Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120–220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120–220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.     

脂肪组织甘油三酯水解酶参与脂肪分解调控

循环中游离脂肪酸增高与肥胖、胰岛素抵抗和2型糖尿病密切相关,其主要来源于脂肪细胞内甘油三酯水解.调控脂肪分解的脂肪酶主要包括激素敏感脂肪酶(hormone-sensitive lipase,HSL)和最近发现的脂肪组织甘油三酯水解酶(adipose triglyceride lipase,ATGL),后者主要分布在脂肪组织,特异水解甘油三酯为甘油二酯,其转录水平受多种因素调控.CGI-58(属于α/β水解酶家族蛋白),可以活化ATGL,基础条件下该蛋白和脂滴包被蛋白(perilipin)紧密结合于脂滴表面,蛋白激酶A激活刺激脂肪分解时,CGI-58与perilipin分离,进而活化ATGL.     

Relative contribution of adipose triglyceride lipase and hormone-sensitive lipase to tumor necrosis factor-α (TNF-α)-induced lipolysis in adipocytes

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to β-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis.     

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.     

Eicosapentaenoic acid inhibits tumour necrosis factor-α-induced lipolysis in murine cultured adipocytes

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with beneficial effects in obesity and insulin resistance. High levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α) in obesity promote lipolysis in adipocytes, leading to the development of insulin resistance. Thus, the aims of the present study were to analyze the potential antilipolytic properties of EPA on cytokine-induced lipolysis and to investigate the possible mechanisms involved. The EPA effects on basal and TNF-α-induced lipolysis were determined in both primary rat and 3T3-L1 adipocytes. Treatment of primary rat adipocytes with EPA (100 and 200 μM) significantly decreased basal glycerol release (P<.01) and prevented cytokine-induced lipolysis in a dose-dependent manner (P<.001). Moreover, EPA decreased TNF-α-induced activation of nuclear factor-κB and extracellular-related kinase 1/2 phosphorylation. In addition, the antilipolytic action of EPA was stimulated by the AMP-kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside and blocked by the AMPK-inhibitor compound C. Moreover, we found that EPA stimulated hormone-sensitive lipase (HSL) phosphorylation on serine-565, which further supports the involvement of AMPK in EPA's antilipolytic actions. Eicosapentaenoic acid treatment (24 h), alone and in the presence of TNF-α,? also decreased adipose triglyceride lipase (ATGL) protein content in cultured adipocytes. However, oral supplementation with EPA for 35 days was able to partially reverse the down-regulation of HSL and ATGL messenger RNA observed in retroperitoneal adipose tissue of high-fat-diet-fed rats. These findings suggest that EPA inhibits proinflammatory cytokine-induced lipolysis in adipocytes. This effect might contribute to explain the insulin-sensitizing properties of EPA.     

Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.     

Sympathetic tone dictates the impact of lipolysis on FABP4 secretion

Levels of circulating fatty acid binding protein 4 (FABP4) protein are strongly associated with obesity and metabolic disease in both mice and humans, and secretion is stimulated by β-adrenergic stimulation both in vivo and in vitro. Previously, lipolysis-induced FABP4 secretion was found to be significantly reduced upon pharmacological inhibition of adipose triglyceride lipase (ATGL) and was absent from adipose tissue explants from mice specifically lacking ATGL in their adipocytes (ATGL^AdpKO). Here, we find that upon activation of β-adrenergic receptors in vivo, ATGL^AdpKO mice unexpectedly exhibited significantly higher levels of circulating FABP4 as compared with ATGL^fl/fl controls, despite no corresponding induction of lipolysis. We generated an additional model with adipocyte-specific deletion of both FABP4 and ATGL (ATGL/FABP4^AdpKO) to evaluate the cellular source of this circulating FABP4. In these animals, there was no evidence of lipolysis-induced FABP4 secretion, indicating that the source of elevated FABP4 levels in ATGL^AdpKO mice was indeed from the adipocytes. ATGL^AdpKO mice exhibited significantly elevated corticosterone levels, which positively correlated with plasma FABP4 levels. Pharmacological inhibition of sympathetic signaling during lipolysis using hexamethonium or housing mice at thermoneutrality to chronically reduce sympathetic tone significantly reduced FABP4 secretion in ATGL^AdpKO mice compared with controls. Therefore, activity of a key enzymatic step of lipolysis mediated by ATGL, per se, is not required for in vivo stimulation of FABP4 secretion from adipocytes, which can be induced through sympathetic signaling.     

