Nelfinavir did not alter mRNA content of perilipin but rather decreased perilipin proteins t½ from >70 to 12 h. Protein degradation of perilipin in both control and nelfinavir-treated adipocytes could be prevented by inhibiting lysosomal proteolysis using leupeptin or NH4Cl but not by the proteasome inhibitor MG-132. We propose that proteolysis of perilipin involving the lysosomal protein degradation machinery may constitute a novel mechanism for enhancing adipocyte lipolysis. [ABSTRACT FROM AUTHOR]

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormone-sensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Pun and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs. [ABSTRACT FROM AUTHOR]

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the ‘downstream’ lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (fonskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a ‘phosphomimetic’ (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Pen A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Pen A regulates ATGL-dependent lipolysis and identify serine 517 as the Pen A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphonylated or phosphomimetic serine 517. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase (HSL) is the predominant lipase effector of catecholamine-stimulated lipolysis in adipocytes. HSL-dependent lipolysis in response to catecholamines is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin A (Peri A), an essential lipid droplet (LD)-associated protein. It is believed that perilipin phosphorylation is essential for the translocation of HSL from the cytosol to the LD, a key event in stimulated lipolysis. Using adipocytes retrovirally engineered from murine embryonic fibroblasts of periipin null mice (Pen-/- MEF), we demonstrate by cell fractionation and confocal microscopy that up to 50% of cellular HSL is LD-associated in the basal state and that PKA-stimulated HSL translocation is fully supported by adenoviral expression of a mutant perilipin lacking all six PKA sites (Peri AΔ1-6). PKA-stimulated HSL translocation was confirmed in differentiated brown adipocytes from perilipin null mice expressing an adipose-specific Peri AΔ1-6 transgene. Thus, PKA-induced HSL translocation was independent of perilipin phosphorylation. However, Peri AΔ1-6 failed to enhance PKA-stimulated lipolysis in either MEF adipocytes or differentiated brown adipocytes. Thus, the lipolytic action(s) of HSL at the LD surface requires PKA-dependent perilipin phosphorylation. In Peri-/- MEF adipocytes, PKA activation significantly enhanced the amount of HSL that could be cross-linked to and co-immunoprecipitated with ectopic Peri A. Notably, this enhanced cross-linking was blunted in Peri-/- MEF adipocytes expressing Peri AΔ1-6. This suggests that PKA-dependent perilipin phosphorylation facilitates (either direct or indirect) perilipin interaction with LD-associated HSL. These results redefine and expand our understanding of how perilipin regulates HSL-mediated lipolysis in adipocytes. [ABSTRACT FROM AUTHOR]

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMPdependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser[sup 81], Ser[sup 222], and Ser[sup 276]). PKAstimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser[sup 433], Ser[sup 492], and Ser[sup 517]). Peri 301-517 prorooted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and nonHSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity. [ABSTRACT FROM AUTHOR]