Sandra C. Souza

Overexpression of Perilipin A and B Blocks the Ability of Tumor Necrosis Factor α to Increase Lipolysis in 3T3-L1 Adipocytes

Perilipins, a family of phosphoproteins, are specifically located at the surface of intracellular lipid (triacylglycerol) droplets, the site of lipolysis. Stimulation of lipolysis in 3T3-L1 adipocytes by tumor necrosis factor α (TNF-α) is associated with a decrease in total cellular expression of perilipin A and B, consistent with the hypothesis that a decrease in perilipin protein expression is required for TNF-α-induced lipolysis. Adenovirus-mediated overexpression of perilipin A or B maintains perilipin protein levels on the lipid droplet and blocks TNF-α-induced lipolysis. In contrast, overexpression of perilipin A or perilipin B does not inhibit isoproterenol-stimulated lipolysis and does not alter the isoproterenol-induced migration of perilipins from the lipid droplet. These results provide the first evidence of how perilipin functions and suggest that TNF-α regulates lipolysis, in part, by decreasing perilipin protein levels at the lipid droplet surface.

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes

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 (forskolin)-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 Peri 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 Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel “master regulator” of PKA-stimulated adipocyte lipolysis.

Perilipin Promotes Hormone-sensitive Lipase-mediated Adipocyte Lipolysis via Phosphorylation-dependent and -independent Mechanisms

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 perilipin null mice (Peri–/– 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.

AMP-activated Protein Kinase Is Activated as a Consequence of Lipolysis in the Adipocyte: POTENTIAL MECHANISM AND PHYSIOLOGICAL RELEVANCE*

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 physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (isoproterenol, forskolin, and isobutylmethylxanthine), which stimulate lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activation 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-stimulated lipolysis and AMPK activation but not that of PKA. Forskolin 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 isoproterenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activation of AMPK in adipocytes by cAMP-inducing agents is a consequence of lipolysis and not of PKA 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 acylation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis.

Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis

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 AMP-dependent 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 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301–517 (Ser433, Ser492, and Ser517). Peri 301–517 promoted 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 non-HSL, 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.

Regulation of adipocyte lipolysis by degradation of the perilipin protein: nelfinavir enhances lysosome-mediated perilipin proteolysis.

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.

Perilipin overexpression in mice protects against diet-induced obesity

Perilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specific aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholamine-stimulated lipolysis was decreased and glucose tolerance significantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our findings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications.

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting (∼70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase (∼3.0°C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.