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL''s C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.     

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.     

Regulation of triglyceride metabolism. IV. Hormonal regulation of lipolysis in adipose tissue

Triacylglycerol (TAG) stored in adipose tissue can be rapidly mobilized by the hydrolytic action of lipases, with the release of fatty acids (FA) that are used by other tissues during times of energy deprivation. Unlike synthesis of TAG, which occurs not only in adipose tissue but also in other tissues such as liver for very-low-density lipoprotein formation, hydrolysis of TAG, lipolysis, predominantly occurs in adipose tissue. Until recently, hormone-sensitive lipase was considered to be the key rate-limiting enzyme responsible for regulating TAG mobilization. However, recent studies on hormone-sensitive lipase-null mice have challenged such a concept. A novel lipase named desnutrin/ATGL has been recently discovered to play a key role in lipolysis in adipocytes. Lipolysis is under tight hormonal regulation. Although opposing regulation of lipolysis in adipose tissue by insulin and catecholamines is well understood, autocrine/paracrine factors may also participate in its regulation. Intricate cooperation of these endocrine and autocrine/paracrine factors leads to a fine regulation of lipolysis in adipocytes, needed for energy homeostasis. In this review, we summarize and discuss the recent progress made in the regulation of adipocyte lipolysis.     

Chronic TNFα and cAMP pre-treatment of human adipocytes alter HSL, ATGL and perilipin to regulate basal and stimulated lipolysis

We examined the effects of chronic TNFα and dibutyryl-cAMP (Db-cAMP) pre-treatment on the lipolytic machinery of human hMADS adipocytes. TNFα decreased adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content and triglycerides (TG)-hydrolase activity but increased basal lipolysis due to a marked reduction in perilipin (PLIN) protein content. Conversely, Db-cAMP increased ATGL and HSL protein content but prevented PLIN phosphorylation, the net result being accentuated basal lipolysis. In forskolin-stimulated conditions, TNFα and Db-cAMP pre-treatment decreased stimulated TG-hydrolase activity and impaired PLIN phosphorylation. Together, this resulted in a severely attenuated response to forskolin-stimulated lipolysis.     

Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype

While fatty acids (FAs) released by white adipose tissue (WAT) provide energy for other organs, lipolysis is also critical in brown adipose tissue (BAT), generating FAs for oxidation and UCP-1 activation for thermogenesis. Here we show that adipose-specific ablation of desnutrin/ATGL in mice converts BAT to a WAT-like tissue. These mice exhibit severely impaired thermogenesis with increased expression of WAT-enriched genes but decreased BAT genes, including UCP-1 with lower PPARα binding to its promoter, revealing the requirement of desnutrin-catalyzed lipolysis for maintaining a BAT phenotype. We also show that desnutrin is phosphorylated by AMPK at S406, increasing TAG hydrolase activity, and provide evidence for increased lipolysis by AMPK phosphorylation of desnutrin in adipocytes and in?vivo. Despite adiposity and impaired BAT function, desnutrin-ASKO mice have improved hepatic insulin sensitivity with lower DAG levels. Overall, desnutrin is phosphorylated/activated by AMPK to increase lipolysis and brings FA oxidation and UCP-1 induction for thermogenesis.     

Lipolysis in adipocytes

Lipolysis in adipocytes, the hydrolysis of triacylglycerol (TAG) to release fatty acids (FAs) and glycerol for use by other organs, is a unique function of white adipose tissue. Lipolysis in adipocytes occurs at the surface of cytosolic lipid droplets, which have recently gained much attention as dynamic organelles integral to lipid metabolism. Desnutrin/ATGL is now established as a bona fide TAG hydrolase and mutations in human desnutrin/ATGL/PNPLA2, as well as in its activator, comparative gene identification 58, are associated with Neutral Lipid Storage Disease. Furthermore, recent identification of AdPLA as the major adipose phospholipase A₂, has led to the discovery of a dominant autocrine/paracrine regulation of lipolysis through PGE₂. Here, we review emerging concepts in the key players in lipolysis and the regulation of this process. We also examine recent findings in mouse models and humans with alterations/mutations in genes involved in lipolysis and discuss activation of lipolysis in adipocytes as a potential therapeutic target.     

Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Lipolysis in hMADS Adipocytes

Lipolysis is the catabolic pathway by which triglycerides are hydrolyzed into fatty acids. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) have the capacity to hydrolyze in vitro the first ester bond of triglycerides, but their respective contributions to whole cell lipolysis in human adipocytes is unclear. Here, we have investigated the roles of HSL, ATGL, and its coactivator CGI-58 in basal and forskolin-stimulated lipolysis in a human white adipocyte model, the hMADS cells. The hMADS adipocytes express the various components of fatty acid metabolism and show lipolytic capacity similar to primary cultured adipocytes. We show that lipolysis and fatty acid esterification are tightly coupled except in conditions of stimulated lipolysis. Immunocytochemistry experiments revealed that acute forskolin treatment promotes HSL translocation from the cytosol to small lipid droplets and redistribution of ATGL from the cytosol and large lipid droplets to small lipid droplets, resulting in enriched colocalization of the two lipases. HSL or ATGL overexpression resulted in increased triglyceride-specific hydrolase capacity, but only ATGL overexpression increased whole cell lipolysis. HSL silencing had no effect on basal lipolysis and only partially reduced forskolin-stimulated lipolysis. Conversely, silencing of ATGL or CGI-58 significantly reduced basal lipolysis and essentially abolished forskolin-stimulated lipolysis. Altogether, these results suggest that ATGL/CGI-58 acts independently of HSL and precedes its action in the sequential hydrolysis of triglycerides in human hMADS adipocytes.Adipose tissue fat stores in humans are mainly dependent upon fatty acid (FA)² supply, FA esterification to triglycerides (TG), and TG breakdown, or lipolysis. Adipose tissue lipolysis is governed by three lipases. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) both have the capacity to initiate TG degradation by cleaving the first ester bond, but HSL is unique in its capacity to break down the second ester bond, converting diglycerides (DG) to monoglycerides (1–3). The non-rate-limiting monoglyceride lipase completes lipolysis by cleaving the last ester bond from a monoglyceride molecule, leading to glycerol release (4). Adipose tissue lipolysis has received much attention over the past 10 years because of its altered regulation in obesity (5).HSL resides freely in the cytosol and can associate with lipid droplets (LD). It is regulated by hormones such as catecholamines, insulin, and natriuretic peptides. Catecholamines bind to β-adrenoceptors on adipocyte cell membranes and activate cyclic AMP-dependent protein kinase. Similarly, natriuretic peptides bind to type A receptors and activate cyclic GMP-dependent protein kinase (6). The protein kinase action in stimulated lipolysis is 2-fold: 1) phosphorylation of HSL, leading to its translocation from the cytosol to LD (7, 8), and 2) phosphorylation of perilipin A (6, 9, 10), the predominant perilipin isoform in adipocytes, enhancing interaction between HSL and LD. The importance of HSL activity in stimulating complete lipolysis is indisputable, particularly given its unique capacity to hydrolyze DG. However, lipolysis is not exclusively dependent upon HSL because HSL null mice revealed residual TG lipase activity in adipose tissue (2, 11). Another adipose tissue lipase was identified (3, 12, 13). ATGL, also known as desnutrin or patatin-like phospholipase domain-containing protein 2, shows affinity toward TG only (3, 14). ATGL is activated by CGI-58, an esterase/thioesterase/lipase subfamily protein devoid of TG hydrolase enzymatic activity (15, 16). The role of HSL and ATGL has been investigated in murine fat cell lipolysis, but the relative importance of these lipases in basal and protein kinase A-stimulated human fat cell lipolysis has remained elusive.Increased fat mass is associated with defects in adipose tissue metabolism. In obesity, resistance to catecholamine-induced lipolysis is observed (17–19). This inhibition of lipolysis may be naturally occurring as an adaptive protective mechanism to minimize FA release and its deleterious consequences on metabolism. Indeed, decreased expression of HSL and ATGL has been observed in isolated adipocytes and differentiated preadipocytes of obese subjects and adipose tissue of insulin-resistant subjects, respectively (20–23). However, by virtue of its mass, adipose tissue basal lipolysis elevates circulating levels of FAs in obese subjects, thereby increasing the risk of insulin resistance. Therefore, the use of pharmacological lipid-lowering agents that act through inhibition of lipolysis has been a promising research avenue leading to the development of several series of HSL inhibitors (24).Herein, we sought to examine the respective contributions of HSL and ATGL to lipolysis and re-esterification in fat cells derived from human adipose tissue derived-multipotent stem cells (termed hMADS cells). These cells, which exhibit at a clonal level normal karyotype, self-renewal ability, and no tumorigenicity, are able to differentiate into functional adipocytes (25, 26). We investigated the localization of HSL and ATGL in basal and stimulated lipolytic conditions and studied lipase activities and whole cell lipolysis in adipocytes with altered expression levels of HSL, ATGL, and its coactivator CGI-58. Our results provide novel insights into ATGL localization and its critical role with coactivator CGI-58 in DG provision to HSL during basal and stimulated lipolysis.     

Cytoplasmic receptor-interacting protein 140 (RIP140) interacts with perilipin to regulate lipolysis

Receptor-interacting protein 140 (RIP140) is abundantly expressed in mature adipocyte and modulates gene expression involved in lipid and glucose metabolism. Protein kinase C epsilon and protein arginine methyltransferase 1 can sequentially stimulate RIP140 phosphorylation and then methylation, thereby promoting its export to the cytoplasm. Here we report a lipid signal triggering cytoplasmic accumulation of RIP140, and a new functional role for cytoplasmic RIP140 in adipocyte to regulate lipolysis. Increased lipid content, particularly an elevation in diacylglycerol levels, promotes RIP140 cytoplasmic accumulation and increased association with lipid droplets (LDs) by its direct interaction with perilipin. By interacting with RIP140, perilipin more efficiently recruits hormone-sensitive lipase (HSL) to LDs and enhances adipose triglyceride lipase (ATGL) forming complex with CGI-58, an activator of ATGL. Consequentially, HSL can more readily access its substrates, and ATGL is activated, ultimately enhancing lipolysis. In adipocytes, blocking cytoplasmic RIP140 accumulation reduces basal and isoproterenol-stimulated lipolysis and the pro-inflammatory potential of their conditioned media (i.e. activating NF-κB and inflammatory genes in macrophages). These results show that in adipocytes with high lipid contents, RIP140 increasingly accumulates in the cytoplasm and enhances triglyceride catabolism by directly interacting with perilipin. The study suggests that reducing nuclear export of RIP140 might be a useful means of controlling adipocyte lipolysis.     

Adipose-selective overexpression of ABHD5/CGI-58 does not increase lipolysis or protect against diet-induced obesity

Adipose triglyceride lipase (ATGL) catalyzes the first step of triacylglycerol hydrolysis in adipocytes. Abhydrolase domain 5 (ABHD5) increases ATGL activity by an unknown mechanism. Prior studies have suggested that the expression of ABHD5 is limiting for lipolysis in adipocytes, as addition of recombinant ABHD5 increases in vitro TAG hydrolase activity of adipocyte lysates. To test this hypothesis in vivo, we generated transgenic mice that express 6-fold higher ABHD5 in adipose tissue relative to wild-type (WT) mice. In vivo lipolysis increased to a similar extent in ABHD5 transgenic and WT mice following an overnight fast or injection of either a β-adrenergic receptor agonist or lipopolysaccharide. Similarly, basal and β-adrenergic-stimulated lipolysis was comparable in adipocytes isolated from ABHD5 transgenic and WT mice. Although ABHD5 expression was elevated in thioglycolate-elicited macrophages from ABHD5 transgenic mice, Toll-like receptor 4 (TLR4) signaling was comparable in macrophages isolated from ABHD5 transgenic and WT mice. Overexpression of ABHD5 did not prevent the development of obesity in mice fed a high-fat diet, as shown by comparison of body weight, body fat percentage, and adipocyte hypertrophy of ABHD5 transgenic to WT mice. The expression of ABHD5 in mouse adipose tissue is not limiting for either basal or stimulated lipolysis.